WO2014048942A1

WO2014048942A1 - Probes for pten, pik3ca, met, and top2a, and method for using the probes

Info

Publication number: WO2014048942A1
Application number: PCT/EP2013/069896
Authority: WO
Inventors: Christopher LANIGAN; Larry Morrison; Bryce PORTIER; Raymond Tubbs
Original assignee: Ventana Medical Systems, Inc.; F. Hoffmann-La Roche Ag; Cleveland Clinic Found.
Priority date: 2012-09-25
Filing date: 2013-09-25
Publication date: 2014-04-03

Abstract

Disclosed herein are probes comprising a plurality of segments of uniquely specific nucleic acid sequences. In some examples, the probes are gene locus probes and/or chromosomal enumeration pericentromeric probes. In particular disclosed embodiments, the gene locus probe may be a nucleic acid probe that includes or consists of a nucleic acid sequence set forth as any one of SEQ ID NOs: 1-50. The disclosed probes are useful for detecting presence of a target nucleic acid in a sample. In some embodiments, the probes are useful for detecting presence of phosphatase and tensin homolog (PTEN), phosphatidylinositol 3-kinase p 1 10 (PIK3CA), Met proto-oncogene (MET), or topoisomerase II alpha (TOP2A) in a sample. The probes and probe sets also can be used to assess neoadjuvant breast cancer treatment prognosis.

Description

PROBES FOR PTEN, PIK3CA, MET, AND TOP2A,

AND METHOD FOR USING THE PROBES

FIELD

This disclosure relates to probes for in vitro detection of a target nucleic acid, a method for detecting nucleic acid target sequences (e.g., genomic DNA or RNA) using the probes, with a particular embodiment concerning identifying subjects likely to benefit from neoadjuvant breast cancer treatment.

RELATED APPLICATION

This application is related to subject matter disclosed in Assignee's co-pending

United States provisional application No. 61/645,347, filed on May 10, 2012, and entitled Uniquely Specific Probes for PTEN, PIK3CA, MET, TOP2a, and MDM2, which is incorporated herein by reference in its entirety. BACKGROUND

Molecular cytogenetics methods are based on hybridization of a nucleic acid probe to its complementary nucleic acid within a cell. A probe for a specific chromosomal region will recognize and hybridize to its complementary sequence on a metaphase chromosome or within an interphase nucleus (for example in a tissue sample). Probes have been developed for a variety of diagnostic and research purposes. Hybridization of chromosome or gene-specific probes has made possible detection of chromosomal abnormalities associated with numerous diseases and syndromes, including constitutive genetic anomalies, such as microdeletion syndromes, chromosome translocations, gene amplification and aneuploidy syndromes, neoplastic diseases, as well as pathogen infections. Most commonly, these techniques are applied to standard cytogenetic preparations on microscope slides. In addition, these procedures can be used on slides of formalin- fixed tissue, blood or bone marrow smears, and directly fixed cells or other nuclear isolates.

Many cancers are characterized by genetic changes that lead to aberrant control of cellular processes, or to uncontrolled growth and proliferation of cells. These genetic changes include gain or loss of function (for example, including amplification or deletion of all or a portion of a gene), gene rearrangement, and changes in sequence (for example, substitution, addition, or deletion or one or more bases). Such changes are known to occur in the genetic regions associated with various genes, including phosphatase and tensin homolog (PTEN), phosphatidylinositol 3-kinase pi 10 (PIK3CA), Met proto-oncogene

(MET), and topoisomerase II alpha (TOP2A) genes. Detection of genetic changes in these regions can provide diagnostic and prognostic information for patients and inform treatment decisions.

Breast cancer is one example where prognostic information is desirable to assess candidates for neoadjuvant treatments. Large breast tumors are difficult to excise. For appropriate candidates, neoadjuvant therapies, such as chemotherapy, immunotherapy, hormone therapy and radiation, can be used first to produce a surgically removable tumor. Neoadjuvant therapies also facilitate delineating tumor and surrounding tissue boundaries. Additionally, neoadjuvant therapy may reduce the extent of local surgery required for patients, so that surgeries like a radical mastectomy need not be carried out. Rather, the patient can undergo breast-conservation surgery, such as a quadrantectomy,

segmentectomy, or lumpectomy. Patient survival need not be jeopardized because the extent of local surgery does not appear to influence major patient outcomes, such as survival or disease-free survival.

However, not all patients respond, or respond well, to neoadjuvant therapy. Some patients do not tolerate the toxicity associated with chemotherapy, and potentially may react so adversely that further surgical treatments are precluded. Identifying subjects that are most likely to respond to neoadjuvant therapy spares patients from ineffective therapies and allows initiating the most suitable therapy at an earlier date. Accordingly, new methods for assessing response to neoadjuvant treatments are desirable.

SUMMARY

Certain disclosed embodiments concern a method for predicting pathologic response in patients being considered for neoadjuvant breast cancer treatment. The method comprises contacting a biological sample from a human patient with a gene locus probe selected from a probe to chromosome 7q31, a probe to chromosome 3q26, a probe to chromosome 10q23, a probe to chromosome 17q21, a chromosomal enumeration pericentromeric probe selected from a chromosome 7 pericentromeric probe, a chromosome 3 pericentromeric probe, a chromosome 10 pericentromeric probe, a chromosome 17 pericentromeric probe, or combinations thereof, under conditions sufficient to enable hybridization of the probe to target sequences; and analyzing probe hybridization information to facilitate determining neoadjuvant breast cancer treatment in the subject based upon the probe hybridization information. The method may further comprise performing immunohistochemistry analysis. The immunohistochemistry analysis can involve using a Ki-67 antibody to detect the Ki-67 protein. The gene locus probe used in the disclosed method may be selected from a probe to a region containing MET, a probe to a region containing PIK3CA, a probe to a region containing PTEN, a probe to a region containing TOP2A, or combinations thereof. In particular disclosed embodiments, the gene locus probe is an isolated nucleic acid probe, comprising: (a) a nucleic acid molecule consisting of a sequence according to any one of SEQ ID NOs: 1-20 and 31-50; or (b) a nucleic acid molecule consisting of at least 250 contiguous nucleotides of any one of SEQ ID NOs: 1-20 and 31-50. The gene locus probe may be an isolated nucleic acid probe, comprising: (a) a nucleic acid molecule having at least 90% sequence identity with a sequence according to any one of SEQ ID NOs: 1-20 and 31-50; or (b) a nucleic acid molecule having at least 90% sequence identity with at least 250 contiguous nucleotides of any one of SEQ ID NOs: 1-20 and 31-50. In other disclosed embodiments, the gene locus probe may be an isolated nucleic acid probe, comprising: (a) a nucleic acid molecule having at least 95% sequence identity with a sequence according to any one of SEQ ID NOs: 1-20 and 31-50; or (b) a nucleic acid molecule having at least 95% sequence identity with at least 250 contiguous nucleotides of any one of SEQ ID NOs: 1-20 and 31-50. In further embodiments, the gene locus probe may be an isolated nucleic acid probe, comprising: (a) a nucleic acid molecule having at least 99% sequence identity to a sequence according to any one of SEQ ID NOs; 1-20 and 31-50; or (b) a nucleic acid molecule having at least 99% sequence identity with at least 250 contiguous nucleotides of any one of SEQ ID NOs: 1-20 and 31-50.

Further embodiments of the disclosed method concern a gene locus probe that is a nucleic acid molecule with at least 90% sequence identity to at least 400 contiguous of any one of SEQ ID NOs: 1-20 and 31-50; or at least 90% sequence identity to at least 500 contiguous nucleotides of any one of SEQ ID NOs: 1-20 and 31-50; or at least 90% sequence identity to at least 1000 contiguous nucleotides of any one of SEQ ID NOs: 1-20 and 31-50; or at least 90% sequence identity to at least 2500 contiguous nucleotides of any one of SEQ ID NOs: 1-20 and 31-50.

As disclosed herein, the biological sample may be contacted with both the gene locus probe and the chromosomal enumeration pericentromeric probe independently or as a probe set. The probe set may further comprise a Ki-67 antibody. In particular disclosed embodiments, the probe set comprises MET + CEN7, Ki-67, TOP2A + CEN17, PTEN + CEN10, PIK3CA + CEN3, or combinations thereof.

The analyzing step of the disclosed method may comprise using MET/CEN7 gain or loss, AND Ki-67, or PIK3CA/CEN3 gain AND MET/CEN7 gain or loss, or

PIK3CA/CEN3 gain OR Ki-67, or PIK3CA/CEN3 gain AND Ki-67, or MET/CEN7 gain AND PTEN/CEN10 gain or loss, or MET/CEN7 gain and loss, and Ki-67, and

PIK3CA/CEN3 gain (at least 2 positive), or MET/CEN7 gain AND PTEN/CEN10 gain or loss to facilitate determining neoadjuvant breast cancer treatment.

According to the disclosed method, each probe can be separately detected. Also, the pathologic response may be complete response, or it may be partial response. The patient may be a HER2 -positive patient. In particular disclosed embodiments, the HER2- positive patient may be a gene amplification-positive patient or a HER-2 expression positive patient. The biological sample first is determined to be from a patient with HER2 elevated breast cancer. The biological sample may comprise a breast tissue biopsy, resection, or a cytology sample. The contacting step of the disclosed method may comprise performing FISH, CISH, SISH, IHC, and combinations thereof. Also, the probe may include a chromogenic label. The method may also comprise using unique chromogens for each probe used. The disclosed method is also capable of having the contacting step performed using an automated system.

In particular disclosed embodiments, the disclosed method further comprises determining a treatment protocol based on a prognosis, and treating the patient according to the protocol. In particular disclosed embodiments, the determining step may comprise selecting criteria by which to judge a low or high parameter state associated with pCR, testing the patient with the gene locus probe, chromosomal enumeration pericentromeric probe, or combinations thereof, and then obtaining a positive or negative response. The positive response indicates that the patient is likely to experience pathologic complete response, and the negative response indicates that the patient is unlikely to experience pathologic complete response. The criteria are selected from AUC from an ROC curve, DFI from an ROC curve, χ² probabilities, Fischer's Exact Test probabilities, optimal cutoff values, or combinations thereof. In particular disclosed embodiments, the AUC from an ROC curve, DFI from an ROC curve, χ2 probability, Fischer's Exact Test probabilities, optimal cut-off values, and combinations thereof are determined using a statistically significant sample for probe hybridization results. The ROC AUC may be 0.5 to 1.0, more typically the ROC AUC is 0.9 or greater. The sensitivity may range from about 0.8 to about 1.0. In particular disclosed embodiments, the specificity ranges from 0.5 to 1.0. The χ2 probability value may be 0.05 or less; more typically, the χ2 probability value ranges from 0.0000001 to 0.05. The optimal cut-off value may range from about 5% to about 70%; more typically, the optimal cut-off value ranges from about 8% to about 68%.

In particular disclosed embodiments, the hybridization information obtained in the method is (i) MET/CEN7 gain or loss, (ii) MET/CEN7 gain or loss AND Ki-67, each having a sensitivity of 1.00. In other disclosed embodiments, the hybridization information is (i) MET/CEN7 gain and loss, and Ki-67, and PIK3CA/CEN3 gain (at least two positives), and (ii) MET/CEN7 gain AND PTEN/CEN10 gain or loss, each having a specificity of 1.00. The method may also comprise using two factor combinations and the two factor combinations have an ROC AUC of greater than 0.90, a sensitivity of 0.8 or greater, a specificity of 0.8 or greater and a χ2 probability value of 0.00006 or less.

In certain disclosed embodiments, the method may further comprise using a first probe set comprising a first gene locus probe and a first chromosomal enumeration pericentromeric probe to perform probe hybridization analysis on a first biological sample; and using a second probe set comprising a second gene locus probe and a second chromosomal enumeration pericentromeric probe to perform probe hybridization analysis on a second biological sample.

Additionally, the disclosed method may further comprise contacting a second biological sample from the human patient with a gene locus probe selected from a probe to chromosome 7q31, a probe to chromosome 3q26, a probe to chromosome 10q23, a probe to chromosome 17q21 , a chromosomal enumeration pericentromeric probe, selected from a chromosome 7 pericentromeric probe, a chromosome 3 pericentromeric probe, a chromosome 10 pericentromeric probe, a chromosome 17 pericentromeric probe, or combinations thereof, under conditions sufficient to enable hybridization of the gene locus probe and/or the chromosomal enumeration pericentromeric probe to target sequences, wherein the gene locus probe and/or chromosomal enumeration pericentromeric probe used for the second biological sample are different from the gene locus probe and/or the chromosomal enumeration pericentromeric probe used for the first biological sample. The probes may be hybridized to the same specimen sample, and each labeled with a distinguishing label.

In particular disclosed embodiments, the method comprises the following steps, not in any particular order: obtaining a first biological sample from a human patient first determined to be from a patient with HER2 elevated breast cancer, the sample comprising a breast tissue biopsy, a breast cancer resection, or a cytology sample; contacting the first biological sample with a first probe set comprising at least a first gene locus probe selected from a probe to chromosome 7q31, a probe to chromosome 3q26, a probe to chromosome 10q23, a probe to chromosome 17q21, and a first chromosomal enumeration

pericentromeric probe, selected from a chromosome 7 pericentromeric probe, a chromosome 3 pericentromeric probe, a chromosome 10 pericentromeric probe, a chromosome 17 pericentromeric probe, or combinations thereof under conditions sufficient to enable hybridization of the probes to a target region; analyzing probe hybridization information to facilitate determining neoadjuvant breast cancer treatment in the subject based upon the probe hybridization information; determining neoadjuvant breast cancer treatment prognosis in the subject based upon the analysis of the probe hybridization information; determining a treatment protocol based on the prognosis; and treating the patient according to the protocol.

According to this method, a second biological sample from the human patient may be contacted with a second probe set comprising (i) a second gene locus probe selected from a probe to chromosome 7q31, a probe to chromosome 3q26, a probe to chromosome 10q23, a probe to chromosome 17q21, or combinations thereof, wherein the second gene locus probe is different from the first gene locus probe, and (ii) a second chromosomal enumeration pericentromeric probe corresponding to the same chromosome as the gene locus probe, under conditions sufficient to enable hybridization of the second gene locus probe and second chromosomal enumeration pericentromeric probe to target sequences. In particular embodiments, the first and second gene locus probes may be selected from a probe to a region containing MET, a probe to a region containing PIK3CA, a probe to a region containing PTEN, a probe to a region containing TOP2A, or combinations thereof. In certain disclosed embodiments, the first and second gene locus probe are nucleic acid probes selected from the group consisting of SEQ ID NOs: 1-10, a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 1-10, SEQ ID NOs: 1 1-

20, nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 1 1-20, SEQ ID NOs: 31-40, a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 31-40, SEQ ID NOs: 41-50, and a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 41-50.

The treating step of the disclosed method may comprise treating in situ hybridization positive patients with a HER2 targeted neoadjuvant therapy, and treating in situ hybridization negative patients with a neoadjuvant chemotherapy or no neoadjuvant therapy. The contacting step may also be carried out on using an automated system.

Also disclosed herein is a probe for predicting pathologic response in patients being considered for neoadjuvant breast cancer treatment selected from a gene locus probe to chromosome 7q31, a probe to chromosome 3q26, a probe to chromosome 10q23, a probe to chromosome 17q21, a chromosomal enumeration pericentromeric probe, selected from a chromosome 7 pericentromeric probe, a chromosome 3 pericentromeric probe, a chromosome 10 pericentromeric probe, a chromosome 17 pericentromeric probe, or combinations thereof. In particular disclosed embodiments, the probe may be selected from a probe to a region containing MET, a probe to a region containing PIK3CA, a probe to a region containing PTEN, a probe to a region containing TOP2A, or combinations thereof.

Particular embodiments concern a probe that is a nucleic acid probe selected from the group consisting of SEQ ID NOs: 1-10 and a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 1- 10; a nucleic acid probe selected from the group consisting of SEQ ID NOs: 1 1-20 and a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 1 1-20; a nucleic acid probe selected from the group consisting of SEQ ID NOs: 31-40 and a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 31-40; and/or a nucleic acid probe selected from the group consisting of SEQ ID NOs: 41-50 and a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 41-50.

Additionally, a probe set for predicting pathologic response in patients being considered for neoadjuvant breast cancer treatment is described herein. The probe set typically comprises at least two probes, wherein the two probes are selected from: a gene locus probe selected from a probe to chromosome 7q31, a probe to chromosome 3q26, a probe to chromosome 10q23, a probe to chromosome 17q21, or combinations thereof; and a chromosomal enumeration pericentromeric probe corresponding to the same chromosome as the gene locus probe, wherein the chromosomal enumeration

pericentromeric probe is selected from chromosome 3, chromosome 7, chromosome 10, and chromosome 17. In particular disclosed embodiments, the gene locus probe is selected from a probe to a region containing MET, a probe to a region containing PIK3CA, a probe to a region containing PTEN, a probe to a region containing TOP2A, or combinations thereof. In other disclosed embodiments, the gene locus probe is a nucleic acid probe selected from the group consisting of SEQ ID NOs: 1- 10 and a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 1-10, or a nucleic acid probe selected from the group consisting of SEQ ID NOs: 1 1-20 and a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 1 1-20, or a nucleic acid probe selected from the group consisting of SEQ ID NOs: 31-40 and a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 31-40, or a nucleic acid probe selected from the group consisting of SEQ ID NOs: 41-50 and a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 41-50.

Disclosed herein is a kit for predicting pathologic response in patients being considered for neoadjuvant breast cancer treatment, comprising a gene locus probe to chromosome 7q31, a probe to chromosome 3q26, a probe to chromosome 10q23, a probe to chromosome 17q21 , a chromosomal enumeration pericentromeric probe, selected from a chromosome 7 pericentromeric probe, a chromosome 3 pericentromeric probe, a chromosome 10 pericentromeric probe, a chromosome 17 pericentromeric probe, or combinations thereof. In particular disclosed embodiments, the gene locus probe is selected from a probe to a region containing MET, a probe to a region containing PIK3CA, a probe to a region containing PTEN, a probe to a region containing TOP2A, or combinations thereof. In other disclosed embodiments, the gene locus probe is a nucleic acid probe selected from the group consisting of SEQ ID NOs: 1-10 and a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 1-10, or a nucleic acid probe selected from the group consisting of SEQ ID NOs: 1 1-20 and a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 1 1-20, or a nucleic acid probe selected from the group consisting of SEQ ID NOs: 31-40 and a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 31-40, or a nucleic acid probe selected from the group consisting of SEQ ID NOs: 41-50 and a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 41-50.

Also disclosed is a kit for predicting pathologic response in patients being considered for neoadjuvant breast cancer treatment, comprising a gene locus probe selected from (a) a nucleic acid molecule having at least 90% sequence identity with the sequence according to any one of SEQ ID NOs: 1-20 and 31-50, or (b) a nucleic acid molecule having at least 90% sequence identity with at least 250 contiguous nucleotides of any one of SEQ ID NOs: 1-20 and 31-50; and a chromosomal enumeration pericentromeric probe corresponding to the same chromosome as the gene locus probe, wherein the chromosomal enumeration pericentromeric probe is selected from chromosome 3, chromosome 7, chromosome 10, and chromosome 17.

The foregoing and other features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 is a digital image of dual in situ hybridization (ISH) of breast tissue using the PTEN probe set SEQ ID NOs: 1-10 and a chromosome 10 centromere probe. FIG. 2 is a digital image of dual ISH of breast tissue using the PIK3CA probe set SEQ ID NOs: 11-20 and a chromosome 3 centromere probe.

FIG. 3 is a digital image of dual ISH of liposarcoma using the MDM2 probe set SEQ ID NOs: 21-30 and a chromosome 12 centromere probe.

FIG. 4 is a digital image of dual ISH of lung tissue using the MET probe set SEQ

ID NOs: 31-40 and a chromosome 7 centromere probe.

FIG. 5 is a digital image of dual ISH of breast tissue using the TOP2A probe set SEQ ID NOs: 41-50 and a chromosome 17 centromere probe.

FIG. 6 is an ROC curve comparing neoadjuvant breast cancer treatment prognostic results associated with single ISH or IHC parameters, and ISH and/or IHC parameters in multiple combinations, for MET/CEN7 gain or loss, Ki-67, AND

PIK3CA/CEN3 gain.

FIG. 7 is an ROC curve comparing neoadjuvant breast cancer treatment prognostic results associated with single ISH, and ISH parameters in multiple

combinations, for MET/CEN7 gain, CEN7 gain, and PTEN/CEN 10 gain or loss.

SEQUENCES

The nucleic acid sequences provided herein are shown using standard letter abbreviations for nucleotide bases, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the provided sequences:

SEQ ID NOs: 1-10 are nucleic acid sequences of probes including linked uniquely specific nucleic acid segments complementary to the human PTEN gene.

SEQ ID NOs: 11-20 are nucleic acid sequences of probes including linked uniquely specific nucleic acid segments complementary to the human PIK3CA gene.

SEQ ID NOs: 21-30 are nucleic acid sequences of probes including linked uniquely specific nucleic acid segments complementary to the human MDM2gene.

SEQ ID NOs: 31-40 are nucleic acid sequences of probes including linked uniquely specific nucleic acid segments complementary to the human MET gene.

SEQ ID NOs: 41-50 are nucleic acid sequences of probes including linked uniquely specific nucleic acid segments complementary to the human TOP2A gene. PROBES AND PROBE SEQUENCES

A. PTEN

Disclosed herein are isolated nucleic acid probes including linked uniquely specific nucleic acid segments complementary to the human PTEN gene {e.g., Gene ID No. 5728; NC_000010.10 (89623195..89728532), incorporated herein by reference as present in GENBANK® on April 30, 2012). In some embodiments, the nucleic acid probes include or consist of nucleic acid molecules with at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or more sequence identity to SEQ ID NOs: 1-10.

In some embodiments, the nucleic acid probe can have a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to one of the nucleotide sequences set forth in SEQ ID NOs: 1-10. In other embodiments, the nucleic acid probe can include a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to at least 250 contiguous nucleotides (such as at least 400, at least 500, at least 750, at least 1000, at least 2000, at least 3000, at least 4000, or at least 5000 contiguous nucleotides) of any one of SEQ ID NOs: 1-10. In some examples, the nucleic acid probes include or consist of the sequences set forth in SEQ ID NOs: 1-10. In one example, the probe includes or consists of a contiguous nucleic acid molecule comprising two or more of SEQ ID NOs: 1- 10 (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NOs: 1-10) in a single nucleic acid molecule.

B. PIK3CA

Disclosed herein are isolated nucleic acid probes including linked uniquely specific nucleic acid segments complementary to the human PIK3CA gene (e.g., Gene ID No. 5290; NC_000003.1 1 (17886631 1..178952500), incorporated herein by reference as present in GENBANK® on April 30, 2012). In some embodiments, the isolated nucleic acid probes include or consist of nucleic acid molecules with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more sequence identity to SEQ ID NOs: 1 1-20.

In some embodiments, the nucleic acid probe can have a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to one of the nucleotide sequences set forth in SEQ ID NOs: 1 1-20. In other embodiments, the nucleic acid probe can include a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to at least 250 contiguous nucleotides (such as at least 400, at least 500, at least 750, at least 1000, at least 2000, at least 3000, at least 4000, or at least 5000 contiguous nucleotides) of any one of SEQ ID NOs: 1 1-20. In some examples, the nucleic acid probes include or consist of the sequences set forth in SEQ ID NOs: 1 1-20. In one example, the probe includes or consists of a contiguous nucleic acid molecule comprising two or more of SEQ ID NOs: 1 1-20 (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NOs: 1 1-20) in a single nucleic acid molecule.

C. MDM2

Disclosed herein are isolated nucleic acid probes including linked uniquely specific nucleic acid segments complementary to the human MDM2 gene (e.g., Gene ID No. 4193; NC 000012.1 1 (69201971..69239212), incorporated herein by reference as present in GENBANK® on April 30, 2012). In some embodiments, the isolated nucleic acid probes include or consist of nucleic acid molecules with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more sequence identity to SEQ ID NOs: 21-30.

In some embodiments, the nucleic acid probe can have a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to one of the nucleotide sequences set forth in SEQ ID NOs: 21-30. In other embodiments, the nucleic acid probe can include a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to at least 250 contiguous nucleotides (such as at least 400, at least 500, at least 750, at least 1000, at least 2000, at least 3000, at least 4000, or at least 5000 contiguous nucleotides) of any one of SEQ ID NOs: 21-30. In some examples, the nucleic acid probes include or consist of the sequences set forth in SEQ ID

NOs: 21-30. In one example, the probe includes or consists of a contiguous nucleic acid molecule comprising two or more of SEQ ID NOs: 21-30 (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NOs: 21-30) in a single nucleic acid molecule. D. MET

Disclosed herein are isolated nucleic acid probes including linked uniquely specific nucleic acid segments complementary to the human MET gene (e.g., Gene ID No. 4233; NC 000007.13 (1 16312459..1 16438440), incorporated herein by reference as present in GENBANK® on April 30, 2012). In some embodiments, the isolated nucleic acid probes include or consist of nucleic acid molecules with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more sequence identity to SEQ ID NOs: 31-40.

In some embodiments, the nucleic acid probe can have a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to one of the nucleotide sequences set forth in SEQ ID NOs: 31-40. In other embodiments, the nucleic acid probe can include a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to at least 250 contiguous nucleotides (such as at least 400, at least 500, at least 750, at least 1000, at least 2000, at least 3000, at least 4000, or at least 5000 contiguous nucleotides) of any one of SEQ ID NOs: 31-40. In some examples, the nucleic acid probes include or consist of the sequences set forth in SEQ ID NOs: 31-40. In one example, the probe includes or consists of a contiguous nucleic acid molecule comprising two or more of SEQ ID NOs: 31-40 (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NOs: 31-40) in a single nucleic acid molecule.

E. TOP2A

Disclosed herein are isolated nucleic acid probes including linked uniquely specific nucleic acid segments complementary to the human TOP2A gene (e.g., Gene ID No.7153; NC_000017.10 (38544773..38574202, complement), incorporated herein by reference as present in GENBANK® on April 30, 2012). In some embodiments, the isolated nucleic acid probes include or consist of nucleic acid molecules with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more sequence identity to SEQ ID NOs: 41-50.

DETAILED DESCRIPTION

Abbreviations

aCGH array comparative genomic hybridization

AP alkaline phosphatase

CGH comparative genomic hybridization

CISH chromogenic in situ hybridization

DNP 2,4-dinitrophenyl

FISH fluorescent in situ hybridization

HRP horseradish peroxidase

ISH in situ hybridization MDM2 murine double minute 2

MET Met proto-oncogene

pCR pathological complete response

PIK3CA phosphatidylinositol 3 -kinase (pi 10)

PTEN phosphatase and tension homolog

SISH silver in situ hybridization

TOP2A topoisomerase II alpha

II. Terms

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which a disclosed invention belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. "Comprising" means "including." Hence "comprising A or B" means "including A" or "including B" or

"including A and B."

Suitable methods and materials for the practice and/or testing of embodiments of the disclosure are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described herein can be used. For example, conventional methods well known in the art to which the disclosure pertains are described in various general and more specific references, including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001 ; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to

2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor

Laboratory Press, 1999.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. In case of conflict, the present specification, including explanations of terms, will control.

Although methods and materials similar or equivalent to those described herein can be used to practice or test the disclosed technology, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

Contact: To bring one agent into close proximity to another agent, thereby permitting the agents to interact. For example, an antibody can be applied to a microscope slide or other surface containing a biological sample, thereby permitting detection of proteins in the sample that are specifically recognized by the antibody.

Detectable label: A compound or composition that is conjugated directly or indirectly to another molecule (such as a nucleic acid probe) to facilitate detection of that molecule. Specific, non- limiting examples of labels include fluorescent and fluorogenic moieties, chromogenic moieties, haptens, affinity tags, and radioactive isotopes. The label can be directly detectable (e.g., optically detectable) or indirectly detectable (for example, via interaction with one or more additional molecules that are in turn detectable).

Exemplary labels in the context of the probes disclosed herein are described below.

Methods for labeling nucleic acids, and guidance in the choice of labels useful for various purposes, are discussed, e.g., in Sambrook and Russell, in Molecular Cloning: A

Laboratory Manual, 3^rd Ed., Cold Spring Harbor Laboratory Press (2001) and Ausubel et ah, in Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley- Intersciences (1987, and including updates).

Fisher's Exact Test: A statistical significance test used to determine the deviation from a null hypothesis exactly, rather than relying on an approximation that becomes exact in the limit as the sample size grows to infinity. Most uses of the Fisher test involve a 2 x 2 contingency table, and the p-value from the test is computed as if the margins of the table are fixed. For the present situation, by way of example and with reference to MET/CEN7, the 2 X 2 table has two rows with the first row being MET/CEN7 ratio high, the second row being MET/CEN7 ratio low, and two columns where the first column is pCR and the second column is not pCR. If, for example, all the numbers are equal, then there is no correlation between that marker and responder/non-responder prognosis. Conversely, if most of the responders have a high ratio, then this marker can be used identify a responder. The Fisher's exact test provides a p value, a probability that the numbers result from chance. The cutoff value often is set at 0.05, or 5%, indicating that the probability that the correlation resulted by chance is 5% or less.

Hybridization: To form base pairs between complementary regions of two strands of DNA, RNA, or between DNA and RNA, thereby forming a duplex molecule. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (such as the Na⁺ concentration) of the hybridization buffer will determine the stringency of hybridization. The presence of a chemical which decreases hybridization (such as formamide) in the hybridization buffer will also determine the stringency (Sadhu et al, J. Biosci. 6:817-821, 1984). Calculations regarding hybridization conditions for attaining particular degrees of stringency are discussed in Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, NY (chapters 9 and 1 1). Hybridization conditions for ISH are also discussed in Landegent et al, Hum. Genet. 77:366-370, 1987; Lichter et al, Hum. Genet. 80:224-234, 1988; and Pinkel et al, Proc. Natl. Acad. Sci. USA 85:9138-9142, 1988.

Intron: An intron is any nucleotide region or sequence within a gene that is removed by RNA splicing during generation of a final mature RNA product of a gene. The term intron may refer to both the DNA sequence within a gene or the corresponding sequence in unprocessed RNA transcripts. Introns are found in the genes of most organisms, and can be located in a wide range of genes, including those that generate proteins, ribosomal RNA (rRNA), and transfer RNA (tRNA). When proteins are generated from intron-containing genes, RNA splicing takes place as part of the RNA processing pathway that follows transcription and precedes translation. The length of intron sequences is highly variable, ranging from less than 100 base pairs to tens of thousands or even hundreds of thousands of base pairs.

Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein, or cell) has been substantially separated or purified away from other biological components in a preparation, a cell of an organism, or the organism itself, in which the component occurs, such as other chromosomal and extra-chromosomal DNA and RNA, proteins and cells. Nucleic acid molecules and proteins that have been "isolated" include nucleic acid molecules and proteins purified by standard purification methods. The term also embraces nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acid molecules and proteins. In some examples, the nucleic acid probes disclosed herein are isolated nucleic acid probes.

Met proto-oncogene (MET): Also known as c-Met, HGFR, AUTS9, RCCP2, or MNNG HOS Transforming gene. MET is a proto-oncogene that encodes a protein known as hepatocyte growth factor receptor (HGFR). The human MET proto-oncogene (GenelD: 4233) usually has a total length of 125,982 bp, and it is located in the 7q31 locus of chromosome 7. Human MET is transcribed into a 6,641 bp mature mRNA, which is then translated into a 1,390 amino-acid MET protein. The sequence of the human MET proto- oncogene was disclosed as early as 1987 by Park et al. (Proc. Natl. Acad. Sci. USA 84:6379-6383, 1987).

Mouse Double Minute 2 (MDM2): The human gene is also known as p53 E3 ubiquitin protein ligase homolog (mouse). The MDM2 gene was originally identified by virtue of its amplification in a spontaneously transformed derivative of mouse BALB/c cells, and the MDM2 protein was subsequently shown to bind to p53 in rat cells transfected with p53 genes. Momand et al. (Cell 69: 1237-1245, 1992) characterized, purified, and identified a cellular phosphoprotein with an apparent molecular mass of 90 kD that formed a complex with both mutant and wild-type p53 protein. The protein was identified as a product of the murine double minute 2 gene (mdm2). Oliner et al. (Nature 358:80-83, 1992) cloned the human MDM2 gene. By fluorescence in situ hybridization onto simultaneously DAPI -banded metaphase chromosomes and interphase nuclei, Mitchell et al. (Chromosome Res. 3:261-262, 1995) mapped MDM2 to human 12ql4.3- ql5 distal to CDK4 and flanked by Genethon microsatellites D12S80 and D12S83.

Momand et al. found that the MDM2 gene enhances the tumorigenic potential of cells when it is overexpressed and encodes a putative transcription factor. Forming a tight complex with the p53 gene, the MDM2 oncogene can inhibit p53-mediated transactivation. Because the 12ql3-ql4 region, to which the MDM2 gene maps, is often aberrant in human sarcomas, Oliner et al. performed Southern blot analysis on DNA from such cancers. A striking amplification of MDM2 sequences was found in 17 of 47 sarcomas, including common bone and soft tissue forms. The results were considered consistent with the hypothesis that MDM2 binds to p53 and that amplification of MDM2 in sarcomas leads to escape from p53-regulated growth control. This mechanism of tumorigenesis parallels that for virus-induced tumors in which viral oncogene products bind to and functionally inactivate p53.

Neoadjuvant: Refers generally to administering a therapeutic agent or treatment before a main treatment. One example concerns reducing the size or extent of a tumor before surgery; thus, making the surgical procedure easier and more likely to succeed. Neoadjuvant treatment also may reduce the consequences of a more extensive treatment technique that would be required if the tumor was not reduced in size. Systemic therapies, such as chemotherapy, immunotherapy and/or hormone therapy can be used first. Non- systemic treatments, such as radiation therapy, also can be used, particularly for locally advanced tumors where clinicians plan an operation at a later stage. Neoadjuvant therapy can turn an untreatable tumor to a treatable tumor by decreasing the tumor volume. Also, it may not be clear if surrounding structures are directly involved in the disease and/or are exhibiting signs of inflammation. Neoadjuvant therapy can help make this distinction. Unfortunately, not everyone is suitable for certain neoadjuvant therapies as a result of, for example, the associated toxicity. Some patients react so adversely that further treatments, especially surgery, are precluded.

Pericentromeric: The region around or near a centromere of a nuclear chromosome.

Pathologic Complete Response: The absence of any detectable in situ or invasive carcinoma or nodal metastases.

Pathologic Partial Response: Any response less than pathologic complete response. Typically involves a reduction in in situ or invasive carcinoma or nodal metastases.

Phosphatase and tensin homolog (PTEN): Also referred to as MMAC1 (mutated in multiple advanced cancers). A gene that, in humans, encodes a ubiquitously expressed tumor suppressor dual-specificity phosphatase that antagonizes the

phosphatidylinositol 3 -kinase signaling pathway through its lipid phosphatase activity and negatively regulates the MAP kinase pathway through its protein phosphatase activity (Pezzolesi et al, Hum. Mol. Genet. 16: 1058-1071, 2007). PTEN acts as a tumor suppressor gene through the action of its phosphatase protein product. This phosphatase is involved in the regulation of the cell cycle, preventing cells from growing and dividing too rapidly. Mutations of this gene are a step in the development of many cancers. The sequence of human PTEN was disclosed as early as 1997 by Li et al. (J. Biol. Chem. 272:29403-29406, 1997).

Phosphatidylinositol 3-kinase, pllO subunit (PIK3CA): Also known as phosphoinositide-3 -kinase, catalytic, alpha polypeptide. Human phosphatidylinositol 3- kinase (EC 2.7.1.137) is composed of 85-kD and 1 10-kD subunits. The 85-kD subunit lacks phosphatidylinositol 3-kinase activity and acts as an adaptor, coupling the 1 10-kD subunit (pi 10) to activated protein tyrosine kinases. The human pi 10 subunit is referred to herein as PIK3CA. Hiles et al. (Cell 70:419-429, 1992) found that the human cDNA for pi 10 predicts a 1,068-amino acid protein related to a protein which in S. cerevisiae is involved in the sorting of proteins to the vacuole. In COS-1 cells, pi 10 was catalytically active only when complexed with p85-alpha. Volinia et al. (Genomics 24:472-477 ^', 1994) contributed to the structural and functional understanding of phosphatidylinositol 3-kinase by purifying, cloning, and subsequently elucidating the expression of the bovine enzyme. cDNA for the human PIK3CA encodes a protein 99% identical to the bovine pi 10. The chromosomal localization of the gene for human PIK3CA is shown to be at 3q21-qter as determined using somatic cell hybrids. In situ hybridization performed using Alu-PCR from the YAC DNA located the human gene in 3q26.3 [Chromosome 3: 178,865,902- 178,957,881]. The sequence for the human PIK3CA gene was disclosed as early as 1994 by Volinia et al.

Probe: A nucleic acid molecule that is capable of hybridizing with a target nucleic acid molecule (e.g., genomic target nucleic acid molecule) and, when hybridized to the target, is capable of being detected either directly or indirectly. Thus probes permit the detection, and in some examples quantification, of a target nucleic acid molecule. In particular examples, a probe includes at least two segments complementary to uniquely specific nucleic acid sequences of a target nucleic acid molecule and are thus capable of specifically hybridizing to at least a portion of the target nucleic acid molecule. Generally, once at least one segment or portion of a segment has (and remains) hybridized to the target nucleic acid molecule other portions of the probe may (but need not) be physically constrained from hybridizing to those other portions' cognate binding sites in the target (e.g., such other portions are too far distant from their cognate binding sites); however, other nucleic acid molecules present in the probe can bind to one another, thus amplifying signal from the probe. A probe can be referred to as a "labeled nucleic acid probe," indicating that the probe is coupled directly or indirectly to a detectable moiety or "label," which renders the probe detectable.

Sample: A specimen containing DNA (for example, genomic DNA), RNA (including mRNA), protein, or combinations thereof, obtained from a subject. Examples include, but are not limited to, chromosomal preparations, peripheral blood, urine, saliva, tissue biopsy, fine needle aspirate, surgical specimen, bone marrow, amniocentesis samples, and autopsy material. In one example, a sample includes genomic DNA. In some examples, the sample is a cytogenetic preparation, for example which can be placed on microscope slides. In particular examples, samples are used directly, or can be manipulated prior to use, for example, by fixing (e.g., using formalin).

Sensitivity and specificity: Statistical measurements of the performance of a binary classification test. Sensitivity measures the proportion of actual positives which are correctly identified (e.g., the percentage of tumors that are identified as being HER2- positive that are identified as HER2 -positive by another method). Specificity measures the proportion of negatives which are correctly identified (e.g., the percentage of tumors identified as HER2 -negative that are identified as HER2 -negative by another method). Sequence identity: The identity (or similarity) between two or more nucleic acid sequences is expressed in terms of the identity or similarity between the sequences.

Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are.

Methods of alignment of sequences for comparison are well known in the art.

Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981 ; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5: 151-3, 1989; Corpet et al, Nuc. Acids Res. 16: 10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8: 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al, J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al, J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biotechnology and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.

BLASTN may be used to compare nucleic acid sequences, while BLASTP may be used to compare amino acid sequences. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned

sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.

The BLAST-like alignment tool (BLAT) may also be used to compare nucleic acid sequences (Kent, Genome Res. 12:656-664, 2002). BLAT is available from several sources, including Kent Informatics (Santa Cruz, CA) and on the Internet

(genome.ucsc.edu).

Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a nucleic acid sequence that has 1 166 matches when aligned with a test sequence having 1554 nucleotides is 75.0 percent identical to the test sequence (1 166÷1554* 100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.1 1, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer. In another example, a target sequence containing a 20-nucleotide region that aligns with 15 consecutive nucleotides from an identified sequence as follows contains a region that shares 75 percent sequence identity to that identified sequence (that is, 15÷20* 100=75).

Subject: Any multi-cellular vertebrate organism, such as human or non-human mammals (e.g., veterinary subjects).

Target nucleic acid sequence or molecule: A defined region or particular portion of a nucleic acid molecule, for example a portion of a genome (such as a gene or a region of mammalian genomic DNA containing a gene of interest). In an example where the target nucleic acid sequence is a target genomic sequence, such a target can be defined by its position on a chromosome (e.g., in a normal cell), for example, according to cytogenetic nomenclature by reference to a particular location on a chromosome; by reference to its location on a genetic map; by reference to a hypothetical or assembled contig; by its specific sequence or function; by its gene or protein name; or by any other means that uniquely identifies it from among other genetic sequences of a genome. In some examples, the target nucleic acid sequence is mammalian genomic sequence (for example human genomic sequence).

In some examples, alterations of a target nucleic acid sequence (e.g., genomic nucleic acid sequence) are "associated with" a disease or condition. In some examples, detection of the target nucleic acid sequence can be used to infer the status of a sample with respect to the disease or condition. For example, the target nucleic acid sequence can exist in two (or more) distinguishable forms, such that a first form correlates with absence of a disease or condition and a second (or different) form correlates with the presence of the disease or condition. The two different forms can be qualitatively distinguishable, such as by polynucleotide polymorphisms, and/or the two different forms can be quantitatively distinguishable, such as by the number of copies of the target nucleic acid sequence that are present in a cell.

Therapeutically effective amount: A dose sufficient to prevent advancement, delay progression, or to cause regression of a disease, or which is capable of reducing symptoms caused by the disease, such as cancer, for example breast cancer. Topoisomerase II alpha (TOP2A): DNA topoisomerases (EC 5.99.1.3) are enzymes that control and alter the topologic states of DNA in both prokaryotes and eukaryotes. Topoisomerase II from eukaryotic cells catalyzes the relaxation of supercoiled DNA molecules, catenation, decatenation, knotting, and unknotting of circular DNA. The reaction catalyzed by topoisomerase II likely involves the crossing-over of two DNA segments. The gene encoding topoisomerase II in humans is present at cytogenetic location: 17q21.2 and has genomic coordinates (GRCh37): 17:38,544,772 - 38,574,201. Tsai-Pflugfelder et al. determined the entire coding sequence of the human TOP2 gene as early as 1988 (Proc. Natl. Acad. Sci. USA 85:7177-7181, 1988). Lang et al. reported the complete structures of the human TOP2A and TOP2B genes in 1998 (Gene 221 :255-266,

1998). The human TOP2A gene spans approximately 30 kb and contains 35 exons.

Uniquely specific sequence: A nucleic acid sequence (for example, a sequence of at least of at least 20 bp (such as at least 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, or more) that is present only one time in a haploid genome of an organism. In a particular example, a uniquely specific nucleic acid sequence is a nucleic acid sequence from a target nucleic acid that has 100% sequence identity with the target nucleic acid and has no significant identity to any other nucleic acid sequences present in the specific haploid genome that includes the target nucleic acid.

Vector: Any nucleic acid that acts as a carrier for other ("foreign") nucleic acid sequences that are not native to the vector. When introduced into an appropriate host cell a vector may replicate itself (and, thereby, the foreign nucleic acid sequence) or express at least a portion of the foreign nucleic acid sequence. In one context, a vector is a linear or circular nucleic acid into which a nucleic acid sequence of interest is introduced (for example, cloned) for the purpose of replication (e.g., production) and/or manipulation using standard recombinant nucleic acid techniques (e.g., restriction digestion). A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. Common vectors include, for example, plasmids, cosmids, phage, phagemids, artificial chromosomes (e.g., BAC, PAC, HAC, YAC), and hybrids that incorporate features of more than one of these types of vectors. Typically, a vector includes one or more unique restriction sites (and in some cases a multi-cloning site) to facilitate insertion of a target nucleic acid sequence. III. Uniquely Specific Nucleic Acid Probes

Disclosed herein are gene locus probes and chromosomal enumeration pericentromeric probes suitable for use in the disclosed method. In particular disclosed embodiments, a gene locus probe to chromosome 7q31, a probe to chromosome 3q26, a probe to chromosome 10q23, a probe to chromosome 17q21, a chromosomal enumeration pericentromeric probe, selected from a chromosome 7 pericentromeric probe, a chromosome 3 pericentromeric probe, a chromosome 10 pericentromeric probe, a chromosome 17 pericentromeric probe or combinations thereof may be used. In particular disclosed embodiments, the probe may be selected from a probe to a region containing MET, a probe to a region containing PIK3CA, a probe to a region containing PTEN, a probe to a region containing TOP2A, or combinations thereof.

In certain disclosed embodiments the gene locus probes are uniquely specific nucleic acid probes complementary to target nucleic acid molecules of interest, including PTEN, PIK3CA, MDM2, MET and TOP2A. Each of the disclosed probes includes a plurality of uniquely specific nucleic acid segments, which were designed, and synthesized utilizing the methods described in U.S. Pat. App. Publ. No. 201 1/0160076 and

International Pat. App. Publ. No. WO 201 1/062293, both of which are incorporated herein by reference in their entirety. The nucleic acid segments included in each probe are each uniquely specific (occur only once in the haploid human genome) and are from non- contiguous portions of the human genome.

Surprisingly, many of the segments are located in introns in the human genome. While not being bound by theory, it is generally believed in the field that the intronic regions of the human genome evolve at a faster rate than coding regions. In illustrative embodiments, the present probes have sequences that are exon-free or substantially exon- free. In one embodiment, the sequences are selected from non-coding gene regions. The development and use of intronic probes is counter-intuitive as most of the field is concerned with genetic abnormalities associated with the protein coding region of the gene. The present probes provide researchers and clinicians with unique and distinct data heretofore unavailable. It is unexpected that introns include uniquely specific sequences due to the belief in the field that introns are not as highly conserved as the exon-containing

DNA sequences. The identification of uniquely specific sequences in intronic regions indicates that some of these regions may be highly conserved. The present probes provide a means to study these highly-conserved intronic regions of these very important genes. A. PTEN

Disclosed herein are isolated nucleic acid probes including linked uniquely specific nucleic acid segments complementary to the human PTEN gene {e.g., Gene ID No. 5728; NC_000010.10 (89623195..89728532), incorporated herein by reference as present in GENBANK® on April 30, 2012). In some embodiments, the nucleic acid probes include or consist of nucleic acid molecules with at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or more sequence identity to the sequences identified in provisional application No. 61/645,347 and disclosed in the sequence listing.

B. PIK3CA

Disclosed herein are isolated nucleic acid probes including linked uniquely specific nucleic acid segments complementary to the human PIK3CA gene {e.g., Gene ID

No. 5290; NC_000003.1 1 (17886631 1..178952500), incorporated herein by reference as present in GENBANK® on April 30, 2012). In some embodiments, the isolated nucleic acid probes include or consist of nucleic acid molecules with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more sequence identity the sequences identified in provisional application No. 61/645,347 and disclosed in the sequence listing.

In some embodiments, the nucleic acid probe can have a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to one of the nucleotide sequences set forth in SEQ ID NOs: 1 1-20. In other embodiments, the nucleic acid probe can include a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to at least 250 contiguous nucleotides (such as at least 400, at least 500, at least 750, at least 1000, at least 2000, at least 3000, at least 4000, or at least 5000 contiguous nucleotides) of any one of SEQ ID NOs: 1 1-20. In some examples, the nucleic acid probes include or consist of the sequences set forth in SEQ ID

NOs: 1 1-20. In one example, the probe includes or consists of a contiguous nucleic acid molecule comprising two or more of SEQ ID NOs: 1 1-20 (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NOs: 1 1-20) in a single nucleic acid molecule. C. MET

Disclosed herein are isolated nucleic acid probes including linked uniquely specific nucleic acid segments complementary to the human MET gene {e.g., Gene ID No. 4233; NC 000007.13 (1 16312459..1 16438440), incorporated herein by reference as present in GENBANK® on April 30, 2012). In some embodiments, the isolated nucleic acid probes include or consist of nucleic acid molecules with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more sequence identity the sequences identified in provisional application No. 61/645,347 and disclosed in the sequence listing.

In some embodiments, the nucleic acid probe can have a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to one of the nucleotide sequences set forth in SEQ ID NOs: 31-40. In other embodiments, the nucleic acid probe can include a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to at least 250 contiguous nucleotides (such as at least 400, at least 500, at least 750, at least 1000, at least 2000, at least 3000, at least

4000, or at least 5000 contiguous nucleotides) of any one of SEQ ID NOs: 31-40. In some examples, the nucleic acid probes include or consist of the sequences set forth in SEQ ID NOs: 31-40. In one example, the probe includes or consists of a contiguous nucleic acid molecule comprising two or more of SEQ ID NOs: 31-40 (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NOs: 31-40) in a single nucleic acid molecule.

D. TOP2A

Disclosed herein are isolated nucleic acid probes including linked uniquely specific nucleic acid segments complementary to the human TOP2A gene (e.g., Gene ID No.7153; NC_000017.10 (38544773..38574202, complement), incorporated herein by reference as present in GENBANK® on April 30, 2012). In some embodiments, the isolated nucleic acid probes include or consist of nucleic acid molecules with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or more sequence identity to the sequences identified in provisional application No. 61/645,347 and disclosed in the sequence listing.

In some embodiments, the nucleic acid probe can have a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to one of the nucleotide sequences set forth in SEQ ID NOs: 41-50. In other embodiments, the nucleic acid probe can include a sequence with at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity (for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to at least 250 contiguous nucleotides (such as at least 400, at least 500, at least 750, at least 1000, at least 2000, at least 3000, at least 4000, or at least 5000 contiguous nucleotides) of any one of SEQ ID NOs: 41-50. In some examples, the nucleic acid probes include or consist of the sequences set forth in SEQ ID

NOs: 41-50. In one example, the probe includes or consists of a contiguous nucleic acid molecule comprising two or more of SEQ ID NOs: 41-50 (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NOs: 41-50) in a single nucleic acid molecule.

In some embodiments, the disclosed probes have a length of at least 250 contiguous nucleotides (such as at least 300, at least 400, at least 500, at least 750, at least

1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000, at least 12,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, or more contiguous nucleotides). In other embodiments, the disclosed probes have a length of about 250-50,000 nucleotides (for example, about 500-50,000, about 1000-

40,000, about 5000-25,000, about 7000-20,000, or about 10,000 to 15,000 nucleotides). The probes can include all or a portion of one or more of SEQ ID NOs: 1-50, or all or a portion of a nucleic acid having at least 70% sequence identity (for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) to one of more of SEQ ID NOs: 1-50. In some examples, the probe is a contiguous nucleic acid molecule comprising up to 10 of the disclosed sequences (such as SEQ ID NOs: 1- 10, SEQ ID NOs: 1 1-20, SEQ ID NOs: 21-30, SEQ ID NOs: 31-40, or SEQ ID NOs: 41-50).

In other examples, the probe is a contiguous nucleic acid molecule including two or more portions or segments, wherein the first portion includes at least 250 contiguous nucleotides at least 70% identical (for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the corresponding portion of any one of SEQ ID NOs: 1-50 and the second portion includes at least 250 contiguous nucleotides at least 70% identical (for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the corresponding portion of any one of SEQ ID NOs: 1-50, where the first and second portions are different.

Each of the disclosed sequences of SEQ ID NOs: 1-50 includes a plurality of 100 bp uniquely specific nucleic acid segments, which were designed and synthesized utilizing the methods described in U.S. patent application publication No. 201 1/0160076 and international patent publication No. WO 201 1/062293, both of which are incorporated herein by reference in their entirety. The disclosed probes are exemplary, and it is understood that in some examples, at least one (such as at least two, at least three, at least four, at least five, or more) of the 100 bp segments (for example nucleotides 1-100, 101-

200, 201-300, and so on) or portions thereof could be removed from the disclosed sequences to provide additional nucleic acid probes for PTEN, PIK3CA, MET, or TOP2A. In other examples, the order of at least one (such as at least two, at least three, at least four, at least five, or more) of the 100 bp segments (for example nucleotides 1-100, 101 -200, 201-300, and so on) could be rearranged from the order in the disclosed probes to provide additional nucleic acid probes for PTEN, PIK3CA, MET, or TOP2A. One of ordinary skill in the art could make and test such modified probes, for example by synthesizing a modified probe and assessing its hybridization to samples known to contain or not to contain the target nucleic acid.

In some examples, the individual uniquely specific segments are produced (for example by oligonucleotide synthesis or by amplification of the sequences from the genomic target nucleic acid) and joined together. In other examples, the nucleic acid probe is synthesized as a series of oligonucleotides (such as individual oligonucleotides of about 100 bp), which are joined together. For example, the segments may be joined or ligated to one another enzymatically (e.g., using a ligase). For example, the segments can be joined in a blunt-end ligation or at a restriction site. In another example, the segments may be synthesized with complementary nucleic acid overhangs (such as at least a 3 bp overhang), annealed, and joined to one another, for example with a ligase. Chemical ligation and amplification can also be used to join the uniquely specific segments. In another example, the entire nucleic acid probe is synthesized and the individual uniquely specific segments are directly joined during synthesis.

In some embodiments, the disclosed nucleic acid probes are included in a vector, such as a plasmid or an artificial chromosome (e.g., yeast artificial chromosome (YAC), PI based artificial chromosome (PAC), or bacterial artificial chromosome (BAC)). In some examples, any one of SEQ ID NOs: 1-50 disclosed herein are introduced into a plasmid vector, for example to allow replication and/or labeling of the nucleic acid probe by standard molecular biology techniques. In one example, the vector is a pUC plasmid vector. In other examples, two or more of the disclosed sequences (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the disclosed sequences) are introduced into a vector, for example to allow replication and/or labeling of the nucleic acid probe by standard molecular biology techniques. The two or more sequences can be introduced into a vector in any order. One of ordinary skill in the art can determine whether the sequences the overlap the junctions between the individual sequences (for example, a window of about 100 bp) are uniquely specific, for example, utilizing the teachings of U.S. Pat. App. Publ. No. 201 1/0160076, and International Pat. Publ. No. WO 2011/062293. If a sequence that is not uniquely specific is introduced, the sequences can be reordered and reanalyzed in order to select an order that does not produce any non-uniquely specific sequences. In particular examples, all of SEQ ID NOs: 1-10 are introduced into a single vector, all of SEQ ID NOs: 1 1-20 are introduced into a single vector, all of SEQ ID NOs: 21-30 are introduced into a single vector, all of SEQ ID NOs: 31-40 are introduced into a single vector, or all of SEQ ID NOs: 41-50 are introduced into a single vector. In one example, the vector is an artificial chromosome (such as a YAC, BAC or PAC).

IV. Probe Sets

In some embodiments, the disclosed probes are included in a set of probes, for example as set of two or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the disclosed probes. In some examples, a probe set includes two or more probes specific for a particular target nucleic acid molecule (such as two or more probes for PTEN, PIK3CA, MET, or TOP2A). In some examples, a probe set includes probes specific for two or more target nucleic acid molecules (such as probes specific for two or more of PTEN, PIK3CA, MET, and TOP2A).

One exemplary probe set includes two or more probes specific for human PTEN. The probe set includes two or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) probes selected from probes including or consisting of the nucleic acid sequence having at least at least 70%, at least 75%, at least 80%, at least 85%, or 90% sequence identity with SEQ ID NOs: 1-10 or at least 250 contiguous nucleotides of any one of SEQ ID NOs: 1-10. In one example, a probe set for PTEN includes probes including or consisting of nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with each of SEQ ID NOs: 1-10. In a specific example, the probe set includes each of SEQ ID NOs: 1-10.

Another exemplary probe set includes probes specific for human PIK3CA. The probe set includes two or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) probes selected from probes including or consisting of the nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with any one of

SEQ ID NOs: 1 1-20 or at least 250 contiguous nucleotides of any one of SEQ ID NOs: 1 1- 20. In one example, a probe set for PIK3CA includes probes including or consisting of nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with each of SEQ ID NOs: 1 1-20. In a particular example, the probe set includes each of SEQ ID NOs: 1 1-20.

A further exemplary probe set includes probes specific for human MDM2. The probe set includes two or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) probes selected from probes including or consisting of the nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with any one of SEQ ID NOs: 21 -30 or at least 250 contiguous nucleotides of any one of SEQ ID NOs: 21 -

30. In one example, a probe set for MDM2 includes probes including or consisting of nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with each of SEQ ID NOs: 21-30. In a particular example, the probe set includes each of SEQ ID NOs: 21-30.

A still further exemplary probe set includes probes specific for human MET. The probe set includes two or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) probes selected from probes including or consisting of the nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with any one of SEQ ID NOs: 31-40 or at least 250 contiguous nucleotides of any one of SEQ ID NOs: 31- 40. In one example, a probe set for MET includes probes including or consisting of nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with each of SEQ ID NOs: 31-40. In a particular example, the probe set includes each of SEQ ID NOs: 31-40.

An additional exemplary probe set includes probes specific for human TOP2A. The probe set includes two or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) probes selected from probes including or consisting of the nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with any one of SEQ ID NOs: 41-50 or at least 250 contiguous nucleotides of any one of SEQ ID NOs: 41-50. In one example, a probe set for TOP2A includes probes including or consisting of nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity with each of SEQ ID NOs: 41-50. In a particular example, the probe set includes each of SEQ ID NOs: 41-50.

In particular disclosed embodiments, the probe set may comprise one or more antibodies suitable for immunohistochemistry analysis, such as Ki-67.

V. Kits

Also disclosed are kits including one or more of the disclosed gene locus or chromosomal enumeration pericentromeric probes (for example, one or more of SEQ ID NOs: 1-50). For example, kits can include at least one probe (such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more probes) or at least one probe set (such as at least 1, 2, 3, 4, or 5 probe sets) as described herein. In one example, the kit includes probes including the nucleic acid sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or all of SEQ ID NOs: 1-10 (or sequences at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NOs: 1-10) or one or more nucleic acids including at least 250 contiguous nucleotides of any one of sequences at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NOs: 1- 10. In another example, the kit includes probes including the nucleic acid sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or all of SEQ ID NOs: 1 1-20 (or sequences at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NOs: 1 1-20) or one or more nucleic acids including at least 250 contiguous nucleotides of any one of sequences at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NOs: 1 1-20. In a further example, the kit includes probes including the nucleic acid sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or all of SEQ ID NOs: 21-30 (or sequences at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NOs: 21-30) or one or more nucleic acids including at least 250 contiguous nucleotides of any one of sequences at least 70%, at least 75%, at least

80%, at least 85%, or at least 90% identical to SEQ ID NOs: 21-30. In a still further example, the kit includes probes including the nucleic acid sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or all of SEQ ID NOs: 31-40 (or sequences at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NOs: 31-40) or one or more nucleic acids including at least 250 contiguous nucleotides of any one of sequences at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NOs: 31-40. In an additional example, the kit includes probes including the nucleic acid sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or all of SEQ ID NOs: 41-50 (or sequences at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NOs: 41-50) or one or more nucleic acids including at least 250 contiguous nucleotides of any one of sequences at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to SEQ ID NOs: 41-50. In additional examples, the kit includes one or more chromosomal enumeration pericentromeric probes to any one of chromosomes 3, 7, 10, 12, and 17. In some examples, the probes are present in separate containers. In other examples, the probes (or the probe set) are in a single container.

In one particular embodiment, the kit includes ten plasmids, each plasmid including one of SEQ ID NOs: 1-10. The plasmids are pooled and labeled (for example, labeled with DNP) and provided in a vial at a concentration of about 60 μg/ml.

In another particular embodiment, the kit includes ten plasmids, each plasmid including one of SEQ ID NOs: 1 1-20. The plasmids are pooled and labeled (for example, labeled with DNP) and provided in a vial at a concentration of about 60 μg/ml.

In a further particular embodiment, the kit includes ten plasmids, each plasmid including one of SEQ ID NOs: 21-30. The plasmids are pooled and labeled (for example, labeled with DNP) and provided in a vial at a concentration of about 60 μg/ml.

In another particular embodiment, the kit includes ten plasmids, each plasmid including one of SEQ ID NOs: 31-40. The plasmids are pooled and labeled (for example, labeled with DNP) and provided in a vial at a concentration of about 70 μg/ml.

In an additional particular embodiment, the kit includes ten plasmids, each plasmid including one of SEQ ID NOs: 41-50. The plasmids are pooled and labeled (for example, labeled with DNP) and provided in a vial at a concentration of about 60 μg/ml.

The kits can also include one or more reagents for detecting a target nucleic acid molecule in a sample (for example, by in situ hybridization or CGH assay), or for producing a detectably labeled probe. For example, a kit can include at least one of the disclosed nucleic acid probes or probe sets, along with one or more buffers, labeled dNTPs, a labeling enzyme (such as a polymerase), primers, nuclease free water, and instructions for producing a labeled probe. In another example, the kit includes one or more of the disclosed nucleic acid probes (unlabeled or labeled) along with buffers and other reagents for performing in situ hybridization. For example, if one or more unlabeled probes are included in the kit, labeling reagents can also be included, along with specific detection agents (for example, fluorescent, chromogenic, luminescent and/or radiometric) and other reagents for performing an in situ hybridization assay, such as paraffin pretreatment buffer, protease(s) and protease buffer, prehybridization buffer, hybridization buffer, wash buffer, counterstain(s), mounting medium, or combinations thereof. In some examples, such kit components are present in separate containers. The kit can optionally further include control slides (such as positive or negative controls, such as those known to contain or not contain the target sequence(s), such as PTEN, PIK3CA, MET, or TOP2A nucleic acid sequences) for assessing hybridization and signal of the probe(s).

In certain examples, the kits include avidin, antibodies, and/or receptors (or other anti-ligands). Optionally, one or more of the detection agents (including a primary detection agent, and optionally, secondary, tertiary or additional detection reagents) are labeled, for example, with a hapten or fluorophore (such as a fluorescent dye or quantum dot). In some instances, the detection reagents are labeled with different detectable moieties (for example, different fluorescent dyes, spectrally distinguishable quantum dots, different haptens, etc). For example, a kit can include two or more nucleic acid probes or probe sets that correspond to and are capable of hybridizing to different target nucleic acids (for example, any of the target nucleic acids disclosed herein). The first probe or probe set can be labeled with a first detectable label (e.g., hapten, fluorophore, etc.), the second probe or probe set can be labeled with a second detectable label, and any additional probes or probe sets (e.g., third, fourth, fifth, etc.) can be labeled with additional detectable labels. The first, second, and any subsequent probes or probe sets can be labeled with different detectable labels, although other detection schemes are possible. If the probe(s) are labeled with indirectly detectable labels, such as haptens, the kits can include detection agents (such as labeled avidin, antibodies or other specific binding agents) for some or all of the probes. In one embodiment, the kit includes probes and detection reagents suitable for multiplex ISH.

In one example, the kit also includes an antibody conjugate, such as an antibody conjugated to a label (e.g., an enzyme, fluorophore, or fluorescent nanoparticle). In some examples, the antibody is conjugated to the label through a linker, such as PEG, 6X-His, streptavidin, or GST.

In another example, the kit includes one or more of the disclosed nucleic acid probes affixed to a solid support (such as an array) along with buffers and other reagents for performing CGH. Reagents for labeling sample and control DNA can also be included, along with other reagents for performing an aCGH assay, prehybridization buffer, hybridization buffer, wash buffer, or combinations thereof. The kit can optionally further include control slides for assessing hybridization and signal of the labeled DNAs. VI. Detectable Labels and Methods of Labeling

The probes disclosed herein can include one or more labels, for example to permit detection of a target nucleic acid molecule. In various applications, such as in situ hybridization procedures, a probe includes a label (e.g., a detectable label). A "detectable label" is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample. Thus, a labeled nucleic acid molecule provides an indicator of the presence or quantity (for example, gene copy number) of a target nucleic acid (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample. The disclosure is not limited to the use of particular labels, although examples are provided.

A label associated with one or more nucleic acid molecules (such as the disclosed probes) can be detected either directly or indirectly. A label can be detected by any known or yet to be discovered mechanism including absorption, emission and/or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons). Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.

Particular examples of detectable labels include fluorescent molecules (or fluorochromes). Numerous fluorochromes are known to those of ordinary skill in the art, and can be selected, for example from Life Technologies, e.g. , see, The Handbook— A Guide to Fluorescent Probes and Labeling Technologies. Examples of particular fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Patent No. 5,866,366 to Nazarenko et al, such as 4-acetamido-4'-isothiocyanatostilbene- 2,2'disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS), 4-amino-N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino- 1 - naphthyl)maleimide, anthranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trifluoromethylcouluarin (Coumarin 151); cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5', 5"-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7- diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'- diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene- l-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4' -isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5- carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'- dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein

isothiocyanate (FITC), and QFITC (XRITC); 2', 7'-difluorofluorescein (OREGON

GREEN®); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho-cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B- phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 -pyrene butyrate; Reactive Red 4 (Cibacron Brilliant Red 3B-A);

rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);

N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;

tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives.

Other suitable fluorophores include thiol-reactive europium chelates which emit at approximately 617 nm (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol.

Chem. 274:3315-22, 1999), as well as GFP, Lissamine™, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Patent No. 5,800,996 to Lee et al.) and derivatives thereof. Other fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Carlsbad, CA) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Patent Nos. 5,696,157, 6, 130, 101 and 6, 716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Patent Nos. 4,774,339, 5, 187,288, 5,248,782, 5,274,1 13, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative of the sulfonated pyrene described in U.S. Patent No. 5, 132,432) and Marina Blue (U.S. Patent No.

5,830,912).

In addition to the fluorochromes described above, a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a quantum dot (obtained, for example, from Life Technologies); see also, U.S. Patent Nos. 6,815,064;

6,682596; and 6,649,138). Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties. When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs of a frequency that corresponds to the bandgap of the semiconductor material used in the semiconductor nanocrystal. This emission can be detected as colored light of a specific wavelength or fluorescence. Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. patent No. 6,602,671. Semiconductor nanocrystals that can be coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et ah, Science 281 :2013-2016, 1998; Chan et a/., Science 281 :2016-2018, 1998; and U.S. Patent

No. 6,274,323.

Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Patent Nos. 6,927,069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225,198; 6,207,392; 6,1 14,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018; 5,505,928; 5,262,357, and in U.S. Patent Publication No. 2003/0165951, as well as PCT

Publication No. WO 1999/026299. Separate populations of semiconductor nanocrystals can be produced that are identifiable based on their different spectral characteristics. For example, semiconductor nanocrystals can be produced that emit light of different colors based on their composition, size or size and composition. For example, quantum dots that emit light at different wavelengths based on size (565 nm, 655 nm, 705 nm, or 800 nm emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlsbad, CA).

Additional labels include, for example, radioisotopes (such as ³H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd³⁺, and liposomes.

Detectable labels that can be used with nucleic acid molecules (such as the disclosed probes) also include enzymes, for example horseradish peroxidase (HRP), alkaline phosphatase (AP), acid phosphatase, glucose oxidase, β-galactosidase, β- glucuronidase, or β-lactamase. Where the detectable label includes an enzyme, a chromogen, fluorogenic compound, or luminogenic compound can be used in combination with the enzyme to generate a detectable signal (numerous of such compounds are commercially available, for example, from Life Technologies). Particular examples of chromogenic compounds include diaminobenzidine (DAB), 4-nitrophenylphosphate (pNPP), fast red, fast blue, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), BCIP/NBT, AP Orange, AP blue, tetramethylbenzidine (TMB), 2,2'-azino-di-[3- ethylbenzothiazoline sulphonate] (ABTS), o-dianisidine, 4-chloronaphthol (4-CN), nitrophenyl- -D-galactopyranoside (ONPG), o-phenylenediamine (OPD), 5-bromo-4- chloro-3-indolyl- -galactopyranoside (X-Gal), methylumbelliferyl- -D-galactopyranoside (MU-Gal), p-nitrophenyl-a-D-galactopyranoside (PNP), 5-bromo-4-chloro-3-indolyl- β - D-glucuronide (X-Gluc), 3-amino-9-ethyl carbazol (AEC), fuchsin, iodonitrotetrazolium

(INT), tetrazolium blue, and tetrazolium violet.

Alternatively, an enzyme can be used in a metallographic detection scheme. For example, silver in situ hybridization (SISH) procedures involve metallographic detection schemes for identification and localization of a hybridized genomic target nucleic acid sequence. Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redox-active agent reduces the metal ion, causing it to form a detectable precipitate. (See, for example, U.S. Patent Application Publication No. 2005/0100976, PCT Publication No. 2005/003777 and U.S. Patent Application Publication No. 2004/0265922). Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate. (See, for example, U.S. Patent No. 6,670, 113).

In non-limiting examples, the disclosed probes are labeled with dNTPs covalently attached to hapten molecules (such as a nitro-aromatic compound {e.g., 2,4-dinitrophenyl

(DNP)), biotin, fluorescein, digoxigenin, etc.). Additional haptens suitable for labeling the disclosed probes include nitropyrazole, 3 -hydroxy quinoxaline, thiazolesulfonamide, nitrocinnamic acid, rotenone, 7-(diethylamino)coumarin-3-carboxylic acid,

benzodiazepine, or benzofuran haptens (see, e.g., International Pat. Publ. No. WO

2012/003476. incorporated herein by reference). Methods for conjugating haptens and other labels to dNTPs {e.g., to facilitate incorporation into labeled probes) are well known in the art. For examples of procedures, see, e.g., U.S. Patent Nos. 5,258,507, 4,772,691, 5,328,824, and 4,71 1,955. Indeed, numerous labeled dNTPs are available commercially, for example from Life Technologies (Carlsbad, CA). A label can be directly or indirectly attached to a dNTP at any location on the dNTP, such as a phosphate (e.g., a, β or γ phosphate) or a sugar.

Detection of labeled nucleic acid molecules can be accomplished by contacting the hapten-labeled nucleic acid molecules bound to the genomic target nucleic acid with a primary anti-hapten antibody. In one example, the primary anti-hapten antibody (such as a mouse anti-hapten antibody) is directly labeled with an enzyme. In another example, a secondary anti-antibody (such as a goat anti-mouse IgG antibody) conjugated to an enzyme is used for signal amplification. In CISH a chromogenic substrate is added, for SISH, silver ions and other reagents as outlined in the referenced patents/applications are added.

In some examples, a probe is labeled by incorporating one or more labeled dNTPs using an enzymatic (polymerization) reaction. For example, the disclosed probes (for example, incorporated into a plasmid vector) can be labeled by nick translation (using, for example, biotin, DNP, digoxigenin, etc.) or by random primer extension with terminal transferase (e.g., 3' end tailing). In some examples, the nucleic probe is labeled by a modified nick translation reaction where the ratio of DNA polymerase I to

deoxyribonuclease I (DNase I) is modified to produce greater than 100% of the starting material. In particular examples, the nick translation reaction includes DNA polymerase I to DNase I at a ratio of at least about 800: 1, such as at least 2000: 1, at least 4000: 1, at least 8000: 1, at least 10,000: 1, at least 12,000: 1, at least 16,000: 1, such as about 800: 1 to 24,000: 1 and the reaction is carried out overnight (for example, for about 16-22 hours) at a substantially isothermal temperature, for example, at about 16°C to 25°C (such as room temperature). If the probe is included in a probe set (for example, multiple plasmids, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more plasmids), the plasmids may be mixed in an equal molar ratio prior to performing the labeling reaction (such as nick translation or modified nick translation).

In other examples, chemical labeling procedures can also be employed. Numerous reagents (including hapten, fluorophore, and other labeled nucleotides) and other kits are commercially available for enzymatic labeling of nucleic acids, including the disclosed probes. As will be apparent to those of ordinary skill in the art, any of the labels and detection procedures disclosed above are applicable in the context of labeling a probe, e.g., for use in in situ hybridization reactions. For example, the Amersham MULTIPRIME® DNA labeling system, various specific reagents and kits available from Molecular Probes/Life Technologies, or any other similar reagents or kits can be used to label the nucleic acids disclosed herein. In particular examples, the disclosed probes can be directly or indirectly labeled with a hapten, a ligand, a fluorescent moiety (e.g., a fluorophore or a semiconductor nanocrystal), a chromogenic moiety, or a radioisotope. For example, for indirect labeling, the label can be attached to nucleic acid molecules via a linker (e.g., PEG or biotin). Additional methods that can be used to label probe nucleic acid molecules are provided in U.S. Application Pub. No. 2005/0158770.

VII. Methods of Detecting a Target Nucleic Acid

The disclosed probes can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH). Exemplary uses are discussed below.

In some embodiments, the disclosed methods of detecting a target nucleic acid include comparing the signal or gene copy number detected in a sample utilizing one or more of the disclosed probes or probe sets in a sample with a control or reference value. In some examples, a change in signal from a probe or probe set relative to a control (such as an increase of about 1.5-fold, 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, or more relative to a control sample or reference value) indicates the presence, expression, or gene copy number of a target nucleic acid (such as PTEN, PIK3CA, MET, and/or TOP2A) in the sample. In some examples, change in expression of a target nucleic acid (such as PTEN, PIK3CA, MET, and/or TOP2A) compared to a control (such as an increase or decrease of about 1.5- fold, 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, or more relative to a control sample or reference value) indicates presence or prognosis of a tumor. In other examples, the gene copy number of a target nucleic acid (such as PTEN, PIK3CA, MET, and/or TOP2A) is determined and an increase in gene copy number (such as a gene copy number greater than about 2, 3, 4, 5, 10, 20, or more) indicates the presence or prognosis of a tumor.

In other examples, the probes may be used to analyze copy number changes associated with autism (e.g., cultured peripheral blood lymphocytes as described by van Daalen et al, Neurogenetics 12:315-323, 2011). In another example, the probes may be used to evaluate signaling pathways associated with leptins and metabolism (e.g., Donato et al., Arq. Bras. Endocrinol. Metab. 54(7):591-602, 2010 and Williams et al, J. Neurosci. 31(37): 13147- 13156, 201 1).

A. In Situ Hybridization

In situ hybridization (ISH) involves contacting a sample containing a target nucleic acid (e.g., a genomic target nucleic acid) in the context of a metaphase or interphase chromosome preparation (such as a cell or tissue sample mounted on a slide) with a labeled probe specifically hybridizable or specific for the target nucleic acid (for example, one or more of the probes disclosed herein). The slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization. The chromosome sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids. The probe (formulated in a suitable hybridization buffer) and the sample are combined, under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium). The chromosome preparation is washed to remove excess probe, and detection of specific labeling of the target is performed using standard techniques.

For example, a biotinylated probe can be detected using fluorescein-labeled avidin or avidin-alkaline phosphatase. For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)-conjugated avidin. Amplification of the FITC signal can be effected, if necessary, by incubation with biotin-conjugated goat anti-avidin antibodies, washing and a second incubation with FITC-conjugated avidin. For detection by enzyme activity, samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre- equilibrated (e.g., in alkaline phosphatase (AP) buffer). The enzyme reaction can be performed in, for example, AP buffer containing NBT/BCIP and stopped by incubation in 2 X SSC. For a general description of in situ hybridization procedures, see, e.g., U.S. Patent No. 4,888,278.

Numerous procedures for FISH, CISH, and SISH are known in the art. For example, procedures for performing FISH are described in U.S. Patent Nos. 5,447,841 ; 5,472,842; and 5,427,932; and for example, in Pinkel et al, Proc. Natl. Acad. Sci.

83:2934-2938, 1986; Pinkel et al, Proc. Natl. Acad. Sci. 85:9138-9142, 1988; and Lichter et al, Proc. Natl. Acad. Sci. 85:9664-9668, 1988. CISH is described in, e.g., Tanner et al, Am. J. Pathol. 157: 1467- 1472, 2000 and U.S. Patent No. 6,942,970. Additional detection methods are provided in U.S. Patent No. 6,280,929.

Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties. As discussed above, probes labeled with fluorophores (including fluorescent dyes and quantum dots) can be directly optically detected when performing FISH.

Alternatively, the probe can be labeled with a non- fluorescent molecule, such as a hapten (such as the following non-limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin- based compounds, and combinations thereof), ligand or other indirectly detectable moiety. Probes labeled with such non- fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody

(or receptor, or other specific binding partner) specific for the chosen hapten or ligand. The detection reagent can be labeled with a fluorophore (e.g., quantum dot) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can in turn be labeled with a fluorophore. Optionally, the detectable label is attached directly to the antibody, receptor

(or other specific binding agent). Alternatively, the detectable label is attached to the binding agent via a linker, such as a hydrazide thiol linker, a polyethylene glycol linker, or any other flexible attachment moiety with comparable reactivities. For example, a specific binding agent, such as an antibody, a receptor (or other anti-ligand), avidin, or the like can be covalently modified with a fluorophore (or other label) via a heterobifunctional polyalkyleneglycol linker such as a heterobifunctional polyethyleneglycol (PEG) linker. A heterobifunctional linker combines two different reactive groups selected, e.g., from a carbonyl-reactive group, an amine -reactive group, a thiol-reactive group and a photo- reactive group, the first of which attaches to the label and the second of which attaches to the specific binding agent.

In other examples, the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH). As indicated above, the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/01 17153.

In further examples, a signal amplification method is utilized, for example, to increase sensitivity of the probe. For example, CAtalyzed Reporter Deposition (CARD), also known as Tyramide Signal Amplification (TSA™) may be utilized. In one variation of this method a biotinylated probe detects the presence of a target by binding thereto. Next a streptavidin-peroxidase conjugate is added. The streptavidin binds to the biotin. A substrate of biotinylated tyramide (tyramine is 4-(2- aminoethyl)phenol) is used, which presumably becomes a free radical when interacting with the peroxidase enzyme. The phenolic radical then reacts quickly with the surrounding material, thus depositing or fixing biotin in the vicinity. This process is repeated by providing more substrate (biotinylated tyramide) and building up more localized biotin. Finally, the "amplified" biotin deposit is detected with streptavidin attached to a fluorescent molecule.

Alternatively, the amplified biotin deposit can be detected with avidin-peroxidase complex, that is then fed 3,3'-diaminobenzidine to produce a brown color. It has been found that tyramide attached to fluorescent molecules also serve as substrates for the enzyme, thus simplifying the procedure by eliminating steps.

In other examples, the signal amplification method utilizes branched DNA (bDNA) signal amplification. In some examples, target-specific oligonucleotides (label extenders and capture extenders) are hybridized with high stringency to the target nucleic acid. Capture extenders are designed to hybridize to the target and to capture probes, which are attached to a microwell plate. Label extenders are designed to hybridize to contiguous regions on the target and to provide sequences for hybridization of a preamplifier oligonucleotide. Signal amplification then begins with preamplifier probes hybridizing to label extenders. The preamplifier forms a stable hybrid only if it hybridizes to two adjacent label extenders. Other regions on the preamplifier are designed to hybridize to multiple bDNA amplifier molecules that create a branched structure. Finally, alkaline phosphatase (AP)-labeled oligonucleotides, which are complementary to bDNA amplifier sequences, bind to the bDNA molecule by hybridization. The bDNA signal is the chemiluminescent product of the AP reaction See, e.g., Tsongalis, Microbiol. Inf. Dis. 126:448-453, 2006; U.S. Pat. No. 7,033,758.

In further examples, the signal amplification method utilizes polymerized antibodies. In some examples, the labeled probe is detected by using a primary antibody to the label (such as an anti-DIG or anti-DNP antibody). The primary antibody is detected by a polymerized secondary antibody (such as a polymerized HRP-conjugated secondary antibody or an AP-conjugated secondary antibody). The enzymatic reaction of AP or HRP leads to the formation of strong signals that can be visualized.

It will be appreciated by those of ordinary skill in the art that by appropriately selecting labeled probe-specific binding agent pairs, multiplex detection schemes can be produced to facilitate detection of multiple target nucleic acids (e.g., genomic target nucleic acids) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample). For example, a first probe that corresponds to a first target nucleic acid can be labeled with a first hapten, such as biotin, while a second probe that corresponds to a second target nucleic acid can be labeled with a second hapten, such as DNP. Following exposure of the sample to the probes, the bound probes can be detected by contacting the sample with a first specific binding agent (in this case avidin labeled with a first fluorophore, for example, a first spectrally distinct quantum dot, e.g., that emits at 585 nm) and a second specific binding agent (in this case an anti-DNP antibody, or antibody fragment, labeled with a second fluorophore (for example, a second spectrally distinct quantum dot, e.g., that emits at 705 nm). Additional probes/binding agent pairs can be added to the multiplex detection scheme using other spectrally distinct

fluorophores. Numerous variations of direct, and indirect (one step, two step or more) can be envisioned, all of which are suitable in the context of the disclosed probes and assays.

Additional details regarding certain detection methods, e.g., as utilized in CISH and SISH procedures, can be found in Bourne, The Handbook of Immunoper oxidase Staining Methods , published by Dako Corporation, Santa Barbara, CA.

In some embodiments, the disclosed probes can be used in methods of determining the copy number of a target nucleic acid (such as PTEN, PIK3CA, MET, or TOP2A) in a biological sample (such as a tissue sample). Methods of determining the copy number of a gene or chromosomal region are well known to those of ordinary skill in the art. In some examples, the methods include in situ hybridization (such as fluorescent, chromogenic, or silver in situ hybridization), comparative genomic hybridization, or polymerase chain reaction (such as real-time quantitative PCR). In some examples, methods of determining gene copy number include counting the number of ISH signals (such as fluorescent, colored, or silver spots) for the target nucleic acid in one or more individual cells. The methods may also include counting the number of ISH signals (such as fluorescent, colored, or silver spots) for a reference (such as a chromosome-specific probe) in the cells. In particular examples, the number of copies of the gene (or chromosome) may be estimated by the person (or computer, in the case of an automated method) scoring the slide. In some examples, an increased copy number relative to a control (such as an increase of about 1.5-fold, 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, or more relative to a control sample or reference value) indicates an increase in the target nucleic acid copy number.

In some examples, the method includes counting the number of copies per cell or nucleus of a reference, such as a chromosomal locus known not to be abnormal, for example a centromere. In some examples, the reference is on the same chromosome as the gene of interest. Exemplary reference chromosomes that can be used for particular human genes of interest are provided in Table 1. In particular examples, the reference locus is detected by using a pericentromeric specific probe. Such probes are known in the art and are commercially available, for example, Vysis CEP probes (Abbott Molecular, Des Plaines, IL) and SPOTLIGHT centromeric probes (Invitrogen, Carlsbad, CA). In some examples, a ratio of target nucleic acid copy number to reference copy number greater than about two (such as greater than about 2, 3, 4, 5, 10, 20, or more), indicates an increase in the target nucleic acid copy number.

Table 1

Exemplary reference chromosomes for particular target nucleic acids

B. Microarray Applications

Comparative genomic hybridization (CGH) is a molecular-cytogenetic method for the analysis of copy number changes (gain/loss) in the DNA content of cells. The contribution of genome structural variation to human disease is found in rare genomic disorders (for example, Trisomy 21 , Prader-Willi Syndrome) and a broad range of human diseases, such as genetic diseases, autism, schizophrenia, cancers, and autoimmune diseases. In one example, the method is based on the hybridization of differently fluorescently labeled sample DNA (for example, labeled with fluorescein-FITC) and normal DNA (for example, labeled with rhodamine or Texas red) to normal human metaphase preparations. Using methods known in the art, such as epifluorescence microscopy and quantitative image analysis, regional differences in the fluorescence ratio of sample versus control DNA can be detected and used for identifying abnormal regions in the sample cell genome. CGH detects unbalanced chromosomes changes (such as increase or decrease in DNA copy number). See, e.g., Kallioniemi et al, Science 258:818-

821, 1992; U.S. Pat. Nos. 5,665,549 and 5,721,098.

Genomic DNA copy number may also be determined by array CGH (aCGH). See, e.g., Pinkel and Albertson, Nat. Genet. 37:S 11-S17, 2005; Pinkel et al, Nat. Genet.

20:207-211, 1998; Pollack et al, Nat. Genet. 23:41-46, 1999. Similar to standard CGH, sample and reference DNA are differentially labeled and mixed. However, for aCGH, the

DNA mixture is hybridized to a slide containing hundreds or thousands of defined DNA probes (such as probes that specifically hybridize to a genomic target nucleic acid of interest). The fluorescence intensity ratio at each probe in the array is used to evaluate regions of DNA gain or loss in the sample, which can be mapped in finer detail than CGH, based on the particular probes which exhibit altered fluorescence intensity.

In general, CGH (and aCGH) does not provide information as to the exact number of copies of a particular genomic DNA or chromosomal region. Instead, CGH provides information on the relative copy number of one sample (such as a tumor sample) compared to another (such as a reference sample, for example a non-tumor cell or tissue sample). Thus, CGH is most useful to determine whether genomic DNA copy number of a target nucleic acid is increased or decreased as compared to a reference sample thereby determining the copy number variation of a target nucleic acid sample relative to a reference sample.

In a particular example, the disclosed probes may be utilized for aCGH. For example, an unlabeled probe disclosed herein (such as one or more of any one of SEQ ID NOs: 1-50 or a portion thereof) may be immobilized on a solid surface (such as nitrocellulose, nylon, glass, cellulose acetate, plastics (for example, polyethylene, polypropylene, or polystyrene), paper, ceramics, metals, and the like). Methods of immobilizing nucleic acids on a solid surface are well known in the art (see, e.g., Bischoff et αΙ., ΑηαΙ. Biochem. 164:336-344, 1987; Kremsky et al, Nuc. Acids Res. 15:2891-2910, 1987). As discussed above, differently fluorescently labeled sample DNA (for example, labeled with fluorescein-FITC) and reference DNA (for example, labeled with rhodamine or Texas red) is hybridized to the probe array and regional differences in the fluorescence ratio of sample versus reference DNA can be detected and used for identifying abnormal regions in the sample cell genome.

In another example, disclosed probes are synthesized in situ on a solid surface (such as nitrocellulose, nylon, glass, cellulose acetate, plastics (for example, polyethylene, polypropylene, or polystyrene), paper, ceramics, metals, and the like). For example, the probes are utilized for printing, in situ, on a solid support utilizing computer based microarray printing methodologies, such as those described in U.S. Pat. Nos. 6,315,958; 6,444, 175; and 7,083,975 and U.S. Pat. Application Nos. 2002/0041420, 2004/0126757, 2007/0037274, and 2007/0140906. In some examples, using a mask-less array synthesis

(MAS) instrument, oligonucleotides synthesized in situ on the microarray are under software control resulting in individually customized arrays based on the particular needs of an investigator. The number of probes synthesized on a microarray varies, for example presently anywhere from 50,000 to 2.1 million probes, in various configurations, can be synthesized on a single microarray slide (for example, Roche NimbleGen CGH

microarrays contain from 385,000 to 4 million or more probes/array).

Probe sequences are synthesized either in situ by MAS instruments, or alternatively by utilizing photolithographic methods as described in U.S. Pat. Nos.

5,143,854; 5,424, 186; 5,405,783; and 5,445,934. Utilizing the disclosed probes for microarray applications is not limited by their method of manufacture, and a skilled artisan will understand additional methods of creating microarrays with uniquely specific oligonucleotide probes thereon that are equally applicable. For example, historical methods of spotting nucleic acid sequences onto solid supports are also contemplated, such that historically utilized probes are replaced by uniquely specific probes as described herein. Regardless of method used to place probes on a microarray, the uniquely specific probes can be used to target one or more nucleic acid samples, either individually or on the same array.

Applications of the probes disclosed herein that are in situ synthesized or otherwise immobilized on a microarray slide can be utilized for aCGH as well as other microarray based genomic target enrichment applications such as those described in U.S. Pat. Publication Nos. 2008/0194413, 2008/0194414, 2009/0203540, and 2009/0221438. Utilizing uniquely specific probes for generating in situ synthesized microarrays provides many improvements over current microarray probe designs. For example, use of uniquely specific probes allows for more specific binding of target sequences as compared to current probes, therefore not as many probes are needed per target and/or in conjunction more can be added to capture additional targets. Further, the need for blocking DNA (for example, Cot-1™ DNA) typically utilized in microarray experiments is reduced or eliminated when utilizing uniquely specific oligonucleotide probes.

For CGH applications, typically both target and reference genomic DNA are hybridized on one array for comparison on one microarray substrate. The CGH Analysis User's Guide (version 5.1, Roche NimbleGen, Madison, WI; available on the World Wide Web at nimblegen.com) describes methods for performing CGH analysis utilizing microarrays. In general, two genomic DNA samples, a target sample and a reference sample, are fragmented and labeled with different detection moieties (for example, Cy-3 and Cy-5 fluorescent moieties). The two labeled samples are mixed and hybridized to a microarray support, in this case a microarray comprising uniquely specific oligonucleotide probes, and the microarray is subsequently assayed for both detection moieties. The microarrays are scanned and detection data captured, for example by scanning a microarray with a microarray scanner (for example, a MS200 Microarray Scanner; Roche NimbleGen). The data is analyzed using analysis software (for example, NimbleScan; Roche NimbleGen). The target genomic sequence data is compared to the reference and DNA copy number gains and losses in target samples are thereby characterized. The target genomic sequences can be, for example, from targeted region(s) of one or more chromosome(s), one whole chromosome, or the total genomic complement of an organism (for example, a eukaryotic genome, such as a mammalian genome, for example a human genome).

For genomic enrichment (also known as sequence capture), typically a genomic sample is hybridized to a microarray support comprising targeted sequence specific probes for specific target enrichment prior to downstream applications, such as sequencing. The Sequence Capture User's Guide (version 3.1, Roche NimbleGen, incorporated by reference herein) describes methods for performing genomic enrichment. In general, a genomic DNA sample is prepared for hybridization to a microarray support, in this case a microarray comprising the disclosed uniquely specific oligonucleotide probes designed to capture targeted sequences from a genomic sample for enrichment. The captured genomic sequences are then eluted from the microarray support and sequenced, or used for other applications.

C. Blocking DNA

Genome-specific blocking DNA (such as human DNA, for example, total human placental DNA or Cot-1™ DNA) is usually included in a hybridization solution (such as for in situ hybridization) to suppress probe hybridization to repetitive DNA sequences or to counteract probe hybridization to highly homologous (frequently identical) off target sequences when a probe complementary to a human genomic target nucleic acid is utilized. In hybridization with standard probes, in the absence of genome-specific blocking DNA, an unacceptably high level of background staining (for example, non-specific binding, such as hybridization to non-target nucleic acid sequence) is usually present, even when a "repeat- free" probe is used. The disclosed probes exhibit reduced background staining, even in the absence of blocking DNA. In particular examples, the hybridization solution including the disclosed probes does not include genome- specific blocking DNA (for example, total human placental DNA or Cot-1™ DNA, if the probe is complementary to a human genomic target nucleic acid). This advantage is derived from the uniquely specific nature of the target sequences included in the probe; each labeled probe sequence binds only to the cognate uniquely specific genomic sequence. This results in dramatic increases in signal to noise ratios for ISH techniques. In some examples the hybridization solution may contain carrier DNA from a different organism (for example, salmon sperm DNA or herring sperm DNA, if the genomic target nucleic acid is a human genomic target nucleic acid) to reduce non-specific binding of the probe to non-DNA materials (for example to reaction vessels or slides) with high net positive charge which can non-specifically bind to the negatively charged probe DNA.

VIII. Response Prediction in Neoadjuvant Breast Cancer Treatment

Certain disclosed embodiments concern a method for predicting pathologic response in patients being considered for neoadjuvant breast cancer treatment. Assays with disclosed probes and probe sets are used to evaluate biological samples from the human patient, such as a biopsy sample, a resection sample, a cytology sample, etc.

Regions of a specimen may be first selected for evaluation using conventional methods, such as stains containing hematoxylin and eosin. For example, a section of a paraffin- embedded specimen can be first analyzed with a conventional technique, such as a hematoxylin/eosin stain, to identify a region as probably cancerous. Cells within that identified portion are then evaluated.

In particular examples, one or more biological samples are contacted with a gene locus probe selected from a probe to chromosome 7q31, a probe to chromosome 3q26, a probe to chromosome 10q23, a probe to chromosome 17q21, a chromosomal enumeration pericentromeric probe selected from a chromosome 7 pericentromeric probe, a chromosome 3 pericentromeric probe, a chromosome 10 pericentromeric probe, a chromosome 17 pericentromeric probe, or combinations thereof, under conditions sufficient to enable hybridization of the probe to target sequences. The probes may be used as probe sets, where a first biological sample is contacted with a first probe set, a second biological sample is contacted with a second probe set, a third biological sample is contacted with a third probe set, such that the number of biological samples may correspond to the number of different probe sets used for a particular analysis. The gene locus probes may be selected from a probe to a region containing MET, a probe to a region containing PIK3CA, a probe to a region containing PTEN, a probe to a region containing TOP2A, or combinations thereof. More typically, the gene locus probes are selected from (a) a nucleic acid molecule having at least 70% sequence identity, preferably at least 80% sequence identity, and even more preferably at least 90% sequence identity, with a sequence according to any one of SEQ ID NOs: 1-50, or (b) a nucleic acid molecule having at least 70% sequence identity, preferably at least 80% sequence identity, and even more preferably at least 90% sequence identity, with at least 250 contiguous nucleotides of any one of SEQ ID NOs: 1-50. In particular disclosed embodiments, the nucleic acid molecule is any one of SEQ ID NOs: 1-20 and 31-50.

The appropriate chromosomal enumeration pericentromeric probe typically is selected from a chromosome 3 pericentromeric probe (a CEN3 probe), a chromosome 7 pericentromeric probe (a CEN7 probe), a chromosome 10 pericentromeric probe (a CEN10 probe), a chromosome 12 pericentromeric probe (a CEN12 probe), and a chromosome 17 pericentromeric probe (a CEN17 probe), and even more typically is selected from a CEN3 probe, a CEN7 probe, and a CEN10 probe. The appropriate chromosomal probe is used for various reasons, including identifying chromosome copy number abnormalities.

Chromosome enumeration probes hybridize to a chromosomal region, usually a repeat sequence region, and indicate the presence or absence of a target sequence on

chromosomes 3, 7, 10, 12, and 17. A chromosome enumeration probe can hybridize to a repetitive sequence, located either near or removed from a centromere, or can hybridize to a unique sequence located at any position on a chromosome. Probes that hybridize with centromeric DNA are available commercially, such as from Molecular Probes, Inc.

(Eugene, Oregon). Alternatively, probes can be made non-commercially using well known techniques. The region of interest can be isolated through cloning or by site-specific amplification via the polymerase chain reaction (PCR). The starting human DNA used to manufacture useful loci specific probes can be obtained by obtaining bacterial cultures containing human DNA sequences matching the desired regions of the human genome. For example, bacterial cells containing bacterial artificial chromosomes (BAC) which comprise specific human DNA sequences, typically 80 to 200 kilobases in length, can be obtained from cell culture repositories. BAC clones spanning the human genome can be obtained from commercial sources to essentially any desired region of the human genome.

Each probe includes a detectable label. The probes can comprise any detectable label that facilitates detecting the probe when hybridized to a target. Effective detection moieties include both direct and indirect labels as described herein, with exemplary detectable labels including fluorophores, chromophores, enzymes and haptens. In certain embodiments the detectable label uniquely identifies the probe to which it is coupled.

The biological sample is contacted with the probes under conditions sufficient to enable probe hybridization to an appropriate target, if any exists, in the sample. Any suitable hybridization method can be used. In situ hybridization techniques, including silver in situ hybridization (SISH), fluorescent in situ hybridization (FISH), and chromogenic in situ hybridization (CISH), and immunohistochemical (IHC) techniques, are suitable techniques for practicing the disclosed embodiments. In general, in situ hybridization is a preferred method, and typically includes the steps of fixing a biological sample, hybridizing one or more probes to target DNA contained within the fixed sample, washing to remove non-specifically bound probe, and detecting the hybridized probe. The biological sample may be contacted with both the gene locus probe and the chromosomal enumeration pericentromeric probe independently, or the biological sample can be contacted with the gene locus probe, the chromosomal enumeration pericentromeric probe as a probe set, as disclosed herein.

Probes are annealed under hybridizing conditions that facilitate annealing between a probe and target sequence. Hybridization conditions vary, depending on the

concentrations, base compositions, complexities, and lengths of the probes, as well as salt concentrations, temperatures, and length of incubation.

Non-specific binding of probes to DNA outside of the target region can be removed by a series of salt washes. Temperature and salt concentration in each wash depend on the desired stringency.

In situ hybridization can also be carried out with the specimen cells from the biological sample in liquid suspension, followed by detection by flow cytometry.

Prior to in situ hybridization, chromosomal probes and chromosomal DNA contained within the cell each are denatured. Denaturation typically is performed by incubating in the presence of high pH, heat (e.g., temperatures from about 70 °C to about 95 °C), organic solvents such as formamide, and combinations thereof. If the

chromosomal probes are prepared as a single- stranded nucleic acid, then denaturation is not required.

In certain embodiments, portions of ISH and/or IHC procedures, or substantially the entire procedure, are performed by an automated staining system. Suitable automated systems include the BenchMark systems sold by Ventana Medical Systems, Inc.

Particular disclosed embodiments of the method concern analyzing probe hybridization information to facilitate determining neoadjuvant breast cancer treatment in a subject. After hybridization, the probe hybridization results can be analyzed and determined by microscopy. Any suitable microscopic imaging method, such as standard bright-field microscopy techniques for analyzing chromogen (CISH) probes and dark-field microscopy for analyzing FISH probes, can be used to analyze probe hybridization results. Automated digital imaging systems also can be used. Probe hybridization results are analyzed statistically to determine neoadjuvant breast cancer treatment prognosis factors. Various factors, or criteria, were identified from the probe results, particularly when certain factors are considered in combination, that strongly correlate to suitability for neoadjuvant breast cancer treatment.

The following factors were evaluated:

1) the average number of gene locus or centromere copies per cell (gene locus/cell or centromere/cell, e.g. MET/cell or CEN7/cell);

2) the percentage of cells with greater than 2 gene locus or centromere signals (gene locus gain or centromere gain, e.g. MET gain or CEN7 gain);

3) the percentage of cells with less than 2 gene locus or centromere signals (gene locus loss or centromere loss, e.g. MET loss or CEN7 loss);

4) the percentage of cells with either greater than 2 or less than 2 gene locus or centromere signals (gene locus or centromere gain or loss, e.g. MET gain or loss or CEN7 gain or loss);

5) the average number of gene locus copies per corresponding centromere copies (gene locus/centromere, e.g. MET/CEN7);

6) the percentage of cells with more copies of the gene locus than corresponding centromere (gene locus/centromere gain, e.g. MET/CEN7 gain);

7) the percentage of cells with fewer copies of the gene locus than corresponding centromere (gene locus/centromere loss, e.g. MET/CEN7 loss); and

8) the percentage of cells with either gene locus gain or gene locus loss (e.g. MET/CEN7 gain or loss).

A range of cutoffs was applied to parameters in a combination. The combination of parameters was considered positive at each combination of cutoffs if one or more of the parameters were positive according to the criteria applied, as described below:

1) for combinations of 2 parameters, positivity required both parameters to be positive for an AND relationship;

2) for combinations of 2 parameters, positivity required either parameter to be positive for an OR relationship;

3) for combinations of 3 parameters, positivity required all 3 parameters to be positive for an AND relationship;

4) for combinations of 3 parameters, positivity required at least 2 parameters to be positive for a 2-parameter positive relationship; and

5) for combinations of 3 parameters, positivity required at least 1 parameter to be positive for a 1 -parameter positive relationship.

Probe hybridization results were characterized using the probes, or probe sets, and factors described above. For example, a copy number abnormality pattern in a sample was assessed by examining the hybridization pattern of the chromosomal probe (e.g., the number of signals for each probe) in the cell, and recording the number of signals.

Aneusomy refers to abnormal copy number generally, either of the whole chromosome or a locus on a chromosome; monosomy refers to one copy; nullsomy refers to zero copies; and polysomy refers to greater than 2 copies.

In specific embodiments, probes to loci containing the HER2, TOP2A, MET, PIK3CA and PTEN genes, and to the centromeric regions (CEN) of appropriate corresponding associated chromosomes 17, 7, 3, and 10, were used to identify genomic copy numbers of the respective loci in tumor specimens from patients who underwent neoadjuvant treatment (HER2 targeted therapies, such as trastuzumab, in combination with chemotherapy) for breast cancer. The patient may be a HER2 -positive patient, and more typically is either a HER2-expression positive or a HER2 gene amplification-positive patient. Analysis of the copy number data has revealed significant improvement in the ability to identify patients who achieved a complete pathological response (pCR), and patients who did not achieve a complete pathological response, based on combinations of the copy number data from two or more of the tested loci. Particularly important were the copy numbers provided by, either alone or in combination, MET/CEN7, Ki-67,

PIK3CA/CEN3, PTEN/CEN10. These factors provided statistically significant associations, such as defined as p-values less than 0.05 as determined by χ² probabilities.

Accordingly, in particular disclosed embodiments, the method may further comprise determining a treatment protocol based on the prognosis, and treating the patient according to the protocol wherein determining comprises selecting a criteria by which to judge a low or high parameter state associated with pCR, testing the patient with the gene locus probe, chromosomal enumeration pericentromeric probe, or combinations thereof, and then obtaining a positive or negative response. In particular disclosed embodiments, the positive response indicates that the patient is likely to experience pathologic complete response and the negative response indicates that the patient is unlikely to experience pathologic complete response. As disclosed herein the criteria used in the method of determining the treatment protocol may be selected from AUC from an ROC curve, DFI from an ROC curve, χ² probabilities, Fischer's Exact Test probabilities, optimal cut-off values, or combinations thereof. The criteria typically are determined using a statistically significant sample for probe hybridization results.

Table 2 provides ROC and contingency table analyses of various parameters and their combinations as determined generally above and by the methods described in Example 3 below. Each row of Table 2 represents a single parameter or combinations of parameters that were analyzed.

Table 2

Analyses of Various Parameters and Combinations thereof for

Response Prediction of Neoadjuvant Breast Cancer Treatment

os.

Column 1 of Table 2 lists number of parameters considered in combination, ranging from a single parameter to 3 parameters in combination, Column 2 of Table 2 the ISH or IHC parameter analyzed in each row and the relationship between the parameters for specimen positivity (AND, OR, or number of positive parameters required for positive specimen). Column 3 lists the number of patients with pCR. Column 4 lists the number of other patients (non-pCR). The total number of patients tested is the sum of the patient numbers reported in columns 3 and 4. Column 5 lists the parameter state that is associated with pCR (high parameter is parameter value >= cutoff; low parameter is parameter value < cutoff).

Column 6 of Table 2 lists the area under the curve (AUC) for a corresponding receiver operator characteristics ROC curve, or ROC AUC. An ROC curve is a plot of the sensitivity (the fraction of patients detected that had a pCR) versus 1 -specificity (where specificity is the fraction of non-responders for which the marker was negative). Once an ROC curve is plotted, the area under a particular curve also can be determined. An AUC of 1 means that the analysis perfectly distinguished responders from non-responders. As a result, the higher the AUC number, the higher the correlation between that marker and assessing prognosis for responding or not responding to neoadjuvant breast cancer treatment.

Column 7 of Table 2 lists the optimal cutoff determined from the ROC curve to separate responders from non-responders. Solely by way of example, the optimal cutoff for the MET/CEN7 ratio was determined to be 1.1. At this cutoff, the sensitivity was 0.56, meaning that 56% of the responders had a ratio equal to or above 1.1, the specificity was 69%, and the p value was 0.23. These results indicate that the MET/CEN7 ratio, by itself, was not overly useful as a prognostic for assessing neoadjuvant breast cancer treatment.

The sensitivity and specificity for detecting pCR using the optimal cutoff are listed in columns 8 and 9, respectively. DFI, or distance from ideal, for a particular result is provided in Column 10 for the result achieved using the indicated optimal cutoffs. The χ² probability, calculated from a contingency table at the optimal cutoff, is listed in column 1 1.

Several ROC curves are plotted in FIGS. 6 and 7. FIGS. 6 and 7 illustrate the improved ability to distinguish patients with pCR and non-pCR as parameters are combined.

Based on these analyses, certain disclosed embodiments of the present invention establish a prognostic correlation between a single marker, a ratio of a single marker and gain or loss of a corresponding chromosome marker, or multiple such markers, if:

(a) the ROC AUC is 0.5 or greater, preferably 0.6 or greater, more preferably 0.7 or greater, even more preferably 0.8 or greater, even more preferably 0.9 or greater, with certain disclosed embodiments, such as combination of MET/CEN7 gain and loss, Ki-67, and PIK3CA/CEN3 gain (at least two positives), having an AUC of 1.0;

(b) the sensitivity is 0.5 or greater, preferably 0.6 or greater, even more preferably 0.7 or greater, even more preferably 0.8 or greater, even more preferably 0.9 or greater, and with particularly effective factors, such as MET/CEN7 gain or loss, MET/CEN7 gain or loss AND Ki-67, and MET/CEN7 gain and loss, Ki-67, and PIK3CA/CEN3 gain (at least two positives), each having a sensitivity of 1.00;

(c) the specificity is 0.6 or greater, preferably 0.7 or greater, even more preferably 0.8 or greater, even more preferably 0.9 or greater, and with particularly effective factors, such as (i) MET/CEN7 gain or loss, Ki-67, and PIK3CA/CEN3 gain (at least two positives), and (ii) MET/CEN7 gain AND PTEN/CEN10 gain or loss having a specificity of 1.00;

(d) the DFI is 0.3 or less, preferably 0.2 or less, even more preferably 0.1 or less, and for certain factors, such as MET/CEN7 gain or loss, Ki-67, and PIK3CA/CEN3 gain (two positives), having a DFI of 0.0; and

(e) the χ² probability is 0.05 or less, preferably 0.005 or less, more preferably 0.0005 or less, even more preferably 0.00005 or less, even more preferably 0.000005 or less, and even more preferably 0.0000005 or less.

With reference to particular single factors, particularly effective factors included: the MET/CEN7 gain or loss, having an ROC AUC of 0.824, a sensitivity of 1.00, a specificity of 0.69, a DFI of 0.313 (at optimal cutoff), and a χ2 probability value of 0.00089 (at optimal cutoff); the MET/CEN7 gain having an ROC AUC of 0.808, a sensitivity of 0.78, a specificity of 0.81, a DFI of 0.291, and a χ2 probability value of 0.0038; MET/cell having an ROC AUC of 0.769, a sensitivity of 0.78, a specificity of 0.69, and a χ2 probability value of 0.025; CEN7 gain having an ROC AUC of 0.701 , a sensitivity of 0.78, a specificity of 0.75, a DFI of 0.334, and a χ2 probability value of 0.01 1 ; Ki-67 having an ROC AUC of 0.838, a sensitivity of 0.77, a specificity of 0.67, a DFI of 0.405, and a χ2 probability value of 0.017; and PTEN/CEN10 gain or less having an ROC AUC of 0.743, a sensitivity of 0.85, a specificity of 0.58, a DFI of 0.444, and a χ2 probability value of 0.025.

However, Table 2 illustrates that using combinations of factors is particularly useful in assessing likelihood of breast cancer patients benefitting from neoadjuvant treatments. For example, particularly effective factors include: MET/CEN7 gain or loss AND Ki-67, having an ROC AUC of 0.967, a sensitivity of 1.00, a specificity of 0.88, and a χ2 probability value of 0.000023; PIK3CA/CEN3 gain AND MET/CEN7 gain or loss, having an ROC AUC of 0.937, a sensitivity of 0.89, a specificity of 0.93, and a χ2 probability value of 0.000056; PIK3CA/CEN3 gain OR Ki-67, having an ROC AUC of 0.877, a sensitivity of 0.92, a specificity of 0.69, and a χ2 probability value of 0.0014; PIK3CA/CEN3 gain AND Ki-67, having an ROC AUC of 0.837, a sensitivity of 0.92, a specificity of 0.69, and a χ2 probability value of 0.0014; MET/CEN7 gain AND

PTEN/CEN10 gain or loss, having an ROC AUC of 0.899, a sensitivity of 0.75, a specificity of 1.00, a DFI of 0.250, and a χ2 probability value of 0.00052; MET/CEN7 gain and loss, Ki-67,and PIK3CA/CEN3 gain (2 pos.), having an ROC AUC of 1.000, a sensitivity of 1.00, a specificity of 1.00, a DFI of 0.0, and a χ2 probability value of 9.6x10^" ⁷; and MET/CEN7 gain, CEN7 gain, and PTEN/CEN10 gain or loss (2 pos.), having an ROC AUC of 0.994, a sensitivity of 1.00, a specificity of 0.91, a DFI of 0.091, and a l probability value of 0.000089.

Based on these results, two or more factor combinations are particularly effective with: an ROC AUC of 0.90 or greater, preferably greater than 0.93, and even more preferably 1.0; a sensitivity of 0.8 or greater, preferably 0.85 or greater, more preferably 0.90 or greater, even more preferably 0.95 or greater, and in certain embodiments 1.00; a specificity of 0.8 or greater, preferably 0.85 or greater, even more preferably 0.90 or greater, and in certain embodiments 1.00; and a χ2 probability value of 0.001 or less, preferably 0.0001 or less, preferably 0.00001 or less, and even more preferably 0.000001 or less.

Disclosed embodiments provide significantly improved information for prognosis of neoadjuvant breast cancer treatment. A person of ordinary skill in the art will appreciate that disclosed embodiments can be used in combination with other, known techniques for assessing neoadjuvant breast cancer treatment prognosis.

The probes and probe sets useful with the methods of the invention can be packaged with other reagents into kits to be used in carrying out the methods of the invention. Probe kits for breast cancer treatment prognosis can include, by way of example, disclosed embodiments of probes, including ISH and IHC probes, labeled with a detectable label.

IX. Methods of Treatment

The disclosed methods can further include selecting subjects for treatment with a HER2 therapy, for example if the sample from the subject is scored as HER2 -positive. Additionally, the disclosed methods can further include administering an HER2 therapy to the subject if the sample from the subject is scored as HER2 -positive. In some examples, the HER2 therapy is trastuzumab.

In some examples, the disclosed methods include providing a therapeutically effective amount of the HER2 therapy to the subject (such as a subject having HER2- positive tumor). Methods and therapeutic dosages of such agents and treatments are known to those of ordinary skill in the art, and for example, can be determined by a skilled clinician. In a non-limiting example, a therapeutically effective amount of trastuzumab is administered to a subject having a breast cancer tumor that is identified as HER2 -positive. In some examples, a therapeutically effective amount of trastuzumab can be about 50-2000 mg/day (such as about 50, 100, 150, 200, 250, 300, 400, 500, 600, 700 ,800, 900, 1000, 1 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 mg/day), administered orally in one or two doses per day. In some examples, the methods include orally administering 200 mg of trastuzumab to the subject once or twice per day if the sample from the subject is scored as HER2-positive. In other examples, the methods include orally administering 250 mg of trastuzumab to the subject once or twice per day if the sample from the subject is scored as HER2-positive. Dosages and dosing schedules of trastuzumab for a subject can be determined by a skilled clinician, taking into account additional factors such as tumor site, tumor stage, tumor grade, patient treatment history, patient performance and nutritional status, concomitant health problems, social and logistic factors, previous primary tumors, and patient preference. Trastuzumab may be administered on a continuous dosing schedule or administered for one or more cycles (for example, one or more cycles of 21-28 days). Treatment may repeat every 21-28 days if administered in cycles.

In some examples, the subject is also administered one or more additional chemotherapeutic agents in combination with the HER2 therapy. Additional

chemotherapeutic agents include, but are not limited to alkylating agents, such as nitrogen mustards (for example, chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (for example, carmustine, fotemustine, lomustine, and streptozocin), platinum compounds (for example, carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan, dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites, such as folic acid (for example, methotrexate, pemetrexed, and raltitrexed), purine (for example, cladribine, clofarabine, fludarabine, mercaptopurine, and tioguanine), pyrimidine (for example, capecitabine), cytarabine, fluorouracil, and gemcitabine; plant alkaloids, such as podophyllum (for example, etoposide, and teniposide), taxane (for example, docetaxel and paclitaxel), vinca (for example, vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic/antitumor antibiotics, such as anthracycline family members (for example, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, rifampicin, hydroxyurea, and mitomycin; topoisomerase inhibitors, such as topotecan and irinotecan; monoclonal antibodies, such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab, and trastuzumab; photosensitizers, such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and verteporfm; and other agents , such as alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin, masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, vemuraflnib, vandetanib, and tretinoin. Selection and therapeutic dosages of such agents are known to those skilled in the art, and can be determined by a skilled clinician.

X. Working Examples

The disclosure is further illustrated by the following non-limiting examples. These examples are provided to illustrate certain features of exemplary working embodiments and are not intended to limit the scope of the disclosure.

Example 1

ISH Detection of PTEN, PIK3CA, MDM2, MET, and TOP2A

This example describes detection of target nucleic acids in tissue samples by ISH using the disclosed PTEN, PIK3CA, MDM2, MET, and TOP2A probes.

Probes were labeled with DNP for ISH. For each target nucleic acid (PIK3CA, PTEN, TOP2A, MET, or MDM2), a set of 10 plasmids including the appropriate probe sequences (PIK3CA - SEQ ID NOs: 1 1-20; PTEN - SEQ ID NOs: 1-10; TOP2A - SEQ ID NOs: 41-50; MET - SEQ ID NOs: 31-40; MDM2 - SEQ ID NOs: 21-30) were pooled, nick translation labeled with DNP, and bulked in Hybrisol® and TE. FFPE tissues were processed for ISH using a BENCHMARK ULTRA system (Ventana Medical Systems, Tucson, AZ) with the conditions for each probe as shown in Table 3. In addition to the gene locus probe, the ISH reactions also each included a pericentromeric probe for the chromosome where the target nucleic acid is located (e.g., PIK3CA - chromosome 3; PTEN - chromosome 10; TOP2A - chromosome 17; MET - chromosome 7; MDM2 - chromosome 12).

All gene probes were labeled with DNP and all reference (pericentromeric) probes were labeled with DIG. DNP-labeled probes were detected using SISH, which produces black staining. DIG-labeled probes were detected using a red chromogen. These were carried out with the following reagents: ultraView™ Red ISH DIG Detection Kit (Ventana Medical Systems) and ultraView™ SISH DNP Detection Kit (Ventana Medical Systems). Table 3

ISH Conditions

Representative digital images of the ISH reactions are shown in FIGS. 1-5. Each probe provided sensitive and specific staining results (Table 4). Tissue samples exhibiting the expected readable results (by a qualified reader; e.g., an appropriately trained pathologist) were designated as "pass." Factors that may contribute to readability include: sufficiently dark staining so as to be apparent, staining across the entire tissue such that all regions of the tissue exhibit the expected result, low background so as to eliminate detecting a false signal, and the biological meaning conferred by the signal matching the expected signal. The characteristics of the signal may vary according to tissue type.

Table 4

ISH results

Example 2

Detection of a Target Nucleic Acid

This example describes particular embodiments of a method that can be used for detecting a target nucleic acid in a sample utilizing probes described herein. However, a person of ordinary skill in the art will appreciate that methods that deviate from this example can also be used to successfully detect a target nucleic acid.

A sample, such as a tissue sample, is obtained from a subject. Tissue samples are prepared for ISH, including deparaffinization and protease digestion. The sample, such as a tissue or cell sample present on a substrate (such as a microscope slide) is incubated with a PTEN probe set (for example, a probe set including each of SEQ ID NOs: 1-10). The hybridization is carried out in the absence of human DNA blocking reagent (for example, in the absence of Cot-1™ DNA). Hybridization of the PTEN probe set to the sample is detected, for example, using microscopy. Detection of hybridization indicates presence of PTEN nucleic acid in the sample. In some examples, gene copy number is detected.

A person of ordinary skill in the art will understand that similar methods can be used to detect the presence of other target nucleic acids in a sample. For example, a probe set (for example, a probe set including each of SEQ ID NOs: 1 1-20) may be used to detect presence of PIK3CA in a sample, a probe set (for example, a probe set including each of SEQ ID NOs: 21-30) may be used to detect presence of MDM2 in a sample, a probe set (for example, a probe set including each of SEQ ID NOs: 31-40) may be used to detect presence of MET in a sample, or a probe set (for example, a probe set including each of SEQ ID NOs: 41-50) may be used to detect presence of TOP2A in a sample. Example 3

Thirty four formalin-fixed paraffin-embedded (FFPE) tissues obtained as needle biopsy patient specimens taken from the archives of the Cleveland Clinic were utilized in the study. Subsequent to biopsy, patients demonstrating HER2 gene amplification by fluorescence in situ hybridization (FISH) were treated in the neoadjuvant setting with either trastuzumab alone, or in combination with a chemotherapeutic drug, and characterized as either (1) demonstrating pathologic complete response (pCR), or (2) not demonstrating pCR.

ISH procedures were performed using techniques known to a person of ordinary skill in the art, and as further disclosed herein. ISH was performed on each specimen using the dual ISH procedure (dual color dual hapten DNA in situ hybridization for the Ventana Benchmark system). Ki-67 immunohistochemistry (IHC) was performed using techniques known to a person of ordinary skill in the art, and as further disclosed herein. Ki-67 automated IHC was performed on each specimen using Ventana primary antibody 30-9 with iVIEW detection on the Benchmark XT.

Probes were hybridized in the following pairs, each comprising a gene locus probe, referred to by the name of a gene contained within the targeted region (e.g. MET), and a pericentromeric (CEN) probe for the appropriate associated chromosome on which the gene locus lies (e.g. CEN7): (1) MET + CEN7; (2) TOP2A + CEN 17; (3) PTEN + CENIO; and (4) PIK3CA + CEN3. Suitable probes, ISH and IHC reagents for performing the methods, and automated systems for performing ISH and IHC techniques, such as BenchMark systems, are commercially available from Ventana Medical Systems, Inc. (Tucson, AZ).

Hybridized and coverslipped specimens were viewed with bright field microscopy. SISH (black) gene locus signals and CISH (red) centromere signals were enumerated on a cell-by-cell basis. Only invasive carcinoma tumor cells were selected for cell-by-cell signal enumeration using a 40X objective in combination with 10X eyepieces.

In general, 50 cells were enumerated per specimen except in several specimens that contained fewer than 50 cells with good hybridization signals. Gene locus and centromere copy number statuses were assessed using multiple measures, including:

1) the average number of gene locus or centromere copies per cell (gene locus/cell or centromere/cell, e.g. MET/cell or CEN7cell),

2) the percentage of cells with greater than 2 gene locus or centromere signals (gene locus gain or centromere gain, e.g. MET gain or CEN7 gain, 3) the percentage of cells with less than 2 gene locus or centromere signals (gene locus loss or centromere loss, e.g. MET loss or CEN7 loss,

4) the percentage of cells either greater than 2 or less than 2 gene locus or centromere signals (gene locus or centromere gain or loss, e.g. MET gain or loss or CEN7 gain or loss),

5) the average number of gene locus copies per corresponding centromere copies (gene locus/centromere, e.g. MET/CEN7),

6) the percentage of cells with more copies of the gene locus than

corresponding centromere (gene locus/centromere gain, e.g. MET/CEN7 gain),

7) the percentage of cells with fewer copies of the gene locus than

corresponding centromere (gene locus/centromere loss, e.g. MET/CEN7 loss), and

8) the percentage of cells with either gene locus gain or gene locus loss (e.g.

MET/CEN7 gain or loss).

The average number of gene loci or centromeres per cell was calculated by

summing the number of gene loci or centromere signals over all the cells enumerated within a specimen, and dividing by the number of cells enumerated. The average number of gene loci per centromeres was calculated by summing the number of gene loci over all the cells enumerated and dividing by the sum of all corresponding centromere signals counted in all the cells enumerated.

For Ki-67 30-9 IHC staining, interpretation was performed only for invasive tumor cells and was evaluated as follows: the area of invasive carcinoma with the highest

proliferation rate was assessed and expressed as the percentage of approximately 50

invasive tumor cell nuclei positive for Ki67 expression.

Values for each parameter for each specimen in the study are listed in Table 5.

Table 5

path TOP2A TOP2A TOP2A

Sample Date tissue resp % gain % loss TOP2A/cell cell total

NeolOO 6/7/2012 Breast pCR 4.00 82.00 1.26 50

NeolOl 6/7/2012 Breast pCR 100.00 0.00 10.38 50

Neol02 6/6/2012 Breast pCR 92.00 0.00 4.82 50

Neol05 6/6/2012 Breast pCR 78.00 4.00 3.90 50

Neol06 6/6/2012 Breast pCR 98.00 0.00 6.54 50

Neol08 6/6/2012 Breast pCR 2.00 58.00 1.46 50

Neol09 6/6/2012 Breast pCR 24.24 30.30 2.03 33

Neol lO 6/8/2012 Breast pCR

Neol l l 6/7/2012 Breast pCR 72.00 4.00 3.16 50

Neol l2 6/8/2012 Breast pCR 82.00 6.00 3.62 50 path TOP2A TOP2A TOP2A

Sample Date tissue resp % gain % loss TOP2A/cell cell total

Neol l4 6/7/2012 Breast pCR 100.00 0.00 9.18 50

Neol l5 6/7/2012 Breast pCR 82.00 0.00 4.22 50

Neol l6 6/7/2012 Breast pCR

Neol l 8 6/7/2012 Breast pCR 44.00 12.00 2.54 50

Neol20 6/7/2012 Breast pCR 12.00 40.00 1.72 50

Neo202 6/7/2012 Breast not pCR 88.00 0.00 3.94 50

Neo203 6/7/2012 Breast not pCR 98.00 0.00 5.30 50

Neo204 6/7/2012 Breast not pCR 6.00 36.00 1.72 50

Neo205 6/7/2012 Breast not pCR 0.00 78.00 1.16 50

Neo206 6/7/2012 Breast not pCR 92.00 0.00 5.30 50

Neo207 6/7/2012 Breast not pCR 52.00 24.00 2.62 50

Neo208 6/7/2012 Breast not pCR 100.00 0.00 15.50 50

Neo209 6/7/2012 Breast not pCR

Neo210B 6/7/2012 Breast not pCR 98.00 2.00 14.66 50

Neo21 1 6/7/2012 Breast not pCR

Neo212 6/7/2012 Breast not pCR 20.00 24.00 2.06 50

Neo213 6/7/2012 Breast not pCR 74.00 4.00 3.02 50

Neo214 6/8/2012 Breast not pCR 100.00 0.00 15.90 50

Neo217 6/7/2012 Breast not pCR 24.00 42.00 1.98 50

Neo218 6/7/2012 Breast not pCR 94.00 0.00 9.46 50

Neo220 6/7/2012 Breast not pCR 76.00 8.00 3.42 50

Neo301 6/7/2012 Breast not pCR 98.00 0.00 4.98 50

Neo304 6/7/2012 Breast not pCR

Neo305 6/7/2012 Breast not pCR 68.00 6.00 2.98 50

Table 5 (Continued)

CEN17- CEN17- path T % CEN17- CEN17- T cell

Sample Date tissue resp gain T % loss T/cell total

NeolOO 6/7/2012 Breast pCR 0.00 90.00 1.10 50

NeolOl 6/7/2012 Breast pCR 8.00 40.00 1.70 50

Neol02 6/6/2012 Breast pCR 74.00 6.00 3.34 50

Neol05 6/6/2012 Breast pCR 78.00 2.00 3.64 50

Neol06 6/6/2012 Breast pCR 18.00 28.00 1.98 50

Neol08 6/6/2012 Breast pCR 8.00 28.00 1.80 50

Neol09 6/6/2012 Breast pCR 30.30 24.24 2.27 33

Neol lO 6/8/2012 Breast pCR

Neol l l 6/7/2012 Breast pCR 68.00 2.00 2.86 50

Neol l2 6/8/2012 Breast pCR 60.00 6.00 2.98 50

Neol l4 6/7/2012 Breast pCR 24.00 38.00 1.86 50

Neol l5 6/7/2012 Breast pCR 60.00 6.00 2.88 50

Neol l6 6/7/2012 Breast pCR

Neol l 8 6/7/2012 Breast pCR 4.00 44.00 1.60 50

Neol20 6/7/2012 Breast pCR 22.00 30.00 1.96 50 CEN17- CEN17- path T % CEN17- CEN17- T cell

Sample Date tissue resp gain T % loss T/cell total

Neo202 6/7/2012 Breast not pCR 42.00 12.00 2.40 50

Neo203 6/7/2012 Breast not pCR 16.00 44.00 1.72 50

Neo204 6/7/2012 Breast not pCR 30.00 18.00 2.26 50

Neo205 6/7/2012 Breast not pCR 0.00 48.00 1.52 50

Neo206 6/7/2012 Breast not pCR 0.00 96.00 1.04 50

Neo207 6/7/2012 Breast not pCR 58.00 24.00 2.78 50

Neo208 6/7/2012 Breast not pCR 12.00 24.00 1.96 50

Neo209 6/7/2012 Breast not pCR

Neo210B 6/7/2012 Breast not pCR 6.00 46.00 1.60 50

Neo21 1 6/7/2012 Breast not pCR

Neo212 6/7/2012 Breast not pCR 30.00 22.00 2.18 50

Neo213 6/7/2012 Breast not pCR 48.00 26.00 2.30 50

Neo214 6/8/2012 Breast not pCR 0.00 28.00 1.72 50

Neo217 6/7/2012 Breast not pCR 0.00 90.00 1.10 50

Neo218 6/7/2012 Breast not pCR 18.00 20.00 2.06 50

Neo220 6/7/2012 Breast not pCR 46.00 6.00 2.56 50

Neo301 6/7/2012 Breast not pCR 8.00 50.00 1.58 50

Neo304 6/7/2012 Breast not pCR

Neo305 6/7/2012 Breast not pCR 8.00 48.00 1.60 50

Table 5 (Continued) path PTEN PTEN % PTEN

Sample Date tissue resp % gain loss PTEN/cell cell total

NeolOO 6/7/2012 Breast pCR 0.00 91.00 1.04 100

NeolOl 6/7/2012 Breast pCR 66.00 12.00 2.88 50

Neol02 6/6/2012 Breast pCR 10.00 60.00 1.48 50

Neol05 6/6/2012 Breast pCR 86.00 0.00 4.24 50

Neol06 6/6/2012 Breast pCR 40.00 10.00 2.34 50

Neol08 6/6/2012 Breast pCR

Neol09 6/6/2012 Breast pCR 10.00 50.00 1.64 50

Neol lO 6/8/2012 Breast pCR 8.70 56.52 1.52 23

Neol l l 6/7/2012 Breast pCR 84.00 4.00 3.14 50

Neol l2 6/8/2012 Breast pCR

Neol l4 6/7/2012 Breast pCR 6.00 84.00 1.22 50

Neol l5 6/7/2012 Breast pCR 72.00 2.00 2.82 50

Neol l6 6/7/2012 Breast pCR 86.00 4.00 3.64 50

Neol l 8 6/7/2012 Breast pCR 20.00 32.00 1.88 50

Neol20 6/7/2012 Breast pCR 12.00 36.00 1.76 50

Neo202 6/7/2012 Breast not pCR 22.00 32.00 1.92 50

Neo203 6/7/2012 Breast not pCR

Neo204 6/7/2012 Breast not pCR 40.00 14.00 2.32 50 path PTEN PTEN % PTEN

Sample Date tissue resp % gain loss PTEN/cell cell total

Neo205 6/7/2012 Breast not pCR

Neo206 6/7/2012 Breast not pCR

Neo207 6/7/2012 Breast not pCR

Neo208 6/7/2012 Breast not pCR 44.00 10.00 2.42 50

Neo209 6/7/2012 Breast not pCR 26.00 22.00 2.06 50

Neo210B 6/7/2012 Breast not pCR 32.14 32.14 2.04 28

Neo21 1 6/7/2012 Breast not pCR 44.00 18.00 2.38 50

Neo212 6/7/2012 Breast not pCR 14.00 38.00 1.80 50

Neo213 6/7/2012 Breast not pCR 28.00 44.00 1.88 50

Neo214 6/8/2012 Breast not pCR

Neo217 6/7/2012 Breast not pCR 12.00 44.00 1.76 50

Neo218 6/7/2012 Breast not pCR 21.88 18.75 2.03 32

Neo220 6/7/2012 Breast not pCR 2.00 80.00 1.22 50

Neo301 6/7/2012 Breast not pCR

Neo304 6/7/2012 Breast not pCR 0.00 92.00 0.34 50

Neo305 6/7/2012 Breast not pCR

Table 5 (Continued) path CENIO CENIO CENIO

Sample Date tissue resp % gain % loss CENlO/cell cell total

NeolOO 6/7/2012 Breast pCR 5.00 54.00 1.51 100

NeolOl 6/7/2012 Breast pCR 56.00 12.00 2.56 50

Neol02 6/6/2012 Breast pCR 84.00 0.00 3.66 50

Neol05 6/6/2012 Breast pCR 80.00 4.00 4.16 50

Neol06 6/6/2012 Breast pCR 46.00 6.00 2.58 50

Neol08 6/6/2012 Breast pCR

Neol09 6/6/2012 Breast pCR 14.00 28.00 1.88 50

Neol lO 6/8/2012 Breast pCR 8.70 47.83 1.61 23

Neol l l 6/7/2012 Breast pCR 44.00 6.00 2.50 50

Neol l2 6/8/2012 Breast pCR

Neol l4 6/7/2012 Breast pCR 76.00 6.00 3.38 50

Neol l5 6/7/2012 Breast pCR 40.00 6.00 2.36 50

Neol l6 6/7/2012 Breast pCR 76.00 4.00 3.10 50

Neol l 8 6/7/2012 Breast pCR 38.00 22.00 2.18 50

Neol20 6/7/2012 Breast pCR 2.00 40.00 1.62 50

Neo202 6/7/2012 Breast not pCR 52.00 6.00 2.66 50

Neo203 6/7/2012 Breast not pCR

Neo204 6/7/2012 Breast not pCR 8.00 44.00 1.64 50

Neo205 6/7/2012 Breast not pCR

Neo206 6/7/2012 Breast not pCR path CENIO CENIO CENIO

Sample Date tissue resp % gain % loss CENlO/cell cell total

Neo207 6/7/2012 Breast not pCR

Neo208 6/7/2012 Breast not pCR 60.00 6.00 2.70 50

Neo209 6/7/2012 Breast not pCR 28.00 10.00 2.24 50

Neo210B 6/7/2012 Breast not pCR 42.86 25.00 2.32 28

Neo21 1 6/7/2012 Breast not pCR 28.00 24.00 2.06 50

Neo212 6/7/2012 Breast not pCR 18.00 36.00 1.84 50

Neo213 6/7/2012 Breast not pCR 66.00 10.00 2.98 50

Neo214 6/8/2012 Breast not pCR

Neo217 6/7/2012 Breast not pCR 10.00 46.00 1.66 50

Neo218 6/7/2012 Breast not pCR 62.50 18.75 2.72 32

Neo220 6/7/2012 Breast not pCR 36.00 12.00 2.28 50

Neo301 6/7/2012 Breast not pCR

Neo304 6/7/2012 Breast not pCR 90.00 6.00 4.10 50

Neo305 6/7/2012 Breast not pCR

Table 5 (Continued) path

PIK3CA PIK3CA PIK3CA resp

Sample Date tissue % gain % loss PIK3CA/cell cell total

NeolOO 6/7/2012 Breast pCR

NeolOl 6/7/2012 Breast pCR 30.00 28.00 2.10 50

Neol02 6/6/2012 Breast pCR 86.00 8.00 4.06 50

Neol05 6/6/2012 Breast pCR 86.00 4.00 3.92 50

Neol06 6/6/2012 Breast pCR 48.00 18.00 2.36 50

Neol08 6/6/2012 Breast pCR 3.33 10.00 1.93 30

Neol09 6/6/2012 Breast pCR 2.38 90.48 1.12 42

Neol lO 6/8/2012 Breast pCR

Neol l l 6/7/2012 Breast pCR 58.00 4.00 3.00 50

Neol l2 6/8/2012 Breast pCR

Neol l4 6/7/2012 Breast pCR 2.00 68.00 1.30 50

Neol l5 6/7/2012 Breast pCR 70.00 8.00 3.06 50

Neol l6 6/7/2012 Breast pCR 72.00 10.00 3.30 50

Neol l 8 6/7/2012 Breast pCR 98.00 0.00 7.82 50

Neol20 6/7/2012 Breast pCR 58.00 20.00 2.52 50

Neo202 6/7/2012 Breast not pCR 86.00 4.00 3.50 50

Neo203 6/7/2012 Breast not pCR 86.00 0.00 3.68 50

Neo204 6/7/2012 Breast not pCR 40.00 26.00 2.32 50

Neo205 6/7/2012 Breast not pCR

Neo206 6/7/2012 Breast not pCR 10.00 36.00 1.74 50

Neo207 6/7/2012 Breast not pCR 44.00 8.00 2.52 50

Neo208 6/7/2012 Breast not pCR 92.00 2.00 4.66 50 path

PIK3CA PIK3CA PIK3CA resp

Sample Date tissue % gain % loss PIK3CA/cell cell total

Neo209 6/7/2012 Breast not pCR 0.00 66.67 1.27 15

Neo210B 6/7/2012 Breast not pCR 16.00 20.00 1.98 50

Neo21 1 6/7/2012 Breast not pCR 98.00 0.00 4.42 50

Neo212 6/7/2012 Breast not pCR 42.00 12.00 2.40 50

Neo213 6/7/2012 Breast not pCR 58.00 12.00 2.68 50

Neo214 6/8/2012 Breast not pCR

Neo217 6/7/2012 Breast not pCR 34.00 12.00 2.24 50

Neo218 6/7/2012 Breast not pCR 74.00 2.00 3.14 50

Neo220 6/7/2012 Breast not pCR 48.00 6.00 2.52 50

Neo301 6/7/2012 Breast not pCR 2.00 32.00 1.70 50

Neo304 6/7/2012 Breast not pCR 82.00 0.00 3.58 50

Neo305 6/7/2012 Breast not pCR 40.00 10.00 2.50 50

Table 5 (Continued) path CEN3 CEN3 CEN3

Sample Date tissue resp % gain % loss CEN3/cell cell total

NeolOO 6/7/2012 Breast pCR

NeolOl 6/7/2012 Breast pCR 18.00 34.00 1.90 50

Neol02 6/6/2012 Breast pCR 76.00 0.00 3.36 50

Neol05 6/6/2012 Breast pCR 82.00 0.00 3.70 50

Neol06 6/6/2012 Breast pCR 30.00 24.00 2.10 50

Neol08 6/6/2012 Breast pCR 6.67 23.33 1.83 30

Neol09 6/6/2012 Breast pCR 0.00 92.86 1.07 42

Neol lO 6/8/2012 Breast pCR

Neol l l 6/7/2012 Breast pCR 70.00 6.00 3.30 50

Neol l2 6/8/2012 Breast pCR

Neol l4 6/7/2012 Breast pCR 28.00 30.00 1.98 50

Neol l5 6/7/2012 Breast pCR 44.00 10.00 2.52 50

Neol l6 6/7/2012 Breast pCR 62.00 10.00 2.72 50

Neol l 8 6/7/2012 Breast pCR 8.00 46.00 1.64 50

Neol20 6/7/2012 Breast pCR 16.00 30.00 1.88 50

Neo202 6/7/2012 Breast not pCR 10.00 46.00 1.66 50

Neo203 6/7/2012 Breast not pCR 72.00 4.00 3.02 50

Neo204 6/7/2012 Breast not pCR 34.00 30.00 2.12 50

Neo205 6/7/2012 Breast not pCR

Neo206 6/7/2012 Breast not pCR 14.00 24.00 1.92 50

Neo207 6/7/2012 Breast not pCR 24.00 6.00 2.24 50

Neo208 6/7/2012 Breast not pCR 24.00 32.00 1.96 50

Neo209 6/7/2012 Breast not pCR 6.67 6.67 2.07 15

Neo210B 6/7/2012 Breast not pCR 20.00 20.00 2.00 50 path CEN3 CEN3 CEN3

Sample Date tissue resp % gain % loss CEN3/cell cell total

Neo21 1 6/7/2012 Breast not pCR 54.00 16.00 2.60 50

Neo212 6/7/2012 Breast not pCR 28.00 22.00 2.14 50

Neo213 6/7/2012 Breast not pCR 48.00 14.00 2.42 50

Neo214 6/8/2012 Breast not pCR

Neo217 6/7/2012 Breast not pCR 16.00 36.00 1.80 50

Neo218 6/7/2012 Breast not pCR 42.00 16.00 2.34 50

Neo220 6/7/2012 Breast not pCR 38.00 10.00 2.34 50

Neo301 6/7/2012 Breast not pCR 0.00 50.00 1.50 50

Neo304 6/7/2012 Breast not pCR 86.00 0.00 3.22 50

Neo305 6/7/2012 Breast not pCR 34.00 34.00 2.08 50

Table 5 (Continued) path MET % MET % MET

Sample Date tissue resp gain loss MET/cell cell total

NeolOO 6/7/2012 Breast pCR

NeolOl 6/7/2012 Breast pCR

Neol02 6/6/2012 Breast pCR 80.00 2.00 3.78 50

Neol05 6/6/2012 Breast pCR 92.00 0.00 4.22 50

Neol06 6/6/2012 Breast pCR 94.00 0.00 4.40 50

Neol08 6/6/2012 Breast pCR 40.00 12.00 2.38 50

Neol09 6/6/2012 Breast pCR 30.00 10.00 2.24 50

Neol lO 6/8/2012 Breast pCR

Neol l l 6/7/2012 Breast pCR 44.00 12.00 2.42 50

Neol l2 6/8/2012 Breast pCR

Neol l4 6/7/2012 Breast pCR 48.00 18.00 2.40 50

Neol l5 6/7/2012 Breast pCR 98.00 0.00 4.70 50

Neol l6 6/7/2012 Breast pCR

Neol l 8 6/7/2012 Breast pCR 16.00 24.00 1.92 50

Neol20 6/7/2012 Breast pCR

Neo202 6/7/2012 Breast not pCR 48.00 6.00 2.50 50

Neo203 6/7/2012 Breast not pCR 76.00 6.00 3.10 50

Neo204 6/7/2012 Breast not pCR

Neo205 6/7/2012 Breast not pCR 14.00 28.00 1.88 50

Neo206 6/7/2012 Breast not pCR 12.00 56.00 1.56 50

Neo207 6/7/2012 Breast not pCR 44.00 16.00 2.34 50

Neo208 6/7/2012 Breast not pCR 40.00 4.00 2.38 50

Neo209 6/7/2012 Breast not pCR 34.00 22.00 2.16 50

Neo210B 6/7/2012 Breast not pCR 28.00 32.00 2.00 50

Neo21 1 6/7/2012 Breast not pCR 98.00 0.00 5.24 50

Neo212 6/7/2012 Breast not pCR 34.00 18.00 2.24 50

Neo213 6/7/2012 Breast not pCR 32.00 28.00 2.10 50 Neo214 6/8/2012 Breast not pCR

Neo217 6/7/2012 Breast not pCR 2.00 38.00 1.64 50

Neo218 6/7/2012 Breast not pCR 6.00 46.00 1.62 50

Neo220 6/7/2012 Breast not pCR 18.00 32.00 1.88 50

Neo301 6/7/2012 Breast not pCR 2.00 38.00 1.66 50

Neo304 6/7/2012 Breast not pCR 94.00 2.00 5.18 50

Neo305 6/7/2012 Breast not pCR

Table 5 (Continued) path CEN7 CEN7 CEN7

Sample Date tissue resp % gain % loss CEN7/cell cell total

NeolOO 6/7/2012 Breast pCR

NeolOl 6/7/2012 Breast pCR

Neol02 6/6/2012 Breast pCR 82.00 0.00 3.92 50

Neol05 6/6/2012 Breast pCR 84.00 2.00 3.50 50

Neol06 6/6/2012 Breast pCR 56.00 2.00 2.70 50

Neol08 6/6/2012 Breast pCR 48.00 6.00 2.60 50

Neol09 6/6/2012 Breast pCR 4.00 40.00 1.64 50

Neol lO 6/8/2012 Breast pCR

Neol l l 6/7/2012 Breast pCR 48.00 16.00 2.46 50

Neol l2 6/8/2012 Breast pCR

Neol l4 6/7/2012 Breast pCR 58.00 12.00 2.62 50

Neol l5 6/7/2012 Breast pCR 98.00 0.00 4.22 50

Neol l6 6/7/2012 Breast pCR

Neol l 8 6/7/2012 Breast pCR 6.00 40.00 1.70 50

Neol20 6/7/2012 Breast pCR

Neo202 6/7/2012 Breast not pCR 60.00 4.00 2.68 50

Neo203 6/7/2012 Breast not pCR 66.00 10.00 2.88 50

Neo204 6/7/2012 Breast not pCR

Neo205 6/7/2012 Breast not pCR 30.00 38.00 1.94 50

Neo206 6/7/2012 Breast not pCR 12.00 48.00 1.64 50

Neo207 6/7/2012 Breast not pCR 30.00 26.00 2.10 50

Neo208 6/7/2012 Breast not pCR 26.00 18.00 2.08 50

Neo209 6/7/2012 Breast not pCR 40.00 24.00 2.16 50

Neo210B 6/7/2012 Breast not pCR 24.00 24.00 2.02 50

Neo21 1 6/7/2012 Breast not pCR 22.00 12.00 2.12 50

Neo212 6/7/2012 Breast not pCR 30.00 20.00 2.10 50

Neo213 6/7/2012 Breast not pCR 16.00 30.00 1.90 50

Neo214 6/8/2012 Breast not pCR

Neo217 6/7/2012 Breast not pCR 4.00 48.00 1.56 50

Neo218 6/7/2012 Breast not pCR 86.00 2.00 3.82 50

Neo220 6/7/2012 Breast not pCR 16.00 22.00 1.94 50

Neo301 6/7/2012 Breast not pCR 10.00 46.00 1.66 50 path CEN7 CEN7 CEN7

Sample Date tissue resp % gain % loss CEN7/cell cell total

Neo304 6/7/2012 Breast not pCR 52.00 2.00 2.88 50

Neo305 6/7/2012 Breast not pCR

Table 5 (Continued)

TOP2A/ path TOP2A/CEN17 TOP2A/CEN17 TOP2A/ CEN17

Sample Date tissue resp % gain % loss CEN17 cell total

NeolOO 6/7/2012 Breast pCR 16.00 6.00 1.15 50

NeolOl 6/7/2012 Breast pCR 100.00 0.00 6.1 1 50

Neol02 6/6/2012 Breast pCR 66.00 12.00 1.44 50

Neol05 6/6/2012 Breast pCR 36.00 30.00 1.07 50

Neol06 6/6/2012 Breast pCR 96.00 0.00 3.30 50

Neol08 6/6/2012 Breast pCR 6.00 34.00 0.81 50

Neol09 6/6/2012 Breast pCR 39.39 30.30 0.89 33

Neol lO 6/8/2012 Breast pCR

Neol l l 6/7/2012 Breast pCR 28.00 14.00 1.10 50

Neol l2 6/8/2012 Breast pCR 42.00 18.00 1.21 50

Neol l4 6/7/2012 Breast pCR 100.00 0.00 4.94 50

Neol l5 6/7/2012 Breast pCR 62.00 14.00 1.47 50

Neol l6 6/7/2012 Breast pCR

Neol l 8 6/7/2012 Breast pCR 62.00 8.00 1.59 50

Neol20 6/7/2012 Breast pCR 18.00 28.00 0.88 50

Neo202 6/7/2012 Breast not pCR 76.00 4.00 1.64 50

Neo203 6/7/2012 Breast not pCR 98.00 2.00 3.08 50

Neo204 6/7/2012 Breast not pCR 10.00 44.00 0.76 50

Neo205 6/7/2012 Breast not pCR 4.00 34.00 0.76 50

Neo206 6/7/2012 Breast not pCR 98.00 0.00 5.10 50

Neo207 6/7/2012 Breast not pCR 20.00 32.00 0.94 50

Neo208 6/7/2012 Breast not pCR 100.00 0.00 7.91 50

Neo209 6/7/2012 Breast not pCR

Neo210B 6/7/2012 Breast not pCR 98.00 2.00 9.16 50

Neo21 1 6/7/2012 Breast not pCR

Neo212 6/7/2012 Breast not pCR 26.00 34.00 0.94 50

Neo213 6/7/2012 Breast not pCR 50.00 4.00 1.31 50

Neo214 6/8/2012 Breast not pCR 100.00 0.00 9.24 50

Neo217 6/7/2012 Breast not pCR 52.00 4.00 1.80 50

Neo218 6/7/2012 Breast not pCR 94.00 2.00 4.59 50

Neo220 6/7/2012 Breast not pCR 58.00 18.00 1.34 50

Neo301 6/7/2012 Breast not pCR 98.00 0.00 3.15 50

Neo304 6/7/2012 Breast not pCR

Neo305 6/7/2012 Breast not pCR 78.00 2.00 1.86 50 Table 5 (Continued)

Table 5 (Continued)

path PIK3CA/CEN3 PIK3CA/CEN3 PIK3CA/CEN3

Sample Date tissue resp % gain % loss PIK3CA/CEN3 cell total

NeolOO 6/7/2012 Breast pCR

NeolOl 6/7/2012 Breast pCR 28.00 12.00 1.11 50

Neol02 6/6/2012 Breast pCR 50.00 26.00 1.21 50

Neol05 6/6/2012 Breast pCR 34.00 24.00 1.06 50 path PIK3CA/CEN3 PIK3CA/CEN3 PIK3CA/CEN3

Sample Date tissue resp % gain % loss PIK3CA/CEN3 cell total

Neol06 6/6/2012 Breast pCR 32.00 12.00 1.12 50

Neol08 6/6/2012 Breast pCR 23.33 13.33 1.05 30

Neol09 6/6/2012 Breast pCR 9.52 4.76 1.04 42

Neol lO 6/8/2012 Breast pCR

Neol l l 6/7/2012 Breast pCR 26.00 38.00 0.91 50

Neol l2 6/8/2012 Breast pCR

Neol l4 6/7/2012 Breast pCR 8.00 62.00 0.66 50

Neol l5 6/7/2012 Breast pCR 50.00 14.00 1.21 50

Neol l6 6/7/2012 Breast pCR 54.00 22.00 1.21 50

Neol l 8 6/7/2012 Breast pCR 98.00 0.00 4.77 50

Neol20 6/7/2012 Breast pCR 52.00 8.00 1.34 50

Neo202 6/7/2012 Breast not pCR 86.00 4.00 2.11 50

Neo203 6/7/2012 Breast not pCR 58.00 12.00 1.22 50

Neo204 6/7/2012 Breast not pCR 36.00 26.00 1.09 50

Neo205 6/7/2012 Breast not pCR

Neo206 6/7/2012 Breast not pCR 10.00 24.00 0.91 50

Neo207 6/7/2012 Breast not pCR 38.00 26.00 1.13 50

Neo208 6/7/2012 Breast not pCR 96.00 4.00 2.38 50

Neo209 6/7/2012 Breast not pCR 0.00 60.00 0.61 15

Neo210B 6/7/2012 Breast not pCR 16.00 16.00 0.99 50

Neo211 6/7/2012 Breast not pCR 86.00 6.00 1.70 50

Neo212 6/7/2012 Breast not pCR 38.00 16.00 1.12 50

Neo213 6/7/2012 Breast not pCR 38.00 18.00 1.11 50

Neo214 6/8/2012 Breast not pCR

Neo217 6/7/2012 Breast not pCR 40.00 4.00 1.24 50

Neo218 6/7/2012 Breast not pCR 60.00 4.00 1.34 50

Neo220 6/7/2012 Breast not pCR 36.00 24.00 1.08 50

Neo301 6/7/2012 Breast not pCR 28.00 8.00 1.13 50

Neo304 6/7/2012 Breast not pCR 40.00 20.00 1.11 50

Neo305 6/7/2012 Breast not pCR 46.00 8.00 1.20 50

Table 5 (Continued)

path MET/CEN7 MET/CEN7 MET/CEN7 Ki67

Sample Date tissue resp % gain % loss MET/CEN7 cell total IHC

NeolOO 6/7/2012 Breast pCR 20.00

NeolOl 6/7/2012 Breast pCR 10.00

Neol02 6/6/2012 Breast pCR 34.00 48.00 0.96 50 90.00

Neol05 6/6/2012 Breast pCR 56.00 14.00 1.21 50 10.00

Neol06 6/6/2012 Breast pCR 82.00 6.00 1.63 50 95.00

Neol08 6/6/2012 Breast pCR 18.00 34.00 0.92 50 30.00

Neol09 6/6/2012 Breast pCR 52.00 0.00 1.37 50 20.00

Neol lO 6/8/2012 Breast pCR

Neol l l 6/7/2012 Breast pCR 32.00 34.00 0.98 50 50.00 path MET/CEN7 MET/CEN7 MET/CEN7 Ki67

Sample Date tissue resp % gain % loss MET/CEN7 cell total IHC

Neol l2 6/8/2012 Breast pCR

Neol l4 6/7/2012 Breast pCR 26.00 38.00 0.92 50 10.00

Neol l5 6/7/2012 Breast pCR 52.00 14.00 1.11 50 50.00

Neol l6 6/7/2012 Breast pCR 50.00

Neol l 8 6/7/2012 Breast pCR 34.00 16.00 1.13 50 90.00

Neol20 6/7/2012 Breast pCR 20.00

Neo202 6/7/2012 Breast not CR 18.00 30.00 0.93 50 5.00

Neo203 6/7/2012 Breast not CR 30.00 22.00 1.08 50 20.00

Neo204 6/7/2012 Breast not pCR

Neo205 6/7/2012 Breast not CR 20.00 30.00 0.97 50 1.00

Neo206 6/7/2012 Breast not pCR 12.00 22.00 0.95 50 2.00

Neo207 6/7/2012 Breast not pCR 30.00 12.00 1.11 50 20.00

Neo208 6/7/2012 Breast not pCR 32.00 10.00 1.14 50 60.00

Neo209 6/7/2012 Breast not pCR 16.00 16.00 1.00 50 10.00

Neo210B 6/7/2012 Breast not pCR 10.00 12.00 0.99 50 20.00

Neo211 6/7/2012 Breast not pCR 96.00 0.00 2.47 50 30.00

Neo212 6/7/2012 Breast not pCR 26.00 20.00 1.07 50 30.00

Neo213 6/7/2012 Breast not pCR 28.00 12.00 1.11 50 2.00

Neo214 6/8/2012 Breast not pCR 5.00

Neo217 6/7/2012 Breast not pCR 22.00 12.00 1.05 50 5.00

Neo218 6/7/2012 Breast not pCR 2.00 90.00 0.42 50 2.00

Neo220 6/7/2012 Breast not pCR 18.00 24.00 0.97 50 1.00

Neo301 6/7/2012 Breast not pCR 22.00 18.00 1.00 50 10.00

Neo304 6/7/2012 Breast not pCR 80.00 2.00 1.80 50 1.00

Neo305 6/7/2012 Breast not pCR 5.00

For each parameter, a range of cutoffs was evaluated, such that specimens with the parameter value greater than or equal to a cutoff were considered 'high' for that parameter, and specimens with the parameter value less than the cutoff were considered 'low' for that 5 parameter. Analyses were performed with either the high parameter being considered

positive (associated with pCR) or the low parameter being considered positive (associated with pCR). A range of cutoffs were applied to each parameter in a combination, and the combination of parameters was considered positive at each combination of cutoffs if one or more of the parameters were positive according to the criteria applied, as described below:

10 1) for combinations of 2 parameters, positivity required both parameters to be

positive for an AND relationship,

2) for combinations of 2 parameters, positivity required either parameter to be positive for an OR relationship, 3) for combinations of 3 parameters, positivity required all 3 parameters to be positive for an AND relationship,

4) for combinations of 3 parameters, positivity required at least 2 parameters to be positive for a 2-parameter positive relationship, and

Sensitivities and specificities for detecting patients with pCR, based on either the high parameter being positive for pCR, or conversely the low parameter being based on patients without pCR, were calculated for parameter and each cutoff.

Receiver Operator Characteristics (ROC) curves were generated as sensitivity versus 1 -specificity over all cutoffs tested. Area Under the Curve (AUC) was calculated for each curve. AUC serves as one measure of a parameter's ability to distinguish patients with pCR from patients without pCR. AUC = 1 is an ideal result for distinguishing patients, with progressively lower values being less favorable.

In addition to AUC, Distance from Ideal (DFI) was calculated for each point on the ROC curves. DFI serves as a measure of the optimal cutoff for that parameter, where DFI = [(1 -specificity)² + (1 -specificity)²]¹⁷². Lower DFI values indicate more optimal cutoffs, with DFI = 0 being ideal (100% sensitivity and 100% specificity; e.g. see

Statistical Analysis section of Heselmeyer-Haddad, American Journal of Pathology 163(4) 1405-1416, 2003, which is incorporated herein by reference).

In addition to ROC analysis, 2X2 contingency tables were evaluated at each cutoff. This analysis provides χ² probabilities and/or probabilities from Fischer's Exact Test, χ² probabilities and/or Fischer's Exact Test probabilities also are useful for measuring a parameter's ability to differentiate patients with pCR from patients without pCR.

The results of this Example 3 are provided above in Table 2.

Example 4

Molecular Morphology Based Genomic Signatures of Moderate Complexity Predict Pathologic Complete Response in HER2 Molecular Breast Carcinoma Class Patients

Treated with Trastuzumab-based Preoperative Therapy.

Preoperative chemotherapy is an effective approach for "downstaging" some breast cancer patients, some of whom achieve a pathologic complete response (pCR), especially for non-luminal B HER2 positive molecular class (HNL). Determination of which HNL patients are more or not likely to achieve pCR would allow a more personalized, predictive approach to neoadjuvant management.

Formalin fixed biopsy specimens from 34 patients with invasive ductal carcinoma treated with trastuzumab4oased therapy prior to definitive resection and pathologic staging were evaluated by dual color dual hapten bright field in situ hybridization (dual ISH) using repeat depleted locus specific probes (designation based on the name of a gene included within the targeted region) and a reference centromeric locus probe (CEN) on the same chromosome. Probe pairs included MET+CEN7, TOP2A+CEN17, PTEN+CEN10, and PIK3CA+CEN3. Only invasive carcinoma tumor cells were scored. In addition to assessing genomic gains and losses by the average number of gene loci or centromeres per cell and the ratio of gene loci to CEN,the percentage of cells with >2 gene or CEN signals (gene locus gain or CEN gain), <2 signals (gene locus loss or CEN loss), gene locus signals > CEN signals (gene locus/CEN gain), and gene locus signals < CEN signals (gene locus/CEN loss) were calculated for each parameter and cut point. The percentage of cells with either gene locus/CEN gain or loss was also evaluated. Sensitivities and specificities for detecting patients with pCR, based on either the high parameter being positive for pCR, and conversely based on the low parameter being positive for pCR, were calculated for each parameter and each cutoff. Receiver Operator Characteristics (ROC) curves were generated as sensitivity versus 1 -specificity over all cutoffs tested, and Area Under the

Curve (AUC) was calculated as one measure of a parameter's ability to distinguish patients with pCR from patients without pCR, with AUC = 1 being ideal and progressively lower values being less favorable. 2X2 contingency tables were evaluated at each cutoff to provide chi square probabilities as another measure of a parameter's ability to differentiate patients with pCR. Combinations of parameters were also evaluated by ROC and contingency table analyses.

MET/CEN7 gain or loss was predictive of pCR (AUC=0.824, N=24) achieving 100% sensitivity and 69% specificity at a cutoff of 50% (X2 p<0.00089). Combining this parameter with PIK3CA/CEN3 gain provided further improvement (AUC 0.937, N=24) with 89% sensitivity and 93% specificity achieved at respective cutoffs of 50 and 55% .

Genomic signatures of moderate complexity generated from dual ISH evaluation of invasive breast carcinoma predict pathologic complete response in HER2 molecular class breast carcinoma patients treated with trastuzumab-based preoperative therapy. These findings require validation in additional patient cohorts.

In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

Patent Claims

1. A method for predicting pathologic response in patients being considered for neoadjuvant breast cancer treatment, comprising:

contacting a biological sample from a human patient with a gene locus probe selected from a probe to chromosome 7q31, a probe to chromosome 3q26, a probe to chromosome 10q23, a probe to chromosome 17q21, a chromosomal enumeration pericentromeric probe selected from a chromosome 7 pericentromeric probe, a chromosome 3 pericentromeric probe, a chromosome 10 pericentromeric probe, a chromosome 17 pericentromeric probe, or combinations thereof, under conditions sufficient to enable hybridization of the probe to target sequences; and

analyzing probe hybridization information to facilitate determining neoadjuvant breast cancer treatment in the subject based upon the probe hybridization information.

2. The method according to claim 1, further comprising performing immunohistochemistry analysis.

3. The method according to claim 2 wherein the immunohistochemistry analysis uses a Ki-67 antibody to detect the Ki-67 protein.

4. The method according to claim 1, wherein the gene locus probe is selected from a probe to a region containing MET, a probe to a region containing PIK3CA, a probe to a region containing PTEN, a probe to a region containing TOP2A, or combinations thereof.

5. The method according to claim 1, wherein the gene locus probe is an isolated nucleic acid probe, comprising:

(a) a nucleic acid molecule consisting of a sequence according to any one of SEQ ID NOs: 1-20 and 31-50; or

(b) a nucleic acid molecule consisting of at least 250 contiguous nucleotides of any one of SEQ ID NOs: 1-20 and 31-50.

6. The method according to claim 1, wherein the gene locus probe is an isolated nucleic acid probe, comprising: (a) a nucleic acid molecule having at least 90% sequence identity with a sequence according to any one of SEQ ID NOs: 1-20 and 31-50; or

(b) a nucleic acid molecule having at least 90% sequence identity with at least 250 contiguous nucleotides of any one of SEQ ID NOs: 1-20 and 31-50.

7. The method according to claim 1, wherein the gene locus probe is an isolated nucleic acid probe, comprising:

(a) a nucleic acid molecule having at least 95% sequence identity with a sequence according to any one of SEQ ID NOs: 1-20 and 31-50; or

(b) a nucleic acid molecule having at least 95% sequence identity with at least 250 contiguous nucleotides of any one of SEQ ID NOs: 1-20 and 31-50.

8. The method according to claim 1, wherein the gene locus probe is an isolated nucleic acid probe, comprising:

(a) a nucleic acid molecule having at least 99% sequence identity to a sequence according to any one of SEQ ID NOs; 1-20 and 31-50; or

(b) a nucleic acid molecule having at least 99% sequence identity with at least 250 contiguous nucleotides of any one of SEQ ID NOs: 1-20 and 31-50.

9. The method according to claim 1, wherein the gene locus probe is a nucleic acid molecule with at least 90% sequence identity to at least 400 contiguous of any one of SEQ ID NOs: 1-20 and 31-50.

10. The method according to claim 1, wherein the gene locus probe is a nucleic acid molecule with at least 90% sequence identity to at least 500 contiguous nucleotides of any one of SEQ ID NOs: 1-20 and 31-50.

1 1. The method according to claim 1 , wherein the gene locus probe is a nucleic acid molecule with at least 90% sequence identity to at least 1000 contiguous nucleotides of any one of SEQ ID NOs: 1-20 and 31-50.

12. The method according to claim 1, wherein the gene locus probe is a nucleic acid molecule with at least 90% sequence identity to at least 2500 contiguous nucleotides of any one of SEQ ID NOs: 1-20 and 31-50.

13. The method according to claim 1, wherein the biological sample is contacted with both the gene locus probe and the chromosomal enumeration

pericentromeric probe independently.

14. The method according to claim 1, wherein the biological sample is contacted with the gene locus probe, the chromosomal enumeration pericentromeric probe as a probe set.

15. The method according to claim 14, further comprising adding a Ki-67 antibody with the probe set.

16. The method according to claim 15, wherein the probe set comprises MET + CEN7, Ki-67, TOP2A + CEN17, PTEN + CENIO, PIK3CA + CEN3, or combinations thereof.

17. The method according to claim 1 or 15, wherein analyzing comprises using MET/CEN7 gain or loss AND Ki-67 to facilitate determining neoadjuvant breast cancer treatment.

18. The method according to claim 1, wherein analyzing comprises using

PIK3CA/CEN3 gain AND MET/CEN7 gain or loss to facilitate determining neoadjuvant breast cancer treatment.

19. The method according to claim 1 or 15, wherein analyzing comprises using PIK3CA/CEN3 gain OR Ki-67 to facilitate determining neoadjuvant breast cancer treatment.

20. The method according to claim 1 or 15, wherein analyzing comprises using PIK3CA/CEN3 gain AND Ki-67 to facilitate determining neoadjuvant breast cancer treatment.

21. The method according to claim 1 , wherein analyzing comprises using MET/CEN7 gain AND PTEN/CEN10 gain or loss to facilitate determining neoadjuvant breast cancer treatment.

22. The method according to claim 1 or 15, wherein analyzing comprises using MET/CEN7 gain and loss, and Ki-67, and PIK3CA/CEN3 gain (at least two positive) to facilitate determining neoadjuvant breast cancer treatment.

23. The method according to claim 1, wherein analyzing comprises using

MET/CEN7 gain, and CEN7 gain, and PTEN/CEN10 gain or loss (at least two positive) to facilitate determining neoadjuvant breast cancer treatment.

24. The method according to claim 1, wherein each probe can be separately detected.

25. The method according to claim 1, wherein the pathologic response is complete response.

26. The method according to claim 1, wherein the pathologic response is partial response.

27. The method according to claim 1, wherein the patient is a HER2 -positive patient.

28. The method according to claim 27, wherein the HER2-positive patient is a gene amplification-positive patient.

29. The method according to claim 27, wherein the HER2 -positive patient is a HER-2 expression positive patient.

30. The method according to claim 1, wherein the biological sample first is determined to be from a patient with HER2 elevated breast cancer.

31. The method according to claim 1 , wherein the biological sample comprises a breast tissue biopsy, resection, or a cytology sample.

32. The method according to claim 1, further comprising:

determining neoadjuvant breast cancer treatment prognosis in the subject based upon the analysis of the probe hybridization information; determining a treatment protocol based on the prognosis; and treating the patient according to the protocol.

33. The method according to claim 1, wherein contacting comprises performing FISH, CISH, SISH, IHC, and combinations thereof.

34. The method according to claim 1, wherein the probe includes a chromogenic label.

35. The method according to claim 1, comprising using unique chromogens for each probe used.

36. The method according claim 1, wherein contacting is performed using an automated system.

37. The method according to claim 32, wherein determining comprises selecting criteria by which to judge a low or high parameter state associated with pathological complete response (pCR), testing the patient with the gene locus probe, chromosomal enumeration pericentromeric probe, or combinations thereof, and then obtaining a positive or negative response.

38. The method according to claim 37 wherein the positive response indicates that the patient is likely to experience pathologic complete response and the negative response indicates that the patient is unlikely to experience pathologic complete response.

39. The method according to claim 37 wherein the criteria are selected from AUC from an ROC curve, DFI from an ROC curve, χ² probabilities, Fischer's Exact Test probabilities, optimal cut-off values, or combinations thereof.

40. The method according to claim 39, wherein the AUC from an ROC curve, DFI from an ROC curve, χ2 probability , Fischer's Exact Test probabilities, optimal cut-off values, and combinations thereof are determined using a statistically significant sample for probe hybridization results.

41. The method according to claim 39, wherein the ROC AUC is 0.5 to 1.0.

42. The method according to claim 41, wherein ROC AUC is 0.9 or greater.

43. The method according to claim 39, wherein the sensitivity is from about 0.8 to about 1.0.

44. The method according to claim 39, wherein the specificity is from 0.5 to

1.0.

45. The method according to claim 39, wherein the χ2 probability value is 0.05 or less.

46. The method according to claim 39, wherein the χ2 probability value is from 0.0000001 to 0.05.

47. The method according to claim 39, wherein the optimal cut-off value ranges from about 5% to about 70%.

48. The method according to claim 39, wherein the optimal cut-off value ranges from about 8% to about 68%.

49. The method according to claim 1, wherein the hybridization information is (i) MET/CEN7 gain or loss, (ii) MET/CEN7 gain or loss AND Ki-67, each having a sensitivity of 1.00.

50. The method according to claim 1, wherein the hybridization information is (i) MET/CEN7 gain and loss, and Ki-67, and PIK3CA/CEN3 gain (at least two positives), and (ii) MET/CEN7 gain AND PTEN/CEN10 gain or loss, each having a specificity of 1.00.

51. The method according to claim 1 , comprising using two factor combinations and the two factor combinations have an ROC AUC of greater than 0.90, a sensitivity of 0.8 or greater, a specificity of 0.8 or greater, and a χ2 probability value of 0.00006 or less.

52. The method according to claim 1, further comprising:

using a first probe set comprising a first gene locus probe and a first chromosomal enumeration pericentromeric probe to perform probe hybridization analysis on a first biological sample; and

using a second probe set comprising a second gene locus probe and a second chromosomal enumeration pericentromeric probe to perform probe hybridization analysis on a second biological sample.

53. The method according to claim 1, further comprising:

contacting a second biological sample from the human patient with a gene locus probe selected from a probe to chromosome 7q31, a probe to chromosome 3q26, a probe to chromosome 10q23, a probe to chromosome 17q21, a chromosomal enumeration pericentromeric probe, selected from a chromosome 7 pericentromeric probe, a chromosome 3 pericentromeric probe, a chromosome 10 pericentromeric probe, a chromosome 17 pericentromeric probe, or combinations thereof, under conditions sufficient to enable hybridization of the gene locus probe and/or chromosomal enumeration pericentromeric probe to target sequences, wherein the gene locus probe and/or chromosomal enumeration pericentromeric probe used for the second biological sample are different from the gene locus probe and/or chromosomal enumeration pericentromeric probe used for the first biological sample.

54. The method of claim 53 wherein all selected probes are hybridized to the same specimen sample, each labeled with a distinguishing label.

55. A method for predicting pathologic response in patients being considered for neoadjuvant breast cancer treatment, comprising:

obtaining a first biological sample from a human patient first determined to be from a patient with HER2 elevated breast cancer, the sample comprising a breast tissue biopsy, a breast cancer resection, or a cytology sample;

contacting the first biological sample with a first probe set comprising at least a first gene locus probe selected from a probe to chromosome 7q31, a probe to chromosome 3q26, a probe to chromosome 10q23, a probe to chromosome 17q21, and a first chromosomal enumeration pericentromeric probe, selected from a chromosome 7 pericentromeric probe, a chromosome 3 pericentromeric probe, a chromosome 10 pericentromeric probe, a chromosome 17 pericentromeric probe, or combinations thereof under conditions sufficient to enable hybridization of the probes to a target region;

analyzing probe hybridization information to facilitate determining neoadjuvant breast cancer treatment in the subject based upon the probe hybridization information; determining neoadjuvant breast cancer treatment prognosis in the subject based upon the analysis of the probe hybridization information;

determining a treatment protocol based on the prognosis; and

treating the patient according to the protocol.

56. The method according to claim 55, further comprising contacting a second biological sample from the human patient with a second probe set comprising (i) a second gene locus probe selected from a probe to chromosome 7q31 , a probe to chromosome 3q26, a probe to chromosome 10q23, a probe to chromosome 17q21, or combinations thereof, wherein the second gene locus probe is different from the first gene locus probe, and (ii) a second chromosomal enumeration pericentromeric probe corresponding to the same chromosome as the gene locus probe, under conditions sufficient to enable hybridization of the second gene locus probe and second chromosomal enumeration pericentromeric probe to target sequences.

57. The method according to claim 56, wherein the first and second gene locus probes are selected from a probe to a region containing MET, a probe to a region containing PIK3CA, a probe to a region containing PTEN, a probe to a region containing TOP2A, or combinations thereof.

58. The method according to claim 56, wherein the first and second gene locus probe are nucleic acid probes selected from the group consisting of SEQ ID NOs: 1-10, a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 1- 10, SEQ ID NOs: 11-20, nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 1 1-20, SEQ ID NOs: 31-40, a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 31 -40, SEQ ID NOs: 41 -

50, and a nucleic acid molecule comprising at least 250 contiguous nucleotides of SEQ ID NOs: 41-50.

59. The method according to claim 55, wherein treating the patient comprises treating in situ hybridization positive patients with a HER2 targeted neoadjuvant therapy, and treating in situ hybridization negative patients with a neoadjuvant chemotherapy or no neoadjuvant therapy.

60. The method according to claim 55, wherein contacting is performed on an automated instrument.

61. A probe for predicting pathologic response in patients being considered for neoadjuvant breast cancer treatment selected from a gene locus probe to chromosome

7q31, a probe to chromosome 3q26, a probe to chromosome 10q23, a probe to

chromosome 17q21 , a chromosomal enumeration pericentromeric probe, selected from a chromosome 7 pericentromeric probe, a chromosome 3 pericentromeric probe, a chromosome 10 pericentromeric probe, a chromosome 17 pericentromeric probe or combinations thereof.

62. A probe set for predicting pathologic response in patients being considered for neoadjuvant breast cancer treatment comprising at least two probes, wherein the two probes are selected from:

a gene locus probe selected from a probe to chromosome 7q31 , a probe to chromosome 3q26, a probe to chromosome 10q23, a probe to chromosome 17q21, or combinations thereof; and

a chromosomal enumeration pericentromeric probe corresponding to the same chromosome as the gene locus probe, wherein the chromosomal enumeration

pericentromeric probe is selected from chromosome 3, chromosome 7, chromosome 10, and chromosome 17.

63. A kit for predicting pathologic response in patients being considered for neoadjuvant breast cancer treatment, comprising a gene locus probe to chromosome 7q31 , a probe to chromosome 3q26, a probe to chromosome 10q23, a probe to chromosome 17q21, a chromosomal enumeration pericentromeric probe, selected from a chromosome 7 pericentromeric probe, a chromosome 3 pericentromeric probe, a chromosome 10 pericentromeric probe, a chromosome 17 pericentromeric probe, or combinations thereof.

64. A kit for predicting pathologic response in patients being considered for neoadjuvant breast cancer treatment, comprising: a gene locus probe selected from (a) a nucleic acid molecule having at least 90% sequence identity with the sequence according to any one of SEQ ID NOs: 1-20 and 31-50, or (b) a nucleic acid molecule having at least 90% sequence identity with at least 250 contiguous nucleotides of any one of SEQ ID NOs: 1-20 and 31-50; and