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WO2011072205A2 - Marqueurs génétiques indicatifs d'une réponse d'un patient atteint d'un cancer au trastuzumab (herceptine) - Google Patents

Marqueurs génétiques indicatifs d'une réponse d'un patient atteint d'un cancer au trastuzumab (herceptine) Download PDF

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WO2011072205A2
WO2011072205A2 PCT/US2010/059846 US2010059846W WO2011072205A2 WO 2011072205 A2 WO2011072205 A2 WO 2011072205A2 US 2010059846 W US2010059846 W US 2010059846W WO 2011072205 A2 WO2011072205 A2 WO 2011072205A2
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marker
subject
copy number
human
markers
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WO2011072205A3 (fr
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James B. Hics
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Cold Spring Harbor Laboratory
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/142—Toxicological screening, e.g. expression profiles which identify toxicity

Definitions

  • the invention relates to the fields of therapeutics and identifying candidates for therapy, in particular to a method of identifying candidates for trastuzumab (Herceptin®) therapy in a patient presenting with breast cancer based on the presence or absence of specific genetic markers in a tumor sample from said patient.
  • trastuzumab Herceptin®
  • IHC immunohistochemistry
  • FISH fluorescent in situ hybridization
  • FISH using entire cells e.g., cultured cells, pulverized tissue, or imprint touch specimens from tumors
  • entire cells e.g., cultured cells, pulverized tissue, or imprint touch specimens from tumors
  • tissue sections complicates the quantitative nature of FISH due to nuclear truncation (i.e., due to the slicing of the tissues during their preparation for staining).
  • CISH Chromogenic in situ hybridization
  • Trastuzumab (Herceptin®) was developed as a targeted therapy to combat Her2 overexpression.
  • Trastuzumab is a humanized monoclonal antibody directed against the extracellular domain of Her2 and is therefore specific for its target.
  • Trastuzumab is approved for the adjuvant treatment of Her2-overexpressing node-positive or node-negative breast cancer.
  • Trastuzumab in combination with paclitaxel is approved for the first-line treatment of Her2-overexpressing metastatic breast cancer.
  • Trastuzumab as a single agent is approved for treatment of Her2- overexpressing breast cancer in patients who have received one or more chemotherapy regimens for metastatic disease.
  • Trastuzumab has been shown to be effective across all of its approved uses (C.
  • trastuzumab monotherapies only lead to tumor regression in approximately 30% of patients.
  • Complicating the issue further is the fact that administration of trastuzumab with anthracyclines and cyclophosphamide, while effective, leads to cardiac events in 28% of the patients.
  • a trastuzumab-containing treatment regimen is expensive, costing up to $100,000 per year.
  • trastuzumab treatment decisions are currently based on the detection of elevated Her2 levels. The discovery of additional markers could improve treatment decisions and identify Her2- subjects who could benefit from receiving trastuzumab, while also refining the number of Her2+ subjects to those who could best benefit from treatment.
  • the invention relates to a process of producing information indicative of whether a human cell has a deletion at chromosome region 17q35.42-q56.76 (marker Al), a deletion at human chromosome region 17q32.010-q32.34.215 (marker A2) or an amplification at human chromosome region 4ql69.81-ql85.239 (marker B), comprising determining by apparatus a copy number for marker Al, marker A2 or marker B, thereby producing information indicative of whether human cell has a deletion at marker Al, a deletion at human marker A2 or an amplification at marker B.
  • the invention in a second aspect, relates to a process for identifying a human subject as a candidate for trastuzumab therapy comprising determining by apparatus a copy number of human chromosome region 17q35.42-q56.76 (marker Al), human chromosome region 17q32.010-q32.34.215 (marker A2) or human chromosome region 4ql69.81-ql85.239 (marker B) in a cancer cell of the subject; and identifying the subject as a candidate for trastuzumab therapy if the copy number data is indicative of a deletion at marker Al, a deletion at marker A2 or an amplification at marker B.
  • the invention in a third aspect, relates to a method of treating a human subject afflicted with cancer comprising obtaining information indicative of whether the human subject is a candidate for trastuzumab therapy, the information based on a copy number of human chromosome region 17q35.42-q56.76 (marker Al), human chromosome region 17q32.010-q32.34.215 (marker A2) or human chromosome region 4ql69.81-ql85.239 (marker B), wherein the subject is a candidate for trastuzumab therapy if the copy number data is indicative of a deletion at marker Al, a deletion at marker A2 or an amplification at marker B; and if the information indicates that the human subject is a candidate for trastuzumab therapy, then administering to the human subject an amount of trastuzumab effective to treat the human subject afflicted with cancer.
  • the invention in a fourth aspect, relates to a kit for identifying a subject's sensitivity to trastuzumab therapy, said kit comprising one or more nucleic acid probes each of which selectively bind to a target polynucleotide sequence of the chromosome region comprising one of human chromosome region 17q35.42-q56.76 (marker Al), human chromosome region 17q32.010-q32.34.215 (marker A2) or human chromosome region 4ql69.81-ql85.239 (marker B) under conditions in which the probe forms a stable hybridization complex with the target polynucleotide sequence.
  • a target polynucleotide sequence of the chromosome region comprising one of human chromosome region 17q35.42-q56.76 (marker Al), human chromosome region 17q32.010-q32.34.215 (marker A2) or human chromosome region 4ql69.81-ql85.239
  • Figure 1 Comparison of aCGH measures of Her2 copy number with Her2 FISH and mRNA expression.
  • FIG. 1A Her2 CGH values (y axis) plotted against Her2 FISH values (x axis). Threshold values (gray lines) for positivity are 1.3 for CGH and 2.0 for FISH.
  • Figure IB Plot of trastuzumab response measured as overall survival (treated vs. untreated) (y axis) over time in months (x axis) for patients assayed as positive by FISH (blue and red) or by CGH (green and orange).
  • Figure 1C Scatterplot of Her2 mRNA expression (y axis) compared to FISH copy number (x axis). Threshold value for overexpression set at 10 (gray line).
  • Figure ID Plot of trastuzumab response measured as overall survival (treated vs. untreated) (y axis) over time in months (x axis) for patients with Her2 expression >10 (blue and red) compared to patients with expression ⁇ 10 (green and orange).
  • Figure 2. Plot of trastuzumab response (treated vs. untreated) plotted as overall survival (y axis) over time in months (x axis) for patients assayed as ERBB2 amplified (blue and red) or non- amplified (green and orange) by FISH ( Figure 2A) or aCGH ( Figure 2B).
  • FIG. 3 Plots of trastuzumab response (treated vs. untreated) plotted as overall survival (y axis) over time in months (x axis) for various combinations of markers demonstrating that Marker Al or Marker B can increase the number of patients sensitive to trastuzumab.
  • Figure 3A Plot of trastuzumab response measured as overall survival (treated vs. untreated) for patients assayed as positive for Her2 amplification by aCGH (blue and red) or negative for Her2 amplification by aCGH (green and orange).
  • Figure 3B Plot of trastuzumab response measured as overall survival (treated vs. untreated) for patients assayed as positive for Her2 amplification by aCGH plus those negative for Her2 amplification but carrying Marker A1 (blue and red) compared to those negative for Her2 amplification by aCGH and not carrying Marker A1 (green and orange).
  • FIG 3C Plot of trastuzumab response measured as overall survival (treated vs. untreated) for patients assayed as negative for Her2 amplification by aCGH (blue and red) and carrying Marker Al or negative for Her2 amplification by aCGH not carrying Marker A 1 (green and orange).
  • Figure 3D Plot of trastuzumab response measured as overall survival (treated vs. untreated) for patients assayed as negative for Her2 amplification by aCGH (blue and red) and carrying Marker B or negative for Her2 amplification by aCGH and not carrying Marker B (green and orange).
  • FIG. 4A CGH (y) v. DASL (x) Figure 4B. FISH (y) v. DASL (x) Figure 4C. CGH (y) v. FISH (x)
  • FIG 6A Trastuzumab extends survival (y axis) measured over months (x axis) for both ERBB2 amplified (blue/red) and NON-amplified cases (green/orange).
  • Figure 6B Deletions of BRCA1 identify response to Trastuzumab among cases NOT amplified for ERBB2. Survival (y axis) measured over months (x axis).
  • Figure 6C Duplication of 4q(term) identifies increased Trastuzumab response among cases NOT amplified for ERBB2. Survival (y axis) measured over months (x axis).
  • Figure 6D Combining ERBB2 amp and NONamp carrying second marker treats more cases(87) than ERBB2 amp alone (53). Survival (y axis) measured over months (x axis).
  • the term “amplification” or “amplified” when each refers to a genomic region indicates that such genomic region is present in the genomic DNA at a higher copy number than the mean copy number of the remainder of the genome.
  • the term “candidate for trastuzumab therapy” refers to a human that is suspected of having cancer that may be evaluated for suitability for trastuzumab treatment. Examples of candidate subjects include, but are not limited to, human women suspected of having breast cancer and human men suspected of having breast cancer.
  • sample as used herein, relates to any sample which can be obtained from the patient.
  • the present method can be applied to any type of biological sample from a patient, such as a biopsy sample, tissue, cell or fluid (serum, saliva, semen, sputum, cerebral spinal fluid (CSF), tears, mucus, sweat, milk, brain extracts and the like).
  • a biopsy sample tissue, cell or fluid
  • said sample is a tumour tissue sample or portion thereof.
  • said tumor tissue sample is a breast tumor tissue sample from a patient suffering from breast cancer.
  • Said sample can be obtained by conventional methods, e.g., biopsy, by using methods well known to those of ordinary skill in the related medical arts. Methods for obtaining the sample from the biopsy include gross apportioning of a mass, or microdissection or other art-known cell-separation methods.
  • Tumour cells can additionally be obtained from fine needle aspiration cytology. In order to simplify conservation and handling of the samples, these can be formalin-fixed and paraffin-embedded or first frozen and then embedded in a cryosolidifiable medium, such as OCT-Compound, through immersion in a highly cryogenic medium that allows for rapid freeze.
  • the samples may be obtained from subjects previously diagnosed with breast cancer (patients), or from subjects who have not been previously diagnosed with breast cancer, or from patients diagnosed with breast cancer who are undergoing treatment, or from subjects diagnosed with breast cancer who have been previously treated.
  • copy number refers to the actual number of these sequences per single cell. Copy number may be reported for one single cell, or reported as the average number in a group of cells (e.g., tissue sample).
  • tissue sample e.g., tissue sample.
  • copy number can be represented as the ratio of hybridization signal at any locus in the genome comparing a tumor DNA sample to a DNA sample from a standard diploid reference.
  • the term "Her2" refers to a nucleic acid sequence encoding the Human epidermal growth factor receptor 2 (Her2) protein, and includes both the wild-type sequence and naturally occurring variations, truncations, and mutations.
  • Her2 is also known as "HER2,” “Her-2,” “Her- 2/neu,” “neu,” “ErbB-2,” and “ERBB2,” all of which terms are encompassed herein in “Her2.”
  • the term “apparatus” refers to any methods, assays or equipment used to determine copy number of one or more genomic regions of a chromosome.
  • Methods may include polymerase chain reaction (PCR), quantitative PCR, Southern Blotting, in situ hybridization techniques (ISH) (e.g., fluorescent in situ hybridization (FISH) or chromogenic in situ hybridization (CISH)), comparative genomic hybridization (CGH), array comparative genomic hybridization (aCGH), representational oligonucleotide microarray analysis (ROMA) and any other techniques used to determine copy number readily available and known to those of skill in the art.
  • ISH in situ hybridization techniques
  • FISH fluorescent in situ hybridization
  • CISH chromogenic in situ hybridization
  • CGH comparative genomic hybridization
  • aCGH array comparative genomic hybridization
  • ROMA representational oligonucleotide microarray analysis
  • Equipment may include DIC microscope, fluorescent microscope, microarrays, slide scanners, computer software, computer algorithms and any other equipment used to determine copy number of a genomic region.
  • the term "indicative of the subject's sensitivity to trastuzumab therapy” refers to a subject that is either Her2+, has a deletion at marker Al, a deletion at marker A2 or an amplification at marker B, has all 4 events, or has any combination or permutation of one, two or three of the events.
  • amplification when used in reference to copy number refers to the condition in which the copy number of a nucleic acid sequence (e.g., Her2) is greater than the copy number of a control sequence (e.g., chromosome 17). In other words, amplification indicates that the ratio of a particular nucleic acid sequence (e.g., Her2) is greater than 1:1 when compared to a control sequence (e.g., 1.1 :1, 1.2:1, or 1.3:1).
  • the term "deletion" when used in reference to copy number refers to the condition in which the copy number of a nucleic acid sequence (e.g., Her2) is less than the copy number of a control sequence (e.g., chromosome 17).
  • amplification indicates that the ratio of a particular nucleic acid sequence (e.g., Her2) is less than 1:1 when compared to a control sequence (e.g., 0.25:1, 0.50:1, or 0.85:1).
  • nucleic acid refers to any nucleic acid containing molecule including, but not limited to DNA or RNA.
  • the term encompasses sequences that include any of the known base analogs of DNA and RNA including, but not limited to, 4-acetylcytosine, 8-hydroxy-N-6- methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5- fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5- carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1- methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-mefhylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladen
  • hybridization is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the melting temperature of the formed hybrid, and the G:C ratio within the nucleic acids.
  • probe refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by amplification (e.g. PCR), which is capable of hybridizing to another oligonucleotide of interest.
  • Probes useful in the present invention may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences (e.g., Her2, marker A1, marker A2 or marker B).
  • any probe used in the present invention may be labeled with any "reporter molecule,” so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme- based immunohistochemical assays), fluorescent (e.g., FISH), radioactive, mass spectroscopy, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.
  • the term "label” refers to any molecule which may be detected.
  • labels include, but are not limited to, 32P, 14C, 1251, 3H, 35S, biotin, digoxigenin, avidin, fluorescent or enzymatic molecules.
  • a process is provided of producing information indicative of whether a human cell has a deletion at chromosome region 17q35.42-q56.76 (marker Al), a deletion at human chromosome region 17q32.010-q32.34.215 (marker A2) or an amplification at human chromosome region 4ql69.81- q185.239 (marker B), comprising determining by apparatus a copy number for marker A1, marker A2 or marker B, thereby producing information indicative of whether human cell has a deletion at marker A1, a deletion at human marker A2 or an amplification at marker B.
  • the instant process comprises determining the copy number for two of the markers A1, A2 or B, thereby producing information indicative of whether the human cell has two of the markers A1, A2 or B,
  • the instant process comprises determining the copy number for all three of the markers A1, A2 and B, thereby producing information indicative of whether the human cell has all three of the markers A1, A2 and B.
  • a process for identifying a human subject as a candidate for trastuzumab therapy comprising:
  • An embodiment of the process comprises determining by apparatus the copy number of two of the markers Al, A2 or B, and identifying the subject as a candidate for trastuzumab therapy if the copy number data of the two markers is indicative of the subject's sensitivity to trastuzumab therapy.
  • the copy number can be determined for any combination or permutation of two of the three markers, including but not limited to A1/A2, A1/B or A2/B.
  • the process comprises determining by apparatus the copy number of all three of the markers Al, A2 or B, and identifying the subject as a candidate for trastuzumab therapy if the copy number data of the three markers is indicative of the subject's sensitivity to trastuzumab therapy.
  • the copy number of each marker can be determined in any order.
  • the human subject has cancer and is Her 2-. In an embodiment of the process, the human subject has cancer and is Her 2+.
  • the apparatus is a hybridization-based assay.
  • the detecting step of the method of the invention comprises contacting the nucleic acid sample with one or more nucleic acid probes each of which selectively binds to a target polynucleotide sequence on the chromosome region at marker Al, marker A2 or marker B, under conditions in which the probe forms a stable hybridization complex with the target polynucleotide sequence; and detecting the hybridization complex.
  • the nucleic acid probes used in the method of the present invention are labeled with a fluorophore.
  • the step of detecting the hybridization complex comprises determining the copy number of the target polynucleotide sequence, thereby determining a deletion of marker Al, a deletion of marker A2or an amplification of marker B.
  • said apparatus is selected from the group consisting of Southern blot, LOH (loss of heterozygosity), PCR, in situ hybridization (ISH) fluorescence ISH (FISH) and comparative genomic hybridization (CGH).
  • the apparatus is a comparative genomic hybridization apparatus.
  • genomic hybridization In an embodiment, once the sample has been obtained and the total DNA has been extracted, genome-wide analysis of DNA copy number changes by comparative genomic hybridization (CGH) is carried out.
  • CGH comparative genomic hybridization
  • total genomic DNA is isolated from test and reference cell populations, differentially labeled and hybridized to a representation of the genome that allows the binding of sequences at different genomic locations to be distinguished.
  • Methods describing representations of genomes are described in US 7,531,307 B2, issued May 12, 2009, Use of representations for DNA genetic analysis, the disclosure of which is incorporated by reference.
  • Hybridization reactions can be performed under conditions of different stringency. The stringency of a hybridization reaction includes the difficulty with which any two nucleic acid molecules will hybridize to one another.
  • stringency can be varied by manipulation of three factors: temperature, salt concentration, and formamide concentration.
  • High temperature and low salt increases stringency.
  • Formamide decreases melting point of DNA, thus lowering the temperature at which a hybrid between two nucleic acid molecules forms.
  • each hybridizing polynucleotide hybridizes to its corresponding polynucleotide under reduced stringency conditions, more preferably stringent conditions, and most preferably highly stringent conditions.
  • the amount of specimen DNA is frequently a constraint on CGH measurements. Typical array CGH procedures use 300 ng to 3 ug of specimen DNA in the labeling reaction, equivalent to approximately 50,000 to 500,000 mammalian cells.
  • random primer labeling protocols are employed, which also amplifies the DNA, so that several micrograms are used in the hybridization.
  • array CGH has been implemented using a wide variety of techniques. On example of a genome- wide method is described in WO/2008/016374, published February 7, 2008, Methods For Assessing Probabilistic Measures Of Clinical Outcome Using Genomic Profiling, the disclosure of which is incorporated herein by reference.
  • array CGH is carried out using arrays from large-insert genomic clones such as bacterial artificial chromosomes (BACs).
  • BACs bacterial artificial chromosomes
  • array CGH The major technical challenge of array CGH is generating hybridization signals that are sufficiently intense and specific so that copy number changes can be detected.
  • the signal intensity on an array element is affected by a number of factors including the base composition, the proportion of repetitive sequence content, and the amount of DNA in the array element available for hybridization.
  • Array elements made from genomic BAC clones typically provide more intense signals than elements employing shorter sequences such as cDNAs, PCR products, and oligonucleotides. The higher signals form the more complex array elements result in better measurement precision, allowing detection of single-copy transition boundaries even in specimens with a high proportion of normal cells.
  • the apparatus is representational oligonucleotide microarray analysis (ROMA).
  • ROMA has an increased resolution over standard COH and is more fully described in US 2007-0207481 Al, September 6,2007, Use of ROMA for characterizing genomic rearrangements; Lucito R et al. Representational oligonucleotide microarray analysis; a high- resolution method to detect genome copy number variation. Genome Res. 2003 Oct;13(10):2291-305; and Hicks et al. Novel patterns of genome rearrangement and their association with survival in breast cancer. Genome Res. 2006 Dec; 16( 12): 1465-79, the disclosures of which are incorporated herein by reference.
  • the apparatus is fluorescent in situ hybridization (FISH) apparatus.
  • FISH fluorescent in situ hybridization
  • FISH is a cytogenetic technique used to detect and localize the presence or absence of specific DNA sequences on chromosomes.
  • FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence similarity.
  • probe is understood any ribopolymicleotide or desoxiribopolynucleotide sequence that specifically binds to only those parts of the chromosome with which they show a high degree of sequence similarity.
  • the probe must be large enough to hybridize specifically with its target but not so large as to impede the hybridization process.
  • Many different FISH probes can be used in the present invention without limitation, e.g.
  • FISH probes derived from bacterial artificial chromosomes (BACs), Tiling Oligonucleotide Probes (TOPs), etc.
  • the design of FISH probes is well know for a person skilled in the art (Bayani J, Squire JA..Curr Protoc Cell Biol. 2004 Sep; Chapter 22:Unit 22.4; Bayani J, Squire J.Curr Protoc Cell Biol. 2004 Oct;Chapter 22:Unit 22.5.; Navin, N. et al. Bioinformatics, Volume 22, Number 19, 1 October 2006 , pp. 2437-2438(2)) Publisher: Oxford University Press).
  • the probe can be tagged directly with fluorophores, with targets for antibodies, or with biotin. Tagging can be done in various ways, such as nick translation, or PCR using tagged nucleotides.
  • the sample can be fixed and paraffin embedded. Thus, an additional step of deparaffination may be performed.
  • an interphase or metaphase chromosome preparation may be produced.
  • the chromosomes are firmly attached to a substrate, usually glass. Repetitive DNA sequences must be blocked by adding short fragments of DNA to the sample.
  • the probe is then applied to the chromosome DNA and incubated for approximately 12 hours while hybridizing. Several wash steps remove all unhybridized or partially-hybridized probes. After standard post hybridization washes the slides are stained with the DNA staining probe such DAPI and mounted with a mounting agent such as antifade.
  • the present invention is not limited by these examples and any DNA staining probe and/or mounting agent readily known to those of skill in the art may be used.
  • results are then visualized and quantified using a microscope that is capable of exciting the dye and recording images. If the fluorescent signal is weak, amplification of the signal may be necessary in order to exceed the detection threshold of the microscope. Fluorescent signal strength depends on many factors such as probe labeling efficiency, the type of probe, and the type of dye. Fluorescently-tagged antibodies or streptavidin are bound to the dye molecule. These secondary components are selected so that they have a strong signal. In an embodiment, prior to imaging all slides are evaluated by a pathologist and regions of interest are identified based on histopathologic and quality criteria including, without excluding others, tumor content, appropriate fixation, necrosis and vascularity.
  • sample has been previously defined and can be applied to any type of biological sample from a patient, such as a biopsy sample, tissue, cell or fluid (serum, saliva, semen, sputum, cerebral spinal fluid (CSF), tears, mucus, sweat, milk, brain extracts and the like).
  • sample is a tumour tissue sample or portion thereof.
  • said tumor tissue sample is a breast tumor tissue sample from a patient suffering from breast cancer or a formalin embedded breast tissue sample.
  • determining if the copy number is indicative of a deletion of marker A1, a deletion of marker A2, and/or an amplification of marker B comprises comparing the copy number of the selected marker or markers to the copy number of a diploid reference.
  • Copy number can then be defined as the ratio of hybridization signal at any locus in the genome comparing a tumor DNA sample to a DNA sample from the standard diploid reference.
  • a method is provided of treating a human subject afflicted with cancer comprising:
  • the information is based on a copy number of two of the markers A1, A2 or B, the copy number data of the two markers being indicative of the subject's sensitivity to trastuzumab therapy.
  • the copy number can be determined for any combination or permutation of two of the three markers, including but not limited to A1/A2, A1/B or A2/B.
  • the information is based on a copy number of all three of the markers A1, A2 and B, the copy number data of the three markers being indicative of the subject's sensitivity to trastuzumab therapy.
  • the subject is Her2-. In an embodiment, the subject is Her2+.
  • the methods provided herein to identify a subject as a candidate for trastuzumab therapy can also be used to determine the sensitivity of a subject to trastuzumab therapy and/or predict the responsiveness of a subject to trastuzumab therapy, wherein the subject has cancer.
  • the copy number of each marker can be determined in any order.
  • the subject has breast cancer.
  • the subject has breast cancer cells.
  • the candidate subject has been previously diagnosed as having cancer cells from diseases including, but not limited to, leukemia, brain cancer, kidney cancer, lymphoma, eye cancer, connective tissue cancer, Hodgkin's disease, bone cancer, testicular cancer, cervical cancer, thyroid cancer, melanoma, skin cancer, uterine cancer, lung cancer, colon cancer, rectal cancer, ovarian cancer, bladder cancer, larynx cancer, prostate cancer, stomach cancer, breast cancer, and pancreatic cancer.
  • the subject has a cancer affected by changes in the Human Epidermal growth factor Receptor 2 (Her2) pathway.
  • trastuzumab therapy is administered as a monotherapy. In another embodiment, trastuzumab therapy is administered as an adjuvant therapy.
  • a sample is obtained from the subject under study.
  • said sample is a tumour tissue sample or portion thereof.
  • said tumor tissue sample is a breast tumor tissue sample from a patient suffering from breast cancer.
  • Said sample can be obtained by conventional methods, e.g., biopsy, by using methods well known to those of ordinary skill in the related medical arts. Methods for obtaining the sample from the biopsy include gross apportioning of a mass, or microdissection or other art-known cell-separation methods.
  • Tumour cells can additionally be obtained from fine needle aspiration cytology.
  • Samples can be obtained from subjects previously diagnosed or not diagnosed with breast cancer, or from subjects who are receiving or have previously received anti-breast cancer treatment. In a particular embodiment, samples can be obtained from patients who have not previously received any anti-breast cancer treatment.
  • these can be formalin-fixed and paraffin-embedded or first frozen and then embedded in a cryosolidifiable medium, such as OCT-Compound, through immersion in a highly cryogenic medium that allows for rapid freeze.
  • a marker including but not limited to A1, A2, B or Her2, is determined using nucleic acids obtained from fresh tissue from a biopsy or fine needle aspiration cytology.
  • Nucleic acid may be isolated from an archival pathological sample or biopsy sample which is first deparaffinized.
  • An exemplary deparaffinization method involves washing the paraffinized sample with an organic solvent, such as xylene, for example.
  • Deparaffinized samples can be rehydrated with an aqueous solution of a lower alcohol. Suitable lower alcohols, include, for example, methanol, ethanol, propanols, and butanols.
  • Deparaffinized samples may be rehydrated with successive washes with lower alcoholic solutions of decreasing concentration, for example.
  • the sample may be simultaneously deparaffinized and rehydrated.
  • the sample is then lysed and nucleic acid is extracted from the sample.
  • tissue selected for fixation and paraffin embedding can be fixed in 10% buffered formalin for 16 hours to 48 hours.
  • the copy number of a marker is determined using nucleic acids obtained from a biopsy tissue sample or fine needle aspiration cytology.
  • the sample size required for analysis may range from 1, 10, 50, 100, 200, 300, 500, 1,000, 5,000, 10,000, to 50,000 or more cells. The appropriate sample size may be determined based on the cellular composition and condition of the biopsy or cytology, and the standard preparative steps for this determination and subsequent isolation of the nucleic acid for use in the invention are well known to one of ordinary skill in the art.
  • the biological sample may be treated to physically or mechanically disrupt tissue or cell structure, to release intracellular components into an aqueous or organic solution to prepare nucleic acids for further analysis.
  • the nucleic acids may be extracted from the sample by procedures known to the skilled person and commercially available.
  • the total DNA extracted from tissue samples represents the working material suitable for subsequent detection of the genetic marker of interest.
  • amplification of nucleic acid may be carried out in order to produce sufficient sample material for further detection procedures.
  • Several techniques can be used for producing sufficient starting material. These techniques include polymerase chain reaction (PCR), degenerate primer PCR using one or several sets of primers, rolling circle amplification, etc.
  • the amplification of the DNA is carried out by means of PCR.
  • the general principles and conditions for amplification and detection of nucleic acids, such as using PCR, are well known for the skilled person in the art.
  • the instant invention provides a kit for identifying a subject's sensitivity to trastuzumab therapy, said kit comprising one or more nucleic acid probes each of which selectively bind to a target polynucleotide sequence of the chromosome region comprising one of human chromosome region 17q35.42-q56.76 (marker A1), human chromosome region 17q32.010-q32.34.215 (marker A2) or human chromosome region 4q169.81-q185.239 (marker B) under conditions in which the probe forms a stable hybridization complex with the target polynucleotide sequence.
  • kits contains one or more nucleic acid probes which selectively bind to target polynucleotide sequences of the chromosome regions comprising two of markers A1, A2 or B.
  • kits contains one or more nucleic acid probes which selectively bind to target polynucleotide sequences of the chromosome regions comprising all three of markers A1, A2 and B.
  • kits contains one or more nucleic acid probes which selectively bind to a target polynucleotide sequence of the chromosome region comprising Her2/neu.
  • Probes can be obtained from commercial sources or they can be made non-commercially using well known techniques. A technique to design probes is disclosed in US 2005-0032095 Al, published February 10, 2005, Virtual representations of nucleotide sequences, the disclosure of which is incorporated by reference.
  • Sources of DNA for use in constructing DNA probes include genomic DNA, cloned DNA sequences such as bacterial artificial chromosomes (BAC), somatic cell hybrids that contain one or a part of a human chromosome along with the normal chromosome complement of the host, and chromosomes purified by flow cytometry or microdissection.
  • the region of interest can be isolated through cloning or by site-specific amplification via the polymerase chain reaction (PCR).
  • oligomeric DNA or PNA probes can also be used.
  • the size of the chromosomal region detected by the probes used in the invention can vary.
  • probes of at least 100,000 bases in complexity may be used, and unlabeled blocking nucleic acid may be used, as disclosed in U.S. Pat. No. 5,756, 696, herein incorporated by reference, to avoid non-specific binding of the probe. It is also possible to use unlabeled, synthesized oligomeric nucleic acid or protein nucleic acid as the blocking nucleic acid.
  • the probes may span approximately the entire genomic coding locus of the gene.
  • Chromosomal probes can contain any detection moiety that facilitates the detection of the probe when hybridized to a chromosome. Effective detection moieties include both direct and indirect labels as described below.
  • An embodiment of the instant kit contains a nucleic acid probe or probes which are directly labeled.
  • An embodiment of the instant kit contains a nucleic acid probe or probes which are indirectly labeled.
  • Chromosomal probes can be directly labeled with a detectable label.
  • detectable labels include fluorophores (i.e., organic molecules that fluoresce after absorbing light), radioactive isotopes (e.g., 32p, and 3H) and chromophores (e.g., enzymatic markers that produce a visually detectable marker).
  • Fluorophores are preferred and can be directly labeled following covalent attachment to a nucleotide by incorporating the labeled nucleotide into the probe with standard techniques such as nick translation, random priming, and PCR labeling.
  • deoxycytidine nucleotides within the probe can be transaminated with a linker.
  • the fluorophore can then be covalently attached to the transaminated deoxycytidine nucleotides. See, e.g., U.S. Pat. No. 5,491,224 to Bittner, et al., which is incorporated herein by reference.
  • Useful probe labeling techniques are described in Molecular Cytogenetics: Protocols and Applications, Y.-S. Fan, Ed., Chap. 2, "Labeling Fluorescence In Situ Hybridization Probes for Genomic Targets", L. Morrison et. al., p. 21-40, Humana Press, ⁇ 2002 (hereafter cited as "Morrison 2002”), incorporated herein by reference.
  • fluorophores examples include: 7-amino-4- methylcoumarin-3 -acetic acid (AMCA), Texas RedTM (Molecular Probes, Inc., Eugene, Oreg.); 5-(and-6)-carboxy-X-rhodamine, lissamine rhodamine B, 5-(and-6)-carboxyfluorescein; fluorescein-5-isothiocyanate (FITC); 7-diethylaminocoumarin-3-carboxylic acid, tetramethylrhodamine-5-(and-6)-isothiocyanate; 5-(and -6)-carboxytetramethylrhodamine; 7- hydroxycoumarin-3-carboxylic acid; 6- [fluorescein 5-(and-6)-carboxamido]hexanoic acid; N - (4,4-difluoro-5, 7-dimethyl-4-bora-3a,4a diaza-3-indace
  • AMCA 7-amino-4
  • fluorophores of different colors can be chosen such that each chromosomal probe in the set can be distinctly visualized.
  • the probe panel of the invention will comprise two or three separate probes, each labeled with a separate fluorophore.
  • Use of four probes may be preferred as providing the best balance between clinical sensitivity (sensitivity can increase with added probes) and imaging/detection complexity (complexity can increase with added probes).
  • Probes can be viewed with a fluorescence microscope and an appropriate filter for each fluorophore, or by using dual or triple band-pass filter sets to observe multiple fluorophores. See, e.g., U.S. Pat. No. 5,776,688 to Bittner, et al., which is incorporated herein by reference. Any suitable microscopic imaging method can be used to visualize the hybridized probes, including automated digital imaging systems, such as those available from MetaSystems or Applied Imaging. Alternatively, techniques such as flow cytometry can be used to examine the hybridization pattern of the chromosomal probes.
  • Probes can also be labeled indirectly, e.g., with biotin or digoxygenin by means well known in the art. However, secondary detection molecules or further processing are then required to visualize the labeled probes.
  • a probe labeled with biotin can be detected by avidin conjugated to a detectable marker, e.g., a fluorophore.
  • avidin can be conjugated to an enzymatic marker such as alkaline phosphatase or horseradish peroxidase. Such enzymatic markers can be detected in standard calorimetric reactions using a substrate for the enzyme.
  • Substrates for alkaline phosphatase include 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium.
  • Diaminobenzoate can be used as a substrate for horseradish peroxidase.
  • 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.
  • Useful probe sets and kits can comprise probes to Her2 and probes to one or more of marker A1, marker A2 or marker B. Additionally, probe sets and kits may only include a probe or probes to one or more of marker A1, marker A2 or marker B. Alternatively, one or more reference probes may be included in the kits already mentioned.
  • an array-based format can be used in which the nucleic acids or the nucleic acid probes of the invention are attached to a solid surface.
  • the attached probe or probes comprise a nucleic acid array.
  • this type of format a large number of different hybridization reactions can be run essentially "in parallel.” This provides rapid, essentially simultaneous, evaluation of a large number of nucleic acid probes.
  • Methods for immobilizing the polynucleotides on the surface and derivatizing the surface are known in the art; see, for example, U.S. Pat. No. 6,664,057, and are also described above. These arrays can be used in CGH or ROMA analysis.
  • the kit comprises instructional material which teaches that the detection of one or more of deletion at marker Al, deletion at marker A2or an amplification at marker B indicates that a subject is likely to be sensitive to trastuzumab therapy.
  • the invention relates to the use of a kit of the invention for identifying a subject as a candidate for trastuzumab therapy, wherein if said components of the kit detect a deletion of marker Al, a deletion of marker A2 or an amplification of marker B, then the subject will be considered a candidate for trastuzumab therapy.
  • Cancer and Leukemia Group B Protocol 9342 was initiated to determine the optimal dose of pachtaxel administered as a 3-hour infusion every 3 weeks to women with metastatic breast cancer.
  • Weekly paclitaxel is more effective than every-3-weeks administration for MBC. Trastuzumab did not improve efficacy for Her2 nonoverexpressors. Neurotoxicity is a treatment-limiting toxicity for weekly paclitaxel.
  • DNA was obtained from formalin-fixed paraffin-embedded (FFPE) tumor tissue samples from patients that had been enrolled in clinical trials (CALGB9840 and CALGB9342). These trials were originally designed to test response to taxane chemotherapy along with response to Herceptin® (trastuzumab) therapy. DNA samples were anonymized, but associated with complete clinical information, including long term (>5 years) followup for disease progression, survival, histopathology and had been tested for Her2/neu (ERBB2) gene amplification by the clinical standard technique known as fluorescence in situ hybridization (FISH). mRNA gene expression data was also available for Her2/neu plus 700 additional genes. The sample set contained approximately equal numbers of four test categories; 1. Her2 FISH negative, untreated with Herceptin; 2. Her2 FISH positive (FISH ratio >2), untreated with Herceptin; 3. Her2 FISH negative, treated with Herceptin; 4. Her2 FISH positive, treated with Herceptin. DNA preparation.
  • FFPE formalin-fixed paraffin-embedded
  • DNA isolated from these samples was extracted using commercially available techniques for purifying DNA from FFPE samples. DNA was stored frozen at -20° C. Genome Copy Number Analysis
  • Kaplan-Meier style curves for plotting survival were obtained using the "survfit” and “survplot” functions in the statistical package that comes standard in the S-plus 2000 mathematical analysis software (sold by MathSoft, Inc.). Mulitvariate and univariate analysis of marker combinations was performed with the S-plus 2000 package or the freeware package known as "R".
  • a specific set of CGH values from the Her2 locus as well as certain locations in the genome not linked in any way to Her2 can be used in combination with the Her2 amplification value to identify a subset of patients, not previously anticipated to respond to trastuzumab (that is, those with low levels of Her2 mRNA and no amplification at the ERBB2 genetic locus) do, in fact, respond to trastuzumab in a statistically significant manner (Figure 2C, D). Significance is expressed by p-values in the figures. Hazard ratios are described below.
  • trastuzumab treatment There is a significant benefit of trastuzumab treatment in the entire cohort.
  • the non- treatment hazard is 1.504, meaning that the non-treated patients are 1.504 many times likely to fail within a month from now provided that they have survived till now.
  • the non-treatment hazard is 2.09.
  • Marker Al A CGH ratio (when compared to a known diploid reference genome) of 0.85 or less for any genomic location bounded by the following chromosomal positions (in megabasepairs) on human chromosome 17q (defined by the reference genome in the UCSC genome browser reflecting freeze HG18:
  • Marker A2 A ratio (when compared to a known diploid reference genome) of 0.85 or less for any genomic location bounded by the following chromosomal positions on human chromosome 17q (defined by the reference genome in the UCSC genome browser reflecting freeze HQ18:
  • Marker B A ratio of 1.1 or greater (when compared to a known diploid reference genome) for any genomic location bounded by the following chromosomal positions on human chromosome 4q (defined by the reference genome in the UCSC genome browser reflecting freeze HG18;
  • Table 1 shows the chromosomal coordinates for ErbB2 and markers Al, A2 and B, in addition to the P-values obtained when patients positive for the respective markers were treated with trastuzumab. The number of patients affected and hazard ratios are also displayed.

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Abstract

L'invention concerne le domaine thérapeutique et l'identification de candidats pour une thérapie, et en particulier un procédé pour identifier des candidats pour la thérapie par trastuzumab (Herceptine®) chez un patient qui souffre d'un cancer du sein, sur la base de la présence ou de l'absence de marqueurs génétiques spécifiques dans un échantillon de tumeur prélevé chez ledit patient.
PCT/US2010/059846 2009-12-11 2010-12-10 Marqueurs génétiques indicatifs d'une réponse d'un patient atteint d'un cancer au trastuzumab (herceptine) Ceased WO2011072205A2 (fr)

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US20090280493A1 (en) * 2006-09-08 2009-11-12 Siemens Healthcare Diagnostics Inc. Methods and Compositions for the Prediction of Response to Trastuzumab Containing Chemotherapy Regimen in Malignant Neoplasia
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US11753641B2 (en) 2010-04-23 2023-09-12 Cold Spring Harbor Laboratory Structurally designed shRNAs
US12351802B2 (en) 2010-04-23 2025-07-08 Cold Spring Harbor Laboratory Structurally designed shRNAs

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