US20130149704A1 - Materials and methods for diagnosis of malignant melanoma and prognosis of metastasis of malignant melanoma - Google Patents

Materials and methods for diagnosis of malignant melanoma and prognosis of metastasis of malignant melanoma Download PDF

Info

Publication number
US20130149704A1
US20130149704A1 US13/709,082 US201213709082A US2013149704A1 US 20130149704 A1 US20130149704 A1 US 20130149704A1 US 201213709082 A US201213709082 A US 201213709082A US 2013149704 A1 US2013149704 A1 US 2013149704A1
Authority
US
United States
Prior art keywords
copy number
ccnd1
myc
metastasis
probe
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US13/709,082
Other languages
English (en)
Inventor
Susan Jewell
Ekaterina Pestova
Gu Li
Carl Slenk
Pedram Gerami
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Abbott Molecular Inc
Original Assignee
Abbott Molecular Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Abbott Molecular Inc filed Critical Abbott Molecular Inc
Priority to US13/709,082 priority Critical patent/US20130149704A1/en
Publication of US20130149704A1 publication Critical patent/US20130149704A1/en
Assigned to ABBOTT MOLECULAR INC. reassignment ABBOTT MOLECULAR INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LI, Gu, PESTOVA, EKATERINA, JEWELL, SUSAN, GERAMI, Pedram, SLENK, Carl
Priority to US15/354,854 priority patent/US10227658B2/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/16—Assays for determining copy number or wherein the copy number is of special importance
    • 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
    • C12Q2600/118—Prognosis of disease development
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • the present disclosure relates to the diagnosis of malignant melanoma, the prognosis of metastasis of malignant melanoma, the determination of copy numbers of genes and/or regions of chromosomes, and in situ hybridization, as well as sets of one or more probes and kits useful for the diagnosis of malignant melanoma and the prognosis of metastasis thereof.
  • ACS American Cancer Society
  • 68,720 new cases of invasive melanoma, and 8,650 deaths were reported in the United States (ACS (2009), supra).
  • histology is recognized as the gold standard for the diagnosis of melanoma, and it is, therefore, the current gold standard for predicting clinical behavior. Histology is essentially a surrogate marker for predicting clinical outcome. However, histology is the method of choice by default, and it has not been completely validated. Numerous epidemiologic and clinical studies illustrate the limitations of histology.
  • indolent melanoma a small percentage of melanomas, which invade the skin to a shallow Breslow's depth, behave in a very malignant and aggressive manner, resulting in metastasis and death.
  • Sentinel lymph node biopsy is performed in melanoma patients with a high risk for metastases to evaluate the lymph node for metastatic involvement by melanoma.
  • patients with a melanoma of Breslow's depth greater than about 0.75 mm are biopsied. Such patients have a poor prognosis based on histological factors such as high mitotic rate, ulceration, or Clark's level IV or V.
  • the sentinel lymph node status is the strongest prognostic indicator of melanoma.
  • Those patients in whom the sentinel lymph node is not involved in melanoma are considered to be a cohort of patients with significantly better prognosis compared to those patients in whom the sentinel lymph node is involved in melanoma.
  • Frequent chromosomal copy number losses include deletions at 9p (82%), 10q (63%), 6q (28%), and 8p (22%). Frequent copy number gains may occur at 7q (50%), 8q (34%), 6p (28%), and 1q (25%), among others (Bastian et al. (1998), supra). Melanomas on acral sites reportedly have significantly more aberrations involving chromosomes 5p, 11q, 12q, and 15, as well as focused gene amplifications (Bastian et al., Amer. J. Path. 163: 1765-1770 (2003)).
  • FISH fluorescent in situ hybridization
  • Copy number gains of specific oncogenes have been linked to prognosis in a number of cancers. For example, amplification of Her-2/neu has been associated with poor prognosis in breast cancers (Tovey et al., Br. J. Cancer 100(5): 680-683 (Mar. 10, 2009)), while elevated copy numbers of the epidermal growth factor receptor (EGFR) gene are highly associated with likely response and survival benefit of non-small cell lung cancer treated with EGFR tyrosine kinase inhibitors (Dahabreh et al., Clin. Cancer Res. 16(1): 291-303 (Jan. 1, 2010)).
  • EGFR epidermal growth factor receptor
  • the present disclosure seeks to provide a method for prognosing malignant melanoma, including atypical Spitz tumors, in a patient.
  • the present disclosure seeks to provide a method for diagnosing malignant melanoma in a patient. It is well-accepted among pathologists that there are several subsets of melanocytic tumors that can be difficult to classify clearly as either benign or malignant (Barnhill et al., Hum. Pathol. 30(5): 513-520 (1999); Corona et al., J. Clin. Oncol. 14(4): 1218-1223 (1996); and McGinnis et al., Arch. Dermatol. 138(5): 617-621 (2002)).
  • the differential diagnosis includes an entirely benign lesion, such as a spitz nevus, as opposed to a highly lethal, malignant lesion, such as melanoma with spitzoid morphological features.
  • a highly lethal, malignant lesion such as melanoma with spitzoid morphological features.
  • Added advantages of differential diagnosis include the avoidance of undue psychological burden resulting from an incorrect diagnosis, the determination of appropriate surgical management with or without sentinel lymph node biopsy, and the determination of appropriate systemic therapy.
  • FISH fluorescence in situ hybridization
  • a method of prognosing metastasis of malignant melanoma in a patient comprises determining in a sample of malignant melanoma obtained from the patient (i) (a) and/or (b), (ii) (c) and/or (d), (iii) (a) and/or (d), or (iv) (b) and/or (c), wherein:
  • (a) is a copy number ratio of CCND1/control centromere or a copy number of CCND1, wherein a copy number ratio of CCND1/control centromere greater than about 1.55 per cell or a copy number of CCND1 greater than about 2.81 per cell indicates that metastasis will likely occur,
  • (b) is a copy number of MYC, wherein a copy number of MYC greater than about 2.48 per cell indicates that metastasis will likely occur,
  • (c) is a percentage of cells having a gain of CCND1/control centromere or a percentage of cells having a gain of CCND1, wherein a percentage of cells of greater than or equal to about 30% having a gain of CCND1 or a percentage of cells of greater than or equal to about 54% having a gain of CCND1/control centromere indicates that metastasis will likely occur, and
  • (d) is a percentage of cells having a gain of MYC, wherein a percentage of cells of greater than about 20% having a gain of MYC indicates that metastasis will likely occur.
  • a method of prognosing metastasis of malignant melanoma in a patient having a melanoma with a Breslow's depth less than about 1 mm comprises determining in a sample of malignant melanoma obtained from the patient a copy number ratio of CCND1/control centromere and a copy number of MYC.
  • a copy number ratio of CCND1/control centromere greater than about 1.55 per cell or a copy number of MYC greater than about 2.48 per cell indicates that metastasis will likely occur.
  • the method comprises determining in a sample of malignant melanoma obtained from the patient a copy number ratio of CCND1/control centromere and a copy number of MYC.
  • a copy number ratio of CCND1/control centromere greater than about 1.38 per cell and a copy number of MYC greater than about 2.36 per cell indicates that metastasis will likely occur.
  • the method comprises determining in a sample of malignant melanoma obtained from the patient a copy number ratio of CCND1/control centromere. A copy number ratio of CCND1/control centromere greater than about 1.55 per cell indicates that metastasis will likely occur.
  • the method can further comprise detecting in the patient a copy number of MYC, wherein a copy number of MYC greater than about 2.60 per cell also indicates that metastasis will likely occur.
  • the method comprises determining in a sample of malignant melanoma obtained from the patient a copy number ratio of CCND1/control centromere or a copy number of MYC.
  • a copy number ratio of CCND1/control centromere greater than about 1.55 per cell indicates that metastasis will likely occur.
  • a copy number of MYC greater than about 2.22 per cell indicates that metastasis will likely occur.
  • the above methods can comprise determining the copy number ratio of CCND1/control centromere and/or the copy number of MYC by in situ hybridization.
  • the in situ hybridization can be fluorescent in situ hybridization (FISH).
  • a kit comprising (a) a set of one or more probes that enables prognosis of metastasis of malignant melanoma in a patient and (b) instructions for prognosing metastasis of malignant melanoma in the patient.
  • the set of one or more probes comprises (i′) a probe for CCND1, alone or in further combination with a probe for a control centromere, and/or (ii′) a probe for MYC.
  • the instructions can comprise determining in a sample of malignant melanoma obtained from the patient (i) (a) and/or (b), (ii) (c) and/or (d), (iii) (a) and/or (d), or (iv) (b) and/or (c), wherein:
  • (a) is a copy number ratio of CCND1/control centromere or a copy number of CCND1, wherein a copy number ratio of CCND1/control centromere greater than about 1.55 per cell or a copy number of CCND1 greater than about 2.81 per cell indicates that metastasis will likely occur,
  • (b) is a copy number of MYC, wherein a copy number of MYC greater than about 2.48 per cell indicates that metastasis will likely occur,
  • (c) is a percentage of cells having a gain of CCND1/control centromere or a percentage of cells having a gain of CCND1, wherein a percentage of cells of greater than or equal to about 30% having a gain of CCND1 or a percentage of cells of greater than or equal to about 54% having a gain of CCND1/control centromere indicates that metastasis will likely occur, and
  • (d) is a percentage of cells having a gain of MYC, wherein a percentage of cells of greater than about 20% having a gain of MYC indicates that metastasis will likely occur.
  • a method of diagnosing malignant melanoma in a patient comprises determining in a number of nuclei in a diagnostic sample, which comprises nucleated cells, obtained from the patient a copy number of RREB1, a copy number of MYC, a copy number of CCND1, and a copy number of CDKN2A, wherein an increase in the copy number of RREB1, an increase in the copy number of MYC, an increase in the copy number of CCND1, and a decrease in the copy number of CDKN2A indicates that the sample comprises a malignant melanoma.
  • the number of nuclei can be about 30, wherein, when increases in copy numbers of RREB1, MYC, and CCND1 and a homozygous deletion of CDKN2A are detected in greater than or equal to 27% of the nuclei, the sample comprises a malignant melanoma.
  • the number of nuclei can be about 30, wherein, when increases in copy numbers of RREB1, MYC, and CCND1 and a homozygous deletion of CDKN2A are detected in greater than or equal to 8 nuclei, the sample comprises a malignant melanoma.
  • the set comprises a probe for RREB1, a probe for MYC, a probe for CCND1, and a probe for CDKN2A.
  • the kit comprises (a) a set of probes that enables diagnosis and prognosis of malignant melanoma in a patient, wherein the set of probes comprises a probe for RREB1, a probe for MYC, a probe for CCND1, and a probe for CDKN2A, and (b) instructions for diagnosing malignant melanoma in the patient, wherein the instructions comprise determining in a diagnostic sample obtained from the patient a copy number of RREB1, a copy number of MYC, a copy number of CCND1, and a copy number of CDKN2A, wherein an increase in the copy number of RREB1, an increase in the copy number of MYC, an increase in the copy number of CCND1, and a decrease in the copy number of CDKN2A indicates that the patient has malignant melanoma, and/or instructions for prognosing metastasis of malignant melanoma in the patient, wherein the instructions comprise determining in a diagnostic sample obtained from the patient
  • the method comprises determining in a number of nuclei in a sample, which comprises nucleated cells, obtained from the patient a copy number of RREB1, a copy number of MYC or ZNF217, a copy number of CCND1, and a copy number of CDKN2A, wherein an increase in the copy number of RREB1, an increase in the copy number of MYC or ZNF217, an increase in the copy number of CCND1, and a decrease in the copy number of CDKN2A indicates that metastasis will likely occur.
  • the number of nuclei can be about 30, wherein, when increases in copy numbers of RREB1, MYC or ZNF217, and CCND1 and a homozygous deletion of CDKN2A are detected in greater than or equal to 27% of the nuclei, metastasis will likely occur.
  • the number of nuclei can be about 30, wherein, when increases in copy numbers of RREB1, MYC or ZNF217, and CCND1 and a homozygous deletion of CDKN2A are detected in greater than or equal to 8 nuclei, metastasis will likely occur.
  • a set of probes that enables prognosis of metastasis of malignant melanoma is also provided.
  • the set comprises a probe for RREB1, a probe for MYC or ZNF217, a probe for CCND1, and a probe for CDKN2A.
  • a kit comprises (a) a set of probes that enables prognosis of metastasis of malignant melanoma in a patient, wherein the set of probes comprises a probe for RREB1, a probe for MYC or ZNF217, a probe for CCND1, and a probe for CDKN2A, and (b) instructions for prognosing malignant melanoma in the patient, wherein the instructions comprise determining in a sample obtained from the patient a copy number of RREB1, a copy number of MYC or ZNF217, a copy number of CCND1, and a copy number of CDKN2A, wherein an increase in the copy number of RREB1, an increase in the copy number of MYC or ZNF217, an increase in the copy number of CCND1, and a decrease in the copy number of CDKN2A indicates that metastasis will likely occur.
  • a method of prognosing metastasis of atypical Spitz tumor in a patient comprises determining in a sample of tumor from the patient a copy number of RREB1, CCND1, and/or CDKN2A, wherein an increase in copy number of RREB1 or an increase in copy number of CCND1 or a homozygous deletion of CDKN2A indicates that aggressive metastasis will likely occur and homozygous deletion of CDKN2A indicates that even more aggressive metastasis will likely occur.
  • the set comprises a probe for RREB1, a probe for CCND1, and a probe for CDKN2A.
  • the kit comprises (a) a set of probes that enables prognosis of metastasis of atypical Spitz tumor in a patient, wherein the set of probes comprises a probe for RREB1, a probe for CCND1, and a probe for CDKN2A, and (b) instructions for prognosing metastasis of atypical Spitz tumor in the patient, wherein the instructions comprise determining in a sample of tumor from the patient a copy number of RREB1, CCND1, and/or CDKN2A, wherein an increase in copy number of RREB1 or an increase in copy number of CCND1 or a homozygous deletion of CDKN2A indicates that aggressive metastasis will likely occur and homozygous deletion of CDKN2A indicates that even more aggressive metastasis will likely occur.
  • FIG. 1 a is a graph of sensitivity vs. 1-specificity for CCND1/chromosome 6, CCND1/cell, and MYC/cell.
  • FIG. 1 b is a graph of the cumulative probability of metastasis-free survival vs. time (months) for CCND1/chromosome 6 ⁇ 1.55, all patients, and CCND1/chromosome 6>1.55.
  • FIG. 1 c is a graph of the cumulative probability of metastasis-free survival vs. time (months) for MYC/cell ⁇ 2.48, all patients, and MYC/cell>2.48.
  • FIG. 1 d is a graph of the cumulative probability of metastasis-free survival vs. time (months) for CCND1/chromosome 6 ⁇ 1.55 and MYC/cell ⁇ 2.48, all patients, and CCND1/chromosome 6>1.55 or MYC/cell>2.48.
  • FIG. 2 a is a graph of cumulative probability of survival vs. time (months) for CCND1/chromosome 6 ⁇ 1.55, all patients, and CCND1/chromosome 6>1.55.
  • FIG. 2 b is a graph of cumulative probability of survival vs. time (months) for MYC/cell ⁇ 2.48, all patients, and MYC/cell>2.48.
  • FIG. 3 a is a graph of cumulative probability of metastasis-free survival vs. time (months) for Breslow's depths ⁇ 1.0 mm and CCND1/chromosome 6 ⁇ 1.55, all Breslow's depths ⁇ 1.0 mm, Breslow's depths>1.0 mm and CCND1/chromosome 6 ⁇ 1.55, all Breslow's depths>1.0 mm, all Breslow's depths>1.0 mm and CCND1/chromosome 6>1.55, and Breslow's depths ⁇ 1.0 mm and CCND1/chromosome 6>1.55.
  • FIG. 3 b is a graph of cumulative probability of metastasis-free survival vs. time (months) for Breslow's depths ⁇ 1.0 mm and MYC/cell ⁇ 2.48, all Breslow's depths ⁇ 1.0 mm, Breslow's depths>1.0 mm and MYC/cell ⁇ 2.48, all Breslow's depths>1.0 mm, all Breslow's depths>1.0 mm and MYC/cell>2.48, and Breslow's depths ⁇ 1.0 mm and MYC/cell>2.48.
  • FIG. 3 c is a graph of cumulative probability of metastasis-free survival vs. time (months) for Breslow's depths ⁇ 2.0 mm and CCND1/chromosome 6 ⁇ 1.55, all Breslow's depths ⁇ 2.0 mm, Breslow's depths>2.0 mm and CCND1/chromosome 6 ⁇ 1.55, all Breslow's depths>2.0 mm, Breslow's depths ⁇ 2.0 mm and CCND1/chromosome 6>1.55, and all Breslow's depths>2.0 mm and CCND1/chromosome 6>1.55.
  • FIG. 3 d is a graph of cumulative probability of metastasis-free survival vs. time (months) for Breslow's depths ⁇ 2.0 mm and MYC/cell ⁇ 2.48, all Breslow's depths ⁇ 2.0 mm, Breslow's depths ⁇ 2.0 mm and MYC/cell>2.48, all Breslow's depths>2.0 mm and MYC/cell ⁇ 2.48, all Breslow's depths>2.0 mm, and Breslow's depths>2.0 mm and MYC/cell>2.48.
  • FIG. 4 a is a graph of cumulative probability of metastasis-free survival vs. time (months (mo)) for Breslow's depths 1-4 mm and CCND1/chromosome 6 (cen6) ⁇ 1.55, all Breslow's depths 1-4 mm, and Breslow's depths 1-4 mm and CCND1/chromosome 6 (cen6)>1.55.
  • FIG. 4 b is a graph of cumulative probability of survival vs. time (months (mo)) for CCND1/chromosome 6 (cen6) ⁇ 1.55, all patients, and CCND1/chromosome 6 (cen6)>1.55.
  • FIG. 5 is a bar graph of average in specimen category vs. parameter, which shows the proportion of cells with chromosomal abnormalities (gain or loss) in the melanoma group and the nevus group for representative parameters calculated for each of the probes CDKN2A, MYC, CEP9, ZNF217, Cox2, BRAF and CEP10.
  • FIG. 6 is a graph of sensitivity vs. 1-specificity for CDKN2A % homozygous deletion ( ⁇ ), CCND1% gain ( ⁇ ), RREB1% gain ( ⁇ ), MYC % gain ( ⁇ ), and all probes ( ⁇ ).
  • FIG. 7 is a graph of sensitivity vs. 1-specificity for CCND1/CEP6 ( ⁇ ), CCND1/CEP6% gain ( ⁇ ), CCND1% gain ( ⁇ ), CCND1/cell (x), MYC/cell (*), and MYC % gain ( ⁇ ).
  • FIG. 8 is a graph of sensitivity vs. 1-specificity for CDNK2A % homozygous loss, RREB1% gain, MYC % gain, and CCND1% gain.
  • a prognosis for malignant melanoma in a patient can be made based on (i) a determination of the copy number ratio of CCND1/control centromere or the copy number of CCND1 and/or the copy number of MYC in a sample of malignant melanoma obtained from the patient or (ii) a determination of the percentage of cells having a gain of CCND1/control centromere or the percentage of cells having a gain of CCND1 and/or the percentage of cells having a gain of MYC.
  • the likelihood of metastasis occurring in the patient can be prognosticated.
  • the present disclosure provides methods of prognosing disease progression, such as metastasis of malignant melanoma, in a patient, a set of one or more probes that enables prognosis of disease progression, such as metastasis of malignant melanoma, and a kit comprising a set of one or more such probes and instructions for prognosing disease progression, such as metastasis of malignant melanoma, in a patient.
  • the present disclosure is also based on the surprising and unexpected discovery that a diagnosis of malignant melanoma in a patient can be made based on a determination of the copy numbers of RREB1, MYC, a copy number of CCND1, and a copy number of CDKN2A.
  • the present disclosure provides a method of diagnosing malignant melanoma in a patient, a set of probes that enables diagnosis of malignant melanoma, and a kit comprising a set of such probes and instructions for diagnosing melanoma in a patient.
  • the method can be used to classify better histologically ambiguous melanocytic tumors and to identify more selectively those histologically ambiguous melanocytic tumors with the greatest likelihood of resulting in distant metastasis or death of a patient.
  • the method also can be used to prognosticate disease progression, such as metastasis of malignant melanoma.
  • the present disclosure is also based on the discovery that a prognosis of metastasis of malignant melanoma in a patient can be made based on a determination of the copy numbers of RREB1, MYC or ZNF217, a copy number of CCND1, and a copy number of CDKN2A.
  • the present disclosure provides a method of prognosing metastasis of malignant melanoma in a patient, a set of probes that enables prognosis of metastasis of malignant melanoma, and a kit comprising a set of such probes and instructions for prognosing metastasis of malignant melanoma.
  • Breslow's depth is considered to be one of the three most important prognostic factors of melanoma. The other factors are T stage and ulceration. Breslow's depth is determined by using an ocular micrometer at a right angle to the skin. The depth from the granular layer of the epidermis to the deepest point of invasion to which tumor cells have invaded the skin is directly measured.
  • Cancer diagnosis generally refers to an identification of a type of cancer.
  • the diagnosis can be differential in nature, e.g., distinguishing histologically ambiguous malanocytic tumors, such as a chronic melanoma from mitotically active nevi, a blue nevus-like metastasis from an epithelioid blue nevus, or a conjunctival melanoma from a conjunctival nevus.
  • Cancer prognosis generally refers to a forecast or prediction of the probable course (e.g., disease progression) or outcome (e.g., metastasis or death) of the cancer.
  • cancer prognosis includes the forecast or the prediction of the progression of melanoma, including the metastasis of malignant melanoma and death.
  • Chrosome enumeration probe is any probe that enables the number of specific chromosomes in a cell to be enumerated.
  • a chromosome enumeration probe typically recognizes and binds to a region near to (referred to as “peri-centromeric”) or at the centromere of a specific chromosome, typically a repetitive DNA sequence (e.g., alpha satellite DNA).
  • the centromere of a chromosome is typically considered to represent that chromosome, since the centromere is required for faithful segregation during cell division.
  • Deletion or amplification of a particular chromosomal region can be differentiated from loss or gain of the whole chromosome (aneusomy), within which it normally resides, by comparing the number of signals corresponding to the particular locus (copy number) to the number of signals corresponding to the centromere.
  • One method for making this comparison is to divide the number of signals representing the locus by the number of signals representing the centromere. Ratios of less than one indicate relative loss or deletion of the locus, and ratios greater than one indicate relative gain or amplification of the locus.
  • comparison can be made between two different loci on the same chromosome, for example on two different arms of the chromosome, to indicate imbalanced gains or losses within the chromosome.
  • a chromosomal arm probe may alternately be used to approximate whole chromosomal loss or gain.
  • such probes are not as accurate at enumerating chromosomes, since the loss of signals for such probes may not always indicate a loss of the entire chromosome.
  • Examples of chromosome enumeration probes include CEP® probes commercially available from Abbott Molecular, Inc., Des Plaines, Ill. (formerly Vysis, Inc., Downers Grove, Ill.).
  • Level I involves the epidermis.
  • Level II involves the epidermis and upper dermis.
  • Level III involves the epidermis, upper dermis, and lower dermis.
  • Level IV involves the epidermis, upper dermis, lower dermis, and subcutis.
  • Codon number is a measurement of DNA, whether of a single locus, one or more loci, or an entire genome.
  • a “copy number” of two is “wild-type” in a human (because of diploidy, except for sex chromosomes).
  • a “copy number” of other than two in a human (except for sex chromosomes) deviates from wild-type. Such deviations include amplifications, i.e., increases in copy numbers, and deletions, i.e., decreases in copy numbers and even the absence of copy numbers.
  • “Fixatives” include, but are not limited to, alcohol solutions, acid acetone solutions, aldehydes (such as formaldehyde, paraformaldehyde, and glutaraldehyde), methanol/acetic acid, and formalin.
  • Label and “detectably labeled” are used interchangeably herein to indicate that an entity (e.g., a probe) can be detected.
  • Label and “detectable label” mean a moiety attached to an entity to render the entity detectable, such as a moiety attached to a probe to render the probe detectable upon binding to a target sequence. The moiety, itself, may not be detectable but may become detectable upon reaction with yet another moiety. Use of the term “detectably labeled” is intended to encompass such labeling.
  • the detectable label can be selected such that the label generates a signal, which can be measured and the intensity of which is proportional to the amount of bound entity.
  • Labeled nucleic acids can be prepared by incorporating or conjugating a label that is directly or indirectly detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical or other means.
  • Suitable detectable labels include radioisotopes, fluorophores, chromophores, chemiluminescent agents, microparticles, enzymes, magnetic particles, electron dense particles, mass labels, spin labels, haptens, and the like. Fluorophores and chemiluminescent agents are preferred herein.
  • “Locus-specific probe” refers to a probe that selectively binds to a specific locus in a region on a chromosome, e.g., a locus that has been determined to undergo gain/loss in metastasis.
  • a probe can target coding or non-coding regions, or both, including exons, introns, and/or regulatory sequences, such as promoter sequences and the like.
  • Nucleic acid sample refers to a sample comprising nucleic acid in a form suitable for hybridization with a probe, such as a sample comprising nuclei or nucleic acids isolated or purified from such nuclei.
  • the nucleic acid sample may comprise total or partial (e.g., particular chromosome(s)) genomic DNA, total or partial mRNA (e.g., particular chromosome(s) or gene(s)), or selected sequence(s).
  • Condensed chromosomes (such as are present in interphase or metaphase) are suitable for use as targets in in situ hybridization, such as FISH.
  • Percentage gain and “% gain” refer generally to the percentage of cells having an increased number of copies a particular gene, whereas “percentage loss” and “% loss” refer generally to the percentage of cells having a decreased number of copies of a particular gene. For example, a normal or wild-type cell contains two copies of each gene. The percentage gain/loss can be determined as follows:
  • Predetermined cutoff and predetermined level refer generally to a cutoff value that is used to assess diagnostic/prognostic/therapeutic efficacy results by comparing the assay results against the predetermined cutoff/level, where the predetermined cutoff/level already has been linked or associated with various clinical parameters (e.g., severity of disease, progression/nonprogression/improvement, etc.).
  • the present disclosure provides exemplary predetermined levels for prognosing metastasis in patients with malignant melanoma. While cutoff values may vary with the manner of assay, the correlations as described herein should remain generally applicable.
  • Probe in the context of the present disclosure, is an oligonucleotide or polynucleotide that can selectively hybridize to at least a portion of a target sequence (e.g., the gene CCND1, the gene MYC, or the centromere of chromosome 6, such as the alpha satellite DNA located at the centromere of chromosome 6) under conditions that allow for or promote selective hybridization.
  • a probe can be complementary to the coding or sense (+) strand of DNA or complementary to the non-coding or anti-sense ( ⁇ ) strand of DNA (sometimes referred to as “reverse-complementary”). Probes can vary significantly in length. A length of about 10 to about 100 nucleotides, such as about 15 to about 75 nucleotides, e.g., about 15 to about 50 nucleotides, can be preferred.
  • tissue sample is a single part or piece of a tissue sample, e.g., a thin slice of tissue or cells cut from a tissue sample. Two or more sections of tissue samples may be taken and analyzed. If desired, a single section can be analyzed at various levels, e.g., morphological and molecular (e.g., nucleic acid and protein).
  • tissue sample e.g., a thin slice of tissue or cells cut from a tissue sample. Two or more sections of tissue samples may be taken and analyzed. If desired, a single section can be analyzed at various levels, e.g., morphological and molecular (e.g., nucleic acid and protein).
  • “Selectively hybridize to” refers to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions.
  • stringent conditions refers to conditions under which a probe will hybridize preferentially to its target sequence, and to a lesser extent to, or not at all to, other non-target sequences.
  • a “stringent hybridization” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization are sequence-dependent, and differ under different conditions.
  • Very stringent conditions are selected to be equal to the T m for a particular probe.
  • An example of stringent hybridization conditions for hybridization of complementary nucleic acids, which have more than 100 complementary residues, on an array or on a filter in a Southern or Northern blot is 42° C. using standard hybridization solutions (see, e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd ed., Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY (2001)).
  • Target sequence refers to a nucleotide sequence that resides at a specific chromosomal location whose loss and/or gain is being determined.
  • a method of prognosing metastasis of malignant melanoma in a patient comprises determining in a sample of malignant melanoma obtained from the patient (i) (a) and/or (b), (ii) (c) and/or (d), (iii) (a) and/or (d), or (iv) (b) and/or (c), wherein:
  • (a) is a copy number ratio of CCND1/control centromere or a copy number of CCND1, wherein a copy number ratio of CCND1/control centromere greater than about 1.55 per cell or a copy number of CCND1 greater than about 2.81 per cell indicates that metastasis will likely occur,
  • (b) is a copy number of MYC, wherein a copy number of MYC greater than about 2.48 per cell indicates that metastasis will likely occur,
  • (c) is a percentage of cells having a gain of CCND1/control centromere or a percentage of cells having a gain of CCND1, wherein a percentage of cells of greater than or equal to about 30% having a gain of CCND1 or a percentage of cells of greater than or equal to about 54% having a gain of CCND1/control centromere indicates that metastasis will likely occur, and
  • (d) is a percentage of cells having a gain of MYC, wherein a percentage of cells of greater than about 20% having a gain of MYC indicates that metastasis will likely occur.
  • a method of prognosing metastasis of malignant melanoma in a patient having a melanoma with a Breslow's depth less than about 1 mm, such as less than 1 mm, is also provided.
  • the method comprises determining in a sample of malignant melanoma obtained from the patient a copy number ratio of CCND1/control centromere and a copy number of MYC.
  • the method comprises determining in a sample of malignant melanoma obtained from the patient a copy number ratio of CCND1/control centromere and a copy number of MYC.
  • the method comprises determining in a sample of malignant melanoma obtained from the patient a copy number ratio of CCND1/control centromere.
  • the method can further comprise determining in a sample of malignant melanoma obtained from the patient a copy number of MYC, wherein a copy number of MYC greater than about 2.60, such as greater than 2.60, also indicates that metastasis will likely occur.
  • the method comprises determining in a sample of malignant melanoma obtained from the patient a copy number ratio of CCND1/control centromere or a copy number of MYC.
  • the method comprises determining in a number of nuclei in a diagnostic sample, which comprises nucleated cells, obtained from the patient a copy number of RREB1, a copy number of MYC, a copy number of CCND1, and a copy number of CDKN2A, wherein an increase in the copy number of RREB1, an increase in the copy number of MYC, an increase in the copy number of CCND1, and a decrease in the copy number of CDKN2A indicates that the sample comprises a malignant melanoma.
  • the diagnostic sample can comprise cells, which comprise nuclei, wherein the copy number of RREB1, the copy number of MYC, the copy number of CCND1, and the copy number of CDKN2A are determined in a number of nuclei.
  • the number of nuclei can be about 30, wherein, when increases in copy numbers of RREB1, MYC, and CCND1 and a homozygous deletion of CDKN2A are detected in greater than or equal to 27% of the nuclei, the sample comprises a malignant melanoma.
  • the number of nuclei can be about 30, wherein, when increases in copy numbers of RREB1, MYC, and CCND1 and a homozygous deletion of CDKN2A are detected in greater than or equal to 8 nuclei, the sample comprises a malignant melanoma.
  • the method comprises determining in a number of nuclei in a sample, which comprises nucleated cells, obtained from the patient a copy number of RREB1, a copy number of MYC or ZNF217, a copy number of CCND1, and a copy number of CDKN2A, wherein an increase in the copy number of RREB1, an increase in the copy number of MYC or ZNF217, an increase in the copy number of CCND1, and a decrease in the copy number of CDKN2A indicates that metastasis will likely occur.
  • the sample can comprise cells, which comprise nuclei, wherein the copy number of RREB1, the copy number of MYC or ZNF217, the copy number of CCND1, and the copy number of CDKN2A are determined in a number of nuclei.
  • the number of nuclei can be about 30, wherein, when increases in copy numbers of RREB1, MYC or ZNF217, and CCND1 and a homozygous deletion of CDKN2A are detected in greater than or equal to 27% of the nuclei, metastasis will likely occur.
  • the number of nuclei can be about 30, wherein, when increases in copy numbers of RREB1, MYC or ZNF217, and CCND1 and a homozygous deletion of CDKN2A are detected in greater than or equal to 8 nuclei, metastasis will likely occur.
  • the method comprises determining in a sample of tumor from the patient a copy number of RREB1, CCND1, and/or CDKN2A, wherein an increase in copy number of RREB1 or an increase in copy number of CCND1 or a homozygous deletion of CDKN2A indicates that aggressive metastasis will likely occur and homozygous deletion of CDKN2A indicates that even more aggressive metastasis will likely occur.
  • the above prognostic methods can comprise determining the copy number ratio of CCND1/control centromere and/or the copy number of MYC by in situ hybridization, in which each probe is detectably labeled and, when two or more probes are hybridized simultaneously or sequentially to the same sample, each probe is detectably labeled with a distinct label.
  • the in situ hybridization can be fluorescent in situ hybridization (FISH), in which each probe is detectably labeled and, when two or more probes are hybridized simultaneously or sequentially to the same sample, each probe is detectably labeled with a distinct fluorophore.
  • FISH fluorescent in situ hybridization
  • the copy number of the CCND1 gene can be determined by using the probe Vysis Locus Specific Identifier (LSI) CCND1.
  • the copy number of a control centromere can be determined by using a probe that hybridizes to the alpha satellite DNA located at the centromere of a chromosome. This probe functions as a control, thereby enabling accounting of differences in efficiency of hybridization between samples as necessary.
  • An example of a probe that hybridizes to the alpha satellite DNA located at the centromere of a chromosome is a Chromosome Enumerator Probe (Cep).
  • a probe that hybridizes to the alpha satellite DNA located at the centromere of chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 can be used.
  • a preferred control centromere probe is one that hybridizes to the alpha satellite DNA of chromosome 11, such as Cep11.
  • the copy number of MYC can be determined by using the probe Vysis LSI MYC.
  • the above diagnostic methods can comprise determining the copy numbers of RREB1, MYC, CCND1, and CDKN2A by in situ hybridization, in which each probe is detectably labeled and, when two or more probes are hybridized simultaneously or sequentially to the same sample, each probe is detectably labeled with a distinct label.
  • the in situ hybridization can be fluorescent in situ hybridization (FISH), in which each probe is detectably labeled and, when two or more probes are hybridized simultaneously or sequentially to the same sample, each probe is detectably labeled with a distinct fluorophore.
  • FISH fluorescent in situ hybridization
  • the copy number of the RREB1 gene can be determined by using the probe Vysis LSI RREB1.
  • the copy number of the MYC gene can be determined by using the probe Vysis LSI MYC.
  • the copy number of the CCND1 gene can be determined by using the probe Vysis LSI CCND1.
  • the copy number of the CDKN2A gene can be determined by using the probe Vysis LSI CDKN2A.
  • the above prognostic methods can comprise determining the copy numbers of RREB1, MYC or ZNF217, CCND1, and CDKN2A by in situ hybridization, in which each probe is detectably labeled and, when two or more probes are hybridized simultaneously or sequentially to the same sample, each probe is detectably labeled with a distinct label.
  • the in situ hybridization can be fluorescent in situ hybridization (FISH), in which each probe is detectably labeled and, when two or more probes are hybridized simultaneously or sequentially to the same sample, each probe is detectably labeled with a distinct fluorophore.
  • FISH fluorescent in situ hybridization
  • the copy number of the RREB1 gene can be determined by using the probe Vysis LSI RREB1.
  • the copy number of the MYC gene can be determined by using the probe Vysis LSI MYC.
  • the copy number of the ZNF217 gene can be determined using the probe Vysis LSI ZNF217.
  • the copy number of the CCND1 gene can be determined by using the probe Vysis LSI CCND1.
  • the copy number of the CDKN2A gene can be determined by using the probe Vysis LSI CDKN2A.
  • the nature/size of the probe will depend, at least in part, on the method used to determine a particular parameter, e.g., copy number, copy number ratio, or percentage gain of a gene of interest.
  • a particular parameter e.g., copy number, copy number ratio, or percentage gain of a gene of interest.
  • the probe can be relatively large.
  • the probe can be smaller, even substantially smaller, than the probe used for in situ hybridization, such as FISH, in which case the probe preferably hybridizes to a sequence within the gene of interest.
  • a probe for detecting a parameter involving CCND1, such as the copy number of CCND1, a copy number ratio involving CCND1, or the percentage gain of CCND1, by in situ hybridization, such as FISH, preferably hybridizes to the 11q13 region of chromosome 11, which comprises the CCND1 gene.
  • the probe also can hybridize to an adjacent region, such as the STS (sequence-tagged site) marker D11S1076, which is located on the centromeric side, the FGF4 gene, which is located on the telomeric side, or both of D11S1076 and FGF4.
  • a probe for detecting a parameter involving CCND1 by another method can be smaller, even substantially smaller, than the probe used for in situ hybridization, such as FISH, in which case the probe preferably hybridizes to a sequence within the CCND1 gene (sequence information is available online from sources such as GenBank (www.ncbi.nlm.nih.gov/genbank) and GeneCards® (www.genecards.org)).
  • CCND1 is used herein to refer to any and all probes that can be used to determine a parameter involving CCND1, whether copy number, copy number ratio, percentage gain, and the like, irrespective of the particular method used to determine the parameter.
  • a probe for detecting a parameter involving MYC such as the copy number of MYC, a copy number ratio involving MYC, or the percentage gain of MYC, by in situ hybridization, such as FISH, preferably hybridizes to the 8q24 region of chromosome 8, which comprises the MYC gene.
  • the probe also can hybridize to an adjacent region located on the centromeric side of 8q24, an adjacent region located on the telomeric side of 8q24, or both.
  • a preferred probe covers approximately 820 kb, such as 821 kb, of 8q24 and is centered on the MYC gene.
  • a probe for detecting a parameter involving MYC by another method can be smaller, even substantially smaller, than the probe used for in situ hybridization, such as FISH, in which case the probe preferably hybridizes to a sequence within the MYC gene (sequence information is available online from sources such as GenBank (www.ncbi.nlm.nih.gov/genbank) and GeneCards® (www.genecards.org)).
  • MYC is used herein to refer to any and all probes that can be used to determine a parameter involving MYC, whether copy number, copy number ratio, percentage gain, and the like, irrespective of the particular method used to determine the parameter.
  • a probe for detecting a parameter involving CDKN2A such as the copy number of CDKN2A, a copy number ratio involving CDKN2A, or the percentage gain of CDKN2A, by in situ hybridization, such as FISH, preferably hybridizes to the 9p21 region of chromosome 9, which comprises the CDKN2A gene.
  • the probe also can hybridize to an adjacent region, such as the STS marker D9S1749, which is located on the centromeric side of 9p21, and the STS marker D9S1752, which is located on the telomeric side, or both of D9S1749 and D9S1752.
  • a preferred probe covers approximately 190 kb of 9p21.
  • a probe for detecting a parameter involving CDKN2A by another method can be smaller, even substantially smaller, than the probe used for in situ hybridization, such as FISH, in which case the probe preferably hybridizes to a sequence within the CDKN2A gene (sequence information is available online from sources such as GenBank (www.ncbi.nlm.nih.gov/genbank) and GeneCards® (www.genecards.org)).
  • CDKN2A is used herein to refer to any and all probes that can be used to determine a parameter involving CDKN2A, whether copy number, copy number ratio, percentage gain, and the like, irrespective of the particular method used to determine the parameter.
  • a probe for detecting a parameter involving RREB1, such as the copy number of RREB1, a copy number ratio involving RREB1, or the percentage gain of RREB1, by in situ hybridization, such as FISH preferably hybridizes to the 6q25 region of chromosome 6, which comprises the RREB1 gene.
  • the probe also can hybridize to an adjacent region, such as the STS marker SHGC-140278, which is located on the centromeric side, and the STS marker RH61070, which is located on the telomeric side, or both of SHGC-140278 and RH61070.
  • a probe for detecting a parameter involving RREB1 by another method can be smaller, even substantially smaller, than the probe used for in situ hybridization, such as FISH, in which case the probe preferably hybridizes to a sequence within the RREB1 gene (sequence information is available online from sources such as GenBank (www.ncbi.nlm.nih.gov/genbank) and GeneCards® (www.genecards.org)).
  • RREB1 is used herein to refer to any and all probes that can be used to determine a parameter involving RREB1, whether copy number, copy number ratio, percentage gain, and the like, irrespective of the particular method used to determine the parameter.
  • a probe for detecting a parameter involving ZNF217 such as the copy number of ZNF217, a copy number ratio involving ZNF217, or the percentage gain of ZNF217, by in situ hybridization, such as FISH, preferably hybridizes to the 20q13 region of chromosome 20, which comprises the ZNF217 gene.
  • the probe also can hybridize to an adjacent region, such as the STS marker RI-29727, which is located on the centromeric side, and the STS marker SHGC-83153, which is located on the telomeric side, or both of RI-29727 and SHGC-83153.
  • a probe for detecting a parameter involving ZNF217 by another method can be smaller, even substantially smaller, than the probe used for in situ hybridization, such as FISH, in which case the probe preferably hybridizes to a sequence within the ZNF217 gene (sequence information is available online from sources such as GenBank (www.ncbi.nlm.nih.gov/genbank) and GeneCards® (www.genecards.org)).
  • ZNF217 is used herein to refer to any and all probes that can be used to determine a parameter involving ZNF217, whether copy number, copy number ratio, percentage gain, and the like, irrespective of the particular method used to determine the parameter.
  • each probe is detectably (and distinctly, if more than one probe is used simultaneously or sequentially on the same sample) labeled, such as by FISH, in which each probe is detectably (and distinctly, if more than one probe is used simultaneously or sequentially on the same sample) labeled with a fluorophore
  • the methods are typically carried out on a sample of a melanoma, which is fresh (fresh cells can be cultured for 1-3 days and a blocker, such as Colcemid, can be added to the culture to block the cells in metaphase, during which chromosomes are highly condensed and can be visualized), frozen, or fixed (e.g., fixed in formalin and embedded in paraffin), treated (e.g., with RNase and pepsin) to increase accessibility of target nucleic acid (e.g., DNA) and reduce non-specific binding, and then subjected to hybridization with one or more probes, washing to remove any unbound probes, and
  • target nucleic acid e.g.,
  • a cell suspension can be applied as a single layer onto a slide, and the cell density can be measured by a light or phase contrast microscope.
  • a section (approximately 5 ⁇ m in thickness) of a formalin-fixed, paraffin-embedded sample of melanoma can be mounted onto a slide, such as a SuperFrost Plus positively charged slide (available from ThermoShandon, Pittsburgh, Pa.), baked at 56° C. overnight, de-paraffinized, submerged in 1 ⁇ saline sodium citrate, pH 6.3, at 80° C. for 35 minutes, and washed in water for three minutes. After protease digestion (4 mg pepsin/mL and 0.2 N HCl) at 37° C.
  • the section can be rinsed in water for three minutes, passed through graded ethanol, and dried.
  • hybridization with one or more probes as described above is carried out at 37° C. for 16-18 hours in an automated co-denaturation oven (HYBrite or ThermoBrite Denaturation/Hybridization System, Abbot Molecular, Inc., Des Plaines, Ill.) according to the manufacturer's instructions (such methods typically involve denaturation of probes and target nucleic acids).
  • the section is preferably placed in washing buffer (2 ⁇ saline sodium citrate/0.3% NP40; available from Abbott Molecular, Inc.) at room temperature for 2-10 minutes to remove the coverslip and then immersed in 73° C.
  • DAPI 4′6′-diamidino-2-phenylindole dihydrochloride hydrate I antifade solution
  • the slide is analyzed with an epi-fluorescence microscope equipped with single band-pass filters (Abbott Molecular, Inc.).
  • cell samples Prior to detection, cell samples may be optionally pre-selected based on apparent cytologic abnormalities. Pre-selection identifies suspicious cells, thereby allowing the screening to be focused on those cells. Pre-selection allows for faster screening and increases the likelihood that a positive result will not be missed.
  • pre-selection cells from a biological sample can be placed on a microscope slide and visually scanned for cytologic abnormalities commonly associated with dysplastic and neoplastic cells.
  • Such abnormalities include abnormalities in nuclear size, nuclear shape, and nuclear staining, as assessed by counterstaining nuclei with nucleic acid stains or dyes such as propidium iodide or 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) usually following hybridization of probes to their target DNAs.
  • nucleic acid stains or dyes such as propidium iodide or 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) usually following hybridization of probes to their target DNAs.
  • DAPI 4,6-diamidino-2-phenylindole dihydrochloride
  • neoplastic cells harbor nuclei that are enlarged, irregular in shape, and/or show a mottled staining pattern.
  • Propidium iodide typically used at a concentration of about 0.4 ⁇ g/ml to about 5 ⁇ g/ml, is a red-fluorescing DNA-specific
  • DAPI typically used at a concentration of about 125 ng/ml to about 1,000 ng/ml, is a blue fluorescing DNA-specific stain that can be observed at an emission peak wavelength of 452 nm with a DAPI filter at low magnification.
  • pre-selected cells on the order of at least 20, and more preferably at least 30-40, in number are chosen for assessing chromosomal losses and/or gains.
  • a tumor-bearing area can be localized using the DAPI filter at low magnification and thoroughly inspected for the presence of nuclei harboring abnormal copy numbers of any probe. In a normal cell, two copies of a given probe will be detected. In an abnormal cell, more or less copies of a given probe will be detected.
  • Areas with the most significant copy number changes are preferably selected for enumeration. Wherever possible, three abnormal areas are selected and, within each abnormal area, 10 random nuclei are analyzed under high power (64 ⁇ or 100 ⁇ objective). Preferably, nuclei are non-overlapping and harbor sufficiently bright signals.
  • cells for detection may be chosen independent of cytologic or histologic features. For example, all non-overlapping cells in a given area or areas on a microscope slide may be assessed for chromosomal losses and/or gains. As a further example, cells on the slide, e.g., cells that show altered morphology, on the order of at least about 50, and more preferably at least about 100, in number that appear in consecutive order on a microscope slide may be chosen for assessing chromosomal losses and/or gains.
  • the copies of CCND1 are counted, and the copy number of CCND1 is determined or the ratio of CCND1/control centromere is determined.
  • the copies of MYC are counted.
  • the (i) copy number of CCND1 or the copy number ratio of CCND1/control centromere and/or (ii) the copy number of MYC is/are then compared to the appropriate predetermined cutoff(s) set forth herein.
  • a copy number of CCND1 or a copy number ratio of CCND1/control centromere greater than the predetermined cutoff indicates that metastasis will likely occur.
  • a copy number of MYC greater than the predetermined cutoff indicates that metastasis will likely occur.
  • the copies of CCND1 are counted and the percentage of cells having a gain of CCND1 or the percentage of cells having a gain of CCND1/control centromere is/are determined. Alternatively or additionally, the percentage of cells having a gain of MYC is determined. The percentage of cells having a gain of CCND1 or the percentage of cells having a gain of CCND1/control centromere is then compared to the appropriate predetermined cutoff(s) as set forth herein.
  • a percentage of cells having a gain of CCND1/control centromere or a percentage of cells having a gain of CCND1 greater than the predetermined cutoff indicates that metastasis will likely occur.
  • a percentage of cells having a gain of MYC greater than the predetermined cutoff indicates that metastasis will likely occur.
  • the copies of RREB1, MYC, CCND1, and CDKN2A are counted.
  • An increase in the copy number of RREB1, an increase in the copy number of MYC, an increase in the copy number of CCND1, and a decrease in the copy number of CDKN2A indicates that the sample comprises a malignant melanoma.
  • the diagnostic sample can comprise cells, which comprise nuclei, wherein the copy number of RREB1, the copy number of MYC, the copy number of CCND1, and the copy number of CDKN2A are determined in a number of nuclei.
  • the number of nuclei can be about 30, wherein, when increases in copy numbers of RREB1, MYC, and CCND1 and a homozygous deletion of CDKN2A are detected in greater than or equal to 27% of the nuclei, the sample comprises a malignant melanoma.
  • the number of nuclei can be about 30, wherein, when increases in copy numbers of RREB1, MYC, and CCND1 and a homozygous deletion of CDKN2A are detected in greater than or equal to 8 nuclei, the sample comprises a malignant melanoma.
  • the copies of RREB1, MYC or ZNF217, CCND1, and CDKN2A are counted.
  • An increase in the copy number of RREB1, an increase in the copy number of MYC or ZNF217, an increase in the copy number of CCND1, and a decrease in the copy number of CDKN2A indicates that the sample comprises a malignant melanoma.
  • the diagnostic sample can comprise cells, which comprise nuclei, wherein the copy number of RREB1, the copy number of MYC or ZNF217, the copy number of CCND1, and the copy number of CDKN2A are determined in a number of nuclei.
  • the number of nuclei can be about 30, wherein, when increases in copy numbers of RREB1, MYC or ZNF217, and CCND1 and a homozygous deletion of CDKN2A are detected in greater than or equal to 27% of the nuclei, metastasis is likely to occur.
  • the number of nuclei can be about 30, wherein, when increases in copy numbers of RREB1, MYC or ZNF217, and CCND1 and a homozygous deletion of CDKN2A are detected in greater than or equal to 8 nuclei, metastasis is likely to occur.
  • such methods comprise contacting a sample of malignant melanoma from a patient, e.g., a nucleic acid sample, with at least one probe that binds selectively to a target nucleic acid sequence (i.e., for prognostic methods, such as those involving characterization of Breslow's depth, CCND1, alone or in further combination with a control centromere, and, alternatively or additionally, and simultaneously or sequentially, in either order, MYC; for diagnostic methods, RREB1, MYC, CCND1, and CDKN2A; and, for other prognostic methods, such as those not involving characterization of Breslow's depth, RREB1, MYC or ZNF217, CCND1, and CDKN2A) under conditions that allow (or promote) the probe to bind selectively with its target nucleic acid sequence and form a stable hybridization complex.
  • Such methods further comprise detecting the formation of the hybridization complex and counting the number of hybridization complexes.
  • the prognostic methods further comprise determining the copy number of CCND1 or the copy number ratio of CCND1/control centromere and comparing the copy number or the copy number ratio to the appropriate predetermined cutoff, wherein a copy number of a copy number ratio greater than the appropriate predetermined cutoff indicates that metastasis will likely occur.
  • the prognostic methods further comprise determining the copy number of MYC and comparing the copy number to the predetermined cutoff, wherein a copy number greater than the predetermined cutoff indicates that metastasis will likely occur.
  • the prognostic methods further comprise determining the percentage of cells having a gain of CCND1 or CCND1/control centromere and comparing the percentage of cells having a gain to the appropriate predetermined cutoff, wherein a percentage of cells having a gain greater than the appropriate predetermined cutoff indicates that metastasis will likely occur.
  • the prognostic methods further comprise determining the percentage of cells having a gain of MYC and comparing the percentage of cells having a gain to the predetermined cutoff, wherein a percentage of cells having a gain greater than the appropriate predetermined cutoff indicates that metastasis will likely occur.
  • the diagnostic methods further comprise determining the copy numbers of RREB1, MYC, CCND1, and CDKN2A, wherein an increase in the copy numbers of RREB1, MYC, CCND1, and a decrease in the copy number of CDKN2A indicates that the sample comprises malignant melanoma.
  • the prognostic methods which do not involve characterization of Breslow's depth, further comprise determining the copy numbers of RREB1, MYC or ZNF217, CCND1, and CDKN2A, wherein an increase in the copy numbers of RREB1, MYC or ZNF217, CCND1, and a decrease in the copy number of CDKN2A indicates that metastasis will likely occur.
  • a copy number of a gene or a copy number ratio e.g., of two genes, of two chromosomes, or of a gene and a chromosome
  • Such methods may necessitate the use of a sample of malignant melanoma that is other than a section of a malignant melanoma that is fixed in formalin and embedded in paraffin, e.g., a fresh or frozen section of a malignant melanoma, homogenized cells from a malignant melanoma, lysed cells from a malignant melanoma, or isolated or purified nucleic acids (e.g., a “nucleic acid sample” such as DNA) from a malignant melanoma (“sample of malignant melanoma” as used herein is intended to encompass all forms of a sample of malignant melanoma that enable the determination of the copy number ratio).
  • a sample of malignant melanoma that is other than a section of a malignant melanoma that is fixed in formalin and embedded in paraffin, e.g., a fresh or frozen section of a malignant melanoma, homo
  • a touch preparation (a monolayer of cells obtained by pressing fresh or frozen tissue against a slide) prepared from an uncultured primary tumor can be used (see, e.g., Kallioniemi et al., Cytogenet. Cell Genet. 60: 190-193 (1992)).
  • Touch preparations contain intact nuclei and do not suffer from the truncation artifact of sectioning.
  • the monolayer of cells in a touch preparation may be fixed, e.g., in alcohol, such as ethanol, or alcoholic solution, such as 3:1 methanol:acetic acid.
  • Nuclei also can be extracted from thick sections of paraffin-embedded specimens to reduce truncation artifacts and eliminate extraneous embedded material.
  • biological samples, once obtained, are harvested and processed prior to hybridization using standard methods known in the art. Such processing typically includes protease treatment and additional fixation in an aldehyde solution, such as formaldehyde.
  • Examples of methods that can be used herein include, but are not limited to, quantitative polymerase chain reaction (Q-PCR), real-time Q-PCR (Applied Biosystems, Foster City, Calif.), densitometric scanning of PCR products, digital PCR, optionally with pre-amplification of the gene(s) and/or chromosomal region(s) for which copy number(s) is/are to be determined (see, e.g., Vogelstein et al., PNAS USA 96: 9236-9241 (1999); U.S. Pat. App. Pub. No. 2005/0252773; and U.S. Pat. App. Pub. No.
  • CGH comparative genomic hybridization
  • MSH multiplex amplifiable probe hybridization
  • MLPA multiplex ligation-dependent probe amplification
  • Denaturation of nucleic acid targets for analysis by in situ hybridization and similar methods typically is done in such a manner as to preserve cell morphology.
  • chromosomal DNA can be denatured by high pH, heat (e.g., temperatures from about 70-95° C.), organic solvents (e.g., formamide), and combinations thereof.
  • Probes can be denatured by heat in a matter of minutes.
  • Conditions for specifically hybridizing the probes to their nucleic acid targets generally include the combinations of conditions that are employable in a given hybridization procedure to produce specific hybrids, the conditions of which may easily be determined by one of ordinary skill in the art. Such conditions typically involve controlled temperature, liquid phase, and contact between a probe and a target. Hybridization conditions vary depending upon many factors including probe concentration, target length, target and probe G-C content, solvent composition, temperature, and duration of incubation. At least one denaturation step may precede contact of the probes with the targets. Alternatively, the probe and the target may be subjected to denaturing conditions together while in contact with one another, or with subsequent contact of the probe with the biological sample.
  • Hybridization may be achieved with subsequent incubation of the probe/sample in, for example, a liquid phase of about a 50:50 volume ratio mixture of 2-4 ⁇ SSC and formamide, at a temperature in the range of about 25 to about 55° C. for a time that is illustratively in the range of about 0.5 to about 96 hours, or more preferably at a temperature of about 32 to about 40° C. for a time in the range of about 2 to about 16 hours.
  • a blocking agent such as unlabeled blocking nucleic acid, as described in U.S. Pat. No.
  • non-specific binding of chromosomal probes to sample DNA may be removed by a series of washes. Temperature and salt concentrations are suitably chosen for a desired stringency. The level of stringency required depends on the complexity of a specific probe sequence in relation to the genomic sequence, and may be determined by systematically hybridizing probes to samples of known genetic composition. In general, high stringency washes may be carried out at a temperature in the range of about 65 to about 80° C. with about 0.2 ⁇ to about 2 ⁇ SSC and about 0.1% to about 1% of a non-ionic detergent such as Nonidet P-40 (NP40). If lower stringency washes are required, the washes may be carried out at a lower temperature with an increased concentration of salt.
  • a non-ionic detergent such as Nonidet P-40 (NP40).
  • the detection method can involve fluorescence microscopy, flow cytometry, or other means for determining probe hybridization. Any suitable microscopic imaging method may be used in conjunction with the methods described herein for observing multiple fluorophores. In the case where fluorescence microscopy is employed, hybridized samples may be viewed under light suitable for excitation of each fluorophore and with the use of an appropriate filter or filters. Automated digital imaging systems such as the MetaSystems, BioView or Applied Imaging systems may alternatively be used.
  • a digital image analysis system can be used to facilitate the display of results and to improve the sensitivity of detecting small differences in fluorescence intensity.
  • An exemplary system is QUIPS (an acronym for quantitative image processing system), which is an automated image analysis system based on a standard fluorescence microscope equipped with an automated stage, focus control and filter wheel (Ludl Electronic Products, Ltd., Hawthorne, N.Y.). The filter wheel is mounted in the fluorescence excitation path of the microscope for selection of the excitation wavelength. Special filters (Chroma Technology, Brattleboro, Vt.) in the dichroic block allow excitation of the multiple dyes without image registration shift.
  • the microscope has two camera ports, one of which has an intensified CCD camera (Quantex Corp., Sunnyvale, Calif.) for sensitive high-speed video image display which is used for finding interesting areas on a slide as well as for focusing.
  • the other camera port has a cooled CCD camera (model 200 by Photometrics Ltd., Arlington, Ariz.), which is used for the actual image acquisition at high resolution and sensitivity.
  • the cooled CCD camera is interfaced to a SUN 4/330 workstation (SUN Microsystems, Inc., Mountain View, Calif.) through a VME bus.
  • the entire acquisition of multicolor images is controlled using an image processing software package SCIL-Image (Delft Centre for Image Processing, Delft, Netherlands).
  • array CGH In array CGH (aCGH) the probes are immobilized at distinct locations on a substrate and are not labeled (see, e.g., Int'l Pat. App. Pub. No. WO 96/17958). Instead, sample nucleic acids, which comprise target nucleic acid(s), are labeled. Either the sample nucleic acids are labeled prior to hybridization or the hybridization complexes are detectably labeled. In dual- or multi-color aCGH the probe array is simultaneously or sequentially hybridized to two or more collections of differently labeled target nucleic acids.
  • a set of one or more probes that enables prognosis of metastasis of malignant melanoma.
  • the set comprises, or consists of, (a) a probe for CCND1, along or in further combination with a probe for a control centromere, and/or (b) a probe for MYC.
  • the probe for CCND1 can be Vysis LSI CCND1.
  • the probe for a control centromere hybridizes to the alpha satellite DNA located at the centromere of a chromosome.
  • An example of a probe for a control centromere is a Chromosome Enumerator Probe (Cep).
  • This probe functions as a control, thereby enabling accounting of differences in efficiency of hybridization between samples as necessary.
  • a probe that hybridizes to the alpha satellite DNA located at the centromere of chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 can be used.
  • a preferred control centromere probe is one that hybridizes to the alpha satellite DNA of chromosome 11, such as Cep11.
  • the probe for MYC can be Vysis LSI MYC.
  • the set comprises, or consists of, a probe for RREB1, a probe for MYC, a probe for CCND1, and a probe for CDKN2A.
  • the probe for RREB1 can be Vysis LSI RREB1.
  • the probe for MYC can be Vysis LSI MYC.
  • the probe for CCND1 can be Vysis LSI CCND1.
  • the probe for CDKN2A can be Vysis LSI CDKN2A.
  • the set comprises, or consists of, a probe for RREB1, a probe for MYC or ZNF217, a probe for CCND1, and a probe for CDKN2A.
  • the probe for RREB1 can be Vysis LSI RREB1.
  • the probe for MYC can be Vysis LSI MYC.
  • the probe for ZNF217 can be Vysis LSI ZNF217.
  • the probe for CCND1 can be Vysis LSI CCND1.
  • the probe for CDKN2A can be Vysis LSI CDKN2A.
  • the set comprises, or consists of, a probe for RREB1, a probe for CCND1, and a probe for CDKN2A.
  • the probe for RREB1 can be Vysis LSI RREB1.
  • the probe for CCND1 can be Vysis LSI CCND1.
  • the probe for CDKN2A can be Vysis LSI CDKN2A.
  • Chromosome enumerator probes and locus-specific probes that target a chromosome region or sub-region can be obtained commercially or readily prepared by those in the art.
  • Such probes can be commercially obtained from Abbott Molecular, Inc. (Des Plaines, Ill.), Molecular Probes, Inc. (Eugene, Oreg.), or Cytocell (Oxfordshire, UK).
  • Chromosomal probes can be prepared, for example, from protein nucleic acids (PNA), cloned human DNA such as plasmids, bacterial artificial chromosomes (BACs), and P1 artificial chromosomes (PACs) that contain inserts of human DNA sequences.
  • PNA protein nucleic acids
  • BACs bacterial artificial chromosomes
  • PACs P1 artificial chromosomes
  • a region of interest can be obtained via PCR amplification or cloning.
  • chromosomal probes can be prepared synthetically in accordance with methods known in the art.
  • a locus-specific probe can be designed to hybridize to an oncogene or tumor suppressor gene, the genetic aberration of which is correlated with metastasis, e.g., CCND1 or MYC.
  • probes are detectably labeled, and, when two or more probes are used simultaneously or sequentially on the same sample, each probe is distinctly labeled.
  • the probes are detectably labeled with fluorophores, and, when two or more probes are used simultaneously or sequentially on the same sample, each probe is distinctly labeled.
  • fluorophores examples include, but are not limited to, 7-amino-4-methylcoumarin-3-acetic acid (AMCA), 5-carboxy-X-rhodamine, 6-carboxy-X-rhodamine, lissamine rhodamine B, 5-carboxyfluorescein, 6-carboxyfluorescein, fluorescein-5-isothiocyanate (FITC), 7-diethylaminocoumarin-3-carboxylic acid, tetramethylrhodamine-5-isothiocyanate, tetramethylrhodamine-6-isothiocyanate, 5-carboxyltetramethylrhodamine, 6-carboxytetramethylrhodamine, 7-hydroxycoumarin-3-carboxylic acid, N-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacenepropionic acid, eosin-5-is
  • the particular label used is not critical; desirably, however, the particular label does not interfere with in situ hybridization of the probe.
  • the label desirably is detectable in as low copy number as possible to maximize the sensitivity of the assay and be detectable above any background signal.
  • the label provides a highly localized signal, thereby providing a high degree of spatial resolution.
  • Fluorophores can be covalently attached to a particular nucleotide, for example, and the labeled nucleotide incorporated into the probe using standard techniques such as nick translation, random priming (Rigby et al., J. Mol. Biol. 113:237 (1997)), PCR labeling, direct labeling by chemical modification of particular residues, such as cytosine residues (U.S. Pat. No. 5,491,224), and the like.
  • the fluorophore can be covalently attached via a linker to the deoxycytidine nucleotides of the probe that have been transaminated.
  • Luminescent agents include, for example, radioluminescent, chemiluminescent, bioluminescent, and phosphorescent label containing moieties.
  • detection moieties that are visualized by indirect means can be used.
  • probes can be labeled with biotin or digoxygenin using routine methods known in the art, and then further processed for detection. Visualization of a biotin-containing probe can be achieved via subsequent binding of avidin conjugated to a detectable marker.
  • the detectable marker may be a fluorophore, in which case visualization and discrimination of probes can be achieved as described below.
  • Chromosomal probes hybridized to target regions may alternatively be visualized by enzymatic reactions of label moieties with suitable substrates for the production of insoluble color products. Each probe may be discriminated from other probes within the set by choice of a distinct label moiety.
  • a biotin-containing probe within a set may be detected via subsequent incubation with avidin conjugated to alkaline phosphatase (AP) or horseradish peroxidase (HRP) and a suitable substrate.
  • AP alkaline phosphatase
  • HRP horseradish peroxidase
  • NBT 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium
  • a kit comprising, or consisting of, (a) a set of one or more probes that enables prognosis of metastasis of malignant melanoma in a patient and (b) instructions for prognosing metastasis of malignant melanoma in the patient.
  • the set of one or more probes comprises (i′) a probe for CCND1, alone or in further combination with a probe for a control centromere, and/or (ii′) a probe for MYC.
  • the instructions comprise (i′) determining in a sample of malignant melanoma obtained from the patient (i) (a) and/or (b), (ii) (c) and/or (d), (iii) (a) and/or (d), or (iv) (b) and/or (c), wherein:
  • (a) is a copy number ratio of CCND1/control centromere or a copy number of CCND1, wherein a copy number ratio of CCND1/control centromere greater than about 1.55 per cell or a copy number of CCND1 greater than about 2.81 per cell indicates that metastasis will likely occur,
  • (b) is a copy number of MYC, wherein a copy number of MYC greater than about 2.48 per cell indicates that metastasis will likely occur,
  • (c) is a percentage of cells having a gain of CCND1/control centromere or a percentage of cells having a gain of CCND1, wherein a percentage of cells of greater than or equal to about 30% having a gain of CCND1 or a percentage of cells of greater than or equal to about 54% having a gain of CCND1/control centromere indicates that metastasis will likely occur, and
  • (d) is a percentage of cells having a gain of MYC, wherein a percentage of cells of greater than about 20% having a gain of MYC indicates that metastasis will likely occur.
  • the probe for CCND1 can be Vysis LSI CCND1.
  • a probe for a centromere control hybridizes to the alpha satellite DNA located at the centromere of a chromosome.
  • An example of a centromere control probe is a Chromosome Enumerator Probe (Cep). This probe functions as a control, thereby enabling accounting of differences in efficiency of hybridization between samples as necessary.
  • a probe that hybridizes to the alpha satellite DNA located at the centromere of chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 can be used.
  • a preferred control centromere probe is one that hybridizes to the alpha satellite DNA of chromosome 11, such as Cep11.
  • the probe for MYC can be Vysis LSI MYC.
  • the kit may further comprise, or consist of, blocking agents or probes, various labels or labeling agents to facilitate detection of the probes, reagents for hybridization (e.g., buffers), a metaphase spread, and the like.
  • a kit comprising, or consisting of, (a) a set of one or more probes that enables diagnosis and prognosis of malignant melanoma in a patient and (b) instructions for diagnosing malignant melanoma and/or instructions for prognosing metastasis of malignant melanoma in a patient is also provided.
  • the set of probes comprises a probe for RREB1, a probe for MYC, a probe for CCND1, and a probe for CDKN2A, and (b) either instructions for diagnosing malignant melanoma in the patient, wherein the instructions comprise determining in a diagnostic sample obtained from the patient a copy number of RREB1, a copy number of MYC, a copy number of CCND1, and a copy number of CDKN2A, wherein an increase in the copy number of RREB1, an increase in the copy number of MYC, an increase in the copy number of CCND1, and a decrease in the copy number of CDKN2A indicates that the patient has malignant melanoma and/or instructions for prognosing metastasis of malignant melanoma in the patient, wherein the instructions comprise determining in a sample of malignant melanoma obtained from the patient a copy number of RREB1, a copy number of MYC, a copy number of CCND1, and
  • the probe for RREB1 can be Vysis LSI RREB1.
  • the probe for MYC can be Vysis LSI MYC.
  • the probe for CCND1 can be Vysis LSI CCND1.
  • the probe for CDKN2A can be Vysis LSI CDKN2A.
  • the kit may further comprise, or consist of, blocking agents or probes, various labels or labeling agents to facilitate detection of the probes, reagents for hybridization (e.g., buffers), a metaphase spread, and the like.
  • kits comprising, or consisting of, (a) a set of probes that enables prognosis of metastasis of malignant melanoma in a patient, wherein the set of probes comprises a probe for RREB1, a probe for MYC or ZNF217, a probe for CCND1, and a probe for CDKN2A, and (b) instructions for prognosing malignant melanoma in the patient, wherein the instructions comprise determining in a sample obtained from the patient a copy number of RREB1, a copy number of MYC or ZNF217, a copy number of CCND1, and a copy number of CDKN2A, wherein an increase in the copy number of RREB1, an increase in the copy number of MYC or ZNF217, an increase in the copy number of CCND1, and a decrease in the copy number of CDKN2A indicates that metastasis will likely occur.
  • the probe for RREB1 can be Vysis LSI RREB1.
  • the probe for MYC can be Vysis LSI MYC.
  • the probe for CCND1 can be Vysis LSI CCND1.
  • the probe for CDKN2A can be Vysis LSI CDKN2A.
  • the probe for ZNF217 can be Vysis LSI ZNF217.
  • the kit may further comprise, or consist of, blocking agents or probes, various labels or labeling agents to facilitate detection of the probes, reagents for hybridization (e.g., buffers), a metaphase spread, and the like.
  • kits comprising, or consisting of, (a) a set of probes that enables prognosis of metastasis of atypical Spitz tumor in a patient, wherein the set of probes comprises a probe for RREB1, a probe for CCND1, and a probe for CDKN2A, and (b) instructions for prognosing metastasis of atypical Spitz tumor in the patient, wherein the instructions comprise determining in a sample of tumor from the patient a copy number of RREB1, CCND1, and/or CDKN2A, wherein an increase in copy number of RREB1 or an increase in copy number of CCND1 or a homozygous deletion of CDKN2A indicates that aggressive metastasis will likely occur and homozygous deletion of CDKN2A indicates that even more aggressive metastasis will likely occur.
  • the probe for RREB1 can be Vysis LSI RREB1.
  • the probe for CCND1 can be Vysis LSI CCND1.
  • the probe for CDKN2A can be Vysis LSI CDKN2A.
  • the kit may further comprise, or consist of, blocking agents or probes, various labels or labeling agents to facilitate detection of the probes, reagents for hybridization (e.g., buffers), a metaphase spread, and the like.
  • This example describes the evaluation of CCND1, alone or in further combination with a control centromere, and MYC in the prognosis of metastasis in patients with malignant melanoma.
  • the number of patients with Breslow's depths ⁇ 1 mm was 12, 1 mm ⁇ Breslow's depths ⁇ 4 mm was 24, and Breslow's depths>4 mm was six. Only cases in which the slides, tissue block and clinical course were all available were included in the study. The histopathology of all cases was verified by a dermatopathologist. Along with the clinical course, Breslow's depth, age, sex, site, presence/absence of ulceration, mitotic count, and Clark's level were recorded.
  • the 31 melanomas and the 35 nevus controls were then evaluated with probes for eight additional loci, namely 9p21 (CDKN2A), Cen 9 (centromere 9), 8q24 (MYC), 7q34 (BRAF), Cen 17 (centromere 17), Cen 10 (centromere 10), 20q13 (ZNF217), and 1q25 (Cox2).
  • 8 loci, along with 6p25, 6q23, Cen 6, and 11q13, were among the top 12 loci originally identified by combinatorial analysis of CGH data for the most frequently aberrant loci in melanoma (Gerami et al. (2009), supra).
  • the probes were arranged in two panels.
  • the first panel included 9p21, Cen 9, 1q25, and Cen 17.
  • the second panel included 8q24, 7q34, Cen 10, and 20q13.
  • the hybridizations were all performed on formalin-fixed, paraffin-embedded sections as previously described (Gerami et al. (2009), supra).
  • a reviewer blinded to the case diagnosis enumerated the cases using the protocol described below.
  • a discriminatory analysis looking at the most frequently aberrant loci in the melanoma group relative to the nevus control group was performed to identify the most complementary and additive additional targets.
  • the hybridization procedure was performed as previously described (Gerami et al. (2009), supra).
  • the slides were analyzed with an epi-fluorescence microscope equipped with single band-pass filters (Abbott Molecular, Inc., Des Plaines, Ill.).
  • the analyses were performed by a trained technician and a dermatopathologist. All analyses were performed blinded of the specimens' diagnoses. Tumor-bearing areas were localized using the DAPI filter at low magnification.
  • the tumor area was then thoroughly inspected for the presence of nuclei harboring abnormal copy numbers of any probe. Areas with the most significant copy number changes were selected for enumeration. Wherever possible, three abnormal areas were selected, and, within each area, ten random nuclei were analyzed under high power (60 ⁇ objective). Nuclei had to be nonoverlapping and harbor sufficiently bright signals. Nuclei that showed no signals for more than one probe were not analyzed. Thirty cells were enumerated in each specimen.
  • the following parameters were calculated for each probe for each specimen: the average signal number per nucleus, the percentage of nuclei with signal counts greater than (percent gain), less than (percent loss), or different (percent aberrant) from two signals.
  • a broad number of parameters were separately calculated for the metastasizing and non-metastasizing cases and compared by the Student's t-test.
  • Varying thresholds independently for each parameter generated a field of points on the graph, and the points with the highest sensitivity value at each specificity value were used to define the curve.
  • Optimal criteria for distinguishing metastatic versus non-metastatic cases were identified, and Kaplan Meier curves for cases meeting the criteria versus those cases not meeting the criteria, as well as the Kaplan Meier curve for the entire group, were then plotted.
  • ROC curves were also calculated according to Breslow's depth as well as depth combined with other statistically significant FISH parameters.
  • Kaplan Meier curves for the optimally discriminating criteria combining Breslow's depth and FISH were then plotted. This analysis was repeated within Breslow subgroups including cases with Breslow's depth ⁇ 1 mm, 1 mm ⁇ Breslow's depth ⁇ 4 mm, and Breslow's depth>4 mm.
  • P-values for differences in Kaplan Meier curves were calculated using the Log-rank test.
  • the loci 9p21, 8q24, and 20q13 were identified as the most frequently altered loci in the data set for cohort 1 and, therefore, the most complementary targets.
  • An average CCND1/chromosome 6 of greater than about 1.55 and an average MYC copy number of greater than about 2.48 were identified as the two parameters most highly associated with metastasis in the data set for cohort 2 (see FIG. 1 a ).
  • the CCND1/chromosome 6 criterion was 95% specific and 38% sensitive for metastasis.
  • the positive predictive value (PPV) was 91%, and the negative predictive value (NPV) was 54%.
  • a Kaplan Meier curve was plotted for cases meeting this criterion versus those that did not and for the entire group (see FIG. 1 b ).
  • the difference between the metastasizing group and the non-metastasizing group resulted in a p-value of 6.48 ⁇ 10 ⁇ 6 using a Log-rank test.
  • the MYC criterion was 90% specific and 32% sensitive for metastasis, with a PPV of 80% and an NPV of 53%.
  • a Kaplan Meier curve also demonstrated the difference between those cases meeting the MYC criterion, those that did not, and the entire group (see FIG. 1 c ).
  • the difference between the metastasizing group and the non-metastasizing group resulted in a p-value of 5.61 ⁇ 10 ⁇ 3 by the Log rank test, which also reached significance. Hence, patients had a high likelihood of being in the metastatic group if either of the two criteria were met.
  • a combined criterion, which allowed for meeting a specific CCND1/chromosome 6 value or MYC average copy number value was also highly associated with metastasis.
  • the p-value for the difference in the Kaplan Meier curve for those cases with a CCND1/chromosome 6 value greater than 1.59 or a MYC average copy number greater than 2.48 versus those cases meeting neither of these criteria was 8.9 ⁇ 10 ⁇ 7 (see FIG. 1 d ).
  • the CCND1/chromosome 6 cutoff value of 1.55 was 100% specific for metastasis among patients with a Breslow's depth of ⁇ 1 mm, with 2/2 patients meeting this criterion having metastasis.
  • the CCND1/chromosome 6 cutoff criterion was 50% sensitive, identifying 2/4 metastatic patients in the group.
  • the MYC cutoff value of 2.48 was also 100% specific for metastasis among patients with a Breslow's depth of ⁇ 1 mm, with 2/2 patients meeting this criterion having metastasis.
  • the MYC cutoff criterion was 67% sensitive, identifying 2/3 metastatic patients in the group.
  • Using a combined criterion of patients having a CCND1/chromosome 6 value greater than 1.55 or a MYC average copy number of greater than 2.48 was 100% sensitive and 100% specific for patients with a Breslow's depth of ⁇ 1 mm. While these numbers are too small for a more detailed statistical analysis, these preliminary results suggest that these markers may be able to identify high risk patients among patients with thin melanomas.
  • FIG. 2 a shows a Kaplan Meier curve for all patients with Breslow's depth of ⁇ 2 mm, those with CCND1/chromosome 6 greater than 1.55, and those with CCND1/chromosome 6 below 1.55.
  • FIG. 2 b shows the Kaplan Meier curve for all patients with Breslow's depth of ⁇ 2 mm and for those meeting and not meeting the MYC criterion. The difference in the curve for those patients meeting and not meeting the criterion was highly statistically significant with a p-value of 8.24 ⁇ 10 ⁇ 3 .
  • a CCND1/chromosome 6 cutoff of greater than 1.55 retained the greatest discriminatory value.
  • a CCND1/chromosome 6 value was available for 61 patients in this Breslow category, and MYC average copy number was available for 58 patients in this Breslow category.
  • a CCND1/chromosome 6 value was 92% specific and 43% sensitive for metastasis in this Breslow group.
  • the p-value for the difference in the Kaplan Meier curve for those patients meeting this criterion versus those patients not meeting this criterion was 5.57 ⁇ 10 ⁇ 4 by the Log-rank test (see FIG. 3 a ).
  • the MYC cutoff of average copy number greater than 2.60 was 91% specific and 17% sensitive. While cases above this value clearly had a high tendency for metastasis, the p-value for the difference in the Kaplan-Meier curve between those patients meeting this criterion and those not meeting this criterion did not reach statistical significance because of the low sensitivity (see FIG. 3 b ).
  • the Kaplan Meier curve shows the difference in the curve for those cases with a CCND1/chromosome 6 value greater than 1.55 and Breslow's depths ⁇ 2.0 mm versus those cases with a CCND1/chromosome 6 less than 1.55 and Breslow's depths greater 2.0, those with CCND1/chromosome 6 greater than 1.55 and Breslow's depths ⁇ 2.0, as well as those with a CCND1/chromosome 6 value less than 1.55 and Breslow's depths less than 2.0.
  • Patients with a CCND1/chromosome 6 value ⁇ 2.0 and a CCND1/chromosome 6 greater than 1.55 had a significantly higher likelihood for metastasis than those with a CCND1/chromosome 6 ⁇ 1.55.
  • the p-value by Log-rank test for the difference in the Kaplan Meier curves for these cases was ⁇ 0.001, which was highly statistically significant.
  • the difference in the Kaplan Meier curve for these two groups was statistically significant by the Log-Rank and Wilcoxon test with a p-value of ⁇ 0.0001 (see FIG. 4 b ).
  • a log regression analysis (see FIG. 5 a ), including average MYC value, CCND1/chromosome 6 value, presence/absence of ulceration, Clark's level, Breslow's depth, sex, age, site, mitotic count, identified average MYC value, and CCND1/chromosome 6 value as independent prognostic parameters, was performed.
  • the multivariate analysis showed CCND1/chromosome 6 to have the highest prognostic power among all variables, and the MYC average value to have the second highest prognostic power.
  • the MYC criterion of having an average copy number of greater than about 2.48 was highly specific (90%) and had a PPV of 80%.
  • the CCND1/chromosome 6 and MYC average copy number were more statistically powerful than other currently recognized AJCC prognosticators, such as Breslow's depth, Clark's level, ulceration status, sex, site and age.
  • FIG. 4 a shows how the combination of the Breslow's depth and the CCND1/chromosome 6 value results in four potential distinct curves with those patients with a Breslow's depth>2 mm and a CCND1/chromosome 6 ⁇ 1.55 having the best prognosis and those patients with a Breslow's depth>2 mm and a CCND1/chromosome 6>1.55 having the worse prognosis.
  • patients with a Breslow's depth>2 mm and with an average MYC copy number >2.48 had significantly worse prognosis than those patients with a Breslow's depth>2 mm and a MYC copy number ⁇ 2.48.
  • 4 b demonstrates four distinct KM curves depending on the Breslow's depth and the MYC copy number with patients with a Breslow's depth>2 mm and an average MYC copy number >2.22 having the worse prognosis, and those with a Breslow's depth ⁇ 2 mm and an average MYC copy number ⁇ 2.22 having the best prognosis.
  • the logistic regression analysis (see Tables 1 and 2) examining CCND1/chromosome 6 and MYC, as well as the Breslow's depth, the Clark's level, the presence/absence of ulceration, sex, age, site, and mitotic count further confirms that these FISH parameters are independent prognostic factors. Additionally, the multivariate analysis shows that the CCND1/chromosome 6 and MYC are first and second, respectively, in their prognostic power in comparison to the other prognosticators, which are listed above and which are currently used by the AJCC.
  • reference category is second category in parentheses **odds of metastases in category of interest divided by odds of metastases in reference category ***odds ratio is fold-change in odds due to a one-unit change in factor
  • ROC Receiver operator characteristic
  • the cutoffs were selected to favor specificity, i.e., to minimize the chance of calling a patient with no metastasis “positive,” with the optimal cutoff considered to be that which yielded a specificity of greater than about 90% and a sensitivity of greater than about 30%. It was discovered that such percentages were prognostic.
  • This example describes the determination of a probe set useful in the diagnosis of malignant melanoma.
  • Cohort 1 consisted of 31 cases, which were identified by searching a database of melanomas for cases with an unequivocal histologic diagnosis of melanoma but a negative result for FISH with probe set 1 (RREB1 (ras-responsive element binding protein 1; 6p25), MYB (6q23), Cep6 (centromere 6), and CCND1 (11q13)). Also selected were 34 cases of unequivocally benign nevi with varying degrees of atypia, including mild, moderate and severe atypia. This cohort was used to develop the best combinations of probes for further analyses. Cases in cohort 2, 3 and 4 had not been previously studied by FISH. Cohort 2 consisted of 49 melanomas and 51 nevi.
  • Cohort 3 consisted of 72 additional melanoma and 85 nevi and was used to develop cutoffs for the “best” probe combinations.
  • FISH with the multi-colored probe sets described below was performed as previously described (Busam et al., J. Cutan. Pathol 37(2): 196-203 (2010)). The slides were analyzed with an epi-fluorescence microscope equipped with single band-pass filters (Abbott Molecular Inc., Des Plaines, Ill.). The analyses were performed by a trained technician and a dermatopathologist. All analyses were performed blinded of the specimens' diagnoses. Tumor-bearing areas were localized using the DAPI filter at low magnification. The tumor area was then thoroughly inspected for the presence of nuclei harboring abnormal copy numbers of any probe. Areas with the most significant copy number changes were selected for enumeration.
  • nuclei were analyzed under high-power (60 ⁇ objective). To qualify, nuclei had to be non-overlapping and harbor sufficiently bright signals. Nuclei that showed no signals for more than two probes were not analyzed. Thirty cells were enumerated in each specimen. The enumeration was done by a technician highly experienced in FISH enumeration and histopathology.
  • the DFI parameter was calculated as follows:
  • DFI ⁇ square root over ((1 ⁇ SENS) 2 +(1 ⁇ SPEC) 2 ) ⁇ square root over ((1 ⁇ SENS) 2 +(1 ⁇ SPEC) 2 ) ⁇ .
  • DFI represents the minimum distance from the ROC curve to the value of a sensitivity of 1 and a false positive rate (1-specificity) of 0.
  • the DFI ranges from 0-1 with 0 being the ideal.
  • probes used in probe set 1 (Busam et al. (2009), supra).
  • the following eight additional probes were selected: CDKN2A (9p21), centromere 9 (Cep9), MYC (8q24), BRAF (7q34), centromere 17 (Cep17), centromere 10 (Cep10), ZNF217 (zinc finger protein 17; 20q13), and Cox2 (1q25) and arranged in two probe sets.
  • probe set 2 This included CDKN2A (9p21), centromere 9 (Cep9), Cox2 (1q25) and centromere 17 (Cep17) in the first probe set and MYC (8q24), BRAF (7q34), centromere 10 (Cep10) and ZNF217 (20q13) in the second probe set. Both probe sets were applied to the 31 melanomas and 35 nevi from cohort 1. Analyses were carried out to determine which loci were most frequently gained or lost in melanoma specimens as compared to nevi specimens to select the four top-performing probes for further evaluation (referred to as probe set 2). The mean and standard deviation of each FISH parameter were calculated separately for the benign and malignant cases of each cohort and compared by the student t test.
  • Cohort 3 consisted of 157 specimens, including 72 melanomas and 85 nevi, which were evaluated using the final selected probe set targeting RREB1 (6p25), p16 (9p21), CCND1 (11q13) and MYC (8q24). Building on the analyses from Cohort 2, the Cohort 3 data set was used to examine various cutoffs for probe parameters in order to select the optimal cutoff for positivity for the final probe set. Cutoffs were calculated on an individual probe basis within the combination of four probes, and also as a fixed value across four probes. Two additional rules were applied to the signal counts to reduce the influence of tetraploid cells and cells with sub-optimal hybridization.
  • cells with 3-4 signals for CDKN2A, RREB1, CCND1 and MYC were not included in the numerator used to calculate percentages (loss, gain, imbalance or homozygous), but were included in the denominator.
  • cells with zero signals for any three or more of the four probes were excluded from the calculation of percentages (loss, gain, imbalance or homozygous).
  • the ROC curves were constructed, and the DFI and AUC values were calculated.
  • FIG. 5 shows the results of the evaluation of the Cohort 1 analysis including 31 melanoma and 35 nevi with probes targeting CDKN2A (9p21), Cep9 (centromere 9), MYC (8q24), BRAF (7q34), Cep17, Cep10, ZNF217 (20q13), and Cox 2 (1q25).
  • the plot illustrates the average proportion of cells with chromosomal abnormalities (gain or loss) in the melanoma and nevus groups for representative parameters calculated for each of the eight probes.
  • Nevi specimens exhibited zero or very few cells with chromosomal gains and approximately 18-30% of cells with apparent chromosomal losses mostly related to nuclear truncation on FFPE specimens.
  • Melanoma specimens demonstrated significant chromosomal copy number gains, as well as an elevated number of cells with deletions at the CDKN2A (9p21) locus.
  • Table 5 shows the top ten best parameters selected as a result of the discriminant analysis of the loci listed above, as judged by the highest AUC and DFI.
  • the discriminant analysis identified CDKN2A (9p21), Cep9, ZNF217 (20q13) and MYC (8q24) as the most complementary set of probes to probe set 1.
  • Each of the probes in the set showed a highly significant p value when comparing the average enumeration values between the melanoma group and the nevus group.
  • the DFI analysis from Cohort 2 identified two potential sets of probe targets among the top four probe combinations. These two combinations included CDKN2A (9p21), RREB1 (6p25), MYC (8q24) and CCND1 (11q13) in the first set and CDKN2A (9p21), RREB1 (6p25), MYC (8q24) and ZNF217 (20q13) in the second set. Both sets had excellent DFI with only a marginal difference, 0.145 versus 0.113, respectively. Since a previous prognostic study showed significant prognostic value of the 8q24 (Barnhill et al. (1999), supra) and 11q13 loci, the first combination was selected in order to maximize the prognostic potential of the assay. Therefore, the final probe set selected included CDKN2A (9p21), RREB1 (6p24.3), MYC (8q24) and CCND1 (11q13.3). This was labeled as probe set 3.
  • Cutoffs were determined using Cohort 3. ROC curves for the 157 specimens of Cohort 3 tested with probe set 3 are shown in FIG. 6 . A plot for each individual probe parameter is shown (CDKN2A Percent Homozygous, RREB1 Percent Gain, MYC Percent Gain, and CCND1 Percent Gain), as well as the combination of four parameters. In the targeted area of the curve with specificity in the 95% or greater region, several cutoff combinations for four probes were available, represented by the black-filled circles on the plot.
  • FIG. 6 is the ROC plot for individual FISH parameters (CDKN2A Percent Homozygous, RREB1 Percent Gain, MYC Percent Gain, and CCND1 Percent Gain) and the four-parameter combination. Parameters were calculated from the FISH evaluation of the 72 melanoma and 85 nevi specimens (Cohort 3). Highest sensitivity and specificity are shown at the point of minimum DFI. Performance with the conservatively selected set of cutoffs is shown by the arrow. The selected cutoff of 27% for each probe is shown as an error on the plot. The AUC highlighted by blackened circles indicate a targeted region of sensitivity and specificity.
  • probe set 1 In addition to cutoff selection, the performance of a new probe set was compared to that of probe set 1. Table 7 shows performance of probe set 1 using published evaluation criteria and cutoffs for positivity (Gerami et al., Am. J. Surg. Pathol. 33(12): 1783-1788 (2009)) compared to performance of the new probe set with two different sets of cutoffs: the one resulting in highest sensitivity and specificity on the ROC curve and one chosen conservatively. Probe set 1 resulted in a sensitivity of 72% and a specificity of 98%, while the new probe set known as probe set 3, with conservatively selected cutoff, demonstrated a sensitivity of 83% and a specificity of 100%.
  • probe set 1 targets three loci from chromosome 6 and one locus from chromosome 11, distinguishing tetraploid cells from cells with whole chromosome 6 gains can prove challenging.
  • the four loci targeted by probe set 4 originate from four distinct loci and include 9p21, which is typically deleted, as a target.
  • 9p21 which is typically deleted, as a target.
  • probability highly favors that a cell showing balanced gains in all four probes, including 9p21, is a tetraploid cell.
  • a formula can be used to eliminate tetraploidy as a source of false positives. The formula eliminates tetraploid cells from being including in the numerator when calculating the percentage of aberrant cells but maintains them in the denominator.
  • Receiver operator characteristic (ROC) curves were constructed to define cutoffs for CDKN2A (9p21), RREB1 (6p25), MYC (8q24), and CCND1 (11q13) for Cohort 3 (see FIG. 8 ; see also Table 10). A total of 30 cells was analyzed per specimen, and the percentage of abnormal cells was calculated. Cells that had three or four FISH signals for all of the probes except for CDKN2A but did not have a CDKN2A (9p21) deletion were considered tetraploid and were not counted as abnormal cells; instead, they were counted as normal cells for the purpose of calculating the percentage of abnormal cells.
  • ROC Receiver operator characteristic
  • tetraploidy rule increased specificity of discrimination of benign tetraploid nevi (the cells of which had 2 ⁇ gain of all chromosomes in the genome) from melanoma (the cells of which had locus-specific chromosomal abnormalities).
  • the cutoffs were selected to favor specificity, i.e., to minimize the chance of calling a patient with no metastasis “positive,” with the optimal cutoff considered to be that which yielded a specificity of greater than about 90% and a sensitivity of greater than about 80%. It was discovered that such percentages were diagnostic.
  • Receiver operator characteristic (ROC) curves were constructed to define cutoffs for CDKN2A (9p21), RREB1 (6p25), either MYC (8q24) or CCND1 (11q13), and ZNF217 for Cohort 2 (see Table 11). A total of 30 cells was analyzed per specimen, and the percentage of abnormal cells was calculated. Cells that had three or four FISH signals for all of the probes except for CDKN2A but did not have a CDKN2A (9p21) deletion were considered tetraploid and were not counted as abnormal cells; instead, they were counted as normal cells for the purpose of calculating the percentage of abnormal cells.
  • ROC Receiver operator characteristic
  • Cells that had three or four FISH signals for all of the probes except for CDKNA2 (9p21) and had a CDKN2A (9p21) deletion were counted as abnormal cells.
  • Application of the tetraploidy rule increased specificity of discrimination of benign tetraploid nevi (the cells of which had 2 ⁇ gain of all chromosomes in the genome) from melanoma (the cells of which had locus-specific chromosomal abnormalities).
  • the cutoffs were selected to favor specificity, i.e., to minimize the chance of calling a patient with no metastasis “positive,” with the optimal cutoff considered to be that which yielded a specificity of greater than about 90% and a sensitivity of greater than about 80%. It was discovered that such percentages were diagnostic.
  • This example describes the evaluation of RREB1, CCND1, CDKN2A and MYC in the prognosis of metastasis in patients with atypical Spitz tumors.
  • Spitz tumor by the submitting pathologist. All cases were then reviewed by a minimum of three experienced dermatopathologists, who agreed with the diagnosis. Histologic features used to qualify a lesion as an atypical Spitz tumor included, but were not limited to, greater than typical nuclear atypia, expansile nodular or sheet-like growth, frequent, deep or atypical mitoses, lack of maturation, epidermal consumption or ulceration or necrosis, large size (>1 cm), or deep extension into the subcutaneous fat.
  • For the second probe set positive criteria were: greater than 29% of enumerated cells with homozygous deletion of CDKN2A (9p21) or more than 29% of cells with more than two copies of RREB1 (6p25), MYC (8q24) or CCND1 (11q13).
  • a total of 75 atypical Spitz tumors with known clinical follow-up from 75 patients was analyzed using the two probe sets.
  • the patients' ages ranged from two to 58 years with an average age of 20.
  • 11 had evidence of tumor spread beyond a sentinel lymph node and in three of these cases the patients developed distant metastasis and death (group 4) (Table 14).
  • Three of four of these sentinel node-positive patients also had tumor within non-sentinel nodes upon completion lymphadenectomy while one patient of two years of age did not undergo completion dissection.
  • FISH overall positivity is defined as any of the individual FISH probes being positive.
  • ⁇ circumflex over ( ) ⁇ FISH probe set 1 6p, 6p CEP 6, 6q, 11q ⁇ circumflex over ( ) ⁇ circumflex over ( ) ⁇ FISH probe set 2: 6p, 9p, 11q, 8q ⁇ cytogenetic high risk: 9p positive; cytogenetic intermediate risk: 9p negative and (6p or 11q) positive; cytogenetic low risk: 9p and 6p and 11q negative
  • CI confidence interval
  • OR odds ratio
  • Spitzoid melanoma with homozygous CDKN2A (9p21) deletion to describe this subtype of melanoma, which is likely of a lower grade than conventional melanoma, is more frequently seen in children than in adults, has spitzoid cytomorphology with homozygous CDKN2A (9p21) deletions in the vast majority of cells, frequently results in transit metastasis, and often has lymph node involvement including non-sentinel lymph nodes.
  • Patient 7 has over eight years of clinical follow-up and remains disease-free following surgery and treatment with interferon. The remaining three patients have more limited follow-up time. Further follow-up studies of these patients are needed to determine the likelihood, frequency and time to development of distant metastasis and death.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Hospice & Palliative Care (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
US13/709,082 2011-12-10 2012-12-10 Materials and methods for diagnosis of malignant melanoma and prognosis of metastasis of malignant melanoma Abandoned US20130149704A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US13/709,082 US20130149704A1 (en) 2011-12-10 2012-12-10 Materials and methods for diagnosis of malignant melanoma and prognosis of metastasis of malignant melanoma
US15/354,854 US10227658B2 (en) 2011-12-10 2016-11-17 Methods for the analysis of spitz tumor samples

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201161569245P 2011-12-10 2011-12-10
US13/709,082 US20130149704A1 (en) 2011-12-10 2012-12-10 Materials and methods for diagnosis of malignant melanoma and prognosis of metastasis of malignant melanoma

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US15/354,854 Division US10227658B2 (en) 2011-12-10 2016-11-17 Methods for the analysis of spitz tumor samples

Publications (1)

Publication Number Publication Date
US20130149704A1 true US20130149704A1 (en) 2013-06-13

Family

ID=47430109

Family Applications (2)

Application Number Title Priority Date Filing Date
US13/709,082 Abandoned US20130149704A1 (en) 2011-12-10 2012-12-10 Materials and methods for diagnosis of malignant melanoma and prognosis of metastasis of malignant melanoma
US15/354,854 Expired - Fee Related US10227658B2 (en) 2011-12-10 2016-11-17 Methods for the analysis of spitz tumor samples

Family Applications After (1)

Application Number Title Priority Date Filing Date
US15/354,854 Expired - Fee Related US10227658B2 (en) 2011-12-10 2016-11-17 Methods for the analysis of spitz tumor samples

Country Status (4)

Country Link
US (2) US20130149704A1 (ja)
EP (2) EP3211099A1 (ja)
JP (3) JP2015500035A (ja)
WO (1) WO2013086478A1 (ja)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8822159B2 (en) * 2012-02-01 2014-09-02 Vanderbilt University Method for differentiating spitz nevi from spitzoid malignant melanoma
EP3199641A1 (en) * 2016-01-27 2017-08-02 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Means and methods for staging, typing and treating a cancerous disease

Families Citing this family (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018108243A (ja) * 2016-12-30 2018-07-12 株式会社三洋物産 遊技機
JP2020146309A (ja) * 2019-03-14 2020-09-17 株式会社三洋物産 遊技機
JP2020146305A (ja) * 2019-03-14 2020-09-17 株式会社三洋物産 遊技機
JP2020146301A (ja) * 2019-03-14 2020-09-17 株式会社三洋物産 遊技機
JP2020146316A (ja) * 2019-03-14 2020-09-17 株式会社三洋物産 遊技機
JP2020146317A (ja) * 2019-03-14 2020-09-17 株式会社三洋物産 遊技機
JP2020146307A (ja) * 2019-03-14 2020-09-17 株式会社三洋物産 遊技機
JP2020146298A (ja) * 2019-03-14 2020-09-17 株式会社三洋物産 遊技機
JP2020146315A (ja) * 2019-03-14 2020-09-17 株式会社三洋物産 遊技機
JP2020146306A (ja) * 2019-03-14 2020-09-17 株式会社三洋物産 遊技機
JP2020146312A (ja) * 2019-03-14 2020-09-17 株式会社三洋物産 遊技機
JP2020146313A (ja) * 2019-03-14 2020-09-17 株式会社三洋物産 遊技機
JP2020146308A (ja) * 2019-03-14 2020-09-17 株式会社三洋物産 遊技機
JP2020146302A (ja) * 2019-03-14 2020-09-17 株式会社三洋物産 遊技機
JP2020146299A (ja) * 2019-03-14 2020-09-17 株式会社三洋物産 遊技機
JP2020146303A (ja) * 2019-03-14 2020-09-17 株式会社三洋物産 遊技機
JP2020146314A (ja) * 2019-03-14 2020-09-17 株式会社三洋物産 遊技機
JP2020146300A (ja) * 2019-03-14 2020-09-17 株式会社三洋物産 遊技機
JP2020146311A (ja) * 2019-03-14 2020-09-17 株式会社三洋物産 遊技機
JP2020146310A (ja) * 2019-03-14 2020-09-17 株式会社三洋物産 遊技機
JP2020146297A (ja) * 2019-03-14 2020-09-17 株式会社三洋物産 遊技機
JP2020146318A (ja) * 2019-03-14 2020-09-17 株式会社三洋物産 遊技機

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6582908B2 (en) * 1990-12-06 2003-06-24 Affymetrix, Inc. Oligonucleotides
US20070059747A1 (en) * 2005-09-02 2007-03-15 Regents Of The University Of California Methods and probe combinations for detecting melanoma

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5756696A (en) 1986-01-16 1998-05-26 Regents Of The University Of California Compositions for chromosome-specific staining
US5491224A (en) 1990-09-20 1996-02-13 Bittner; Michael L. Direct label transaminated DNA probe compositions for chromosome identification and methods for their manufacture
EP1134293A3 (en) 1992-03-04 2004-01-07 The Regents of The University of California Comparative genomic hybridization (CGH)
US5830645A (en) 1994-12-09 1998-11-03 The Regents Of The University Of California Comparative fluorescence hybridization to nucleic acid arrays
US7604965B2 (en) 2003-04-03 2009-10-20 Fluidigm Corporation Thermal reaction device and method for using the same
KR101518085B1 (ko) 2007-09-07 2015-05-07 플루이다임 코포레이션 카피수 변이 측정, 방법 및 시스템
EP2359284A2 (en) 2008-10-31 2011-08-24 Abbott Laboratories Method for genomic classification of malignant melanoma based on patterns of gene copy number alterations

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6582908B2 (en) * 1990-12-06 2003-06-24 Affymetrix, Inc. Oligonucleotides
US20070059747A1 (en) * 2005-09-02 2007-03-15 Regents Of The University Of California Methods and probe combinations for detecting melanoma

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array. Affymetrix Package Insert for the HG-U133 Plus 2.0 Array, 2003, available via url: ) *
Ahern, H. The Scientist. July 1995. 9(15): 20-25. *
GeneCards GeneAnnot search for CCND1, available via url: , printed on July 21, 2014 *
GeneCards GeneAnnot search for CDKN2A, available via url: , printed on July 21, 2014 *
GeneCards GeneAnnot search for RREB1, available via url: , printed on July 21, 2014 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8822159B2 (en) * 2012-02-01 2014-09-02 Vanderbilt University Method for differentiating spitz nevi from spitzoid malignant melanoma
EP3199641A1 (en) * 2016-01-27 2017-08-02 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Means and methods for staging, typing and treating a cancerous disease
WO2017129753A1 (en) * 2016-01-27 2017-08-03 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Means and methods for staging, typing and treating a cancerous disease
CN108770360A (zh) * 2016-01-27 2018-11-06 弗劳恩霍夫应用研究促进协会 对癌性疾病进行分期、分型和治疗的手段和方法
US11702701B2 (en) 2016-01-27 2023-07-18 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Means and methods for staging, typing and treating a cancerous disease

Also Published As

Publication number Publication date
JP2017093446A (ja) 2017-06-01
EP3211099A1 (en) 2017-08-30
JP2019047798A (ja) 2019-03-28
EP2788768A1 (en) 2014-10-15
US10227658B2 (en) 2019-03-12
US20170067126A1 (en) 2017-03-09
WO2013086478A1 (en) 2013-06-13
JP2015500035A (ja) 2015-01-05
JP6430471B2 (ja) 2018-11-28

Similar Documents

Publication Publication Date Title
US10227658B2 (en) Methods for the analysis of spitz tumor samples
US7232655B2 (en) Method and probe set for detecting cancer
US10302645B2 (en) Materials and methods for diagnosis, prognosis and assessment of therapeutic/prophylactic treatment of prostate cancer
US9994909B2 (en) Diagnostic methods for determining prognosis of non-small cell lung cancer
EP2758544B1 (en) Materials and methods for prognosis of progression of barrett's esophagus
US20160160298A1 (en) Diagnostic methods for determining prognosis of non-small cell lung cancer
US20180291465A1 (en) Materials and methods for assessment of colorectal adenoma
US9290814B2 (en) Materials and methods for diagnosis of bladder cancer and monitoring recurrence thereof
EP2569624A1 (en) Detection of chromosomal abnormalities associated with endometrial cancer
US8785131B2 (en) Prognostic test for early stage non small cell lung cancer (NSCLC)

Legal Events

Date Code Title Description
AS Assignment

Owner name: ABBOTT MOLECULAR INC., ILLINOIS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:JEWELL, SUSAN;PESTOVA, EKATERINA;LI, GU;AND OTHERS;SIGNING DATES FROM 20130416 TO 20130627;REEL/FRAME:030721/0389

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION