Methods and probe combinations for detecting melanoma

Abstract

The present invention is based on the discovery of methods and combinations of probes to chromosomal regions that are gained or lost or imbalanced in melanoma that provide highly specific and sensitive assays for the detection of melanoma cells.

1 . A method of detecting the presence of melanoma cells in a biological sample from a patient, the method comprising: 
 a) contacting the sample with a combination of at least two probes, wherein the probes are selected from group consisting of a chromosome 6 enumerator probe, a chromosome 10 enumerator probe, a probe that targets chromosome region 6p, a probe that targets chromosome region 6q, a probe that targets chromosome region 7q, a probe that targets chromosome region 11q, a probe that targets chromosome region 17q, a probe that targets chromosome region 1q, and a probe that targets chromosome regions 20q;    b) incubating each probe of the set with the sample under conditions in which each probe binds selectively with a polynucleotide sequence on its target chromosome or chromosomal region to form a stable hybridization complex;    c) detecting the hybridization pattern of the combination of probes, wherein a hybridization pattern showing at least one gain or loss or imbalance at a chromosomal region targeted by the probes is indicative of melanoma.    
   
   
       2 . The method of  claim 1 , wherein the combination of at least two probes has a difference from ideal (DFI) value of 0.29 or less.  
   
   
       3 . The method of  claim 1 , wherein the combination of at least two probes has a DFI value of about 0.20 or less.  
   
   
       4 . The method of  claim 1 , wherein one of the probes in the combination of at least two probes is a probe that targets chromosome subregion 6p25.  
   
   
       5 . The method of  claim 4 , wherein a second probe in the combination of at least two probes is selected from the group consisting of a chromosome 10 enumerator probe, a probe that targets subregion 11q13, and a probe that targets chromosome subregion 6q23.  
   
   
       6 . The method of  claim 1 , wherein the combination is a combination of at least three probes selected from the group set forth in  claim 1 .  
   
   
       7 . The method of  claim 6 , wherein one of the three probes is a probe that targets chromosome subregion 6p25.  
   
   
       8 . The method of  claim 7 , wherein the combination of at least three probes has two probes selected from the group of two probes consisting of: 
 a) a probe that targets chromosome subregion 6q23 and a chromosome 6 enumerator probe;    b) a probe that targets chromosome subregion 1q31 and a probe that targets chromosome subregion 17q25;    c) a probe that targets chromosome subregion 11q13 and a chromosome 10 enumerator probe;    d) a probe that targets chromosome subregion 11q3 and a probe that targets chromosome subregion 17q25;    e) a probe that targets chromosome subregion 17q25 and a chromosome 10 enumerator probe;    f) a probe that targets chromosome subregion 1q31 and a chromosome 10 enumerator probe;    g) a probe that targets chromosome subregion 1q31 and a probe that targets chromosome subregion 11q13;    h) a probe that targets chromosome subregion 7q34 and a probe that targets chromosome subregion 17q25;    i) a probe that targets chromosome subregion 20q13 and a probe that targets chromosome subregion 17q25;    j) a probe that targets chromosome subregion 20q13 and a probe that targets chromosome subregion 11q13;    k) a probe that targets chromosome subregion 20q13 and a probe that targets chromosome subregion 6q23;    l) a probe that targets chromosome subregion 11q13 and a chromosome 10 enumerator probe;    m) a probe that targets chromosome subregion 7q34 and a chromosome 10 enumerator probe; and    n) a probe that targets chromosome subregion 20q13 and a chromosome 10 enumerator probe.    
   
   
       9 . The method of  claim 6 , wherein the combination of at least three probes has a probe that targets chromosome subregion 1q31, a probe that targets chromosome subregion 11q13, and a probe that targets chromosome subregions 17q25.  
   
   
       10 . The method of  claim 1 , wherein the combination of probes is a combination of at least four probes selected from the group set forth in  claim 1 .  
   
   
       11 . The method of  claim 10 , wherein one of the four probes is a probe to chromosome subregion 6p25.  
   
   
       12 . The method of  claim 11 , further wherein the combination of four probes has three probes selected from the group of three probes consisting of: 
 a) a probe to chromosome subregion 6q23, a chromosome 6 enumerator probe, and a chromosome 10 enumerator probe;    b) a probe to chromosome subregion 6q23, a probe to chromosome subregion 11q13, and a chromosome 6 enumerator probe;    c) a probe to chromosome subregion 1q31, a probe to chromosome subregion 11q13, and a probe to chromosome subregion 17q25;    d) a probe to chromosome subregion 6q23, a probe to chromosome subregion 11q13, and a chromosome 10 enumerator probe;    e) a probe to chromosome subregion 6q23, a probe to chromosome subregion 1q31, and a chromosome 10 enumerator probe;    f) a probe to chromosome subregion 1q31, a probe to chromosome subregion 17q25, and a chromosome 10 enumerator probe;    g) a probe to chromosome subregion 17q25, a probe to chromosome subregion 6q23, and a chromosome 6 enumerator probe;    h) a probe to chromosome subregion 20q13, a probe to chromosome subregion 6q23, and a chromosome 6 enumerator probe;    i) a probe to chromosome subregion 7q34, a probe to chromosome subregion 6q23, and a chromosome 6 enumerator probe;    j) a probe to chromosome subregion 7q34, a probe to chromosome subregion 1q31, and a probe to chromosome subregion 17q25;    k) a probe to chromosome subregion 20q13, a probe to chromosome subregion 1q31, and a probe to chromosome subregion 17q25;    l) a probe to chromosome subregion 7q34, a probe to chromosome subregion 6q23, and a probe to chromosome subregion 17q25;    m) a probe to chromosome subregion 20q13, a probe to chromosome subregion 6q23, and a probe to chromosome subregion 17q25;    n) a probe to chromosome subregion 7q34, a probe to chromosome subregion 11q13, and a chromosome 10 enumerator probe;    o) a probe to chromosome subregion 20q13, a probe to chromosome subregion 11q13, and a chromosome 10 enumerator probe;    p) a probe to chromosome subregion 7q34, a probe to chromosome subregion 17q25, and a probe to chromosome subregion 11q13;    q) a probe to chromosome subregion 20q13, a probe to chromosome subregion 17q25, and a probe to chromosome subregion 11q13;    r) a probe to chromosome subregion 7q34, a probe to chromosome subregion 17q25, and a chromosome 10 enumerator probe;    s) a probe to chromosome subregion 20q13, a probe to chromosome subregion 17q25, and a chromosome 10 enumerator probe;    t) a probe to chromosome subregion 7q34, a probe to chromosome subregion 20q13, and a probe to chromosome subregion 17q25;    u) a probe to chromosome subregion 6q23, a probe to chromosome subregion 17q25, and a chromosome 10 enumerator probe; and    v) a probe to chromosome subregion 17q25, a probe to chromosome subregion 11q13, and a chromosome 10 enumerator probe.    
   
   
       13 . The method of  claim 10 , wherein the combination of at least four probes has a probe to chromosome subregion 17q25, a probe to chromosome subregion 7q34, a probe to chromosome subregion 6q23, and a chromosome 6 enumerator probe.  
   
   
       14 . The method of  claim 1 , wherein the biological sample is a skin biopsy.  
   
   
       15 . The method of  claim 14 , wherein the biological sample is a formalin-fixed, paraffin-embedded sample.  
   
   
       16 . The method of  claim 1 , wherein the probes are fluorescently labeled.  
   
   
       17 . A method of detecting the presence or absence of melanoma cells in a biological sample from a patient, the method comprising: 
 a) contacting the sample with a combination of at least two probes, wherein the probes are selected from group consisting of a chromosome 6 enumerator probe, a chromosome 10 enumerator probe, a probe that targets chromosome region 6p, a probe that targets chromosome region 6q, a probe that targets chromosome region 7q, a probe that targets chromosome region 11q, a probe that targets chromosome region 17q, a probe that targets chromosome region 1q, and a probe that targets chromosome regions 20q;    b) incubating each probe of the set with the sample under conditions in which each probe binds selectively with a polynucleotide sequence on its target chromosome or chromosomal region to form a stable hybridization complex;    c) detecting and analyzing the hybridization pattern of the combination of probes for the presence or absence of melanoma.    
   
   
       18 . A combination of probes comprising at least two probes selected from the group set forth in  claim 1 , wherein the combination of probes has a difference from ideal (DFI) value of less than about 0.29 for melanoma.  
   
   
       19 . The combination of probes of  claim 18 , wherein the combination of probes has a DFI value of less than about 0.20.  
   
   
       20 . The combination of probes of  claim 18 , wherein the two probes are selected from the group consisting of: 
 a) a probe to chromosome subregion 6p25 and a chromosome 10 enumerator probe;    b) a probe to chromosome subregion 6p25 and a probe to chromosome subregions 11q13; and    c) a probe to chromosome subregion 6p25 and a probe to chromosome subregions 6q23.    
   
   
       21 . A combination of probes comprising at least three probes selected from the group set forth in  claim 1 , wherein the combination of probes has a difference from ideal (DFI) value of less than about 0.29 for melanoma.  
   
   
       22 . The combination of  claim 21 , wherein the three probes are selected from the group of probes consisting of: 
 a) a probe that targets chromosome subregions 6p25, a probe that targets chromosome subregion 6q23 and a chromosome 6 enumerator probe;    b) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 1q31 and a probe that targets chromosome subregion 17q25;    c) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 11q13 and a chromosome 10 enumerator probe;    d) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 11q13 and a probe that targets chromosome subregion 17q25;    e) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 17q25 and a chromosome 10 enumerator probe;    f) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 1q31 and a chromosome 10 enumerator probe;    g) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 1q31 and a probe that targets chromosome subregion 11q13;    h) a probe that targets chromosome subregion 1q31, a probe that targets chromosome subregion 11q13 and a probe that targets chromosome subregion 17q25;    i) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 7q34 and a probe that targets chromosome subregion 17q25;    j) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 20q13 and a probe that targets chromosome subregion 17q25;    k) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 20q13 and a probe that targets chromosome subregion 11q3;    l) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 20q13 and a probe that targets chromosome subregion 6q23;    m) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 11q13 and a chromosome 10 enumerator probe;    n) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 7q34 and a chromosome 10 enumerator probe; and    o) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 20q13 and a chromosome 10 enumerator probe.    
   
   
       23 . A combination of probes that has four probes selected from the group set forth in  claim 1 , wherein the combination of probes has a difference from ideal (DFI) value of less than about 0.29 for melanoma.  
   
   
       24 . The combination of  claim 23 , wherein the four probes are selected from the group consisting of: 
 a) a probe to chromosome subregions 6p25; a probe to chromosome subregion 6q23, a chromosome 6 enumerator probe, and a chromosome 10 enumerator probe;    b) a probe to chromosome subregions 6p25; a probe to chromosome subregion 6q23, a probe to chromosome subregion 11q13, and a chromosome 6 enumerator probe;    c) a probe to chromosome subregions 6p25; a probe to chromosome subregion 1q31, a probe to chromosome subregion 11q13, and a probe to chromosome subregion 17q25;    d) a probe to chromosome subregions 6p25; a probe to chromosome subregion 6q23, a probe to chromosome subregion 11q13, and a chromosome 10 enumerator probe; and    e) a probe to chromosome subregions 6p25; a probe to chromosome subregion 6q23, a probe to chromosome subregion 1q31, and a chromosome 10 enumerator probe;    f) a probe to chromosome subregions 6p25, probe to chromosome subregion 1q31, a probe to chromosome subregion 17q25, and a chromosome 10 enumerator probe;    g) a probe to chromosome subregions 6p25, a probe to chromosome subregion 17q25, a probe to chromosome subregion 6q23, and a chromosome 6 enumerator probe;    h) a probe to chromosome subregions 6p25, a probe to chromosome subregion 20q13, a probe to chromosome subregion 6q23, and a chromosome 6 enumerator probe;    i) a probe to chromosome subregions 6p25, a probe to chromosome subregion 7q34, a probe to chromosome subregion 6q23, and a chromosome 6 enumerator probe;    j) a probe to chromosome subregions 6p25, a probe to chromosome subregion 7q34, a probe to chromosome subregion 1q31, and a probe to chromosome subregion 17q25;    k) a probe to chromosome subregions 6p25, a probe to chromosome subregion 20q13, a probe to chromosome subregion 1q31, and a probe to chromosome subregion 17q25;    l) a probe to chromosome subregions 6p25, a probe to chromosome subregion 7q34, a probe to chromosome subregion 6q23, and a probe to chromosome subregion 17q25;    m) a probe to chromosome subregions 6p25, a probe to chromosome subregion 20q13, a probe to chromosome subregion 6q23, and a probe to chromosome subregion 17q25;    n) a probe to chromosome subregions 6p25, a probe to chromosome subregion 7q34, a probe to chromosome subregion 11q13, and a chromosome 10 enumerator probe;    o) a probe to chromosome subregions 6p25, a probe to chromosome subregion 20q13, a probe to chromosome subregion 11q13, and a chromosome 10 enumerator probe;    p) a probe to chromosome subregions 6p25, a probe to chromosome subregion 7q34, a probe to chromosome subregion 17q25, and a probe to chromosome subregion 11q13;    q) a probe to chromosome subregions 6p25, a probe to chromosome subregion 20q13, a probe to chromosome subregion 17q25, and a probe to chromosome subregion 11q13;    r) a probe to chromosome subregions 6p25, a probe to chromosome subregion 7q34, a probe to chromosome subregion 17q25, and a chromosome 10 enumerator probe;    s) a probe to chromosome subregions 6p25, a probe to chromosome subregion 20q13, a probe to chromosome subregion 17q25, and a chromosome 10 enumerator probe;    t) a probe to chromosome subregions 6p25, a probe to chromosome subregion 7q34, a probe to chromosome subregion 20q13, and a probe to chromosome subregion 17q25;    u) a probe to chromosome subregions 6p25, a probe to chromosome subregion 6q23, a probe to chromosome subregion 17q25, and a chromosome 10 enumerator probe;    v) a probe to chromosome subregions 6p25, a probe to chromosome subregion 17q25, a probe to chromosome subregion 11q13, and a chromosome 10 enumerator probe; and    x) a probe to chromosome subregion 17q25, a probe to chromosome subregion 7q34, a probe to chromosome subregion 6q23, and a chromosome 6 enumerator probe.    
   
   
       25 . A combination of two probes selected from the group consisting of a chromosome 6 enumerator probe, a chromosome 10 enumerator probe, a probe that targets chromosome region 6p, a probe that targets chromosome region 6q, a probe that targets chromosome region 7q, a probe that targets chromosome region 11q, a probe that targets chromosome region 17q, a probe that targets chromosome region 1q, and a probe that targets chromosome regions 20q.  
   
   
       26 . A combination of three probes selected from the group set forth in  claim 25 .  
   
   
       27 . A combination of four probes selected from the group set forth in  claim 25 .  
   
   
       28 . A kit comprising a combination at least two probes selected from the group set forth in  claim 1 , wherein the combination of probes has a difference from ideal (DFI) value of less than about 0.29 for melanoma.  
   
   
       29 . The kit of  claim 28 , wherein the combination of probes has a DFI value of less than about 0.20.  
   
   
       30 . The kit of  claim 28 , wherein the two probes are selected from the group consisting of: 
 a) a probe to chromosome subregion 6p25 and a chromosome 10 enumerator probe;    b) a probe to chromosome subregion 6p25 and a probe to chromosome subregions 11q13; and    c) a probe to chromosome subregion 6p25 and a probe to chromosome subregions 6q23.    
   
   
       31 . A kit comprising a combination of at least three probes selected from the group of probes set forth in  claim 1 .  
   
   
       32 . The kit of  claim 31 , wherein the three probes are selected from the group of probes consisting of: 
 a) a probe that targets chromosome subregions 6p25, a probe that targets chromosome subregion 6q23 and a chromosome 6 enumerator probe;    b) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 1q31 and a probe that targets chromosome subregion 17q25;    c) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 11q13 and a chromosome 10 enumerator probe;    d) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 11q13 and a probe that targets chromosome subregion 17q25;    e) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 17q25 and a chromosome 10 enumerator probe;    f) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 1q31 and a chromosome 10 enumerator probe;    g) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 1q31 and a probe that targets chromosome subregion 11q13;    h) a probe that targets chromosome subregion 1q31, a probe that targets chromosome subregion 11q13 and a probe that targets chromosome subregion 17q25;    i) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 7q34 and a probe that targets chromosome subregion 17q25;    j) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 20q13 and a probe that targets chromosome subregion 17q25;    k) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 20q13 and a probe that targets chromosome subregion 11q13;    l) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 20q13 and a probe that targets chromosome subregion 6q23;    m) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 11q13 and a chromosome 10 enumerator probe;    n) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 7q34 and a chromosome 10 enumerator probe; and    o) a probe that targets chromosome subregion 6p25, a probe that targets chromosome subregion 20q13 and a chromosome 10 enumerator probe.    
   
   
       33 . A kit comprising a combination of at least four probes selected from the group of probes set forth in  claim 1 .  
   
   
       34 . The kit of  claim 33 , wherein the four probes are selected from the group consisting of: 
 a) a probe to chromosome subregions 6p25; a probe to chromosome subregion 6q23, a chromosome 6 enumerator probe, and a chromosome 10 enumerator probe;    b) a probe to chromosome subregions 6p25; a probe to chromosome subregion 6q23, a probe to chromosome subregion 1q13, and a chromosome 6 enumerator probe;    c) a probe to chromosome subregions 6p25; a probe to chromosome subregion 1q31, a probe to chromosome subregion 11q13, and a probe to chromosome subregion 17q25;    d) a probe to chromosome subregions 6p25; a probe to chromosome subregion 6q23, a probe to chromosome subregion 11q13, and a chromosome 10 enumerator probe; and    e) a probe to chromosome subregions 6p25; a probe to chromosome subregion 6q23, a probe to chromosome subregion 1q31, and a chromosome 10 enumerator probe;    f) a probe to chromosome subregions 6p25, probe to chromosome subregion 1q31, a probe to chromosome subregion 17q25, and a chromosome 10 enumerator probe;    g) a probe to chromosome subregions 6p25, a probe to chromosome subregion 17q25, a probe to chromosome subregion 6q23, and a chromosome 6 enumerator probe;    h) a probe to chromosome subregions 6p25, a probe to chromosome subregion 20q13, a probe to chromosome subregion 6q23, and a chromosome 6 enumerator probe;    i) a probe to chromosome subregions 6p25, a probe to chromosome subregion 7q34, a probe to chromosome subregion 6q23, and a chromosome 6 enumerator probe;    j) a probe to chromosome subregions 6p25, a probe to chromosome subregion 7q34, a probe to chromosome subregion 1q31, and a probe to chromosome subregion 17q25;    k) a probe to chromosome subregions 6p25, a probe to chromosome subregion 20q13, a probe to chromosome subregion 1q31, and a probe to chromosome subregion 17q25;    l) a probe to chromosome subregions 6p25, a probe to chromosome subregion 7q34, a probe to chromosome subregion 6q23, and a probe to chromosome subregion 17q25;    m) a probe to chromosome subregions 6p25, a probe to chromosome subregion 20q13, a probe to chromosome subregion 6q23, and a probe to chromosome subregion 17q25;    n) a probe to chromosome subregions 6p25, a probe to chromosome subregion 7q34, a probe to chromosome subregion 11q13, and a chromosome 10 enumerator probe;    o) a probe to chromosome subregions 6p25, a probe to chromosome subregion 20q13, a probe to chromosome subregion 11q13, and a chromosome 10 enumerator probe;    p) a probe to chromosome subregions 6p25, a probe to chromosome subregion 7q34, a probe to chromosome subregion 17q25, and a probe to chromosome subregion 11q3;    q) a probe to chromosome subregions 6p25, a probe to chromosome subregion 20q13, a probe to chromosome subregion 17q25, and a probe to chromosome subregion 1q13;    r) a probe to chromosome subregions 6p25, a probe to chromosome subregion 7q34, a probe to chromosome subregion 17q25, and a chromosome 10 enumerator probe;    s) a probe to chromosome subregions 6p25, a probe to chromosome subregion 20q13, a probe to chromosome subregion 17q25, and a chromosome 10 enumerator probe;    t) a probe to chromosome subregions 6p25, a probe to chromosome subregion 7q34, a probe to chromosome subregion 20q13, and a probe to chromosome subregion 17q25;    u) a probe to chromosome subregions 6p25, a probe to chromosome subregion 6q23, a probe to chromosome subregion 17q25, and a chromosome 10 enumerator probe;    v) a probe to chromosome subregions 6p25, a probe to chromosome subregion 17q25, a probe to chromosome subregion 11q13, and a chromosome 10 enumerator probe; and    x) a probe to chromosome subregion 17q25, a probe to chromosome subregion 7q34, a probe to chromosome subregion 6q23, and a chromosome 6 enumerator probe.    
   
   
       35 . A kit that contains a probe set for diagnosing melanoma, wherein the probe set is a two-probe combination selected from the group of probes consisting of a chromosome 6 enumerator probe, a chromosome 10 enumerator probe, a probe that targets chromosome region 6p, a probe that targets chromosome region 6q, a probe that targets chromosome region 7q, a probe that targets chromosome region 11q, a probe that targets chromosome region 17q, a probe that targets chromosome region 1q, and a probe that targets chromosome regions 20q.  
   
   
       36 . A kit that contains a probe set for diagnosing melanoma, wherein the probe set is a three-probe combination selected from the group of probes set forth in  claim 35 .  
   
   
       37 . A kit that contains a probe set for diagnosing melanoma, wherein the probe set is a four-probe combination selected from the group of probes set forth in  claim 35.