Abstract:
The methods and compositions described herein address the need for diagnostic method that could be offered to women during yearly checkups to allow for early detection, diagnosis and classification, and treatment of endometrial cancer. In addition, these methods and compositons addresse the current need for improving diagnostic accuracy of biopsy procedures in symptomatic patients.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of U.S. provisional application No. 61/395,303, filed May 10, 2010, which is hereby incorporated by reference in its entirety. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates generally to the area of detecting, diagnosing, and monitoring of endometrial hyperplasia and carcinoma. 
       BACKGROUND OF THE INVENTION 
       [0003]    Uterine cancer is the fourth most common malignancy diagnosed in women in the United States (estimated 42,793 cases in 2009) and is the seventh most common cause of cancer death among U.S. women. Over 95% of all uterine cancers are cancers of the endometrium (lining of the body of the uterus). Lifetime probability of developing cancer of the uterus is 1 in 40 (U.S.). 35-50% of women ages 35-70 present with one or more risk factors for endometrial cancer. 
         [0004]    Two different clinicopathologic subtypes are recognized based on light microscopic appearance, clinical behavior, and epidemiology: the estrogen-related (type I, endometrioid) and the non-estrogen-related types (type II, nonendometrioid such as papillary serous and clear cell). Despite it aggressiveness, endometrial cancer is difficult to diagnose, thus many patients present with symptoms of the late-stage cancer. 
       SUMMARY OF THE INVENTION 
       [0005]    In particular embodiments, the present invention provides a method of detecting the presence of endometrial carcinoma in a biological sample from a subject. The method entails contacting the sample with one or more probes for one or more chromosome regions selected from the group consisting of: 1q, 2p, 2q, 3p, 3q, 7p, 8p, 8q, 9p, 9q, the centromeric region of chromosome 10, 10q, 15q, 16q, 17p, the centromeric region of chromosome 18, 18q, 19p, 20q, and 22q. The one or more probes are incubated 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. Hybridization of the one or more probes is detected, wherein a hybridization pattern showing at least one gain or loss or imbalance at a chromosomal region targeted by the probes is indicative of endometrial carcinoma. 
         [0006]    In certain embodiments, a hybridization pattern showing a gain in one or more chromosome regions selected from the group consisting of: 1q, 2p, 3q, 8q, 10q, and 20q is indicative of endometrial carcinoma. In specific embodiments, a hybridization pattern showing a gain in one or more chromosome regions selected from the group consisting of: 1q, 10p, and 10q is indicative of endometrioid carcinoma. In other specific embodiments, a hybridization pattern showing a gain in one or more chromosome regions selected from the group consisting of: 3q, 8q, 18q, and 20q is indicative of non-endometrioid carcinoma. In an illustrative embodiment, a hybridization pattern showing a gain in 1q31-qtel is indicative of endometrial carcinoma. 
         [0007]    In certain embodiments, a hybridization pattern showing a loss in one or more chromosome regions selected from the group consisting of: 9p, 9q, 15q, 16q, 17p, 18q, 19p, and 22q is indicative of endometrial carcinoma. In illustrative embodiments, a hybridization pattern showing a loss in one or more chromosome regions selected from the group consisting of: 15q11-q13, 18q21, and 19 ptel is indicative of endometrial carcinoma. 
         [0008]    In certain embodiments, the one or more probes are for one or more chromosome subregions selected from the group consisting of: 1q25, 2p24, 2q26, 3p21, 3q27-q29, 7p21, 8p11, 8q24, 9q34, the centromeric region of chromosome 10, 10q23, 10q26, 15q11-q13, 16q24, the centromeric region of chromosome 18, 18q21, 20q12 and 20q13. 
         [0009]    In particular embodiments, the sample is contacted with a combination of at least 3 probes for a set of chromosome subregions selected from the group consisting of
       1q25, 8q24, 15q1′-q13;   1q25, 10q26, 15q11-q13;   1q25, 2p24, 8q24   1q25, 8q24, 10q26;   1q25, 8p11, 15q11-q13;   1q25, 2p24, 8p11;   1q25, 8p11, 10q26;   1q25, 8p11, 8q24;   1q25, 2p24, 10q26;   1q25, 2p24, 15q11-q13;   8q24, 10q26, 15q11-q13;   1q25, 8p11, 20q13;   1q25, 8q24, 20q13;   1q25, 15q11-q13, 20q13;   1q25, 10q26, 20q13;   8p11, 10q26, 15q11-q13;   1q25, 2p24, 20q13;   2p24, 8p11, 10q26;   2p24, 8q24, 10q26;   2p24, 10q26, 15q11-q13;   2p24, 8q24, 15q11-q13;   10q26, 15q11-q13, 20q13;   1q25, 8p11, 18q21;   1q25, 8q24, 18q21;   1q25, 10q26, 18q21;   1q25, 15q11-q13, 18q21;   8p11, 8q24, 10q26;   8q24, 10q26, 20q13;   1q25, 2p24, 18q21;   2p24, 8p11, 8q24;   2p24, 8p11, 15q11-q13;   8p11, 8q24, 15q11-q13;   2p24, 10q26, 20q13;   8p11, 10q26, 20q13;   2p24, 8p11, 20q13;   2p24, 8q24, 20q13;   8q24, 15q11-q13, 20q13; and   8p11, 15q11-q13, 20q13.       
 
         [0048]    In certain embodiments, the sample is contacted with a combination of at least 3 probes for a set of chromosome subregions selected from the group consisting of:
       1q25, 18q21, CEP18, 8q24;   2p24, 2q26, 10q26, 2q13; and   10q23, CEP10, and 8p11.       
 
         [0052]    In particular embodiments, the sample is contacted with a combination of at least 2 probes for a set of chromosome subregions selected from the group consisting of:
       18q21, 1q24, 8q24, CEP18;   1q24, 8q24, 10q26, CEP18;   18q21, 1q24, 10q26, CEP18;   1q24, 8q24, CEP18, 3q27-q29;   18q21, 1q24, 8q24, 10q26;   1q24, 2p24, 10q26, CEP18;   1q24, 10q26, CEP18, 3q27-q29;   1q24, 10q26, CEP18, 20q13;   1q24, CEP18, 3q27-q29, 20q13;   1q24, 2p24, CEP18, 3q27-q29;   18q21, 1q24, 10q26, 20q13;   1q24, 8q24, CEP18;   18q21, 1q24, CEP18; and   1q24, CEP18.       
 
         [0067]    In illustrative embodiments, the sample is contacted with a combination of at least 4 probes for a set of chromosome subregions selected from the group consisting of:
       1q24, 8q24, CEP18, 20q13;   1q24, CEP18, 3q27-q29, 20q13;   1q24, CEP18, 20q13, 10q26;   CEP10, 8q24, CEP18, 1q24;   10q26, CEP10, 1q24, 8q24;   8q24, 1q24, 20q13, CEP18; 10q26;   20q13, CEP10, 1q24, 10q26,
 
wherein a hybridization pattern showing a gain in one or more of these chromosome subregions is indicative of endometrial carcinoma.
       
 
         [0075]    In specific embodiments, one or more of a gain at one of more of 1q24, 8q24, CEP18, and 20q13 are indicative of endometrial carcinoma. In alternative specific embodiments, one or more of a 20q13 gain, a 1q24 gain, a CEP10 imbalance, and a 10q26 gain are indicative of endometrial carcinoma. 
         [0076]    In particular embodiments, the sample is contacted with a combination of at least 2 probes for a set of chromosome subregions selected from the group consisting of:
       18q21, 1q25, 8q24, CEP18;   1q25, 8q24, 10q26, CEP18;   18q21, 1q25, 10q26, CEP18;   1q25, 8q24, CEP18, 3q27-q29;   18q21, 1q25, 8q24, 10q26;   1q25, 2p24, 10q26, CEP18;   1q25, 10q26, CEP18, 3q27-q29;   1q25, 10q26, CEP18, 20q13;   1q25, CEP18, 3q27-q29, 20q13;   1q25, 2p24, CEP18, 3q27-q29;   18q21, 1q25, 10q26, 20q13;   1q25, 8q24, CEP18;   18q21, 1q25, CEP18; and   1q25, CEP18.       
 
         [0091]    In illustrative embodiments, the sample is contacted with a combination of at least 4 probes for a set of chromosome subregions selected from the group consisting of
       1q25, 8q24, CEP18, 20q13;   1q25, CEP18, 3q27-q29, 20q13;   1q25, CEP18, 20q13, 10q26;   CEP10, 8q24, CEP18, 1q25;   10q26, CEP10, 1q25, 8q24;   8q24, 1q25, 20q13, CEP18; 10q26;   20q13, CEP10, 1q25, 10q26,
 
wherein a hybridization pattern showing a gain in one or more of these chromosome subregions is indicative of endometrial carcinoma.
       
 
         [0099]    In specific embodiments, one or more of a gain at one of more of 1q25, 8q24, CEP18, and 20q13 are indicative of endometrial carcinoma. In alternative specific embodiments, one or more of a 20q13 gain, a 1q25 gain, a CEP10 imbalance, and a 10q26 gain are indicative of endometrial carcinoma. 
         [0100]    In variations of any of the preceding embodiments, the probe combination can distinguish samples including endometrial carcinoma from samples that do not include endometrial carcinoma with a sensitivity of at least 93% and a specificity of at least 90%. For example, the sensitivity can be at least 95% and the specificity can be at least 90.4%. In specific embodiments, the sensitivity is least 96% and the specificity is at least 91%. 
         [0101]    In variations of any of the preceding embodiments, the probe combination can include between 2 and 10 probes. In particular embodiments, the probe combination includes between 3 and 8 probes. In an illustrative embodiment, the probe combination includes 4 probes. 
         [0102]    In any of preceding embodiments, the method can be carried out by array comparative genomic hybridization (aCGH) to probes immobilized on a substrate. Alternatively, the method can be carried out by fluorescence in situ hybridization, and each probe in the probe combination can be labeled with a different fluorophore. 
         [0103]    In any of the preceding embodiments, the sample can be an endometrial brushing specimen or an endometrial biopsy specimen. 
         [0104]    In any of the preceding claims, when the results of the method indicate endometrial carcinoma, the method can additionally include treating the subject for endometrial carcinoma. 
         [0105]    The invention also provides, in certain embodiments, a combination of probes including between 2 and 10 probes selected from any of the groups set forth above, wherein the combination of probes has a sensitivity of at least 93% and a specificity of at least 90% for distinguishing samples including endometrial carcinoma from samples that do not include endometrial carcinoma. In particular embodiments, the combination of probes has a sensitivity of at least 95% and a specificity of at least 90.4%. In illustrative embodiments, the combination of probes has a sensitivity of at least 96% and a specificity of at least 91%. In various embodiments, the probe combination includes between 3 and 8 probes, e.g., 4 probes. 
         [0106]    Another aspect of the invention includes a kit for diagnosing endometrial carcinoma, wherein the kit includes a combination of probes including between 2 and 10 probes selected from any of the groups set forth above, wherein the combination of probes has a sensitivity of at least 93% and a specificity of at least 90% for distinguishing samples including endometrial carcinoma from samples that do not include endometrial carcinoma. In particular embodiments, the combination of probes has a sensitivity of at least 95% and a specificity of at least 90.4%. In illustrative embodiments, the combination of probes has a sensitivity of at least 96% and a specificity of at least 91%. In various embodiments, the probe combination includes between 3 and 8 probes, e.g., 4 probes. 
         [0107]    In various embodiments, a chromosomal gain, loss, or imbalance detected by at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 probes is indicative of endometrial carcinoma. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0108]      FIG. 1A-C : The frequency of genomic changes in all cancers from CGH data. 
           [0109]    FIG.  2 A(1-3)-B(1-3): The frequency of genomic changes in A, endometrioid cancers; B, non-endometrioid cancers from CGH data. 
           [0110]      FIG. 3 : Complementation of selected genomic array clones. A, Abnormal (gain or loss); NC, no change in copy number. 
           [0111]      FIG. 4A-I : Probe sets 1 and 2 shown on the aCGH data output. 
           [0112]      FIG. 5 : Representative example of cells with FISH signals (amplification). 
           [0113]      FIG. 6A-C : Probe sets 1 and 2. Average % abnormal cells in all specimens evaluated. A, % Cells with Gain; B, % Cells with Loss; C, % Cells with Gains and Losses. 
           [0114]      FIG. 7 : ROC Curve for CEP18+1q24+MYC+DCC, % Abnormal (% cells with either copy number gain or copy number loss for at least 1 of 4 loci). 
           [0115]      FIG. 8 : Individual Probes and Combinations (ROC Curves, % Abnormal). 
           [0116]      FIG. 9 : ROC Curves, 4-Probe Combination, Gains of 1q24, MYC, CEP18 and 20q13.2. 
           [0117]      FIG. 10 : Evaluation of additional probes to improve sensitivity of endometrial cancer detection. 
       
    
    
     DETAILED DESCRIPTION 
       [0118]    The present invention provides a method of detection of chromosomal abnormalities associated with endometrial carcinoma of both endometrioid and non-endometrioid types, as well as probe combinations and diagnostic kits. The methods can utilize techniques known as Comparative Genomic Hybridization on a microarray (aCGH) and in situ hybridization (e.g., Fluorescence In Situ Hybridization (FISH)) using a combination of Locus Specific Identifier (LSI) and Chromosome Enumerator (CEP) probes to detect cells that have chromosomal abnormalities consistent with a diagnosis of endometrial cancer. The methods described herein can be used to detect endometrial cancer in various types of specimens (e.g., endometrial brushing specimen or endometrial biopsy specimen) obtained in the doctors office or operating room. 
         [0119]    There is currently no cytological diagnostic test for the early detection of endometrial cancer available today, and there is no test or procedure to routinely screen women at risk for endometrial cancer. Endometrial biopsy is recommended as the initial evaluation of women with abnormal uterine bleeding. The disadvantages of biopsy are that it an invasive and uncomfortable procedure for the patient. Moreover, tumors comprising &lt;50% of the endometrium may be inadequately sampled by endometrial biopsy. Inadequate sampling by biopsy may result in false negative results and necessitate additional endometrial biopsies to determine the cause of persistent abnormal uterine bleeding. As endometrial cancers are relatively fast growing, patients often present after the cancers have already developed and spread locally. 
         [0120]    Conventional cytology collected with an endometrial sampling device such as the Tao brush offers the advantage of being relatively non-invasive and therefore more comfortable for the patient. In addition, endometrial sampling for cytology is less likely than biopsy to result in false negative results due to inadequate sampling. The problem with conventional cytology is that most pathologists do not have experience with interpreting endometrial cytology and many consider it difficult to interpret. Furthermore, even experienced cytopathologists find that there are significant fraction of cases that cannot be definitely diagnosed as either positive or negative for cancer and which must be categorized as indeterminate for the presence of cancer. 
         [0121]    The methods and compositions described herein will provide means for screening and improved diagnosis of endometrial cancer. Specifically, the methodology described herein can provide one or more of the following benefits: distinguish cancer from difficult benign conditions; distinguish benign tissue from pre-cancerous lesions and pre-cancerous lesions from cancer; distinguish endometrioid and non-endometrioid tumors; provide an early screening tool for outpatient tests on cytology specimens; aid in diagnosis of endometrial cancer in biopsy or surgical specimens (aid histological tissue evaluation); and provide an aid in monitoring of cancer and pre-cancerous conditions during therapy. 
         [0122]    Advantages of the methods described herein can include one or more of the following: use of stable DNA for detection of chromosomal abnormalities (deletion, amplification, aneusomy, translocation); rapid detection: results could be obtained in 18-36 hours; implementation possibilities include multiplexed methods (e.g., microarray) and multicolor FISH; use as stand-alone test or as adjuncts to other tests (histology, PSA, nomogram, methylation, mutation); use on cytology specimens or biopsy (fresh-frozen or FFPE); combination of several probes increases sensitivity and specificity as compared to a single-analyte assay; increased sensitivity compared to conventional cytology. 
       DEFINITIONS 
       [0123]    Terms used in the claims and specification are defined as set forth below unless otherwise specified. 
         [0124]    The term “endometrial carcinoma” refers to a malignant neoplasm of the endometrium, which is the mucous membrane lining the uterus. Two different clinicopathologic subtypes are recognized based on light microscopic appearance, clinical behavior, and epidemiology: the estrogen-related (type I, “endometrioid”) and the non-estrogen-related types (type II, “nonendometrioid”, such as papillary serous and clear cell). 
         [0125]    The terms “tumor” or “cancer” in an animal refer to the presence of cells possessing characteristics such as atypical growth or morphology, including uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Often, cancer cells will be in the form of a tumor, but such cells may exist alone within an animal. The term tumor includes both benign and malignant neoplasms. The term “neoplastic” refers to both benign and malignant atypical growth. 
         [0126]    The term “biological sample” or “specimen” is intended to mean a sample obtained from a subject suspected of having, or having endometrial carcinoma. In some embodiments, the sample includes a formalin-fixed paraffin-embedded biopsy. In addition to subjects suspected of having endometrial carcinoma, the biological sample may further be derived from a subject that has been diagnosed with endometrial carcinoma for confirmation of diagnosis or establishing that all of the tumor was removed (“clear margin”). The sample may be derived from a endometrial brushing specimen or endometrial biopsy specimen. 
         [0127]    The terms “nucleic acid” or “polynucleotide,” as used herein, refer to a deoxyribonucleotide or ribonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides which have similar or improved binding properties, for the purposes desired. The term also encompasses nucleic-acid-like structures with synthetic backbones. DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino), 3′-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs); see Oligonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Research and Applications (1993, CRC Press). PNAs contain non-ionic backbones, such as N-(2-aminoethyl)glycine units. Phosphorothioate linkages are described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197. Other synthetic backbones encompassed by the term include methyl-phosphonate linkages or alternating methylphosphonate and phosphodiester linkages (Strauss-Soukup (1997) Biochemistry 36: 8692-8698), and benzylphosphonate linkages (Samstag (1996) Antisense Nucleic Acid Drug Dev 6: 153-156). 
         [0128]    The terms “hybridizing specifically to,” “specific hybridization,” and “selectively hybridize to,” as used herein, refer to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions. The term “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 sequences. A “stringent hybridization” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization (e.g., as in array, Southern or Northern hybridization, or FISH) are sequence-dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in, e.g., Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes part I, Ch. 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays,” Elsevier, N.Y. (“Tijssen”). Generally, highly stringent hybridization and wash conditions are selected to be about 5° C. lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. The T m  is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. 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 (2001) Molecular Cloning: A Laboratory Manual (3rd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, and detailed discussion, below). 
         [0129]    A “chromosomal probe” or “chromosomal probe composition” refers to one or more polynucleotides that specifically hybridize to a region of a chromosome. The target sequences to which the probe can bind vary in length, e.g., from about 25,000 nucleotides to about 800,000 nucleotides. Smaller probes, e.g., that hybridize to a region of less than 100,000 nucleotides, or to a region of less than 10,000 nucleotides, can also be employed. Thus, in various embodiments, the probe can hybridize target sequences that are 25,000 nucleotides, 30,000 nucleotides, 50,000 nucleotides, 100,000 nucleotides, 150,000 nucleotides, 200,000 nucleotides, 250,000 nucleotides, 300,000 nucleotides, 350,000 nucleotides, 400,000 nucleotides, 450,000 nucleotides, 500,000 nucleotides, 550,000 nucleotides, 600,000 nucleotides, 650,000 nucleotides, 700,000 nucleotides, 750,000 nucleotides, or 800,00 nucleotides in length or that have a length falling in any range having any of these values as endpoints. A probe to a particular chromosomal region can include multiple polynucleotide fragments, e.g., ranging in size from about 50 to about 1,000 nucleotides in length. 
         [0130]    A chromosome enumeration probe (CEP) is any probe able to enumerate the number of specific chromosomes in a cell. 
         [0131]    The term “label containing moiety” or “detection moiety” generally refers to a molecular group or groups associated with a chromosomal probe, either directly or indirectly, that allows for detection of that probe upon hybridization to its target. 
         [0132]    The term “target region” or “nucleic acid target” refers to a nucleotide sequence that resides at a specific chromosomal location whose loss and/or gain is indicative of the presence of endometrial carcinoma. 
       INTRODUCTION 
       [0133]    The methods described herein are based, in part, on the identification of highly sensitive and specific chromosomal probe combinations that can be used to selectively detect endometrial carcinoma. The probe combinations provide higher sensitivity and specificity than individual probes. The probes encompass locus-specific probes as well as chromosome enumeration probes (CEPs), which typically hybridize to centromeric regions. The methods are carried by hybridizing one or more probes to nucleic acids from, e.g., cytology specimens (uterine brushings, washings, swabs) or cells from frozen specimens or fixed specimens, such as formalin-fixed, paraffin-embedded tissue. 
       Chromosomal Probes 
       [0134]    Probes for use in the invention are used for hybridization to nucleic acids that are present in biological samples from subjects where there is some degree of suspicion of endometrial carcinoma. In certain embodiments, the probes are labeled with detectable labels, e.g., fluorescent labels. 
         [0135]    Chromosome Enumeration Probe 
         [0136]    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. The centromere of a chromosome is typically considered to represent that chromosome entity 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 for the corresponding 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. Similarly, 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. 
         [0137]    In lieu of a centromeric probe for a chromosome, one of skill in the art will recognize that a chromosomal arm probe may alternately be used to approximate whole chromosomal loss or gain. However, 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 chromosomes. Examples of chromosome enumeration probes include CEP® probes (e.g., CEP® 12 and X/Y probes) commercially available from Abbott Molecular, DesPlaines, Ill. (formerly Vysis, Inc., Downers Grove, Ill.). 
         [0138]    Chromosome enumerator probes and locus-specific probes that target a chromosome region or subregion can readily be prepared by those in the art or can be obtained commercially, e.g., from Abbott Molecular, Molecular Probes, Inc. (Eugene, Oreg.), or Cytocell (Oxfordshire, UK). Such probes are prepared using standard techniques. Chromosomal probes may be prepared, for example, from protein nucleic acids, cloned human DNA such as plasmids, bacterial artificial chromosomes (BACs), and P1 artificial chromosomes (PACs) that contain inserts of human DNA sequences. A region of interest may be obtained via PCR amplification or cloning. Alternatively, chromosomal probes may be prepared synthetically. 
         [0139]    Locus-Specific Probes 
         [0140]    Probes that can be used in the method described herein include probes that selectively hybridize to chromosome regions (e.g., 1q, 2p, 2q, 3p, 3q, 7p, 8p, 8q, 9p, 9q, 10q, 15q, 16q, 17p, 18q, 19p, 20q, and 22q) or subregions of the chromosome regions (e.g., 1q25, 2p24, 2q26, 3p21, 3q27-q29, 7p21, 8p11, 8q24, 9q34, 10q23, 10q26, 15q11-q13, 16q24, 18q21, 20q12 and 20q13). (The subregion designations as used herein include the designated band and typically about 10 megabases of genomic sequence to either side.) Such probes are also referred to as “locus-specific probes.” A locus-specific probe selectively binds to a specific locus at a chromosomal region that is known to undergo gain or loss in endometrial carcinoma. 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. 
         [0141]    When targeting of a particular gene locus is desired, probes that hybridize along the entire length of the targeted gene are preferred in some embodiments, although not required. In specific embodiments, a locus-specific probe can be designed to hybridize to an oncogene or tumor suppressor gene, the genetic aberration of which is correlated with endometrial carcinoma. 
         [0142]    Probes useful in the methods described herein generally include a collection of one or more nucleic acid fragments whose hybridization to a target can be detected. Probes can be produced from a source of nucleic acids from one or more particular (preselected) portions of the genome, for example one or more clones, an isolated whole chromosome or chromosome fragment, or a collection of polymerase chain reaction (PCR) amplification products. Probes useful in the method described herein can be produced from nucleic acids found in the regions described herein. The probe may be processed in some manner, for example, by blocking or removal of repetitive nucleic acids or enrichment with unique nucleic acids. 
         [0143]    In certain embodiments, e.g., in in situ hybridization (e.g., FISH)-based embodiments, locus-specific probe targets preferably include at least 100,000 nucleotides. For cells of a given sample, relative to those of a control, increases or decreases in the number of signals for a probe indicate a gain or loss, respectively, for the corresponding region. 
         [0144]    Probes may also be employed as isolated nucleic acids immobilized on a solid surface (e.g., nitrocellulose), as in aCGH. In some embodiments, the probes may be members of an array of nucleic acids as described, for instance, in WO 96/17958, which is hereby incorporated by reference it its entirety and specifically for its description of array CGH. Techniques capable of producing high density arrays are well-known (see, e.g., Fodor et al. Science 767-773 (1991) and U.S. Pat. No. 5,143,854), both of which are hereby incorporated by reference for this description. 
         [0145]    As described in detail below, loci that were putatively associated with endometrial cancer were identified and the sensitivity and specificity of this association was examined in detail using array Comparative Genomic Hybridization (aCGH) and Fluoresence In Situ Hybridization (FISH). The clones selected from aCGH analysis included: LAMC2 (1q25-q31), MYCN (2p24.1), RASSF (3p21.3), TP63 (3q27-q29), IL6 (7p21), FGFR1 (8p11.2-p11.1), MYC (8q24), TSC1 (9q34), PTEN (10q23.3), FGFR2 (10q26), UBE3A/D15S10 (15q11-q13), FANCA (16q24.3), DCC (18q21.3), NCOA3 (20q12), and ZNF217 (20q13.2). The probes in FISH analysis included: 1q25, PTEN (10q23.3), DCC (18q21.2), CEP10, CEP18, FGFR1 (8p11.2), MYC (8q24), MYCN (2p24.3), PIK3CA (2q26.32), FGFR2 (10q26.13), and ZNF217 (20q13.2). New FISH probes were developed for NMYC, FGFR1, and FGFR2. 
         [0146]    Probe Selection Methods 
         [0147]    Probe combinations can be selected for their ability to simply detect endometrial carcinoma, but are typically selected for the ability to discriminate between endometrial carcinoma and other conditions. Thus, analyses of probe combinations are typically performed to determine the DFI values of different probe combinations for discriminating between endometrial carcinoma and other conditions or normal tissue. In particular embodiments, probe combinations can be analyzed to discriminate between endometriod and non-endometrioid types of endometrial carcinoma. 
         [0148]    Probe combinations for use in the methods of the present invention can be selected using the principles described in the examples. Combinations of chromosomal probes within a probe combination are chosen for sensitivity, specificity, and detectability regarding endometrial carcinoma. Sensitivity refers to the ability of a test (e.g. FISH) to detect disease (e.g. endometrial carcinoma) when it is present. More precisely, sensitivity is defined as True Positives/(True Positives+False Negatives). A test with high sensitivity has few false negative results, while a test with low sensitivity has many false negative results. In particular embodiments, the combination of probes has a sensitivity of least about: 93, 94, 95, 96, 97, 98, 99, or 100%, or a sensitivity falling in a range with any of these values as endpoints. 
         [0149]    Specificity, on the other hand, refers to the ability of test (e.g. FISH) to give a negative result when disease is not present. More precisely, specificity is defined as True Negatives/(True Negatives+False Positives). A test with high specificity has few false positive results, while a test with a low specificity has many false positive results. In certain embodiments, the combination of probes has a specificity of at about: 88, 89, 90, 91, 92, 93, 94, or 95%, or a specificity falling in a range with any of these values as endpoints. 
         [0150]    In general, chromosomal probe combinations with the highest combined sensitivity and specificity for the detection of endometrial carcinoma are preferred. In exemplary embodiments the combination of probes has a sensitivity and specificity of at least about: 93% and 88%, 95% and 90%, 96% and 91%, 97% and 92%, respectively, or any combination of sensitivity and specificity based on the values given above for each of these parameters. 
         [0151]    The combined sensitivity and specificity of a probe combination can be represented by the parameter distance from ideal (DFI), defined as [(1-sensitivity) 2 +(1-specificity) 2 ] 1/2  DFI values range from 0 to 1.414, with 0 representing a probe combination having 100% sensitivity and 100% specificity and 1.414 representing a probe combination with 0% sensitivity and 0% specificity. 
         [0152]    There is no limit to the number of probes that can be employed in a combination, although, in certain embodiments, no more than ten probes are combined. Additionally, in some embodiments, the number of probes within a set that is to be viewed by a human observer (and not with computer assisted imaging techniques) may be restricted for practical reasons, e.g., by the number of unique fluorophores that provide visually distinguishable signals upon hybridization. For example, typically four or five unique fluorophores (e.g., which appear as red, green, aqua, and gold signals to the human eye) can be conveniently employed in a single probe combination. Generally, the sensitivity of an assay increases as the number of probes within a set increases. However, the increases in sensitivity become smaller and smaller with the addition of more probes and at some point the inclusion of additional probes to a probe combination is not associated with significant increases in the sensitivity of the assay (“diminishing returns”). Increasing the number of probes in a probe combination may decrease the specificity of the assay. Accordingly, a probe combination of the present invention typically includes two, three, or four chromosomal probes, as necessary to provide optimal balance between sensitivity and specificity. 
         [0153]    Individual probes can be chosen for inclusion in a probe combination based on their ability to complement other probes within the combination. Specifically, they are targeted to chromosomes or chromosomal subregions that are not frequently altered simultaneously within a given endometrial carcinoma. Thus, each probe in a probe combination complements the other(s), i.e., identifies endometrial carcinoma where the other probes in the combination sometime fail to identify. One method for determining which probes complement one another is to identify single probes with the lowest DFI values for a group of tumor specimens. Then additional probes can be tested on the tumor samples that the initial probe failed to identify, and the probe with the lowest DFI value measured in combination with the initial probe(s) is added to the set. This may then be repeated until a full set of chromosomal probes with the desired DFI value is achieved. 
         [0154]    Discrimination analysis is one method that can be used to determine which probes are best able to detect endometrial carcinoma. This method assesses if individual probes are able to detect a statistically different percentage of abnormal cells in test specimens (e.g., endometrial carcinoma) when compared to normal specimens. The detection of cells with chromosomal (or locus) gains or chromosomal (or locus) losses can both be used to identify neoplastic cells in endometrial carcinoma patients. However, chromosomal losses sometimes occur as an artifact in normal cells because of random signal overlap and/or poor hybridization. In sections of formalin-fixed paraffin-embedded material, commonly used to assess biopsies, truncation of nuclei in the sectioning process can also produce artifactual loss of chromosomal material. Consequently, chromosomal gains are often a more reliable indicator of the presence of neoplastic cells. 
         [0155]    Cutoff values for individual chromosomal gains and losses must be determined when choosing a probe combination. The term “cutoff value” is intended to mean the value of a parameter associated with chromosomal aberration that divides a population of specimens into two groups—those specimens above the cutoff value and those specimens below the cutoff value. For example, the parameter may be the absolute number or percentage of cells in a population that have genetic aberrations (e.g., losses or gains for target regions). If the number or percentage of cells in the specimen harboring losses or gains for a particular probe is higher than the cutoff value, the sample is determined to be positive for endometrial carcinoma. 
         [0156]    Useful probe combinations are discussed in detail in the Example below. In exemplary combinations, one or more of a gain at one of more of 1q24, 8q24, CEP18, and 20q13 are indicative of endometrial carcinoma, as are (i) one or more of a gain at one of more of 1q25, 8q24, CEP18, and 20q13 and (ii) one or more of a 20q13 gain, a 1q24 gain, a CEP10 imbalance, and a 10q26 gain. Also of note are that different genomic changes were observed when comparing endometrioid and non-endometrioid subtypes. Gains in chromosomal arms 1q, 10p and 10q were common in endometrioid carcinomas. Multiple gains across the genome were identified in non-endometrioid carcinomas with the most common gains seen in 3q, 8q and 20q. 
       Probe Hybridization 
       [0157]    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 skill in the art. Such conditions typically involve controlled temperature, liquid phase, and contact between a chromosomal 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, both the probe and nucleic acid 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.times. 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. In order to increase specificity, use of a blocking agent such as unlabeled blocking nucleic acid as described in U.S. Pat. No. 5,756,696 (the contents of which are herein incorporated by reference in their entirety, and specifically for the description of the use of blocking nucleic acid), may be used in conjunction with the methods of the present invention. Other conditions may be readily employed for specifically hybridizing the probes to their nucleic acid targets present in the sample, as would be readily apparent to one of skill in the art. 
         [0158]    Upon completion of a suitable incubation period, 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. 
       Detection of Probe Hybridization Patterns 
       [0159]    The hybridization probes can be detected using any means known in the art. Label-containing moieties can be associated directly or indirectly with chromosomal probes. Different label-containing moieties can be selected for each individual probe within a particular combination so that each hybridized probe is visually distinct from the others upon detection. Where FISH is employed, the chromosomal probes can conveniently be labeled with distinct fluorescent label-containing moieties. In such embodiments, fluorophores, organic molecules that fluoresce upon irradiation at a particular wavelength, are typically directly attached to the chromosomal probes. A large number of fluorophores are commercially available in reactive forms suitable for DNA labeling. 
         [0160]    Attachment of fluorophores to nucleic acid probes is well known in the art and may be accomplished by any available means. 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, PCR labeling, and the like. Alternatively, the fluorophore can be covalently attached via a linker to the deoxycytidine nucleotides of the probe that have been transaminated. Methods for labeling probes are described in U.S. Pat. No. 5,491,224 and Molecular Cytogenetics: Protocols and Applications (2002), Y.-S. Fan, Ed., Chapter 2, “Labeling Fluorescence In Situ Hybridization Probes for Genomic Targets,” L. Morrison et al., p. 21-40, Humana Press, both of which are herein incorporated by reference for their descriptions of labeling probes. 
         [0161]    Exemplary fluorophores that can be used for labeling probes include TEXAS RED (Molecular Probes, Inc., Eugene, Oreg.), CASCADE blue aectylazide (Molecular Probes, Inc., Eugene, Oreg.), SPECTRUMORANGE™ (Abbott Molecular, Des Plaines, Ill.) and SPECTRUMGOLD™ (Abbott Molecular). 
         [0162]    One of skill in the art will recognize that other agents or dyes can be used in lieu of fluorophores as label-containing moieties. Suitable labels that can be attached to probes include, but are not limited to, radioisotopes, fluorophores, chromophores, mass labels, electron dense particles, magnetic particles, spin labels, molecules that emit luminescence, electrochemically active molecules, enzymes, cofactors, and enzyme substrates. Luminescent agents include, for example, radioluminescent, chemiluminescent, bioluminescent, and phosphorescent label containing moieties. Alternatively, detection moieties that are visualized by indirect means can be used. For example, 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 may be achieved as described above for FISH. 
         [0163]    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. 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium (NBT) serve as substrates for alkaline phosphatase, while diaminobenzidine serves as a substrate for HRP. 
         [0164]    In embodiments where fluorophore-labeled probes or probe compositions are used, 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 of the present invention 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. 
         [0165]    In array CGH, the probes are not labeled, but rather are immobilized at distinct locations on a substrate, as described in WO 96/17958. In this context, the probes are often referred to as the “target nucleic acids.” The sample nucleic acids are typically labeled to allow detection of hybridization complexes. The sample nucleic acids used in the hybridization may be detectably labeled prior to the hybridization reaction. Alternatively, a detectable label may be selected which binds to the hybridization product. In dual- or mult-color aCGH, the target nucleic acid array is hybridized to two or more collections of differently labeled nucleic acids, either simultaneously or serially. For example, sample nucleic acids (e.g., from endometrial carcinoma biopsy) and reference nucleic acids (e.g., from normal endometrium) are each labeled with a separate and distinguishable label. Differences in intensity of each signal at each target nucleic acid spot can be detected as an indication of a copy number difference. Although any suitable detectable label can be employed for aCGH, fluorescent labels are typically the most convenient. 
         [0166]    Preferred methods of visualizing signals are described in WO 93/18186, which is hereby incorporated by reference for this description. To facilitate the display of results and to improve the sensitivity of detecting small differences in fluorescence intensity, a digital image analysis system can be used. 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 filterwheel (Ludl Electronic Products, Ltd., Hawthorne, N.Y.). The filterwheel 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., Tucson, 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). 
       Screening and Diagnosis of Patients for Endometrial Carcinoma 
       [0167]    The detection methods of the invention include obtaining a biological sample from a subject having endometrial carcinoma or suspected of having endometrial carcinoma. The biological sample can be a cytology specimen, (e.g, uterine brushing, washing, or swab). In particular embodiments, the biological sample is a frozen or fixed specimen, such as formalin-fixed and paraffin embedded specimen. The sample is contacted with one or more chromosomal probe(s) to selectively detect endometrial carcinoma in the sample, if any, under conditions for specifically hybridizing the probes to their nucleic acid targets present in the sample. Probes of a combination can be hybridized concurrently or sequentially with the results of each hybridization imaged digitally, the probe or probes stripped, and the sample thereafter hybridized with the remaining probe or probes. Multiple probe combinations can also be hybridized to the sample in this manner. 
         [0168]    The biological sample can be from a patient suspected of having endometrial carcinoma or from a patient diagnosed with endometrial carcinoma, e.g., for confirmation of diagnosis or establishing a clear margin, or for the detection of endometrial carcinoma cells in other tissues such as lymph nodes. The biological sample can also be from a subject with an ambiguous diagnosis in order to clarify the diagnosis. The biological sample can also be from a subject with a histopathologically benign lesion to confirm the diagnosis. Biological samples can be obtained using any of a number of methods known in the art. 
         [0169]    As noted, a biological sample can be treated with a fixative such as formaldehyde and embedded in paraffin and sectioned for use in the methods of the invention. Alternatively, fresh or frozen tissue can be pressed against glass slides to form monolayers of cells known as touch preparations, which contain intact nuclei and do not suffer from the truncation artifact of sectioning. These cells may be fixed, e.g., in alcoholic solutions such as 100% ethanol or 3:1 methanol:acetic acid. Nuclei can also be extracted from thick sections of paraffin-embedded specimens to reduce truncation artifacts and eliminate extraneous embedded material. Typically, 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. 
       Prescreening of Samples 
       [0170]    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. During 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. Typically, 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.mu.g/ml to about 5.mu.g/ml, is a red-fluorescing DNA-specific dye that can be observed at an emission peak wavelength of 614 nm. DAPI, typically used at a concentration of about 125 ng/ml to about 1000 ng/ml, is a blue fluorescing DNA-specific stain that can be observed at an emission peak wavelength of 452 nm. In this case, only those cells pre-selected for detection are subjected to counting for chromosomal losses and/or gains. Preferably, 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. Preselection of a suspicious region on a tissue section may be performed on a serial section stained by conventional means, such as H&amp;E or PAP staining, and the suspect region marked by a pathologist or otherwise trained technician. The same region can then be located on the serial section stained by FISH and nuclei enumerated within that region. Within the marked region, enumeration may be limited to nuclei exhibiting abnormal characteristics as described above. 
         [0171]    Alternatively, 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. 
       Hybridization Pattern 
       [0172]    In Situ Hybridization 
         [0173]    The hybridization pattern for the set of chromosomal probes to the target regions is detected and recorded for cells chosen for assessment of chromosomal losses and/or gains. Hybridization is detected by the presence or absence of the particular signals generated by each of the chromosomal probes. The term “hybridization pattern” is intended to refer to the quantification of chromosomal losses/gains for those cells chosen for such assessment, relative to the number of the same in an evenly matched control sample, for each probe throughout a chosen cell sample. The quantification of losses/gains can include determinations that evaluate the ratio of one locus to another on the same or a different chromosome. Once the number of target regions within each cell is determined, as assessed by the number of regions showing hybridization to each probe, relative chromosomal gains and/or losses may be quantified. 
         [0174]    The relative gain or loss for each probe is determined by comparing the number of distinct probe signals in each cell to the number expected in a normal cell, i.e., where the copy number should be two. Non-neoplastic cells in the sample, such as keratinocytes, fibroblasts, and lymphocytes, can be used as reference normal cells. More than the normal number of probe signals is considered a gain, and fewer than the normal number is considered a loss. Alternatively, a minimum number of signals per probe per cell can be required to consider the cell abnormal (e.g., 5 or more signals). Likewise for loss, a maximum number of signals per probe can be required to consider the cell abnormal (e.g., 0 signals, or one or fewer signals). 
         [0175]    The percentages of cells with at least one gain and/or loss are to be recorded for each locus. A cell is considered abnormal if at least one of the identified genetic aberrations identified by a probe combination of the present invention is found in that cell. A sample may be considered positive for a gain or loss if the percentage of cells with the respective gain or loss exceeds the cutoff value for any probes used in an assay. Alternatively, two or more genetic aberrations can be required in order to consider the cell abnormal with the effect of increasing specificity. For example, wherein gains are indicative of a endometrial carcinoma, a sample is considered positive if it contains, for example, at least four cells showing gains of at least two or more probe-containing regions. 
         [0176]    aCGH 
         [0177]    Array CGH can be carried out in single-color or dual- or multi-color mode. In single-color mode, only the sample nucleic acids are labeled and hybridized to the nucleic acid array. Copy number differences can be detected by detecting a signal intensity at a particular target nucleic acid spot on the array that differs significantly from the signal intensity observed at one or more spots corresponding to one or more loci that are present in the sample nucleic acids at a normal copy number. To facilitate this determination, the array can include target elements for one or more loci that are not expected to show copy number difference(s) in endometrial carcinoma. 
         [0178]    In dual- or multi-color mode, signal corresponding to each labeled collection of nucleic acids (e.g., sample nucleic acids and normal, reference nucleic acids) is detected at each target nucleic acid spot on the array. The signals at each spot can be compared, e.g., by calculating a ratio. For example, if the ratio of sample nucleic acid signal to reference nucleic acid signal exceeds I, this indicates a gain in the sample nucleic acids at the locus corresponding to the target nucleic acid spot on the array. Conversely, if t if the ratio of sample nucleic acid signal to reference nucleic acid signal is less than 1, this indicates a loss in the sample nucleic acids at the corresponding locus. 
         [0000]    Other Methods of Detecting Copy Number Variations Associated with Endometrial Carcinoma 
         [0179]    Those of skill in the art appreciate that copy number variations at any of the loci described herein can be detected using other methods, including amplification-based methods and high-throughput DNA sequencing. 
         [0180]    Amplification-Based Detection 
         [0181]    In amplification-based assays, the target nucleic acids act as template(s) in amplification reaction(s) (e.g., Polymerase Chain Reaction (PCR)). In a quantitative amplification, the amount of amplification product is proportional to the amount of template in the original sample. Detailed protocols for quantitative PCR are provided in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.). A number of commercial quantitative PCR systems are available, for example the TaqMan system from Applied Biosystems. 
         [0182]    Other suitable amplification methods include, but are not limited to, ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560; Landegren et al. (1988) Science 241: 1077; and Barringer et al. (1990) Gene 89: 117), multiplex ligation-dependent probe amplification (MLPA), transcription amplification (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication (Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapter PCR, etc. 
         [0183]    Amplification is typically carried out using primers that specifically amplify one or more loci within each chromosome or chromosomal subregion to be queried. Detection can be carried out by any standard means, including a target-specific probe, a universal probe that binds, e.g., to a sequence introduced into all amplicons via one or both primers, or a double-stranded DNA-binding dye (such as, e.g., SYBR Green). In illustrative embodiments, padlock probes or molecular inversion probes are employed for detection. 
         [0184]    High-Throughput DNA Sequencing 
         [0185]    In particular embodiments, amplification methods are employed to produce amplicons suitable for high-throughput (i.e., automated) DNA sequencing. Generally, amplification methods that provide substantially uniform amplification of target nucleotide sequences are employed in preparing DNA sequencing libraries having good coverage. In the context of automated DNA sequencing, the term “coverage” refers to the number of times the sequence is measured upon sequencing. The counts obtained are typically normalized relative to a reference sample or samples to determine relative copy number. Thus, upon performing automated sequencing of a plurality of target amplicons, the normalized number of times the sequence is measured reflects the number of target amplicons including that sequence, which, in turn, reflects the number of copies of the target sequence in the sample DNA. 
         [0186]    Amplification for sequencing may involve emulsion PCR isolates in which individual DNA molecules along with primer-coated beads are present in aqueous droplets within an oil phase. Polymerase chain reaction (PCR) then coats each bead with clonal copies of the DNA molecule followed by immobilization for later sequencing. Emulsion PCR is used in the methods by Marguilis et al. (commercialized by 454 Life Sciences), Shendure and Porreca et al. (also known as “Polony sequencing”) and SOLiD sequencing, (developed by Agencourt, now Applied Biosystems). Another method for in vitro clonal amplification for sequencing is bridge PCR, where fragments are amplified upon primers attached to a solid surface, as used in the Illumina Genome Analyzer. Some sequencing methods do not require amplification, for example, the single-molecule method developed by the Quake laboratory (later commercialized by Helicos). This method uses bright fluorophores and laser excitation to detect pyrosequencing events from individual DNA molecules fixed to a surface. Pacific Biosciences has also developed a single molecule sequencing approach that does not require amplification. 
         [0187]    After in vitro clonal amplification (if necessary), DNA molecules that are physically bound to a surface are sequenced. Sequencing by synthesis, like dye-termination electrophoretic sequencing, uses a DNA polymerase to determine the base sequence. Reversible terminator methods (used by Illumina and Helicos) use reversible versions of dye-terminators, adding one nucleotide at a time, and detect fluorescence at each position in real time, by repeated removal of the blocking group to allow polymerization of another nucleotide. Pyrosequencing (used by 454) also uses DNA polymerization, adding one nucleotide species at a time and detecting and quantifying the number of nucleotides added to a given location through the light emitted by the release of attached pyrophosphates. 
         [0188]    Pacific Biosciences Single Molecule Real Time (SMRT™) sequencing relies on the processivity of DNA polymerase to sequence single molecules and uses phospholinked nucleotides, each type labeled with a different colored fluorophore. As the nucleotides are incorporated into a complementary DNA strand, each is held by the DNA polymerase within a detection volume for a greater length of time than it takes a nucleotide to diffuse in and out of that detection volume. The DNA polymerase then cleaves the bond that previously held the fluorophore in place and the dye diffuses out of the detection volume so that fluorescence signal returns to background. The process repeats as polymerization proceeds. 
         [0189]    Sequencing by ligation uses a DNA ligase to determine the target sequence. Used in the Polony method and in the SOLiD technology, this method employs a pool of all possible oligonucleotides of a fixed length, labeled according to the sequenced position. Oligonucleotides are annealed and ligated; the preferential ligation by DNA ligase for matching sequences results in a signal informative of the nucleotide at that position. 
         [0000]    Probe Combinations and Kits for Use in Diagnostic and/or Prognostic Applications 
         [0190]    The invention includes highly specific and sensitive combinations of probes, as described herein, that can be used to detect endometrial carcinoma and kits for use in diagnostic, research, and prognostic applications. Kits include probe combinations and can also include reagents such as buffers and the like. The kits may include instructional materials containing directions (i.e., protocols) for the practice of the methods of this invention. While the instructional materials typically include written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials. 
       REFERENCES 
       [0000]    
       
         1. Int J Mol. Med. 2003 November; 12(5):727-31. 11q23-24 loss is associated with chromosomal instability in endometrial cancer. Kawasaki K, Suehiro Y, Umayahara K, Morioka H, Ito T, Saito T, Tsukamoto N, Sugino N, Kato H, Sasaki K. 
         2. Hum Genet. 1997 October; 100 (5-6):629-36. Frequent gains on chromosome arms 1q and/or 8q in human endometrial cancer. Suzuki A, Fukushige S, Nagase S, Ohuchi N, Satomi S, Horii A. 
         3. Cancer Genet Cytogenet. 2008 February; 181 (1):25-30. Recurrent gene amplifications in human type I endometrial adenocarcinoma detected by fluorescence in situ hybridization. Samuelson E, Levan K, Adamovic T, Levan G, Horvath G. 
         4. Cytogenet Genome Res. 2006; 115 (1):16-22. Chromosomal alterations in 98 endometrioid adenocarcinomas analyzed with comparative genomic hybridization. Levan K, Partheen K, Osterberg L, Helou K, Horvath G. 
         5. Eur J Obstet Gynecol Reprod Biol. 2005 May 1; 120 (1):107-14. Genetic imbalances in endometrial hyperplasia and endometrioid carcinoma detected by comparative genomic hybridization. Muslumanoglu H M, Oner U, Ozalp S, Acikalin M F, Yalcin O T, Ozdemir M, Artan S. 
         6. Am J Clin Pathol. 2004 October; 122 (4):546-51. Overrepresentation of 8q in carcinosarcomas and endometrial adenocarcinomas. Schulten H J, Gunawan B, Enders C, Donhuijsen K, Emons G, Füzesi L. 
         7. Hum Genet. 1997 October; 100 (5-6):629-36. Frequent gains on chromosome arms 1q and/or 8q in human endometrial cancer. Suzuki A, Fukushige S, Nagase S, Ohuchi N, Satomi S, Horii A. 
         8. Genes Chromosomes Cancer. 1997 February; 18 (2):115-25. Detection of DNA gains and losses in primary endometrial carcinomas by comparative genomic hybridization. Sonoda G, du Manoir S, Godwin A K, Bell D W, Liu Z, Hogan M, Yakushiji M, Testa J R. 
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         14. Kipp B R, Medeiros F, Campion M B, Distad T J, Peterson L M, Keeney G L, et al. Direct uterine sampling with the Tao brush sampler using a liquid-based preparation method for the detection of endometrial cancer and atypical hyperplasia: a feasibility study. Cancer 2008; 114 (4):228-35. 
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         [0208]    All publications cited herein are explictly incorporated by reference. 
       EXAMPLES 
       [0209]    The following examples are offered to illustrate, but not to limit the claimed invention. 
       Example 1 
     Array CGH Studies 
       [0210]    Study Design 
         [0211]    In a collaborative study of Abbott Molecular and Mayo Clinic carried out in 2007-2009, fresh-frozen, biopsy from proven endometrial carcinoma specimens from 62 patients were obtained with informed consent at the time of surgery from Rochester Methodist Hospital at Mayo Clinic, Rochester, Minn. Serial 5-μm sections were cut from the collected tissue and sections were stained with hematoxylin and eosin (H&amp;E) for histological analysis. Specimens with greater than 70% tumor were selected for aCGH analysis and remaining sections were left unstained for future clinical studies. 
         [0212]    DNA from the selected specimens was extracted and amplified prior to CGH analysis by the GenoSensor™ system. Vysis GenoSensor DNA microarray slides obtained from Abbott Molecular (Des Plaines, Ill.) were used for CGH analysis. The microarray slides contained 287 DNA targets consisting of known oncogenes, tumor suppressor genes, and regions of gain, loss, or loss of heterozygosity commonly associated with cancer (Attachment 1). The targets were arrayed in triplicate from BAC libraries. 
         [0213]    Test and normal reference DNA samples were random-prime labeled, using Vysis GeneSensor labeling reagents, with Cyanine-3-dCTP, and Cyanine-5-dCTP (Perkin Elmer/NEN), respectively. Random priming refers to the process whereby synthetic DNA octamers of random sequence bind to complementary DNA sequences (along a DNA template) and serve as templates for DNA synthesis and elongation by DNA Polymerase I (Klenow Fragment). Following additional purification, test and reference DNA were mixed in equal proportion (in hybridization buffer), denatured, and hybridized against the Vysis GenoSensor™ Array 300 human genomic DNA microarray. Hybridization proceeded at 30° C. for 72 hours, followed by washing and scanning of arrays. Array images were analyzed with GenoSensor™ software, which segments and identifies each target using the blue (DAPI) image plane. The software measures mean intensities from the green and red image planes, subtracts background, determines mean ratio of green/red signal, and calculates the ratio most representative of the modal DNA copy number of the sample DNA. Gender mismatched (male/female or female/male) hybridizations provided control with respect to the detection of autosomal copy number imbalances. Arrays were hybridizes at Mayo Clinic and aCGH data obtained was analyzed by Abbott Molecular. 
         [0214]    In total, data for 57 Cancer specimens (44 endometrioid and 13 non-endometrioid) and 9 normal specimens was available for analysis. 
         [0215]    Results 
         [0216]    The CGH data revealed several promising genomic targets for these investigational FISH probe sets ( FIG. 1 ). The most frequent gains observed in all endometrial carcinomas included areas on chromosomal arms 1q, 2p, 3q, 8q, and 20q. Most common losses seen in all carcinomas included 9p, 9q, 16q, 17p, 18q, and 22q. Interestingly, different genomic changes were observed when comparing endometrioid and non-endometrioid subtypes. Gains in chromosomal arms 1q, 10p and 10q were common in endometrioid carcinomas. Multiple gains across the genome were identified in non-endometrioid carcinomas with the most common gains seen in 3q, 8q and 20q. 
         [0217]    Gross analysis of chromosomal changes demonstrated that most frequent changes, of all cancers analyzed, were as follows: 
         [0218]    1. Gains on 1q31-qtel (20-30%) 
         [0219]    2. Gains on 3q, 8q and 10q (about 20%) 
         [0220]    3. Gain on 2p (about 15%) 
         [0221]    4. Losses on 9q (about 18%) 
         [0222]    5. Losses on 15q11-q13 (about 18%) 
         [0223]    6. Losses on 18q21.3 and 19 ptel (about 18-19%) 
         [0224]    7. Losses 16q and 17p (about 12%) 
         [0225]    Table 1 provides details of the most frequently affected loci (by array clone). 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Loci with the frequently of genomic copy number imbalance of &gt;10% in cancer specimens. 
               
             
          
           
               
                   
                   
                   
                   
                   
                 FREQ 
                 FREQ 
                 FREQ 
                 FREQ 
                 FREQ 
                 FREQ 
                 FREQ 
               
               
                   
                   
                   
                 FREQ 
                 FREQ 
                 GAIN 
                 LOSS 
                 IMBAL 
                 GAIN 
                 LOSS 
                 GAIN 
                 LOSS 
               
               
                 Clone 
                   
                 Cyto 
                 GAIN 
                 LOSS 
                 Ca 
                 Ca 
                 Ca 
                 EN 
                 EN 
                 NE 
                 NE 
               
               
                 # 
                 Clone Name 
                 Location 
                 (%) 
                 (%) 
                 (%) 
                 (%) 
                 (%) 
                 (%) 
                 (%) 
                 (%) 
                 (%) 
               
               
                   
               
             
          
           
               
                 8 
                 AMC2 
                 q25-q31 
                 7.27 
                 .00 
                 1.58 
                 .00 
                 1.58 
                 4.09 
                 .00 
                 3.08 
                 .00 
               
               
                 0 
                 GFB2 
                 q41 
                 2.73 
                 .00 
                 6.32 
                 .00 
                 6.32 
                 1.82 
                 .00 
                 .69 
                 .00 
               
               
                 7 
                 I-5663, 
                 q21 
                 1.21 
                 .00 
                 4.56 
                 .00 
                 4.56 
                 7.27 
                 .00 
                 5.38 
                 .00 
               
               
                   
                 I-13414 
               
               
                 1 
                 KT3 
                 q44 
                 2.73 
                 .00 
                 4.56 
                 .00 
                 4.56 
                 9.55 
                 .00 
                 .69 
                 .00 
               
               
                 2 
                 HGC-18290 
                 q tel 
                 2.73 
                 .00 
                 4.56 
                 .00 
                 4.56 
                 9.55 
                 .00 
                 .69 
                 .00 
               
               
                 36 
                 29F16/SP6 
                 9p tel 
                 .03 
                 6.67 
                 .51 
                 9.30 
                 2.81 
                 .27 
                 8.18 
                 .69 
                 3.08 
               
               
                 3 
                 QTEL10 
                 q tel 
                 6.67 
                 .00 
                 9.30 
                 .00 
                 9.30 
                 5.00 
                 .00 
                 .00 
                 .00 
               
               
                 6 
                 YCN(N-myc) 
                 p24.1 
                 2.12 
                 .55 
                 4.04 
                 .26 
                 9.30 
                 .82 
                 .82 
                 8.46 
                 .00 
               
               
                 4 
                 FI2 
                 q tel 
                 6.67 
                 .00 
                 9.30 
                 .00 
                 9.30 
                 1.36 
                 .00 
                 6.15 
                 .00 
               
               
                 10 
                 YC 
                 q24.12- 
                 6.67 
                 .00 
                 9.30 
                 .00 
                 9.30 
                 .82 
                 .00 
                 1.54 
                 .00 
               
               
                   
                   
                 q24.13 
               
               
                 11 
                 TK2 
                 q24-qter 
                 6.67 
                 .00 
                 9.30 
                 .00 
                 9.30 
                 .82 
                 .00 
                 1.54 
                 .00 
               
               
                 22 
                 SC1 
                 q34 
                 .52 
                 5.15 
                 .75 
                 7.54 
                 9.30 
                 .27 
                 .09 
                 .00 
                 6.15 
               
               
                 35 
                 GFR2 
                 0q26 
                 6.67 
                 .00 
                 9.30 
                 .00 
                 9.30 
                 0.45 
                 .00 
                 5.38 
                 .00 
               
               
                 82 
                 BE3A, 
                 5q11-q13 
                 .52 
                 5.15 
                 .75 
                 7.54 
                 9.30 
                 .27 
                 3.64 
                 .00 
                 0.77 
               
               
                   
                 15S10 
               
               
                 31 
                 CC 
                 8q21.3 
                 .52 
                 6.67 
                 .75 
                 7.54 
                 9.30 
                 .27 
                 .82 
                 .00 
                 3.85 
               
               
                 3 
                 P63 
                 q27-q29 
                 5.15 
                 .00 
                 7.54 
                 .00 
                 7.54 
                 .09 
                 .00 
                 6.15 
                 .00 
               
               
                 06 
                 GFR1 
                 p11.2- 
                 2.12 
                 .03 
                 4.04 
                 .51 
                 7.54 
                 .09 
                 .27 
                 0.77 
                 .69 
               
               
                   
                   
                 p11.1 
               
               
                 21 
                 BCCR1 
                 q33.2 
                 .52 
                 3.64 
                 .75 
                 5.79 
                 7.54 
                 .27 
                 .82 
                 .00 
                 6.15 
               
               
                 6 
                 QTEL11 
                 q tel 
                 0.61 
                 .55 
                 0.53 
                 .26 
                 5.79 
                 3.64 
                 .27 
                 .00 
                 5.38 
               
               
                 02 
                 8S596 
                 p tel 
                 .58 
                 .06 
                 .77 
                 .02 
                 5.79 
                 .82 
                 .55 
                 5.38 
                 5.38 
               
               
                 96 
                 RA16D 
                 6q23.2 
                 .03 
                 0.61 
                 .51 
                 2.28 
                 5.79 
                 .00 
                 .09 
                 5.38 
                 3.08 
               
               
                 70 
                 F2 
                 2q12.2 
                 .58 
                 .06 
                 .77 
                 .02 
                 5.79 
                 .82 
                 .82 
                 5.38 
                 .69 
               
               
                 71 
                 DGFB(SIS) 
                 2q13.1 
                 .69 
                 .15 
                 .77 
                 .02 
                 5.79 
                 .82 
                 .27 
                 5.38 
                 3.08 
               
               
                 9 
                 TGS2(COX2) 
                 q31.1 
                 3.64 
                 .00 
                 5.79 
                 .00 
                 5.79 
                 0.45 
                 .00 
                 .00 
                 .00 
               
               
                 7 
                 SH2, KCNK12 
                 p22.3- 
                 3.64 
                 .00 
                 5.79 
                 .00 
                 5.79 
                 3.64 
                 .00 
                 3.08 
                 .00 
               
               
                   
                   
                 2p22.1 
               
               
                 09 
                 XT1 
                 q24.11- 
                 3.64 
                 .00 
                 5.79 
                 .00 
                 5.79 
                 .82 
                 .00 
                 6.15 
                 .00 
               
               
                   
                   
                 q24.13 
               
               
                 12 
                 HGC-3110 
                 q tel 
                 3.64 
                 .00 
                 5.79 
                 .00 
                 5.79 
                 .55 
                 .00 
                 3.85 
                 .00 
               
               
                 6 
                 1S2465, 
                 p12 
                 0.61 
                 .52 
                 2.28 
                 .75 
                 4.04 
                 .82 
                 .27 
                 0.77 
                 .00 
               
               
                   
                 1S3402 
               
               
                 99 
                 ANCA 
                 6q24.3 
                 .52 
                 0.61 
                 .75 
                 2.28 
                 4.04 
                 .27 
                 .09 
                 .00 
                 3.08 
               
               
                 0 
                 L6 
                 p21 
                 2.12 
                 .00 
                 4.04 
                 .00 
                 4.04 
                 1.36 
                 .00 
                 3.08 
                 .00 
               
               
                 01 
                 8S504 
                 p tel 
                 .06 
                 .06 
                 .02 
                 .02 
                 4.04 
                 .82 
                 .55 
                 .69 
                 5.38 
               
               
                 37 
                 tSG27915 
                 0q tel 
                 2.12 
                 .00 
                 4.04 
                 .00 
                 4.04 
                 5.91 
                 .00 
                 .69 
                 .00 
               
               
                 97 
                 DH13 
                 6q24.2- 
                 .00 
                 2.12 
                 .00 
                 4.04 
                 4.04 
                 .00 
                 .09 
                 .00 
                 0.77 
               
               
                   
                   
                 q24.3 
               
               
                 5 
                 PTEL27 
                 p tel 
                 0.61 
                 .00 
                 2.28 
                 .00 
                 2.28 
                 1.36 
                 .00 
                 5.38 
                 .00 
               
               
                 8 
                 2S447 
                 q tel 
                 .06 
                 .55 
                 .02 
                 .26 
                 2.28 
                 .09 
                 .27 
                 .00 
                 5.38 
               
               
                 5 
                 ASSF1 
                 p21.3 
                 0.61 
                 .00 
                 2.28 
                 .00 
                 2.28 
                 .09 
                 .00 
                 3.08 
                 .00 
               
               
                 7 
                 4S2930 
                 q tel 
                 .03 
                 .58 
                 .51 
                 .77 
                 2.28 
                 .55 
                 .27 
                 .00 
                 0.77 
               
               
                 8 
                 84C11/T3 
                 p tel 
                 .09 
                 .52 
                 0.53 
                 .75 
                 2.28 
                 .09 
                 .00 
                 5.38 
                 .69 
               
               
                 2 
                 TR1B 
                 q13 
                 .09 
                 .52 
                 0.53 
                 .75 
                 2.28 
                 .09 
                 .27 
                 5.38 
                 .00 
               
               
                 36 
                 MBT1 
                 0q25.3- 
                 .09 
                 .52 
                 0.53 
                 .75 
                 2.28 
                 3.64 
                 .00 
                 .00 
                 .69 
               
               
                   
                   
                 q26.1 
               
               
                 78 
                 GH(D14S308) 
                 4q tel 
                 2.12 
                 .00 
                 2.28 
                 .00 
                 2.28 
                 .09 
                 .00 
                 3.08 
                 .00 
               
               
                 08 
                 17S125, 
                 7p12-p11.2 
                 .00 
                 0.61 
                 .00 
                 2.28 
                 2.28 
                 .00 
                 .55 
                 .00 
                 8.46 
               
               
                   
                 17S61 
               
               
                 10 
                 LGL1 
                 7p12- 
                 .00 
                 0.61 
                 .00 
                 2.28 
                 2.28 
                 .00 
                 .82 
                 .00 
                 0.77 
               
               
                   
                   
                 17p11.2 
               
               
                 48 
                 OP1 
                 0q12-q13.1 
                 .09 
                 .52 
                 0.53 
                 .75 
                 2.28 
                 .55 
                 .27 
                 0.77 
                 .00 
               
               
                 49 
                 COA3(AIB1) 
                 0q12 
                 0.61 
                 .00 
                 2.28 
                 .00 
                 2.28 
                 .27 
                 .00 
                 6.15 
                 .00 
               
               
                 57 
                 PD52L2, TOM 
                 0q tel 
                 0.61 
                 .00 
                 2.28 
                 .00 
                 2.28 
                 .55 
                 .00 
                 8.46 
                 .00 
               
               
                 69 
                 CR 
                 2q11.23 
                 .55 
                 .58 
                 .51 
                 .77 
                 2.28 
                 .00 
                 .82 
                 5.38 
                 5.38 
               
               
                   
               
             
          
         
       
     
         [0226]    As evident from  FIG. 2 , Non-Endometrioid (NE) tumors had overall greater number of genomic changes as compared to Endometrioid (EN) tumors. In addition, the pattern of changes was different between EN and NE tumors. For example, changes at 1q and 10q loci were more prevalent in EN tumors, while changes at 8q and 18q appeared more prevalent in NE tumors. 
         [0227]    Sensitivity and Specificity Analysis 
         [0228]    Analysis was carried out to determine changes in which loci are most abundant in cancers (both NE and EN) and give the highest sensitivity and specificity in detecting cancer. 
         [0229]    First, analysis has been carried out on individual loci and the loci with the best sensitivity and specificity value as represented by the DFI parameter calculated as: 
         [0000]      DFI=√{square root over ((1−SENS) 2 +(1−SPEC) 2 )}{square root over ((1−SENS) 2 +(1−SPEC) 2 )}
 
         [0000]    were selected. As shown below, the best performance was demonstrated by loci located on the long arm of chromosome 1. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                   
                 case # 
                   
                 SPEC vs 
                 DFI vs 
               
               
                 PROBE 1 
                 specimens 
                 SENS 
                 norm 
                 norm 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 LAMC2:18:1q25-q31 
                 57 
                 0.3158 
                 1.0000 
                 0.6842 
               
               
                 TGFB2:20:1q41 
                 57 
                 0.2632 
                 1.0000 
                 0.7368 
               
               
                 WI-5663, WI- 
                 57 
                 0.2456 
                 1.0000 
                 0.7544 
               
               
                 13414:17:1q21 
               
               
                 SHGC-18290:22:1q tel 
                 57 
                 0.2456 
                 1.0000 
                 0.7544 
               
               
                 AKT3:21:1q44 
                 57 
                 0.2456 
                 0.8889 
                 0.7625 
               
               
                   
               
             
          
         
       
     
         [0230]    Then, we considered additional clones, and chosen a representative probe for a segment where several probes were located in one apparent contiguous region of rearrangement. It is evident (Table 2) that the sensitivity and specificity of the individual loci is low. Therefore, combinations of the loci listed below were evaluated. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Individual clone performance for selected clones. 
               
             
          
           
               
                   
                 control 
                 case 
                   
                   
                   
               
             
          
           
               
                   
                 # 
                   
                 # 
                   
                 Case 
                 Control 
                   
               
             
          
           
               
                   
                   
                 speci- 
                   
                 speci- 
                   
                 SPEC 
                 DFI 
                 marker 
                 marker 
                 marker 
                 marker 
                   
               
               
                 Clone 
                   
                 mens 
                 SENS 
                 mens 
                 SENS 
                 vs norm 
                 vs norm 
                 (s)+ 
                 (s)− 
                 (s)+ 
                 (s)− 
                 chi sq prob 
               
               
                   
               
             
          
           
               
                 LAMC2:18:1q25-q31 
                 abnorm 
                 9 
                 0.0000 
                 57 
                 0.3158 
                 1.0000 
                 0.6842 
                 18 
                 39 
                 0 
                 9 
                 4.8060E−02 
               
               
                 MYCN(N-myc):26:2p24.1 
                 abnorm 
                 9 
                 0.0000 
                 57 
                 0.1930 
                 1.0000 
                 0.8070 
                 11 
                 46 
                 0 
                 9 
                 1.4883E−01 
               
               
                 RASSF1:45:3p21.3 
                 abnorm 
                 9 
                 0.0000 
                 57 
                 0.1228 
                 1.0000 
                 0.8772 
                 7 
                 50 
                 0 
                 9 
                 2.6617E−01 
               
               
                 TP63:53:3q27-q29 
                 abnorm 
                 9 
                 0.0000 
                 57 
                 0.1754 
                 1.0000 
                 0.8246 
                 10 
                 47 
                 0 
                 9 
                 1.7252E−01 
               
               
                 IL6:90:7p21 
                 abnorm 
                 9 
                 0.0000 
                 57 
                 0.1404 
                 1.0000 
                 0.8596 
                 8 
                 49 
                 0 
                 9 
                 2.3056E−01 
               
               
                 FGFR1:106:8p11.2-p11.1 
                 abnorm 
                 9 
                 0.0000 
                 57 
                 0.1754 
                 1.0000 
                 0.8246 
                 10 
                 47 
                 0 
                 9 
                 1.7252E−01 
               
               
                 MYC:110:8q24.12-q24.13 
                 abnorm 
                 9 
                 0.0000 
                 57 
                 0.1930 
                 1.0000 
                 0.8070 
                 11 
                 46 
                 0 
                 9 
                 1.4883E−01 
               
               
                 TSC1:122:9q34 
                 abnorm 
                 9 
                 0.0000 
                 57 
                 0.1930 
                 1.0000 
                 0.8070 
                 11 
                 46 
                 0 
                 9 
                 1.4883E−01 
               
               
                 PTEN:134:10q23.3 
                 abnorm 
                 9 
                 0.2222 
                 57 
                 0.0702 
                 0.7778 
                 0.9560 
                 4 
                 53 
                 2 
                 7 
                 1.4034E−01 
               
               
                 FGFR2:135:10q26 
                 abnorm 
                 9 
                 0.0000 
                 57 
                 0.1930 
                 1.0000 
                 0.8070 
                 11 
                 46 
                 0 
                 9 
                 1.4883E−01 
               
               
                 UBE3A, D15S10:182:15q11-q13 
                 abnorm 
                 9 
                 0.0000 
                 57 
                 0.1930 
                 1.0000 
                 0.8070 
                 11 
                 46 
                 0 
                 9 
                 1.4883E−01 
               
               
                 FANCA:199:16q24.3 
                 abnorm 
                 9 
                 0.0000 
                 57 
                 0.1404 
                 1.0000 
                 0.8596 
                 8 
                 49 
                 0 
                 9 
                 2.3056E−01 
               
               
                 DCC:231:18q21.3 
                 abnorm 
                 9 
                 0.1111 
                 57 
                 0.1930 
                 0.8889 
                 0.8146 
                 11 
                 46 
                 1 
                 8 
                 5.5398E−01 
               
               
                 NCOA3(AIB1):249:20q12 
                 abnorm 
                 9 
                 0.0000 
                 57 
                 0.1228 
                 1.0000 
                 0.8772 
                 7 
                 50 
                 0 
                 9 
                 2.6617E−01 
               
               
                 ZNF217(ZABC1):254:20q13.2 
                 abnorm 
                 9 
                 0.0000 
                 57 
                 0.0702 
                 1.0000 
                 0.9298 
                 4 
                 53 
                 0 
                 9 
                 4.1224E−01 
               
               
                   
               
             
          
         
       
     
         [0231]    Complementation between the selected loci, in all of the tested specimens is represented in  FIG. 3 . 
         [0232]    Sensitivity and specificity in tumor detection was then evaluated using JMP 8.0 statistical analysis software (SAS Institute), utilizing Fit X by Y contingency table analysis. In this analysis, all 13 loci, and groups of 10, 9 and 8 complimentary clones were evaluated. A sample was called positive when either one of the loci (at least one locus) in the group was positive. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
               
               
               
               
             
               
               
               
             
               
             
               
               
             
               
               
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
               
               
               
               
             
               
               
               
             
               
             
               
               
             
               
               
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
               
               
               
               
             
               
               
               
             
               
             
               
               
             
               
               
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
               
               
               
               
             
               
               
               
             
           
               
                   
               
             
             
               
                 1. A set of all 14 clones: 
               
             
          
           
               
                   
                 Pos/Neg 14 Loci 
                   
               
             
          
           
               
                   
                   
                 Row % 
                 NEG 
                 POS 
               
               
                   
                   
               
               
                   
                 Tumor           
                 NORMAL 
                 88.89 
                 11.11 
               
               
                   
                   
                 TUMOR 
                 28.07 
                 71.93 
               
               
                   
                   
               
             
          
           
               
                 Tests 
               
             
          
           
               
                   
                 N 
                 DF 
                 −LogLike 
                 RSquare (U) 
               
               
                   
                   
               
               
                   
                 990 
                 1 
                 94.293904 
                 0.1453 
               
               
                   
                   
               
             
          
           
               
                   
                 Test 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
                   
               
               
                   
                 Likelihood Ratio 
                 188.588 
                 &lt;.0001* 
               
               
                   
                 Pearson 
                 186.366 
                 &lt;.0001* 
               
               
                   
                   
               
             
          
           
               
                 Fisher&#39;s 
                   
                   
               
               
                 Exact Test 
                 Prob 
                 Alternative Hypothesis 
               
               
                   
               
               
                 Left 
                 1.0000 
                 Prob(Pos/Neg 14 Loci = POS) is greater for 
               
               
                   
                   
                 Tumor? = NORMAL than TUMOR 
               
               
                 Right 
                 &lt;.0001* 
                 Prob(Pos/Neg 14 Loci = POS) is greater for 
               
               
                   
                   
                 Tumor? = TUMOR than NORMAL 
               
               
                 2-Tail 
                 &lt;.0001* 
                 Prob(Pos/Neg 14 Loci = POS) is different 
               
               
                   
                   
                 across Tumor? 
               
               
                   
               
             
          
           
               
                 2. A representative subset of 10 clones: 
               
             
          
           
               
                   
                 DCC 
               
               
                   
                 FGFR2 
               
               
                   
                 IL6 
               
               
                   
                 LAMC2 
               
               
                   
                 MYC 
               
               
                   
                 MYCN(N-myc) 
               
               
                   
                 PTEN 
               
               
                   
                 RASSF1 
               
               
                   
                 TSC1 
               
               
                   
                 UBE3A, D15S10 
               
             
          
           
               
                   
                 Pos/Neg 10 
                   
               
             
          
           
               
                   
                   
                 Row % 
                 NEG 
                 POS 
               
               
                   
                   
               
               
                   
                            umor? 
                 NORMAL 
                 66.67 
                 33.33 
               
               
                   
                   
                 TUMOR 
                 26.32 
                 73.68 
               
               
                   
                   
               
             
          
           
               
                 Tests 
               
             
          
           
               
                   
                 N 
                 DF 
                 −LogLike 
                 RSquare (U) 
               
               
                   
                   
               
               
                   
                 924 
                 1 
                 37.838597 
                 0.0655 
               
               
                   
                   
               
             
          
           
               
                   
                 Test 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
                   
               
               
                   
                 Likelihood Ratio 
                 75.677 
                 &lt;.0001* 
               
               
                   
                 Pearson 
                 81.670 
                 &lt;.0001* 
               
               
                   
                   
               
             
          
           
               
                 Fisher&#39;s 
                   
                   
               
               
                 Exact Test 
                 Prob 
                 Alternative Hypothesis 
               
               
                   
               
               
                 Left 
                 1.0000 
                 Prob(Pos/Neg 10 = POS) is greater for 
               
               
                   
                   
                 Tumor? = NORMAL than TUMOR 
               
               
                 Right 
                 &lt;.0001* 
                 Prob(Pos/Neg 10 = POS) is greater for 
               
               
                   
                   
                 Tumor? = TUMOR than NORMAL 
               
               
                 2-Tail 
                 &lt;.0001* 
                 Prob(Pos/Neg 10 = POS) is different 
               
               
                   
                   
                 across Tumor? 
               
               
                   
               
             
          
           
               
                 3. A representative subset of 9 clones 
               
             
          
           
               
                   
                 DCC 
               
               
                   
                 FGFR2 
               
               
                   
                 IL6 
               
               
                   
                 LAMC2 
               
               
                   
                 MYC 
               
               
                   
                 MYCN(N-myc) 
               
               
                   
                 RASSF1 
               
               
                   
                 TSC1 
               
               
                   
                 UBE3A, D15S10 
               
             
          
           
               
                   
                 Pos/Neg 9 
                   
               
             
          
           
               
                   
                   
                 Row % 
                 NEG 
                 POS 
               
               
                   
                   
               
               
                   
                 Tumor           
                 NORMAL 
                 88.89 
                 11.11 
               
               
                   
                   
                 TUMOR 
                 28.07 
                 71.93 
               
               
                   
                   
               
             
          
           
               
                 Tests 
               
             
          
           
               
                   
                 N 
                 DF 
                 −LogLike 
                 RSquare (U) 
               
               
                   
                   
               
               
                   
                 924 
                 1 
                 88.007643 
                 0.1453 
               
               
                   
                   
               
             
          
           
               
                   
                 Test 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
                   
               
               
                   
                 Likelihood Ratio 
                 176.015 
                 &lt;.0001* 
               
               
                   
                 Pearson 
                 173.942 
                 &lt;.0001* 
               
               
                   
                   
               
             
          
           
               
                 Fisher&#39;s 
                   
                   
               
               
                 Exact Test 
                 Prob 
                 Alternative Hypothesis 
               
               
                   
               
               
                 Left 
                 1.0000 
                 Prob(Pos/Neg 9 = POS) is greater for 
               
               
                   
                   
                 Tumor? = NORMAL than TUMOR 
               
               
                 Right 
                 &lt;.0001* 
                 Prob(Pos/Neg 9 = POS) is greater for 
               
               
                   
                   
                 Tumor? = TUMOR than NORMAL 
               
               
                 2-Tail 
                 &lt;.0001* 
                 Prob(Pos/Neg 9 = POS) is different 
               
               
                   
                   
                 across Tumor? 
               
               
                   
               
             
          
           
               
                 4. A representative subset of 8 clones 
               
             
          
           
               
                   
                 FGFR2 
               
               
                   
                 IL6 
               
               
                   
                 LAMC2 
               
               
                   
                 MYC 
               
               
                   
                 MYCN(N-myc) 
               
               
                   
                 RASSF1 
               
               
                   
                 TSC1 
               
               
                   
                 UBE3A, D15S10 
               
             
          
           
               
                   
                 Pos/Neg 8 
                   
               
             
          
           
               
                   
                   
                 Row % 
                 NEG 
                 POS 
               
               
                   
                   
               
               
                   
                            umor? 
                 NORMAL 
                 100.0 
                 0.00 
               
               
                   
                   
                 TUMOR 
                 29.82 
                 70.18 
               
               
                   
                   
               
             
          
           
               
                 Tests 
               
             
          
           
               
                   
                 N 
                 DF 
                 −LogLike 
                 RSquare (U) 
               
               
                   
                   
               
               
                   
                 924 
                 1 
                 133.24371 
                 0.2151 
               
               
                   
                   
               
             
          
           
               
                   
                 Test 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
                   
               
               
                   
                 Likelihood Ratio 
                 266.487 
                 &lt;.0001* 
               
               
                   
                 Pearson 
                 224.453 
                 &lt;.0001* 
               
               
                   
                   
               
             
          
           
               
                 Fisher&#39;s 
                   
                   
               
               
                 Exact Test 
                 Prob 
                 Alternative Hypothesis 
               
               
                   
               
               
                 Left 
                 1.0000 
                 Prob(Pos/Neg 8 = POS) is greater for 
               
               
                   
                   
                 Tumor? = NORMAL than TUMOR 
               
               
                 Right 
                 &lt;.0001 * 
                 Prob(Pos/Neg 8 = POS) is greater for 
               
               
                   
                   
                 Tumor? = TUMOR than NORMAL 
               
               
                 2-Tail 
                 &lt;.0001 * 
                 Prob(Pos/Neg 8 = POS) is different 
               
               
                   
                   
                 across Tumor? 
               
               
                   
               
               
                             indicates data missing or illegible when filed 
               
             
          
         
       
     
         [0233]    FISH Probe Set Selection 
         [0234]    To select 4-color probe sets for further FISH experiments, we evaluated the selected loci in greater detail supplementing statistical analysis with literature review. From the further analysis, for the probe selection, we excluded loci that were previously implicated in benign endometrial diseases. In addition, we considered NCOA3 and ZNF217 to be potentially located on one segment of rearrangement and thus picked available probe, ZNF 217, for further FISH studies. 
         [0000]    
       
         
               
               
             
           
               
                   
               
               
                 Clone on array: 
                 Vysis FISH probe availability: 
               
               
                   
               
             
             
               
                 1q25-q31, LAMC2 
                 1q25 probe 
               
               
                 18q21.3, DCC: loss 
                 probe available 
               
               
                 1q24, MYC 
                 probe available 
               
               
                 10q26, FGFR2: gain (10q23 in 
                 no probe/clone PTENq23.3 
               
               
                 literature) 
               
               
                 15q11, UBE3A: deletion 
                 PWS probe 
               
               
                 3q27-q29, TP63: gain (3q24-26.4 
                 2 clones or substitute by PIK3CA 
               
               
                 reported in literature) 
               
               
                 3p21.3, RASSF1: gain 
                 2 clones 
               
               
                 7p21, IL6: gain 
                 1 clone 
               
               
                 2p24.1, MYCN: gain 
                 SG, SO probes 
               
               
                 8p11.2, FGFR1: gain/deletion 
                 2 clones 
               
               
                 9q33-34 (TSC1): loss (found in 
               
               
                 endometriosis also) 
               
               
                 20q12-q13 (NCOA3?): gain (in 
                 20q11.2 HIRA, 20q13.2 ZNF217 
               
               
                 literature q13.2) 
               
               
                 16q24.2-q24.3: loss (16q literature) 
               
               
                   
               
             
          
         
       
     
         [0235]    As evident from the results outlined above, CGH data produced by the Genosenor array yielded several preliminary chromosomal targets which included LAMC2 (1q25), NMYC (2p24.1), PIK3CA (3q27-q29), MYC (8q24), FGFR2 (10q26), centromeric region of chromosome 18 (CEP18), DCC (18q21), and ZNF217 (20q13). Of these eight probes, numerous potential four-probe combinations have been identified and probes were grouped in probe sets. 
         [0236]    Contingency table analysis was used to assess probe combinations, and the combinations were ranked by a chi square p value and by the DFI value. As at least some of the loci (such as DCC) represent deletions, we first assessed 3-probe combinations to allow one Chromosome Enumerator Probe (CEP) to be added to the probe set if required, as a control for a deletion probe. Shown below is the list of best 3-probe combination in detecting endometrial cancer (Table 3) with chi square p value of &lt;0.05. 
         [0237]    The following probe sets were selected and used in FISH experiments using Table 3 above as a guide. New probes were designed and manufactured in Abbott Molecular R&amp;D as indicated below. 
         [0000]    
       
         
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
           
               
                   
               
             
             
               
                 Probe Set #1 
               
             
          
           
               
                   
                 1q25 
                 SpectrumGold 
               
               
                   
                 DCC (18q21.2) 
                 SpectrumRed 
               
               
                   
                 CEP18 
                 SpectrumGreen 
               
               
                   
                 MYC (8q24) 
                 SpectrumAqua 
               
             
          
           
               
                 Probe Set #2 
               
             
          
           
               
                   
                 MYCN (2p24.3) 
                 SpectrumGreen (new probe) 
               
               
                   
                 PIK3CA (2q26.32) 
                 SpectrumGold 
               
               
                   
                 FGFR2 (10q26.13) 
                 SpectrumAqua (new probe) 
               
               
                   
                 ZNF217 (20q13.2) 
                 pectrumRed 
               
             
          
           
               
                 Probe Set #3 
               
             
          
           
               
                   
                 PTEN (10q23.3) 
                 SpectrumOrange 
               
               
                   
                 CEP10 
                 SpectrumGreen 
               
               
                   
                 FGFR1 (8p11.2) 
                 SpectrumAqua (new 
               
               
                   
                   
                 probe) 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 Selected best 3-probe combinations (p &lt; 0.05) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Normal 
                 Cancer 
               
             
          
           
               
                   
                   
                   
                 # 
                 # spec- 
                   
                 marker 
                 marker 
                 # spec- 
               
               
                 PROBE 1 
                 PROBE 2 
                 PROBE 3 
                 Probes 
                 imens 
                 SENS 
                 (s)+ 
                 (s)− 
                 imens 
               
               
                   
               
               
                 LAMC2:18:1q25-q31 
                 MYC:110:8q24.12-q24.13 
                 UBE3A, D15S10:182:15q11- 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 LAMC2:18:1q25-q31 
                 FGFR2:135:10q26 
                 UBE3A, D15S10:182:15q11- 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 LAMC2:18:1q25-q31 
                 MYCN(N-myc):26:2p24.1 
                 MYC:110:8q24.12-q24.13 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 LAMC2:18:1q25-q31 
                 MYC:110:8q24.12-q24.13 
                 FGFR2:135:10q26 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 LAMC2:18:1q25-q31 
                 FGFR1:106:8p11.2-p11.1 
                 UBE3A, D15S10:182:15q11- 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 LAMC2:18:1q25-q31 
                 MYCN(N-myc):26:2p24.1 
                 FGFR1:106:8p11.2-p11.1 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 LAMC2:18:1q25-q31 
                 FGFR1:106:8p11.2-p11.1 
                 FGFR2:135:10q26 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 LAMC2:18:1q25-q31 
                 FGFR1:106:8p11.2-p11.1 
                 MYC:110:8q24.12-q24.13 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 LAMC2:18:1q25-q31 
                 MYCN(N-myc):26:2p24.1 
                 FGFR2:135:10q26 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 LAMC2:18:1q25-q31 
                 MYCN(N-myc):26:2p24.1 
                 UBE3A, D15S10:182:15q11- 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 MYC:110:8q24.12-q24.1 
                 FGFR2:135:10q26 
                 UBE3A, D15S10:182:15q11- 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 LAMC2:18:1q25-q31 
                 FGFR1:106:8p11.2-p11.1 
                 ZNF217(ZABC1):254:20q13 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 LAMC2:18:1q25-q31 
                 MYC:110:8q24.12-q24.13 
                 2NF217(ZABC1):254:20q13 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 LAMC2:18:1q25-q31 
                 UBE3A, D15S10:182:15q11 
                 ZNF217(ZABC1):254:20q13 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 LAMC2:18:1q25-q31 
                 FGFR2:135:10q26 
                 ZNF217(ZABC1):254:20q13 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 FGFR1:106:8p11.2-p11 
                 FGFR2:135:10q26 
                 UBE3A, D15S10:182:15q11- 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 LAMC2:18:1q25-q31 
                 MYCN(N-myc):26:2p24.1 
                 ZNF217(ZABC1):254:20q13 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 MYCN(N-myc):26:2p24. 
                 FGFR1:106:8p11.2-p11.1 
                 FGFR2:135:10q26 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 MYCN(N-myc):26:2p24. 
                 MYC:110:8q24.12-q24.13 
                 FGFR2:135:10q26 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 MYCN(N-myc):26:2p24. 
                 FGFR2:135:10q26 
                 UBE3A, D15S10:182:15q11- 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 MYCN(N-myc):26:2p24. 
                 MYC:110:8q24.12-q24.13 
                 UBE3A, D15S10:182:15q11- 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 FGFR2:135:10q26 
                 UBE3A, D15S10:182:15q11 
                 ZNF217(ZABC1):254:20q13 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 LAMC2:18:1q25-q31 
                 FGFR1:106:8p11.2-p11.1 
                 DCC:231:18q21.3 
                 3 
                 9 
                 0.1111 
                 1 
                 8 
                 57 
               
               
                 LAMC2:18:1q25-q31 
                 MYC:110:8q24.12-q24.13 
                 DCC:231:18q21.3 
                 3 
                 9 
                 0.1111 
                 1 
                 8 
                 57 
               
               
                 LAMC2:18:1q25-q31 
                 FGFR2:135:10q26 
                 DCC:231:18q21.3 
                 3 
                 9 
                 0.1111 
                 1 
                 8 
                 57 
               
               
                 LAMC2:18:1q25-q31 
                 UBE3A, D15S10:182:15q11 
                 DCC:231:18q21.3 
                 3 
                 9 
                 0.1111 
                 1 
                 8 
                 57 
               
               
                 FGFR1:106:8p11.2-p11. 
                 MYC:110:8q24.12-q24.13 
                 FGFR2:135:10q26 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 MYC:110:8q24.12-q24.1 
                 FGFR2:135:10q26 
                 ZNF217(ZABC1):254:20q13 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 LAMC2:18:1q25-q31 
                 MYCN(N-myc):26:2p24.1 
                 DCC:231:18q21.3 
                 3 
                 9 
                 0.1111 
                 1 
                 8 
                 57 
               
               
                 MYCN(N-myc):26:2p24. 
                 FGFR1:106:8p11.2-p11.1 
                 MYC:110:8q24.12-q24.13 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 MYCN(N-myc):26:2p24. 
                 FGFR1:106:8p11.2-p11.1 
                 UBE3A, D15S10:182:15q11- 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 FGFR1:106:8p11.2-p11. 
                 MYC:110:8q24.12-q24.13 
                 UBE3A, D15S10:182:15q11- 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 MYCN(N-myc):26:2p24. 
                 FGFR2:135:10q26 
                 ZNF217(ZABC1):254:20q13 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 FGFR1:106:8p11.2-p11. 
                 FGFR2:135:10q26 
                 ZNF217(ZABC1):254:20q13 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 MYCN(N-myc):26:2p24. 
                 FGFR1:106:8p11.2-p11.1 
                 ZNF217(ZABC1):254:20q13 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 MYCN(N-myc).26:2p24. 
                 MYC:110:8q24.12-q24.13 
                 ZNF217(ZABC1):254:20q13 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 MYC:110:8q24.12-q24.1 
                 UBE3A, D15S10:182:15q11 
                 ZNF217(ZABC1):254:20q13 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                 FGFR1:106:8p11.2-p11. 
                 UBE3A, D15S10:182:15q11 
                 ZNF217(ZABC1):254:20q13 
                 3 
                 9 
                 0.0000 
                 0 
                 9 
                 57 
               
               
                   
               
             
          
           
               
                   
                 Cancer 
                   
               
             
          
           
               
                   
                   
                   
                   
                 SPEC 
                 DFI vs 
                 marker 
                 marker 
                 chi sq 
               
               
                 PROBE 1 
                 PROBE 2 
                 PROBE 3 
                 SENS 
                 vs norm 
                 norm 
                 (s)+ 
                 (s)− 
                 prob 
               
               
                   
               
               
                 LAMC2:18:1q25-q31 
                 MYC:110:8q24.12-q24.13 
                 UBE3A, D15S10:182:15q11- 
                 0.5439 
                 1.0000 
                 0.4561 
                 31 
                 26 
                 0.0024 
               
               
                 LAMC2:18:1q25-q31 
                 FGFR2:135:10q26 
                 UBE3A, D15S10:182:15q11- 
                 0.5439 
                 1.0000 
                 0.4561 
                 31 
                 26 
                 0.0024 
               
               
                 LAMC2:18:1q25-q31 
                 MYCN(N-myc):26:2p24.1 
                 MYC:110:8q24.12-q24.13 
                 0.5263 
                 1.0000 
                 0.4737 
                 30 
                 27 
                 0.0032 
               
               
                 LAMC2:18:1q25-q31 
                 MYC:110:8q24.12-q24.13 
                 FGFR2:135:10q26 
                 0.5263 
                 1.0000 
                 0.4737 
                 30 
                 27 
                 0.0032 
               
               
                 LAMC2:18:1q25-q31 
                 FGFR1:106:8p11.2-p11.1 
                 UBE3A, D15S10:182:15q11- 
                 0.5263 
                 1.0000 
                 0.4737 
                 30 
                 27 
                 0.0032 
               
               
                 LAMC2:18:1q25-q31 
                 MYCN(N-myc):26:2p24.1 
                 FGFR1:106:8p11.2-p11.1 
                 0.5088 
                 1.0000 
                 0.4912 
                 29 
                 28 
                 0.0043 
               
               
                 LAMC2:18:1q25-q31 
                 FGFR1:106:8p11.2-p11.1 
                 FGFR2:135:10q26 
                 0.5088 
                 1.0000 
                 0.4912 
                 29 
                 28 
                 0.0043 
               
               
                 LAMC2:18:1q25-q31 
                 FGFR1:106:8p11.2-p11.1 
                 MYC:110:8q24.12-q24.13 
                 0.4912 
                 1.0000 
                 0.5088 
                 28 
                 29 
                 0.0056 
               
               
                 LAMC2:18:1q25-q31 
                 MYCN(N-myc):26:2p24.1 
                 FGFR2:135:10q26 
                 0.4912 
                 1.0000 
                 0.5088 
                 28 
                 29 
                 0.0056 
               
               
                 LAMC2:18:1q25-q31 
                 MYCN(N-myc):26:2p24.1 
                 UBE3A, D15S10:182:15q11- 
                 0.4737 
                 1.0000 
                 0.5263 
                 27 
                 30 
                 0.0072 
               
               
                 MYC:110:8q24.12-q24.1 
                 FGFR2:135:10q26 
                 UBE3A, D15S10:182:15q11- 
                 0.4737 
                 1.0000 
                 0.5263 
                 27 
                 30 
                 0.0072 
               
               
                 LAMC2:18:1q25-q31 
                 FGFR1:106:8p11.2-p11.1 
                 ZNF217(ZABC1):254:20q13 
                 0.4737 
                 1.0000 
                 0.5263 
                 27 
                 30 
                 0.0072 
               
               
                 LAMC2:18:1q25-q31 
                 MYC:110:8q24.12-q24.13 
                 2NF217(ZABC1):254:20q13 
                 0.4737 
                 1.0000 
                 0.5263 
                 27 
                 30 
                 0.0072 
               
               
                 LAMC2:18:1q25-q31 
                 UBE3A, D15S10:182:15q11 
                 ZNF217(ZABC1):254:20q13 
                 0.4737 
                 1.0000 
                 0.5263 
                 27 
                 30 
                 0.0072 
               
               
                 LAMC2:18:1q25-q31 
                 FGFR2:135:10q26 
                 ZNF217(ZABC1):254:20q13 
                 0.4561 
                 1.0000 
                 0.5439 
                 26 
                 31 
                 0.0093 
               
               
                 FGFR1:106:8p11.2-p11 
                 FGFR2:135:10q26 
                 UBE3A, D15S10:182:15q11- 
                 0.4386 
                 1.0000 
                 0.5614 
                 25 
                 32 
                 0.0117 
               
               
                 LAMC2:18:1q25-q31 
                 MYCN(N-myc):26:2p24.1 
                 ZNF217(ZABC1):254:20q13 
                 0.4386 
                 1.0000 
                 0.5614 
                 25 
                 32 
                 0.0117 
               
               
                 MYCN(N-myc):26:2p24. 
                 FGFR1:106:8p11.2-p11.1 
                 FGFR2:135:10q26 
                 0.4211 
                 1.0000 
                 0.5789 
                 24 
                 33 
                 0.0147 
               
               
                 MYCN(N-myc):26:2p24. 
                 MYC:110:8q24.12-q24.13 
                 FGFR2:135:10q26 
                 0.4211 
                 1.0000 
                 0.5789 
                 24 
                 33 
                 0.0147 
               
               
                 MYCN(N-myc):26:2p24. 
                 FGFR2:135:10q26 
                 UBE3A, D15S10:182:15q11- 
                 0.4035 
                 1.0000 
                 0.5965 
                 23 
                 34 
                 0.0182 
               
               
                 MYCN(N-myc):26:2p24. 
                 MYC:110:8q24.12-q24.13 
                 UBE3A, D15S10:182:15q11- 
                 0.3860 
                 1.0000 
                 0.6140 
                 22 
                 35 
                 0.0225 
               
               
                 FGFR2:135:10q26 
                 UBE3A, D15S10:182:15q11 
                 ZNF217(ZABC1):254:20q13 
                 0.3860 
                 1.0000 
                 0.6140 
                 22 
                 35 
                 0.0225 
               
               
                 LAMC2:18:1q25-q31 
                 FGFR1:106:8p11.2-p11.1 
                 DCC:231:18q21.3 
                 0.5088 
                 0.8889 
                 0.5036 
                 29 
                 28 
                 0.0260 
               
               
                 LAMC2:18:1q25-q31 
                 MYC:110:8q24.12-q24.13 
                 DCC:231:18q21.3 
                 0.5088 
                 0.8889 
                 0.5036 
                 29 
                 28 
                 0.0260 
               
               
                 LAMC2:18:1q25-q31 
                 FGFR2:135:10q26 
                 DCC:231:18q21.3 
                 0.5088 
                 0.8889 
                 0.5036 
                 29 
                 28 
                 0.0260 
               
               
                 LAMC2:18:1q25-q31 
                 UBE3A, D15S10:182:15q11 
                 DCC:231:18q21.3 
                 0.5088 
                 0.8889 
                 0.5036 
                 29 
                 28 
                 0.0260 
               
               
                 FGFR1:106:8p11.2-p11. 
                 MYC:110:8q24.12-q24.13 
                 FGFR2:135:10q26 
                 0.3684 
                 1.0000 
                 0.6316 
                 21 
                 36 
                 0.0274 
               
               
                 MYC:110:8q24.12-q24.1 
                 FGFR2:135:10q26 
                 ZNF217(ZABC1):254:20q13 
                 0.3684 
                 1.0000 
                 0.6316 
                 21 
                 36 
                 0.0274 
               
               
                 LAMC2:18:1q25-q31 
                 MYCN(N-myc):26:2p24.1 
                 DCC:231:18q21.3 
                 0.4912 
                 0.8889 
                 0.5208 
                 28 
                 29 
                 0.0327 
               
               
                 MYCN(N-myc):26:2p24. 
                 FGFR1:106:8p11.2-p11.1 
                 MYC:110:8q24.12-q24.13 
                 0.3509 
                 1.0000 
                 0.6491 
                 20 
                 37 
                 0.0333 
               
               
                 MYCN(N-myc):26:2p24. 
                 FGFR1:106:8p11.2-p11.1 
                 UBE3A, D15S10:182:15q11- 
                 0.3509 
                 1.0000 
                 0.6491 
                 20 
                 37 
                 0.0333 
               
               
                 FGFR1:106:8p11.2-p11. 
                 MYC:110:8q24.12-q24.13 
                 UBE3A, D15S10:182:15q11- 
                 0.3509 
                 1.0000 
                 0.6491 
                 20 
                 37 
                 0.0333 
               
               
                 MYCN(N-myc):26:2p24. 
                 FGFR2:135:10q26 
                 ZNF217(ZABC1):254:20q13 
                 0.3509 
                 1.0000 
                 0.6491 
                 20 
                 37 
                 0.0333 
               
               
                 FGFR1:106:8p11.2-p11. 
                 FGFR2:135:10q26 
                 ZNF217(ZABC1):254:20q13 
                 0.3509 
                 1.0000 
                 0.6491 
                 20 
                 37 
                 0.0333 
               
               
                 MYCN(N-myc):26:2p24. 
                 FGFR1:106:8p11.2-p11.1 
                 ZNF217(ZABC1):254:20q13 
                 0.3333 
                 1.0000 
                 0.6667 
                 19 
                 38 
                 0.0401 
               
               
                 MYCN(N-myc).26:2p24. 
                 MYC:110:8q24.12-q24.13 
                 ZNF217(ZABC1):254:20q13 
                 0.3333 
                 1.0000 
                 0.6667 
                 19 
                 38 
                 0.0401 
               
               
                 MYC:110:8q24.12-q24.1 
                 UBE3A, D15S10:182:15q11 
                 ZNF217(ZABC1):254:20q13 
                 0.3333 
                 1.0000 
                 0.6667 
                 19 
                 38 
                 0.0401 
               
               
                 FGFR1:106:8p11.2-p11. 
                 UBE3A, D15S10:182:15q11 
                 ZNF217(ZABC1):254:20q13 
                 0.3158 
                 1.0000 
                 0.6842 
                 18 
                 39 
                 0.0481 
               
               
                   
               
             
          
         
       
     
         [0238]    Probe sets #1 and 2 in relation to aCGH data are shown in  FIG. 4 . The plot illustrates that the probes were chosen in such a manner as to be able to detect both endometrioid and non-endometrioid tumors. 
       Fluorescence In Situ Hybridization Studies 
       [0239]    Study Design 
         [0240]    A probe selection study is currently being performed to determine which four-probe combination of the eight FISH probes can most accurately detect endometrial carcinoma. 
         [0241]    Archived formalin-fixed paraffin embedded endometrial tissue specimens taken from patients undergoing hysterectomy or Pipelle biopsy during 2000 to 2006 were utilized for this probe selection study. A variety of specimens diagnosed as endometrial carcinoma (endometrioid type and non-endometrioid type), simple and complex hyperplasia and normal endometrial epithelium were selected for FISH analysis (Table 4). 
         [0242]    Ten histologically negative specimens from patients without a history of endometrial carcinoma were also evaluated as a normal value study. Six paraffin sectioned slides were prepared for each case, one was stained with hematoxylin and eosin (H&amp;E) while five unstained slides were prepared for FISH analysis. The H&amp;E slide was microscopically evaluated by a gynecologic pathologist and areas of interest (tumor in cancer specimens; normal epithelium in benign specimens) for FISH analysis were identified. This area of interest was concurrently marked on the unstained tissue slide and was hybridized with each of the two FISH probe sets (separate slides used for each of the two probe sets). 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Patient Population 
               
             
          
           
               
                   
                 Genosensor 
                 Non Geno- 
                   
               
               
                   
                 cases 
                 sensor 
                 Total 
               
               
                   
                   
               
             
          
           
               
                   
                 Normal 
                 0 
                 10 
                 10 
               
               
                   
                 Simple Hyperplasia 
                 0 
                 5 
                 5 
               
               
                   
                 Complex Hyperplasia 
                 2 
                 4 
                 6 
               
               
                   
                 Endometrioid Grade 1 
                 8 
                 7 
                 15 
               
               
                   
                 Endometrioid Grade 2 
                 7 
                 3 
                 10 
               
               
                   
                 Endometrioid Grade 3 
                 6 
                 4 
                 10 
               
               
                   
                 Serous 
                 6 
                 3 
                 9 
               
               
                   
                 Clear Cell 
                 1 
                 1 
                 2 
               
               
                   
                 MMMT 
                 2 
                 1 
                 3 
               
               
                   
                 Total 
                 32 
                 38 
                 70 
               
               
                   
                   
               
             
          
         
       
     
         [0243]    Hybridized slides were evaluated using a fluorescence microscope. The area of interest was identified, 50 cells were evaluated (50 tumor cells for the cancer or pre-neoplastic cases and 50 normal cells for the normal cases) and the number of signals from each of the four probes was recorded. A representative example of endometrial cancer cells exhibiting multiple gains for these probes is shown in  FIG. 5 . A statistical analysis was performed by Abbott Molecular, Inc. using the signal patterns from all recorded cells. The ten histologically negative specimens were used to calculate the number of signals present in normal endometrial tissue. ROC curve analyses were performed to determine the optimal cutoff values used to discriminate chromosomal abnormalities (gains and losses) from cells with normal chromosomal content for each probe analyzed. Numerous different four-probe combinations were evaluated using this technique to determine the best probe combination to distinguish endometrial cancer from normal endometrial tissue and precursor lesions. 
         [0244]    Results 
         [0245]    Probe Sets #1 and #2 
         [0246]    Two probe sets, #1 and #2, were evaluated on all specimens.  FIG. 6  illustrates the proportion of cells with chromosomal abnormalities by histologic subtype for each of the eight probes. Normal endometrial specimens exhibited zero or very few cells with chromosomal gains and approximate 5-20% of cells showed a form of chromosomal loss. In additional very few (&lt;10%) cells with chromosomal gains were identified in hyperplasia specimens. 
         [0247]    The following FISH parameters were analyzed:
       1. % Gain, percent of cells with a copy number gain (&gt;2 copies per cell) of a locus out of 50 cells counted (50=100%)   2. % Loss, percent of cells with a copy number loss (&lt;2 copies per cell) of a locus out of 50 cells counted (50=100%)   3. % Abnormal, percent of cells with either a copy number loss (&lt;2 copies per cell) OR a copy number gain (&gt;2 copies per cell) of a locus out of 50 cells counted (50=100%)       
 
         [0251]    The conclusions drawn from  FIG. 6  are: No gains were observed in Normal specimens for DCC, CEP18, MYC and 1q24. The increase in % cells with gains was observed from normal to hyperplasia to cancer, with most gains in Non-Endometrioid tumors. With losses, there is no clear separation between normal, hyperplasia and cancer. A trend is observed in the increase of % Abnormal from normal to hyperplasia to cancer, with the greatest number of abnormalities in Non-Endometrioid tumors. MYC and 1q24 appear to have the largest range of difference. 
         [0252]    Probes directed to 1q25 and 8q24 had the highest percentage of abnormal cells in specimens with EN and NE cancers. All other probes detected approximately the same proportion of cells with gains in EN and NE cancers. For chromosomal losses, 18q (DCC) and CEP 18 exhibited the most cells with loss in EN and NE cancers, as well as normal and hyperplasia specimens. 
         [0253]    A multivariate analysis was performed to determine which four-probe combination yielded the great combination of sensitivity and specificity for NE and NE caners. Using JMP 8.0 Statistical Analysis software (SAS Institute, 2008) 70 4-probe combinations of 8 probes were tested. “% Abnormal” was chosen as FISH parameter for analysis (see definitions) and Nominal Logistic Regression platform was utilized with the Test specimens chosen as “all cancers” and Control specimens as hyperplasia+normal. ROCs were constructed, and 20 best combinations with the highest AUC were selected. The combinatorial Excel program was run on the selected combinations to obtain high-resolution data on sensitivity and specificity. The program constructed contingency tables and calculated DFI values for the 4-probe combinations at each cutoff level of % “abnormal” for each probe. 
         [0254]    The results of these analyses revealed numerous possible candidate four-probe sets (Table 5). The best probe set combination included DCC, 1q24, MYC, and CEP18 which had a AUC of 0.952 with a sensitivity of 1.000 and a specificity of 0.9048. Three other combinations had a sensitivity of 0.979 and a specificity of 0.900 which included (set 2) 1q24, MYC, FGFR2, CEP18, (set 3) DCC, 1q24, FGFR2, CEP18, and (set 4) 1q24, MYC, CEP18 and PIK3CA. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Probe combinations that discriminate 
               
               
                 Cancer from Normal and Hyperplasia. 
               
             
          
           
               
                   
                   
                   
                   
                   
                 Best 
                 Best 
               
               
                 Locus 
                 Locus 
                 Locus 
                 Locus 
                 AUC 
                 Sens 
                 Spec 
               
               
                 1 
                 2 
                 3 
                 4 
                 (JMP) 
                 LM 
                 LM 
               
               
                   
               
             
          
           
               
                 DCC 
                 1q24 
                 MYC 
                 CEP18 
                 0.95238 
                 1.0000 
                 0.9048 
               
               
                 1q24 
                 MYC 
                 FGFR2 
                 CEP18 
                 0.95918 
                 0.9796 
                 0.9000 
               
               
                 DCC 
                 1q24 
                 FGFR2 
                 CEP18 
                 0.95867 
                 0.9796 
                 0.9000 
               
               
                 1q24 
                 MYC 
                 CEP18 
                 PIK3CA 
                 0.95306 
                 0.9796 
                 0.9000 
               
               
                 DCC 
                 1q24 
                 MYC 
                 FGFR2 
                 0.95714 
                 0.9592 
                 0.9000 
               
               
                 1q24 
                 NMYC 
                 FGFR2 
                 CEP18 
                 0.95714 
                 0.9592 
                 0.9000 
               
               
                 1q24 
                 FGFR2 
                 CEP18 
                 PIK3CA 
                 0.95714 
                 0.9592 
                 0.9000 
               
               
                 1q24 
                 FGFR2 
                 CEP18 
                 20q13 
                 0.95612 
                 0.9592 
                 0.9000 
               
               
                 1q24 
                 CEP18 
                 PIK3CA 
                 20q13 
                 0.95612 
                 0.9592 
                 0.9000 
               
               
                 1q24 
                 NMYC 
                 CEP18 
                 PIK3CA 
                 0.9551 
                 0.9592 
                 0.9000 
               
               
                 DCC 
                 1q24 
                 FGFR2 
                 20q13 
                 0.95408 
                 0.9388 
                 0.9000 
               
               
                   
                 1q24 
                 MYC 
                 CEP18 
                   
                 0.9796 
                 0.9048 
               
               
                 DCC 
                 1q24 
                   
                 CEP18 
                   
                 0.9796 
                 0.9048 
               
               
                   
                 1q24 
                   
                 CEP18 
                   
                 0.9592 
                 0.9048 
               
               
                   
               
               
                 Analyzed % Cells with any abnormality (% abnormal) using JMP8.0 with combinatorial Excel program (contingency table analysis). Test = all cancers; Control = hyperplasia + normal 
               
             
          
         
       
     
         [0255]      FIGS. 7 and 8  present Receiver Operator curves for the selected probe combination. It is evident from the figures that adding probes to a single-probe FISH assay improves sensitivity and specificity. 
         [0256]    The distribution by tumor type and grade is shown below (Table 6): 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Tumor Type and Grade By FISH (4Best) % Abnormal: 
               
               
                 POS or NEG. Cutoffs used as listed in FIG. 7. 
               
             
          
           
               
                   
                 Count 
                   
                   
                   
               
               
                   
                 Row % 
                 NEG 
                 POS 
                 Total 
               
               
                   
                   
               
             
          
           
               
                   
                 Normal 
                 10 
                 0 
                 10 
               
               
                   
                   
                 100.00 
                 0.00 
               
               
                   
                 Simple hyperplasia 
                 4 
                 1 
                 5 
               
               
                   
                   
                 80.00 
                 20.00 
               
               
                   
                 Complex hyperplasia 
                 5 
                 1 
                 6 
               
               
                   
                   
                 83.33 
                 16.67 
               
               
                   
                 Endometrioid Grade 1 
                 0 
                 15 
                 15 
               
               
                   
                   
                 0.00 
                 100.00 
               
               
                   
                 Endometrioid Grade 2 
                 0 
                 10 
                 10 
               
               
                   
                   
                 0.00 
                 100.00 
               
               
                   
                 Endometrioid Grade 3 
                 0 
                 10 
                 10 
               
               
                   
                   
                 0.00 
                 100.00 
               
               
                   
                 Carcinosarcoma/MMMT 
                 0 
                 3 
                 3 
               
               
                   
                   
                 0.00 
                 100.00 
               
               
                   
                 Clear cell 
                 0 
                 2 
                 2 
               
               
                   
                   
                 0.00 
                 100.00 
               
               
                   
                 Serous 
                 0 
                 9 
                 9 
               
               
                   
                   
                 0.00 
                 100.00 
                   
               
               
                   
                   
                 19 
                 51 
                 70 
               
               
                   
                   
               
             
          
         
       
     
         [0257]    Detection of positives in the complex hyperplasia specimens could be due to heterogeneity in this category and could reflect risk of progression to cancer. 
         [0258]    Comparison of FISH to CGH Array Data 
         [0259]    Performance of the 1q24, CEP16, DCC and MYC FISH probe set was compared by contingency analysis in JMP 8.0 to the aCGH Probe Selection Set #1: 
         [0260]    DCC (18q21.3)—frequency of loss in all cancers ˜17% 
         [0261]    Cep18—included for FISH probe set (detection of deletions) 
         [0262]    LAMC2 (1q25)—frequency of gain in all cancers &gt;20% 
         [0263]    MYC (8q24.12-q24.13)—frequency of gain in all cancers ˜17% 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                 Contingency Table Array (Ca vs N) 
               
               
                 One of Loci Changed 
               
             
          
           
               
                   
                 Count 
                   
                   
                   
               
               
                   
                 Row % 
                 NEG 
                 POS 
               
               
                   
                   
               
             
          
           
               
                   
                 Ca/NoCa 
                 Ca 
                 28 
                 29 
                 57 
               
               
                   
                   
                   
                 49.12 
                 50.88 
               
               
                   
                   
                 N 
                 8 
                 1 
                 9 
               
               
                   
                   
                   
                 88.89 
                 11.11 
                   
               
               
                   
                   
                   
                 36 
                 30 
                 66 
               
               
                   
                   
               
               
                   
                 Sensitivity = 50.88% (29 out of 57) 
               
               
                   
                 Specificity = 88.89% (8 out of 9) 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                 Contingency Table FISH (Ca vs N + Hyperplasia) 
               
               
                 FISH (4Best) % Imbal: POS or NE           
               
             
          
           
               
                   
                 Count 
                   
                   
                   
               
               
                   
                 Row % 
                 NEG 
                 POS 
               
               
                   
                   
               
             
          
           
               
                   
                 Test or Ref? 
                 Ca 
                 0 
                 49 
                 49 
               
               
                   
                   
                   
                 0.00 
                 100.00 
               
               
                   
                   
                 N + Hyperpl 
                 19 
                 2 
                 21 
               
               
                   
                   
                   
                 90.48 
                 9.52 
                   
               
               
                   
                   
                   
                 19 
                 51 
                 70 
               
               
                   
                   
               
               
                   
                 Sensitivity = 100% (49 out of 49) 
               
               
                   
                 Specificity = 90.48% (19 out of 21) 
               
               
                   
                             indicates data missing or illegible when filed 
               
             
          
         
       
     
         [0264]    It is apparent that FISH assay with probe designed based on microarray results has significantly improved on array performance. This is possibly due to influence of benign cells in the macro-dissected tumors that dilute the analyte tumor DNA and thus lead to lower sensitivity. This problem in microarray experiments could be overcome by careful selection of specimens with high percentage of tumor cells and by micro-dissection of the tumor area. 
         [0265]    Analysis of Copy Number Gains Only, Best Combinations: Cancer Vs Normal+Hyperplasia 
         [0266]    For a practical FISH application, an alternative probe combination was evaluated that avoids technically challenging detection of losses (looses and gains are considered in “% abnormal” parameter). 
         [0267]    Following the procedure outlined above, all 4-probe combinations were analyzed, and best were selected (preliminary analysis using SD of % Cells in excel), Table 7 and  FIG. 9 . 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Best 4-probe combinations, Gains only. 
               
             
          
           
               
                 PROBE 
                 PROBE 
                 PROBE 
                 PROBE 
                   
                   
                   
                 CUTOFF 
                 CUTOFF 
                 CUTOFF 
                 CUTOFF 
               
               
                 1 
                 2 
                 3 
                 4 
                 SENS 
                 SPEC 
                 DFI 
                 1 
                 2 
                 3 
                 4 
               
               
                   
               
             
          
           
               
                 1q24 
                 MYC 
                 CEP18 
                 20q13.2 
                 0.84 
                 0.95 
                 0.17 
                 41.30 
                 8.36 
                 7.91 
                 4.46 
               
               
                 1q24 
                 CEP18 
                 PIK3CA 
                 20q13.2 
                 0.84 
                 0.95 
                 0.17 
                 8.36 
                 7.91 
                 18.47 
                 4.46 
               
               
                 1q24 
                 CEP18 
                 20q13.2 
                 FGFR2 
                 0.84 
                 0.90 
                 0.19 
                 8.36 
                 7.91 
                 4.46 
                 15.71 
               
               
                   
               
             
          
         
       
     
         [0268]    Interestingly, the combinations listed above have improved on specificity of cancer detection but have decreased sensitivity, especially to early-stage endometrioid tumors ( FIG. 9  and Table 8). 
         [0269]    The distribution of positive and negative specimens using the 1q24, MYC, CEP18 and 20q13 probe set is shown in Table 8. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Tumor Type and Grade By FISH: Gains of MYC, CEP18, 1q24, 
               
               
                 20q13. Cutoffs used as listed in FIG. 9. 
               
             
          
           
               
                   
                 Count 
                   
                   
                   
               
               
                   
                 Row % 
                 NEG 
                 POS 
                 Total 
               
               
                   
                   
               
             
          
           
               
                   
                 Normal 
                 10 
                 0 
                 10 
               
               
                   
                   
                 100.00 
                 0.00 
               
               
                   
                 Simple hyperplasia 
                 4 
                 1 
                 5 
               
               
                   
                   
                 80.00 
                 20.00 
               
               
                   
                 Complex hyperplasia 
                 5 
                 1 
                 6 
               
               
                   
                   
                 83.33 
                 16.67 
               
               
                   
                 Endometrioid Grade 1 
                 3 
                 12 
                 15 
               
               
                   
                   
                 20.00 
                 80.00 
               
               
                   
                 Endometrioid Grade 2 
                 1 
                 9 
                 10 
               
               
                   
                   
                 10.00 
                 90.00 
               
               
                   
                 Endometrioid Grade 3 
                 0 
                 10 
                 10 
               
               
                   
                   
                 0.00 
                 100.00 
               
               
                   
                 Carcinosarcoma/MMMT 
                 0 
                 3 
                 3 
               
               
                   
                   
                 0.00 
                 100.00 
               
               
                   
                 Clear cell 
                 0 
                 2 
                 2 
               
               
                   
                   
                 0.00 
                 100.00 
               
               
                   
                 Serous 
                 0 
                 9 
                 9 
               
               
                   
                   
                 0.00 
                 100.00 
                   
               
               
                   
                   
                 23 
                 47 
                 70 
               
               
                   
                   
               
             
          
         
       
     
         [0270]    As evident from the table, this set (FISH gains) has lower sensitivity towards low-grade endometrial tumors. 
         [0271]    Probe Set #3 Evaluation 
         [0272]    Probe set #3 was evaluated on 16 early-stage endometrioid cancer specimens and 12 benign (normal and hyperplasia) specimens to determine whether it improves on sensitivity of detection of early-stage (grade 1 and 2) endometrioid tumors with the FISH assay that evaluates gains of MYC, CEP18, 1q24, 20q13. 
         [0273]    The analysis of the FISH data has demonstrated that the addition of the PTEN loss to the selected probe set at a cutoff of 14% could increase the sensitivity of detection of endometrial cancer to 100%, however decreasing the specificity ( FIG. 10 ). This finding is in agreement with aCGH data discussed above. In contrast, an addition of Chromosome 10 “% abnormal cells” to the selected probes at a cutoff of 10-12% of cells with any abnormality (gain or loss), allowed for increased sensitivity without sacrificing the specificity. Interestingly, a combination of CEP10 with MYC, CEP18, and 1q24 yielded the same 100% sensitivity for the 16 cancer specimens and 92% specificity against 12 normal+hyperplasia specimens. Therefore, an added assessment of aneusomy 10 to the FISH probe set could be a beneficial. Addition of FGFR1 did not significantly improve cancer detection, however, when used in combination with CEP10, 1q24 and MYC probes, FGFR1 gain at a cutoff of 2% cells yielded 100% sensitivity for the 16 cancer specimens and 92% specificity against 12 normal+hyperplasia specimens. However, when all the 70 specimens are considered (with limited data included for the probe set #3), the only probe that did not result in decrease in specificity when combined with the MYC, 1q24, 20q13, and CEP18 gain probes was FGFR1 at the cutoff of ≧2% cells with gain of the probe: 
         [0274]    Test or Ref? By MYC gain, 1q24 gain, 20q13 gain, CEP18 gain, FGFR1 gain: 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Count 
                   
                   
                   
               
               
                   
                 Row % 
                 NEG 
                 POS 
                 Total 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Ca 
                 2 
                 47 
                 49 
               
               
                   
                   
                 4.08 
                 95.92 
               
               
                   
                 N + Hyperpl 
                 19 
                 2 
                 21 
               
               
                   
                   
                 90.48 
                 9.52 
                   
               
               
                   
                   
                 21 
                 49 
                 70 
               
               
                   
                   
               
             
          
         
       
     
         [0275]    Interestingly though, a combination of 20q13.2, 1q24, CEP10 and FGFR1 on all 70 specimens (with the data available thus far) demonstrated sensitivity and specificity of 96% and 91%, as shown below: 
         [0276]    Test or Ref? By 20q13, CEP10, 1q24, FGFR1 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Count 
                   
                   
                   
               
               
                   
                 Row % 
                 NEG 
                 POS 
                 Total 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Ca 
                 2 
                 47 
                 49 
               
               
                   
                   
                 4.08 
                 95.92 
               
               
                   
                 N + Hyperpl 
                 19 
                 2 
                 21 
               
               
                   
                   
                 90.48 
                 9.52 
                   
               
               
                   
                   
                 21 
                 49 
                 70 
               
               
                   
                   
               
             
          
         
       
     
         [0277]    As data for all of the specimens is unavailable at this point for Probe Set #3, it appears feasible that the combination of 4 probes that evaluate 20q13 gain, 1q24 gain, CEP10 imbalance, and FGFR1 gain could prove to be superior to the 1q24, MYC, CEP8 and 20q13.2 probe set in future experiments. 
         [0278]    Probe Set #4 Evaluation 
         [0279]    Probe set #4 was evaluated on the same set of endometrioid cancer specimens, normal and hyperplasia specimens described above to determine whether it improves on sensitivity of detection of low grade (grade 1 and 2) endometrioid tumors above that obtained with the FISH assay that evaluates gains of MYC, CEP18, 1q25, 20q13. 
         [0280]    The analysis of the FISH data demonstrates that the substitution of the FGFR1 gain (with a cutoff of 4%) for 20q in the MYC, 1q25, and CEP probe set provided a sensitivity of 90%, which was similar to the MYC, 1q25, CEP18 and 20q probe set. However, the addition of FGFR1, resulted in one additional complex hyperplasia specimen to be diagnosed as positive. (Table 9). 
         [0281]    Interestingly, further analyses revealed that FGFR1 could significantly increase the sensitivity of the four probe set over 20q. When the cutoff of FGFR1 was reduced to 2%, the probe combination of FGFR1, MYC, 1q25, and CEP18 had a sensitivity and specificity of 96% and 81% respectively. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Evaluation of additional probes (probe set 3) to improve sensitivity of endometrial 
               
               
                 cancer detection over that obtained with MYC, CEP 18, 1q25 and 20q13. 
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                   
                   
                 DCC/ 
                 PTEN/ 
                   
                   
                 FGFR1 % 
               
               
                 ID 
                 Type and 
                 MYC % 
                 CEP18 % 
                 1q25 % 
                 20q13.2 % 
                 FGFR1 % 
                 FGFR1 % 
                 CEP18 % 
                 CEP10 % 
                 PTEN % 
                 CEP10 % 
                 Gain OR 
               
               
                 # 
                 Grade 
                 Gain &gt;4 
                 Gain &gt;4 
                 Gain &gt;6 
                 Gain &gt;4 
                 Gain &gt;4 
                 Gain &gt;2 
                 Loss &gt;16 
                 Loss &gt;12 
                 Loss &gt;18 
                 Loss &gt;10 
                 Loss &gt;10 
               
               
                   
               
             
          
           
               
                 101 
                 N 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 102 
                 N 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 POS 
               
               
                 103 
                 N 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 104 
                 N 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 105 
                 N 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 106 
                 N 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 107 
                 N 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 108 
                 N 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 109 
                 N 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 110 
                 N 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 POS 
                 POS 
                 NEG 
               
               
                 86 
                 SH 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 POS 
                 NEG 
                 POS 
                 POS 
                 NEG 
               
               
                 88 
                 SH 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 96 
                 SH 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 97 
                 SH 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 100 
                 SH 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 85 
                 CH 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
               
               
                 87 
                 CH 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 89 
                 CH 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 90 
                 CH 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 POS 
               
               
                 91 
                 CH 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 POS 
                 NEG 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 93 
                 CH 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 2 
                 EG1 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 POS 
                 NEG 
                 POS 
                 POS 
                 POS 
               
               
                 3 
                 EG1 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 POS 
                 NEG 
               
               
                 4 
                 EG1 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 5 
                 EG1 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 POS 
                 NEG 
               
               
                 14 
                 EG1 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 POS 
               
               
                 15 
                 EG1 
                 NEG 
                 NEG 
                 NEG 
                 POS 
                 POS 
                 NEG 
                 POS 
                 NEG 
                 NEG 
                 POS 
                 POS 
               
               
                 16 
                 EG1 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 POS 
                 POS 
                 POS 
                 NEG 
                 POS 
                 POS 
                 NEG 
               
               
                 18 
                 EG1 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 56 
                 EG1 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 POS 
                 NEG 
                 POS 
                 POS 
                 NEG 
               
               
                 57 
                 EG1 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 POS 
                 POS 
                 NEG 
               
               
                 58 
                 EG1 
                 NEG 
                 POS 
                 NEG 
                 POS 
                 NEG 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 POS 
                 NEG 
               
               
                 59 
                 EG1 
                 NEG 
                 NEG 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 POS 
                 POS 
                 NEG 
               
               
                 61 
                 EG1 
                 NEG 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 POS 
                 NEG 
                 NEG 
                 POS 
                 NEG 
               
               
                 63 
                 EG1 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 65 
                 EG1 
                 NEG 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 POS 
                 NEG 
                 NEG 
                 POS 
                 POS 
                 NEG 
               
               
                 7 
                 EG2 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 8 
                 EG2 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 33 
                 EG2 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 POS 
                 NEG 
                 POS 
                 POS 
                 NEG 
               
               
                 34 
                 EG2 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 POS 
               
               
                 35 
                 EG2 
                 POS 
                 POS 
                 POS 
                 NEG 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 POS 
               
               
                 36 
                 EG2 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 POS 
                 NEG 
                 NEG 
                 POS 
               
               
                 37 
                 EG2 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 POS 
                 POS 
                 NEG 
               
               
                 66 
                 EG2 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 POS 
                 NEG 
                 POS 
                 POS 
                 NEG 
               
               
                 67 
                 EG2 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 70 
                 EG2 
                 POS 
                 POS 
                 POS 
                 NEG 
                 POS 
                 POS 
                 POS 
                 NEG 
                 POS 
                 POS 
                 POS 
               
               
                 9 
                 EG3 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 POS 
                 POS 
                 NEG 
                 POS 
                 POS 
                 NEG 
               
               
                 42 
                 EG3 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 POS 
               
               
                 43 
                 EG3 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 POS 
               
               
                 44 
                 EG3 
                 POS 
                 POS 
                 POS 
                 NEG 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
               
               
                 45 
                 EG3 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 POS 
               
               
                 47 
                 EG3 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 POS 
               
               
                 75 
                 EG3 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 POS 
               
               
                 76 
                 EG3 
                 POS 
                 NEG 
                 POS 
                 POS 
                 NEG 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
               
               
                 77 
                 EG3 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 POS 
                 NEG 
                 NEG 
                 POS 
               
               
                 83 
                 EG3 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 POS 
                 POS 
               
               
                 11 
                 C/M 
                 POS 
                 NEG 
                 NEG 
                 POS 
                 POS 
                 POS 
                 NEG 
                 POS 
                 POS 
                 POS 
                 POS 
               
               
                 12 
                 C/M 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
               
               
                 78 
                 C/M 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 POS 
               
               
                 54 
                 Clear 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 POS 
                 NEG 
                 NEG 
                 POS 
               
               
                   
                 cell 
               
               
                 81 
                 Clear 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 POS 
               
               
                   
                 cell 
               
               
                 1 
                 S 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 POS 
                 POS 
               
               
                 6 
                 S 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 POS 
               
               
                 10 
                 S 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 POS 
               
               
                 49 
                 S 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 POS 
               
               
                 50 
                 S 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 NEG 
                 NEG 
                 POS 
               
               
                 51 
                 S 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 POS 
               
               
                 79 
                 S 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 POS 
               
               
                 80 
                 S 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 NEG 
                 POS 
               
               
                 94 
                 S 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 POS 
                 NEG 
                 POS 
                 POS 
               
               
                   
               
               
                 Table Abbreviations: N = Normal; SH = Simple hyperplasia; CH = Complex hyperplasia; EG1 = Endometrioid Grade 1; EG2 = Endometrioid Grade 2; EG3 = Endometrioid Grade 3; Carcinosarcoma/MMMT = CM; S = Serous; POS = Number of cells with abnormal sign patterns identified met or exceeded the threshold for the probe; NEG = Number of cells with abnormal sign patterns identified was less than the threshold for the probe 
               
             
          
         
       
     
         [0282]    Analyses of other probes to increase the sensitivity of MYC, 1q25 and CEP18 are shown in Table 10. The addition of DCC, CEP10, PTEN, or FGFR1 increase the sensitivity of the combination probe set to 94-98%. However, the increase in sensitivity was achieved at the expense of decreased specificity (76-81%). 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 10 
               
             
             
               
                   
               
               
                 Analysis of adding DCC, CEP10, PTEN or FGFR1 to 
               
               
                 a probe set of MYC, 1q25 and CEP18 to increase 
               
               
                 the sensitivity of endometrial cancer detection 
               
             
          
           
               
                   
                 Gains 
                 Losses 
                 Sensi- 
                 Speci- 
               
             
          
           
               
                   
                 Probe 1 
                 Probe 2 
                 Probe 3 
                 Probe 4 
                 tivity 
                 ficity 
               
               
                   
                   
               
             
          
           
               
                 Cut- 
                 MYC &gt;4 
                 1q25 &gt;6 
                 CEP18 &gt;4 
                 DCC/ 
                 98% 
                 76% 
               
               
                 off 
                   
                   
                   
                 CEP18 &gt;16 
               
               
                 Cut- 
                 MYC &gt;4 
                 1q25 &gt;6 
                 CEP18 &gt;4 
                 CEP10 &gt;10 
                 98% 
                 81% 
               
               
                 off 
               
               
                 Cut- 
                 MYC &gt;4 
                 1q25 &gt;6 
                 CEP18 &gt;4 
                 PTEN &gt;18 
                 96% 
                 81% 
               
               
                 off 
               
               
                 Cut- 
                 MYC &gt;4 
                 1q25 &gt;6 
                 CEP18 &gt;4 
                 FGFR1 &gt;10 
                 94% 
                 81% 
               
               
                 off 
                   
                   
                   
                 (gains and 
               
               
                   
                   
                   
                   
                 loss) 
               
               
                   
               
             
          
         
       
     
         [0283]    Additional analyses were performed to determine the optimal probe set with cutoffs to attain very high specificity for endometrial carcinoma. Results from this analysis can be seen in Table 11. Numerous probe and abnormality combinations achieved at least 95% specificity. Those that produced the highest sensitivity with greater than 95% specificity are highlighted in yellow at the top of Table 11. All of these combinations included the evaluation of locus loss or combination of loss and gains (imbal). The best performing probe set that only evaluated gains had a sensitivity of 84% and specificity of 95%, which included FGFR1, 1q25, CEP18 and 20q13 with cutoffs of 8%, 8%, 8% and 8% respectively (highlighted in blue). Two other probe sets containing 1q25, MYC, CEP18 and FGFR1 or 20q13 using cutoffs of 12%, 12%, 12%, and 12%, respectively, achieved a sensitivity of 80% and specificity of 95% (highlighted in green). 
         [0284]    The most appealing probe set included 4 LSI probes of 1q25, MYC, FGFR1 and 20q13 using similar cutoffs also had a sensitivity and specificity of 80% and 95%, respectively (seen in red). This probe set is ideal due because it performs nearly as well as other probe sets while only analyzing specimens for chromosomal gains. Chromosomal gains are easier to identify by technologists and would likely have a higher inter-observer reproducibility than when evaluating chromosomal losses. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                 Analysis of different probes and cutoffs to achieve specificity of &gt;95% 
               
               
                 for endometrial cancer detection 
               
             
          
           
               
                 PROBE 1 
                 PROBE 2 
                 PROBE 3 
                 PROBE 4 
                   
                   
               
             
          
           
               
                   
                   
                 Cut 
                   
                   
                 Cut 
                   
                   
                 Cut 
                   
                   
                 Cut 
                   
                   
               
               
                 Probe 
                 Abn 
                 off 
                 Probe 
                 Abn 
                 off 
                 Probe 
                 Abn 
                 off 
                 Probe 
                 Abn 
                 off 
                 SENS 
                 SPEC 
               
               
                   
               
               
                 PTEN/ 
                 loss 
                 12 
                 FGFR1 
                 imbal 
                 12 
                 1q25 
                 gain 
                 12 
                 MYC 
                 gain 
                 12 
                 0.857 
                 0.952 
               
               
                 CEP10 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 FGFR1 
                 imbal 
                 12 
                 FGFR1 
                 loss 
                 12 
                 1q25 
                 gain 
                 12 
                 MYC 
                 gain 
                 12 
                 0.857 
                 0.952 
               
               
                 FGFR1 
                 imbal 
                 12 
                 FGFR1 
                 gain 
                 12 
                 1q25 
                 gain 
                 12 
                 MYC 
                 gain 
                 12 
                 0.857 
                 0.952 
               
               
                 FGFR1 
                 imbal 
                 12 
                 1q25 
                 gain 
                 12 
                 MYC 
                 gain 
                 12 
                 CEP18 
                 gain 
                 12 
                 0.857 
                 0.952 
               
               
                 FGFR1 
                 gain 
                 8 
                 1q25 
                 gain 
                 8 
                 CEP18 
                 gain 
                 8 
                 20q13.2 
                 gain 
                  8 
                 0.837 
                 0.952 
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                 PTEN/ 
                 loss 
                 12 
                 FGFR1 
                 imbal 
                 12 
                 1q25 
                 gain 
                 12 
                 CEP18 
                 gain 
                 12 
                 0.816 
                 0.952 
               
               
                 CEP10 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 FGFR1 
                 loss 
                 12 
                 1q25 
                 gain 
                 12 
                 MYC 
                 gain 
                 12 
                 CEP18 
                 gain 
                 12 
                 0.816 
                 0.952 
               
               
                 PTEN/ 
                 loss 
                 12 
                 FGFR1 
                 gain 
                 12 
                 1q25 
                 gain 
                 12 
                 MYC 
                 gain 
                 12 
                 0.796 
                 0.952 
               
               
                 CEP10 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 FGFR1 
                 gain 
                 28 
                 CEP10 
                 imbal 
                 28 
                 1q25 
                 gain 
                 28 
                 MYC 
                 gain 
                 28 
                 0.796 
                 0.952 
               
               
                 PTEN/ 
                 loss 
                 12 
                 1q25 
                 gain 
                 12 
                 MYC 
                 gain 
                 12 
                 CEP18 
                 gain 
                 12 
                 0.796 
                 0.952 
               
               
                 CEP10 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                 PTEN/ 
                 loss 
                 12 
                 1q25 
                 gain 
                 12 
                 MYC 
                 gain 
                 12 
                 20q13.2 
                 gain 
                 12 
                 0.796 
                 0.952 
               
               
                 CEP10 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 Abbreviations: 
               
               
                 Probe = locus of interest; 
               
               
                 Abn = Type of abnormality; 
               
               
                 loss = evaluates only loss of locus; 
               
               
                 gain = evaluates only gain of locus; 
               
               
                 imbal = evaluates gains or loss; 
               
               
                 cutoff = % of cells with abnormality to consider a specimen as positive. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
             
           
               
                   
               
             
             
               
                 ATTACHMENT 1. GeneSensor ™ 300 Array Clone List 
               
             
          
           
               
                   
                   
                 Cyto Loc, 
                   
               
               
                   
                   
                 Locus Link 
                 Utility 
               
               
                   
               
               
                 1 
                 CEB108/T7 
                 1p tel 
                 Sub Tel 
               
               
                 2 
                 1PTEL06 
                 1p tel 
                 Sub Tel 
               
               
                 3 
                 CDC2L1(p58) 
                 1p36 
                 u DEL 
               
               
                 4 
                 PRKCZ 
                 1p36.33 
                 u DEL 
               
               
                 5 
                 TP73 
                 1p36.33 
                 u DEL/LOH 
               
               
                 6 
                 D1S2660 
                 1p36.32 
                 u DEL/LOH 
               
               
                 7 
                 D1S214 
                 1p36.31 
                 u DEL/LOH 
               
               
                 8 
                 D1S1635 
                 1p36.22 
                 LOH 
               
               
                 9 
                 D1S199 
                 1p36.13 
                 LOH 
               
               
                 10 
                 FGR(SRC2) 
                 1p36.2-p36.1 
                 AMP(1) 
               
               
                 11 
                 MYCL1(LMYC) 
                 1p34.3 
                 AMP(1) 
               
               
                 12 
                 D1S427, FAF1 
                 1p32.3 
                 LOH 
               
               
                 13 
                 D1S500 
                 1p31.1 
                 LOH 
               
               
                 14 
                 D1S418 
                 1p13.1 
                 LOH 
               
               
                 15 
                 NRAS 
                 1p13.2 
                 AMP(1) 
               
               
                 16 
                 D1S2465, D1S3402 
                 1p12 
               
               
                 17 
                 WI-5663, WI-13414 
                 1q21 
               
               
                 18 
                 LAMC2 
                 1q25-q31 
                 AMP(1) 
               
               
                 19 
                 PTGS2(COX2) 
                 1q31.1 
               
               
                 20 
                 TGFB2 
                 1q41 
               
               
                 21 
                 AKT3 
                 1q44 
                 AMP 
               
               
                 22 
                 SHGC-18290 
                 1q tel 
                 Sub Tel 
               
               
                 23 
                 1QTEL10 
                 1q tel 
                 Sub Tel 
               
               
                 24 
                 U32389 
                 2p tel 
                 Sub Tel 
               
               
                 25 
                 2PTEL27 
                 2p tel 
                 Sub Tel 
               
               
                 26 
                 MYCN(N-myc) 
                 2p24.1 
                 AMP(1) 
               
               
                 27 
                 MSH2, KCNK12 
                 2p22.3-2p22.1 
                 LOH 
               
               
                 28 
                 REL 
                 2p13-p12 
                 AMP(1) 
               
               
                 29 
                 GNLY 
                 2p12-q11 
                 M 
               
               
                 30 
                 SGC34236 
                 2q13 
                 M 
               
               
                 31 
                 BIN1 
                 2q14 
                 pTSG 
               
               
                 32 
                 LRP1B 
                 2q21.2 
                 pTSG 
               
               
                 33 
                 TBR1 
                 2q23-q37 
                 M 
               
               
                 34 
                 ITGA4 
                 2q31-q32 
                 M 
               
               
                 35 
                 CASP8 
                 2q33-q34 
                 LOH 
               
               
                 36 
                 ERBB4(HER-4) 
                 2q33.3-q34 
                 HER-2 homol 
               
               
                 37 
                 WI-6310 
                 2q tel 
                 Sub Tel 
               
               
                 38 
                 D2S447 
                 2q tel 
                 Sub Tel 
               
               
                 39 
                 3PTEL25 
                 3p tel 
                 Sub Tel 
               
               
                 40 
                 3PTEL01, CHL1 
                 3p tel 
                 Sub Tel 
               
               
                 41 
                 VHL 
                 3p25-p26 
                 TSG 
               
               
                 42 
                 RAF1 
                 3p25 
                 AMP(1) 
               
               
                 43 
                 THRB 
                 3p24.3 
                 LOH 
               
               
                 44 
                 MLH1 
                 3p21.3-p23 
                 Del 
               
               
                 45 
                 RASSF1 
                 3p21.3 
                 pTSG 
               
               
                 46 
                 FHIT 
                 3p14.2 
                 pTSG 
               
               
                 47 
                 p44S10 
                 3p14.1 
               
               
                 48 
                 D3S1274, ROBO1 
                 3p12-3p13 
                 LOH 
               
               
                 49 
                 RBP1, RBP2 
                 3q21-q22 
               
               
                 50 
                 TERC 
                 3q26 
                 AMP(1) 
               
               
                 51 
                 EIF5A2 
                 3q26.2 
               
               
                 52 
                 PIK3CA 
                 3q26.3 
                 AMP(1) 
               
               
                 53 
                 TP63 
                 3q27-q29 
                 TSG 
               
               
                 54 
                 MFI2 
                 3q tel 
                 Sub Tel 
               
               
                 55 
                 3QTEL05 
                 3q tel 
                 Sub Tel 
               
               
                 56 
                 GS10K2/T7 
                 4p tel 
                 Sub Tel 
               
               
                 57 
                 SHGC4-207 
                 4p tel 
                 Sub Tel 
               
               
                 58 
                 D4S114 
                 4p16.3 
                 u DEL 
               
               
                 59 
                 WHSC1 
                 4p16.3 
                 u DEL 
               
               
                 60 
                 DDX15 
                 4p15.3 
                 M 
               
               
                 61 
                 KIT 
                 4q11-q12 
                 ONC 
               
               
                 62 
                 PDGFRA 
                 4q11-q13 
                 AMP(1) 
               
               
                 63 
                 EIF4E 
                 4q24 (by 
                 AMP 
               
               
                   
                   
                 ucsc) 
               
               
                 64 
                 PGRMC2 
                 4q26 
               
               
                 65 
                 PDZ-GEF1 
                 4q32.1 
                 M 
               
               
                 66 
                 4QTEL11 
                 4q tel 
                 Sub Tel 
               
               
                 67 
                 D4S2930 
                 4q tel 
                 Sub Tel 
               
               
                 68 
                 C84C11/T3 
                 5p tel 
                 Sub Tel 
               
               
                 69 
                 D5S23 
                 5p15.2 
                 u DEL 
               
               
                 70 
                 D5S2064 
                 5p15.2 
                 u DEL 
               
               
                 71 
                 DAB2 
                 5p13 
                 pTSG 
               
               
                 72 
                 DHFR, MSH3 
                 5q11.2-q13.2 
                 gain/loss Ca 
               
               
                 73 
                 APC 
                 5q21-q22 
                 Del 
               
               
                 74 
                 EGR1 
                 5q31.1 
                 Del 
               
               
                 75 
                 CSF1R 
                 5q33-q35 
                 Del 
               
               
                 76 
                 NIB1408 
                 5q tel 
                 Sub Tel 
               
               
                 77 
                 5QTEL70 
                 5q tel 
                 Sub Tel 
               
               
                 78 
                 6PTEL48 
                 6p tel 
                 Sub Tel 
               
               
                 79 
                 PIM1 
                 6p21.2 
                 M 
               
               
                 80 
                 CCND3 
                 6p21 
                 AMP 
               
               
                 81 
                 D6S414 
                 6p12.1-p21.1 
               
               
                 82 
                 HTR1B 
                 6q13 
                 M 
               
               
                 83 
                 D6S434 
                 6q16.3 
                 Del 
               
               
                 84 
                 D6S268 
                 6q16.3-q21 
                 LOH 
               
               
                 85 
                 MYB 
                 6q22-q23 
                 AMP(1) 
               
               
                 86 
                 D6S311 
                 6q23-24 
                 LOH 
               
               
                 87 
                 ESR1 
                 6q25.1 
                 AMP(1) 
               
               
                 88 
                 6QTEL54 
                 6q tel 
                 Sub Tel 
               
               
                 89 
                 G31341 
                 7p tel 
                 Sub Tel 
               
               
                 90 
                 IL6 
                 7p21 
                 M 
               
               
                 91 
                 EGFR 
                 7p12.3-p12.1 
                 AMP(1) 
               
               
                 92 
                 ELN 
                 7q11.23 
                 u DEL 
               
               
                 93 
                 RFC2, CYLN2 
                 7q11.23 
                 u DEL 
               
               
                 94 
                 ABCB1(MDR1) 
                 7q21.1 
                 AMP(1) 
               
               
                 95 
                 CDK6 
                 7q21-q22 
                 AMP 
               
               
                 96 
                 SERPINE1 
                 7q21.3-q22 
                 pTSG 
               
               
                 97 
                 MET 
                 7q31 
                 AMP(1) 
               
               
                 98 
                 TIF1 
                 7q32-q34 
                 M 
               
               
                 99 
                 stSG48460 
                 7q tel 
                 Sub Tel 
               
               
                 100 
                 7QTEL20 
                 7q tel 
                 Sub Tel 
               
               
                 101 
                 D8S504 
                 8p tel 
                 Sub Tel 
               
               
                 102 
                 D8S596 
                 8p tel 
                 Sub Tel 
               
               
                 103 
                 CTSB 
                 8p22 
                 AMP(1) 
               
               
                 104 
                 PDGRL 
                 8p22-p21.3 
                 Del 
               
               
                 105 
                 LPL 
                 8p22 
                 Del 
               
               
                 106 
                 FGFR1 
                 8p11.2-p11.1 
                 AMP(1) 
               
               
                 107 
                 MOS 
                 8q11 
                 AMP(1) 
               
               
                 108 
                 E2F5 
                 8p22-q21.3 
                 M 
               
               
                 109 
                 EXT1 
                 8q24.11-q24.13 
                 TSG, uDel 
               
               
                 110 
                 MYC 
                 8q24.12-q24.13 
                 AMP(1) 
               
               
                 111 
                 PTK2 
                 8q24-qter 
                 AMP 
               
               
                 112 
                 SHGC-31110 
                 8q tel 
                 Sub Tel 
               
               
                 113 
                 U11829 
                 8q tel 
                 Sub Tel 
               
               
                 114 
                 AF170276 
                 9p tel 
                 Sub Tel 
               
               
                 115 
                 D9S913 
                 9ptel 
                 Sub Tel 
               
               
                 116 
                 MTAP 
                 9p21.3 
                 LOH 
               
               
                 117 
                 CDKN2A(p16), MTAP 
                 9p21 
                 TSG 
               
               
                 118 
                 AFM137XA11 
                 9p11.2 
                 M 
               
               
                 119 
                 D9S166 
                 9p12-q21 
               
               
                 120 
                 PTCH 
                 9q22.3 
                 TSG 
               
               
                 121 
                 DBCCR1 
                 9q33.2 
                 TSG 
               
               
                 122 
                 TSC1 
                 9q34 
                 TSG 
               
               
                 123 
                 ABL1 
                 9q34.1 
                 AMP(1) 
               
               
                 124 
                 H18962 
                 9q tel 
                 Sub Tel 
               
               
                 125 
                 D9S325 
                 9q tel 
                 Sub Tel 
               
               
                 126 
                 10PTEL006 
                 10p tel 
                 Sub Tel 
               
               
                 127 
                 SHGC-44253 
                 10p tel 
                 Sub Tel 
               
               
                 128 
                 D10S249, D10S533 
                 10p15 
                 TSG 
               
               
                 129 
                 GATA3 
                 10p15 
               
               
                 130 
                 WI-2389, D10S1260 
                 10p14-p13 
                 u DEL 
               
               
                 131 
                 BMI1 
                 10p13 
                 gain 
               
               
                 132 
                 D10S167 
                 10p11-10q11 
                 near Cen 
               
               
                 133 
                 EGR2 
                 10q21.3 
                 M 
               
               
                 134 
                 PTEN 
                 10q23.3 
                 TSG 
               
               
                 135 
                 FGFR2 
                 10q26 
                 AMP(1) 
               
               
                 136 
                 DMBT1 
                 10q25.3-q26.1 
               
               
                 137 
                 stSG27915 
                 10q tel 
                 Sub Tel 
               
               
                 138 
                 10QTEL24 
                 10q tel 
                 Sub Tel 
               
               
                 139 
                 11PTEL03 
                 11p tel 
                 Sub Tel 
               
               
                 140 
                 INS 
                 11p tel 
                 Sub Tel 
               
               
                 141 
                 HRAS 
                 11p15.5 
                 AMP(1) 
               
               
                 142 
                 CDKN1C(p57) 
                 11p15.5 
                 TSG 
               
               
                 143 
                 WT1 
                 11p13 
                 TSG 
               
               
                 144 
                 KAI1 
                 11p11.2 
               
               
                 145 
                 D11S461 
                 11q12.2 
                 near cen 
               
               
                 146 
                 MEN1 
                 11q13 
               
               
                 147 
                 CCND1 
                 11q13 
                 AMP(1) 
               
               
                 148 
                 FGF4, FGF3 
                 11q13 
                 AMP(1) 
               
               
                 149 
                 EMS1 
                 11q13 
                 AMP(1) 
               
               
                 150 
                 GARP 
                 11q13.5-q14 
                 AMP(1) 
               
               
                 151 
                 PAK1 
                 11q13-q14 
                 AMP(1) 
               
               
                 152 
                 RDX 
                 11q22.3 
                 LOH 
               
               
                 153 
                 ATM 
                 11q22.3 
                 LOH 
               
               
                 154 
                 MLL 
                 11q23 
                 AMP(1) 
               
               
                 155 
                 WI-6509 
                 11q tel 
                 Sub Tel 
               
               
                 156 
                 AF240622 
                 11q tel 
                 Sub Tel 
               
               
                 157 
                 8M16/SP6 
                 12p tel 
                 Sub Tel 
               
               
                 158 
                 SHGC-5557 
                 12p tel 
                 Sub Tel 
               
               
                 159 
                 CCND2 
                 12p13 
                 AMP(1) 
               
               
                 160 
                 CDKN1B(p27) 
                 12p13.1-p12 
                 TSG 
               
               
                 161 
                 KRAS2 
                 12p11.2 
                 AMP(1) 
               
               
                 162 
                 WNT1(INT1) 
                 12q12-q13 
                 AMP(1) 
               
               
                 163 
                 CDK2, ERBB3 
                 12q13 
                 AMP 
               
               
                 164 
                 GLI 
                 12q13.2-q13.3 
                 AMP(1) 
               
               
                 165 
                 SAS, CDK4 
                 12q13-q14 
                 AMP(1) 
               
               
                 166 
                 MDM2 
                 12q14.3-q15 
                 AMP(1) 
               
               
                 167 
                 DRIM, ARL1 
                 12q23 
               
               
                 168 
                 stSG8935 
                 12q tel 
                 Sub Tel 
               
               
                 169 
                 U11838 
                 12q tel 
                 Sub Tel 
               
               
                 170 
                 BRCA2 
                 13q12-q13 
                 TSG 
               
               
                 171 
                 RB1 
                 13q14 
                 TSG 
               
               
                 172 
                 D13S319 
                 13q14.2 
                 LOH 
               
               
                 173 
                 D13S25 
                 13q14.3 
                 LOH 
               
               
                 174 
                 D13S327 
                 13q tel 
                 Sub Tel 
               
               
                 175 
                 PNN(DRS) 
                 14q13 
               
               
                 176 
                 TCL1A 
                 14q32.1 
                 gain/loss 
               
               
                 177 
                 AKT1 
                 14q32.32 
                 AMP(1) 
               
               
                 178 
                 IGH(D14S308) 
                 14q tel 
                 Sub Tel 
               
               
                 179 
                 IGH(SHGC-36156) 
                 14q tel 
                 Sub Tel 
               
               
                 180 
                 D15S11 
                 15q11-q13 
                 u DEL 
               
               
                 181 
                 SNRPN 
                 15q12 
                 u DEL 
               
               
                 182 
                 UBE3A, D15S10 
                 15q11-q13 
                 u DEL 
               
               
                 183 
                 GABRB3 
                 15q11.2-q12 
                 u DEL 
               
               
                 184 
                 MAP2K5 
                 15q23 
               
               
                 185 
                 FES 
                 15q26.1 
                 AMP(1) 
               
               
                 186 
                 IGF1R 
                 15q25-q26 
                 AMP(1) 
               
               
                 187 
                 PACE4C 
                 15q tel 
                 Sub Tel 
               
               
                 188 
                 WI-5214 
                 15q tel 
                 Sub Tel 
               
               
                 189 
                 16PTEL03 
                 16p tel 
                 Sub Tel 
               
               
                 190 
                 stSG48414 
                 16p tel 
                 Sub Tel 
               
               
                 191 
                 CREBBP 
                 16p13.3 
                 u DEL 
               
               
                 192 
                 EMP2 
                 16p13.3 
               
               
                 193 
                 ABCC1(MRP1) 
                 16p13.1 
                 AMP(1) 
               
               
                 194 
                 CYLD 
                 16q12-q13 
                 TSG 
               
               
                 195 
                 CDH1 
                 16q22.1 
                 LOH 
               
               
                 196 
                 FRA16D 
                 16q23.2 
               
               
                 197 
                 CDH13 
                 16q24.2-q24.3 
                 LOH 
               
               
                 198 
                 LZ16 
                 16q24.2 
                 Del 
               
               
                 199 
                 FANCA 
                 16q24.3 
               
               
                 200 
                 stSG30213 
                 16q tel 
                 Sub Tel 
               
               
                 201 
                 16QTEL013 
                 16q tel 
                 Sub Tel 
               
               
                 202 
                 282M15/SP6 
                 17p tel 
                 Sub Tel 
               
               
                 203 
                 WI-14673 
                 17p tel 
                 Sub Tel 
               
               
                 204 
                 HIC1 
                 17p13.3 
                 LOH 
               
               
                 205 
                 D17S379, MNT 
                 17p13.3 
                 u DEL/LOH 
               
               
                 206 
                 PAFAH1B1(LIS1) 
                 17p13.3 
                 u DEL 
               
               
                 207 
                 TP53(p53) 
                 17p13.1 
                 TSG 
               
               
                 208 
                 D17S125, D17S61 
                 17p12-p11.2 
                 u Del/u Dup 
               
               
                 209 
                 D17S1296, D17S1523 
                 17p12-p11.2 
                 u Del/u Dup 
               
               
                 210 
                 LLGL1 
                 17p12-17p11.2 
                 u DEL 
               
               
                 211 
                 FLI, TOP3A 
                 17p12-17p11.2 
                 u DEL 
               
               
                 212 
                 NF1 5′ 
                 17q11.2 
               
               
                 213 
                 NF1 3′ 
                 17q11.2 
               
               
                 214 
                 BRCA1 
                 17q21 
                 TSG 
               
               
                 215 
                 PPARBP(PBP) 
                 17q12 
                 AMP 
               
               
                 216 
                 ERBB2(HER-2) 
                 17q11.2-17q12 
                 AMP(1) 
               
               
                 217 
                 THRA 
                 17q11.2 
                 AMP 
               
               
                 218 
                 TOP2A 
                 17q21-q22 
                 AMP 
               
               
                 219 
                 NME1(NME23) 
                 17q21.3 
                 LOH? 
               
               
                 220 
                 RPS6KB1(STK14A) 
                 17q23 
                 AMP(1) 
               
               
                 221 
                 D17S1670 
                 17q23 
               
               
                 222 
                 TK1 
                 17q23.2-q25.3 
                 AMP? 
               
               
                 223 
                 SHGC-103396 
                 17q tel 
                 Sub Tel 
               
               
                 224 
                 AFM217YD10 
                 17q tel 
                 Sub Tel 
               
               
                 225 
                 D18S552 
                 18p tel 
                 Sub Tel 
               
               
                 226 
                 SHGC17327 
                 18p tel 
                 Sub Tel 
               
               
                 227 
                 YES1 
                 18p11.31-p11.21 
                 AMP(1) 
               
               
                 228 
                 TYMS(TS) 
                 18p11.32 
                 AMP 
               
               
                 229 
                 LAMA3 
                 18q11.2 
               
               
                 230 
                 FRA18A(D18S978) 
                 18q12.3 
               
               
                 231 
                 DCC 
                 18q21.3 
                 Del 
               
               
                 232 
                 MADH4(DPC4) 
                 18q21.1 
                 TSG 
               
               
                 233 
                 BCL2 3′ 
                 18q21.3 
                 AMP 
               
               
                 234 
                 CTDP1, SHGC-145820 
                 18q tel 
                 Sub Tel 
               
               
                 235 
                 18QTEL11 
                 18q tel 
                 Sub Tel 
               
               
                 236 
                 129F16/SP6 
                 19p tel 
                 Sub Tel 
               
               
                 237 
                 stSG42796 
                 19p tel 
                 Sub Tel 
               
               
                 238 
                 INSR 
                 19p13.2 
                 AMP(1) 
               
               
                 239 
                 JUNB 
                 19p13.2 
                 AMP(1) 
               
               
                 240 
                 CCNE1 
                 19q12 
                 AMP(1) 
               
               
                 241 
                 AKT2 
                 19q13.1-q13.2 
                 LOH 
               
               
                 242 
                 GLTSCR2, SULT2A1 
                 19q13.32 
               
               
                 243 
                 D19S238E 
                 19 q tel 
                 Sub Tel 
               
               
                 244 
                 20PTEL18 
                 20p tel 
                 Sub Tel 
               
               
                 245 
                 SOX22 
                 20p tel 
                 Sub Tel 
               
               
                 246 
                 JAG1 
                 20p12.1-p11.23 
                 uDel 
               
               
                 247 
                 MKKS, SHGC-79896 
                 20p12.1-p11.23 
                 uDel 
               
               
                 248 
                 TOP1 
                 20q12-q13.1 
                 AMP(1) 
               
               
                 249 
                 NCOA3(AIB1) 
                 20q12 
                 AMP(1) 
               
               
                 250 
                 MYBL2 
                 20q13.1 
                 AMP(1) 
               
               
                 251 
                 CSE1L(CAS) 
                 20q13 
                 AMP(1) 
               
               
                 252 
                 PTPN1 
                 20q13.1-q13.2 
                 AMP(1) 
               
               
                 253 
                 STK6(STK15) 
                 20q13.2-q13.3 
                 AMP(1) 
               
               
                 254 
                 ZNF217(ZABC1) 
                 20q13.2 
                 AMP(1) 
               
               
                 255 
                 CYP24 
                 20q13.2 
                 AMP 
               
               
                 256 
                 TNFRSF6B(DCR3) 
                 20q13 
                 AMP 
               
               
                 257 
                 TPD52L2, TOM 
                 20q tel 
                 Sub Tel 
               
               
                 258 
                 20QTEL14 
                 20q tel 
                 Sub Tel 
               
               
                 259 
                 D21S378 
                 21q11.2 
                 M 
               
               
                 260 
                 RUNX1(AML1) 
                 21q22.3 
                 AMP(1) 
               
               
                 261 
                 DYRK1A 
                 21q22 
                 gain 
               
               
                 262 
                 D21S341, D21S342 
                 21q22.3 
                 gain 
               
               
                 263 
                 PCNT2(KEN) 
                 21q tel 
                 Sub Tel 
               
               
                 264 
                 21QTEL08 
                 21q tel 
                 Sub Tel 
               
               
                 265 
                 D22S543 
                 22q11 
                 M 
               
               
                 266 
                 GSCL 
                 22q11.21 
                 u DEL 
               
               
                 267 
                 HIRA(TUPLE1) 
                 22q11.21 
                 u DEL 
               
               
                 268 
                 TBX1 
                 22q11.2 
                 u DEL 
               
               
                 269 
                 BCR 
                 22q11.23 
                 AMP(1) 
               
               
                 270 
                 NF2 
                 22q12.2 
                 TSG 
               
               
                 271 
                 PDGFB(SIS) 
                 22q13.1 
                 AMP(1) 
               
               
                 272 
                 ARHGAP8 
                 22q13.3 
               
               
                 273 
                 ARSA 
                 22q tel 
                 Sub Tel 
               
               
                 274 
                 22QTEL31 
                 22q tel 
                 Sub Tel 
               
               
                 275 
                 DXYS129 
                 X/Yp tel 
                 Sub Tel 
               
               
                 276 
                 STS 3′ 
                 Xp22.3 
                 u DEL 
               
               
                 277 
                 STS 5′ 
                 Xp22.3 
                 u DEL 
               
               
                 278 
                 KAL 
                 Xp22.3 
                 u DEL 
               
               
                 279 
                 DMD exon 45-51 
                 Xp21.1 
               
               
                 280 
                 DXS580 
                 Xp11.2 
               
               
                 281 
                 DXS7132 
                 Xq12 
               
               
                 282 
                 AR 3′ 
                 Xq11-q12 
                 AMP(1) 
               
               
                 283 
                 XIST 
                 Xq13.2 
               
               
                 284 
                 OCRL1 
                 Xq25 
               
               
                 285 
                 EST CDY16c07 
                 X/Yq tel 
                 Sub Tel 
               
               
                 286 
                 SRY 
                 Yp11.3 
               
               
                 287 
                 AZFa region 
                 Yq11 
               
               
                   
               
             
          
           
               
                 Sub Tel 
                 Single copy sequence near the telomere 
               
               
                 AMP(1) 
                 Cancer Amplicon previously placed on AmpliOncl 
               
               
                   
                 Chip 
               
               
                 AMP 
                 Cancer Amplicon not previously placed on 
               
               
                   
                 AmpliOncl Chip 
               
               
                 u DEL 
                 Region lost in microdeletion syndrome 
               
               
                 TSG 
                 Tumor Supressor Gene 
               
               
                 pTSG 
                 putqtive Tumor Supressor Gene 
               
               
                 M 
                 Marker added to reduce genomic gaps 
               
               
                 gain 
                 Region gained in cancer 
               
               
                 u Dup 
                 Micro Duplication 
               
               
                 LOH 
                 Region of Loss of Heterozygosity 
               
               
                 Del 
                 Deletion Region 
               
               
                 near Cen 
                 Single copy sequence near the centromere