Abstract:
Disclosed in this specification is a method of defining chromosome regions of prognostic value by summarizing the significance of all SNPs (single nucleotide polymorphism) in a predetermined section of a chromosome to define chromosome regions of prognostic value. Based on the SNPs in specified genes, a more accurate prognosis for breast cancer may be provided.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims priority to and the benefit of co-pending U.S. provisional patent application Ser. No. 61/007,650, filed Dec. 14, 2007, which application is incorporated herein by reference in its entirety. 
     
    
     FIELD OF THE INVENTION 
       [0002]    This invention relates, in one embodiment, to a method of providing a prognosis for breast cancer by determining the number of single nucleotide polymorphisms (SNPs) in specified genes. 
       BACKGROUND OF THE INVENTION 
       [0003]    Breast cancer is a heterogeneous disease that exhibits a wide variety of clinical presentations, histological types and growth rates. In patients with no detectable lymph node involvement (a population thought to be at low-risk) between 20-30% of the patients develop recurrent disease after five to ten years of follow-up. Identification of individuals in this group who are at risk for recurrence cannot be done reliably at present. 
         [0004]    DNA copy number alterations (CNAs) or copy number polymorphisms (CNPs), such as deletions, insertion and amplifications, are believed to be one of the major genomic alterations that contribute to the carcinogenesis. Both conventional and array-based comparative genomic hybridizations have revealed chromosomal regions that are altered in breast tumors. There is no study, however, that used a high throughput, high resolution platform to investigate the relationship of DNA copy number alterations with breast cancer prognosis. 
       SUMMARY OF THE INVENTION 
       [0005]    The methods disclosed herein make it feasible to use copy number alterations (CNAs) to predict patient prognostic outcome. When combined with gene expression based signatures for prognosis, copy number signature (CNS) refines risk classification and can identify those breast cancer patients who have a significantly worse outlook in prognosis and a potential differential response to chemotherapeutic drugs. 
         [0006]    In the examples discussed herein a high-throughput and high-resolution oligo-nucleotide based single nucleotide polymorphism (SNP) array technology was used to analyze the CNAs for more than 100,000 SNP loci in the breast cancer genome. In a large cohort of 313 LNN (lymph node negative) breast cancer patients CNAs were identified that were correlated with a subset of patients with a very high probability of developing distant metastasis. The prognostic power of the CNAs was validated in two independent patient cohorts. In addition, using published predictive gene signatures, the identified patient subgroups with different prognosis were tested for putative drug efficacy. The results indicate that combining DNA copy number analysis and gene expression analysis provides an additional and better means for risk assessment for breast cancer patients. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS  
         [0007]    The present invention is disclosed with reference to the accompanying drawings, wherein: 
           [0008]      FIG. 1  is an analysis workflow to identify the genes (SNPs) with prognostic copy number alterations (CNAs); 
           [0009]      FIGS. 2A and 2B  depict the chromosomal regions with prognostic CNAs; 
           [0010]      FIG. 3  shows distant metastasis-free survival as a function of CNS; 
           [0011]      FIG. 4  illustrations the sensitivity to chemotherapeutic compounds; 
           [0012]      FIG. 5  graphically depicts the differentiation of ER-positive and ER-negative tumors; and 
           [0013]      FIG. 6  illustrates certain data of ER-negative tumors. 
       
    
    
       [0014]    The examples set out herein illustrate several embodiments of the invention but should not be construed as limiting the scope of the invention in any manner. 
       DETAILED DESCRIPTION  
       [0015]    Specific DNA copy number alterations (CNAs), such as deletions and amplifications, are major genomic alterations that contribute to the carcinogenesis and tumor progression through reduced apoptosis, unchecked proliferation, increased motility and angiogenesis. Because a significant proportion of genomic aberrations are unrelated to cancer biology and merely due to random neutral events, it is a challenge to identify those causative gene CNAs that are responsible for gene expression regulation that ultimately leads to malignant transformation and progression. Both fluorescence in situ hybridization and comparative genomic hybridizations (CGH) have revealed chromosomal regions that showed CNAs in breast tumors. In a recent study including 51 breast tumors, a high-resolution SNP array was used together with gene-expression profiling to refine breast cancer amplicon boundaries and narrow the list of potential driver genes. However, only a limited number of studies investigated the CNAs in relation to their prognostic significance while the sample sizes of these studies were too small to draw firm conclusions. In addition, fewer studies investigated breast cancer prognosis using combined analysis of CNAs and gene expression profiling with sufficient sample size and a technology that had appropriate coverage and mapping resolution of the human genome. 
         [0016]    This specification describes the analysis of DNA copy numbers for over 100,000 SNP loci across the human genome in genomic DNA from 313 lymph node-negative (LNN) primary breast tumors for which genome-wide gene-expression data were also available. Combining these two data sets allowed the identification of genomic loci, and their mapped genes, that have high correlation with distance metastasis. The identified patient subgroups were further tested for putative drug efficacy based on published predictive signatures. 
         [0017]    A combined analysis of DNA copy number and gene expression was performed on a large cohort of 313 LNN breast cancer patients who received no adjuvant systemic therapy. To our knowledge, this is the largest such study to analyze CNAs for breast cancer prognosis using the high-density SNP array technology that has much higher resolution than aCGH. A signature of 81 genes that showed CNAs and concordant gene expression regulation were identified from a training set of 200 LNN patients. This CNS was validated in the independent 113 LNN patients, as well as in an external aCGH data set of 116 LNN patients. Preliminary clinical utility has been demonstrated since the very poor prognostic group with a particularly rapid relapse identified by the 81-gene CNS actually constituted a subset of the poor prognostic patients predicted by the 76-gene GES alone. Thus by applying CNS in addition to GES, risk classification for breast cancer patients&#39; prognosis is clearly improved. Furthermore, by using previously reported gene signature profiles for sensitivity to chemotherapeutic compounds, it was shown that this very poor prognostic group might be much more resistant to preoperative T/FAC combination chemotherapy, particularly against the cyclophosphamide and doxorubicin compounds, while benefiting from etoposide and topotecan. This may suggest that patients belonging to this category should be closely monitored and be managed with different chemotherapy regimes compared with other patient groups, and that the 81 genes of the CNS also play an important role in chemo sensitivity. 
         [0018]    Previous studies investigating the association between gene amplification and breast cancer prognosis considered different breast cancer subtypes such as ER positive and ER negative as a single homogenous cohort. However, it is well known that these tumors are pathologically and biologically very different as evidenced by tremendous distinct global gene expression profiles. This dichotomy also extended to the global pattern of the DNA copy numbers. Therefore, the analysis needed to be performed separately for ER-positive and ER-negative (estrogen-receptor positive and negative) tumors. Indeed, the prognostic chromosomal regions identified from the ER-positive tumors share little in common with those from the ER-negative tumors. For example, chromosome region 8q is a widely known site of DNA amplification that is associated with poor prognosis in breast cancer. The region 8q was indeed a hotspot for amplification in ER-positive tumors, but contained no significant amplified areas for ER-negative tumors. Because ER-negative tumors constitute only a small percentage (˜25%) of the LNN breast cancers, it is reasonable to speculate that those studies that did not separate the two types of breast tumors in their analysis may had their conclusions overwhelmed by the results from the majority of the samples of ER-positive tumors. Another apparent difference between the two types of tumors observed from our analysis was at chromosome region 20q13.2-13.3. A gain in copy number of this region in ER-positive tumors, but by contrast, a loss in copy number of this region in ER-negative tumors, was related to an early recurrence. Taken together, these results re-emphasize that ER-positive and ER-negative tumors follow different biological pathways for cancer development and progression. 
       Identification of Prognostic Chromosomal Regions 
       [0019]    The median of the mean copy numbers computed from each SNP&#39;s interquartile copy number estimates was 2.1, consistent with the general assumption that the majority of the genome is diploid. Unsupervised analysis using PCA on all 313 tumors showed that chromosomal copy number variations displayed a clear trend of separation between ER-positive and ER-negative tumors ( FIG. 5 ). Therefore, these two types of breast tumors not only differ on global gene expression profiles as indicated by many studies before, but also have distinct chromosomal variations on the DNA level. Therefore, it is necessary that the subsequent analysis be performed separately for ER-positive and ER-negative tumors. The patients were randomly divided into a training set of 200 patients (133 for ER-positive and 67 for ER-negative tumors) and a testing set of 113 patients (66 for ER-positive and 47 for ER negative tumors) (Table 1 and  FIG. 1 ) in an approximate 2:1 ratio. The training set was used to identify prognostic chromosome regions and the mapped genes, and to construct a CNS to predict distance metastasis; the testing set was set aside solely for validation purpose. 
         [0020]    First, chromosome regions were identified whose CNAs were correlated with patients&#39; DMFS. For ER-positive tumors, 45 chromosomal regions distributed over 17 chromosomes were identified as having CNAs that correlated with DMFS ( FIG. 2A  and Table 7), for ER-negative tumors there were 56 regions distributed over 19 chromosomes (Table 8). The total of these region sizes for ER-positive and ER-negative tumors were 521 (Table 4) and 496 Mb (Table 5), respectively. The prognostic chromosomal regions identified from the ER-positive tumors share little in common with those from the ER-negative tumors ( FIGS. 2A and 2B ). 
         [0021]    In the training set of 200 patients an 81-gene prognostic copy number signature (CNS) was constructed that identified a subgroup of patients with a high probability of distant metastasis in the independent testing set of 113 patients (hazard ratio [HR]:2.8, 95% confidence interval [CI]:1.4-5.6,p=0.0036), and in an external data set of 116 patients (HR: 3.7, 95 CI: 1.3-10.6,p=0.0102). These high-risk patients constituted a subset of the high-risk patients predicted by our previously established 76-gene expression signature (GES). This very poor prognostic group identified by CNS and GES was putatively more resistant to preoperative paclitaxel and 5-FU-doxorubicin-cyclophosphamide (T/FAC) combination chemotherapy (p=0.0003), particularly against the doxorubicin and cyclophosphamide compound, while potentially benefiting from etoposide and topotecan. 
       Patient Samples 
       [0022]    Frozen tumor specimens of 313 LNN breast cancer patients selected from the tumor bank at the Erasmus Medical Center (Rotterdam, Netherlands) were used in this study. None of these patients did receive any systemic (neo)adjuvant therapy. The guidelines for local primary treatment were the same. Among these specimens, 273 were used to develop a 76-gene signature for the prediction of distant metastasis using Affymetrix U133A chips. The remaining 40 patients were used to study prognostic biological pathways. The study was approved by the Medical Ethics Committee of the Erasmus MC Rotterdam, The Netherlands (MEC 02.953), and was conducted in accordance to the Code of Conduct of the Federation of Medical Scientific Societies in the Netherlands (http://www.fmwv.nl/), and where ever possible the Reporting Recommendations for Tumor Marker Prognostic Studies REMARK was followed. 
         [0023]    A sampling of 199 tumors were classified as ER positive and 114 as ER negative, using previously described ER (and PgR) cutoffs. Median age of patients at the time of surgery (breast conserving surgery: 230 patients; modified radical mastectomy: 83 patients) was 54 years (range, 26-83 years). The median follow-up time for surviving patients (n=220) was 99 months (range, 20-169 months). A total of 114 patients (36%) developed distant metastasis and were counted as failures in the analysis of DMFS. Of the 93 patients who died, 7 died without evidence of disease and were censored at last follow-up in the analysis of DMFS; 86 patients died after a previous relapse. The clinicopathological characteristics of the patients are given in Table 1. The data set containing the clinical and SNP data has been submitted to Gene Expression Omnibus database with accession number 10099 (http://www.ncbi.nlm.nih.gov/geo, username: jyu8; password: jackxyu). 
         [0024]    The external array CGH (aCGH) data set of 116 LNN patients used in this study as an independent validation was downloaded from http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE8757. The clinical data (Table 1) related to this data set were kindly provided by Dr. Teschendorff, University of Cambridge, UK. 
       DNA Isolation, Hybridization and DNA Copy Number Analysis 
       [0025]    Genomic DNA was isolated from 5 to 10 30 μm tumor cryostat sections (10-25 mg) with QIAamp DNA mini kit (Qiagen, Venlo, Netherlands) according to the protocol provided by the manufacturer. Genomic DNA from each patient sample was allelo-typed using the Affymetrix GeneChip® Mapping 100K Array Set (Affymetrix, Santa Clara, Calif.) in accordance with the standard protocol. Briefly, 250 ng of genomic DNA was digested with either Hind III or XbaI, and then ligated to adapters that recognize the cohesive four base pair (bp) overhangs. A generic primer that recognizes the adapter sequence was used to amplify adapter-ligated DNA fragments with PCR conditions optimized to preferentially amplify fragments ranging from 250 to 2000 bp size using DNA Engine (MJ Research, Watertown, Mass.). After purification with the Qiagen MinElute 96 UF PCR purification system, a total of 40 μg of PCR product was fragmented and about 2.9 μg was visualized on a 4% TBE agarose gel to confirm that the average size of DNA fragments was smaller than 180 bp. The fragmented DNA was then labeled with biotin and hybridized to the Affymetrix GeneChip® Human Mapping 100K Array Set for 17 hours at 480 C in a hybridization oven. The arrays were washed and stained using Affymetrix Fluidics Station, and scanned with GeneChip Scanner 3000 G7 and GeneChip® Operating software (GCOS) (Affymetrix). GTYPE (Affymetrix) software was used to generate a SNP call for each probe set on the array. SNP call was determined for 96.6% of the probe sets across the study, with a standard deviation of 2.6%. CCNT 3.0 software was then used to generate a value representing the copy number of each probe set. This was done by comparing the hybridized intensities of each chip to a manufacturer provided reference set of intensity measurements for over 100 normal individuals of various ethnicities. The copy number measurements were then smoothed using the genomic smoothing function of CCNT with a window size of 0.5 Mb. The Affymetrix GeneChip@Human Mapping 100K Array Set contains 115,353 probe sets for which the exact mapping positions were defined. The median length of the interval between the probe sets was 8.6 kb, 75% of the intervals were less than 28 kb and 95% were less than 94.5 kb. 
         [0000]    Identification of Chromosome Regions with Prognostic Copy Number Alterations 
         [0026]    An integrated analytical method was designed to identify the chromosome regions and the mapped candidate genes whose CNAs were correlated with distance metastasis, by taking advantage of the availability of the genomic data on both RNA gene expression which were generated from our previous studies and DNA copy number from the same cohort of patients that became available in this study ( FIG. 1 ). Our method is very similar in principle to the approach that Adler et al. took and described as stepwise linkage analysis of microarray signatures (SLAMS) to identify genetic regulators of expression signatures by intersecting genome-wide DNA copy number and gene expression data. ER-positive and ER-negative patients were analyzed separately and randomly split the patients, in an approximate 2:1 ratio, into a training set of 200 patients and a testing set of 113 patients ( FIG. 1 ) while balancing on the clinical and pathological parameters including T stage, grade, menopausal status and recurrences. The training set was used to identify prognostic chromosome regions and the mapped genes, and to construct a CNS to predict distance metastasis; the testing set was set aside solely for validation purpose. 
         [0027]    The first step in our analysis was to identify chromosome regions whose copy number alterations were correlated with distance metastasis. Briefly, in the training set the univariate Cox proportional-hazards regression was used to evaluate the statistical significance of the correlation between the copy number of each individual SNP and the time of DMFS. Then, to define prognostic chromosomal regions, chromosomes were scanned in steps of 1 Mb using a sliding window of 5 Mb which contained an average of 250 SNPs to compile the Cox regression p-values of all SNPs within the window and to determine a smoothed p-value of all these SNPs as a whole relative to permutated data sets. Briefly, for a given window of size 5 Mb containing n SNPs, let β i  and P i  denote the Cox regression coefficient and the P value from the Cox regression for the i th  SNP, respectively. A log score S for this window was defined by summarizing the statistical significance of all SNPs within this window as a whole as follows: 
         [0000]    
       
         
           
             
               
                 
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         [0028]    The indicator variable I i  was used to account for and to distinguish the positively correlated copy number changes from the negatively correlated ones, indicated by the signs of the Cox regression coefficients β i . The positive coefficients reflect that relapsing patients had higher copy numbers than disease-free patients and the negative coefficients suggested the opposite. To compute the smoothed p-values from the log scores, permutations were used to derive the null distribution of the log scores. Four hundred permutations were performed by shuffling the clinical information with regard to the patient IDs. From the smoothed p-values, the prognostic chromosomal regions were defined as the chromosomal segments within which the smoothed p-values were all less than 0.05. 
       Construction of CNS and Predictive Model 
       [0029]    Once the prognostic chromosome regions were identified, the well defined genes were mapped with an Entrez Gene ID within those regions using the UCSC Genome Browser (http://genome.ucsc.edu) Human March 2006 (hg18) assembly. Next, two filtering steps were used to select those genes with greater confidence of having prognostic values to build a CNS. First, those genes that have at least one corresponding Affymetrix U133A probe set ID were filtered down. Only those genes that had statistically significant Cox regression p-values (p&lt;0.05) from the gene expression data were followed through. Second, the correlation between the gene expression levels and copy numbers must be greater than 0.5. If the gene contained multiple SNPs inside, then the SNP with the best Cox regression p-value was selected; if contained no SNP, then the nearest SNP was chosen. For U133A probe set, the one with the best Cox p-value was used. 
         [0030]    To build a model using the genes in the CNS to predict distant metastasis, the genes numeric copy number estimates were transformed into discrete values, i.e., amplification, no change, or deletion. In order to do the transformation, the diploid copy numbers for each gene was estimated by performing a normal mixture modeling on the representative SNP&#39;s copy number data and using the main peak of the modeled distribution as the estimate of the diploid copy number. Then for amplification, it was defined as 1.5 units above the diploid copy number estimate to ensure low false positives due to the intrinsic data variability; whereas deletion was defined as 0.5 units below the diploid copy number estimate because of the nature of the alteration and the narrow distribution of the copy number data for copy number loss. Once the copy number data were transformed, the following simple and intuitive algorithm was used to build a predictive model. The algorithm classified a patient as a relapser if at least n genes had copy numbers altered in that patient, and as a non-relapser otherwise. All possible scenarios were examined for n ranging from 1 to all genes in the CNS and determined the value of n by examining the performance of the signature in the training set as measured by a significant log-rank test p-value and setting a lower limit for the percentage of positives (predicted relapsers) to avoid the situation of very small number of positives as n increases. 
       Validation of CNS 
       [0031]    The performance of the CNS was assessed both in the copy number data set of the remaining testing patients and in the external aCGH data set using the same algorithm described above. For the external data set, because it was derived from totally different aCGH technology and the data format was log 2 ratios, the cutoff for amplification was set at 0.45 while the cutoff for deletion was −0.35 to ensure comparable percentage of positives generated as the SNP array technology. As with the construction of the CNS, the validation was done in the ER positive and negative tumors separately using the corresponding subsets of genes in the CNS. The final performance shown, however, represented the combined performance for both ER positive and negative patients in the testing set. 
       Putative Response to Chemotherapy 
       [0032]    To test for putative responses of testing set patients to chemotherapeutic compounds, gene expression signatures in two published studies were used. The original gene expression data set and the R function for the prediction algorithm of diagonal linear discriminant analysis (DLDA) for the 30-gene preoperative paclitaxel, fluorouracil, doxorubicin and cyclophosphamide (T/FAC) response signature was downloaded from http://bioinformatics.mdanderson.org/pubdata.html. The model was trained from the original data set using the provided R function and then tested in our gene expression data set. For each of the seven gene expression signatures that predict sensitivity to individual chemotherapeutic drugs, the predicted probability of sensitivity to each compound using the Bayesian fitting of binary probit regression models was calculated with the help of Drs. Anil Potti and Joseph Nevins (for details see Potti A, Dressman H K, Bild A, Riedel R F, Chan G, Sayer R, et al. Genomic signatures to guide the use of chemotherapeutics. Nat Med. 2006 November; 12(11):1294-300). 
       Statistical Analysis 
       [0033]    Unsupervised analysis using principal component analysis (PCA) was performed on the copy number dataset with all SNPs to examine the potential subclasses of the tumors. Kaplan-Meier survival plots and log-rank tests were used to assess the differences in DMFS of the predicted high and low risk groups. Cox&#39;s proportional-hazard regression was performed to compute the HR and its 95% CI. Due to missing data on grade, multivariate Cox regression analysis was done by multiple imputation using Markov Chain Monte Carlo method under the general location model (Schafer J L. Analysis of incomplete multivariate data. London: Chapman &amp; Hall/CRC Press; 1997). T tests were performed to assess the significance of differential therapeutic responses among the prognostic groups. All statistical analyses were performed using R version 2.6.2. 
       Search for Prognostic Candidate Genes to Construct CNS 
       [0034]    The gene expression profiling data from our previous studies of the same tumors were used (Wang Y, Klijn J G, Zhang Y, Sieuwerts A M, Look M P, Yang F, et al. Gene-expression profiles to predict distant metastasis of lymph-node-negative primary breast cancer. Lancet. 2005 Feb. 19;365(9460):671-9 and Yu J X, Sieuwerts A M, Zhang Y, Martens J W, Smid M, Klijn J G, et al. Pathway analysis of gene signatures predicting metastasis of node-negative primary breast cancer. BMC Cancer. 2007 Sep. 25;7(1):182) to screen for genes that had consistent change patterns between the gene expression profiles and the copy number variations. It was deemed reasonable that the change in copy numbers has to be reflected in the corresponding change in gene expression levels in order to have a phenotypic effect. Within these prognostic regions, a total of 2,833 and 3,656 genes were mapped for ER-positive tumors (Table 4) and ER-negative tumors (Table 5), respectively. For the ER-positive tumors, 122 genes had significant Cox regression p&lt;0.05 in both the gene expression data and the copy number data, and showed the same direction for the changes in DNA copy number and gene expression. For the ER-negative tumors, 78 genes had significant p-values in both data sets, and showed the same direction of alterations ( FIG. 6 ). Of these, 53 (43%) genes for ER-positive and 28 (36%) genes for ER-negative tumors, respectively, had correlation coefficients between gene expression and copy number greater than 0.5. Thus in total 81 prognostic candidate genes were identified which were then used as CNS for prognosis (Table 2 and Table 6A and 6B). 
       Validation of CNS 
       [0035]    The validation was done in the ER positive and negative tumors separately for the testing set using 53 and 28 genes from the CNS, respectively. The final performance shown represented the combined results of the 2 subgroups. In the testing set of 113 independent patients, the Kaplan-Meier analyses of the two patient groups stratified by the 81-gene CNS showed a statistically significant difference in time to distance metastasis ( FIG. 3 , A) with a hazard ratio (HR) of 2.8 (p=0.0036). The estimated rate of distance metastasis at 5 years for the two groups was 27% [95% confidence interval (CI), 17% to 35%] and 67% (95% CI, 32% to 84%), respectively. When used in conjunction with our previously identified 76-gene GES (Wang Y, Klijn J G, Zhang Y, Sieuwerts A M, Look M P, Yang F, et al. Gene-expression profiles to predict distant metastasis of lymph-node-negative primary breast cancer. Lancet. 2005 Feb. 19;365(9460):671-9), the patient group with worse prognosis outcome defined by the 81-gene CNS remained the same with 67% of estimated distance metastasis at 5 years. The 76-gene GES stratified the other patient group with better prognosis further to a good and a poor prognosis group with the 5-year estimated rate of recurrence at 11% and 37%, respectively ( FIG. 3 , B). This result led to three prognostic groups, which were defined as good, poor and very poor groups for GES good/CNS good, GES poor/CNS good, GES poor/CNS poor groups, respectively. Multivariate Cox regression analysis of both signatures together with traditional clinical and pathological factors showed that the combination of the two signatures was the only significant (likelihood ratio test p=0.0003) prognostic factor for DMFS, with HRs of 8.86 comparing the very poor versus good prognostic group, and 3.59 for comparison of the poor versus the good prognostic group (Table 3). 
         [0036]    Next, the CNS were tested in a completely independent external data set of 116 LNN patients (79 ER-positive and 37 ER-negative tumors) derived from a lower resolution aCGH technology (Chin S F, Teschendorff A E, Marioni J C, Wang Y, Barbosa-Morais N L, Thorne N P, et al. High-resolution array-CGH and expression profiling identifies a novel genomic subtype of ER negative breast cancer. Genome Biol. 2007 Oct. 9;8(10):R215). The 81-gene CNS significantly stratified this patient cohort ( FIG. 3 , C) into two prognostic groups with a HR of 3.7 (p=0.0102) and remained to be the only significant prognosticator in a multivariate Cox regression analysis including age, tumor size, grade, ER status (p=0.015). The lower rate of distance metastasis at 5 years (19%) for the poor prognostic group, compared with that of our own data set, was likely due to the smaller tumor sizes (78% smaller than 2 cm) and the fact that over one-third of the patients had received adjuvant hormone and/or chemotherapy in this cohort (Table 1). 
       Response to Chemotherapy 
       [0037]    The chemotherapy response profiles were subsequently investigated for the three prognostic groups determined by the GES and CNS prognostic assays using well-validated gene signatures derived from two studies (Potti A, Dressman H K, Bild A, Riedel R F, Chan G, Sayer R, et al. Genomic signatures to guide the use of chemotherapeutics. Nat Med. 2006 Nov.;12(11):1294-300 and Hess K R, Anderson K, Symmans W F, Valero V, Ibrahim N, Mejia J A, et al. Pharmacogenomic predictor of sensitivity to preoperative chemotherapy with paclitaxel and fluorouracil, doxorubicin, and cyclophosphamide in breast cancer. J Clin Oncol. 2006 Sep. 10;24(26):4236-44) for which follow-up validation studies were also available (Bonnefoi H, Potti A, Delorenzi M, Mauriac L, Campone M, Tubiana-Hulin M, et al. Validation of gene signatures that predict the response of breast cancer to neoadjuvant chemotherapy: a substudy of the EORTC 10994/BIG 00-01 clinical trial. Lancet Oncol. 2007 Dec.;8(12):1071-8 and Peintinger F, Anderson K, Mazouni C, Kuerer H M, Hatzis C, Lin F, et al. Thirty-gene pharmacogenomic test correlates with residual cancer burden after preoperative chemotherapy for breast cancer. Clin Cancer Res. 2007 Jul. 15;13(14):4078-82). Firstly, using a previously published 30-gene signature that predicted pathological complete response (pCR) to preoperative T/FAC chemotherapy (Hess K R, Anderson K, Symmans W F, Valero V, Ibrahim N, Mejia J A, et al. Pharmacogenomic predictor of sensitivity to preoperative chemotherapy with paclitaxel and fluorouracil, doxorubicin, and cyclophosphamide in breast cancer. J Clin Oncol. 2006 Sep. 10;24(26):4236-44), each patient in the different prognostic subgroups was assigned into 2 response groups: either as having pCR or still with residual disease. Only 2 of the 15 patients (13%) in the very poor prognostic group were predicted as having pCR, while 34 of the 60 patients (57%) and 14 of the 38 patients (37%) in the poor and good prognostic groups, respectively, were predicted as having pCR. The chemo response score for the very poor prognostic group was significantly lower than those of the poor prognostic group (p=0.0003), indicating that these patients would be much more resistant to preoperative T/FAC chemotherapy in case these patients would have received pre-operative T/FAC chemotherapy ( FIG. 4 , A). Secondly, response profiles were determined for the three prognostic groups against seven individual chemotherapeutic compounds using expression signatures established on cell lines (Potti A, Dressman H K, Bild A, Riedel R F, Chan G, Sayer R, et al. Genomic signatures to guide the use of chemotherapeutics. Nat Med. 2006 Nov.;12(11):1294-300). For each compound, the predicted probability of sensitivity to the compound was calculated using the Bayesian fitting of binary probit regression models. Compared with the poor prognostic group, the patients in the very poor prognostic group appeared to be more resistant to doxorubicin ( FIG. 4 , D) and cyclophosphamide ( FIG. 4 , E), consistent with the prediction of response to T/FAC by the 30-gene signature ( FIG. 4 , A). On the other hand, the very poor prognosis group was more sensitive to etoposide ( FIG. 4 , G) and topotecan ( FIG. 4 , H). Thus, when combined with gene expression based signatures for prognosis and therapy prediction, CNAs measured by SNP arrays improve risk classification and can identify those breast cancer patients who have a significantly worse outlook in prognosis and a potential differential response to chemotherapeutic drugs. 
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                 TABLE 1 
               
             
             
               
                   
               
               
                 Clinical and pathological characteristics of patients and their tumors 
               
             
          
           
               
                   
                 All patients 
                   
                 Validation set 
                   
               
               
                 Characteristics 
                 (n = 313) 
                 Training set (n = 200) 
                 (n = 113) 
                 External validation set (n = 116) 
               
               
                   
               
             
          
           
               
                 Age, years 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Mean (SD) 
                 54 
                 (12) 
                 54 
                 (12) 
                 54 
                 (12) 
                 57 
                 (10) 
               
               
                 &lt;=40 
                 45 
                 (14%) 
                 30 
                 (15%) 
                 15 
                 (13%) 
                 6 
                 (5%) 
               
               
                 41-55 
                 134 
                 (43%) 
                 84 
                 (42%) 
                 50 
                 (44%) 
                 41 
                 (35%) 
               
               
                 56-70 
                 98 
                 (31%) 
                 62 
                 (31%) 
                 36 
                 (32%) 
                 68 
                 (59%) 
               
               
                 &gt;70 
                 36 
                 (12%) 
                 24 
                 (12%) 
                 12 
                 (11%) 
                 1 
                 (1%) 
               
               
                 Menopausal status 
               
               
                 Premenopausal 
                 152 
                 (49%) 
                 96 
                 (48%) 
                 56 
                 (50%) 
                 38 
                 (33%) 
               
               
                 Postmenopausal 
                 161 
                 (51%) 
                 104 
                 (52%) 
                 57 
                 (50%) 
                 78 
                 (67%) 
               
               
                 T stage 
               
               
                 T1 
                 153 
                 (49%) 
                 97 
                 (49%) 
                 56 
                 (49%) 
                 90 
                 (78%) 
               
               
                 T2 
                 148 
                 (47%) 
                 95 
                 (47%) 
                 53 
                 (47%) 
                 26 
                 (22%) 
               
             
          
           
               
                 T3/4 
                 11 
                 (4%) 
                 8 
                 (4%) 
                 3 
                 (3%) 
                 0 
               
             
          
           
               
                 Unknown 
                 1 
                 (0%) 
                 0 
                 1 
                 (1%) 
                 0 
               
             
          
           
               
                 Grade 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Poor 
                 165 
                 (53%) 
                 111 
                 (56%) 
                 54 
                 (48%) 
                 (48%) 
                 (42%) 
               
               
                 Moderate 
                 45 
                 (14%) 
                 29 
                 (14%) 
                 16 
                 (14%) 
                 (34%) 
                 (29%) 
               
               
                 Good 
                 6 
                 (2%) 
                 3 
                 (2%) 
                 3 
                 (3%) 
                 (34%) 
                 (29%) 
               
             
          
           
               
                 Unknown 
                 97 
                 (31%) 
                 57 
                 (28%) 
                 40 
                 (35%) 
                 0 
               
             
          
           
               
                 ER status 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Positive 
                 199 
                 (64%) 
                 133 
                 (67) 
                 66 
                 (58%) 
                 (79%) 
                 (68%) 
               
               
                 Negative 
                 114 
                 (36%) 
                 67 
                 (33) 
                 47 
                 (42%) 
                 (37%) 
                 (32%) 
               
               
                 PR status 
               
             
          
           
               
                 Positive 
                 156 
                 (50%) 
                 100 
                 (50%) 
                 56 
                 (50%) 
                 NA 
               
               
                 Negative 
                 148 
                 (47%) 
                 92 
                 (46%) 
                 56 
                 (50%) 
                 NA 
               
               
                 Unknown 
                 9 
                 (3%) 
                 8 
                 (4%) 
                 1 
                 (1%) 
                 NA 
               
             
          
           
               
                 Metastasis within 5 years 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Yes 
                 99 
                 (32%) 
                 64 
                 (32%) 
                 35 
                 (31%) 
                 8 
                 (7%) 
               
               
                 No 
                 204 
                 (65%) 
                 127 
                 (64%) 
                 77 
                 (68%) 
                 104 
                 (90%) 
               
               
                 Censored 
                 10 
                 (3%) 
                 9 
                 (4%) 
                 1 
                 (1%) 
                 4 
                 (3%) 
               
               
                 Adjuvant systemic 
               
               
                 therapy 
               
             
          
           
               
                 Yes 
                 0 
                 0 
                 0 
                 43 
                 (37%) 
               
             
          
           
               
                 No 
                 313 
                 (100%) 
                 200 
                 (100%) 
                 113 
                 (100%) 
                 71 
                 (61%) 
               
             
          
           
               
                 Unknown 
                 0 
                 0 
                 0 
                 2 
                 (2%) 
               
               
                   
               
               
                 Grade was assessed by regional pathologists and reflects the current practice during the years the tumors were collected; ER positive and PgR positive: &gt;10 fmol/mg protein or &gt;10% positive tumor cells. NA, not available. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Description of the 81 genes used as the copy number signature (CNS) 
               
             
          
           
               
                   
                 Prognostic genes with copy number alteration 
               
               
                   
                   
               
             
          
           
               
                 Gain in ER+ tumors 
                 SMC4, PDCD10, PREP, CBX3, NUP205, TCEB1, TERF1, TPD52, GGH, TRAM1, 
               
               
                   
                 ZBTB10, YTHDF3, EIF3E, POLR2K, RPL30, CCNE2, RAD54B, MTERFD1, ENY2, 
               
               
                   
                 DPY19L4, ZNF623, SCRIB, SLC39A4, ATP6V1G1, PSMA6, STRN3, CLTC, TRIM37, 
               
               
                   
                 NME1, NME2, RPS6KB1, PPM1D, MED13, SLC35B1, APPBP2, MKS1, C17orf71, 
               
               
                   
                 HEATR6, TMEM49, USP32, ANKRD40, NME1-NME2, ZNF264, ZNF304, ATP5E, 
               
               
                   
                 CSTF1, PPP1R3D, AURKA, RAE1, STX16, C20orf43, RAB22A 
               
               
                 Loss in ER+ tumors 
                 TCTN3 
               
               
                 Gain in ER− tumors 
                 C1orf9, COX5B, EIF5B, DDX18, TSN, p20, METTL5, MGAT1, TUBB2A, RWDD1, 
               
               
                   
                 PGM3, FOXO3, CDC40, REV3L, HDAC2, TSPYL4, C6orf60, ASF1A, MED23, 
               
               
                   
                 TSPYL1, ACTR10, KIAA0247, RARA, KRT10, RIOK3, IMPACT 
               
               
                 Loss in ER− tumors 
                 HDAC1, BSDC1 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Multivariate Cox regression analysis 
               
               
                 of the GES and CNS combination 
               
             
          
           
               
                   
                 Multivariate analysis 
               
             
          
           
               
                   
                 HR 
                 (95% CI) 
                 p 
               
               
                   
                   
               
             
          
           
               
                 Age (per 10-yr increment) 
                 0.77 
                 (0.48-1.22) 
                 0.2573 
               
               
                 Post versus premenopausal 
                 1.34 
                 (0.45-3.97) 
                 0.5920 
               
               
                 Grade 1 and 2 versus 3 
                 0.45 
                 (0.17-1.19) 
                 0.1060 
               
               
                 Tumor size &gt;20 mm vs ≦20 mm 
                 1.02 
                 (0.54-1.92) 
                 0.9583 
               
               
                 ER negative versus positive 
                 1.07 
                 (0.52-2.19) 
                 0.8590 
               
               
                 GES &amp; CNS combination 
               
               
                 poor versus good 
                 3.59 
                 (1.35-9.49) 
                 0.0102 
               
               
                 very poor versus good 
                 8.86 
                 (2.76-28.4) 
                 0.0002 
               
               
                   
               
               
                 HR = hazard ratio; 95% CI = 95% confidence interval. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Chromosome regions with prognostic copy number 
               
               
                 alterations (CNAs) for ER-positive tumors 
               
             
          
           
               
                   
                 Chromosome 
                 No. 
                 Total region size 
                 Total No. 
                   
                 No. SNPs within 
               
               
                 Chromosome 
                 size (Mb) 
                 regions 
                 (Mb) 
                 SNPs 
                 No. genes 
                 genes 
               
               
                   
               
             
          
           
               
                 1 
                 245.12 
                 3 
                 32.64 
                 1257 
                 224 
                 440 
               
               
                 2 
                 242.40 
                 4 
                 12.18 
                 391 
                 69 
                 142 
               
               
                 3 
                 198.70 
                 5 
                 38 
                 1791 
                 183 
                 786 
               
               
                 4 
                 191.09 
                 2 
                 13.67 
                 408 
                 106 
                 141 
               
               
                 5 
                 180.61 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 6 
                 170.82 
                 1 
                 6.23 
                 255 
                 37 
                 128 
               
               
                 7 
                 158.62 
                 3 
                 55.75 
                 3212 
                 237 
                 1294 
               
               
                 8 
                 146.05 
                 5 
                 58.6 
                 2629 
                 264 
                 938 
               
               
                 9 
                 138.17 
                 3 
                 52.57 
                 2178 
                 227 
                 726 
               
               
                 10 
                 135.23 
                 4 
                 57.82 
                 2434 
                 342 
                 1000 
               
               
                 11 
                 134.17 
                 3 
                 55.27 
                 2100 
                 444 
                 825 
               
               
                 12 
                 132.29 
                 3 
                 20.98 
                 959 
                 58 
                 340 
               
               
                 13 
                 114.05 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 14 
                 106.31 
                 4 
                 32.5 
                 1747 
                 172 
                 607 
               
               
                 15 
                 100.18 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 16 
                 88.37 
                 1 
                 1.82 
                 4 
                 2 
                 1 
               
               
                 17 
                 78.18 
                 1 
                 17.64 
                 558 
                 180 
                 201 
               
               
                 18 
                 76.07 
                 1 
                 49.73 
                 2622 
                 145 
                 760 
               
               
                 19 
                 63.46 
                 1 
                 2.25 
                 27 
                 57 
                 17 
               
               
                 20 
                 62.38 
                 1 
                 13.14 
                 441 
                 86 
                 150 
               
               
                 21 
                 46.92 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 22 
                 48.98 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 X 
                 154.41 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Total 
                 3012.60 
                 45 
                 521 
                 23013 
                 2833 
                 8496 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Chromosome regions with prognostic copy number 
               
               
                 alterations (CNAs) for ER-negative tumors 
               
             
          
           
               
                   
                 Chromosome 
                 No. 
                 Total region size 
                 Total No. 
                   
                 No. SNPs within 
               
               
                 Chromosome 
                 size (Mb) 
                 regions 
                 (Mb) 
                 SNPs 
                 No. genes 
                 genes 
               
               
                   
               
             
          
           
               
                 1 
                 245.12 
                 4 
                 27.91 
                 880 
                 278 
                 460 
               
               
                 2 
                 242.40 
                 9 
                 106.87 
                 4185 
                 555 
                 1459 
               
               
                 3 
                 198.70 
                 4 
                 23.92 
                 728 
                 189 
                 248 
               
               
                 4 
                 191.09 
                 3 
                 13.67 
                 657 
                 66 
                 207 
               
               
                 5 
                 180.61 
                 5 
                 21.71 
                 855 
                 127 
                 337 
               
               
                 6 
                 170.82 
                 5 
                 50.78 
                 2679 
                 193 
                 891 
               
               
                 7 
                 158.62 
                 4 
                 14.35 
                 613 
                 107 
                 310 
               
               
                 8 
                 146.05 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 9 
                 138.17 
                 1 
                 10.62 
                 0 
                 1 
                 0 
               
               
                 10 
                 135.23 
                 1 
                 8.83 
                 200 
                 48 
                 85 
               
               
                 11 
                 134.17 
                 3 
                 31.25 
                 977 
                 466 
                 349 
               
               
                 12 
                 132.29 
                 3 
                 14.19 
                 651 
                 41 
                 238 
               
               
                 13 
                 114.05 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 14 
                 106.31 
                 3 
                 22.1 
                 970 
                 146 
                 501 
               
               
                 15 
                 100.18 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 16 
                 88.37 
                 2 
                 28.22 
                 896 
                 265 
                 470 
               
               
                 17 
                 78.18 
                 1 
                 5.88 
                 99 
                 182 
                 28 
               
               
                 18 
                 76.07 
                 2 
                 13.15 
                 611 
                 45 
                 163 
               
               
                 19 
                 63.46 
                 1 
                 15.77 
                 209 
                 360 
                 107 
               
               
                 20 
                 62.38 
                 1 
                 12.41 
                 423 
                 85 
                 143 
               
               
                 21 
                 46.92 
                 1 
                 3.63 
                 76 
                 66 
                 44 
               
               
                 22 
                 48.98 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 X 
                 154.41 
                 3 
                 70.44 
                 1118 
                 436 
                 300 
               
               
                 Total 
                 3012.60 
                 56 
                 496 
                 16827 
                 3656 
                 6340 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 6A 
               
             
             
               
                   
               
               
                 Description of the 81 genes used as the CNS 
               
             
          
           
               
                   
                   
                   
                   
                   
                 100K 
                   
               
               
                   
                   
                   
                   
                 U133A 
                 Array 
               
               
                 gene 
                 chromosome 
                 Entrez 
                   
                 Cox P 
                 SNP ID 
                 SNP Cox 
               
               
                 symbol 
                 location 
                 ID 
                 U133A ID 
                 value 
                 (SNP_A-) 
                 P value 
               
               
                   
               
             
          
           
               
                 SMC4 
                 3q26.1 
                 10051 
                 201664_at 
                 0.0001 
                 1706664 
                 0.0001 
               
               
                 PDCD10 
                 3q26.1 
                 11235 
                 210907_s_at 
                 0.0101 
                 1753577 
                 0.0115 
               
               
                 PREP 
                 6q22 
                 5550 
                 204117_at 
                 0.0288 
                 1692699 
                 0.0116 
               
               
                 CBX3 
                 7p15.2 
                 11335 
                 201091_s_at 
                 0.0058 
                 1674739 
                 0.0003 
               
               
                 NUP205 
                 7q33 
                 23165 
                 212247_at 
                 0.0093 
                 1657909 
                 0.0004 
               
               
                 TCEB1 
                 8q21.11 
                 6921 
                 202823_at 
                 0.0153 
                 1684065 
                 0.0079 
               
               
                 TERF1 
                 8q13 
                 7013 
                 203448_s_at 
                 0.042 
                 1745614 
                 0.0061 
               
               
                 TPD52 
                 8q21 
                 7163 
                 201690_s_at 
                 0.0048 
                 1665579 
                 0.019 
               
               
                 GGH 
                 8q12.3 
                 8836 
                 203560_at 
                 0.0215 
                 1682989 
                 0.0143 
               
               
                 TRAM1 
                 8q13.3 
                 23471 
                 201398_s_at 
                 0.0066 
                 1695245 
                 0.0133 
               
               
                 ZBTB10 
                 8q13-q21.1 
                 65986 
                 219312_s_at 
                 0.0003 
                 1656394 
                 0.005 
               
               
                 YTHDF3 
                 8q12.3 
                 253943 
                 221749_at 
                 0.0056 
                 1719283 
                 0.009 
               
               
                 EIF3E 
                 8q22-q23 
                 3646 
                 208697_s_at 
                 0.0306 
                 1689974 
                 0.0149 
               
               
                 POLR2K 
                 8q22.2 
                 5440 
                 202634_at 
                 0.037 
                 1642344 
                 0.0235 
               
               
                 RPL30 
                 8q22 
                 6156 
                 200062_s_at 
                 0.0498 
                 1747204 
                 0.0185 
               
               
                 CCNE2 
                 8q22.1 
                 9134 
                 205034_at 
                 0.0013 
                 1659515 
                 0.028 
               
               
                 RAD54B 
                 8q21.3-q22 
                 25788 
                 219494_at 
                 0.019 
                 1663487 
                 0.0354 
               
               
                 MTERFD1 
                 8q22.1 
                 51001 
                 219363_s_at 
                 0.0291 
                 1717843 
                 0.0174 
               
               
                 ENY2 
                 8q23.1 
                 56943 
                 218482_at 
                 0.0128 
                 1675508 
                 0.0088 
               
               
                 DPY19L4 
                 8q22.1 
                 286148 
                 213391_at 
                 0.0001 
                 1727257 
                 0.0091 
               
               
                 ZNF623 
                 8q24.3 
                 9831 
                 206188_at 
                 0.0005 
                 1695955 
                 0.0121 
               
               
                 SCRIB 
                 8q24.3 
                 23513 
                 212556_at 
                 0.0323 
                 1695955 
                 0.0121 
               
               
                 SLC39A4 
                 8q24.3 
                 55630 
                 219215_s_at 
                 0.0056 
                 1695955 
                 0.0121 
               
               
                 ATP6V1G1 
                 9q32 
                 9550 
                 208737_at 
                 0.0499 
                 1712044 
                 0.0066 
               
               
                 TCTN3 
                 10q23.33 
                 26123 
                 212123_at 
                 −0.03 
                 1647197 
                 −0.0179 
               
               
                 PSMA6 
                 14q13 
                 5687 
                 208805_at 
                 0.0053 
                 1739239 
                 0.0265 
               
               
                 STRN3 
                 14q13-q21 
                 29966 
                 204496_at 
                 0.002 
                 1657718 
                 0.0021 
               
               
                 CLTC 
                 17q11-qter 
                 1213 
                 200614_at 
                 0.0011 
                 1665731 
                 0.0096 
               
               
                 TRIM37 
                 17q23.2 
                 4591 
                 213009_s_at 
                 0.0036 
                 1740610 
                 0.0025 
               
               
                 NME1 
                 17q21.3 
                 4830 
                 201577_at 
                 0.0478 
                 1735518 
                 0.0006 
               
               
                 NME2 
                 17q21.3 
                 4831 
                 201268_at 
                 0.0422 
                 1665752 
                 0.0002 
               
               
                 RPS6KB1 
                 17q23.1 
                 6198 
                 204171_at 
                 0.0002 
                 1665339 
                 0.0028 
               
               
                 PPM1D 
                 17q23.2 
                 8493 
                 204566_at 
                 0.0015 
                 1738127 
                 0.0035 
               
               
                 MED13 
                 17q22-q23 
                 9969 
                 201987_at 
                 0.0001 
                 1758346 
                 0.0042 
               
               
                 SLC35B1 
                 17q21.33 
                 10237 
                 202433_at 
                 0.0356 
                 1722156 
                 0.003 
               
               
                 APPBP2 
                 17q21-q23 
                 10513 
                 202630_at 
                 0.0117 
                 1707055 
                 0.0045 
               
               
                 MKS1 
                 17q22 
                 54903 
                 218630_at 
                 0.0272 
                 1704909 
                 0.0343 
               
               
                 C17orf71 
                 17q22 
                 55181 
                 218514_at 
                 0.0069 
                 1740610 
                 0.0025 
               
               
                 HEATR6 
                 17q23.1 
                 63897 
                 218991_at 
                 0.0026 
                 1687894 
                 0.0014 
               
               
                 TMEM49 
                 17q23.1 
                 81671 
                 220990_s_at 
                 0.0044 
                 1668378 
                 0.0071 
               
               
                 USP32 
                 17q23.2 
                 84669 
                 211702_s_at 
                 0.0042 
                 1674736 
                 0.0026 
               
               
                 ANKRD40 
                 17q21.33 
                 91369 
                 211717_at 
                 0.0468 
                 1744474 
                 0.046 
               
               
                 NME1- 
                 17q21.3 
                 654364 
                 201268_at 
                 0.0422 
                 1735518 
                 0.0006 
               
               
                 NME2 
               
               
                 ZNF264 
                 19q13.4 
                 9422 
                 205917_at 
                 0.0068 
                 1706627 
                 0.0078 
               
               
                 ZNF304 
                 19q13.4 
                 57343 
                 207753_at 
                 0.0331 
                 1645690 
                 0.0129 
               
               
                 ATP5E 
                 20q13.32 
                 514 
                 217801_at 
                 0.0118 
                 1693246 
                 0.0126 
               
               
                 CSTF1 
                 20q13.31 
                 1477 
                 32723_at 
                 0.0054 
                 1656558 
                 0.0093 
               
               
                 PPP1R3D 
                 20q13.3 
                 5509 
                 204554_at 
                 0.0205 
                 1700634 
                 0.0249 
               
               
                 AURKA 
                 20q13.2-q13.3 
                 6790 
                 204092_s_at 
                 0.0001 
                 1739857 
                 0.0093 
               
               
                 RAE1 
                 20q13.31 
                 8480 
                 201558_at 
                 0.0032 
                 1758638 
                 0.0465 
               
               
                 STX16 
                 20q13.32 
                 8675 
                 221500_s_at 
                 0.0039 
                 1688537 
                 0.0063 
               
               
                 C20orf43 
                 20q13.31 
                 51507 
                 217737_x_at 
                 0.0191 
                 1667932 
                 0.0148 
               
               
                 RAB22A 
                 20q13.32 
                 57403 
                 218360_at 
                 0.001 
                 1645691 
                 0.0077 
               
               
                 HDAC1 
                 1p34 
                 3065 
                 201209_at 
                 −0.0382 
                 1656045 
                 −0.0266 
               
               
                 BSDC1 
                 1p35.1 
                 55108 
                 218004_at 
                 −0.0196 
                 1677842 
                 −0.0266 
               
               
                 C1orf9 
                 1q24 
                 51430 
                 203429_s_at 
                 0.0429 
                 1707822 
                 0.0024 
               
               
                 COX5B 
                 2cen-q13 
                 1329 
                 211025_x_at 
                 0.0145 
                 1705118 
                 0.0018 
               
               
                 EIF5B 
                 2q11.2 
                 9669 
                 201025_at 
                 0.0441 
                 1728008 
                 0.0076 
               
               
                 DDX18 
                 2q14.1 
                 8886 
                 208896_at 
                 0.0143 
                 1696503 
                 0.0061 
               
               
                 TSN 
                 2q21.1 
                 7247 
                 201513_at 
                 0.0416 
                 1673463 
                 0.0455 
               
               
                 p20 
                 2q21.1 
                 130074 
                 212017_at 
                 0.0308 
                 1718104 
                 0.011 
               
               
                 METTL5 
                 2q31.1 
                 29081 
                 221570_s_at 
                 0.0397 
                 1652493 
                 0.0045 
               
               
                 MGAT1 
                 5q35 
                 4245 
                 201126_s_at 
                 0.0156 
                 1683255 
                 0.0185 
               
               
                 TUBB2A 
                 6p25 
                 7280 
                 204141_at 
                 0.0152 
                 1713325 
                 0.0487 
               
               
                 RWDD1 
                 6q13-q22.33 
                 51389 
                 219598_s_at 
                 0.0158 
                 1750430 
                 0.0311 
               
               
                 PGM3 
                 6q14.1-q15 
                 5238 
                 210041_s_at 
                 0.003 
                 1724282 
                 0.0413 
               
               
                 FOXO3 
                 6q21 
                 2309 
                 204131_s_at 
                 0.048 
                 1645067 
                 0.0459 
               
               
                 CDC40 
                 6q21 
                 51362 
                 203376_at 
                 0.0037 
                 1711755 
                 0.0306 
               
               
                 REV3L 
                 6q21 
                 5980 
                 208070_s_at 
                 0.004 
                 1667275 
                 0.0468 
               
               
                 HDAC2 
                 6q21 
                 3066 
                 201833_at 
                 0.0362 
                 1645015 
                 0.0007 
               
               
                 TSPYL4 
                 6q22.1 
                 23270 
                 212928_at 
                 0.0146 
                 1669819 
                 0.0098 
               
               
                 C6orf60 
                 6q22.31 
                 79632 
                 220150_s_at 
                 0.0259 
                 1694717 
                 0.0129 
               
               
                 ASF1A 
                 6q22.31 
                 25842 
                 203427_at 
                 0.0148 
                 1740438 
                 0.0168 
               
               
                 MED23 
                 6q22.33-q24.1 
                 9439 
                 218846_at 
                 0.0453 
                 1661877 
                 0.0186 
               
               
                 TSPYL1 
                 6q22-q23 
                 7259 
                 221493_at 
                 0.0155 
                 1758155 
                 0.0144 
               
               
                 ACTR10 
                 14q23.1 
                 55860 
                 222230_s_at 
                 0.0011 
                 1741052 
                 0.0343 
               
               
                 KIAA0247 
                 14q24.1 
                 9766 
                 202181_at 
                 0.0128 
                 1702018 
                 0.0005 
               
               
                 RARA 
                 17q21 
                 5914 
                 203749_s_at 
                 0.0474 
                 1731414 
                 0.0281 
               
               
                 KRT10 
                 17q21 
                 3858 
                 213287_s_at 
                 0.0309 
                 1735532 
                 0.0251 
               
               
                 RIOK3 
                 18q11.2 
                 8780 
                 202130_at 
                 0.0134 
                 1740064 
                 0.0024 
               
               
                 IMPACT 
                 18q11.2-q12.1 
                 55364 
                 218637_at 
                 0.016 
                 1684789 
                 0.017 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 6B 
               
             
             
               
                   
               
               
                 Description of the 81 genes used as the CNS (continued) 
               
             
          
           
               
                   
                   
                 gene 
                   
                   
                   
               
               
                   
                   
                 expression 
                 diploid 
               
               
                   
                 gain or 
                 &amp; copy 
                 copy 
                 copy 
               
               
                 gene 
                 loss 
                 number 
                 number 
                 number 
               
               
                 symbol 
                 (1 = gain; −1 = loss) 
                 correlation 
                 estimate 
                 cutoff 
                 description 
               
               
                   
               
             
          
           
               
                 SMC4 
                 1 
                 0.519 
                 2.176 
                 3.676 
                 SMC4 structural maintenance of chromosomes 
               
               
                   
                   
                   
                   
                   
                 4-like 1 (yeast) 
               
               
                 PDCD10 
                 1 
                 0.756 
                 2.108 
                 3.608 
                 programmed cell death 10 
               
               
                 PREP 
                 1 
                 0.722 
                 2.133 
                 3.633 
                 prolyl endopeptidase 
               
               
                 CBX3 
                 1 
                 0.585 
                 2.187 
                 3.687 
                 chromobox homolog 3 (HP1 gamma homolog, 
               
               
                   
                   
                   
                   
                   
                   Drosophila ) 
               
               
                 NUP205 
                 1 
                 0.576 
                 2.153 
                 3.653 
                 nucleoporin 205 kDa 
               
               
                 TCEB1 
                 1 
                 0.653 
                 2.348 
                 3.848 
                 transcription elongation factor B (SIII), 
               
               
                   
                   
                   
                   
                   
                 polypeptide 1 (15 kDa, elongin C) 
               
               
                 TERF1 
                 1 
                 0.801 
                 2.729 
                 4.229 
                 telomeric repeat binding factor (NIMA-interacting) 1 
               
               
                 TPD52 
                 1 
                 0.624 
                 1.904 
                 3.404 
                 tumor protein D52 
               
               
                 GGH 
                 1 
                 0.528 
                 2.011 
                 3.511 
                 gamma-glutamyl hydrolase (conjugase, 
               
               
                   
                   
                   
                   
                   
                 folylpolygammaglutamyl hydrolase) 
               
               
                 TRAM1 
                 1 
                 0.618 
                 2.211 
                 3.711 
                 translocation associated membrane protein 1 
               
               
                 ZBTB10 
                 1 
                 0.674 
                 2.027 
                 3.527 
                 zinc finger and BTB domain containing 10 
               
               
                 YTHDF3 
                 1 
                 0.62 
                 1.922 
                 3.422 
                 YTH domain family, member 3 
               
               
                 EIF3E 
                 1 
                 0.544 
                 2.106 
                 3.606 
                 eukaryotic translation initiation factor 3, subunit 6 
               
               
                   
                   
                   
                   
                   
                 48 kDa 
               
               
                 POLR2K 
                 1 
                 0.694 
                 2.216 
                 3.716 
                 polymerase (RNA) II (DNA directed) polypeptide 
               
               
                   
                   
                   
                   
                   
                 K, 7.0 kDa 
               
               
                 RPL30 
                 1 
                 0.698 
                 2.227 
                 3.727 
                 ribosomal protein L30 
               
               
                 CCNE2 
                 1 
                 0.527 
                 2.241 
                 3.741 
                 cyclin E2 
               
               
                 RAD54B 
                 1 
                 0.692 
                 1.954 
                 3.454 
                 RAD54 homolog B ( S. cerevisiae ) 
               
               
                 MTERFD1 
                 1 
                 0.788 
                 2.45 
                 3.95 
                 MTERF domain containing 1 
               
               
                 ENY2 
                 1 
                 0.775 
                 2.009 
                 3.509 
                 enhancer of yellow 2 homolog ( Drosophila ) 
               
               
                 DPY19L4 
                 1 
                 0.58 
                 1.979 
                 3.479 
                 dpy-19-like 4 ( C. elegans ) 
               
               
                 ZNF623 
                 1 
                 0.618 
                 1.837 
                 3.337 
                 zinc finger protein 623 
               
               
                 SCRIB 
                 1 
                 0.735 
                 1.837 
                 3.337 
                 scribbled homolog ( Drosophila ) 
               
               
                 SLC39A4 
                 1 
                 0.64 
                 1.837 
                 3.337 
                 solute carrier family 39 (zinc transporter), 
               
               
                   
                   
                   
                   
                   
                 member 4 
               
               
                 ATP6V1G1 
                 1 
                 0.518 
                 2.214 
                 3.714 
                 ATPase, H+ transporting, lysosomal 13 kDa, V1 
               
               
                   
                   
                   
                   
                   
                 subunit G1 
               
               
                 TCTN3 
                 −1 
                 0.577 
                 2.288 
                 1.788 
                 chromosome 10 open reading frame 61 
               
               
                 PSMA6 
                 1 
                 0.616 
                 2.226 
                 3.726 
                 proteasome (prosome, macropain) subunit, alpha 
               
               
                   
                   
                   
                   
                   
                 type, 6 
               
               
                 STRN3 
                 1 
                 0.503 
                 2.122 
                 3.622 
                 striatin, calmodulin binding protein 3 
               
               
                 CLTC 
                 1 
                 0.883 
                 1.939 
                 3.439 
                 clathrin, heavy polypeptide (Hc) 
               
               
                 TRIM37 
                 1 
                 0.781 
                 2.555 
                 4.055 
                 tripartite motif-containing 37 
               
               
                 NME1 
                 1 
                 0.812 
                 1.805 
                 3.305 
                 non-metastatic cells 1, protein (NM23A) 
               
               
                   
                   
                   
                   
                   
                 expressed in 
               
               
                 NME2 
                 1 
                 0.743 
                 1.624 
                 3.124 
                 non-metastatic cells 2, protein (NM23B) 
               
               
                   
                   
                   
                   
                   
                 expressed in 
               
               
                 RPS6KB1 
                 1 
                 0.758 
                 2.027 
                 3.527 
                 ribosomal protein S6 kinase, 70 kDa, polypeptide 
               
               
                   
                   
                   
                   
                   
                 1 
               
               
                 PPM1D 
                 1 
                 0.85 
                 2.049 
                 3.549 
                 protein phosphatase 1D magnesium-dependent, 
               
               
                   
                   
                   
                   
                   
                 delta isoform 
               
               
                 MED13 
                 1 
                 0.778 
                 2.164 
                 3.664 
                 thyroid hormone receptor associated protein 1 
               
               
                 SLC35B1 
                 1 
                 0.78 
                 2.318 
                 3.818 
                 solute carrier family 35, member B1 
               
               
                 APPBP2 
                 1 
                 0.857 
                 2.063 
                 3.563 
                 amyloid beta precursor protein (cytoplasmic tail) 
               
               
                   
                   
                   
                   
                   
                 binding protein 2 
               
               
                 MKS1 
                 1 
                 0.555 
                 2.13 
                 3.63 
                 Meckel syndrome, type 1 
               
               
                 C17orf71 
                 1 
                 0.86 
                 2.555 
                 4.055 
                 chromosome 17 open reading frame 71 
               
               
                 HEATR6 
                 1 
                 0.782 
                 2.104 
                 3.604 
                 — 
               
               
                 TMEM49 
                 1 
                 0.706 
                 1.913 
                 3.413 
                 transmembrane protein 49 
               
               
                 USP32 
                 1 
                 0.812 
                 2.146 
                 3.646 
                 ubiquitin specific peptidase 32 
               
               
                 ANKRD40 
                 1 
                 0.62 
                 2.157 
                 3.657 
                 ankyrin repeat domain 40 
               
               
                 NME1- 
                 1 
                 0.77 
                 1.805 
                 3.305 
                 — 
               
               
                 NME2 
               
               
                 ZNF264 
                 1 
                 0.557 
                 1.661 
                 3.161 
                 zinc finger protein 264 
               
               
                 ZNF304 
                 1 
                 0.78 
                 1.649 
                 3.149 
                 zinc finger protein 304 
               
               
                 ATP5E 
                 1 
                 0.514 
                 1.99 
                 3.49 
                 ATP synthase, H+ transporting, mitochondrial F1 
               
               
                   
                   
                   
                   
                   
                 complex, epsilon subunit 
               
               
                 CSTF1 
                 1 
                 0.526 
                 1.866 
                 3.366 
                 cleavage stimulation factor, 3′ pre-RNA, subunit 
               
               
                   
                   
                   
                   
                   
                 1, 50 kDa 
               
               
                 PPP1R3D 
                 1 
                 0.601 
                 2.231 
                 3.731 
                 protein phosphatase 1, regulatory subunit 3D 
               
               
                 AURKA 
                 1 
                 0.577 
                 1.866 
                 3.366 
                 aurora kinase A 
               
               
                 RAE1 
                 1 
                 0.676 
                 2.475 
                 3.975 
                 RAE1 RNA export 1 homolog ( S. pombe ) 
               
               
                 STX16 
                 1 
                 0.61 
                 2.179 
                 3.679 
                 syntaxin 16 
               
               
                 C20orf43 
                 1 
                 0.509 
                 1.912 
                 3.412 
                 chromosome 20 open reading frame 43 
               
               
                 RAB22A 
                 1 
                 0.801 
                 2.52 
                 4.02 
                 RAB22A, member RAS oncogene family 
               
               
                 HDAC1 
                 −1 
                 0.551 
                 2.329 
                 1.829 
                 histone deacetylase 1 
               
               
                 BSDC1 
                 −1 
                 0.616 
                 2.259 
                 1.759 
                 BSD domain containing 1 
               
               
                 C1orf9 
                 1 
                 0.532 
                 2.448 
                 3.948 
                 chromosome 1 open reading frame 9 
               
               
                 COX5B 
                 1 
                 0.739 
                 1.846 
                 3.346 
                 cytochrome c oxidase subunit Vb 
               
               
                 EIF5B 
                 1 
                 0.618 
                 1.706 
                 3.206 
                 eukaryotic translation initiation factor 5B 
               
               
                 DDX18 
                 1 
                 0.581 
                 2.186 
                 3.686 
                 DEAD (Asp-Glu-Ala-Asp) box polypeptide 18 
               
               
                 TSN 
                 1 
                 0.626 
                 2.308 
                 3.808 
                 translin 
               
               
                 p20 
                 1 
                 0.537 
                 1.701 
                 3.201 
                 LOC130074 
               
               
                 METTL5 
                 1 
                 0.509 
                 2.158 
                 3.658 
                 methyltransferase like 5 
               
               
                 MGAT1 
                 1 
                 0.848 
                 2.435 
                 3.935 
                 mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N- 
               
               
                   
                   
                   
                   
                   
                 acetylglucosaminyltransferase 
               
               
                 TUBB2A 
                 1 
                 0.563 
                 2.221 
                 3.721 
                 tubulin, beta 2A 
               
               
                 RWDD1 
                 1 
                 0.655 
                 1.996 
                 3.496 
                 RWD domain containing 1 
               
               
                 PGM3 
                 1 
                 0.787 
                 2.052 
                 3.552 
                 phosphoglucomutase 3 
               
               
                 FOXO3 
                 1 
                 0.823 
                 2.259 
                 3.759 
                 forkhead box O3 
               
               
                 CDC40 
                 1 
                 0.715 
                 2.261 
                 3.761 
                 cell division cycle 40 homolog ( S. cerevisiae ) 
               
               
                 REV3L 
                 1 
                 0.614 
                 1.9 
                 3.4 
                 REV3-like, catalytic subunit of DNA polymerase 
               
               
                   
                   
                   
                   
                   
                 zeta (yeast) 
               
               
                 HDAC2 
                 1 
                 0.639 
                 2.034 
                 3.534 
                 histone deacetylase 2 
               
               
                 TSPYL4 
                 1 
                 0.501 
                 1.863 
                 3.363 
                 TSPY-like 4 
               
               
                 C6orf60 
                 1 
                 0.531 
                 1.916 
                 3.416 
                 chromosome 6 open reading frame 60 
               
               
                 ASF1A 
                 1 
                 0.669 
                 1.821 
                 3.321 
                 ASF1 anti-silencing function 1 homolog A ( S . 
               
               
                   
                   
                   
                   
                   
                   cerevisiae ) 
               
               
                 MED23 
                 1 
                 0.564 
                 2.03 
                 3.53 
                 mediator complex subunit 23 
               
               
                 TSPYL1 
                 1 
                 0.529 
                 1.916 
                 3.416 
                 TSPY-like 1 
               
               
                 ACTR10 
                 1 
                 0.635 
                 1.965 
                 3.465 
                 actin-related protein 10 homolog ( S. cerevisiae ) 
               
               
                 KIAA0247 
                 1 
                 0.573 
                 1.913 
                 3.413 
                 KIAA0247 
               
               
                 RARA 
                 1 
                 0.685 
                 2.08 
                 3.58 
                 retinoic acid receptor, alpha 
               
               
                 KRT10 
                 1 
                 0.777 
                 2.085 
                 3.585 
                 keratin 1 
               
               
                 RIOK3 
                 1 
                 0.594 
                 2.021 
                 3.521 
                 RIO kinase 3 (yeast) 
               
               
                 IMPACT 
                 1 
                 0.556 
                 2.242 
                 3.742 
                 Impact homolog (mouse) 
               
               
                   
               
               
                 The top 53 genes are from ER-positive tumors, the bottom 28 are from ER-negative tumors. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Prognostic chromosome regions in ER-positive tumors 
               
             
          
           
               
                   
                 start 
                 end 
                 copy number change 
               
               
                 chromosome 
                 (base) 
                 (base) 
                 (1 = gains; −1 = loss) 
               
               
                   
               
             
          
           
               
                 1 
                 10678225 
                 18511423 
                 −1 
               
               
                 1 
                 28955687 
                 32872286 
                 −1 
               
               
                 1 
                 83788073 
                 104676601 
                 −1 
               
               
                 2 
                 9818363 
                 14413615 
                 −1 
               
               
                 2 
                 24752932 
                 25901745 
                 −1 
               
               
                 2 
                 95284610 
                 95979338 
                 1 
               
               
                 2 
                 130443728 
                 136187793 
                 −1 
               
               
                 3 
                 48603 
                 5655734 
                 1 
               
               
                 3 
                 8147792 
                 11885879 
                 1 
               
               
                 3 
                 49266749 
                 50512778 
                 −1 
               
               
                 3 
                 151441127 
                 172623620 
                 1 
               
               
                 3 
                 173869649 
                 180099794 
                 1 
               
               
                 4 
                 103115 
                 10491185 
                 −1 
               
               
                 4 
                 35641248 
                 38921691 
                 −1 
               
               
                 6 
                 104481650 
                 110713418 
                 1 
               
               
                 7 
                 250149 
                 43854476 
                 1 
               
               
                 7 
                 49374011 
                 54893546 
                 1 
               
               
                 7 
                 132167036 
                 138790478 
                 1 
               
               
                 8 
                 47365080 
                 48965918 
                 1 
               
               
                 8 
                 56155338 
                 90048318 
                 1 
               
               
                 8 
                 91075378 
                 92102438 
                 1 
               
               
                 8 
                 94156558 
                 113670698 
                 1 
               
               
                 8 
                 143455438 
                 146023088 
                 1 
               
               
                 9 
                 42004193 
                 42930351 
                 1 
               
               
                 9 
                 68229855 
                 94387165 
                 1 
               
               
                 9 
                 97218677 
                 122702285 
                 1 
               
               
                 10 
                 25372233 
                 28876308 
                 −1 
               
               
                 10 
                 47564708 
                 48732733 
                 −1 
               
               
                 10 
                 49900758 
                 51068783 
                 −1 
               
               
                 10 
                 82605458 
                 134582570 
                 −1 
               
               
                 11 
                 802188 
                 16154613 
                 −1 
               
               
                 11 
                 68966955 
                 73879731 
                 1 
               
               
                 11 
                 98443611 
                 133447140 
                 −1 
               
               
                 12 
                 42668236 
                 46370371 
                 1 
               
               
                 12 
                 69817226 
                 85859811 
                 1 
               
               
                 12 
                 87093856 
                 88327901 
                 1 
               
               
                 14 
                 22535406 
                 36835923 
                 1 
               
               
                 14 
                 44636205 
                 49836393 
                 1 
               
               
                 14 
                 53736534 
                 60236769 
                 1 
               
               
                 14 
                 83637615 
                 90137850 
                 1 
               
               
                 16 
                 32070490 
                 33891366 
                 −1 
               
               
                 17 
                 42580727 
                 60216632 
                 1 
               
               
                 18 
                 25801802 
                 75535109 
                 −1 
               
               
                 19 
                 61179186 
                 63432439 
                 1 
               
               
                 20 
                 47547185 
                 60690155 
                 1 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Prognostic chromosome regions in ER-negative tumors 
               
             
          
           
               
                   
                 start 
                 end 
                 copy number change 
               
               
                 chromosome 
                 (base) 
                 (base) 
                 (1 = gains; −1 = loss) 
               
               
                   
               
             
          
           
               
                 1 
                 21122489 
                 34177819 
                 −1 
               
               
                 1 
                 115120865 
                 120839024 
                 −1 
               
               
                 1 
                 167342185 
                 175175383 
                 1 
               
               
                 1 
                 224785637 
                 226091170 
                 1 
               
               
                 2 
                 61514948 
                 73003078 
                 −1 
               
               
                 2 
                 82193582 
                 88993415 
                 −1 
               
               
                 2 
                 95284610 
                 101723403 
                 1 
               
               
                 2 
                 108616281 
                 156866427 
                 1 
               
               
                 2 
                 164908118 
                 172949809 
                 1 
               
               
                 2 
                 192479630 
                 195926069 
                 1 
               
               
                 2 
                 215455890 
                 228092833 
                 −1 
               
               
                 2 
                 230390459 
                 239580963 
                 −1 
               
               
                 2 
                 240729776 
                 241304182 
                 −1 
               
               
                 3 
                 31822343 
                 44282633 
                 −1 
               
               
                 3 
                 48020720 
                 50512778 
                 −1 
               
               
                 3 
                 95122010 
                 97861880 
                 1 
               
               
                 3 
                 151441127 
                 157671272 
                 1 
               
               
                 4 
                 30173843 
                 31814064 
                 1 
               
               
                 4 
                 55323906 
                 61884792 
                 −1 
               
               
                 4 
                 71726121 
                 77193526 
                 −1 
               
               
                 5 
                 18740255 
                 19799220 
                 −1 
               
               
                 5 
                 30388870 
                 40978520 
                 −1 
               
               
                 5 
                 47332310 
                 48391275 
                 −1 
               
               
                 5 
                 170172250 
                 176526040 
                 1 
               
               
                 5 
                 177585005 
                 180232417 
                 1 
               
               
                 6 
                 1657478 
                 6850618 
                 1 
               
               
                 6 
                 62010774 
                 66052414 
                 1 
               
               
                 6 
                 76438694 
                 97211254 
                 1 
               
               
                 6 
                 107597534 
                 123176954 
                 1 
               
               
                 6 
                 127331466 
                 132524606 
                 1 
               
               
                 7 
                 91322477 
                 93530291 
                 −1 
               
               
                 7 
                 99049826 
                 100153733 
                 −1 
               
               
                 7 
                 106777175 
                 114504524 
                 −1 
               
               
                 7 
                 136030710 
                 139342431 
                 −1 
               
               
                 9 
                 55453875 
                 66072045 
                 −1 
               
               
                 10 
                 42236621 
                 51068783 
                 −1 
               
               
                 11 
                 34577523 
                 45631269 
                 1 
               
               
                 11 
                 54916541 
                 73879731 
                 −1 
               
               
                 11 
                 93530835 
                 94759029 
                 −1 
               
               
                 12 
                 36498011 
                 45136326 
                 −1 
               
               
                 12 
                 58093798 
                 62412956 
                 1 
               
               
                 12 
                 130285431 
                 131519476 
                 1 
               
               
                 14 
                 35535876 
                 38135970 
                 1 
               
               
                 14 
                 54386557 
                 71937192 
                 1 
               
               
                 14 
                 103138320 
                 105088390 
                 −1 
               
               
                 16 
                 17503482 
                 32070490 
                 −1 
               
               
                 16 
                 73950638 
                 87607208 
                 −1 
               
               
                 17 
                 34742547 
                 40621182 
                 1 
               
               
                 18 
                 16836580 
                 28942853 
                 1 
               
               
                 18 
                 36271972 
                 37318989 
                 1 
               
               
                 19 
                 36393397 
                 52166172 
                 −1 
               
               
                 20 
                 49007515 
                 61420320 
                 −1 
               
               
                 21 
                 41980980 
                 45609552 
                 −1 
               
               
                 23 
                 677050 
                 24691270 
                 −1 
               
               
                 23 
                 34296958 
                 56710230 
                 −1 
               
               
                 23 
                 130353838 
                 154368058 
                 −1