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The most frequent genetic alterations in melanoma are gain-of-function (GOF) mutations in BRAF, which result in RAF–MEK–ERK signaling pathway addiction. Despite therapeutic success of RAF and MEK inhibitors in treating BRAFV600-mutant tumors, a major challenge is the inevitable emergence of drug resistance, which often involves reactivation of the MAPK pathway. Interestingly, resistant tumors are often sensitive to drug withdrawal, suggesting that hyperactivation of the MAPK pathway is not tolerated. To further characterize this phenomenon, isogenic models of inducible MAPK hyperactivation in BRAFV600E melanoma cells were generated by overexpression of ERK2. Using this model system, supraphysiologic levels of MAPK signaling led to cell death, which was reversed by MAPK inhibition. Furthermore, complete tumor regression was observed in an ERK2-overexpressing xenograft model. To identify mediators of MAPK hyperactivation–induced cell death, a large-scale pooled shRNA screen was conducted, which revealed that only shRNAs against BRAF and MAP2K1 rescued loss of cell viability. This suggested that no single downstream ERK2 effector was required, consistent with pleiotropic effects on multiple cellular stress pathways. Intriguingly, the detrimental effect of MAPK hyperactivation could be partially attributed to secreted factors, and more than 100 differentially secreted proteins were identified. The effect of ERK2 overexpression was highly context dependent, as RAS/RAF mutant but not RAS/RAF wild-type melanoma were sensitive to this perturbation.Implications:This vulnerability to MAPK hyperactivation raises the possibility of novel therapeutic approaches for RAS/RAF-mutant cancers.

Inhibitors targeting BRAF and its downstream kinase MEK produce robust response in patients with advanced BRAFV600-mutant melanoma. However, the duration and depth of response vary significantly between patients; therefore, predicting response a priori remains a significant challenge. Here, we utilized the Novartis collection of patient-derived xenografts to characterize transcriptional alterations elicited by BRAF and MEK inhibitors in vivo, in an effort to identify mechanisms governing differential response to MAPK inhibition. We show that the expression of an MITF-high, “epithelial-like” transcriptional program is associated with reduced sensitivity and adaptive response to BRAF and MEK inhibitor treatment. On the other hand, xenograft models that express an MAPK-driven “mesenchymal-like” transcriptional program are preferentially sensitive to MAPK inhibition. These gene-expression programs are somewhat similar to the MITF-high and -low phenotypes described in cancer cell lines, but demonstrate an inverse relationship with drug response. This suggests a discrepancy between in vitro and in vivo experimental systems that warrants future investigations. Finally, BRAFV600-mutant melanoma relies on either MAPK or alternative pathways for survival under BRAF and MEK inhibition in vivo, which in turn predicts their response to further pathway suppression using a combination of BRAF, MEK, and ERK inhibitors. Our findings highlight the intertumor heterogeneity in BRAFV600-mutant melanoma, and the need for precision medicine strategies to target this aggressive cancer.

BRAFV600 mutant melanoma PDXs exhibit varied degrees of MAPK pathway suppression upon BRAF and MEK inhibitor treatment

List of genes from the DEG clustering analysis.

Figures S1, S2, s3, S4, Table S1

The cell surface glycoprotein P-cadherin is highly expressed in a number of malignancies, including those arising in the epithelium of the bladder, breast, esophagus, lung, and upper aerodigestive system. PCA062 is a P-cadherin specific antibody–drug conjugate that utilizes the clinically validated SMCC-DM1 linker payload to mediate potent cytotoxicity in cell lines expressing high levels of P-cadherin in vitro, while displaying no specific activity in P-cadherin–negative cell lines. High cell surface P-cadherin is necessary, but not sufficient, to mediate PCA062 cytotoxicity. In vivo, PCA062 demonstrated high serum stability and a potent ability to induce mitotic arrest. In addition, PCA062 was efficacious in clinically relevant models of P-cadherin–expressing cancers, including breast, esophageal, and head and neck. Preclinical non-human primate toxicology studies demonstrated a favorable safety profile that supports clinical development. Genome-wide CRISPR screens reveal that expression of the multidrug-resistant gene ABCC1 and the lysosomal transporter SLC46A3 differentially impact tumor cell sensitivity to PCA062. The preclinical data presented here suggest that PCA062 may have clinical value for treating patients with multiple cancer types including basal-like breast cancer.

Figure S1. Cellular phenotypes observed in response to ERK2 overexpression in A-375 cells. Figure S2. No paracrine effect was produced by cells dying due to knockdown of the essential gene PSMA3. Figure S3. A-375 xenograft tumors with ERK2 overexpression displayed necrosis and/or fibrosis. Figure S4. Paradoxical activation triggered by low doses of BRAFi in GR-M augmented the growth suppression caused by ERK2 overexpression. Figure S5. Graphical representations of vector constructs used in this study. Table S1. MSigDB Hallmark pathways that were significantly enriched in genes upregulated after 20 h treatment with doxycycline in A-375 ERK2 cells. Table S2. Transcription factor targets (from MSigDB C3 TFT) for known ERK targets were significantly enriched in genes upregulated after 20 h treatment with doxycycline in A-375 ERK2 cells.

Cell line sensitivity table

Purpose:Ductal carcinoma in situ (DCIS) is a nonobligate precursor of invasive breast cancer. Here, we sought to investigate the level of intralesion genetic heterogeneity in DCIS and the patterns of clonal architecture changes in the progression from DCIS to invasive disease.Experimental Design:Synchronous DCIS (n = 27) and invasive ductal carcinomas of no special type (IDC-NSTs; n = 26) from 25 patients, and pure DCIS (n = 7) from 7 patients were microdissected separately and subjected to high-depth whole-exome (n = 56) or massively parallel sequencing targeting ≥410 key cancer-related genes (n = 4). Somatic genetic alterations, mutational signatures, clonal composition, and phylogenetic analyses were defined using validated computational methods.Results:DCIS revealed genetic alterations similar to those of synchronously diagnosed IDC-NSTs and of non-related IDC-NSTs from The Cancer Genome Atlas (TCGA), whereas pure DCIS lacked PIK3CA mutations. Clonal decomposition and phylogenetic analyses based on somatic mutations and copy number alterations revealed that the mechanisms of progression of DCIS to invasive carcinoma are diverse, and that clonal selection might have constituted the mechanism of progression from DCIS to invasive disease in 28% (7/25) of patients. DCIS displaying a pattern of clonal selection in the progression to invasive cancer harbored higher levels of intralesion genetic heterogeneity than DCIS where no clonal selection was observed.Conclusions:Intralesion genetic heterogeneity is a common feature in DCIS synchronously diagnosed with IDC-NST. DCIS is a nonobligate precursor of IDC-NST, whose mechanisms of progression to invasive breast cancer are diverse and vary from case to case.

Supplementary Fig. S3: Evolution of mutational signatures in treatment-naïve patients with de novo synchronous metastatic breast cancer. (A) Heatmap represents the similarity of the observed mutational signatures (blue, see color key) to those previously observed in human cancers (15, 36), separately for the trunk mutations (yellow), the mutations specific to the primary tumor (green) and the mutations specific to the metastatic lesion (pink). (B) Barplots illustrate the mutational signatures of the mutations specific to the primary tumor and the metastatic lesion of Cases 2, 4, 7 and 9. In each panel, the colored barplot illustrates each mutational signature according to the 96 substitution classification defined by the substitution classes (C>A, C>G, C>T, T>A, T>C and T>G bins) and the 5' and 3' sequence context, normalized using the observed trinucleotide frequency in the human exome to that in the human genome. The bars are ordered first by mutation class (C>A/G>T, C>G/G>C, C>T/G>A, T>A/A>T, T>C/A>G, T>G/A >C), then by the 5' flanking base (A, C, G, T) and then by the 3' flanking base (A, C, G, T). *: >20%.

Purpose: Paired primary breast cancers and metachronous metastases after adjuvant treatment are reported to differ in their clonal composition and genetic alterations, but it is unclear whether these differences stem from the selective pressures of the metastatic process, the systemic therapies, or both. We sought to define the repertoire of genetic alterations in breast cancer patients with de novo metastatic disease who had not received local or systemic therapy.Experimental Design: Up to two anatomically distinct core biopsies of primary breast cancers and synchronous distant metastases from nine patients who presented with metastatic disease were subjected to high-depth whole-exome sequencing. Mutations, copy number alterations and their cancer cell fractions, and mutation signatures were defined using state-of-the-art bioinformatics methods. All mutations identified were validated with orthogonal methods.Results: Genomic differences were observed between primary and metastatic deposits, with a median of 60% (range 6%–95%) of shared somatic mutations. Although mutations in known driver genes including TP53, PIK3CA, and GATA3 were preferentially clonal in both sites, primary breast cancers and their synchronous metastases displayed spatial intratumor heterogeneity. Likely pathogenic mutations affecting epithelial-to-mesenchymal transition–related genes, including SMAD4, TCF7L2, and TCF4 (ITF2), were found to be restricted to or enriched in the metastatic lesions. Mutational signatures of trunk mutations differed from those of mutations enriched in the primary tumor or the metastasis in six cases.Conclusions: Synchronous primary breast cancers and metastases differ in their repertoire of somatic genetic alterations even in the absence of systemic therapy. Mutational signature shifts might contribute to spatial intratumor genetic heterogeneity. Clin Cancer Res; 23(15); 4402–15. ©2017 AACR.

Supplementary Methods detailing the methods employed for Copy number alteration (CNA) analysis, Sanger sequencing and supplementary references.

Supplementary Figure S3 shows the copy number profiles of ductal carcinomas in situ (DCIS) and invasive ductal carcinomas of no special type (IDC-NST), and frequency of most commonly mutated genes in DCIS, compared to luminal B IDC-NSTs, and according to ER/HER2 status.

Purpose:Lobular carcinoma in situ (LCIS) is a preinvasive lesion of the breast. We sought to define its genomic landscape, whether intralesion genetic heterogeneity is present in LCIS, and the clonal relatedness between LCIS and invasive breast cancers.Experimental Design: We reanalyzed whole-exome sequencing (WES) data and performed a targeted amplicon sequencing validation of mutations identified in 43 LCIS and 27 synchronous more clinically advanced lesions from 24 patients [9 ductal carcinomas in situ (DCIS), 13 invasive lobular carcinomas (ILC), and 5 invasive ductal carcinomas (IDC)]. Somatic genetic alterations, mutational signatures, clonal composition, and phylogenetic trees were defined using validated computational methods.Results:WES of 43 LCIS lesions revealed a genomic profile similar to that previously reported for ILCs, with CDH1 mutations present in 81% of the lesions. Forty-two percent (18/43) of LCIS were found to be clonally related to synchronous DCIS and/or ILCs, with clonal evolutionary patterns indicative of clonal selection and/or parallel/branched progression. Intralesion genetic heterogeneity was higher among LCIS clonally related to DCIS/ILC than in those nonclonally related to DCIS/ILC. A shift from aging to APOBEC-related mutational processes was observed in the progression from LCIS to DCIS and/or ILC in a subset of cases.Conclusions:Our findings support the contention that LCIS has a repertoire of somatic genetic alterations similar to that of ILCs, and likely constitutes a nonobligate precursor of breast cancer. Intralesion genetic heterogeneity is observed in LCIS and should be considered in studies aiming to develop biomarkers of progression from LCIS to more advanced lesions.

Supplementary Table S1: Clinicopathologic characteristics of breast cancer patients with de novo metastastic disease included in the study.

Solid papillary carcinoma with reverse polarity (SPCRP) is a rare breast cancer subtype with an obscure etiology. In this study, we sought to describe its unique histopathologic features and to identify the genetic alterations that underpin SPCRP using massively parallel whole-exome and targeted sequencing. The morphologic and immunohistochemical features of SPCRP support the invasive nature of this subtype. Ten of 13 (77%) SPCRPs harbored hotspot mutations at R172 of the isocitrate dehydrogenase IDH2, of which 8 of 10 displayed concurrent pathogenic mutations affecting PIK3CA or PIK3R1. One of the IDH2 wild-type SPCRPs harbored a TET2 Q548* truncating mutation coupled with a PIK3CA H1047R hotspot mutation. Functional studies demonstrated that IDH2 and PIK3CA hotspot mutations are likely drivers of SPCRP, resulting in its reversed nuclear polarization phenotype. Our results offer a molecular definition of SPCRP as a distinct breast cancer subtype. Concurrent IDH2 and PIK3CA mutations may help diagnose SPCRP and possibly direct effective treatment. Cancer Res; 76(24); 7118–29. ©2016 AACR.

Supplementary Fig. S1: Repertoire of somatic mutations, clonal composition and phylogenetic tree analysis of cases 1-3 and cases 5-9.

Supplementary Table S6: Comparative analysis of the mutational frequencies of genes affecting selected functional pathways in metaplastic breast cancers and triple-negative invasive ductal carcinomas of no special type and correlations of mutational status with clinicopathologic parameters. p

Excel file containing Supplementary Data File 1: Non-synonymous somatic mutations identified by whole-exome sequencing and amplicon sequencing in the LCIS, DCIS, ILCs and IDCs studied.

Supplementary Table S1: Sequencing statistics.

Document containing figures illustrating results of Sanger sequencing, DNA methylation profiling of SPCRPs, the analysis of IDH2 expression in non-malignant breast epithelial cells and its downstream effects, and tables describing the antibodies used for immunohistochemical analysis, the alterations surveyed by the SNaPshot genotyping assay, Sanger sequencing primers, and statistics and results of the massively parallel sequencing analyses.

Supplementary Figure S4: Morphologic and immunophenotypic features of case MB-T88. Supplementary Figure S5: GATA3 mutational spectrum in male breast cancer and female breast cancer and the impact of selected mutations on the survival of male breast cancer patients.

Purpose: To identify therapeutic targets in ovarian clear cell carcinomas, a chemoresistant and aggressive type of ovarian cancer.Experimental Design: Twelve ovarian clear cell carcinoma cell lines were subjected to tiling path microarray comparative genomic hybridization and genome-wide expression profiling analysis. Regions of high-level amplification were defined and genes whose expression levels were determined by copy number and correlated with gene amplification were identified. The effects of inhibition of PPM1D were assessed using short hairpin RNA constructs and a small-molecule inhibitor (CCT007093). The prevalence of PPM1D amplification and mRNA expression was determined using chromogenic in situ hybridization and quantitative real-time reverse transcription-PCR in a cohort of pure ovarian clear cell carcinomas and on an independent series of unselected epithelial ovarian cancers.Results: Array-based comparative genomic hybridization analysis revealed regions of high-level amplification on 1q32, 1q42, 2q11, 3q24-q26, 5p15, 7p21-p22, 11q13.2-q13.4, 11q22, 17q21-q22, 17q23.2, 19q12-q13, and 20q13.2. Thirty-four genes mapping to these regions displayed expression levels that correlated with copy number gains/amplification. PPM1D had significantly higher levels of mRNA expression in ovarian clear cell carcinoma cell lines harboring gains/amplifications of 17q23.2. PPM1D inhibition revealed that PPM1D expression and phosphatase activity are selectively required for the survival of ovarian clear cell carcinoma cell lines with 17q23.2 amplification. PPM1D amplification was significantly associated with ovarian clear cell carcinoma histology (P = 0.0003) and found in 10% of primary ovarian clear cell carcinomas. PPM1D expression levels were significantly correlated with PPM1D gene amplification in primary ovarian clear cell carcinomas.Conclusion: Our data provide strong circumstantial evidence that PPM1D is a potential therapeutic target for a subgroup of ovarian clear cell carcinomas.

Supplementary Table S7: Comparative analysis of the frequencies of mutations in luminal A-like and luminal B-like male breast cancer. Supplementary Table S8: Comparative analysis of genes affecting selected functional pathways. Supplementary Table S9: Mutational frequency of male breast cancer vs subsets of female breast cancer from the TCGA study. Supplementary Table S10: Potentially targetable genes mutated exclusively in ER-positive/HER2-negative male breast cancer.

Document containing methods on whole exome and targeted massively parallel sequencing analysis, 2-hydroxyglutarate (2HG) assay of cell lines, methylation profiling, and supplementary references.

Supplementary Fig. S2. Repertoire of somatic non-synonymous mutations of metaplastic breast cancers (MBCs) and triple-negative invasive ductal carcinomas of no special type (IDC-NSTs). Clinico-pathologic and immunohistochemical features, and non-synonymous somatic mutations somatic mutations identified in (a) 35 MBCs subjected to whole-exome sequencing, and in 69 triple-negative IDC-NSTs from TCGA breast cancer study (9) and in (b) 16, ten and nine chondroid, spindle and squamous MBCs, respectively, subjected to whole-exome sequencing. The effects of the mutations are colored-coded according to the legend, with hotspots colored in red. Mutations of indeterminate pathogenicity and likely passenger mutations are indicated by hatched boxes. The presence of multiple non-synonymous mutations in the same gene is represented by an asterisk. For MBCs, the presence of loss of heterozygosity of the wild-type allele of a mutated gene is represented by a diagonal bar, and mutations found to be clonal by ABSOLUTE (24) are indicated by a black box. Only recurrently mutated genes are presented (i.e. genes affected by non-synonymous somatic mutations in {greater than or equal to}2 MBCs). Gene names highlighted in purple were (a) significantly more frequently altered in MBCs or (b) differentially altered in the given subtype of MBC. Percentages to the right of the mutation heatmaps indicate the percentage of cases affected by non-synonymous somatic mutations in a given gene. Bar charts (top) indicate the number of non-synonymous and synonymous somatic single nucleotide variants (SNVs), and the number of somatic insertions and deletions (indels) for each sample. The dominant mutational signatures (35, 36) were assigned using deconstructSigs (37). Large-scale transitions (LST)-high and LST-low status was determined in accordance with Popova et al (39). TCGA, The Cancer Genome Atlas.

Supplementary Figure S1; Supplementary Tables S1-S4.

Supplementary Data from PPM1D Is a Potential Therapeutic Target in Ovarian Clear Cell Carcinomas

Purpose: Resistance to anti-HER2 therapies in HER2+ breast cancer can occur through activation of alternative survival pathways or reactivation of the HER signaling network. Here we employed BT474 parental and treatment-resistant cell line models to investigate a mechanism by which HER2+ breast cancer can reactivate the HER network under potent HER2-targeted therapies.Experimental Design: Resistant derivatives to lapatinib (L), trastuzumab (T), or the combination (LR/TR/LTR) were developed independently from two independent estrogen receptor ER+/HER2+ BT474 cell lines (AZ/ATCC). Two derivatives resistant to the lapatinib-containing regimens (BT474/AZ-LR and BT474/ATCC-LTR lines) that showed HER2 reactivation at the time of resistance were subjected to massive parallel sequencing and compared with parental lines. Ectopic expression and mutant-specific siRNA interference were applied to analyze the mutation functionally. In vitro and in vivo experiments were performed to test alternative therapies for mutant HER2 inhibition.Results: Genomic analyses revealed that the HER2L755S mutation was the only common somatic mutation gained in the BT474/AZ-LR and BT474/ATCC-LTR lines. Ectopic expression of HER2L755S induced acquired lapatinib resistance in the BT474/AZ, SK-BR-3, and AU565 parental cell lines. HER2L755S-specific siRNA knockdown reversed the resistance in BT474/AZ-LR and BT474/ATCC-LTR lines. The HER1/2–irreversible inhibitors afatinib and neratinib substantially inhibited both resistant cell growth and the HER2 and downstream AKT/MAPK signaling driven by HER2L755S in vitro and in vivo.Conclusions: HER2 reactivation through acquisition of the HER2L755S mutation was identified as a mechanism of acquired resistance to lapatinib-containing HER2-targeted therapy in preclinical HER2-amplified breast cancer models, which can be overcome by irreversible HER1/2 inhibitors. Clin Cancer Res; 23(17); 5123–34. ©2017 AACR.

File containing the legends for Supplementary Figures S1-S4 and Supplementary Tables S1-S6.

Supplementary Table S11: Comparative analysis of the frequencies of mutations in luminal A-like male breast cancers and subsets of luminal A female breast cancers from the TCGA study. Supplementary Table S12: Comparative analysis of the frequencies of mutations in luminal B-like male breast cancers and subsets of luminal B female breast cancers from the TCGA study.

Supplementary Table S2 shows the non-synonymous somatic mutations identified in synchronous DCIS and IDC-NSTs subjected to whole-exome sequencing or MSK-IMPACT targeted massively parallel sequencing and/or targeted amplicon sequencing.

Purpose: Metaplastic breast carcinoma (MBC) is a rare and aggressive histologic type of breast cancer, predominantly of triple-negative phenotype, and characterized by the presence of malignant cells showing squamous and/or mesenchymal differentiation. We sought to define the repertoire of somatic genetic alterations and the mutational signatures of MBCs.Experimental Design: Whole-exome sequencing was performed in 35 MBCs, with 16, 10, and 9 classified as harboring chondroid, spindle, and squamous metaplasia as the predominant metaplastic component. The genomic landscape of MBCs was compared with that of triple-negative invasive ductal carcinomas of no special type (IDC-NST) from The Cancer Genome Atlas. Wnt and PI3K/AKT/mTOR pathway activity was assessed using a qPCR assay.Results: MBCs harbored complex genomes with frequent TP53 (69%) mutations. In contrast to triple-negative IDC-NSTs, MBCs more frequently harbored mutations in PIK3CA (29%), PIK3R1 (11%), ARID1A (11%), FAT1 (11%), and PTEN (11%). PIK3CA mutations were not found in MBCs with chondroid metaplasia. Compared with triple-negative IDC-NSTs, MBCs significantly more frequently harbored mutations in PI3K/AKT/mTOR pathway–related (57% vs. 22%) and canonical Wnt pathway–related (51% vs. 28%) genes. MBCs with somatic mutations in PI3K/AKT/mTOR or Wnt pathway–related genes displayed increased activity of the respective pathway.Conclusions: MBCs are genetically complex and heterogeneous, and are driven by a repertoire of somatic mutations distinct from that of triple-negative IDC-NSTs. Our study highlights the genetic basis and the importance of PI3K/AKT/mTOR and Wnt pathway dysregulation in MBCs and provides a rationale for the metaplastic phenotype and the reported responses to PI3K/AKT/mTOR inhibitors in these tumors. Clin Cancer Res; 23(14); 3859–70. ©2017 AACR.

File containing the Supplementary Methods.

Purpose: Male breast cancer is rare, and its genomic landscape has yet to be fully characterized. Lacking studies in men, treatment of males with breast cancer is extrapolated from results in females with breast cancer. We sought to define whether male breast cancers harbor somatic genetic alterations in genes frequently altered in female breast cancers.Experimental Design: All male breast cancers were estrogen receptor–positive, and all but two were HER2-negative. Fifty-nine male breast cancers were subtyped by immunohistochemistry, and tumor–normal pairs were microdissected and subjected to massively parallel sequencing targeting all exons of 241 genes frequently mutated in female breast cancers or DNA-repair related. The repertoires of somatic mutations and copy number alterations of male breast cancers were compared with that of subtype-matched female breast cancers.Results: Twenty-nine percent and 71% of male breast cancers were immunohistochemically classified as luminal A–like or luminal B–like, respectively. Male breast cancers displayed a heterogeneous repertoire of somatic genetic alterations that to some extent recapitulated that of estrogen receptor (ER)-positive/HER2-negative female breast cancers, including recurrent mutations affecting PIK3CA (20%) and GATA3 (15%). ER-positive/HER2-negative male breast cancers, however, less frequently harbored 16q losses, and PIK3CA and TP53 mutations than ER-positive/HER2-negative female breast cancers. In addition, male breast cancers were found to be significantly enriched for mutations affecting DNA repair–related genes.Conclusions: Male breast cancers less frequently harbor somatic genetic alterations typical of ER-positive/HER2-negative female breast cancers, such as PIK3CA and TP53 mutations and losses of 16q, suggesting that at least a subset of male breast cancers are driven by a distinct repertoire of somatic changes. Given the genomic differences, caution may be needed in the application of biologic and therapeutic findings from studies of female breast cancers to male breast cancers. Clin Cancer Res; 22(16); 4045–56. ©2016 AACR.

Legends for Supplementary Figures S1-S5

PDF file containing the Supplementary Methods and Supplementary References.

Supplementary Figure 1: Validation of somatic mutations in orthogonal methods. Supplementary Figure S2: Mutations and copy number alterations in ER-positive/HER2-negative male and female breast cancers. Supplementary Figure S3: Cancer cell fraction and clonality of non-synonymous mutations in male breast cancer.

Supplementary Figures S1-S8

supplementary methods and figure legends

Supplementary tables S1-S4

Purpose: Ovarian clear cell carcinomas (OCCC) are a drug-resistant and aggressive type of epithelial ovarian cancer. We analyzed the molecular genetic profiles of OCCCs to determine whether distinct genomic subgroups of OCCCs exist.Experimental design: Fifty pure primary OCCCs were subjected to high-resolution microarray-based comparative genomic hybridization (aCGH). Unsupervised hierarchical clustering using Ward's linkage analysis was performed to identify genomic subgroups of OCCCs. Survival analysis was performed using Kaplan–Meier method and log-rank test. Cox-regression analysis was used to identify independent predictors of outcome. Differentially amplified regions between genomic subgroups of OCCCs were identified using a multi-Fisher's exact test.Results: Hierarchical cluster analysis revealed two distinct clusters of OCCCs with different clinical outcomes. Patients from cluster-1 had a significantly shorter median progression-free survival (PFS) than those from cluster-2 (11 vs. 65 months, P = 0.009), although estimates for ovarian cancer–specific survival (OCS) did not reach statistical significance (P = 0.065). In multivariate analysis, suboptimal debulking surgery and genomic cluster were independently prognostic for PFS. Recurrently amplified genomic regions with a significantly higher prevalence in cluster-1 than cluster-2 OCCCs were identified and validated. HER2 gene amplification and protein overexpression was observed in 14% of OCCCs, suggesting that this may constitute a potential therapeutic target for a subgroup of these tumors.Conclusions: OCCCs constitute a heterogeneous disease at the genomic level despite having similar histological features. The pattern of genomic aberrations in subgroups of OCCCs is of clinical significance. We have identified recurrently amplified regions that may harbor potential therapeutic targets for subgroups of OCCCs. Clin Cancer Res; 17(6); 1521–34. ©2011 AACR.

File containing Supplementary Methods, Legends for Supplementary Tables S1-S2, Legends for Supplementary Figures S1-S4 and Legends for the Supplementary Data File 1.

Supplementary Tables 1-2 from A Mouse Model of Melanoma Driven by Oncogenic KRAS