Maryou B. Lambros

RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors.

BACKGROUND Cutaneous squamous-cell carcinomas and keratoacanthomas are common findings in patients treated with BRAF inhibitors. METHODS We performed a molecular analysis to identify oncogenic mutations (HRAS, KRAS, NRAS, CDKN2A, and TP53) in the lesions from patients treated with the BRAF inhibitor vemurafenib. An analysis of an independent validation set and functional studies with BRAF inhibitors in the presence of the prevalent RAS mutation was also performed. RESULTS Among 21 tumor samples, 13 had RAS mutations (12 in HRAS). In a validation set of 14 samples, 8 had RAS mutations (4 in HRAS). Thus, 60% (21 of 35) of the specimens harbored RAS mutations, the most prevalent being HRAS Q61L. Increased proliferation of HRAS Q61L-mutant cell lines exposed to vemurafenib was associated with mitogen-activated protein kinase (MAPK)-pathway signaling and activation of ERK-mediated transcription. In a mouse model of HRAS Q61L-mediated skin carcinogenesis, the vemurafenib analogue PLX4720 was not an initiator or a promoter of carcinogenesis but accelerated growth of the lesions harboring HRAS mutations, and this growth was blocked by concomitant treatment with a MEK inhibitor. CONCLUSIONS Mutations in RAS, particularly HRAS, are frequent in cutaneous squamous-cell carcinomas and keratoacanthomas that develop in patients treated with vemurafenib. The molecular mechanism is consistent with the paradoxical activation of MAPK signaling and leads to accelerated growth of these lesions. (Funded by Hoffmann-La Roche and others; ClinicalTrials.gov numbers, NCT00405587, NCT00949702, NCT01001299, and NCT01006980.).

FGFR1 Amplification Drives Endocrine Therapy Resistance and Is a Therapeutic Target in Breast Cancer

Amplification of fibroblast growth factor receptor 1 (FGFR1) occurs in approximately 10% of breast cancers and is associated with poor prognosis. However, it is uncertain whether overexpression of FGFR1 is causally linked to the poor prognosis of amplified cancers. Here, we show that FGFR1 overexpression is robustly associated with FGFR1 amplification in two independent series of breast cancers. Breast cancer cell lines with FGFR1 overexpression and amplification show enhanced ligand-dependent signaling, with increased activation of the mitogen-activated protein kinase and phosphoinositide 3-kinase-AKT signaling pathways in response to FGF2, but also show basal ligand-independent signaling, and are dependent on FGFR signaling for anchorage-independent growth. FGFR1-amplified cell lines show resistance to 4-hydroxytamoxifen, which is reversed by small interfering RNA silencing of FGFR1, suggesting that FGFR1 overexpression also promotes endocrine therapy resistance. FGFR1 signaling suppresses progesterone receptor (PR) expression in vitro, and likewise, amplified cancers are frequently PR negative, identifying a potential biomarker for FGFR1 activity. Furthermore, we show that amplified cancers have a high proliferative rate assessed by Ki67 staining and that FGFR1 amplification is found in 16% to 27% of luminal B-type breast cancers. Our data suggest that amplification and overexpression of FGFR1 may be a major contributor to poor prognosis in luminal-type breast cancers, driving anchorage-independent proliferation and endocrine therapy resistance.

Functionally recurrent rearrangements of the MAST kinase and Notch gene families in breast cancer.

Breast cancer is a heterogeneous disease that has a wide range of molecular aberrations and clinical outcomes. Here we used paired-end transcriptome sequencing to explore the landscape of gene fusions in a panel of breast cancer cell lines and tissues. We observed that individual breast cancers have a variety of expressed gene fusions. We identified two classes of recurrent gene rearrangements involving genes encoding microtubule-associated serine-threonine kinase (MAST) and members of the Notch family. Both MAST and Notch-family gene fusions have substantial phenotypic effects in breast epithelial cells. Breast cancer cell lines harboring Notch gene rearrangements are uniquely sensitive to inhibition of Notch signaling, and overexpression of MAST1 or MAST2 gene fusions has a proliferative effect both in vitro and in vivo. These findings show that recurrent gene rearrangements have key roles in subsets of carcinomas and suggest that transcriptome sequencing could identify individuals with rare, targetable gene fusions.

Caveolin 1 Is Overexpressed and Amplified in a Subset of Basal-like and Metaplastic Breast Carcinomas: A Morphologic, Ultrastructural, Immunohistochemical, and In situ Hybridization Analysis

Purpose: The distribution and significance of caveolin 1 (CAV1) expression in different breast cell types and role in breast carcinogenesis remain poorly understood. Both tumor-suppressive and oncogenic roles have been proposed for this protein. The aims of this study were to characterize the distribution of CAV1 in normal breast, benign breast lesions, breast cancer precursors, and metaplastic breast carcinomas; to assess the prognostic significance of CAV1 expression in invasive breast carcinomas; and to define whether CAV1 gene amplification is the underlying genetic mechanism driving CAV1 overexpression in breast carcinomas. Experimental Design: CAV1 distribution in frozen and paraffin-embedded whole tissue sections of normal breast was evaluated using immunohistochemistry, immunofluorescence, and immunoelectron microscopy. CAV1 expression was immunohistochemically analyzed in benign lesions, breast cancer precursors, and metaplastic breast carcinomas and in a cohort of 245 invasive breast carcinomas from patients treated with surgery followed by anthracycline-based chemotherapy. In 25 cases, CAV1 gene amplification was assessed by chromogenic in situ hybridization. Results: In normal breast, CAV1 was expressed in myoepithelial cells, endothelial cells, and a subset of fibroblasts. Luminal epithelial cells showed negligible staining. CAV1 was expressed in 90% of 39 metaplastic breast carcinomas and in 9.4% of 245 invasive breast cancers. In the later cohort, CAV1 expression was significantly associated with ‘basal-like’ immunophenotype and with shorter disease-free and overall survival on univariate analysis. CAV1 gene amplification was found in 13% of cases with strong CAV1 expression. Conclusions: The concurrent CAV1 amplification and overexpression call into question its tumor-suppressive effects in basal-like breast carcinomas.

FGFR1 amplification in breast carcinomas: a chromogenic in situ hybridisation analysis

BackgroundThe amplicon on 8p11.2 is reported to be found in up to 10% of breast carcinomas. It has been demonstrated recently that this amplicon has four separate cores. The second core encompasses important oncogene candidates, including the fibroblast growth factor receptor 1 (FGFR1) gene. Recent studies have demonstrated that specific FGFR1 amplification correlates with gene expression and that FGFR1 activity is required for the survival of a FGFR1 amplified breast cancer cell line.MethodsFGFR1 amplification was analysed in tissue microarrays comprising a cohort of 880 unselected breast tumours by means of chromogenic in situ hybridisation using inhouse-generated FGFR1-specific probes. Chromogenic in situ hybridisation signals were counted in a minimum 30 morphologically unequivocal neoplastic cells. Amplification was defined as >5 signals per nucleus in more than 50% of cancer cells or when large gene copy clusters were seen.ResultsFGFR1 amplification was observed in 8.7% of the tumours and was significantly more prevalent in patients >50 years of age and in tumours that lacked HER2 expression. No association was found with other histological parameters. Survival analysis revealed FGFR1 amplification as an independent prognostic factor for overall survival in the whole cohort. Subgroup analysis demonstrated that the independent prognostic impact of FGFR1 amplification was only seen in patients with oestrogen-receptor-positive tumours, where FGFR1 amplification was the strongest independent predictor of poor outcome.ConclusionGiven that up to 8.7% of all breast cancers harbour FGFR1 amplification and that this amplification is an independent predictor of overall survival, further studies analysing the FGFR1 as a potential therapeutic target for breast cancer patients are warranted.

|[beta]|-Catenin pathway activation in breast cancer is associated with triple-negative phenotype but not with CTNNB1 mutation

Aberrant β-catenin expression as determined by assessment of its subcellular localization constitutes a surrogate marker of Wnt signalling pathway activation and has been reported in a subset of breast cancers. The association of β-catenin/Wnt pathway activation with clinical outcome and the mechanisms leading to its activation in breast cancers still remain a matter of controversy. The aims of this study were to address the distribution of β-catenin expression in invasive breast cancers, the correlations between β-catenin expression and clinicopathological features and survival of breast cancer patients, and to determine whether aberrant β-catenin expression is driven by CTNNB1 (β-catenin encoding gene) activating mutations. Immunohistochemistry was performed on a tissue microarray containing 245 invasive breast carcinomas from uniformly treated patients, using two anti-β-catenin monoclonal antibodies. Selected samples were subjected to CTNNB1 exon 3 mutation analysis by direct gene sequencing. A good correlation between the two β-catenin antibodies was observed (Spearmans r >0.62, P<0.001). Respectively, 31 and 11% of the cases displayed lack/reduction of β-catenin membranous expression and nuclear accumulation. Complete lack of β-catenin expression was significantly associated with invasive lobular carcinoma histological type. Subgroup analysis of non-lobular cancers or non-lobular grade 3 carcinomas revealed that lack/reduction of β-catenin membranous expression and/or nuclear accumulation were significantly associated with oestrogen receptor negativity, absence of HER2 gene amplification and overexpression, lack/reduction of E-cadherin expression and tumours of triple-negative and basal-like phenotype. Univariate survival analysis revealed a significant association between β-catenin nuclear expression and shorter metastasis-free and overall survival in the whole cohort; however, β-catenin nuclear expression was not an independent predictor of outcome in multivariate analysis. No CTNNB1 mutations were identified in the 28 selected breast carcinomas analysed. In conclusion, β-catenin/Wnt pathway activation is preferentially found in triple-negative/basal-like breast carcinomas, is associated with poor clinical outcome and is unlikely to be driven by CTNNB1 mutations in breast cancer.

Pleomorphic lobular carcinoma of the breast: role of comprehensive molecular pathology in characterization of an entity

Immunohistochemical analysis of E‐cadherin has changed the way lobular neoplasia is perceived. It has helped to classify difficult cases of carcinoma in situ with indeterminate features and led to the identification of new variants of lobular carcinoma. Pleomorphic lobular carcinoma (PLC) and pleomorphic lobular carcinoma in situ (PLCIS), recently described variants of invasive and in situ classic lobular carcinoma, are reported to be associated with more aggressive clinical behaviour. Although PLC/PLCIS show morphological features of classic lobular neoplasia and lack E‐cadherin expression, it is still unclear whether these lesions evolve through the same genetic pathway as lobular carcinomas or are high‐grade ductal neoplasms that have lost E‐cadherin. Here we have analysed a case of extensive PLCIS and invasive PLC associated with areas of E‐cadherin‐negative carcinoma in situ with indeterminate features, using immunohistochemistry, chromogenic in situ hybridization, high‐resolution comparative genomic hybridization (CGH) and array‐based CGH. We observed that all lesions lacked E‐cadherin and β‐catenin and showed gain of 1q and loss of 16q, features that are typical of lobular carcinomas but are not seen in high‐grade ductal lesions. In addition, amplifications of c‐myc and HER2 were detected in the pleomorphic components, which may account for the high‐grade features in this case and the reported aggressive clinical behaviour of these lesions. Taken together, these data suggest that at least some PLCs may evolve from the same precursor or through the same genetic pathway as classic lobular carcinomas. Copyright

Does chromosome 17 centromere copy number predict polysomy in breast cancer? A fluorescence in situ hybridization and microarray-based CGH analysis

Approximately 8% of breast cancers show increased copy numbers of chromosome 17 centromere (CEP17) by fluorescence in situ hybridization (FISH) (ie average CEP17 >3.0 per nucleus). Currently, this pattern is believed to represent polysomy of chromosome 17. HER2‐amplified cancers have been shown to harbour complex patterns of genetic aberrations of chromosome 17, in particular involving its long arm. We hypothesized that aberrant copy numbers of CEP17 in FISH assays may not necessarily represent true chromosome 17 polysomy. Eighteen randomly selected CEP17 polysomic cases and a control group of ten CEP17 disomic cases, as defined by dual‐colour FISH, were studied by microarray‐based comparative genomic hybridization (aCGH), which was performed on microdissected samples using a 32K tiling‐path bacterial artificial chromosome microarray platform. Additional FISH probes were employed for SMS (17p11.2) and RARA (17q21.2) genes, as references for chromosome 17 copy number. Microarray‐based comparative genomic hybridization revealed that 11 out of the 18 polysomic cases harboured gains of 17q with involvement of the centromere, one displayed 17q gain sparing the centromeric region, and only one could be defined as polysomic. The remaining five cases displayed amplification of the centromeric region. Among these, one case, showing score 2+ by immunohistochemistry and 8.5 HER2 mean copy number, was classified as not amplified by HER2/CEP17 ratio and as amplified by HER2/SMS ratio. Our results suggest that true chromosome 17 polysomy is likely to be a rare event in breast cancer and that CEP17 copy number greater than 3.0 in FISH analysis is frequently related to gain or amplification of the centromeric region. Larger studies investigating the genetic profiles of CEP17 polysomic cases are warranted. Copyright

Tiling Path Genomic Profiling of Grade 3 Invasive Ductal Breast Cancers

Purpose: To characterize the molecular genetic profiles of grade 3 invasive ductal carcinomas of no special type using high-resolution microarray-based comparative genomic hybridization (aCGH) and to identify recurrent amplicons harboring putative therapeutic targets associated with luminal, HER-2, and basal-like tumor phenotypes. Experimental Design: Ninety-five grade 3 invasive ductal carcinomas of no special type were classified into luminal, HER-2, and basal-like subgroups using a previously validated immunohistochemical panel. Tumor samples were microdissected and subjected to aCGH using a tiling path 32K BAC array platform. Selected regions of recurrent amplification were validated by means of in situ hybridization. Expression of genes pertaining to selected amplicons was investigated using quantitative real-time PCR and gene silencing was done using previously validated short hairpin RNA constructs. Results: We show that basal-like and HER-2 tumors are characterized by “sawtooth” and “firestorm” genetic patterns, respectively, whereas luminal cancers were more heterogeneous. Apart from confirming known amplifications associated with basal-like (1q21, 10p, and 12p), luminal (8p12, 11q13, and 11q14), and HER-2 (17q12) cancers, we identified previously unreported recurrent amplifications associated with each molecular subgroup: 19q12 in basal-like, 1q32.1 in luminal, and 14q12 in HER-2 cancers. PPM1D gene amplification (17q23.2) was found in 20% and 8% of HER-2 and luminal cancers, respectively. Silencing of PPM1D by short hairpin RNA resulted in selective loss of viability in tumor cell lines harboring the 17q23.2 amplification. Conclusions: Our results show the power of aCGH analysis in unraveling the genetic profiles of specific subgroups of cancer and for the identification of novel therapeutic targets.

PTEN Deficiency in Endometrioid Endometrial Adenocarcinomas Predicts Sensitivity to PARP Inhibitors

Endometrioid endometrial cancer cell lines harboring PTEN loss of function have impaired homologous recombination response to DNA double-strand breaks and show an exquisite sensitivity to PARP inhibition. Putting PARP Inhibitors on the Map Targeted drug therapies that interfere with specific oncoproteins in certain cancers like lung cancer and chronic myeloid leukemia have shown great promise, but designing targeted therapies for tumors that have lost a tumor suppressor protein has proved challenging. Building on the idea of targeting a complementary gene or pathway in tumors with an inactive tumor suppressor protein, Dedes and colleagues set out to tackle endometrial cancer. This cancer is the fourth most common malignancy in women and is very difficult to treat particularly in the later stages of the disease. The authors demonstrate that endometrial cancer cell lines that lack the tumor suppressor protein PTEN show defects in the repair of DNA damage and are consequently very sensitive to drugs that block poly(ADP) ribose polymerase (PARP), an enzyme critical for DNA repair. Given that such PARP inhibitors are currently in late-stage clinical trials for treating breast and ovarian cancer, this study opens the door to using PARP inhibitors for treating endometrial cancer. First the authors demonstrated that the majority of endometrial cancer cell lines that they analyzed indeed had lost the PTEN tumor suppressor protein. PTEN both regulates a major growth signaling pathway in cells (the PI3K-AKT-mTOR pathway) and has recently been shown to be important for maintaining genomic stability. Dedes and co-workers then showed that loss of PTEN rendered endometrial cancer cells unable to repair DNA double-strand breaks induced by ionizing radiation. Other types of tumors that cannot repair DNA damage, such as breast and ovarian cancer cells carrying mutations in the BRCA1 and BRCA2 genes, are acutely sensitive to PARP inhibitors. Dedes et al. reasoned that endometrial cancer cells lacking PTEN may also be sensitive to these drugs. This is exactly what they found when they treated their endometrial cancer cell lines with the potent PARP inhibitor, KU0058948. To demonstrate that it was loss of PTEN that rendered the cancer cells highly sensitive to the drug, they re-expressed PTEN in endometrial cancer cell lines lacking this tumor suppressor protein and demonstrated that these cancer cell lines were now able to repair DNA damage and thus were resistant to treatment with the PARP inhibitor. Given that 80% of endometrial cancers lack PTEN, treatment with PARP inhibitors may be an effective way to treat this disease. PTEN (phosphatase and tensin homolog) loss of function is the most common genetic aberration in endometrioid endometrial carcinomas. In addition to its well-described role in cell signaling, PTEN is involved in the maintenance of genomic stability. Loss of PTEN function causes defects in repair of DNA double-strand breaks by homologous recombination and, therefore, sensitizes cells to inhibition of the poly(adenosine diphosphate ribose) polymerase (PARP). Here, we determined the PTEN status of eight endometrioid endometrial carcinoma cell lines and correlated it with in vitro sensitivity to the PARP inhibitor KU0058948. PTEN-deficient cells showed a significantly greater sensitivity to KU0058948 than the two endometrioid endometrial carcinoma cell lines with wild-type PTEN. The cell lines lacking PTEN expression were unable to elicit a homologous recombination damage response as assayed by RAD51 focus function (a marker of competent homologous recombination DNA repair) upon irradiation and treatment with PARP inhibitors. PTEN silencing in PTEN wild-type Hec-1b cells resulted in reduced RAD51 foci formation after DNA damage and increased sensitivity to PARP inhibition. PTEN reexpression in PTEN-null cell lines resulted in enhanced RAD51 foci formation and in relative resistance to KU0058948. Given that up to 80% of endometrioid endometrial cancers lack PTEN expression, our results suggest that PARP inhibitors may be therapeutically useful for a subset of endometrioid endometrial cancers.