Therapeutic strategies in male breast cancer: Clinical implications of chromosome 17 gene alterations and molecular subtypes

Male breast cancer (MBC) is a rare disease. To date, therapy is mainly based on studies and clinical experiences with breast cancer in women. Only little is known about molecular typing of MBC, particularly with regard to potential biological predictors for adjuvant therapy. In female breast cancer tumors with chromosome 17 centromere (CEP17) duplication, HER2 and/or Topoisomerase II alpha (Topo II-alpha) gene alterations have been suggested to be associated with poor prognosis and increased sensitivity to anthracycline-containing regimens. In a well characterized cohort of 96 primary invasive MBC, we studied CEP17, HER2 and Topo II-alpha alterations by fluorescence in-situ hybridization (FISH), and expression of hormone receptors (HR), HER2 and Ki67 by immunohistochemistry to define molecular subtypes. Tumor characteristics and follow-up data were available and correlated with molecular findings. HER2 amplification and Topo II-alpha amplification/deletion were exceptionally rare in MBC (6.3% and 3.1%, respectively). CEP17 polysomy were found in 9.4% of tumors. HER2, Topo II-alpha and CEP17 gene alterations were not correlated to patients outcome. 96.9% of our cases were HR positive. Triple negative tumors were found in only 3.1% of the cases. In nodal negative tumors luminal A subtypes were significantly associated with better overall survival. Our results provide evidence for a predominant male breast cancer phenotype, characterized by HR expression and a lack of HER2/Topo II-alpha alterations and CEP17 duplicates. Therefore, the impact of anthracycline sensitivity linked to HER2/Topo II-alpha alterations as found in female breast cancer has low clinical significance for this specific male breast cancer phenotype. (C) 2013 Elsevier Ltd. All rights reserved

High-resolution melting analysis is a sensitive diagnostic tool to detect imatinib-resistant and imatinib-sensitive PDGFRA exon 18 mutations in gastrointestinal stromal tumors

The mutational status of KIT and PDGFRA is highly relevant for prognosis and therapy prediction in gastrointestinal stromal tumors (GIST). PDGFRA exon 18 mutations have direct therapeutic implications since it is crucial to distinguish mutations associated with sensitivity to tyrosine kinase inhibitors from those causing primary resistance, eg, the most common exon 18 mutation p.D842V. In response to a growing demand for reliable, faster and more sensitive methods we established and validated a high-resolution melting (BRM) assay for PDGFRA exon 18. A total of 159 GIST samples were comparatively analyzed by FIRM and direct Sanger sequencing. We demonstrate that BRM provides highly reliable mutational results with higher sensitivity and shorter time to diagnosis compared to Sanger sequencing. We determined the sensitivity threshold of FIRM at 6% of mutated alleles. PDGFRA exon 18 wild-type status and the most common p.D842V resistance mutation (together representing >90% of the cases) can be detected specifically by FIRM. Other rare mutations can be pre-screened by FIRM and afterwards determined precisely by DNA sequencing. In this way we detected four novel mutations in PDGFRA exon 18, two of which were associated with an aggressive clinical course. Including these new mutations, we provide a comprehensive overview of all 60 currently known subtypes of PDGFRA exon 18 mutations in GIST. Seven of them (accounting for about 75% of all exon 18-mutated GISTs) are reported to be resistant to imatinib. However, there are at least 10 other mutations which are regarded as sensitive to tyrosine kinase inhibitors. (C) 2014 Elsevier Inc. All rights reserved

Fibroblast growth factor receptor 1 (FGFR1) amplification is a potential therapeutic target in small-cell lung cancer

Small-cell lung cancer (SCLC) comprises about 13-15% of all lung cancers, and more than 29400 new cases have been diagnosed in the United States in the year 2012. SCLC is a biologically complex tumor typically occurring in heavy smokers. Its medical treatment has almost remained unchanged over the last decades and selected treatment options have not been established so far, mainly due to the lack of targetable genetic alterations. In this study we analyzed a cohort of 307 SCLC samples for fibroblast growth factor receptor 1 (FGFR1) amplification using a dual color FISH probe. FGFR1 status was correlated with clinical data. FGFR1 amplifications were observed in 5.6% of evaluable pulmonary SCLCs. Most of them (93%) fulfilled the criteria for high-level amplification and only one case showed low-level amplification. Amplification patterns were homogenous in the entire tumor area without occurrence of any 'hot spot' areas. FGFR1 amplification status was not associated with age, sex, stage, smoking status or overall survival. FGFR1 amplification analysis by FISH analysis in SCLC is, under respect of certain technical issues, applicable in the routine clinical setting. However, the FGFR1 amplification patterns in SCLC differs strongly from the previously described FGFR1 amplification pattern in squamous cell carcinoma of the lung, as positive SCLC harbor mostly homogeneous high-level amplifications. We provide evidence that an estimated number of 1640 newly diagnosed FGFR1-positive SCLC cases in the United States annually could benefit from targeted therapy. Therefore, we recommend including SCLC in the screening for ongoing clinical trials with FGFR1 inhibitors

Chromogenic in situ hybridization is a reliable assay for detection of ALK rearrangements in adenocarcinomas of the lung

Reliable detection of anaplastic lymphoma kinase (ALK) rearrangements is a prerequisite for personalized treatment of lung cancer patients, as ALK rearrangements represent a predictive biomarker for the therapy with specific tyrosine kinase inhibitors. Currently, fluorescent in situ hybridization (FISH) is considered to be the standard method for assessing formalin-fixed and paraffin-embedded tissue for ALK inversions and translocations. However, FISH requires a specialized equipment, the signals fade rapidly and it is difficult to detect overall morphology and tumor heterogeneity. Chromogenic in situ hybridization (CISH) has been successfully introduced as an alternative test for the detection of several genetic aberrations. This study validates a newly developed ALK CISH assay by comparing FISH and CISH signal patterns in lung cancer samples with and without ALK rearrangements. One hundred adenocarcinomas of the lung were included in this study, among them 17 with known ALK rearrangement. FISH and CISH were carried out and evaluated according to the manufacturers' recommendations. For both assays, tumors were considered positive if >= 15% of tumor cells showed either isolated 3' signals or break-apart patterns or a combination of both. A subset of tumors was exemplarily examined by using a novel EML4 (echinoderm microtubule-associated protein-like 4) CISH probe. Red, green and fusion CISH signals were clearcut and different signal patterns were easily recognized. The percentage of aberrant tumor cells was statistically highly correlated (P<0.001) between FISH and CISH. On the basis of 86 samples that were evaluable by ALK CISH, we found a 100% sensitivity and 100% specificity of this assay. Furthermore, EML4 rearrangements could be recognized by CISH. CISH is a highly reliable, sensitive and specific method for the detection of ALK gene rearrangements in pulmonary adenocarcinomas. Our results suggest that CISH might serve as a suitable alternative to FISH, which is the current gold standard

MET gene copy number alterations and expression of MET and hepatocyte growth factor are potential biomarkers in angiosarcomas and undifferentiated pleomorphic sarcomas.

Soft tissue sarcomas are a heterogeneous group of tumors with many different subtypes. In 2014 an estimated 12,020 newly diagnosed cases and 4,740 soft tissue sarcoma related deaths can be expected in the United States. Many soft tissue sarcomas are associated with poor prognosis and therapeutic options are often limited. The evolution of precision medicine has not yet fully reached the clinical treatment of sarcomas since therapeutically tractable genetic changes have not been comprehensively studied so far. We analyzed a total of 484 adult-type malignant mesenchymal tumors by MET fluorescence in situ hybridization and MET and hepatocyte growth factor immunohistochemistry. Eleven different entities were included, among them the most common and clinically relevant subtypes and tumors with specific translocations or complex genetic changes. MET protein expression was observed in 2.6% of the cases, all of which were either undifferentiated pleomorphic sarcomas or angiosarcomas, showing positivity rates of 14% and 17%, respectively. 6% of the tumors showed hepatocyte growth factor overexpression, mainly seen in undifferentiated pleomorphic sarcomas and angiosarcomas, but also in clear cell sarcomas, malignant peripheral nerve sheath tumors, leiomyosarcomas and gastrointestinal stromal tumors. MET and hepatocyte growth factor overexpression were significantly correlated and may suggest an autocrine activation in these tumors. MET FISH amplification and copy number gain were present in 4% of the tumors (15/413). Two samples, both undifferentiated pleomorphic sarcomas, fulfilled the criteria for high level amplification of MET, one undifferentiated pleomorphic sarcoma reached an intermediate level copy number gain, and 12 samples of different subtypes were categorized as low level copy number gains for MET. Our findings indicate that angiosarcomas and undifferentiated pleomorphic sarcomas rather than other frequent adult-type sarcomas should be enrolled in screening programs for clinical trials with MET inhibitors. The screening methods should include both in situ hybridization and immunohistochemistry

Copy number variations in atypical fibroxanthomas and pleomorphic dermal sarcomas

Atypical fibroxanthomas (AFX) and pleomorphic dermal sarcomas (PDS) are frequent cutaneous sarcomas typically arising on sun-exposed skin in elderly patients. In contrast to AFX, which generally do not recur after complete excision, PDS locally recur in up to 50% and metastasize in up to 20%. We recently detected characteristic UV-induced TP53 mutations as potential driver mutation in almost all PDS investigated as well as activating PIK3CA and RAS gene mutations in around one third of our tumors representing targets for personalized treatments in patients with unresectable or metastasized PDS. In the present study, we identified amplifications and deletions in a small part of the PDS (6 of 27 cases) but not in AFX suggesting that copy number variations (CNV) might not be an initial event in tumor development but rather important during tumor progression. In addition to BRAF, KNSTRN, IDH1 and PDGFRA amplification, CNV analyses revealed deletions in the CDKN2A, KIT and PDGFRA genes. In cases where an appropriate FISH assay was established, the CNV results could be verified by FISH analysis. Amplification of BRAF, KIT or PDGFRA and/or losses of CDKN2A might represent bad prognostic markers, although larger studies are needed to clarify their association with prognosis or progression in PDS

Comparison of the genomic background of MET-altered carcinomas of the lung: biological differences and analogies

Although non-small-cell lung cancer is a leading cause of cancer-related deaths, the molecular characterization and classification of its genetic alterations has drastically changed treatment options and overall survival within the last few decades. In particular, tyrosine kinase inhibitors targeting specific molecular alterations, among other MET, have greatly improved the prognosis of non-small-cell lung cancer patients. Here, we compare the genomic background of a subset of non-small-cell lung cancer cases harboring either a MET high-level amplification (n = 24) or a MET exon 14 skipping mutation (n = 26), using next-generatison sequencing, fluorescence in situ hybridization, immunohistochemistry, and Nanostring nCounter (R) technology. We demonstrate that the MET-amplified cohort shows a higher genetic instability, compared with the mutant cohort (p < 0.001). Furthermore, MET mutations occur at high allele frequency and in the presence of co-occurring TP53 mutations (n = 7), as well as MDM2 (n = 7), CDK4 (n = 6), and HMGA2 (n = 5) co-amplifications. No other potential driver mutation has been detected. Conversely, in the MET-amplified group, we identify co-occurring pathogenic NRAS and KRAS mutations (n = 5) and a significantly higher number of TP53 mutations, compared with the MET-mutant cohort (p = 0.048). Of note, MET amplifications occur more frequently as subclonal events. Interestingly, despite the significantly (p = 0.00103) older age at diagnosis of stage IIIb/IV of MET-mutant patients (median 77 years), compared with MET high-level amplified patients (median 69 years), MET-mutant patients with advanced-stage tumors showed a significantly better prognosis at 12 months (p = 0.04). In conclusion, the two groups of MET genetic alterations differ, both clinically and genetically: our data strongly suggest that MET exon 14 skipping mutations represent an early driver mutation. In opposition, MET amplifications occur usually in the background of other strong genetic events and therefore MET amplifications should be interpreted in the context of each tumor's genetic background, rather than as an isolated driver event, especially when considering MET-specific treatment options

Prevalence and potential biological role of TERT amplifications in ALK translocated adenocarcinoma of the lung

Aims The advent of specific ALK-targeting drugs has radically changed the outcome of patients with ALK translocated non-small-cell lung cancer (NSCLC). However, emerging resistance to treatment with ALK inhibitors in these patients remains a major concern. In previous studies, we analysed two ALK+ patient cohorts (TP53 wild-type/TP53 mutated) in terms of copy number alterations. All patients belonging to the TP53 wild-type group had mainly genetically stable genomes, with one exception showing chromosomal instability and amplifications of several gene loci, including TERT. Here, we aimed to determine the prevalence of TERT amplifications in these ALK+ lung cancer patients by analysing an independent cohort of 109 ALK translocated cases. We further analysed the copy numbers of numerous cancer-relevant genes and other genetic aberrations. Methods and results The prevalence of TERT amplifications was determined by means of FISH analyses. Copy numbers of 87 cancer-relevant genes were determined by NanoString nCounter(R) technology, FoundationOne(R) and lung-specific NGS panels in some of these TERT-amplified samples, and clinical data on patients with TERT-amplified tumours were collected. Our data revealed that five (4.6%) of all 109 analysed ALK+ patients harboured amplification of TERT and that these patients had genetically unstable genomes. Conclusions Our preliminary study shows that ALK+ adenocarcinomas should be evaluated in the context of their genomic background in order to more clearly understand and predict patients' individual course of disease