Evaluation of 7q31 region improves the accuracy of EGFR FISH assay in non small cell lung cancer

<p>Abstract</p> <p>Background</p> <p>Increase of <it>EGFR </it>gene copy number consequent to gene amplification and/or polysomy of chromosome 7 has been significantly associated with better clinical outcome in Non Small Cell Lung Cancer (NSCLC) patients treated with Tyrosin-Kinase Inhibitors (TKIs).</p> <p>The primary method to detect <it>EGFR </it>copy number is FISH (Fluorescence in Situ Hybridization), that in lung cancer requires a precise standardization due to the presence of intratumor heterogeneity and high frequency of chromosome 7 polysomy.</p> <p>Recommendations and interpretative guidelines to discriminate NSCLC patients into FISH positive (gene amplification and high chromosome 7 polysomy) and FISH negative have been proposed by the University of Colorado Cancer Center (UCCC). However, in a subset of cases the distinction between <it>EGFR </it>amplification and chromosome 7 polysomy can be controversial because of a complex pattern of multiple <it>EGFR </it>and centromere signals.</p> <p>Methods</p> <p>In order to distinguish more accurately these two genetic events, 20 NSCLC FISH positive patients, showing a controversial pattern of <it>EGFR </it>and centromere specific signals, were further evaluated for the status of 7q31 distal region.</p> <p>Results</p> <p>A discrepancy between FISH results obtained with UCCC scoring system and 7q31 control was evidenced in 2 patients (10%).</p> <p>Conclusion</p> <p>Our data strengthen the usefulness of 7q31 region evaluation to discriminate EGFR amplification from chromosome 7 polysomy in controversial <it>EGFR </it>FISH positive cases. Since it has been reported a possible different contribution of amplification and polysomy to TKIs susceptibility in NSCLC, the clear distinction between these two genetic events may be important to identify a subset of patients more responsive to the therapy.</p

Evolving concepts in HER2 evaluation in breast cancer: heterogeneity, HER2-low carcinomas and beyond

Abstract The human epidermal growth factor receptor 2 (HER2) is a well-known negative prognostic factor in breast cancer and a target of the monoclonal antibody trastuzumab as well as of other anti-HER2 compounds. Pioneering works on HER2-positive breast cancer in the 90' launched a new era in clinical research and oncology practice that has reshaped the natural history of this disease. In diagnostic pathology the HER2 status is routinely assessed by using a combination of immunohistochemistry (IHC, to evaluate HER2 protein expression levels) and in situ hybridization (ISH, to assess HER2 gene status). For this purpose, international recommendations have been developed by a consensus of experts in the field, which have changed over the years according to new experimental and clinical data. In this review article we will document the changes that have contributed to a better evaluation of the HER2 status in clinical practice, furthermore we will discuss HER2 heterogeneity defined by IHC and ISH as well as by transcriptomic analysis and we will critically describe the complexity of HER2 equivocal results. Finally, we will introduce the clinical impact of HER2 mutations and we will define the upcoming category of HER2-low breast cancer with respect to emerging clinical data on the efficacy of specific anti-HER2 agents in subgroups of breast carcinomas lacking the classical oncogene addition dictated by HER2 amplification

Pursuit of Gene Fusions in Daily Practice: Evidence from Real-World Data in Wild-Type and Microsatellite Instable Patients

Agnostic biomarkers such as gene fusions allow to address cancer patients to targeted therapies; however, the low prevalence of these alterations across common malignancies poses challenges and needs a feasible and sensitive diagnostic process. RNA-based targeted next generation sequencing was performed on 125 samples of patients affected either by colorectal carcinoma, melanoma, or lung adenocarcinoma lacking genetic alterations in canonical driver genes, or by a colorectal carcinoma with microsatellite instability. Gene fusion rates were compared with in silico data from MSKCC datasets. For NTRK gene fusion detection we also employed a multitarget qRT-PCR and pan-TRK immunohistochemistry. Gene fusions were detected in 7/55 microsatellite instable colorectal carcinomas (12.73%), and in 4/70 of the “gene driver free” population (5.71%: 3/28 melanomas, 10.7%, and 1/12 lung adenocarcinomas, 8.3%). Fusion rates were significantly higher compared with the microsatellite stable and “gene driver positive” MSKCC cohorts. Pan-TRK immunohistochemistry showed 100% sensitivity, 91.7% specificity, and the occurrence of heterogeneous and/or subtle staining patterns. The enrichment of gene fusions in this “real-world” cohort highlights the feasibility of a workflow applicable in clinical practice. The heterogeneous expression in NTRK fusion positive tumours unveils challenging patterns to recognize and raises questions on the effective translation of the chimeric protein

Targeting EGFR/HER2 pathways enhances the antiproliferative effect of gemcitabine in biliary tract and gallbladder carcinomas

<p>Abstract</p> <p>Background</p> <p>Advanced biliary tract carcinomas (BTCs) have poor prognosis and limited therapeutic options. Therefore, it is crucial to combine standard therapies with molecular targeting. In this study EGFR, HER2, and their molecular transducers were analysed in terms of mutations, amplifications and over-expression in a BTC case series. Furthermore, we tested the efficacy of drugs targeting these molecules, as single agents or in combination with gemcitabine, the standard therapeutic agent against BTC.</p> <p>Methods</p> <p>Immunohistochemistry, FISH and mutational analysis were performed on 49 BTC samples of intrahepatic (ICCs), extrahepatic (ECCs), and gallbladder (GBCs) origin. The effect on cell proliferation of different EGFR/HER2 pathway inhibitors as single agents or in combination with gemcitabine was investigated on BTC cell lines. Western blot analyses were performed to investigate molecular mechanisms of targeted drugs.</p> <p>Results</p> <p>EGFR is expressed in 100% of ICCs, 52.6% of ECCs, and in 38.5% of GBCs. P-MAPK and p-Akt are highly expressed in ICCs (>58% of samples), and to a lower extent in ECCs and GBCs (<46%), indicating EGFR pathway activation. HER2 is overexpressed in 10% of GBCs (with genomic amplification), and 26.3% of ECCs (half of which has genomic amplification). EGFR or its signal transducers are mutated in 26.5% of cases: 4 samples bear mutations of PI3K (8.2%), 3 cases (6.1%) in K-RAS, 4 (8.2%) in B-RAF, and 2 cases (4.1%) in PTEN, but no loss of PTEN expression is detected. EGI-1 cell line is highly sensitive to gemcitabine, TFK1 and TGBC1-TKB cell lines are responsive and HuH28 cell line is resistant. In EGI-1 cells, combination with gefitinib further increases the antiproliferative effect of gemcitabine. In TFK1 and TGBC1-TKB cells, the efficacy of gemcitabine is increased with addiction of sorafenib and everolimus. In TGBC1-TKB cells, lapatinib also has a synergic effect with gemcitabine. HuH28 becomes responsive if treated in combination with erlotinib. Moreover, HuH28 cells are sensitive to lapatinib as a single agent. Molecular mechanisms were confirmed by western blot analysis.</p> <p>Conclusion</p> <p>These data demonstrate that EGFR and HER2 pathways are suitable therapeutic targets for BTCs. The combination of gemcitabine with drugs targeting these pathways gives encouraging results and further clinical studies could be warranted.</p

Rituximab Treatment Prevents Lymphoma Onset in Gastric Cancer Patient-Derived Xenografts.

Patient-Derived Xenografts (PDXs), entailing implantation of cancer specimens in immunocompromised mice, are emerging as a valuable translational model that could help validate biologically relevant targets and assist the clinical development of novel therapeutic strategies for gastric cancer. More than 30% of PDXs generated from gastric carcinoma samples developed human B-cell lymphomas instead of gastric cancer. These lymphomas were monoclonal, Epstein Barr Virus (EBV) positive, originated tumorigenic cell cultures and displayed a mutational burden and an expression profile distinct from gastric adenocarcinomas. The ability of grafted samples to develop lymphomas did not correlate with patient outcome, nor with the histotype, the lymphocyte infiltration level, or the EBV status of the original gastric tumor, impeding from foreseeing lymphoma onset. Interestingly, lymphoma development was significantly more frequent when primary rather than metastatic samples were grafted. Notably, the development of such lympho-proliferative disease could be prevented by a short rituximab treatment upon mice implant, without negatively affecting gastric carcinoma engraftment. Due to the high frequency of human lymphoma onset, our data show that a careful histologic analysis is mandatory when generating gastric cancer PDXs. Such care would avoid misleading results that could occur if testing of putative gastric cancer therapies is performed in lymphoma PDXs. We propose rituximab treatment of mice to prevent lymphoma development in PDX models, averting the loss of human-derived samples