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Small cell lung cancer (SCLC) is an aggressive tumor, and despite its sensitivity to chemotherapy and radiotherapy, patients usually have a short survival. There are no clinically relevant predictive factors of responses to therapies, and therapeutic options are still limited.Clinical data and somatic mutations of genes included in the MSK-IMPACT panel were retrieved from cBioPortal for 108 SCLCs and analyzed to identify mutated gene networks. Results were validated in an independent cohort of 54 SCLCs, whose information was also available from cBioPortal.Different networks were observed in tumors of short and long survivors. Degree (K) and betweenness (B) are key features that characterize a gene in its network of related mutations. By comparing their B/K ratio, 2 signatures of mutated genes were identified, describing short (IL-7R, NTRK2, HNF-1A) and long survivors (NBN, PTPN-11, IRS-1, INPP-4A, PIK-3CG, HGF, LATS-2, SMARCA-4, FLT-3, EIF-4A2, SPEN, PAX-5, SH2-D1A, ARID-1A, HOXB-13, ERCC-4, FANCA, FH, FGFR-2, MST-1R, SMAD-4, DDR-2, IGF-1R, PIK-3CB). Patients with at least 1 mutated gene of the short signature had a worse median overall survival of 8 versus 28 months (P.001). Patients with at least 1 mutated gene of the long signature had a better median overall survival of 39 versus 20 months (P = .004). The value of the short signature was further confirmed in an independent cohort of SCLCs.The networks of mutated genes could help subclassify SCLCs based on their somatic mutations and aid in identifying a subset of tumors with poor prognosis.

Background Insights into the mechanism of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) could provide important information for further patient management, including the choice of second-line treatment. The EGFR T790M mutation is the most common mechanism of resistance to first- and second-generation EGFR TKIs. Owing to its biologic relevance in the response of non–small-cell lung cancer (NSCLC) to the selective pressure of treatment, the present study investigated whether the occurrence of T790M at progression differed among patients receiving gefitinib, erlotinib, or afatinib. Patients and Methods The present retrospective study included patients with NSCLC with an EGFR activating mutation, who had received gefitinib, erlotinib, or afatinib as first-line treatment. Plasma samples for the analysis of cell-free DNA were taken at disease progression and analyzed using a digital droplet polymerase chain reaction EGFR mutation assay. Results A total of 83 patients were enrolled; 42 had received gefitinib or erlotinib and 41afatinib. The patient characteristics were comparable across the 2 groups. The median time to progression (TTP) was 14.4 months for the gefitinib and erlotinib group and 10.2 months for the afatinib group (P = .09). Of the 83 patients, 47 (56.6%) were positive for the T790M in plasma. A greater incidence of T790M was observed in patients with progression during gefitinib or erlotinib therapy compared with patients treated with afatinib (33 [79%] vs. 14 [34%], respectively; odds ratio, 7.1; 95% confidence interval, 2.7-18.5; P = .0001). Conclusions Although gefitinib, erlotinib, and afatinib showed a comparable TTP in patients receiving first-line therapy, the incidence of T790M differed among them, as demonstrated by the present study, which could have implications for the choice of second-line treatment.

Molecular heterogeneity characterizes tumours’ evolution and adaptation and, because of its dynamics and continuous changes under external pressure, it is one of the major causes of drug resistance, contributing to therapy failure. Several studies reported evidence of molecular events occuring in individual tumours, including monoclonal or polyclonal resistance, and primary or secondary resistance mechanisms. While primary resistance is a phenomenon already present at the diagnosis of a tumor, the acquired one is strongly related to the selective pressure of treatments administered. Therefore, the pharmacological characteristics of a drug, including its potency, binding affinity and structure, largely influence the mechanism of resistance that will arise at the progression of the disease. As an example, the lung cancer experience clearly demonstrated that the highest is the potency of a drug on its target, the more are the possibilities that the mechanism of resistance will arise independently of the target itself. The present review is focused on tumour heterogeneity and its relation to resistance to targeted-therapy, based on treatment selective pressure across different tumour types, including lung, colorectal, prostate, breast cancer and melanoma. The mechanisms of resistance based on the drug potency and the selective pressure of treatments are discussed, leading to new drug developments or new therapeutic combinations.

Background In patients with anaplastic lymphoma kinase (ALK)-positive non–small-cell lung cancer (NSCLC), disease progression occurs after a median of 9 to 10 months of crizotinib treatment. Several mechanisms of resistance have been identified and include ALK mutations and amplification or the activation of bypassing signaling pathways. Rebiopsy in NSCLC patients represents a critical issue and the analysis of circulating cell-free DNA (cfDNA) has a promising role for the identification of resistance mechanisms. Patients and Methods Twenty patients with advanced ALK-positive NSCLC were enrolled after disease progression during crizotinib treatment; cfDNA was analyzed using digital droplet polymerase chain reaction (BioRad, Hercules, CA) for ALK (p.L1196M, p.G1269A, and p.F1174L) and Kirsten rat sarcoma (KRAS) (codons 12 and 13) mutations. Results ALK secondary mutations (p.L1196M, p.G1269A, and p.F1174L) were identified in 5 patients; 1 patient had 2 ALK mutations (p.L1196M and p.G1269A). Overall, 10 patients presented KRAS mutations (7 p.G12D, 2 p.G12V, and 1 p.G12C mutations, respectively). In 3 patients KRAS mutations were associated with ALK mutations. cfDNA was monitored during the treatment with second-generation ALK inhibitors and the amount of ALK as well as KRAS mutations decreased along with tumor regression. Conclusion ALK and KRAS mutations are associated with acquired resistance to crizotinib in ALK-positive NSCLC. In particular, ALK acquired mutations can be detected in plasma and could represent a promising tumor marker for response monitoring.

Background Insights into the mechanism of resistance to first generation EGFR-TKIs may provide important information for further patient management, including the choice of second-line treatments. T790M is a gatekeeper mutation of the ATP binding pocket of the EGFR kinase domain and is the most common mechanism of resistance to first-generation EGFR-TKIs. Owing to its biologic relevance in the response of NSCLC to the selective pressure of treatments, the present study investigated in circulating cell-free DNA (cfDNA) if the occurrence of T790M at progression differed among gefitinib, afatinib, and erlotinib. Methods This study included patients with NSCLC bearing EGFR activating mutation, and given gefitinib (G), erlotinib (E) or afatinib (A) as first-line treatment. Plasma samples for the analysis of cfDNA were taken at disease progression (PD) and analyzed by a ddPCR using the ddPCR EGFR Mutation Assay. In selected cases, a rebiopsy was performed to confirm the absence of the T790M in negative plasma. Results A total of 83 patients were enrolled; 42 patients received G/E and 41 received A. Patients’ characteristics were comparable across the two groups. Median time to progression (TTP) was 14.4 in G/E vs 10.2 months in A group (p = 0.09). Forty-seven out of 83 patients (56.6%) were positive for the T790M in plasma. There was a higher incidence of the T790M in patients who progressed to G/E than in patients treated with A: 33 (79%) vs 14 (34%), respectively (p = 0.0001). To confirm the absence of the T790M, a rebiopsy was feasible in 7 patients of the G/E group and in 23 of the A group. The analysis of the cytological sample confirmed the absence of the T790M, and PI3K mutation was found in both groups in 1 patient (2%). Three patients (7%) had MET amplification in the A group. Afatinib dosage was reduced in 15 patients to 30 mg; T790M was not correlated with the dose reduction, being detectable in 6 patients who needed the reduction and in 8 who received the full standard dose (p = 0.54). Conclusions In conclusion, even though gefitinib, erlotinib, and afatinib belong to the same class of EGFR-TKIs, differences in the appearance of resistance mutation are demonstrated in the present study and this finding may have implications in the choice of 2nd-line treatment. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

The WHO-classification was shown to be an independent prognostic marker in some but not all retrospective studies possibly due to lack of reproducibility. We investigated the reproducibility of the WHO-classification and its prognostic implication using a large series of resected thymomas.Four independent pathologists histologically classified a surgical series of 129 thymic tumors in a blinded fashion. Fleiss' kappa-coefficient was used to assess the pathologists' overall agreement, and Cohen-Kappa to assess the agreement between two observers. Disease-related-survival (DRS) and progression-free-survival (PFS) curves were generated by Kaplan-Meier method and compared by log-rank test.In 63/129 (48.8%) cases there was a complete agreement; in 43/129 (33.3%) cases 3/4 pathological diagnoses were identical; in 15/129 (11.6%) cases the diagnoses were identical by pair; in 8/129 (6.2%) cases three different pathological diagnoses were on record. The Kappa-correlation coefficient was only moderate (0.53). A following web review carried out on the 23 cases with at least two different diagnoses reached a complete consensus. The histotype showed a statistically significant impact on PFS and DRS in the classification provided by only two pathologists.In this study, the agreement on WHO classification of thymomas was only moderate and this impacted on patients management. Web consensus conference on the diagnosis, more stringent diagnostic criteria or the adoption of referral diagnostic centres may substantially reduce discrepancies.

Introduction Thymic carcinomas represent the most aggressive histotype of thymic epithelial tumors (TETs). The 2004 World Health Organization classification has assigned a subgroup of thymic carcinomas as t(15;19) carcinomas based on the presence of t(15;19), a translocation found in poorly differentiated and highly aggressive NUT midline carcinomas. These tumors are characterized byrearrangement of the NUT (nuclear protein in testis) gene on chromosome 15q14, which in most cases fuses to the bromodomain containing 4 ( BRD 4) gene on chromosome 19 p13.1 through reciprocal t(15;19) translocation, resulting in constitutive BRD4-NUT fusion protein expression. To our knowledge, NUT translocation has been reported only in four thymic carcinomas. Due to the rarity of TETs, the prevalence of NUT rearrangement in TETs has however never been systematically explored. Methods Formalin-fixed paraffin-embedded samples of histologically confirmed TETs were evaluated for NUT expression and rearrangement by immunohistochemistry and fluorescence in situ hybridization, respectively. Results A series of 148 TETs (37 carcinomas and 111 thymomas) were examined for NUT expression and rearrangement. Only one thymic carcinoma (2.7% of thymic carcinomas or 0.68% of TETs) was found positive for NUT expression and rearrangement. Conclusions Rearrangement of NUT is infrequent in TETs. We propose that caution should be taken to distinguish t(15;19) thymic carcinoma from other mediastinal carcinomas, as NUT midline carcinomas are often associated with dreadful prognosis or overt lethality.

Background Overexpression of c-kit, a tyrosine kinase receptor protein encoded by the protooncogene kit, has been previously reported in thymic epithelial tumors and in other neoplasms such as gastrointestinal stromal tumors, myeloproliferative disorders, melanoma, and seminoma. Mutations in the kit gene have been related to response to imatinib in gastrointestinal stromal tumor and one case report of thymic carcinoma. We studied expression of c-kit in a large retrospective series of thymic epithelial malignancies and sequenced the whole gene in a subset of patients. Methods Thymic epithelial tumors from 120 patients (13 thymic carcinomas and 107 thymomas) were examined. Immunohistochemical staining with an antic-kit polyclonal antibody was performed on a tissue microarray. Mutation analyses of exons 1 to 20 were conducted by direct DNA sequencing of polymerase chain reaction products in eight thymic carcinomas, five thymomas, and one thymic carcinoma cell line. Results The percentage of c-kit positive cells was significantly higher in thymic carcinoma (46%) than in thymoma (4%). Decreased disease-related survival and progression-free survival were observed in c-kit positive tumors. No mutations were detected. Conclusion c-kit expression is strongly but not exclusively related to thymic carcinoma histotype, and it is of prognostic value. Mutations are very rare.

Aim: To investigate the molecular aberrations of thymic epithelial tumors (TETs) which, currently, are largely unknown, hampering progress in diagnosis, prognostication and targeted therapy. Methods: Frozen tumor and normal blood were available for 28 TETs with a high proportion of cancer cell content (>80%). Exome capture was performed using Sure Select All Exon (Agilent) and TruSeq Exome Enrichment kit (Illumina) in 21 and 35 samples, respectively. Sequencing was performed using Genome Analyzer-II (Illumina) and HiSeq2000 (Illumina) in 21 and 35 samples, respectively. Reads were aligned using Novoalign (Novocraft) and somatic mutations were detected comparing exome sequencing of tumor and normal DNA of each patients using VarScan2. A single nucleotide missense mutation of GTF2I was the most common in TETs, and was confirmed in an independent cohort of 268 formalin fixed paraffin embedded TETs. Results: Thymic carcinomas had a higher number of mutations than thymomas with a mean of 43.5 and 17.4, respectively (p=0.001). In thymic carcinomas (11), genes with recurrent mutations included TP53 (4), CYLD (3), BAP1 (2), CDKN2A (2) and PBRM1 (2). A single nucleotide mutation of GTF2I (chr7:74146970T/A) was observed in 35% of thymomas (17). The mutation was missense (leucine to histidine), not previously described in cancer or as a polymorphism in dbSNP137 database. The mutation was predicted to alter the structure of the protein or its function according to Poliphen2 and SIFT algorithms. The presence of GTF2I mutation was confirmed in an extended cohort of 268 TETs: 82% of A (56), 74% of AB (54), 32% of B1 (28), 22% of B2 (32), 21% of B3 thymomas (62) and 8% of thymic carcinomas (36). The mutation was more frequent in stages I-II (57%) than in stages III-IV (19%; p Conclusions: GTF2I mutation is frequent in thymic epithelial tumors, especially in A-AB histotypes and is associated with a better outcome. Disclosure: All authors have declared no conflicts of interest.

Aim: Trastuzumab is effective for the treatment of advanced gastric cancers with HER2 amplification. Beside HER2 amplification, several molecular targets are under evaluation in metastatic gastric tumors. We evaluated the role of HER2, EGFR, MET, AKT1 and phospho-mTOR expression in resected stage II-III adenocarcinomas. Methods: 92 patients with resected stomach (63%) or gastro-esophageal adenocarcinomas (27%) were evaluated using immunohistochemistry. Antibodies anti- HER2, EGFR, MET, AKT1 and phospho-mTOR were used for immunostaining of formalin-fixed paraffin-embedded tumors' slides. Using FISH, HER2 amplification was evaluated in cases with a 2+ staining. Results: EGFR overexpression (11%) was a poor prognostic factor for overall survival (3-year OS: 47% vs 77%; Log-Rank p = 0.033). MET overexpression (36%) was associated with a trend for a worse survival (3-year OS: 65% vs 77%; Log-Rank p = 0.084). HER2 amplification/overexpression and mTOR hyper-phosphorylation were observed in 13% and 48% of tumors, respectively. AKT1 overexpression (8%) was mutually exclusive with that of EGFR and together defined a subgroup of tumors with a poor prognosis (3-year OS: 52% vs. 79%, Log-Rank p = 0.005). Conclusions: EGFR was confirmed a poor prognostic factor in resected gastric cancers. We firstly describe a mutually exclusive overexpression of EGFR and AKT1 with potential prognostic implications, suggesting the relevance of this pathway for the growth of gastric cancers. Disclosure: All authors have declared no conflicts of interest.