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Eosinophilic, solid and cystic (ESC) renal cell carcinoma (RCC) is characterized by a solid and cystic architecture with cells showing abundant eosinophilic cytoplasm with hobnail arrangement and a cytokeratin 7-negative/cytokeratin 20-positive immunophenotype. Recent studies have suggested that bi-allelic events affecting TSC genes might play an important role for such tumors. However, only indirect evidence of the clonal origin of TSC mutation has been gathered so far. Therefore, in this paper we aimed to perform multi-regional tumor sampling molecular analysis in four ESC RCC cases that had been completely embedded, three sporadic and one occurring in a patient with tuberous sclerosis complex (TSC). Histologically, the 4 cases showed cystic and solid architecture and cells with abundant eosinophilic cytoplasm with cytoplasmic stippling and round to oval nuclei. Immunohistochemistry showed at least focal expression of cytokeratin 20 in all tissue samples and negative cytokeratin 7, as well as diffuse positivity for S100A1 and at least focal expression of cathepsin K in three out of four cases. The sporadic cases showed the same somatic TSC1 mutations in all tissue samples analyzed, while the TSC-associated case showed the same TSC1 alteration in both normal tissue and all tumor samples analyzed, proving the germline nature of the alteration. In conclusion, our data demonstrate that clonal TSC loss is a key event in ESC RCC and support considering ESC RCC as an entity given its distinct morphologic, immunophenotypical and molecular characteristics.

Background: circulating tumor DNA (ctDNA) is a source of tumor genetic material for EGFR testing in NSCLC. Real-word data about liquid biopsy (LB) clinical practice are lacking. The aim of the study was to describe the LB practice for EGFR detection in North Eastern Italy. Methods: we conducted a multi-regional survey on ctDNA testing practices in lung cancer patients. Results: Median time from blood collection to plasma separation was 50 min (20&ndash, 120 min), median time from plasma extraction to ctDNA analysis was 24 h (30 min&ndash, 5 days) and median turnaround time was 24 h (6 h&ndash, 5 days). Four hundred and seventy five patients and 654 samples were tested. One hundred and ninety-two patients were tested at diagnosis, with 16% EGFR mutation rate. Among the 283 patients tested at disease progression, 35% were T790M+. Main differences in LB results between 2017 and 2018 were the number of LBs performed for each patient at disease progression (2.88 vs. 1.2, respectively) and the percentage of T790M+ patients (61% vs. 26%).

Aside from the CTNNB1 and adenomatous polyposis coli (APC) mutations, the genetic profile of pediatric aggressive fibromatosis (AF) has remained poorly characterized. The aim of this study was to shed more light on the mutational spectrum of pediatric AF, comparing it with its adult counterpart, with a view to identifying biomarkers for use as prognostic factors or new potential therapeutic targets. CTNNB1,APC,AKT1,BRAF TP53, and RET Sanger sequencing and next‐generation sequencing (NGS) with the 50‐gene Ion AmpliSeq Cancer Hotspot Panel v2 were performed on formalin‐fixed samples from 28 pediatric and 33 adult AFs. The prognostic value of CTNNB1,AKT1, and BRAF mutations in pediatric AF patients was investigated. Recurrence‐free survival (RFS) curves were estimated with the Kaplan–Meier method and statistical comparisons were drawn using the log‐rank test. In addition to the CTNNB1 mutation (64%), pediatric AF showed AKT1 (31%), BRAF (19%), and TP53 (9%) mutations, whereas only the CTNNB1 mutation was found in adult AF. The polymorphism Q472H VEGFR was identified in both pediatric (56%) and adult (40%) AF. Our results indicate that the mutational spectrum of pediatric AF is more complex than that of adult AF, with multiple gene mutations involving not only CTNNB1 but also AKT1 and BRAF. This intriguing finding may have clinical implications and warrants further investigations.

IntroductionLittle is known about genotypic and phenotypic correlations in undifferentiated large-cell carcinoma (LCC) of the lung.MethodsThirty LCC were dissected by unsupervised targeted next generation sequencing analysis for 50 cancer-associated oncogenes and tumor suppressor genes. Cell differentiation lineages were unveiled by using thyroid transcription factor-1 (TTF1) for adenocarcinoma (ADC) and p40 for squamous cell carcinoma (SQC), dichotomizing immunohistochemistry (IHC) results for TTF1 as negative or positive (whatever its extent) and for p40 as negative, positive, or focal (if

We present a very interesting case of lung adenocarcinoma carrying an uncommon EGFR exon 19 insertion with concomitant PIK3CA mutation showing dramatic and persisting improvement upon erlotinib therapy, after progression during PI3K inhibitor.

Background There is lack of evidence about systemic treatment of pseudomyxoma peritonei (PMP) relapsing after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy. There is also lack of biomarkers able to predict outcomes beyond known clinical and pathological prognostic features. Methods Fifteen patients with relapsed PMP and progressive disease within the last 6 months were included and received metronomic capecitabine (625 mg/mq/day b.i.d.) and bevacizumab (7.5 mg/Kg three-weekly) until progressive disease/unacceptable toxicity. The primary endpoint was progression-free survival (PFS). Ion Torrent® next generation sequencing technology (Hot-spot Cancer Panel) was used to characterize molecular features. Results At a median follow up of 12 months, median PFS was 8.2 months and 1-year overall survival was 91 %. Partial responses were observed in 20 % of cases, but a significant reduction of tumor markers in up to 79 %. Treatment was very well tolerated without no new safety signals. All tumor samples except one had KRAS mutations. Patients with GNAS mutations had a significantly shorter median PFS as compared to GNAS wild-type ones (5.3 months vs. not reached; p

A new somatic mutation in the coding region of Kirsten rat sarcoma viral oncogene homolog gene ( KRAS ), G48A, has been identified in a patient with non–small cell lung cancer (NSCLC). No other mutations were found by screening several genes known to be mutated in NSCLC. The patient responded to first-line therapy and is still under maintenance treatment 18 months after diagnosis. Normal and cancer cells were engineered to express the KRAS(G48A) mutation. KRAS(G48A) overexpression did not change the growth or the response to treatment compared with KRAS(wild type)-expressing cells. Analysis of the structure of the KRAS(G48A) mutant predicted altered interactions with other proteins. Analysis of KRAS binding to B-Raf proto-oncogene, serine/threonine kinase showed that the KRAS(G48A) mutant behaves more like a wild-type than a classical KRAS(G12) mutant. In conclusion, this new mutation in the coding region of KRAS , found in NSCLC, does not induce phenotypic changes similar to those induced by G12 mutants but presumably affects KRAS binding to proteins other than B-Raf proto-oncogene, serine/threonine kinase.

While pulmonary adenocarcinoma (ADC) is morphologically heterogeneous, little is known about intra-tumor gene mutation heterogeneity (ITH). We therefore subjected 20 ADC nodules, 5 mutated for EGFR and 5 for KRAS, 5 with an ALK translocation, and 5 wild type (WT) for these alterations, to unsupervised next-generation sequencing of tumor regions from diverse architectural patterns. When 2 or more different gene mutations were found in a single tumor, this fulfilled the criteria for ITH. In the 84 studied tumor regions with diverse architecture, 71 gene mutations and 34 WT profiles were found. ITH was observed in 9/15 (60 %) ADC, 3 with an EGFR, 3 with a KRAS, and 3 with an ALK aberration, as reflected in 5, 6, and 9 additional mutations, respectively, detected in these tumors. EGFR mutations were observed in 21/22 and KRAS mutations in 18/22 tumor regions, suggesting that they appear early and have a driver role (dominant or trunk mutations). Branching mutations (in EZH2, PIK3CA, TP53, and EGFR exon 18) occurred in two or more regions, while private mutations (in ABL1, ALK, BRAF, HER2, KDR, LKB1, PTEN, MET, SMAD4, SMARCB1, and SRC) were confined to unique tumor samples of individual lesions, suggesting that they occurred later on during tumor progression. Patients with a tumor showing branching mutations ran a worse clinical course, independent of confounding factors. We conclude that in ADC, ITH exists in a pattern suggesting spatial and temporal hierarchy with dominant, branching, and private mutations. This is consistent with diverse intra-tumor clonal evolution, which has potential implications for patient prognosis or development of secondary therapy resistance.

Little is known about molecular testing on tumor tissue retrieved from stained sections, for which there may be a clinical need. We retrospectively analyzed 112 sections from 56 tumor patients using either fluorescence in situ hybridization (FISH) with different probes (19 sections from 17 patients) or Sanger or targeted next generation sequencing for detection of BRAF, EGFR, KRAS, C-KIT, and TP53 mutations (93 sections from 39 patients). Tumor tissue sections had been stained by hematoxylin and eosin (H&E) (42 sections) or by immunohistochemistry for cytoplasmic or nuclear/nuclear-cytoplasmic markers (70 sections) with a peroxidase (P-IHC, with 3,3'-diaminobenzidine as chromogen) or alkaline phosphatase label (AP-IHC, with Warp Red™ as chromogen). For FISH analysis, the concordance rate between the original diagnosis and that obtained on H&E- or P-IHC-stained tissue sections (AP-IHC was not on record for this set of patients) was 95% (18 out of 19 tumor sections). Only one tumor sample, diffusely positive for MLH1, did not yield any nuclear hybridization signal. For sequencing analysis, the concordance rate was 100% on negative P-IHC and positive AP-IHC-stained sections, regardless of the subcellular localization of the reaction product. Mutations were detected in only 52% of cases expressing nuclear/nuclear-cytoplasmic markers, regardless of the sequencing technology used (p = 0.0002). In conclusion, stained sections may be a valuable resource for FISH or sequencing analysis, but on cases expressing nuclear markers sequencing results need to be interpreted cautiously.

Background: Acquired resistance to anti-EGFR MoAbs (cetuximab and panitumumab) represents a challenge in the treatment of mCRC, but its molecular mechanisms are not completely understood. Even in presence of RAS-BRAF-PI3KCA ”quadruple wt” and HER-2/MET negative status for protein expression and gene amplification, pts who primarily respond to anti-EGFR MoAbs will eventually develop secondary resistance. Prior retrospective and small series uniquely investigated a single biomarker, showing in some cases the emergence of KRAS mutations,HER-2 or MET amplifications. Our study aimed at comprehensively describing all known molecular alterations possibly associated with acquired resistance. Methods: Pts with mCRC were prospectively treated with cetuximab- or panitumumab-based therapy until progressive disease. All archival tumors were defined as RAS-BRAF-PI3KCA”quadruple wt” by Sanger sequencing, as well as HER-2/MET negative by both immunohistochemistry (IHC) and in-situ hybridization (ISH). At the time of disease progression, tumor re-biopsy was performed on the most accessible site of metastasis, as institutional procedure for a wide phase 1 screening program. On both archival tissue and re-biopsy, next generation sequencing of 50 genes' hotspot regions included in the Hotspot Cancer Panel v2 (Life Technologies) was performed by using the Ion Torrent Personal Genome Machine platform (Life Technologies). Moreover,HER-2/MET status were repeated on tumor re-biopsy by IHC and ISH. Results: Seventeen pts were recruited. All had prior objective response to anti-EGFRs. Next-generation sequencing confirmed RAS-BRAF-PI3KCA wt status on archival tumors. The results of our analyses on tumor re-biopsies are shown in the table: IDNGS: Acquired mutations (% mutant alleles)cellularity% mutant alleles normalized for cellularityHER2 ISHMET ISH#1KRAS Q61H (37%)80%46%--#2-Amplified-#3BRAF V600E (13%)90%14%--#4NRAS Q61R (13%)40%32%--#5--Amplified#6KRAS Q61K (4%)70%6% Acquired RAS or BRAF mutations were found in 4 (23%) and 1 (6%) cases, respectively. Acquired HER-2 or MET amplification were found in 4 (23%) and 2 (12%) cases, respectively. As shown for patient #6, some degree of intra-tumor heterogeneity may exist due to concomitant presence of low represented RAS-mutated and HER-2 amplified sub-clones. In some cases (35%), a detectable acquired mechanism of resistance remains unknown. Conclusions: Based on our results, currently known molecular alterations associated with acquired resistance were mainly mutually exclusive. In a relevant subset of cases additional molecular profiling is warranted. Citation Format: Filippo Pietrantonio, Rosa Berenato, Federica Perrone, Annunziata Gloghini, Elena Tamborini, Benedetta Picciani, Adele Busico, Giulio Settanni, Chiara Costanza Volpi, Ambra Vittoria Gualeni, Alessio Pellegrinelli, Massimo Milione, Marta Caporale, Monica Niger, Maria Di Bartolomeo, Filippo de Braud. Comprehensive molecular characterization of acquired resistance to anti-EGFR monoclonal antibodies (MoAbs) in patients with metastatic colorectal cancer (mCRC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B57.

Background Outcome of pseudomyxoma peritonei (PMP) after cytoreductive surgery (CRS) and hypertermic intraperitoneal chemotherapy (HIPEC) is heterogeneous even after adjusting for clinico-pathological prognostic variables. The identification of additional prognostic or even predictive biomarkers is an unmet clinical need. Patients and methods Forty patients with mucinous appendiceal tumors and PMP were clinically eligible and had evaluable tumor samples obtained after CRS and HIPEC. We carried out next-generations sequencing (NGS) of 50 gene's hotspot regions contained in the Hotspot Cancer Panel v2 using the Ion Torrent Personal Genome Machine platform (Life Technologies). Results KRAS and GNAS mutations were found in 72% and 52%, and their allelic frequency was below 10% in 55% and 43% of samples, respectively. KRAS and GNAS mutations were associated with worse progression-free survival (PFS) at univariate analysis (P = 0.006 and 0.011, respectively). At multivariate analysis, only KRAS mutations were independently associated with PFS (P = 0.012); GNAS mutations were not—being significantly associated with other poor prognostic features such as incomplete cytoreduction or KRAS mutations. Validation of results was carried out in an independent bi-institutional cohort of 25 patients and the prognostic effect of KRAS mutations was again confirmed in the multivariate model (P = 0.029). NGS approach allowed the discovery of other potentially druggable mutations such as those in PI3K, AKT, LKB1, FGFR3 and PDGFRA. Conclusions Given the homogeneity of this series and the sensitivity of NGS in this low-cellularity tumor, we demonstrated for the first time a poor prognostic role of KRAS mutations.