Search

Background. Homologous recombination repair (HRR) is the main mechanism of repair of DNA double-strand breaks. Its deficiency (HRD) is a common feature of epithelial ovarian cancers (EOCs). BRCA1/2 mutations and/or other aberrations in genes of HRR are well known causes of HRD and genomic instability. Poly ADP-ribose polymerase inhibitors (PARPi) have revolutionized the management of BRCA mutant EOCs and demonstrated activity in HRD tumor cells. Determining HRD status can provide informations on the magnitude of benefit for PARPi therapy. Myriad MyChoice CDx is a next generation sequencing- based in vitro diagnostic test that assesses the Genomic Instability Score (GIS) which is an algorithmic measurement of loss of heterozygosity, telomeric allelic imbalance, and large-scale state transitions using DNA isolated from formalin-fixed paraffin embedded tumor tissue specimens. However Myriad MyChoice CDx, is a centrally performed and costly assay, with no reimbursement scheduled, at least in Italy. Methods. In this report, we described our experience in performing the HRD Focus AmoyDx (Amoy Diagnostics Ltd, Xiamen, Fujian, China) on the same samples of EOCs evaluated with Myriad MyChoiceCDx assay. Results. The overall percent agreement between AmoyDx and Myriad was 87.8% (65 of 74 tumors tested). All the 36 AmoyDx negative cases were confirmed to be negative by Myriad (negative predictive value, 100%). Conclusions. The concordance of the results with the gold standard Myriad MyChoice CDx assay suggest the feasibility and reliability of HRD testing in diagnostic laboratories with high-throughput NGS platforms and qualified personnel.

Targeted therapy in lung cancer requires the assessment of multiple oncogenic driver alterations, including fusion genes. This retrospective study evaluated the Idylla GeneFusion prototype, an automated and ease-of-use (

AimsPoly (ADP-ribose) polymerase (PARP) inhibitors (PARPIs) represent a standard of care for the clinical management of high-grade serous ovarian cancer (HGSOC). The recognition of homologous recombination deficiency (HRD) has emerged as a predictive biomarker of response for first-line PARPIs treatment in patients with HGOSC. On the other hand, this test is extremely complex and therefore it is often externalised. Regrettably, the reliability of outsourced HRD testing can be troubled by inconclusive results and high rejection rates. In this methodological study, we assessed the technical feasibility, interassay and interlaboratory reproducibility of in-house HRD testing using three different commercially available next-generation sequencing assays.MethodsA total of n=20 epithelial ovarian cancer samples previously analysed with MyChoice CDx were subjected to HRD retesting using three different platforms in three different major pathology laboratories, that is, SOPHiA DDM HRD Solution, HRD focus and Oncomine homologous recombination repair pathway predesigned panel. Concordance was calculated by Cohen’s (dual) and Fleiss (triple) κ coefficients.ResultsIn-houseBRCA1/2molecular testing yielded a concordance rate >90.0% among all participating centres. HRD scores were successfully calculated by each institution with a concordance rate of 76.5%. Concerning the external gold standard test, the overall percentage of agreement ranged from 80.0% to 90.0% with a positive percentage agreement ranging from 75.0% to 80.0% and a negative percentage agreement ranging from 80.0% to 100%.ConclusionsIn-house testing for HRD can be reliably performed with commercially available next-generation sequencing assays.

We present a case of a woman with epidermal growth factor receptor (EGFR)-mutated lung adenocarcinoma who received gefitinib for 2 years and obtained a partial response. The patient then developed liver metastasis and a breast lesion, displaying high estrogen receptor (ER) expression and harboring the same EGFR mutation. From the radiological studies, it was not possible to make a differential diagnosis between primary breast cancer and breast metastasis from lung cancer. After the removal of the breast nodule, thanks to the clinical history, radiology, and above all, molecular and immunohistochemical investigations, a diagnosis of breast metastasis from lung adenocarcinoma was made. This case emphasizes the importance of a comprehensive clinical, pathological, and molecular analysis in the differential diagnosis between primary breast cancer and metastases from extramammary tumor to guide adequate treatment decision making.

In the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, pathologists can be exposed to infection handling surgical specimens. Guidelines related to safety procedures in the laboratory have been released. However, there is a lack of studies performed on biopsy and surgical resection specimens. Here we report the detection of SARS-CoV-2 in formalin-fixed paraffin-embedded samples from surgical resection of tongue squamous cell carcinoma of a patient who developed COVID-19 postsurgery. RNA of SARS-CoV-2 strain was detected in the tumour and the normal submandibular gland samples using real-time PCR-based assay. No viral RNA was found in metastatic and reactive lymph nodes. We demonstrated that SARS-CoV-2 RNA can be detected in routine histopathological samples even before COVID-19 disease development. These findings may give important information on the possible sites of infection or virus reservoir, and highlight the necessity of proper handling and fixation before sample processing.

The establishment of PARP inhibitors in the treatment of epithelial ovarian carcinoma (EOC) has prompt BRCA assessment at the time of diagnosis. We described our five years of experience of tumor BRCA testing, as part of a multidisciplinary workflow for the management of EOC patients. We used a BRCA next-generation sequencing (NGS) test for profiling formalin-fixed, paraffin-embedded (FFPE) EOCs of 762 consecutive patients, with a success rate of 99.7% and a median turnaround time of 12 days. We found 178 (23.4%) cases with pathogenic/likely pathogenic (P/LP) mutations, 74 (9.7%) cases with variants of uncertain significance and 508 (66.8%) wild type tumors. Among 174 patients without P/LP mutations and investigated with multiple-ligation probe-amplification analysis on peripheral blood, two (1.1%) were positive for large rearrangements. Patients with P/LP alterations and/or with positive family history were referred to genetic counselling. Comparing tumor and blood NGS test results of 256 patients, we obtained a tumor test negative predictive value of 100% and we defined 76% of P/LP alterations as germline and 24% as somatic variants. The proposed workflow may successfully identify EOC patients with BRCA1/2 alteration, guiding both therapeutic and risk assessment clinical decisions.

Introduction: Based on the SOLAR-1 study, the PI3Kα-specific inhibitor alpelisib has been approved in combination with fulvestrant for postmenopausal women, and men, with HR+/HER2-, PIK3CA-mutated, advanced or metastatic breast cancer (BC). Despite PIK3CA mutations occurring in ⁓40% of HR+/HER2- BC, PIK3CA molecular testing is a new task to be carried out in clinical practice. Adopting the most appropriate testing strategy (RT-PCR vs. NGS) on the most appropriate biological sample is of capital significance in oncologic pathology. Here, we sought to assess the concordance rate for PIK3CA molecular analysis between different technical platforms in both metastatic and matched primary tumors. Methods: From our Institutional registry, n=16 HR+/HER2- metastatic BC, which were found to harbor PIK3CA mutations via next-generation sequencing (NGS) assays [custom panel and Oncomine Comprehensive Assay (OCA) v3 (Thermo Fisher Scientific, Waltham, MA, USA)], were selected. In half of the cases (n=8), matched primary tumors were available and were subjected to PIK3CA testing with NGS. The analytical performance between NGS and a semi-closed RT-PCR (EasyPGX®, Diatech Pharmacogenetics, Italy) was assessed in 13/16 (81.3%) metastatic samples for which archival slides and blocks and/or residual extracted DNA were available, and all the primary tumors. Results: Overall, upfront testing of PIK3CA mutational status with NGS detected genetic alterations in exons 7, 9, and 20. A concordance rate of 100.0% was observed between primary and metastatic tumors. On the other hand, the analysis of primary tumors (n=8) and 13/16 (81.3%) metastatic samples with RT-PCR revealed a concordance of 42.9%. Analytical performance comparison showed that the two technologies were concordant in 7/13 metastatic cases (53.8%). In two of the discordant cases (15.4%), RT-PCR did not identify the PIK3CA mutations due to their absence from its reference range. Interestingly, visual inspection of the RT-PCR raw data increased the concordance to 76.9%. In primary tumors, consensus between the two testing methods was observed in 5 (62.5%) cases. Discussion: The concordance rate analysis shows that upfront PIK3CA molecular testing with NGS appears to be more efficacious compared with RT-PCR. Our data suggest that primary tissues reflect the PIK3CA mutational status when tested with NGS. RT-PCR is simpler with a shorter turnaround time, and less expensive than NGS approaches, however, trained personnel are required for accurate results interpretation. In addition, the limited reference range of this testing method should be taken into account for potential false negative results. Conclusion: In terms of PIK3CA molecular testing, NGS should be the preferred method in comparison with RT-PCR. Primary tumors represent a valid biospecimen to be used for this analysis in the absence of the metastatic sample. Citation Format: Konstantinos Venetis, Francesco Pepe, Elham Sajjadi, Giuseppina Bonizzi, Mariia Ivanova, Davide Vacirca, Alessandra Rappa, Massimo Barberis, Giuseppe Viale, Elena Guerini-Rocco, Elisabetta Munzone, Umberto Malapelle, Nicola Fusco. Comparison of next-generation sequencing and real-time PCR for PIK3CA testing in hormone receptor-positive/HER2-negative breast cancer on metastatic and matched primary tumor samples [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-04-14.

(1) Background: The development of laryngeal cancer is a multistep process involving structural alterations of the epithelial mucosa, from dysplasia (LDy) to invasive carcinoma. In this study, we define new biomarkers, prognostic for malignant transformation, in patients affected by LDy. (2) Methods: We used targeted next-generation sequencing and immunohistochemical analysis to define the mutational and immunological landscape of 15 laryngeal dysplasia progressing to invasive cancer (progressing dysplasia), as well as 31 cases of laryngeal dysplasia that did not progress to carcinoma (non-progressing dysplasia). Two pathologists independently analyzed the presence of tumor-infiltrating lymphocytes in LDy pre-embedded paraffin-fixed specimens. The RNA-based next-generation sequencing panel OIRRA was used to evaluate the expression of 395 genes related to immune system activation. (3) Results: High TILs are significantly correlated with a higher risk of malignant transformation. The non-brisk pattern was significantly associated with an 86% reduced risk of malignant progression (OR = 0.16, 95% CI: 0.03–0.5, p = 0.008). TILs showed a highly positive correlation with CCR6, CD83, HLA-DPB1, MX1 and SNAI1, and they were inversely correlated with CD48, CIITA, CXCR4, FCER1G, IL1B, LST1 and TLR8. (4) Conclusions: TILs have a great potential to identify high-risk progression dysplasia and thus to define surveillance protocols and prevention programs.

AimsAnalysis of microsatellite instability (MSI) is strongly recommended in endometrial cancer (EC) and colorectal cancer to screen for Lynch syndrome, to predict prognosis and to determine optimal treatment and follow-up. In a large monoinstitutional series of ECs, we evaluated the reliability and accuracy of Idylla assay, a rapid, fully automated system to detect MSI, and we compared its performance with two routine reference methods.MethodsWe evaluated MSI status in 174 formalin-fixed, paraffin-embedded EC tissue samples using immunohistochemistry (IHC) for mismatch repair (MMR) proteins and Idylla assay. Samples with discordant or equivocal results were analysed with a third technique, the Promega MSI kit.ResultsIdylla MSI assay and IHC were highly concordant (overall agreement: 154/170=90.59%, 95% CI 85.26% to 94.12%). However, in four samples, MMR-IHC staining was equivocal; moreover, 16 cases showed discordant results, that is, MMR deficient using IHC and microsatellite stable using Idylla. These 20 samples were reanalysed using the MSI-Promega kit, which showed the same results of Idylla assay in 18/20 cases (overall agreement: 90%, 95% CI 69.90% to 97.21%).ConclusionsOur results suggest that IHC is an efficient method to determine MMR status in ECs. However, the Idylla MSI assay is a rapid and reliable tool to define MSI status, and it could represent a valuable alternative to conventional MSI-PCR methods.

To investigate the role of the altered activation of the immune system in the prognosis of patients affected by laryngeal squamous cell carcinoma (LSCC). We analyzed 56 patients with advanced LSCC divided into two groups according to their prognosis: the first group relapsed within 24 months after treatment, the second group had no evidence of disease at 2 years. The presence of stromal tumor infiltrating lymphocytes (TILs) at the tumor-host border was investigated. In 43 patients we evaluated the expression of 395 genes related to immune system activation through a next generation sequencing panel. Priority-LASSO models and clustering analyses were integrated with multivariate Cox proportional hazard modeling to identify independent genes associated with relapse and estimate hazard ratios in relation to gene expression and TILs. TILs and the expression of genes related with immune system activation (FCGR1A, IFNA17, FCRLA, NCR3, KREMEN1, CD14, CD3G, CD19, CD20 and CD79A) were significantly associated with prognostic factors or disease specific survival. In patients with lymph node metastases and advanced T stage (pT4), the expression of other genes was altered. Low TILs count was highly associated with relapse within 2 years (p 0.001). Low TILs and altered expression of specific genes associated with tumor-immune systems interactions emerged as independent risk factors, associated to poor prognosis and relapse within 2 years in advanced LSCC. Evaluation of patients' immune profile could be useful for prognosis and future therapeutic approaches towards personalized therapy.

e17592 Background: Homologous recombination deficiency (HRD) is a common feature of epithelial ovarian cancers (EOCs) as approximately half of EOCs have molecular aberrations in homologous recombination repair (HRR) genes, including BRCA1/2 gene mutations. Poly ADP-ribose Polymerase inhibitors (PARPi) have reformed the management of BRCA1/2 mutant ovarian cancer patients and demonstrated activity also in tumor with HRD status. The evaluation of HRD together with BRCA1/2 gene alterations is nowadays critical to guide the therapy decision for all patients with EOC. Different methods to assessed HRD have been reported and different assays are being implemented in clinical practice. Here, we sought to evaluate the analytical validity of a commercial in house HRD assay as compared to a reference standard method. Methods: 59 consecutive case of EOCs were included in this study. DNA extracted from representative formalin-fixed paraffin-embedded (FFPE) tissue block of each case were subjected to next-generation sequencing (NGS) analysis of BRCA1/2 genes alterations and HRD status using the AmoyDx HRD Focus Panel (Amoy Diagnostics Ltd, Xiamen, Fujian, China). HRD status was assesses through AmoyDx Genomic Scar Score (GSS) considering the length, type and location of the genome-wide chromosomal copy number variation through SNP probes spread throughout the genome. Cases with a GSS > 50 were considered as HRD positive. For each case the same FFPE tissue block was sent to Myriad Genetic Laboratories for myChoice CDx testing as reference standard. Assay concordance was measured as positive (PPA), negative (NPA) and overall (OPA) percentage of agreement according to the different scores proposed by the manufacturers. Results: HRD status data were available for 52 (88.1%) and 56 (94.9%) samples considering in-house and reference standard assay, respectively, and 49 cases was available for concordance analyses. AmoyDx HRD Focus Panel identified 23/49 samples (46.9%) as HRD positive and 26 (53.1%) as HRD negative. The concordance of HRD status between AmoyDx HRD Focus Panel and myChoice CDx was 100% and 81,2% for positive (PPA) and negative (NPA) cases respectively, with an overall analytical concordance (OPA) of 87.8%. No discordance was seen in BRCA1/2 status between in-house and reference standard assay (OPA: 100%). Conclusions: The HRD status assessed by AmoyDx HRD Focus Panel is highly concordant with that obtained using the current standard reference method. Commercial in-house test may offer a cost-effective, reliable and robust alternative to the centralized assay for the evaluation of HRD status in patient with EOC.

Background The breast cancer genome dynamically evolves during malignant progression and recurrence. We investigated the genomic profiles of primary early-stage breast cancers and matched relapses to elucidate the molecular underpinnings of the metastatic process, focusing on potentially actionable alterations in the recurrences. Methods A mono-institutional cohort of 128 patients with breast cancers (n = 68 luminal B HER2, n = 6 luminal B HER2+, n = 1 HER2+ non-luminal, n = 56 triple negative) and at least one recurrence in a timeframe of 17 years was evaluated. Next-generation sequencing comprehensive genomic profiling was performed on 289 formalin-fixed paraffin-embedded (FFPE) samples, including primary tumors and matched relapses. Correlations of genomic aberrations with clinicopathologic factors and time to breast cancer relapse were analyzed. Results Genomic data were available for 188 of 289 FFPE samples that achieved the sequencing quality parameters (failure rate 34.9%), including 106 primary tumors and 82 relapses. All primary and relapse samples harbored at least one genomic alteration, with a median number of six alterations per sample (range 1–16). The most frequent somatic genomic alterations were mutations of TP53 (primary tumors = 49%, relapses = 49%) and PIK3CA (primary tumors = 33%, relapses = 30%). Distinctive genomic alterations of primary tumors were significantly associated with molecular subtypes. TP53, PIK3R1, and NF1 somatic alterations were more frequently detected in triple negative tumors (p value CCND1, FGF3, and FGFR1 copy number gains were recurrently identified in luminal cases (p value TP53 mutations and MYC amplification were significantly and independently associated with a shorter time to relapse (p value Conclusions Specific genomic aberrations of primary breast cancers were associated with time to relapse. Primary tumors and matched recurrences showed a core of shared driver genomic aberrations but private actionable alterations have been identified in the relapses.

BackgroundWith the approval of the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib for newly diagnosed, breast cancer gene (BRCA)1/2 mutated, ovarian cancer women, the assessment of BRCA1/2 tumour status will be shortly required at the time of diagnosis.AimTo investigate the feasibility of next-generation sequencing (NGS)-based BRCA tumour test on cytological specimens from ovarian cancer ascites.MethodsWe evaluated the BRCA1/2 status on neoplastic ascites and corresponding tumour tissue of 11 patients with ovarian cancer, using the NGS ‘Oncomine BRCA Research Assay’.ResultsThe NGS-based BRCA test on cytological samples had a success rate of 100%, with 11 of 11 concordant BRCA1/2 results between ascites and tumour tissues analyses, including two wild type samples and nine cases harbouring somatic or germline variants.ConclusionBRCA test may be performed on ovarian cancer ascites, reproducing BRCA1/2 tumour status and representing a useful tool for clinical decision-making.

BACKGROUND Molecular testing of cytological lung cancer specimens includes, beyond epidermal growth factor receptor (EGFR), emerging predictive/prognostic genomic biomarkers such as Kirsten rat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral [v-ras] oncogene homolog (NRAS), B-Raf proto-oncogene, serine/threonine kinase (BRAF), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA). Next-generation sequencing (NGS) and other multigene mutational assays are suitable for cytological specimens, including smears. However, the current literature reflects single-institution studies rather than multicenter experiences. METHODS Quantitative cytological molecular reference slides were produced with cell lines designed to harbor concurrent mutations in the EGFR, KRAS, NRAS, BRAF, and PIK3CA genes at various allelic ratios, including low allele frequencies (AFs; 1%). This interlaboratory ring trial study included 14 institutions across the world that performed multigene mutational assays, from tissue extraction to data analysis, on these reference slides, with each laboratory using its own mutation analysis platform and methodology. RESULTS All laboratories using NGS (n = 11) successfully detected the study's set of mutations with minimal variations in the means and standard errors of variant fractions at dilution points of 10% (P = .171) and 5% (P = .063) despite the use of different sequencing platforms (Illumina, Ion Torrent/Proton, and Roche). However, when mutations at a low AF of 1% were analyzed, the concordance of the NGS results was low, and this reflected the use of different thresholds for variant calling among the institutions. In contrast, laboratories using matrix-assisted laser desorption/ionization–time of flight (n = 2) showed lower concordance in terms of mutation detection and mutant AF quantification. CONCLUSIONS Quantitative molecular reference slides are a useful tool for monitoring the performance of different multigene mutational assays, and this could lead to better standardization of molecular cytopathology procedures. Cancer Cytopathol 2017;125:615-26. © 2017 American Cancer Society.

Activating mutations of the RET gene have been described for both papillary (chromosomal rearrangement) and medullary (missense mutations) thyroid carcinomas. Here, we describe a case of a Warthin-like variant of papillary thyroid carcinoma displaying some morphological aspects that mimic the diffuse sclerosing variant. The tumour harboured BRAF V600E mutation and a novel germline point mutation in the RET gene, with unknown clinical and pathological meaning.

The PARP inhibitor olaparib has been approved in the maintenance setting of platinum-sensitive epithelial ovarian cancer patients with germline or somatic BRCA1/2 mutation. Therefore, the availability of a tumor BRCA test has become a clinical need. We report the results of the clinical implementation of a tumor BRCA test within the frame of an institutional workflow for the management of patients with nonmucinous and nonborderline epithelial ovarian cancer. In total, 223 patients with epithelial ovarian cancer were prospectively analyzed. BRCA1/2 status was evaluated on formalin-fixed, paraffin-embedded tumor specimens using next-generation sequencing technology. The tumor BRCA test had a success rate of 99.1% (221 of 223 successfully analyzed cases) and a median turnaround time of 17 calendar days. Among the 221 cases, BRCA1 or BRCA2 pathogenic/likely pathogenic mutations were found in 62 (28.1%) cases and variants of uncertain significance in 25 (11.3%) cases. The concordance rate between tumor BRCA test results and germline BRCA1/2 status was 87%, with five cases harboring pathogenic/likely pathogenic somatic-only mutations. The next-generation, sequencing-based tumor BRCA test showed a high success rate and a turnaround time compatible with clinical purposes. The tumor BRCA test could be implemented in a molecular diagnostic setting and it may guide the clinical management of patients with epithelial ovarian cancer.

Background: Artificial genomic reference standards in a cytocentrifuge/cytospin format with well-annotated genomic data are useful for validating next-generation sequencing (NGS) on routine cytopreparations. Here, reference standards were optimized to be stained by different laboratories before DNA extraction and to contain a lower number of cells (2 × 10 5 ). This was done to better reflect the clinical challenge of working with insufficient cytological material. Methods: A total of 17 worldwide laboratories analyzed customized reference standard slides (slides A-D). Each laboratory applied its standard workflow. The sample slides were engineered to harbor epidermal growth factor receptor (EGFR) c.2235_2249del15 p.E746_A750delELREA, EGFR c.2369C>T p.T790M, Kirsten rat sarcoma viral oncogene homolog (KRAS) c.38G>A p.G13D, and B-Raf proto-oncogene, serine/threonine kinase (BRAF) c.1798_1799GT>AA p.V600K mutations at various allele frequencies (AFs). Results: EGFR and KRAS mutation detection showed excellent interlaboratory reproducibility, especially on slides A and B (10% and 5% AFs). On slide C (1% AF), either the EGFR mutation or the KRAS mutation was undetected by 10 of the 17 laboratories (58.82%). A reassessment of the raw data in a second-look analysis highlighted the mutations (n=10) that had been missed in the first-look analysis. BRAF c.1798_1799GT>AA p.V600K showed a lower concordance rate for mutation detection and AF quantification. Conclusions: The data show that the detection of low-abundance mutations is still clinically challenging and may require a visual inspection of sequencing reads to detect. Genomic reference standards in a cytocentrifuge/cytospin format are a valid tool for regular quality assessment of laboratories performing molecular studies on cytology with low-AF mutations.

Arachidonic acid (AA) stimulates endothelial cell (EC) proliferation through an increase in intracellular Ca2 + concentration ([Ca2 +]i), that, in turn, promotes nitric oxide (NO) release. AA-evoked Ca2 + signals are mainly mediated by Transient Receptor Potential Vanilloid 4 (TRPV4) channels. Circulating endothelial colony forming cells (ECFCs) represent the only established precursors of ECs. In the present study, we, therefore, sought to elucidate whether AA promotes human ECFC (hECFC) proliferation through an increase in [Ca2 +]i and the following activation of the endothelial NO synthase (eNOS). AA induced a dose-dependent [Ca2 +]i raise that was mimicked by its non-metabolizable analogue eicosatetraynoic acid. AA-evoked Ca2 + signals required both intracellular Ca2 + release and external Ca2 + inflow. AA-induced Ca2 + release was mediated by inositol-1,4,5-trisphosphate receptors from the endoplasmic reticulum and by two pore channel 1 from the acidic stores of the endolysosomal system. AA-evoked Ca2 + entry was, in turn, mediated by TRPV4, while it did not involve store-operated Ca2 + entry. Moreover, AA caused an increase in NO levels which was blocked by preventing the concomitant increase in [Ca2 +]i and by inhibiting eNOS activity with NG-nitro- l -arginine methyl ester ( l -NAME). Finally, AA per se did not stimulate hECFC growth, but potentiated growth factors-induced hECFC proliferation in a Ca2 +- and NO-dependent manner. Therefore, AA-evoked Ca2 + signals emerge as an additional target to prevent cancer vascularisation, which may be sustained by ECFC recruitment.

Genetic alterations of leucine-rich repeat kinase 2 (LRRK2), one of the most important contributors to familial Parkinson&rsquo, s disease (PD), have been hypothesized to play a role in cancer development due to demographical and preclinical data. Here, we sought to define the prevalence and prognostic significance of LRRK2 somatic mutations across all types of human malignancies by querying the publicly available online genomic database cBioPortal. Ninety-six different studies with 14,041 cases were included in the analysis, and 761/14,041 (5.4%) showed genetic alterations in LRRK2. Among these, 585 (76.9%) were point mutations, indels or fusions, 168 (22.1%) were copy number variations (CNVs), and 8 (1.0%) showed both types of alterations. One case showed the somatic mutation R1441C. A significant difference in terms of overall survival (OS) was noted between cases harboring somatic LRRK2 whole deletions, amplifications, and CNV-unaltered cases (median OS: 20.09, 57.40, and 106.57 months, respectively, p = 0.0008). These results suggest that both LRRK2 amplifications and whole gene deletions could play a role in cancer development, paving the way for future research in terms of potential treatment with LRRK2 small molecule inhibitors for LRRK2-amplified cases.

AimsAny reference laboratory testing non-small cell lung cancer samples for predictive biomarkers needs to develop and validate a wide range of different molecular techniques, each with a specific time requirement and application. Updated international guidelines suggest that next generation sequencing (NGS) to be the initial procedure. However, in a non-negligible subset of cases, library generation may fail or amplicon coverage may be insufficient. In these NGS ‘invalid’ cases, the Idylla system may represent a viable option for rapid epidermal growth factor receptor (EGFR) genotyping.MethodsThis retrospective study included 68 archival DNA samples previously processed by Ion Torrent NGS assay. Out of these, 43 cases, including 24 EGFR mutant samples, had a valid NGS result, whereas 25/68 (37%) were invalid. All samples were retested by directly pipetting the DNA inside the EGFR Idylla assay cartridge.ResultsIn all 43 cases with a valid NGS result, Idylla confirmed the EGFR mutational status. In particular, 24/24 (100%) of EGFR mutant samples as detected by NGS were confirmed by Idylla. Moreover, a large portion of cases (20/25; 80%) whose assessment by NGS was invalid were adequately processed by Idylla. Noteworthy, in 4/25 (16%) of cases, Idylla detected actionable EGFR mutations.ConclusionsIdylla assay could be very useful to quickly process cases for which NGS does not allow genotyping.

BackgroundMolecular profiling of advanced non-small cell lung cancers (NSCLC) is essential to identify patients who may benefit from targeted treatments. In the last years, the number of potentially actionable molecular alterations has rapidly increased. Next-generation sequencing allows for the analysis of multiple genes simultaneously.AimsTo evaluate the feasibility and the throughput of next-generation sequencing in clinical molecular diagnostics of advanced NSCLC.MethodsA single-institution cohort of 535 non-squamous NSCLC was profiled using a next-generation sequencing panel targeting 22 actionable and cancer-related genes.Results441 non-squamous NSCLC (82.4%) harboured at least one gene alteration, including 340 cases (63.6%) with clinically relevant molecular aberrations. Mutations have been detected in all but one gene (FGFR1) of the panel. Recurrent alterations were observed in KRAS, TP53, EGFR, STK11 and MET genes, whereas the remaining genes were mutated in KRAS or EGFR in association with TP53 alterations.ConclusionsThe study highlights the feasibility of targeted next-generation sequencing in clinical setting. The majority of NSCLC harboured mutations in clinically relevant genes, thus identifying patients who might benefit from different targeted therapies.

Endothelial progenitor cells (EPCs) are mobilized into circulation to replace damaged endothelial cells and recapitulate the vascular network of injured tissues. Intracellular Ca(2+) signals are key to EPC activation, but it is yet to be elucidated whether they are endowed with the same blend of Ca(2+) -permeable channels expressed by mature endothelial cells. For instance, endothelial colony forming cells (ECFCs), the only EPC subset truly committed to acquire a mature endothelial phenotype, lack canonical transient receptor potential channels 3, 5 and 6 (TRPC3, 5 and 6), which are widely distributed in vascular endothelium; on the other hand, they express a functional store-operated Ca(2+) entry (SOCE). The present study was undertaken to assess whether human circulating EPCs possess TRP vanilloid channel 4 (TRPV4), which plays a master signalling role in mature endothelium, by controlling both vascular remodelling and arterial pressure. We found that EPCs express both TRPV4 mRNA and protein. Moreover, both GSK1016790A (GSK) and phorbol myristate acetate and, two widely employed TRPV4 agonists, induced intracellular Ca(2+) signals uniquely in presence of extracellular Ca(2+). GSK- and PMA-induced Ca(2+) elevations were inhibited by RN-1734 and ruthenium red, which selectively target TRPV4 in mature endothelium. However, TRPV4 stimulation with GSK did not cause EPC proliferation, while the pharmacological blockade of TRPV4 only modestly affected EPC growth in the presence of a growth factor-enriched culture medium. Conversely, SOCE inhibition with BTP-2, La(3+) and Gd(3+) dramatically decreased cell proliferation. These data indicate that human circulating EPCs possess a functional TRPV4 protein before their engraftment into nascent vessels.

5572 Background: The PARPi Olaparib has been approved in maintenance setting of recurrent platinum-sensitive and BRCA mutated, ovarian cancer (OC) patients. However, according to the striking results of SOLO 1 trial, it could be shortly proposed even to newly diagnosed BRCA mutated OC women. We report the results of the implementation of tumor BRCA test in diagnostic setting within the frame of institutional workflow for the management of OC patients. Methods: 223 women with OC were consecutively referred over 25 months to tumor BRCA test. The test was requested by gynecologic oncologist at the diagnosis of non-mucinous and non-borderline OC, upon discussion on the implications of the test result and written consent collected for each patient. Formalin-fixed paraffin embedded (FFPE) specimens of OC were tested using the automatized “Oncomine BRCA Research Assay” Next Generation Sequencing (NGS) panel. The tumor BRCA test was also performed on 5 archetypal cytological samples from ascites. Results: All the cases were considered adequate for the NGS analysis according to the tumor cell content (more than 10%) and the DNA yield extracted (more than 10 ng). The tumor BRCA test had a successful rate of 99.1% . The median Turn-Around Time (TAT) was 17 calendar days, from 33 days of the first trimester to 14 days of the last trimester of this analysis. Overall BRCA1 or BRCA2 pathogenic (P)/likely pathogenic (LP) mutations were found in 62 (28.1%) cases and variants of uncertain significance (VUS) in 25 (11.3%) cases, including 3 cases with a BRCA1 P variant and a concurrent BRCA2 VUS alteration. In detail, 47 P/LP variants and 16 VUS were identified in BRCA1 whereas 15 P/LP mutations and 9 VUS occurred in BRCA2. Complete concordance in tumor BRCA test results were seen between ascites cytological samples and matched tumors. Conclusions: The tumor BRCA test could be implemented in routine diagnostic setting, at diagnosis of non-mucinous and non-borderline OC. The test could be performed on FFPE specimens, had an high successful rate and a TAT compatible with clinical needs. The promising data on cytological samples will be confirmed in larger series.

The identification of EGFR mutations and their respectively tyrosine kinase inhibitors (TKIs), changed dramatically treatment and survival of patients with EGFR-positive lung cancer. Nowadays, different EGFR TKIs as afatinib, erlotinib and gefitinib are approved worldwide for the treatment of NSCLC harbouring EGFR mutations, in particular exon 19 deletions or exon 21 (Leu858Arg) substitution EGFR mutations. In first-line setting, when comparing with platinum-based chemotherapy, these target drugs improves progression-free survival, response rate and quality of life. Unfortunately, the development of different mechanism of resistance, limits the long term efficacy of these agents. The most clear mechanism of resistance is the development of EGFR Thr790Met mutation. Against this new target, different third-generation EGFR-mutant-selective TKIs, such as osimertinib, rociletinib and olmutinib, showed a great activity. In this review, we summarize the scientific evidences about biology, evaluation and treatment on NSCLC with EGFR T790M mutation.

Arachidonic acid (AA) stimulates endothelial cell (EC) proliferation through an increase in intracellular Ca

Endothelial progenitor cells (EPCs) are mobilized into circulation to replace damaged endothelial cells and recapitulate the vascular network of injured tissues. Intracellular Ca(2+) signals are key to EPC activation, but it is yet to be elucidated whether they are endowed with the same blend of Ca(2+) -permeable channels expressed by mature endothelial cells. For instance, endothelial colony forming cells (ECFCs), the only EPC subset truly committed to acquire a mature endothelial phenotype, lack canonical transient receptor potential channels 3, 5 and 6 (TRPC3, 5 and 6), which are widely distributed in vascular endothelium; on the other hand, they express a functional store-operated Ca(2+) entry (SOCE). The present study was undertaken to assess whether human circulating EPCs possess TRP vanilloid channel 4 (TRPV4), which plays a master signalling role in mature endothelium, by controlling both vascular remodelling and arterial pressure. We found that EPCs express both TRPV4 mRNA and protein. Moreover, both GSK1016790A (GSK) and phorbol myristate acetate and, two widely employed TRPV4 agonists, induced intracellular Ca(2+) signals uniquely in presence of extracellular Ca(2+). GSK- and PMA-induced Ca(2+) elevations were inhibited by RN-1734 and ruthenium red, which selectively target TRPV4 in mature endothelium. However, TRPV4 stimulation with GSK did not cause EPC proliferation, while the pharmacological blockade of TRPV4 only modestly affected EPC growth in the presence of a growth factor-enriched culture medium. Conversely, SOCE inhibition with BTP-2, La(3+) and Gd(3+) dramatically decreased cell proliferation. These data indicate that human circulating EPCs possess a functional TRPV4 protein before their engraftment into nascent vessels.

BackgroundAn increase in the frequency of circulating endothelial colony forming cells (ECFCs), the only subset of endothelial progenitor cells (EPCs) truly belonging to the endothelial phenotype, occurs in patients affected by primary myelofibrosis (PMF). Herein, they might contribute to the enhanced neovascularisation of fibrotic bone marrow and spleen. Store-operated Ca2+ entry (SOCE) activated by the depletion of the inositol-1,4,5-trisphosphate (InsP3)-sensitive Ca2+ store drives proliferation in ECFCs isolated from both healthy donors (N-ECFCs) and subjects suffering from renal cellular carcinoma (RCC-ECFCs). SOCE is up-regulated in RCC-ECFCs due to the over-expression of its underlying molecular components, namely Stim1, Orai1, and TRPC1.Methodology/principal findingsWe utilized Ca2+ imaging, real-time polymerase chain reaction, western blot analysis and functional assays to evaluate molecular structure and the functional role of SOCE in ECFCs derived from PMF patients (PMF-ECFCs). SOCE, induced by either pharmacological (i.e. cyclopiazonic acid or CPA) or physiological (i.e. ATP) stimulation, was significantly higher in PMF-ECFCs. ATP-induced SOCE was inhibited upon blockade of the phospholipase C/InsP3 signalling pathway with U73111 and 2-APB. The higher amplitude of SOCE was associated to the over-expression of the transcripts encoding for Stim2, Orai2-3, and TRPC1. Conversely, immunoblotting revealed that Stim2 levels remained constant as compared to N-ECFCs, while Stim1, Orai1, Orai3, TRPC1 and TRPC4 proteins were over-expressed in PMF-ECFCs. ATP-induced SOCE was inhibited by BTP-2 and low micromolar La3+ and Gd3+, while CPA-elicited SOCE was insensitive to Gd3+. Finally, BTP-2 and La3+ weakly blocked PMF-ECFC proliferation, while Gd3+ was ineffective.ConclusionsTwo distinct signalling pathways mediate SOCE in PMF-ECFCs; one is activated by passive store depletion and is Gd3+-resistant, while the other one is regulated by the InsP3-sensitive Ca2+ pool and is inhibited by Gd3+. Unlike N- and RCC-ECFCs, the InsP3-dependent SOCE does not drive PMF-ECFC proliferation.