An increasing number of driver genomic alterations with potential targeted treatments have been identified in non-small cell lung cancer (NSCLC). Much less is known about the incidence and different distribution of concurrent alterations, as identified by comprehensive genomic profiling in oncogene-addicted NSCLCs. Genomic data from advanced NSCLC consecutively analyzed using a broad next-generation sequencing panel were retrospectively collected. Tumors harboring at least one main actionable gene alteration were categorized according to the presence/absence of concurrent genomic aberrations, to evaluate different patterns among the main oncogene-addicted NSCLCs. Three-hundred-nine actionable gene alterations were identified in 284 advanced NSCLC patients during the study period. Twenty-five tumor samples (8%) displayed concurrent alterations in actionable genes. Co-occurrences involving any pathogenic variant or copy number variation (CNV) were identified in 82.8% of cases. Overall, statistically significant differences in the number of concurrent alterations, and the distribution of TP53, STK11, cyclines and receptor tyrosin kinase (RTK) aberrations were observed across the eight actionable gene groups. NGS analyses of oncogene-addicted NSCLCs showed a different distribution and pattern of co-alteration profiles. Further investigations are needed to evaluate the prognostic and treatment-related impact of these concurrent alterations, hooked to the main gene aberrations.

Somatic mutations in PIK3CA are present in ~40% breast cancers (BC); their detection in hormone receptor (HR)+/HER2− tumors allows for selecting patients with advanced disease eligible for PIK3CA targeting with alpelisib. The choice of what type of PIK3CA testing approach to adopt and which tissue sample to analyze is a new task in breast pathology. In this methodological study, we sought to assess the performance of next-generation sequencing (NGS) and RT-PCR for PIK3CA testing on archival formalin-fixed paraffin-embedded (FFPE) primary tumors and corresponding metastases. Sixteen HR+/HER2− BC with known PIK3CA-mutated status (ex. 7, 9, and 20) on metastatic samples by means of amplicon-based targeted NGS were selected, and n = 13 of these samples were re-tested with a commercially available CE-IVD RT-PCR assay. All available primary tumors (n = 8) were tested with both methods. NGS detected mutations in all samples, while RT-PCR in n = 2 sample-pairs and overall, in n = 5/8 (62.5%) primary tumors and 7/13 (53.8%) metastases (κ = 0.09; 95% CI, −0.69–0.87). Slight agreement (κ = 0; 95% CI, −0.59–0.59) was observed between NGS and RT-PCR, with the former being generally more sensitive in cases with low DNA quality and quantity. Post hoc visual inspection of the RT-PCR data increased the concordance to 76.9%. Targeted NGS offers reliable and robust PIK3CA testing on both tumor and metastasis FFPE samples; the accuracy of RT-PCR depends on the DNA quantity and quality. In HR+/HER2− BC, both the selection of the PIK3CA testing strategy of FFPE tissues and which sample to analyze should consider several technical parameters and should be tailored for each case.

Background: Actionable driver mutations account for 40–50% of NSCLC cases, and their identification clearly affects treatment choices and outcomes. Conversely, non-actionable mutations are genetic alterations that do not currently have established treatment implications. Among co-occurring alterations, the identification of concurrent actionable genomic alterations is a rare event, potentially impacting prognosis and treatment outcomes. Methods: We retrospectively evaluated the prevalence and patterns of concurrent driver genomic alterations in a large series of NSCLCs to investigate their association with clinicopathological characteristics, to assess the prognosis of patients whose tumor harbors concurrent alterations in the genes of interest and to explore their potential therapeutic implications. Results: Co-occurring driver alterations were identified in 26 out of 1520 patients with at least one gene alteration (1.7%). Within these cases, the incidence of concurrent actionable gene alterations was 39% (0.7% of the overall cohort). Among compound actionable gene mutations, EGFR was the most frequently involved gene (70%). The most frequent association was EGFR mutations with ROS1 rearrangement. Front-line targeted treatments were the preferred approach in patients with compound actionable mutations, with dismal median PFS observed (6 months). Conclusions: Advances in genomic profiling technologies are facilitating the identification of concurrent mutations. In patients with concurrent actionable gene alterations, integrated molecular and clinical data should be used to guide treatment decisions, always considering rebiopsy at the moment of disease progression.

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.

(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.

This article describes how the transcriptional alterations of the innate immune system divide dysplasias into aggressive forms that, despite the treatment, relapse quickly and more easily, and others where the progression is slow and more treatable. It elaborates on how the immune system can change the extracellular matrix, favoring neoplastic progression, and how infections can enhance disease progression by increasing epithelial damage due to the loss of surface immunoglobulin and amplifying the inflammatory response. We investigated whether these dysregulated genes were linked to disease progression, delay, or recovery. These transcriptional alterations were observed using the RNA-based next-generation sequencing (NGS) panel Oncomine Immune Response Research Assay (OIRRA) to measure the expression of genes associated with lymphocyte regulation, cytokine signaling, lymphocyte markers, and checkpoint pathways. During the analysis, it became apparent that certain alterations divide dysplasia into two categories: progressive or not. In the future, these biological alterations are the first step to provide new treatment modalities with different classes of drugs currently in use in a systemic or local approach, including classical chemotherapy drugs such as cisplatin and fluorouracile, older drugs like fenretinide, and new checkpoint inhibitor drugs such as nivolumab and pembrolizumab, as well as newer options like T cell therapy (CAR-T). Following these observed alterations, it is possible to differentiate which dysplasias progress or not or relapse quickly. This information could, in the future, be the basis for determining a close follow-up, minimizing surgical interventions, planning a correct and personalized treatment protocol for each patient and, after specific clinical trials, tailoring new drug treatments.