Beyond Traditional Morphological Characterization of Lung Neuroendocrine Neoplasms: In Silico Study of Next-Generation Sequencing Mutations Analysis across the Four World Health Organization Defined Groups

Lung neuroendocrine neoplasms (LNENs) classes, as proposed by the World Health Organization 2015, do not provide properly prognostic and therapeutic indications. In fact, high-throughput molecular analysis, based on next-generation sequencing, identified novel molecular subgroups, associated with different genomic signatures, that could pave the way for alternative therapeutic approaches. The present review, coupled with in silico molecular analysis, could show the current genomic alterations state in actual LNENS groups. Interestingly our manuscript suggests that the molecular novelties could improve the LNENs therapeutics efficacy. In more detail, we reported the differences of gene alterations and mutational rate between LNENS, confirming the central pathogenetic role given by a different mutational rate in chromatin remodeling genes and tumor suppressors TP53-RB1. In conclusion, our results underlined that a further molecular layer is needed to improve the efficacy of LNENs medical treatment.Lung neuroendocrine neoplasms (LNENs) represent a rare and heterogeneous population of lung tumors. LNENs incidence rate has increased dramatically over the past 30 years. The current World Health Organization LNENs classification (WHO 2015), distinguished four LNENs prognostic categories, according to their morphology, necrosis amount and mitotic count: typical carcinoid (TC), atypical-carcinoid (AC), large cell neuroendocrine carcinoma (LCNEC) and small cell lung cancer (SCLC). At present, due to their rarity and biological heterogeneity there is still no consensus on the best therapeutic approach. Next-generation-sequencing analysis showed that WHO 2015 LNENs classes, could be characterized also by specific molecular alterations: frequently mutated genes involving chromatin remodeling and generally characterized by low mutational burden (MB) are frequently detected in both TC and AC; otherwise, TP53 and RB1 tumor suppressor genes alterations and high MB are usually detected in LCNEC and SCLC. We provide an overview concerning gene mutations in each WHO 2015 LNENs class in order to report the current LNENs mutational status as potential tool to better understand their clinical outcome and to drive medical treatment

Improved Prognostic Prediction in Never-Smoker Lung Cancer Patients by Integration of a Systemic Inflammation Marker with Tumor Immune Contexture Analysis

Almost 25% of lung cancers (LCs) occur in never-smokers. LC inflammatory profile, based on plasma C-reactive protein levels (CRP), predicts mortality, independently by smoking-status. We hypothesized that: CRP could be associated with tumor immune contexture (TIC) in never-smokers and both these two parameters may improve their prognosis. Sixty-eight never-smokers LC patients with high or low CRP were selected. The programmed cell death protein 1 (PD-1) and its ligand (PD-L1), the human leukocyte antigens (HLA-DR and HLA-I), CD8, CD4, CD3, CD33, CD163, and CD68 were evaluated by immunohistochemistry on surgical samples given TIC evaluation. The classification model based on TIC scores was generated by Classification and Regression Tree analysis. Tumor mutational burden was evaluated by targeted next-generation sequencing. Exclusively high CRP (H-CRP) subset showed PD-L1 expression in 35% of LC as well as lower HLA-I and HLA-DR in their stromal cells. CD3, CD4, CD8, HLA-I, HLA-DR tumor cells staining were associated with a "low inflammatory profile" subset. CRP and LC immune profiles drive clinical outcome: 5-year survival 88% against 8% was associated with low and high-risk profiles (p< 0.0001). Clinical outcome prediction in never-smoker LC patients may be improved by both CRP and tumor immune contexture evaluation

Metformin Enhances Cisplatin-Induced Apoptosis and Prevents Resistance to Cisplatin in Co-mutated KRAS/LKB1 NSCLC

Abstract Introduction We hypothesized that activating KRAS mutations and inactivation of the liver kinase B1 (LKB1) oncosuppressor can cooperate to sustain NSCLC aggressiveness. We also hypothesized that the growth advantage of KRAS/LKB1 co-mutated tumors could be balanced by higher sensitivity to metabolic stress conditions, such as metformin treatment, thus revealing new strategies to target this aggressive NSCLC subtype. Methods We retrospectively determined the frequency and prognostic value of KRAS/LKB1 co-mutations in tissue specimens from NSCLC patients enrolled in the TAILOR trial. We generated stable LKB1 knockdown and LKB1-overexpressing isogenic H1299 and A549 cell variants, respectively, to test the in vitro efficacy of metformin. We also investigated the effect of metformin on cisplatin-resistant CD133+ cells in NSCLC patient-derived xenografts. Results We found a trend towards worse overall survival in patients with KRAS/LKB1 co-mutated tumors as compared to KRAS-mutated ones (hazard ratio: 2.02, 95% confidence interval: 0.94–4.35, p = 0.072). In preclinical experiments, metformin produced pro-apoptotic effects and enhanced cisplatin anticancer activity specifically in KRAS/LKB1 co-mutated patient-derived xenografts. Moreover, metformin prevented the development of acquired tumor resistance to 5 consecutive cycles of cisplatin treatment (75% response rate with metformin-cisplatin as compared to 0% response rate with cisplatin), while reducing CD133+ cells. Conclusions LKB1 mutations, especially when combined with KRAS mutations, may define a specific and more aggressive NSCLC subtype. Metformin synergizes with cisplatin against KRAS/LKB1 co-mutated tumors, and may prevent or delay the onset of resistance to cisplatin by targeting CD133+ cancer stem cells. This study lays the foundations for combining metformin with standard platinum-based chemotherapy in the treatment of KRAS/LKB1 co-mutated NSCLC

Management of BRCA Tumour Testing in an Integrated Molecular Tumour Board Multidisciplinary Model

Tumour testing of the BRCA1/2 genes is routinely performed in patients with different cancer histological subtypes. To accurately identify patients with tumour-detected germline pathogenic variants (PVs) is a relevant issue currently under investigation. This study aims at evaluating the performance of the tumour-to-germline diagnostic flowchart model defined at our Institutional Molecular Tumour Board (MTB). Results from tumour BRCA sequencing of 641 consecutive unselected cancer patients were discussed during weekly MTB meetings with the early involvement of clinical geneticists for appropriate referral to genetic counselling. The overall tumour detection rate of BRCA1/2 PVs was 8.7% (56/641), ranging from 24.4% (31/127) in high-grade ovarian cancer to 3.9% (12/304) in tumours not associated with germline BRCA1/2 PVs. Thirty-seven patients with PVs (66%) were evaluated by a clinical geneticist, and in 24 of them (64.9%), germline testing confirmed the presence of the PV in blood. Nine of these patients (37.5%) were not eligible for germline testing according to the criteria in use at our institution. Cascade testing was subsequently performed on 18 relatives. The tumour-to-germline diagnostic pipeline, developed in the framework of our institutional MTB, compared with guideline-based germline testing following genetic counselling, proved to be effective in identifying a higher number of germline BRCA PVs carriers

LKB1 mutations are not associated with the efficacy of first-line and second-line chemotherapy in patients with advanced non-small-cell lung cancer (NSCLC): a post hoc analysis of the TAILOR trial

Purpose In patients with advanced lung adenocarcinoma, the impact ofLKB1mutations on cytotoxic chemotherapy efficacy remains poorly explored. Here, we aimed at investigating the potential impact ofLKB1mutational status on chemotherapy efficacy in advanced non-small-cell lung cancer (NSCLC) patients enrolled in the TArceva Italian Lung Optimisation tRial (TAILOR) trial. Methods The multicenter TAILOR trial randomised patients withEGFR-wild type (wt) advanced NSCLC progressing on/after previous platinum-based chemotherapy to receive docetaxel or erlotinib. Here, we evaluated the impact ofLKB1mutational status on progression-free survival (PFS) and overall survival (OS) in patients treated with second-line docetaxel/erlotinib or during prior platinum-based chemotherapy. Results Out of 222 patients randomised in the TAILOR trial, left-over tumour tissues were available for 188 patients, and 120 patients with evaluableLKB1status were included. Of them, 17 (14.17%) patients hadLKB1-mutated tumours, while 103 (85.83%) hadLKB1-wt disease. During second-line treatment, PFS and OS were not statistically significantly different in patients withLKB1-mutated when compared withLKB1-wt NSCLC (adjusted HR (aHR)=1.29, 95% CI 0.75 to 2.21; p=0.364 and aHR=1.41, 95% CI 0.82 to 2.44; p=0.218, respectively). Similarly, we found no significant association betweenLKB1mutations and patient PFS or OS during prior first-line platinum-based chemotherapy (aHR=1.04, 95% CI 0.55 to 1.97; p=0.910 and aHR=0.83, 95% CI 0.42 to 1.65; p=0.602, respectively). Conclusion Among advanced NSCLC patients receiving two lines of systemic therapy,LKB1mutations were not associated with PFS or OS during second-line docetaxel or prior first-line platinum-based chemotherapy. While larger prospective trials are needed to confirm our findings, cytotoxic chemotherapy remains the backbone of investigational combination strategies in this patient population

Genomic and transcriptomic analyses of thyroid cancers identify DICER1 somatic mutations in adult follicular-patterned RAS-like tumors

BackgroundPapillary thyroid carcinoma (PTC) is the most common type of thyroid cancer (TC). Several genomic and transcriptomic studies explored the molecular landscape of follicular cell-derived TCs, and BRAFV600E, RAS mutations, and gene fusions are well-established drivers. DICER1 mutations were described in specific sets of TC patients but represent a rare event in adult TC patients.MethodsHere, we report the molecular characterization of 30 retrospective follicular cell-derived thyroid tumors, comprising PTCs (90%) and poorly differentiated TCs (10%), collected at our Institute. We performed DNA whole-exome sequencing using patient-matched control for somatic mutation calling, and targeted RNA-seq for gene fusion detection. Transcriptional profiles established in the same cohort by microarray were investigated using three signaling-related gene signatures derived from The Cancer Genome Atlas (TCGA).ResultsThe occurrence of BRAFV600E (44%), RAS mutations (13%), and gene fusions (13%) was confirmed in our cohort. In addition, in two patients lacking known drivers, mutations of the DICER1 gene (p.D1709N and p.D1810V) were identified. DICER1 mutations occur in two adult patients with follicular-pattern lesions, and in one of them a second concurrent DICER1 mutation (p.R459*) is also observed. Additional putative drivers include ROS1 gene (p.P2130A mutation), identified in a patient with a rare solid-trabecular subtype of PTC. Transcriptomics indicates that DICER1 tumors are RAS-like, whereas the ROS1-mutated tumor displays a borderline RAS-/BRAF-like subtype. We also provide an overview of DICER1 and ROS1 mutations in thyroid lesions by investigating the COSMIC database.ConclusionEven though small, our series recapitulates the genetic background of PTC. Furthermore, we identified DICER1 mutations, one of which is previously unreported in thyroid lesions. For these less common alterations and for patients with unknown drivers, we provide signaling information applying TCGA-derived classification

Circulating tumor DNA to anticipate loco-regional recurrence in early-stage breast cancer: a proof-of-concept study

BackgroundLoco-regional recurrence (LRR) poses a clinical challenge for the follow-up of patients treated with curative intent for early-stage breast cancer (EBC). While circulating tumor DNA (ctDNA) has been shown to predict distant metastases, its value for LRR is less characterized.MethodsStarting from an index case with documented LRR and available tumor and plasma samples, we report the analysis of the prospective phase III fenretinide prevention trial, which primarily aimed to assess the incidence of second malignancy in women with T1-T2 N0 EBC. Patients were eligible if they had FFPE and/or frozen tissue from primary or recurrent invasive tumor for next generation sequencing, and at least three serial plasma samples for ctDNA analysis by digital PCR.ResultsThe TP53 R196* mutation was identified in the primary tumor of the index case with a variant allele frequency (VAF) of 29%, and in the LRR with a VAF of 58%. The same mutation was also detected in plasma prior to both the primary and LRR surgeries with VAFs of 0.19% and 0.12%, respectively. Following treatment, the mutation became undetectable in plasma samples during follow-up, consistent with the absence of recurrence. Among 40 eligible patients from the fenretinide prevention trial, 27 (67.5%) had primary tumor somatic variants trackable in plasma. Median age was 55 years (range, 35-78); stage I (16, 59%) and stage II (11, 41%); mostly luminal-like (19, 70%); median follow-up 173 months (range, 98-193); common mutations included PIK3CA (50%), TP53 (30.7%), and PTEN (5.9%). Six patients developed LRR as first event; 4 distant metastases. In all LRR cases, except one, ctDNA was detected prior to surgery and anticipated the clinical diagnosis up to 28 months. Three patients with LRR developed distant metastases 1 to 2 years later.ConclusionThese findings show the potential of ctDNA for the early detection of LRR in EBC, and its promise as a tool for timely interventions and personalized surveillance strategies

Different clinical effects upon separate inhibition of coexisting EGFR and PI3KCA mutations in a lung adenocarcinoma patient

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