KRAS mutations are detected in over one third of lung adenocarcinomas, most frequently in Caucasian and smoker patients. The impact of KRAS mutations on lung adenocarcinoma prognosis is currently subject to debate, as is their impact on the response to chemotherapy and EGFR tyrosine kinase inhibitors. The different methods for KRAS status assessment, based on histological and cytological samples or biological fluids, offer varying sensitivities. Since no treatments are available in clinical routine for KRAS-mutated lung cancer patients, one of the current major challenges in thoracic oncology is developing new dedicated strategic therapies. Different molecules can be developed that act on a post-transcriptional KRAS protein level, blocking its cytoplasmic membrane recruitment. The efficacy of these molecules' targeting of the different signaling pathways activated by the KRAS mutation (such as the MEK and BRAF pathways) is related to the particular KRAS mutation subtype. New therapeutic strategies are currently focused on certain genes linked with KRAS inducing a synthetic lethal interaction. The purpose of this work is to provide an overview of i) the recent epidemiological and molecular findings concerning KRASmutated lung adenocarcinoma, ii) the prognostic impact of KRAS mutations, in particular during response to treatment, iii) the available methods for detecting this mutation, and iv) the current molecules under development for new therapeutic strategies and the clinical trials targeting this genomic alteration.

Circulating tumors cells (CTCs) can be detected in the blood of metastatic melanoma patients (MMPs) both as isolated circulating tumor cells (iCTCs) and circulating tumor microemboli (CTMs), but their clinical significance remains unknown. The aim of this work was to evaluate the prognostic impact in metastatic cutaneous melanoma of CTMs and iCTCs identified by a cytomorphological approach using the isolation by size of tumor cell (ISET) method. We characterized the phenotype of CTCs using anti‐PS100, anti‐SOX10, anti‐CD10, and anti‐TRF2 antibodies. 128 MMPs and 37 control healthy individuals with benign nevi were included in this study. Results were compared to the follow‐up of patients. 109/128 (85%) MMPs showed CTCs, 44/128 (34%) with 2 to 6 CTMs and 65/128 (51%) with 4 to 9 iCTCs. PS100 expression was homogeneous in iCTCs and heterogeneous in CTMs. SOX10, CD10, and TRF2 were mainly expressed in CTMs. None of the control subjects demonstrated circulating malignant tumor cells. Overall survival was significantly decreased in patients with CTMs, independently of the therapeutic strategies. In conclusion, the presence of CTMs is an independent predictor of shorter survival from the time of diagnosis of MMPs.

In this study, we performed microRNA (miRNA) expression profiling on a large series of sporadic and hereditary forms of medullary thyroid carcinomas (MTC). More than 60 miRNAs were significantly deregulated in tumor vs adjacent non-tumor tissues, partially overlapping with results of previous studies. We focused our attention on the strongest up-regulated miRNA in MTC samples, miR-375, the deregulation of which has been previously observed in a variety of human malignancies including MTC. We identified miR-375 targets by combining gene expression signatures from human MTC (TT) and normal follicular (Nthy-ori 3-1) cell lines transfected with an antagomiR-375 inhibitor or a miR-375 mimic, respectively, and from an in silico analysis of thyroid cell lines of Cancer Cell Line Encyclopedia datasets. This approach identified SEC23A as a bona fide miR-375 target, which we validated by immunoblotting and immunohistochemistry of non-tumor and pathological thyroid tissue. Furthermore, we observed that miR-375 overexpression was associated with decreased cell proliferation and synergistically increased sensitivity to vandetanib, the clinically relevant treatment of metastatic MTC. We found that miR-375 increased PARP cleavage and decreased AKT phosphorylation, affecting both cell proliferation and viability. We confirmed these results through SEC23A direct silencing in combination with vandetanib, highlighting the importance of SEC23A in the miR-375-associated increased sensitivity to vandetanib.Since the combination of increased expression of miR-375 and decreased expression of SEC23A point to sensitivity to vandetanib, we question if the expression levels of miR-375 and SEC23A should be evaluated as an indicator of eligibility for treatment of MTC patients with vandetanib.

PD-1/PD-L1 inhibitors demonstrated durable clinical responses in patients with lung squamous cell carcinoma. However, the expression pattern of PD-L1 and the presence of CD8 and PD-1 tumor-infiltrating T cells in the basaloid variant of squamous cell carcinoma remain unknown. immunohistochemistry analysis of PD-L1 expression, with three recently validated monoclonal antibodies used in clinical trials (clones SP142, SP263, and 28-8), and detection of CD8 and PD-1 tumor-infiltrating T cells was performed on whole-tissue sections from 56 patients following surgery for basaloid squamous cell carcinoma. Data were correlated to clinicopathological parameters and outcome. Fair to poor concordance was observed between the SP142 vs SP263 clones, and SP142 vs 28-8 (κ range, 0.018-0.412), while the 28-8 and SP263 demonstrated a strong correlation in both the tumor cell and immune cell compartments (κ=0.883, and κ=0.721). Expression of PD-L1 correlated with a high content of CD8 and PD-1 tumor-infiltrating T cells when using SP142 (P=0.012; P=0.022), but not with SP263 or 28-8 (P=0.314; P=0.611). In the multivariate analysis, we found significantly better disease-free and overall survival rates for high PD-L1 expression with SP142, CD8 and PD-1 tumor-infiltrating T cells (P=0.003; P=0.007). No significant prognosis value was observed for SP263 and 28-8 clones, except a correlation between improved overall survival and SP263 in the univariate analysis (P=0.039), not confirmed in the multivariate model. In conclusion, we report that the expression of PD-L1 and the content of CD8 and PD-1 tumor-infiltrating T cells is an independent indicator of better outcome in basaloid squamous cell carcinoma patients, although the observed effect is dependent on the PD-L1 immunohistochemistry assay.

This study showed that VE1 IHC should be a substitute for molecular biology in the initial assessment of the BRAFV600E status in MPP. This methodology needs to be set up in pathology laboratories in accordance with quality control/quality assurance accreditation procedures. Under these strict conditions the question is to know if BRAFV600E-IHC can serve not only as a prescreening tool, but also as a stand-alone test (at least in cases displaying an unequivocally staining pattern) as well as an alternative predictive test for samples for which the molecular biology failed.

Background: Assessment of actionable EGFR mutations is mandatory for treatment-naïve advanced or metastatic non-squamous lung carcinoma (NSLC), but the results need to be obtained in less than 10 working days. For rapid EGFR testing, an EGFR-specific polymerase chain reaction (PCR) assay is an alternative and simple approach compared to next generation sequencing (NGS). Here, we describe how a rapid EGFR-specific PCR assay can be implemented in a single laboratory center (LPCE, Nice, France) as reflex testing in treatment-naïve NSLC. Methods: A total of 901 biopsies from NSLC with more than 10% of tumor cells were prospectively and consecutively evaluated for EGFR mutation status between November 2017 and December 2019 using the Idylla system (Biocartis NV, Mechelen, Belgium). NGS was performed for nonsmokers with NSLC wild type for EGFR, ALK, ROS1, and BRAF and with less than 50% PD-L1 positive cells using the Hotspot panel (Thermo Fisher Scientific, Waltham, MA, USA). Results: Results were obtained from 889/901 (97%) biopsies with detection of EGFR mutations in 114/889 (13%) cases using the Idylla system. Among the 562 EGFR wild type tumors identified with Idylla, NGS detected one actionable and one nonactionable EGFR mutation. Conclusions: Rapid and targeted assessment of EGFR mutations in treatment-naïve NSLC can be implemented in routine clinical practice. However, it is mandatory to integrate this approach into a molecular algorithm that allows evaluation of potentially actionable genomic alterations other than EGFR mutations.

International guidelines recommend BRAF mutational status assessment in treatment-naive advanced non-squamous non-small cell lung carcinoma (NSCLC) patients since the presence of a BRAFV600 mutation enables specific BRAF inhibitor treatment. For this purpose, the mutational status needs to be obtained in 10 working days. Herein, we prospectively evaluated the feasibility of systematic assessment of the BRAF status using immunohistochemistry (IHC) in a single institution (LPCE, Nice) at baseline for NSCLC diagnosed. Methods: 1317 NSCLC were evaluated using BRAF IHC from 2011 to 2019. Initially the BRAF status was prospectively assessed using NGS and/or pyrosequencing in 618 consecutively diagnosed NSCLC patients from 2012 to 2016; BRAFV600E and BRAF nonV600E mutated tumors detected in this cohort were retrospectively evaluated using BRAF IHC. Secondarily, 699 biopsies of NSCLC were prospectively analyzed between 2017 and 2019 using BRAF IHC. BRAF IHC positive tumors were tested using a rapid BRAF specific PCR based assay. Results: Initially, 21/618 (3%) of tumors (15 early and 6 late stage tumors) were BRAFV600E mutated according to the results of NGS and/or pyrosequencing. BRAF IHC was positive in 21/21 of these cases and negative in 51/ 51 (100 %) BRAF non V600E mutated cases. In the prospective BRAF IHC tested cohort of patients, 24/699 (3%) tumors (13 early and 11 late stage tumors) were positive with VE1 IHC. The BRAF PCR assay was positive in 20/ 24 (83 %) of these cases. Conclusion: BRAFV600E IHC screening of treatment-naïve NSCLC patients is a rapid, specific and very sensitive method which can lead in advanced stage positive NSCLC tumors to a BRAF inhibitor treatment. This test can be routinely integrated into mandatory predictive biomarker 'testing of NSCLC. According to the organization of patient care and the physician's request, this practice can be proposed as an alternative to NGS-based tissue biopsy made at baseline.

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