Use of non-small cell lung cancer multicellular tumor spheroids to study the impact of chemotherapy.

BACKGROUND: Lung cancers represent the main cause of cancer related-death worldwide. Recently, immunotherapy alone or in combination with chemotherapy has deeply impacted the therapeutic care leading to an improved overall survival. However, relapse will finally occur, with no efficient second line treatment so far. New therapies development based on the comprehension of resistance mechanisms is necessary. However, the difficulties to obtain tumor samples before and after first line treatment hamper to clearly understand the consequence of these molecules on tumor cells and also to identify adapted second line therapies. METHODS: To overcome this difficulty, we developed multicellular tumor spheroids (MCTS) using characterized Non-Small Cell Lung Cancer (NSCLC) cell lines, monocytes from healthy donors and fibroblasts. MCTS were treated with carboplatin-paclitaxel or -gemcitabine combinations according to clinical administration schedules. The treatments impact was studied using cell viability assay, histological analyses, 3'RNA sequencing, real-time PCR, flow cytometry and confocal microscopy. RESULTS: We showed that treatments induced a decrease in cell viability and strong modifications in the transcriptomic profile notably at the level of pathways involved in DNA damage repair and cell cycle. Interestingly, we also observed a modification of genes expression considered as hallmarks of response to immune check point inhibitors and immunogenicity, particularly an increase in CD274 gene expression, coding for PD-L1. This result was validated at the protein level and shown to be restricted to tumor cells on MCTS containing fibroblasts and macrophages. This increase was also observed in an additional cell line, expressing low basal CD274 level. CONCLUSIONS: This study shows that MCTS are interesting models to study the impact of first line therapies using conditions close to clinical practice and also to identify more adapted second line or concomitant therapies for lung cancer treatment.

The use of minimal residual disease in thoracic oncology: Gaps between promises and the on-the-ground reality of daily practice.

The assessment of minimal residual disease (MRD) from blood samples of patients with resected non-small cell lung carcinoma (NSCLC) is promising and opens up many opportunities for the optimisation of patient care in daily practice. Notably, this includes the potential for escalation or de-escalation of adjuvant therapies. Thus, the evaluation of MRD status can directly contribute to an increase in the overall survival of early stage NSCLC patients and/or limit therapeutic but also "financial" toxicity. Therefore, several clinical trials recently evaluated MRD in early stage NSCLC by integrating and retrospectively comparing the results of MRD assessments. In this context, there is an urgent need to close the gap between clinical research and the use of the evaluation of MRD in routine daily practice. Further action needs to be taken, particularly in evaluating the pertinence of the detection of MRD in prospective interventional clinical studies. This may be done in part by comparing different parameters, such as the techniques used, the different time points and the cutoffs of MRD assessments. This article investigates the assessment of MRD in non-small cell lung cancers, with a special focus on the issues associated with the various assays and the limitations of using circulating free DNA analyses for MRD assessment in early stage lung cancer. Recommendations and tips for the optimisation of MRD evaluation in non-small cell lung cancers are provided.

Efficacy of imiquimod in the management of lentigo maligna.

BACKGROUND: Lentigo maligna (LM) is a melanocytic proliferation occurring on photo-exposed skin that may progress to LM melanoma. Surgery is recommended as first-line treatment. Excision margins of 5-10 mm remain, without international consensus. Several studies have shown that imiquimod, an immunomodulator, induces LM regression. This study investigated the effect of imiquimod versus placebo in neoadjuvant settings. PATIENTS AND METHODS: We performed a prospective, randomized, multicentre, phase III clinical study. Patients were randomly assigned in 1:1 ratio to receive imiquimod or placebo for 4 weeks, followed by LM excision 4 weeks after the last application of imiquimod or placebo. The primary endpoint was extra-lesional excision, with a 5 mm margin from the residual pigmentation after imiquimod or vehicle. Secondary endpoints included the gain on the surface removed between the two groups; number of revision surgeries to obtain extra-lesional excisions; relapse-free time; and number of complete remissions after treatment. RESULTS: A total of 283 patients participated in this study; 247 patients, 121 patients in the placebo group and 126 in the imiquimod group, accounted for the modified ITT population. The first extralesional extirpation was performed in 116 (92%) imiquimod patients and in 102 (84%) placebo patients; the difference was not significant (p = 0.0743). Regarding the surface of LM, imiquimod reduced the LM surface (4.6-3.1 cm2 ) significantly (p < 0.001) more compared to the placebo (3.9-4.1 cm2 ). CONCLUSION: Imiquimod reduces the lentigo maligna surface after 1 month of treatment, without a higher risk of intralesional excision and with a positive aesthetic outcome.

Multiple Hotspot Mutations Scanning by Single Droplet Digital PCR.

BACKGROUND: Progress in the liquid biopsy field, combined with the development of droplet digital PCR (ddPCR), has enabled noninvasive monitoring of mutations with high detection accuracy. However, current assays detect a restricted number of mutations per reaction. ddPCR is a recognized method for detecting alterations previously characterized in tumor tissues, but its use as a discovery tool when the mutation is unknown a priori remains limited. METHODS: We established 2 ddPCR assays detecting all genomic alterations within KRAS exon 2 and EGFR exon 19 mutation hotspots, which are of clinical importance in colorectal and lung cancer, with use of a unique pair of TaqMan® oligoprobes. The KRAS assay scanned for the 7 most common mutations in codons 12/13 but also all other mutations found in that region. The EGFR assay screened for all in-frame deletions of exon 19, which are frequent EGFR-activating events. RESULTS: The KRAS and EGFR assays were highly specific and both reached a limit of detection of <0.1% in mutant allele frequency. We further validated their performance on multiple plasma and formalin-fixed and paraffin-embedded tumor samples harboring a panel of different KRAS or EGFR mutations. CONCLUSIONS: This method presents the advantage of detecting a higher number of mutations with single-reaction ddPCRs while consuming a minimum of patient sample. This is particularly useful in the context of liquid biopsy because the amount of circulating tumor DNA is often low. This method should be useful as a discovery tool when the tumor tissue is unavailable or to monitor disease during therapy.

PD-L1 expression by tumor cell lines: A predictive marker in melanoma.

Prognostic biomarkers for patients with melanoma after lymph node resection are of clinical relevance and could thus enable the identification of patients who therefore would most benefit from adjuvant treatment. The aim of this work was to determine, using an in vitro model, whether immune-related biomarkers, such as MHC-class I and II, melanoma-associated antigens, IDO1 and PD-L1, could also be relevant to predict the risk of relapse of patients with stage III melanoma after lymph node resection. We established tumor cell lines from metastatic lymph nodes of 50 patients with melanoma. The expression of investigated biomarkers was determined on untreated and IFN-γ treated melanoma cell lines using flow cytometry. Among the selected biomarkers, the IFN-γ-induced expression of PD-L1 and IDO1 was associated with an increased risk of relapse (P = .0001 and P = .013, respectively) and was also associated with death for IDO1 (P = .0005). In the future, this immunologic signature could permit the identification of patients at higher risk of relapse and justifying an adjuvant treatment using immunotherapy.

BRAF mutations might be more common than supposed in vulvar melanomas.

Data on BRAF, NRAS and KIT mutations are scarce in patients with vulvo-vaginal melanomas and are associated with important therapeutic issues. We investigated their prevalence in a cohort of patients with female lower genital tract melanomas between 2003 and 2017. Of the 22 patients, 5 (22.7%) harboured a BRAF mutation, which was much higher than the rate of 5% reported in the literature. One patient, who was tested negative on the primary melanoma, had a NRAS mutation in a cutaneous metastasis. Our data provide a rationale for prospective and repeated mutations testing in female lower genital tract melanomas.

Routine molecular profiling of patients with advanced non-small-cell lung cancer: results of a 1-year nationwide programme of the French Cooperative Thoracic Intergroup (IFCT).

BACKGROUND: The molecular profiling of patients with advanced non-small-cell lung cancer (NSCLC) for known oncogenic drivers is recommended during routine care. Nationally, however, the feasibility and effects on outcomes of this policy are unknown. We aimed to assess the characteristics, molecular profiles, and clinical outcomes of patients who were screened during a 1-year period by a nationwide programme funded by the French National Cancer Institute. METHODS: This study included patients with advanced NSCLC, who were routinely screened for EGFR mutations, ALK rearrangements, as well as HER2 (ERBB2), KRAS, BRAF, and PIK3CA mutations by 28 certified regional genetics centres in France. Patients were assessed consecutively during a 1-year period from April, 2012, to April, 2013. We measured the frequency of molecular alterations in the six routinely screened genes, the turnaround time in obtaining molecular results, and patients' clinical outcomes. This study is registered with ClinicalTrials.gov, number NCT01700582. FINDINGS: 18,679 molecular analyses of 17,664 patients with NSCLC were done (of patients with known data, median age was 64·5 years [range 18-98], 65% were men, 81% were smokers or former smokers, and 76% had adenocarcinoma). The median interval between the initiation of analysis and provision of the written report was 11 days (IQR 7-16). A genetic alteration was recorded in about 50% of the analyses; EGFR mutations were reported in 1947 (11%) of 17,706 analyses for which data were available, HER2 mutations in 98 (1%) of 11,723, KRAS mutations in 4894 (29%) of 17,001, BRAF mutations in 262 (2%) of 13,906, and PIK3CA mutations in 252 (2%) of 10,678; ALK rearrangements were reported in 388 (5%) of 8134 analyses. The median duration of follow-up at the time of analysis was 24·9 months (95% CI 24·8-25·0). The presence of a genetic alteration affected first-line treatment for 4176 (51%) of 8147 patients and was associated with a significant improvement in the proportion of patients achieving an overall response in first-line treatment (37% [95% CI 34·7-38·2] for presence of a genetic alteration vs 33% [29·5-35·6] for absence of a genetic alteration; p=0·03) and in second-line treatment (17% [15·0-18·8] vs 9% [6·7-11·9]; p<0·0001). Presence of a genetic alteration was also associated with improved first-line progression-free survival (10·0 months [95% CI 9·2-10·7] vs 7·1 months [6·1-7·9]; p<0·0001) and overall survival (16·5 months [15·0-18·3] vs 11·8 months [10·1-13·5]; p<0·0001) compared with absence of a genetic alteration. INTERPRETATION: Routine nationwide molecular profiling of patients with advanced NSCLC is feasible. The frequency of genetic alterations, acceptable turnaround times in obtaining analysis results, and the clinical advantage provided by detection of a genetic alteration suggest that this policy provides a clinical benefit. FUNDING: French National Cancer Institute (INCa).

Clinical significance of BRAF mutation status in circulating tumor DNA of metastatic melanoma patients at baseline.

Circulating tumor DNA is a promising non-invasive tool for cancer monitoring. The main objective of our work was to investigate the relationship between mutant BRAF DNA in plasma and clinical response. Thirty-eight stage IV patients with a V600 mutated BRAF melanoma were included prior to any treatment. DNA was extracted from plasma and mutant DNA was detected using the amplification-refractory mutation system method. Before the beginning of any treatment, the corresponding BRAF mutation was detected in 29 of the 38 tested plasma samples (76.3% positive per cent agreement). We observed a strong correlation between the presence of circulating mutated DNA and overall survival (OS; P=.02), and with the number of metastatic sites (P=.01). The presence of circulating mutated DNA was also strongly correlated with serum LDH activity (P<.01) and S100 protein concentration (P<.01). Finally, seven patients presented discordant BRAF status in different tumor sites. In all these patients, the test performed on ctDNA was positive, suggesting that ctDNA analysis might be less sensitive to tumor heterogeneity. Altogether, these results suggest that plasmatic mutant BRAF DNA is a prognostic factor of OS, correlated with tumor burden. In addition, it represents an interesting alternative source of DNA to detect BRAF mutations before treatment.

Activation of the prostaglandin D2 metabolic pathway in Crohn's disease: involvement of the enteric nervous system.

BACKGROUND: Recent works provide evidence of the importance of the prostaglandin D2 (PGD2) metabolic pathway in inflammatory bowel diseases. We investigated the expression of PGD2 metabolic pathway actors in Crohn's disease (CD) and the ability of the enteric nervous system (ENS) to produce PGD2 in inflammatory conditions. METHODS: Expression of key actors involved in the PGD2 metabolic pathway and its receptors was analyzed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in colonic mucosal biopsies of patients from three groups: controls, quiescent and active CD patients. To determine the ability of the ENS to secrete PGD2 in proinflammatory conditions, Lipocalin-type prostaglandin D synthase (L-PGDS) expression by neurons and glial cells was analyzed by immunostaining. PGD2 levels were determined in a medium of primary culture of ENS and neuro-glial coculture model treated by lipopolysaccharide (LPS). RESULTS: In patients with active CD, inflamed colonic mucosa showed significantly higher COX2 and L-PGDS mRNA expression, and significantly higher PGD2 levels than healthy colonic mucosa. On the contrary, peroxysome proliferator-activated receptor Gamma (PPARG) expression was reduced in inflamed colonic mucosa of CD patients with active disease. Immunostaining showed that L-PGDS was expressed in the neurons of human myenteric and submucosal plexi. A rat ENS primary culture model confirmed this expression. PGD2 levels were significantly increased on primary culture of ENS treated with LPS. This production was abolished by AT-56, a specific competitive L-PGDS inhibitor. The neuro-glial coculture model revealed that each component of the ENS, ECG and neurons, could contribute to PGD2 production. CONCLUSIONS: Our results highlight the activation of the PGD2 metabolic pathway in Crohn's disease. This study supports the hypothesis that in Crohn's disease, enteric neurons and glial cells form a functional unit reacting to inflammation by producing PGD2.

Impact of PD-L1 Expression on the Overall Survival of Caucasian Patients with Advanced EGFR-Mutant NSCLC Treated with Frontline Osimertinib.

BACKGROUND: The treatment of advanced non-small cell lung cancer (NSCLC) harboring an oncogenic epidermal growth factor receptor mutation (EGFRm) is currently based on osimertinib, a third-generation tyrosine kinase inhibitor (TKI). High Programmed death ligand 1 (PD-L1) expression ≥ 50% demonstrated to be a negative prognostic factor, mostly among Asian populations treated with 1st/2nd generation TKI. OBJECTIVE: We investigated the impact of PD-L1 expression on the progression free survival (PFS) and overall survival (OS) within a cohort of patients receiving osimertinib as first-line treatment. METHODS: Our bi-centre French retrospective study included all newly diagnosed patients with an advanced EGFRm (common and uncommon) NSCLC, between May 2018 and November 2022, treated with osimertinib. The primary endpoint was OS according to tumor proportion score PD-L1 expression (low/intermediate < 50% vs high ≥ 50%). Survival analyses were performed using Kaplan-Meier method and Cox model for adjusted multivariate analysis. RESULTS: Of 96 patients, median age was 71 (IQR 62-76), 70 were women (72.9%), 81 had a performance status (PS) 0-1 (84.3%). Median follow-up was 22.6 months (95% CI 20.5-24.7). Twenty patients (20.8%) had high PD-L1 expression ≥ 50%. No significant differences in baseline characteristics were observed based on PD-L1 status. Patients with PD-L1 ≥ 50% had significant shorter PFS and OS than those with PD-L1 < 50%, respectively 9.3 vs 17.5 months (p = 0.044 months) and 14.3 vs 26.0 months (p = 0.025). Multivariable adjustment for baseline characteristics found that PS ≥ 2 (HR 2.79, 95% CI 1.12-6.93, p = 0.027), PD-L1 ≥ 50% (HR 2.61, 95% CI 1.31 to 5.22, p = 0.007) and uncommon EGFR mutation (HR 4.59, 95% CI 1.95-10.80, p = <0.001) were associated with a shorter OS. Brain metastases at diagnosis and age ≥ 65 were not, respectively HR 1.66 (95% CI 0.90-3.06, p = 0.11) and HR 0.95 (95% CI 0.50-1.80, p=0.9). CONCLUSIONS: Our study found that PD-L1 expression ≥ 50% was associated with a shorter OS in EGFRm NSCLC patients treated with first line osimertinib. Further research is warranted to understand the underlying molecular and cellular mechanisms of this correlation.

Prognostic Impact of TP53 Mutations in Metastatic Nonsquamous Non-small-cell Lung Cancer.

BACKGROUND: The prognostic impact of TP53 mutations in advanced or metastatic nonsquamous non-small-cell lung cancer (nsNSCLC) patients treated with chemotherapy and/or immune checkpoint inhibitors (ICI) remains unclear. MATERIALS AND METHODS: We retrospectively collected data from patients with nsNSCLC treated in the first line from January 2018 to May 2021. The patient was separated into 2 groups according to their TP53 mutation status (wt vs. mut). Survival was estimated through the Kaplan-Meier method and compared by log-rank test. RESULTS: Of 220 patients included, 126 were in the mutTP53 group, and 94 were in the wtTP53wt group. Median OS (mOS) was not significantly different between the mutTP53 and wtTP53 groups [17.5 months (95% confidence interval (CI), 11.3-21.5) vs. 9.5 months (95% CI, 7.4-14.2), (P = .051)]. In subgroup analyses, the mutTP53 group treated with ICI had a significantly improved mOS compared to the wtTP53 group [(24.7 months (95% CI, 20.8-not reach) vs. 12.0 months (95% CI, 4.7-not reach), (P = .017)] and mPFS [(9.6 months (95% CI, 5.8-not reach) vs. 3.2 months (95% CI, 1.3-13.8) (P = .048)]. There was no difference in terms of mOS and mPFS between the mutTP53 and the wtTP53 group treated by chemotherapy alone or combined with ICI. CONCLUSION: TP53 mutation had no survival impact in the overall population, but is associated with better outcomes with ICI alone. These results suggest that patients with TP53 mutations could be treated with ICI alone, and wild-type patients could benefit from the addition of chemotherapy.

New chondrosarcoma cell lines and mouse models to study the link between chondrogenesis and chemoresistance.

Chondrosarcomas are cartilage-forming, poorly vascularized tumors. They represent the second malignant primary bone tumor of adults after osteosarcoma, but in contrast to osteosarcoma they are resistant to chemotherapy and radiotherapy, surgical excision remaining the only therapeutic option. Few cell lines and animal models are available, and the mechanisms behind their chemoresistance remain largely unknown. Our goal was to establish new cell lines and animal cancer models from human chondrosarcoma biopsies to study their chemoresistance. Between 2007 and 2012, 10 chondrosarcoma biopsies were collected and used for cell culture and transplantation into nude mice. Only one transplanted biopsy and one injected cell line has engrafted successfully leading to conventional central high-grade chondrosarcoma similar to the original biopsies. In culture, two new stable cell lines were obtained, one from a dedifferentiated and one from a grade III conventional central chondrosarcoma biopsy. Their genetic characterization revealed triploid karyotypes, mutations in IDH1, IDH2, and TP53, deletion in CDKN2A and/or MDM2 amplification. These cell lines expressed mesenchymal membrane markers (CD44, 73, 90, 105) and were able to produce a hyaline cartilaginous matrix when cultured in chondrogenic three-dimensional (3D) pellets. Using a high-throughput quantitative RT-PCR approach, we observed that cell lines cultured in monolayer had lost expression of several genes implicated in cartilage development (COL2A1, COMP, ACAN) but restored their expression in 3D cultures. Chondrosarcoma cells in monolayer were sensitive to several conventional chemotherapeutic agents but became resistant to low doses of mafosfamide or doxorubicin when cultured in 3D pellets, in parallel with an altered nucleic accumulation of the drug. Our results indicate that the cartilaginous matrix produced by chondrosarcoma cells may impair diffusion of several drugs and thus contribute to chemoresistance. Therefore, 3D chondrogenic cell pellets constitute a more relevant model to study chondrosarcoma chemoresistance and may be a valuable alternative to animal experimentations.

Implementation of circulating tumour DNA multi-target mutation testing in plasma: a perspective from an external quality assessment providers' survey.

Demand for large-scale tumour profiling across cancer types has increased in recent years, driven by the emergence of targeted drug therapies. Analysing alternations in plasma circulating tumour DNA (ctDNA) for cancer detection can improve survival; ctDNA testing is recommended when tumour tissue is unavailable. An online survey of molecular pathology testing was circulated by six external quality assessment members of IQN Path to registered laboratories and all IQN Path collaborative corporate members. Data from 275 laboratories across 45 countries were collected; 245 (89%) perform molecular pathology testing, including 177 (64%) which perform plasma ctDNA diagnostic service testing. The most common tests were next-generation sequencing-based (n = 113). Genes with known stratified treatment options, including KRAS (n = 97), NRAS (n = 84), and EGFR (n = 130), were common targets. The uptake of ctDNA plasma testing and plans to implement further testing demonstrates the importance of support from a well-designed EQA scheme.

TP53 mutations correlate with the non-coding RNA content of small extracellular vesicles in melanoma.

Non-coding RNAs (ncRNAs) are important regulators of gene expression. They are expressed not only in cells, but also in cell-derived extracellular vesicles (EVs). The mechanisms controlling their loading and sorting remain poorly understood. Here, we investigated the impact of TP53 mutations on the non-coding RNA content of small melanoma EVs. After purification of small EVs from six different patient-derived melanoma cell lines, we characterized them by small RNA sequencing and lncRNA microarray analysis. We found that TP53 mutations are associated with a specific micro and long non-coding RNA content in small EVs. Then, we showed that long and small non-coding RNAs enriched in TP53 mutant small EVs share a common sequence motif, highly similar to the RNA-binding motif of Sam68, a protein interacting with hnRNP proteins. This protein thus may be an interesting partner of p53, involved in the expression and loading of the ncRNAs. To conclude, our data support the existence of cellular mechanisms associate with TP53 mutations which control the ncRNA content of small EVs in melanoma.

High efficacy of alectinib in a patient with advanced lung adenocarcinoma with 2 rare ALK fusion sites: a case report.

Anaplastic lymphoma kinase (ALK) fusions have been identified in approximately 5% of non-small cell lung cancer (NSCLC) cases. ALK-tyrosine kinase inhibitors (TKIs) are the standard first-line treatment for patients with ALK-positive (ALK+) advanced NSCLC. Along with widespread use of next-generation sequencing (NGS) for the molecular diagnosis of lung cancer, an increasing number of ALK fusion partners are being reported, with the majority being effective for ALK-TKIs. Here, we present the case of a 42-year-old female with no smoking history who was diagnosed with stage IVB lung adenocarcinoma. Two rare ALK fusions were detected simultaneously by NGS in this patient: latent transforming growth factor beta-binding protein 1 (LTBP1)-ALK and huntingtin-interacting protein 1 (HIP1)-ALK. HIP1-ALK fusion was also detected by further RNA sequencing, but LTBP1-ALK failed to give a positive signal. The patient received alectinib as first-line therapy and consequently achieved a good response. Progression-free survival (PFS) was more than 19 months, and the treatment with alectinib is ongoing currently. During treatment, clinical symptoms disappeared and no significant adverse events occurred. This is the first case report describing a patient with an NSCLC tumor harboring 2 rare ALK fusions who responded to alectinib. Our report enriches the knowledge of ALK fusion sites and provides an effective clinical basis for the screening of sensitive fusions.

Metastatic pulmonary carcinoids with EML4-ALK fusion response to ALK inhibitors: two case reports and review of literature.

Background: Pulmonary carcinoids (PC), including typical (TC) and atypical carcinoids (AC), are low-grade neuroendocrine tumors (NETs) which account for 1-5% of all lung tumors. Due to the low prevalence of PC and extreme rarity of anaplastic lymphoma kinase (ALK) rearrangements in patients with PC, the advances in targeted therapy development in PC are still limited and there is no standard treatment. Even though in patients with PC harboring ALK rearrangements there is a room for a success in targeted therapy. To our knowledge, case 1 was the first report to detect ALK gene p.I1171N mutation after taking alectinib and sensitive to ceritinib in patients with atypical carcinoid. Case Description: Herein, we report the cases of 2 non-smoking patients, 51 year-old female with tumor in left lower lobe and 49 year-old female with tumor in right upper lobe, both with metastatic PC who harbored EML4-ALK fusion and were sensitive to small-molecule ALK inhibitors. The first patient initially received alectinib, then therapy was switched to ceritinib after developing drug resistance due to the missense mutation of ALK gene p.I1171N mutation in exon 22 detected by next-generation sequencing (NGS), and finally died of intracranial disease progression. The second patient also received alectinib, and her treatment is currently ongoing with good effect and tolerance. After conducting comprehensive review of literature, we found that 14 lung NETs with ALK rearrangements have been reported to date. The clinical outcome was partial response for 6 NETs patients and 5 patients exhibited stable disease after treatment with ALK inhibitors. Conclusions: According to the effectiveness of ALK inhibitors in our cases and previous articles, we recommend alectinib for the first-line treatment of metastatic PC with EML4-ALK fusion and highlight the need for molecular profiling of metastatic lung NETs patients and that ALK inhibitors are feasible in the treatment for metastatic lung NETs patients with ALK rearrangements. Finally, further studies to assess the real prevalence of ALK gene fusions and their spectrum of sensitivity to different ALK inhibitors are needed in larger cohorts.

Mucosal IL-10 and TGF-beta play crucial roles in preventing LPS-driven, IFN-gamma-mediated epithelial damage in human colon explants.

IL-10 is an immunomodulatory cytokine that plays an obligate role in preventing spontaneous enterocolitis in mice. However, little is known about IL-10 function in the human intestinal mucosa. We showed here that IL-10 was constitutively expressed and secreted by the human normal colonic mucosa, including epithelial cells. Depletion of IL-10 in mucosal explants induced both downregulation of the IL-10-inducible, immunosuppressive gene BCL3 and upregulation of IFN-gamma, TNF-alpha, and IL-17. Interestingly, TGF-beta blockade also strongly induced IFN-gamma production. In addition, the high levels of IFN-gamma produced upon IL-10 depletion were responsible for surface epithelium damage and crypt loss, mainly by apoptosis. Polymyxin B, used as a scavenger of endogenous LPS, abolished both IFN-gamma production and epithelial barrier disruption. Finally, adding a commensal bacteria strain to mucosa explant cultures depleted of both IL-10 and LPS reproduced the ability of endogenous LPS to induce IFN-gamma secretion. These findings demonstrate that IL-10 ablation leads to an endogenous IFN-gamma-mediated inflammatory response via LPS from commensal bacteria in the human colonic mucosa. We also found that both IL-10 and TGF-beta play crucial roles in maintaining human colonic mucosa homeostasis.

KRAS mutations in metastatic colorectal cancer: from a de facto ban on anti-EGFR treatment in the past to a potential biomarker for precision medicine.

INTRODUCTION: The high frequency of RAS mutations, particularly KRAS mutations, in colorectal cancer (CRC) and the ineffectiveness of anti-EGFR antibodies in treating this disease has created a significant unmet medical need, especially for treating patients in the metastatic phase of this disease. There are many different types of RAS mutations, the most frequent being G12V (c.35 G > T (p.G12V)), G12D (c.35 G > A (p.G12D)), and G13D (c.38 G > A (p.G13D)). Here, we provide an overview of RAS mutations in CRC and their therapeutic implications. AREAS COVERED: The therapeutic strategies against metastatic CRC with RAS mutations are elaborated according to patient and disease characteristics and integrated into a multiline strategy. The complexity of the molecular structure of RAS and its relationship with the MAPK/ERK pathway partly explain the initial therapeutic failure with MEK or farnesyltransferase inhibitors. Conversely, the development of direct KRAS inhibitors or drugs targeting RAS regulators (e.g. SOS1 and SHP2) has opened new therapeutic fields, requiring the distinction of each KRAS mutation type. EXPERT OPINION: In the future, KRAS inhibitors, including SOS1 and SHP2 inhibitors, might be used in combination with other signal transduction inhibitors, such as MEK inhibitors or anti-EGFR antibodies, which block alternative pathways of activation.

Circulating Tumor DNA Early Kinetics Predict Response of Metastatic Melanoma to Anti-PD1 Immunotherapy: Validation Study.

The ability of early (first weeks of treatment) ctDNA kinetics to identify primary resistance to anti-PD1 immunotherapies was evaluated with a validation cohort of 49 patients treated with anti-PD1 for metastatic BRAF or NRAS-mutated melanoma, alone and pooled with the 53 patients from a previously described derivation cohort. BRAF or NRAS mutations were quantified on plasma DNA by digital PCR at baseline and after two or four weeks of treatment. ctDNA kinetics were interpreted according to pre-established biological response criteria. A biological progression (bP, i.e., a significant increase in ctDNA levels) at week two or week four was associated with a lack of benefit from anti-PD1 (4-month PFS = 0%; 1-year OS = 13%; n = 12/102). Patients without initial bP had significantly better PFS and OS (4-month PFS = 78%; 1-year OS = 73%; n = 26/102), as did patients whose ctDNA kinetics were not evaluable, due to low/undetectable baseline ctDNA (4-month PFS = 80%; 1-year OS = 81%; n = 64/102). ctDNA detection at first-line anti-PD1 initiation was an independent prognostic factor for OS and PFS in multivariate analysis. Overall, early ctDNA quantitative monitoring may allow the detection of primary resistances of metastatic melanoma to anti-PD1 immunotherapies.