Quantitative interactomics in primary T cells unveils TCR signal diversification extent and dynamics.

The activation of T cells by the T cell antigen receptor (TCR) results in the formation of signaling protein complexes (signalosomes), the composition of which has not been analyzed at a systems level. Here, we isolated primary CD4+ T cells from 15 gene-targeted mice, each expressing one tagged form of a canonical protein of the TCR-signaling pathway. Using affinity purification coupled with mass spectrometry, we analyzed the composition and dynamics of the signalosomes assembling around each of the tagged proteins over 600 s of TCR engagement. We showed that the TCR signal-transduction network comprises at least 277 unique proteins involved in 366 high-confidence interactions, and that TCR signals diversify extensively at the level of the plasma membrane. Integrating the cellular abundance of the interacting proteins and their interaction stoichiometry provided a quantitative and contextual view of each documented interaction, permitting anticipation of whether ablation of a single interacting protein can impinge on the whole TCR signal-transduction network.

Quantitative proteomics analysis of signalosome dynamics in primary T cells identifies the surface receptor CD6 as a Lat adaptor-independent TCR signaling hub.

T cell antigen receptor (TCR)-mediated activation of T cells requires the interaction of dozens of proteins. Here we used quantitative mass spectrometry and activated primary CD4(+) T cells from mice in which a tag for affinity purification was knocked into several genes to determine the composition and dynamics of multiprotein complexes that formed around the kinase Zap70 and the adaptors Lat and SLP-76. Most of the 112 high-confidence time-resolved protein interactions we observed were previously unknown. The surface receptor CD6 was able to initiate its own signaling pathway by recruiting SLP-76 and the guanine nucleotide-exchange factor Vav1 regardless of the presence of Lat. Our findings provide a more complete model of TCR signaling in which CD6 constitutes a signaling hub that contributes to the diversification of TCR signaling.

mTert induction in p21-positive cells counteracts capillary rarefaction and pulmonary emphysema.

Lung diseases develop when telomeres shorten beyond a critical point. We constructed a mouse model in which the catalytic subunit of telomerase (mTert), or its catalytically inactive form (mTertCI), is expressed from the p21Cdkn1a locus. Expression of either TERT or TERTCI reduces global p21 levels in the lungs of aged mice, highlighting TERT non-canonical function. However, only TERT reduces accumulation of very short telomeres, oxidative damage, endothelial cell (ECs) senescence and senile emphysema in aged mice. Single-cell analysis of the lung reveals that p21 (and hence TERT) is expressed mainly in the capillary ECs. We report that a fraction of capillary ECs marked by CD34 and endowed with proliferative capacity declines drastically with age, and this is counteracted by TERT but not TERTCI. Consistently, only TERT counteracts decline of capillary density. Natural aging effects are confirmed using the experimental model of emphysema induced by VEGFR2 inhibition and chronic hypoxia. We conclude that catalytically active TERT prevents exhaustion of the putative CD34 + EC progenitors with age, thus protecting against capillary vessel loss and pulmonary emphysema.

Upregulation of IL4-induced gene 1 enzyme by B2 cells during melanoma progression impairs their antitumor properties.

B cells present in human cutaneous melanoma have been associated with protective or detrimental effects on disease progression according to their phenotype. By using the RET model of spontaneous melanoma and adoptive transfer of B16 melanoma cells, we show that immature and follicular B2 (B2-FO) cells exert a protective effect on melanoma progression by promoting the generation of effector memory T cells and limiting the recruitment of polymorphonuclear myeloid-derived suppressor cells. Unfortunately, this beneficial effect progressively wanes as a consequence of enhanced expression of the IL4-induced gene 1 (IL4I1) enzyme by immature B cells and B2-FO cells. Endogenous IL4I1 selectively decreases CXCR5 expression in splenic immature B cells, subverting their trafficking to primary tumors and enhancing the production of IL-10 by B2 cells, thereby promoting an immunosuppressive microenvironment. Accordingly, B2 cells from RET IL4I1KO mice more efficiently controlled B16 melanoma growth than B2 cells from IL4I1-competent RET mice. Collectively, immature B cells and B2-FO cells are key actors in the control of melanoma growth, but their mobility and functions are differently impaired by IL4I1 overexpression during melanoma progression. Thus, our present data strongly urge us to associate an IL4I1 antagonist with current immunotherapy to improve the treatment of metastatic melanoma.

Genomic profiling of small bowel adenocarcinoma: a pooled analysis from 3 databases.

BACKGROUND: Small bowel adenocarcinoma is a rare disease. The genomic profiling tumours according to clinical characteristics and its impact on the prognosis remains unclear. METHODS: A pooled analysis of clinical data, genomic profiling and MisMatch Repair (MMR) status from three databases was performed. RESULTS: A total of 188 tumour samples were analysed. A predisposing disease was reported in 22.3%, mainly Lynch syndrome and Crohn's disease. The tumours were localized in 80.2% and metastatic in 18.8%. The most frequent mutations were KRAS (42.0%) among them 7/79 are G12C, TP53 (40.4%), APC (19.1%), PIK3CA (18.6%), SMAD4 (12.8%) and ERBB2 (9.6%). Mutation distribution differed according to predisposing disease for TP53, ERBB2, IDH1, FGFR3, FGFR1 and KDR. KRAS and SMAD4 mutations were more frequent in metastatic tumour, whereas ERBB2 mutations were absent in metastatic tumour. For localized tumour, APC mutation was independently associated with a poor overall survival (OS) (p = 0.0254). 31.8% of localized tumours and 11.3% of metastatic tumours were dMMR (29.8% of the entire cohort). A dMMR status was associated with a better OS (HR = 0.61 [0.39-0.96], p = 0.0316). CONCLUSIONS: There is a different genomic profile according to the stage and predisposing disease. dMMR and APC mutation in localized tumour predict a better prognosis.

New Biomarkers to Define A Biological Borderline Situation for Pancreatic Adenocarcinoma - Results of An Ancillary Study of the PANACHE01-PRODIGE48 Trial.

OBJECTIVE: To investigate in patients treated for a resectable pancreatic ductal adenocarcinoma (PA), the prognostic value of baseline CA19-9 and circulating tumour DNA (ctDNA) for overall survival (OS), to improve death risk stratification, based on a planned ancillary study from PANACHE01-PRODIGE 48 trial. SUMMARY BACKGROUND DATA: Biological borderline situation that was first used by the MD Anderson, became a standard practice following the international consensus conference in 2016 to manage PA. Regarding the risk of systemic disease especially in the setting of "markedly elevated" CA19-9, neoadjuvant therapy is advised to avoid unnecessary surgery, with risk of early recurrence. To best define biological borderline situations, new biomarkers are needed. METHODS: Characteristics at diagnosis and OS were compared between patients with or without ctDNA status available. OS was estimated with Kaplan Meier method and compared with log-rank test. Restricted cubic spline approach was used to identify optimal threshold for biological parameters for death risk stratification. Univariate and multivariate Cox proportional hazard models were estimated to assess the association of ctDNA status and other parameters with OS. RESULTS: Among the 132 patients from the primary population for analysis in the PANACHE01 -PRODIGE 48 trial, 92(71%) were available for ctDNA status at diagnosis. No selection bias was identified between patients with or without ctDNA status. 14 patients (15%) were ctDNA+ and exhibited a higher risk for death (P=0,0188; HR95% CI: 2.28 (1.12-4.63). In the 92 patients with ctDNA status available among the others parameter analysed only CA19-9 was statically associated with OS in univariate analysis. Patients with log of CA19-9 equal or superior to 4.4 that corresponds to a CA19-9 of 80 UI/mL were identified at higher risk for death (P=0,0143; HR95% CI: 2.2 (1.15-4.19). In multivariate analysis CA19-19 remained independently associated with OS (p-value=0.0323). When combining the two biomarkers, median OS was of 19.4 (IC 95% 3.8-Not reached) months, 30.2 (IC 95% 17.1-NR) months and not reached (IC 95% 39.3-NR) for "CA19-9 high and ctDNA+ group", "CA19-9 high or ctDNA+ group", and "CA19-9 low and ctDNA- group", respectively (logrank P=0,0069). DISCUSSION: Progress in the management of potentially operable PA remains limited, relying solely on strategies to optimize the sequence of complete treatment, based on modern multidrug chemotherapy (FOLFIRINOX, GemNabPaclitaxel) and surgical resection. The identification of risk criteria, such as the existence of systemic disease, is an important issue, currently referred to as "biologic borderline disease". Few data, particularly from prospective studies, allow us to identify biomarkers other than CA19-9. CONCLUSION: Combining ctDNA to CA19-9 could be of interest to best define biological borderline situations in PA.

Bevacizumab plus FOLFIRI after failure of platinum-etoposide first-line chemotherapy in patients with advanced neuroendocrine carcinoma (PRODIGE 41-BEVANEC): a randomised, multicentre, non-comparative, open-label, phase 2 trial.

BACKGROUND: There is no standard second-line treatment after platinum-etoposide chemotherapy for gastroenteropancreatic neuroendocrine carcinoma. We aimed to evaluate the efficacy of FOLFIRI plus bevacizumab, and FOLFIRI alone, in this setting. METHODS: We did a randomised, non-comparative, open-label, phase 2 trial (PRODIGE 41-BEVANEC) at 26 hospitals in France. We included patients aged 18 years or older with locally advanced or metastatic gastroenteropancreatic neuroendocrine carcinoma or neuroendocrine carcinoma of unknown primary origin, documented progressive disease during or after first-line platinum-etoposide chemotherapy, and an Eastern Cooperative Oncology Group performance status of 0-2. Patients were randomly assigned (1:1; block size of three), without stratification, to receive FOLFIRI (irinotecan 180 mg/m2, calcium folinate 400 mg/m2 or levofolinate 200 mg/m2, and fluorouracil 400 mg/m2 bolus then 2400 mg/m2 over 46 h) plus bevacizumab 5 mg/kg or FOLFIRI alone, intravenously, every 2 weeks until disease progression or unacceptable toxicity. Neither patients nor investigators were masked to group assignment. The primary outcome was overall survival at 6 months after randomisation, evaluated in the modified intention-to-treat population (all enrolled and randomly assigned patients who received at least one cycle of FOLFIRI). This study is now complete and is registered with ClinicalTrials.gov, NCT02820857. FINDINGS: Between Sept 5, 2017, and Feb 8, 2022, 150 patients were assessed for eligibility and 133 were enrolled and randomly assigned: 65 to the FOLFIRI plus bevacizumab group and 68 to the FOLFIRI group. 126 patients (59 in the FOLFIRI plus bevacizumab group and 67 in the FOLFIRI group) received at least one cycle of FOLFIRI and were included in the modified intention-to-treat population, 83 (66%) of whom were male and 43 (34%) were female, and the median age of the patients was 67 years (IQR 58-73). The primary tumour location was colorectal in 38 (30%) of 126 patients, pancreatic in 34 (27%), gastro-oesophageal in 22 (17%), and unknown in 23 (18%). After a median follow-up of 25·7 months (95% CI 22·0-38·2), 6-month overall survival was 53% (80% CI 43-61) in the FOLFIRI plus bevacizumab group and 60% (51-68) in the FOLFIRI group. Grade 3-4 adverse events that occurred in at least 5% of patients were neutropenia (eight [14%] patients), diarrhoea (six [10%]), and asthenia (five [8%]) in the FOLFIRI plus bevacizumab group, and neutropenia (seven [10%]) in the FOLFIRI group. One treatment-related death (ischaemic stroke) occurred in the FOLFIRI plus bevacizumab group. INTERPRETATION: The addition of bevacizumab did not seem to increase the benefit of FOLFIRI with regard to overall survival. FOLFIRI could be considered as a standard second-line treatment in patients with gastroenteropancreatic neuroendocrine carcinoma. FUNDING: French Ministry of Health and Roche SAS.

Clinical relevance of circulating ESR1 mutations during endocrine therapy for advanced hormone-dependent endometrial carcinoma.

OBJECTIVE: Endocrine therapy is frequently administered in patients with hormone dependent (HR+) metastatic endometrial cancer. ESR1 mutations have emerged as a key mechanism of aromatase inhibitor (AI) resistance in HR + metastatic breast cancer and can be monitored using circulating tumor DNA (ctDNA). The aim of this study was to explore the incidence and clinical relevance of circulating ESR1 mutations in patients treated by AI or megestrol acetate (M) for advanced endometrial carcinoma. METHODOLOGY: This single-center retrospective study was performed at the Henri Becquerel Center (Rouen) and looked for circulating ESR1 gene mutations by droplet digital PCR (E380Q, L536R, Y537S, Y537N, Y537C, D538G, S463P) in patients with advanced HR + endometrial carcinoma treated between 2008 and 2020 for at least 30 days by AI or M. Analyses were performed before exposure and at progression/during endocrine therapy. RESULTS: Twenty-two patients were included: 13 were treated with AI, 12 of whom progressed; 9 patients were treated with M, 8 of whom progressed. 68.1% of the patients had low-grade endometrial carcinoma and 54.5% had received chemotherapy in the metastatic setting. The median duration of treatment was 152 days (min 47 - max 629) with AI and 155 days (min 91-max 1297) with M. Under AI, there was no ESR1 mutation at baseline, and one Y537C mutation at progression with a variant allele frequency (VAF) of 0.14%. Under M, one patient had a Y537C (VAF 0.2%) at baseline that disappeared during treatment. Another patient had a Y537S mutation emergence at progression after 91 days of treatment (VAF 1.83%). There was no significant difference between the circulating DNA concentration before and after hormone therapy (p = 0.16). CONCLUSION: ESR1 mutations do not seem to be involved in the mechanisms of resistance to AI or M in HR+ endometrial cancer. The clinical relevance of their detection is not demonstrated.

Circulating DNA changes are predictive of disease progression after transarterial chemoembolization.

Transarterial chemoembolization (TACE) is used to treat patients with unresectable hepatocellular carcinoma (HCC). We evaluated the clinical impact of a-fetoprotein (AFP) and circulating cell-free and tumor DNA (cfDNA and ctDNA) changes around the TACE procedure. Our prospective monocentric study enrolled consecutive patients treated with TACE, with samples collected at baseline (D - 1), Day 2 (D + 2) and 1 month (M + 1) after TACE. cfDNA was quantified by the fluorometric method, and ctDNA was quantified by digital polymerase chain reaction designed for two hotspot TERT mutations. Computerized tomography scans or magnetic resonance imaging were performed at M + 1 every 3 months following TACE and independently reviewed. The objective was to identify thresholds of cfDNA, ctDNA and AFP changes associated with progressive disease (PD) using receiver operating characteristic curves. Thirty-eight patients were included from March 2018 to March 2019. All markers significantly increased from D - 1 to D + 2 (P < .005), and cfDNA and ctDNA significantly decreased from D + 2 to M + 1 (P < .0001). The analysis of changes from D - 1 to M + 1 identified thresholds at +31.4% for cfDNA and 0% for ctDNA that were significantly associated with PD at M + 1 (44.4% [>+31.4%] vs 3.8% [≤+31.4%] and 50.0% [>0%] vs 5.0% [≤0%], respectively). No significant threshold was identified for AFP. Using a score combining cfDNA and ctDNA, the patients were classified into high- or low-risk PD groups at M + 1, with PD rates of 80.0% vs 4.3% (P = .001) and median progression-free survival times of 1.3 vs 10.3 months (P = .002). Our study suggests that cfDNA and ctDNA increases around the TACE procedure and are associated with therapeutic failure.

Are patients living far from hospital at higher risk of late adjuvant chemotherapy for colon cancer?

AIMS: Late adjuvant chemotherapy (aCT) administration after colectomy (>56 d) is known to be associated with impaired prognosis. We aim to identify risk factors associated with late aCT, especially the travel time between patients' home and hospital. METHODS: We performed a retrospective monocentre cohort study. Patients included had a colectomy for a stage III or high risk stage II colon cancer between 2009 and 2015 performed at a French university hospital. Risk factors for late aCT were identified using a fractional polynomial logistic regression. RESULTS: Ninety-four patients were included. The risk of late aCT was associated with travel time length, emergent colectomy, the need for scheduled care before aCT, and length of time between colectomy and postoperative multidisciplinary meeting advising aCT. CONCLUSION: Our study suggests that, in patients with colon cancer, factors unrelated to disease severity and complexity could be associated with a higher risk of late aCT.