Prognostic value of BRCA1 promoter methylation for patients with epithelial ovarian cancer.

OBJECTIVE: BRCA1 promoter methylation (BRCA1pm) is suspected to alter prognosis of patients with epithelial ovarian cancer (EOC). We aimed to evaluate the prognostic impact of this epigenetic modification. METHODS: We conducted a retrospective, monocentric study from 11/2006 to 08/2018. Patients with EOC and available status concerning somatic BRCA1/2 mutation and BRCA1pm were included. Three groups were defined: patients without BRCA1/2 mutation or BRCA1pm, patients with BRCA1/2 mutation and patients with BRCA1pm. BRCA1/2 mutations were analyzed in current care settings by next-generation sequencing (NGS). BRCA1pm analysis was assessed and quantified from bisulfite converted DNAs using fluorescent methylation specific polymerase chain reaction (PCR) and fragment analysis. All patients signed a consent form and the study was authorized by a Personal Protection Committee. Descriptive statistics were used to describe groups. Multivariate analysis was performed using the logistic regression model and including the variables that could be known at the time of diagnosis and that were significant at univariate analysis. Survival was compared between the groups. Kaplan-Mayer curves were used to express the differences in survival that were compared using log rank tests. RESULTS: 145 patients were included: 95 (65.5%) patients without BRCA1/2 mutation or BRCA1pm, 32 (22.1%) patients with BRCA1/2 mutation, 18 (12.4%) patients with BRCA1pm. Median survival was decreased in patients with BRCA1pm. Comparison of survival revealed a significant difference in overall survival (p = 0.0078) with a worse prognosis for patients with a BRCA1pm. CONCLUSION: BRCA1pm in patients with EOC is an independent factor associated with a decreased overall survival. SYNOPSIS: BRCA1 promotor methylation in patients with epithelial ovarian cancer is an independent factor associated with a decreased overall survival.

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.

Clinical challenges of consensus molecular subtype CMS4 colon cancer in the era of precision medicine.

Over the past decade, our understanding of the diversity of colorectal cancer (CRC) has expanded significantly, raising hopes of tailoring treatments more precisely for individual patients. A key achievement in this direction was the establishment of the consensus molecular classification, particularly identifying the challenging consensus molecular subtype (CMS) CMS4 associated with poor prognosis. Due to its aggressive nature, extensive research is dedicated to the CMS4 subgroup. Recent years have unveiled molecular and microenvironmental features at the tissue level specific to CMS4 CRC. This has paved the way for mechanistic studies and the development of preclinical models. Simultaneously, efforts have been made to easily identify patients with CMS4 CRC. Reassessing clinical trial results through the CMS classification lens has improved our understanding of the therapeutic challenges linked to this subtype. Exploration of the biology of CMS4 CRC is yielding potential biomarkers and novel treatment approaches. This overview aims to provide insights into the clinico-biological characteristics of the CMS4 subgroup, the molecular pathways driving this subtype, and available diagnostic options. We also emphasize the therapeutic challenges associated with this subtype, offering potential explanations. Finally, we summarize the current tailored treatments for CMS4-CRC emerging from fundamental and preclinical studies.

Wnt, glucocorticoid and cellular prion protein cooperate to drive a mesenchymal phenotype with poor prognosis in colon cancer.

BACKGROUND: The mesenchymal subtype of colorectal cancer (CRC), associated with poor prognosis, is characterized by abundant expression of the cellular prion protein PrPC, which represents a candidate therapeutic target. How PrPC is induced in CRC remains elusive. This study aims to elucidate the signaling pathways governing PrPC expression and to shed light on the gene regulatory networks linked to PrPC. METHODS: We performed in silico analyses on diverse datasets of in vitro, ex vivo and in vivo models of mouse CRC and patient cohorts. We mined ChIPseq studies and performed promoter analysis. CRC cell lines were manipulated through genetic and pharmacological approaches. We created mice combining conditional inactivation of Apc in intestinal epithelial cells and overexpression of the human prion protein gene PRNP. Bio-informatic analyses were carried out in two randomized control trials totalizing over 3000 CRC patients. RESULTS: In silico analyses combined with cell-based assays identified the Wnt-ß-catenin and glucocorticoid pathways as upstream regulators of PRNP expression, with subtle differences between mouse and human. We uncover multiple feedback loops between PrPC and these two pathways, which translate into an aggravation of CRC pathogenesis in mouse. In stage III CRC patients, the signature defined by PRNP-CTNNB1-NR3C1, encoding PrPC, ß-catenin and the glucocorticoid receptor respectively, is overrepresented in the poor-prognosis, mesenchymal subtype and associates with reduced time to recurrence. CONCLUSIONS: An unleashed PrPC-dependent vicious circle is pathognomonic of poor prognosis, mesenchymal CRC. Patients from this aggressive subtype of CRC may benefit from therapies targeting the PRNP-CTNNB1-NR3C1 axis.

Non-metastatic colon cancer: French Intergroup Clinical Practice Guidelines for diagnosis, treatments, and follow-up (TNCD, SNFGE, FFCD, GERCOR, UNICANCER, SFCD, SFED, SFRO, ACHBT, SFP, AFEF, and SFR).

INTRODUCTION: This article is a summary of the French intergroup guidelines regarding the management of non-metastatic colon cancer (CC), revised in November 2022. METHODS: These guidelines represent collaborative work of all French medical and surgical societies involved in the management of CC. Recommendations were graded in three categories (A, B, and C) according to the level of evidence found in the literature published up to November 2022. RESULTS: Initial evaluation of CC is based on clinical examination, colonoscopy, chest-abdomen-pelvis computed tomography (CT) scan, and carcinoembryonic antigen (CEA) assay. CC is usually managed by surgery and adjuvant treatment depending on the pathological findings. The use of adjuvant therapy remains a challenging question in stage II disease. For high-risk stage II CC, adjuvant chemotherapy must be discussed and fluoropyrimidine monotherapy or oxaliplatin-based chemotherapy proposed according to the type and number of poor prognostic features. Oxaliplatin-based chemotherapy (FOLFOX or CAPOX) is the current standard for adjuvant therapy of patients with stage III CC. However, these regimens are associated with significant oxaliplatin-induced neurotoxicity. The results of the recent IDEA study provide evidence that 3 months of treatment with CAPOX is as effective as 6 months of oxaliplatin-based therapy in patients with low-risk stage III CC (T1-3 and N1). A 6-month oxaliplatin-based therapy remains the standard of care for high-risk stage III CC (T4 and/or N2). For patients unfit for oxaliplatin, fluoropyrimidine monotherapy is recommended. CONCLUSION: French guidelines for non-metastatic CC management help to offer the best personalized therapeutic strategy in daily clinical practice. Each individual case must be discussed within a multidisciplinary tumor board and then the treatment option decided with the patient.

Mutational signature, cancer driver genes mutations and transcriptomic subgroups predict hepatoblastoma survival.

BACKGROUND: Hepatoblastoma is the most frequent pediatric liver cancer. The current treatments lead to 80% of survival rate at 5 years. In this study, we evaluated the clinical relevance of molecular features to identify patients at risk of chemoresistance, relapse and death of disease. METHODS: All the clinical data of 86 children with hepatoblastoma were retrospectively collected. Pathological slides were reviewed, tumor DNA sequencing (by whole exome, whole genome or target) and transcriptomic profiling with RNAseq or 300-genes panel were performed. Associations between the clinical, pathological, mutational and transcriptomic data were investigated. RESULTS: High-risk patients represented 44% of our series and the median age at diagnosis was 21.9 months (range: 0-208). Alterations of the WNT/ß-catenin pathway and of the 11p15.5 imprinted locus were identified in 98% and 74% of the tumors, respectively. Other cancer driver genes mutations were only found in less than 11% of tumors. After neoadjuvant chemotherapy, disease-specific survival and poor response to neoadjuvant chemotherapy were associated with 'Liver Progenitor' (p = 0.00049, p < 0.0001) and 'Immune Cold' (p = 0.0011, p < 0.0001) transcriptomic tumor subtypes, SBS35 cisplatin mutational signature (p = 0.018, p = 0.001), mutations in rare cancer driver genes (p = 0.0039, p = 0.0017) and embryonal predominant histological type (p = 0.0013, p = 0.0077), respectively. Integration of the clinical and molecular features revealed a cluster of molecular markers associated with resistance to chemotherapy and survival, enlightening transcriptomic 'Immune Cold' and Liver Progenitor' as a predictor of survival independent of the clinical features. CONCLUSIONS: Response to neoadjuvant chemotherapy and survival in children treated for hepatoblastoma are associated with genomic and pathological features independently of the clinical features.

Prospective observational study on biomarkers of response in pancreatic ductal adenocarcinoma.

Adjuvant chemotherapy benefits patients with resected pancreatic ductal adenocarcinoma (PDAC), but the compromised physical state of post-operative patients can hinder compliance. Biomarkers that identify candidates for prompt adjuvant therapy are needed. In this prospective observational study, 1,171 patients with PDAC who underwent pancreatectomy were enrolled and extensively followed-up. Proteomic profiling of 191 patient samples unveiled clinically relevant functional protein modules. A proteomics-level prognostic risk model was established for PDAC, with its utility further validated using a publicly available external cohort. More importantly, through an interaction effect regression analysis leveraging both clinical and proteomic datasets, we discovered two biomarkers (NDUFB8 and CEMIP2), indicative of the overall sensitivity of patients with PDAC to adjuvant chemotherapy. The biomarkers were validated through immunohistochemistry on an internal cohort of 386 patients. Rigorous validation extended to two external multicentic cohorts-a French multicentric cohort (230 patients) and a cohort from two grade-A tertiary hospitals in China (466 patients)-enhancing the robustness and generalizability of our findings. Moreover, experimental validation through functional assays was conducted on PDAC cell lines and patient-derived organoids. In summary, our cohort-scale integration of clinical and proteomic data demonstrates the potential of proteomics-guided prognosis and biomarker-aided adjuvant chemotherapy for PDAC.

Dihydropyrimidine dehydrogenase gene variants for predicting grade 4-5 fluoropyrimidine-induced toxicity: FUSAFE individual patient data meta-analysis.

BACKGROUND: Dihydropyrimidine dehydrogenase (DPD) deficiency is the main known cause of life-threatening fluoropyrimidine (FP)-induced toxicities. We conducted a meta-analysis on individual patient data to assess the contribution of deleterious DPYD variants *2A/D949V/*13/HapB3 (recommended by EMA) and clinical factors, for predicting G4-5 toxicity. METHODS: Study eligibility criteria included recruitment of Caucasian patients without DPD-based FP-dose adjustment. Main endpoint was 12-week haematological or digestive G4-5 toxicity. The value of DPYD variants *2A/p.D949V/*13 merged, HapB3, and MIR27A rs895819 was evaluated using multivariable logistic models (AUC). RESULTS: Among 25 eligible studies, complete clinical variables and primary endpoint were available in 15 studies (8733 patients). Twelve-week G4-5 toxicity prevalence was 7.3% (641 events). The clinical model included age, sex, body mass index, schedule of FP-administration, concomitant anticancer drugs. Adding *2A/p.D949V/*13 variants (at least one allele, prevalence 2.2%, OR 9.5 [95%CI 6.7-13.5]) significantly improved the model (p < 0.0001). The addition of HapB3 (prevalence 4.0%, 98.6% heterozygous), in spite of significant association with toxicity (OR 1.8 [95%CI 1.2-2.7]), did not improve the model. MIR27A rs895819 was not associated with toxicity, irrespective of DPYD variants. CONCLUSIONS: FUSAFE meta-analysis highlights the major relevance of DPYD *2A/p.D949V/*13 combined with clinical variables to identify patients at risk of very severe FP-related toxicity.

Features of colorectal adenomas among young patients with Lynch syndrome according to path_MMR: Results from the PRED-IdF registry.

BACKGROUND: Lynch syndrome (LS) is the most frequent inherited colorectal cancer syndrome. AIM: To assess the burden of adenoma in LS patients under 50 years-old followed in the PRED-IdF network. METHODS: From January 2010 to January 2019, all patients under 50 years of age with a confirmed LS germline mutation were included. The main objective was the description of adenomas characteristics according to path_MMR. RESULTS: We analyzed data from 708 patients (mean age 34.8 ± 8.6), of which 41.8 % were male. Among these patients, 37.6% had path_MLH1, 45.4% path_MSH2, 13.9% path_MSH6, 2.9% path_PMS2, and 1.2% path_EpCAM. The analysis included 1721 (70.9%) follow-up colonoscopies. A total of 682 adenomas were detected, including 140 (20.5%) advanced adenomas. The adenoma detection rates during the first and follow-up colonoscopies were 19.2% and 20.5%, respectively. Most adenomas were <10 mm (57.9%), located in the proximal colon (334, 48.9%), and presented as non-polypoid lesions (493, 72.3%). The median growth time for adenomas was 23 months (range 9-114) irrespective of the path_MMR mutation (p = 0.62). CONCLUSION: LS patients under 50 years of age have a high burden of adenomas, particularly small non-polypoid adenomas located in the proximal colon. These results highlight the need for intensive screening, with a particular focus on the proximal colon.

International Validation of the Immunoscore Biopsy in Patients With Rectal Cancer Managed by a Watch-and-Wait Strategy.

PURPOSE: No biomarker capable of improving selection and monitoring of patients with rectal cancer managed by watch-and-wait (W&W) strategy is currently available. Prognostic performance of the Immunoscore biopsy (ISB) was recently suggested in a preliminary study. METHODS: This international validation study included 249 patients with clinical complete response (cCR) managed by W&W strategy. Intratumoral CD3+ and CD8+ T cells were quantified on pretreatment rectal biopsies by digital pathology and converted to ISB. The primary end point was time to recurrence (TTR; the time from the end of neoadjuvant treatment to the date of local regrowth or distant metastasis). Associations between ISB and outcomes were analyzed by stratified Cox regression adjusted for confounders. Immune status of tumor-draining lymph nodes (n = 161) of 17 additional patients treated by neoadjuvant chemoradiotherapy and surgery was investigated by 3'RNA-Seq and immunofluorescence. RESULTS: Recurrence-free rates at 5 years were 91.3% (82.4%-100.0%), 62.5% (53.2%-73.3%), and 53.1% (42.4%-66.5%) with ISB High, ISB Intermediate, and ISB Low, respectively (hazard ratio [HR; Low v High], 6.51; 95% CI, 1.99 to 21.28; log-rank P = .0004). ISB was also significantly associated with disease-free survival (log-rank P = .0002), and predicted both local regrowth and distant metastasis. In multivariate analysis, ISB was independent of patient age, sex, tumor location, cT stage (T, primary tumor; c, clinical), cN stage (N, regional lymph node; c, clinical), and was the strongest predictor for TTR (HR [ISB High v Low], 6.93; 95% CI, 2.08 to 23.15; P = .0017). The addition of ISB to a clinical-based model significantly improved the prediction of recurrence. Finally, B-cell proliferation and memory in draining lymph nodes was evidenced in the draining lymph nodes of patients with cCR. CONCLUSION: The ISB is validated as a biomarker to predict both local regrowth and distant metastasis, with a gradual scaling of the risk of pejorative outcome.

Circadian rhythm and circulating cell-free DNA release on healthy subjects.

In the last decade, clinical studies have investigated the clinical relevance of circulating cell-free-DNA (ccfDNA) as a diagnostic and prognosis tool in various diseases including cancers. However, limited knowledge on ccfDNA biology restrains its full development in the clinical practice. To improve our understanding, we evaluated the impact of the circadian rhythm on ccfDNA release in healthy subjects over a 24-h period. 10 healthy female subjects underwent blood sampling at 8am and 20 healthy male subjects underwent serial blood sampling (8:00 AM, 9:00 AM, 12:00 PM, 4:00 PM, 8:00 PM, 12:00 AM, 4 AM (+ 1 Day) and 8 AM (+ 1 Day)). We performed digital droplet-based PCR (ddPCR) assays to target 2 DNA fragments (69 & 243 bp) located in the KRAS gene to determine the ccfDNA concentration and fragmentation profile. As control, half of the samples were re-analyzed by capillary miniaturized electrophoresis (BIAbooster system). Overall, we did not detect any influence of the circadian rhythm on ccfDNA release. Instead, we observed a decrease in the ccfDNA concentration after meal ingestion, suggesting either a post-prandial effect or a technical detection bias due to a higher plasma load in lipids and triglycerides. We also noticed a potential effect of gender, weight and creatinine levels on ccfDNA concentration.

Time to recurrence and its relation to survival after recurrence in patients resected for stage III colon cancer.

BACKGROUND: It is intuitively thought that early relapse is associated with poor survival after recurrence (SAR) in resected colon cancer (CC) patients, but this has never been formally studied. METHODS: We pooled data from stage III patients treated with oxaliplatin-based adjuvant therapy in two phase III trials, to analyse time to recurrence (TTR) and its relationship with SAR. TTR and SAR were also studied according to molecular status (mismatch repair (MMR), RAS, and BRAFV600E). Early relapsing patients were defined as patients having a TTR event within 12 months after starting adjuvant chemotherapy. RESULTS: 4548 stage III CC patients were included in the present analysis. Deficient MMR (dMMR) CC patients experienced fewer recurrences than proficient (p)MMR CC patients (18.8% versus 27.6%) but had a significantly shorter median TTR (mTTR; 0.74 versus 1.40 years, p < 0.0001). In pMMR patients, BRAF and RAS mutations were also associated with earlier mTTR as compared to double wild-type (WT) patients (0.99 versus 1.38 versus 1.54 years, respectively, p < 0.0001). Early recurrence occurred in 397 patients and was associated with a median SAR (2.2 versus 3.3 years, p = 0.0007). However, this association was mainly due to pMMR/RAS and BRAFV600E mutated tumours and was not confirmed in dMMR and pMMR/double WT subgroups. CONCLUSION: In resected stage III CC treated with standard oxaliplatin-based adjuvant therapy, TTR varies between dMMR, pMMR/RAS, or BRAFV600E mutated and pMMR/double WT tumours. In addition, early relapse is associated with poor survival, mainly due to patients resected for a pMMR/RAS or BRAFV600E mutated tumour.

Primary Colorectal Tumor Displays Differential Genomic Expression Profiles Associated with Hepatic and Peritoneal Metastases.

BACKGROUND: Despite improvements in characterization of CRC heterogeneity, appropriate risk stratification tools are still lacking in clinical practice. This study aimed to elucidate the primary tumor transcriptomic signatures associated with distinct metastatic routes. METHODS: Primary tumor specimens obtained from CRC patients with either isolated LM (CRC-Liver) or PM (CRC-Peritoneum) were analyzed by transcriptomic mRNA sequencing, gene set enrichment analyses (GSEA) and immunohistochemistry. We further assessed the clinico-pathological associations and prognostic value of our signature in the COAD-TCGA independent cohort. RESULTS: We identified a significantly different distribution of Consensus Molecular Subtypes between CRC-Liver and CRC-peritoneum groups. A transcriptomic signature based on 61 genes discriminated between liver and peritoneal metastatic routes. GSEA showed a higher expression of immune response and epithelial invasion pathways in CRC-Peritoneum samples and activation of proliferation and metabolic pathways in CRC-Liver samples. The biological relevance of RNA-Seq results was validated by the immunohistochemical expression of three significantly differentially expressed genes (ACE2, CLDN18 and DUSP4) in our signature. In silico analysis of the COAD-TCGA showed that the CRC-Peritoneum signature was associated with negative prognostic factors and poor overall and disease-free survivals. CONCLUSIONS: CRC primary tumors spreading to the liver and peritoneum display significantly different transcriptomic profiles. The implementation of this signature in clinical practice could contribute to identify new therapeutic targets for stage IV CRC and to define individualized follow-up programs in stage II-III CRC.

Avelumab vs Standard Second-Line Chemotherapy in Patients With Metastatic Colorectal Cancer and Microsatellite Instability: A Randomized Clinical Trial.

Importance: Only 1 randomized clinical trial has shown the superiority of immune checkpoint inhibitors in patients with deficient mismatch repair and/or microsatellite instability (dMMR/MSI) metastatic colorectal cancer (mCRC) in the first-line setting. Objectives: To determine whether avelumab (an anti-programmed cell death ligand 1 antibody) improves progression-free survival (PFS) compared with standard second-line chemotherapy in patients with dMMR/MSI mCRC. Design, Setting, and Participants: The SAMCO-PRODIGE 54 trial is a national open-label phase 2 randomized clinical trial that was conducted from April 24, 2018, to April 29, 2021, at 49 French sites. Patients with dMMR/MSI mCRC who experienced progression while receiving standard first-line therapy were included in the analysis. Interventions: Patients were randomized to receive standard second-line therapy or avelumab every 2 weeks until progression, unacceptable toxic effects, or patient refusal. Main Outcome and Measures: The primary end point was PFS according to RECIST (Response Evaluation Criteria in Solid Tumours), version 1.1, evaluated by investigators in patients with mCRC and confirmed dMMR and MSI status who received at least 1 dose of treatment (modified intention-to-treat [mITT] population). Results: A total of 122 patients were enrolled in the mITT population. Median age was 66 (IQR, 56-76) years, 65 patients (53.3%) were women, 100 (82.0%) had a right-sided tumor, and 52 (42.6%) had BRAF V600E-mutated tumors. There was no difference in patients and tumor characteristics between treatment groups. No new safety concerns in either group were detected, with fewer treatment-related adverse events of at least grade 3 in the avelumab group than in the chemotherapy group (20 [31.7%] vs 34 [53.1%]; P = .02). After a median follow-up of 33.3 (95% CI, 28.3-34.8) months, avelumab was superior to chemotherapy with or without targeted agents with respect to PFS (15 [24.6%] vs 5 [8.2%] among patients without progression; P = .03). Rates of PFS rates at 12 months were 31.2% (95% CI, 20.1%-42.9%) and 19.4% (95% CI, 10.6%-30.2%) in the avelumab and control groups, respectively, and 27.4% (95% CI, 16.8%-39.0%) and 9.1% (95% CI, 3.2%-18.8%) at 18 months. Objective response rates were similar in both groups (18 [29.5%] vs 16 [26.2%]; P = .45). Among patients with disease control, 18 (75.7%) in the avelumab group compared with 9 (19.1%) in the control group had ongoing disease control at 18 months. Conclusions: The SAMCO-PRODIGE 54 phase 2 randomized clinical trial showed, in patients with dMMR/MSI mCRC, better PFS and disease control duration with avelumab over standard second-line treatment, with a favorable safety profile. Trial Registration: ClinicalTrials.gov Identifier: NCT03186326.

Downregulation of mitochondrial complex I induces ROS production in colorectal cancer subtypes that differently controls migration.

BACKGROUND: Colorectal cancer (CRC) can be classified into four molecular subtypes (CMS) among which CMS1 is associated with the best prognosis, while CMS4, the mesenchymal subtype, has the worst outcome. Although mitochondria are considered to be hubs of numerous signaling pathways, the study of mitochondrial metabolism has been neglected for many years. Mitochondrial Complex I (CI) plays a dual role, both in energy and reactive oxygen species (ROS) production. However, the possible contribution of CI to tumorigenesis in cancer remains unclear. The purpose of this study was to investigate the CI under the prism of the CMS classification of CRC in ex vivo models. METHODS: Biochemical dosages, bioenergetics analysis and western-blot were used to characterize CI expression, function and redox balance in LoVo and MDST8 cell lines, belonging to CMS1 and CMS4 subgroups, respectively. Cell proliferation and migration were assessed by xCELLigence technology. Overproduction or scavenging of mitochondrial ROS (mtROS) were performed to analyze the effect of mtROS on proliferation, migration, and mesenchymal markers. Focal adhesion kinase (FAK) and its activation were analyzed by immunofluorescence. We assessed the distribution of two CI scores in CRC cohorts according to CMS classification and their relevance for patient survival. RESULTS: We found that CI is downregulated in CMS4 cells and is associated with elevated mtROS. We establish for the first time that in these migrating cells, mtROS production is maintained at optimal levels not only through changes in CI activity but also by inactivation/acetylation of superoxide dismutase 2 (SOD2), a major mitochondrial antioxidant enzyme. We show that promoting or scavenging mtROS both mitigate CMS4 cells' migration. Our results also point to a mtROS-mediated focal adhesion kinase (FAK) activation, which likely sustains their migratory phenotype. Using cohorts of CRC patients, we document that the expression of CI is downregulated in the CMS4 subgroup, and that low CI expression is associated with poor prognosis. Patients' datasets reveal an inverse correlation between CI and the epithelial-mesenchymal transition (EMT) pathway. CONCLUSION: We showed that inhibition of CI contributes to heighten mtROS, which likely foster MDST8 migration and might account for the specific EMT signature of CMS4 tumors. These data reveal a novel role of mitochondrial CI in CRC, with biological consequences that may be targeted with anti- or pro-oxidant drugs in clinical practice.

Molecular-based targeted therapies in patients with hepatocellular carcinoma and hepato-cholangiocarcinoma refractory to atezolizumab/bevacizumab.

BACKGROUND & AIMS: The "French Medicine Genomic program 2025" has been designed to give patients with cancers that are refractory to systemic treatments access to off-label therapies adapted to their specific genomic profile. Herein, we reported the results of this program in patients with advanced hepatocellular carcinoma (HCC) and hepato-cholangiocarcinoma (H-CCK). METHODS: In one center, all patients with HCC or H-CCK who progressed under atezolizumab/bevacizumab with available tumor frozen samples benefited from whole-genome/-exome and RNA sequencing. Targeted therapies were matched to genomic alterations following the recommendations of a molecular tumor board and radiological response and overall survival were assessed. RESULTS: Among 135 patients with HCC and H-CCK treated by atezolizumab/bevacizumab, 20 patients benefited from genomic analysis after progression (16 HCC; 4 H-CCK). Nineteen patients had analyzable data, 70% were male, median age was 57 years, 65% had metastatic disease and 45% had vascular invasion. Among these 19 patients, 14 patients (76%) harbored at least one actionable genomic alteration and 9/14 received an adapted targeted therapy (45%). One patient with H-CCK showing CDK4 amplification was treated with palbociclib and achieved a partial radiological response for 16 months. Another patient with H-CCK, high HER2 overexpression and a high homologous recombination score was treated with trastuzumab/olaparib and had stable disease. One patient with an HCC and bi-allelic inactivation of TSC2 achieved a complete radiological response under everolimus. The remaining six treated patients (all HCC) had progressive disease, including three patients treated with trametinib, two with everolimus and one with olaparib. CONCLUSION: Molecular-based guided therapy is feasible in patients with HCC/H-CCK progressing under atezolizumab/bevacizumab and may be useful in a small subset of patients. IMPACT AND IMPLICATIONS: The use of whole-genome/-exome and RNA sequencing in clinical practice has not been reported in patients with hepatocellular carcinoma and hepato-cholangiocarcinoma. Herein, we performed a pilot study which suggested that whole-genome/-exome and RNA sequencing is feasible on tumor biopsies from patients refractory to atezolizumab/bevacizumab, with a small subset of patients exhibiting at least one actionable genomic alteration and receiving an adapted targeted therapy. This proof-of-concept study suggests that this clinical strategy could benefit a small subset of patients. Finally, validation of this approach will be required in a larger cohort of patients.

Transcriptomic Signatures of MSI-High Metastatic Colorectal Cancer Predict Efficacy of Immune Checkpoint Inhibitors.

PURPOSE: Microsatellite instability (MSI) is currently the only predictive biomarker of efficacy of immune checkpoint inhibitors (ICI) in metastatic colorectal cancers (mCRC). However, 10% to 40% of patients with MSI mCRC will experience a primary resistance to ICI. EXPERIMENTAL DESIGN: In two cohorts of patients with MSI mCRC treated with ICI (exploratory, N = 103; validation, N = 35), 3' RNA sequencing was performed from primary tumors. Previously described single-cell transcriptomic signatures of tumor microenvironment (TME) were analyzed. RESULTS: In the exploratory cohort, the unsupervised clustering allowed the identification of three clusters of tumors with distinct transcriptional profiles: cluster A ("stromalHIGH-proliferationLOW"), cluster B ("stromalHIGH-proliferationMED"), and cluster C ("stromalLOW-proliferationHIGH"), with an enrichment of patients progressing at first disease assessment under ICI in cluster A (30% vs. 12% in cluster B and 8.1% in cluster C; P = 0.074). Progression-free survival (PFS) was also significantly shorter in patients belonging to cluster A, compared with clusters B or C (P < 0.001) with 2-year PFS rates of 33.5%, 80.5%, and 78.3%, respectively. In multivariate analysis, PFS was still significantly longer in patients belonging to cluster B [HR, 0.19; 95% confidence interval (CI), 0.08-0.45; P < 0.001] and cluster C (HR, 0.25; 95% CI, 0.10-0.59; P = 0.02), compared with patients belonging to cluster A. The association of this clustering with PFS under ICI was confirmed in the validation cohort. PFS related to non-ICI-based regimens was not significantly different according to cluster. CONCLUSIONS: This unsupervised transcriptomic classification identified three groups of MSI mCRCs with different compositions of TME cells and proliferative capacities of TME/tumor cells. The "stromalHIGH-proliferationLOW" cluster is associated with a poorer prognosis with ICI treatment.

Circulating tumour DNA at baseline for individualised prognostication in patients with chemotherapy-naïve metastatic colorectal cancer. An AGEO prospective study.

PURPOSE: Baseline circulating tumour DNA (ctDNA) is a potential prognostic marker in metastatic colorectal cancer (mCRC) patients. However, few studies have compared ctDNA with the usual prognostic factors, and no ctDNA cut-off has been proposed for daily use in clinical practice. PATIENTS AND METHODS: Chemotherapy-naive patients with mCRC were prospectively included. Plasma samples were collected at diagnosis and analysed centrally by both NGS and methylation digital PCR. Baseline patient and disease characteristics, treatment regimens, and secondary surgeries were collected. The restricted cubic spline method was used to define the optimal cut-off of ctDNA mutated allelic frequency (MAF). Prognostic values were assessed on overall survival (OS) using Cox models. RESULTS: From July 2015 to December 2016, 412 patients were included. ctDNA was undetectable in 83 patients (20%). ctDNA was an independent prognostic marker for OS considering the whole study population. The optimal cut-off for ctDNA MAF was 20% with median OS of 16.0 and 35.8 months for patients with MAF ≥20% and<20%, respectively (hazard ratio = 0.40; 95% confidence intervals: 0.31-0.51; P < 0.0001). The independent prognostic value of ctDNA MAF at 20% was confirmed in subgroups defined by RAS/BRAF status or resectability of metastases. Combining ctDNA MAF and carcinoembryonic antigen levels allowed us to define three different prognostic groups with median OS of 14.2, 21.1, and 46.4 months (P < 0.0001). CONCLUSION: ctDNA with a MAF cut-off of 20% improves prognostication of chemotherapy-naïve mCRC patients and may be useful in the future for individualised therapeutic decisions and as a stratification factor in clinical trials. TRIAL REGISTRATION: Clinicaltrials.gov, NCT02502656.

New hormone receptor-positive breast cancer mouse cell line mimicking the immune microenvironment of anti-PD-1 resistant mammary carcinoma.

BACKGROUND: Progress in breast cancer (BC) research relies on the availability of suitable cell lines that can be implanted in immunocompetent laboratory mice. The best studied mouse strain, C57BL/6, is also the only one for which multiple genetic variants are available to facilitate the exploration of the cancer-immunity dialog. Driven by the fact that no hormone receptor-positive (HR+) C57BL/6-derived mammary carcinoma cell lines are available, we decided to establish such cell lines. METHODS: BC was induced in female C57BL/6 mice using a synthetic progesterone analog (medroxyprogesterone acetate, MPA) combined with a DNA damaging agent (7,12-dimethylbenz[a]anthracene, DMBA). Cell lines were established from these tumors and selected for dual (estrogen+progesterone) receptor positivity, as well as transplantability into C57BL/6 immunocompetent females. RESULTS: One cell line, which we called B6BC, fulfilled these criteria and allowed for the establishment of invasive estrogen receptor-positive (ER+) tumors with features of epithelial to mesenchymal transition that were abundantly infiltrated by myeloid immune populations but scarcely by T lymphocytes, as determined by single-nucleus RNA sequencing and high-dimensional leukocyte profiling. Such tumors failed to respond to programmed cell death-1 (PD-1) blockade, but reduced their growth on treatment with ER antagonists, as well as with anthracycline-based chemotherapy, which was not influenced by T-cell depletion. Moreover, B6BC-derived tumors reduced their growth on CD11b blockade, indicating tumor sustainment by myeloid cells. The immune environment and treatment responses recapitulated by B6BC-derived tumors diverged from those of ER+ TS/A cell-derived tumors in BALB/C mice, and of ER- E0771 cell-derived and MPA/DMBA-induced tumors in C57BL/6 mice. CONCLUSIONS: B6BC is the first transplantable HR+ BC cell line derived from C57BL/6 mice and B6BC-derived tumors recapitulate the complex tumor microenvironment of locally advanced HR+ BC naturally resistant to PD-1 immunotherapy.

Intratumor heterogeneity and cell secretome promote chemotherapy resistance and progression of colorectal cancer.

The major underlying cause for the high mortality rate in colorectal cancer (CRC) relies on its drug resistance, to which intratumor heterogeneity (ITH) contributes substantially. CRC tumors have been reported to comprise heterogeneous populations of cancer cells that can be grouped into 4 consensus molecular subtypes (CMS). However, the impact of inter-cellular interaction between these cellular states on the emergence of drug resistance and CRC progression remains elusive. Here, we explored the interaction between cell lines belonging to the CMS1 (HCT116 and LoVo) and the CMS4 (SW620 and MDST8) in a 3D coculture model, mimicking the ITH of CRC. The spatial distribution of each cell population showed that CMS1 cells had a preference to grow in the center of cocultured spheroids, while CMS4 cells localized at the periphery, in line with observations in tumors from CRC patients. Cocultures of CMS1 and CMS4 cells did not alter cell growth, but significantly sustained the survival of both CMS1 and CMS4 cells in response to the front-line chemotherapeutic agent 5-fluorouracil (5-FU). Mechanistically, the secretome of CMS1 cells exhibited a remarkable protective effect for CMS4 cells against 5-FU treatment, while promoting cellular invasion. Secreted metabolites may be responsible for these effects, as demonstrated by the existence of 5-FU induced metabolomic shifts, as well as by the experimental transfer of the metabolome between CMS1 and CMS4 cells. Overall, our results suggest that the interplay between CMS1 and CMS4 cells stimulates CRC progression and reduces the efficacy of chemotherapy.