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Background: Immune checkpoint blockade (ICB) in combination with chemotherapy improves outcome of patients with triple-negative breast cancer (TNBC) in metastatic and early settings. The identification of predictive biomarkers able to guide treatment decisions is challenging and currently limited to programmed death-ligand 1 (PD-L1) expression and high tumor mutational burden (TMB) in the advanced setting, with several limitations. Materials and methods: We carried out a retrospective analysis of clinical-pathological and molecular characteristics of tumor samples from 11 patients with advanced TNBC treated with single-agent pembrolizumab participating in two early-phase clinical trials: KEYNOTE-012 and KEYNOTE-086. Clinical, imaging, pathological [i.e. tumor-infiltrating lymphocytes (TILs), PD-L1 status], RNA sequencing, and whole-exome sequencing data were analyzed. We compared our results with publicly available transcriptomic data from TNBC cohorts from TCGA and METABRIC. Results: Response to pembrolizumab was heterogeneous: two patients experienced exceptional long-lasting responses, six rapid progressions, and three relatively slower disease progression. Neither PD-L1 nor stromal TILs were significantly associated with response to treatment. Increased TMB values were observed in tumor samples from exceptional responders compared to the rest of the cohort (P = 3.4 × 10−4). Tumors from exceptional responders were enriched in adaptive and innate immune cell signatures. Expression of regulatory T-cell markers (FOXP3, CCR4, CCR8, TIGIT) was mainly observed in tumors from responders except for glycoprotein-A repetitions predominant (GARP), which was overexpressed in tumors from rapid progressors. GARP RNA expression in primary breast tumors from the public dataset was significantly associated with a worse prognosis. Conclusions: The wide spectrum of clinical responses to ICB supports that TNBC is a heterogeneous disease. Tumors with high TMB respond better to ICB. H, SCOPUS: ar.j, info:eu-repo/semantics/published

G protein-coupled receptors (GPCRs) are seven transmembrane domains proteins that constitute the largest family of membrane receptors. These receptors respond to a wide variety of extracellular stimuli, proteins, peptides, lipids, ions, sugars, and light, which trigger a conformational change in the receptor and initiate downstream signaling cascades. GPCRs are involved in most physiological and pathophysiological processes, making them crucial targets for therapeutic drugs. However, many of them remain orphan without yet identified endogenous ligands. Super conserved receptors expressed in brain (SREB) constitute a subfamily of three orphan GPCRs characterized by their high evolutionary conservation in vertebrates and their high expression in the brain. They include GPR27 (SREB1), GPR85 (SREB2), and GPR173 (SREB3). In this study we aimed to characterize this subfamily by studying their pharmacological and signaling properties. We attempted to deorphanize these receptors using an aequorin-based calcium mobilization assay, screening for their endogenous ligands in fish tissue extracts. We also tested whether they displayed constitutive activity in cell-based assays by overexpressing them in heterologous systems. Then, given their particularly strong conservation of amino acid sequences in intracellular loops, an unusual feature in GPCR subgroups, we hypothesized that SREBs might interact with unusual intracellular partners. We therefore focused our efforts on the identification of such partners, using a BioID2 proximity-labeling approach, in order to shed light on original interaction networks. Although our deorphanization and constitutive activity approaches were not successful, we identified several intracellular interactors of SREBs, the amino acid transporter subunit SLC3A2, the AKAP protein LRBA, and the cytoskeletal protein 4.1G (EPB41L2) and confirmed these physical interactions. We further characterized the interaction between EPB41L2 and SREB1 by showing the, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

To explore whether specific triple-negative breast cancer (TNBC) molecular subtypes are predictive for a benefit from maintenance low-dose cyclophosphamide and methotrexate (CM) in the adjuvant IBCSG 22-00 phase III clinical trial., SCOPUS: ar.j, info:eu-repo/semantics/published

Triple negative breast cancer (TNBC) is a highly heterogeneous disease with a poor prognosis and a paucity of therapeutic options. In recent years, immunotherapy has emerged as a new treatment option for patients with TNBC. However, this therapeutic evolution is paralleled by a growing need for biomarkers which allow for a better selection of patients who are most likely to benefit from this immune checkpoint inhibitor (ICI)-based regimen. These biomarkers will not only facilitate a better optimization of treatment strategies, but they will also avoid unnecessary side effects in non-responders, and limit the increasing financial toxicity linked to the use of these agents. Huge efforts have been deployed to identify predictive biomarkers for the ICI, but until now, the fruits of this labor remained largely unsatisfactory. Among clinically validated biomarkers, only programmed death-ligand 1 protein (PD-L1) expression has been prospectively assessed in TNBC trials. In addition to this, microsatellite instability and a high tumor mutational burden are approved as tumor agnostic biomarkers, but only a small percentage of TNBC fits this category. Furthermore, TNBC should no longer be approached as a single biological entity, but rather as a complex disease with different molecular, clinicopathological, and tumor microenvironment subgroups. This review provides an overview of the validated and evolving predictive biomarkers for a response to ICI in TNBC., SCOPUS: re.j, info:eu-repo/semantics/published

A novel pillar of epigenetics has recently gained the attention of the scientific community: mRNA modifications. This new field, commonly referred to as “epitranscriptomics”, has emerged as a crucial regulatory level of gene expression. Yet, the study of these modifications is still in its early days and has definitely not reached its full potential.The presence of modifications in mitochondrial mRNAs has emerged very recently, and is suggested to play a role in fine-tuning mitochondrial gene expression and function. However, the epitranscriptomic landscape of mitochondrial mRNA remains very poorly explored. The aim of this thesis was to investigate a novel mitochondrial mRNA modification: the N3- methylcytosine (m3C), from its physiological to its pathological relevance.In the first part of this thesis, we show that METTL8 catalyzes m3C on mitochondrial mRNA, specifically on complex I subunits transcripts of the electron transport chain (ETC). Furthermore, we report that METTL8 influences the protein level of its modified targets, and therefore regulates the complex I activity. Finally, we confirm the complex I deficiency in a Mettl8-knockout mouse model, that we established.In the second part of this thesis, we aimed to tackle the pathological impact of this modification. In regard to the emerging crosstalk between cervical cancer and mitochondria, we decided to investigate for the first time, the potential pathological role of m3C in cervical cancer. First, we confirmed that METTL8 expression is higher in patient’s biopsies compared to normal cervical tissues, and correlates with the disease progression. We then showed that METTL8-dependent m3C promotes cell proliferation and migration; and tumor growth in a xenografts model. Furthermore, we decipher that METTL8-driven signature is enriched for mitochondrial functions and complex I activity.In conclusion, our findings improve our understanding of this emerging epitranscriptomic field, and specifically unravel the, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

While circulating tumour (ct)DNA is an indicator of minimal residual disease and negative prognostic factor in stage II-III colon cancer, no study has ever analysed the value of this biomarker in colon cancer patients treated with neoadjuvant chemotherapy. We sought to fill this gap by using prospectively collected plasma samples from 80 stage III colon cancer patients, receiving one cycle of neoadjuvant FOLFOX followed by surgery +/- adjuvant FOLFOX in the PePiTA trial. Samples were collected at baseline, 2 weeks and surgery. NPY and WIF1 were selected as universal methylation markers for ctDNA, and analysed with ddPCR technology. ROC curves were applied for cut-off points, and outcome measures included 5-year disease-free survival (DFS) and 6-year overall survival (OS). After a median follow-up of 52.5 months, baseline circulating-free (cf) DNA was an independent prognostic factor for DFS (HR 3.35, 95% CI: 1.15-9.77, = .03), and a trend towards a similar association was observed for relative cfDNA changes between baseline and surgery (HR 2.57, 95% CI: 0.94-7.05, = .07). Among 60 ctDNA assessable patients, 25 (42%) had detectable ctDNA at baseline. While detection of ctDNA at any pre-operative timepoint was not associated with outcome, patients with ctDNA increase (change of the worst trending methylation marker ≥11%, or mean ctDNA change of NPY and WIF1 ≥ 0%) between baseline and surgery showed a trend towards worse 5-year DFS (HR 3.66, 95% CI: 0.81-16.44, = .09). This is the first study of ctDNA in the neoadjuvant setting of early-stage colon cancer. Results are hypothesis-generating and should be confirmed in larger series.

While circulating tumour (ct)DNA is an indicator of minimal residual disease and negative prognostic factor in stage II-III colon cancer, no study has ever analysed the value of this biomarker in colon cancer patients treated with neoadjuvant chemotherapy. We sought to fill this gap by using prospectively collected plasma samples from 80 stage III colon cancer patients, receiving one cycle of neoadjuvant FOLFOX followed by surgery +/- adjuvant FOLFOX in the PePiTA trial., info:eu-repo/semantics/published

We questioned whether EMT is required for metastasis, and we have analyzed metastasis formation in two distinct SCC models, different in terms of SCC ability to undergo EMT. We demonstrated that in spontaneous mouse model in which tumors presented a wide range of EMT (from well differentiated to fully EMT-like) presented high metastatic incidence: 49% (20/70) of mice presented LN metastasis and 28% (34/70) of mice presented lung metastases. Importantly, metastatic incidence was always associated with EMT-like tumors. However, in the second mouse model, that develops mostly well-differentiated tumors, the metastatic incidence was very low: 12% (5/42) of mice developed lung metastases and 10% (4/42) developed lymph node metastases. Importantly, the rare metastases were associated with tumors presenting some degree of EMT, supporting that EMT is needed for metastases arising from skin SCC.To understand whether EMT can occur at the intermediate step between primary tumor and distant metastasis, we analyzed blood from mice based on YFP reporter. We uncovered that most CTCs were EMT-like: 80% of CTCs were EpCAM-negative. This finding suggests that EpCAM marker, a gold standard used to detect CTCs, is not optimal as it doesn’t detect CTCs of EMT phenotype thus underestimating the real number of CTCs. Importantly, we noticed that the presence of CTCs was always associated with lung metastases and indeed can be used to predict prognosis of oncologic disease. Our immunofluorescent analysis of lung and lymph nodes showed that the majority of metastases were of differentiated phenotype. For the lung metastases, 71% (36/51) of them presented epithelial phenotype YFP+/K14+/Vim-, 14% (7/51) presented differentiated epithelial features but did not express K14 or Vimentin, 12% (6/51) presented an EMT hybrid epithelial phenotype YFP+/K14+/Vim+, whereas only 4% (2/51) were purely mesenchymal YFP+/K14-/Vim+. Histological analyses were confirmed by FACS analysis showing that lung metast, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished