Abstract Immune checkpoint inhibitors (ICIs) have transformed outcomes in microsatellite instability-high (MSI-H) deficient mismatch repair (dMMR) cancers. The influence of germline MMR gene variants on ICI remains unclear. In this single-center study, 93 patients with Lynch syndrome-associated MSI-H/dMMR digestive cancers received ICI monotherapy or combination therapy. Germline MMR variants were classified, and progression-free survival (PFS) was evaluated by gene and variant type. Most patients carried MLH1 or MSH2 variants. At 24 months, estimated PFS was 81.5% for MLH1, 67.2% for MSH2/EPCAM, and 78.6% for MSH6, with no PFS events in MLH1 promoter methylation or PMS2 subgroups. No statistically significant differences in PFS were observed between gene groups. Performance status ≥ 2 was the only factor associated with poorer PFS. Neither MMR gene type nor variant class significantly influenced ICI efficacy in Lynch syndrome-related MSI-H/dMMR digestive cancers, supporting the use of ICIs regardless of germline MMR genotype.

Superficial vascular anomalies are complex disorders characterized by abnormal vascular growth. Next-generation sequencing has recently identified somatic genetic alterations associated with these malformations, offering new insights for targeted treatments. However, tissue biopsies for genetic testing can be invasive and difficult to obtain, especially in arteriovenous malformations (AVM) with hemorrhagic risks. A liquid biopsy, a non-invasive approach, offers a promising solution by detecting genetic mutations in cell-free DNA. This pilot study aimed to evaluate the feasibility of using a liquid biopsy for the genetic analysis of patients with superficial vascular anomalies through cell-free DNA sampling. Additionally, it explored whether specific sampling sites, such as the afferent artery, nidus, and efferent vein, could enhance the sensitivity of detecting pathogenic variants in patients with AVM. A total of 88 patients were enrolled, including 55 with AVM and 33 with lymphatic malformations. For patients with AVM, cell-free DNA samples were collected from peripheral blood, efferent veins, afferent arteries, and the AVM nidus. In patients with lymphatic malformations, cystic lymphatic fluid was collected by a direct puncture during diagnostic procedures. A molecular analysis was performed using a targeted gene panel relevant to somatic alterations in solid tumors. Pathogenic variants were validated by digital polymerase chain reaction for patients with lymphatic malformations. Pathogenic variants were identified in 23.6

TP53 is included in most cancer predisposition multigene panels, especially those exploring Hereditary Breast and Ovarian Cancer (HBOC) predisposition. The purpose of this study was to define the contribution of TP53 pathogenic variants (PV) to the HBOC phenotype by collecting genotypes and phenotypes of 398 patients harboring a TP53 variant identified by 53,085 HBOC panel sequencing in 15 French laboratories. Heterozygous TP53 variants were identified in 0.44% of HBOC panels, evenly distributed between PV and VUS. Breast cancers associated with TP53 were predominantly triple positive, particularly Her2+ breast cancer, in situ cancer, or phyllodes tumors ( p < 0.0001 for both). Interestingly, TP53 PV were identified across all ages in breast cancer patients, with enrichment before 36y. We demonstrated that null variants were linked with the HBOC phenotype, and missense variants, especially with a dominant negative effect, with the LFS phenotype ( p = 0.0096). Patients with breast cancer harboring null variants displayed an earlier age of onset compared to missense ( p = 0.0030). Surprisingly, we identified, in late‐onset cancer patients, TP53 hotspot PV usually identified in classic LFS, which underlines variable penetrance. Thus, this study suggests the existence of two phenotypic entities associated with TP53 PV: clinical LFS and TP53 ‐related breast cancer. The type of TP53 variant, as well as modifying factors reflected in family history, may influence these phenotypes, and both should be considered to define the clinical follow‐up of patients and relatives.

Pancreatic cancer is a rising cause of cancer death. Therapeutic options are scarce and of limited efficacy. Up to 26% of patients with metastatic pancreatic cancer could benefit from targeted therapies. We report here for the first time the case of three patients with metastatic pancreatic ductal adenocarcinoma (PDAC) without KRAS alteration for whom an activating mutation in exon 19 of the epidermal growth factor receptor ( EGFR ) gene was found through mainstreaming NGS. The EGFR variant was confirmed on multiple tumor samples and by circulating tumor DNA (ctDNA) analysis in two patients. The three patients were treated with osimertinib with early molecular, biologic, and morpho-metabolic responses. At the last follow-up, one patient had an ongoing response after 17 months, and disease control had been maintained for 8 and 6 months in the other two. Known resistance mechanisms were observed on ctDNA analysis at progression. These observations demonstrate the benefit of osimertinib for treating EGFR -mutated PDAC and highlight the interest in investigating rare molecular alterations, especially in patients without KRAS alterations.

INTRODUCTION:NSCLC is frequent and associated with poor prognosis among people living with human immunodeficiency virus (PLWHIV); nevertheless, the contributing factors remain unknown. METHODS:We prospectively compared the immunogenomic characteristics of 27 NSCLC samples from 15 PLWHIV and 12 immunocompetent patients (ICs). Tumor whole-exome and RNA sequencing, along with a bioinformatics pipeline, allowed analysis of tumor mutational burden, molecular signatures, tumor microenvironment, and prediction of tumor neoepitopes. We conducted ex vivo interferon gamma (IFNγ) enzyme-linked ImmunoSpot assays and intracellular cytokine staining flow cytometry assays to functionally validate our bioinformatic pipeline for neoepitope prediction and to investigate the antitumor immune response. RESULTS:Tumor mutational burden, molecular profiles, number of predicted neoepitopes, and their major histocompatibility complex (MHC) class I/II predicted restriction were similar in both groups. Nevertheless, T-cell responses to neoepitopes, detectable in 4 out of 11 PLWHIV and 5 out of 11 IC, were exclusively directed against MHC class II-restricted neoepitopes in PLWHIV, whereas it was balanced between MHC class I and class II neoepitopes in IC. Intracellular cytokine staining revealed primarily monofunctional responses, mainly mediated by tumor necrosis factor-α-producing CD4 T-cells against MHC class II-restricted neoepitopes, and by CD8 T-cells producing CD107, tumor necrosis factor-α, or IFNγ against MHC class I-restricted neoepitopes. A low CD4 nadir was associated with the lack of neoepitope-specific responses in PLWHIV. Furthermore, PLWHIV tumor microenvironments displayed lower neutrophil proportions and decreased T-cell function markers. CONCLUSIONS:Our results indicate that despite similar mutational profiles, HIV infection severely impairs both local and systemic antitumor immune responses in patients with NSCLC, particularly to MHC class I-restricted neoepitopes. These findings support the use of MHC-class I-restricted neoepitope-based immunotherapy in this population.

Central nervous system (CNS) dural arteriovenous fistulas (DAVF) have been reported in PTEN -related hamartoma tumor syndrome (PHTS). However, PHTS-associated DAVF remain an underexplored field of the PHTS clinical landscape. Here, we studied cases with a PTEN pathogenic variant identified between 2007 and 2020 in our laboratory ( n  = 58), and for whom brain imaging was available. Two patients had DAVF (2/58, 3.4%), both presenting at advanced stages: a 34-year-old man with a left lateral sinus DAVF at immediate risk of hemorrhage, and a 21-year-old woman with acute intracranial hypertension due to a torcular DAVF. Interestingly, not all patients had 3D TOF/MRA, the optimal sequences to detect DAVF. Early diagnosis of DAVF can be lifesaving, and is easier to treat compared to developed, proliferative, or complex lesions. As a result, one should consider brain MRI with 3D TOF/MRA in PHTS patients at genetic diagnosis, with subsequent surveillance on a case-by-case basis.

Patients with EGFR-mutated non-small cell lung cancer (NSCLC) benefit from treatment with tyrosine kinase inhibitors (TKI) targeting EGFR. Despite improvements in patient care, especially with the 3rd generation TKI osimertinib, disease relapse is observed in all patients. Among the various processes involved in TKI resistance, epithelial-to-mesenchymal transition (EMT) is far from being fully characterized. We hypothesized that the cellular prion protein PrPC could be involved in EMT and EGFR-TKI resistance in NSCLC. Using 5 independent lung adenocarcinoma datasets, including our own cohort, we document that the expression of the PRNP gene encoding PrPC is associated with EMT. By manipulating the levels of PrPC in different EGFR-mutated NSCLC cell lines, we firmly establish that the expression of PrPC is mandatory for cells to maintain or acquire a mesenchymal phenotype. Mechanistically, we show that PrPC operates through an ILK-RBPJ cascade, which also controls the expression of EGFR. Our data further demonstrate that PrPC levels are elevated in EGFR-mutated versus wild-type tumours or upon EGFR activation in vitro. In addition, we provide evidence that PRNP levels increase with TKI resistance and that reducing PRNP expression sensitizes cells to osimertinib. Finally, we found that plasma PrPC levels are increased in EGFR-mutated NSCLC patients from 2 independent cohorts and that their longitudinal evolution mirrors that of disease. Altogether, these findings define PrPC as a candidate driver of EMT-dependent resistance to EGFR-TKI in NSCLC. They further suggest that monitoring plasma PrPC levels may represent a valuable non-invasive strategy for patient follow-up and warrant considering PrPC-targeted therapies for EGFR-mutated NSCLC patients with TKI failure.

Background: Mismatch repair-deficient (dMMR) tumors displaying microsatellite instability (MSI) represent a paradigm for the success of immune checkpoint inhibitor (ICI)-based immunotherapy, particularly in patients with metastatic colorectal cancer (mCRC). However, a proportion of patients with dMMR/MSI mCRC exhibit resistance to ICI. Identification of tools predicting MSI mCRC patient response to ICI is required for the design of future strategies further improving this therapy.Patients and methods: We combined high-throughput DNA and RNA sequencing of tumors from 116 patients with MSI mCRC treated with anti-programmed cell death protein 1 & PLUSMN; anti-cytotoxic T-lymphocyte-associated protein 4 of the NIPICOL phase II trial (C1, NCT03350126, discovery set) and the ImmunoMSI prospective cohort (C2, validation set). The DNA/RNA predictors whose status was significantly associated with ICI status of response in C1 were subsequently validated in C2. Primary endpoint was progression-free survival by immune RECIST (iRECIST) (iPFS).Results: Analyses showed no impact of previously suggested DNA/RNA indicators of resistance to ICI, e.g. MSIsensor score, tumor mutational burden, or specific cellular and molecular tumoral contingents. By contrast, iPFS under ICI was shown in C1 and C2 to depend both on a multiplex MSI signature involving the mutations of 19 microsatellites hazard ratio cohort C2 (HRC2) = 3.63; 95% confidence interval (CI) 1.65-7.99; P = 1.4 x 10e3] and the expression of a set of 182 RNA markers with a non-epithelial transforming growth factor beta (TGFB)-related desmoplastic orientation (HRC2 = 1.75; 95% CI 1.03-2.98; P = 0.035). Both DNA and RNA signatures were independently predictive of iPFS.Conclusions: iPFS in patients with MSI mCRC can be predicted by simply analyzing the mutational status of DNA microsatellite-containing genes in epithelial tumor cells together with non-epithelial TGFB-related desmoplastic RNA markers.

Abstract Background Small-cell lung cancer (SCLC) accounts for approximately 15% of lung cancer and is associated with poor prognosis. In platinum-refractory or -resistant SCLC patients, few treatment options are available. Topotecan is one of the standards of care for these patients, however, due to its high toxicity, several different approaches are employed. FOLFIRI (folinate, 5-fluorouracil and irinotecan) is a chemotherapy regimen used in digestive neuroendocrine carcinoma, which shares pathological similarities with SCLC. In this retrospective study, we evaluated the efficacy and safety of FOLFIRI in patients with platinum-resistant/refractory SCLC. Methods Medical records from all consecutive SCLC patients treated with FOLFIRI in a French University Hospital from 2013 to 2021 were analyzed retrospectively. The primary endpoint was the objective response rate according to RECIST v1.1 or EORTC criteria (ORR); secondary endpoints included duration of response, disease control rate, progression-free survival (PFS), overall survival (OS) and safety profile. Results Thirty-four patients with metastatic platinum-resistant (n = 14) or -refractory (n = 20) SCLC were included. Twenty-eight were evaluable for response, with a partial response observed in 5 patients for an overall ORR in the evaluable population of 17.9% (5/28) and 14.7% (5/34) in the overall population. The disease control rate was 50% (14/28) in the evaluable population. The median PFS and OS were 2.8 months (95%CI, 2.0–5.2 months) and 5.3 months (95%CI, 3.5–8.9 months), respectively. All patients were included in the safety analysis. Grade 3 or 4 adverse events occurred in 13 (38.2%) patients. The most common grade 3 or 4 adverse events were asthenia, neutropenia, thrombopenia and diarrhea. There was no adverse event leading to discontinuation or death. Conclusion FOLFIRI showed some activity for platinum-resistant/refractory SCLC in terms of overall response and had an acceptable safety profile. However, caution is needed in interpreting this result. FOLFIRI could represent a potential new treatment for platinum-resistant/refractory SCLC patients. Further prospective studies are needed to assess the benefits of this chemotherapy regimen. HIGHLIGHTS FOLFIRI showed some activity for platinum-resistant/refractory SCLC in terms of overall response. FOLFIRI was well-tolerated in platinum resistant/refractory SLCL patients. FOLFIRI could represent a potential new treatment for SCLC, prospective studies are needed.

PURPOSE:The Lynch syndrome (LS)-glioma association is poorly documented. As for mismatch repair deficiency (MMRd) in glioma, a hallmark of LS-associated tumors, there are only limited data available. We determined MMRd and LS prevalence in a large series of unselected gliomas, and explored the associated characteristics. Both have major implications in terms of treatment, screening, and prevention.METHODS:Somatic next-generation sequencing was performed on 1,225 treatment-naive adult gliomas referred between 2017 and June 2022. For gliomas with ≥1 MMR pathogenic variant (PV), MMR immunohistochemistry (IHC) was done. Gliomas with ≥1 PV and protein expression loss were considered MMRd. Eligible patients had germline testing. To further explore MMRd specifically in glioblastomas, isocitrate dehydrogenase (IDH)-wild type (wt), we performed IHC, and complementary sequencing when indicated, in a series of tumors diagnosed over the 2007-2021 period.RESULTS:Nine gliomas were MMRd (9/1,225; 0.73%). Age at glioma diagnosis was <50 years for all but one case. Eight were glioblastomas, IDH-wt, and one was an astrocytoma, IDH-mutant. ATRX (n = 5) and TP53 (n = 8) PV were common. There was no TERT promoter PV or EGFR amplification. LS prevalence was 5/1,225 (0.41%). One 77-year-old patient was a known LS case. Four cases had a novel LS diagnosis, with germline PV in MSH2 (n = 3) and MLH1 (n = 1). One additional patient had PMS2-associated constitutional mismatch repair deficiency. Germline testing was negative in three MSH6-deficient tumors. In the second series of glioblastomas, IDH-wt, MMRd prevalence was 12.5% in the <40-year age group, 2.6% in the 40-49 year age group, and 1.6% the ≥50 year age group.CONCLUSION:Screening for MMRd and LS should be systematic in glioblastomas, IDH-wt, diagnosed under age 50 years.

IntroductionALK tyrosine kinase inhibitors (ALK TKIs) have improved prognosis in ALK-rearranged (ALK+) non-small-cell lung cancer (NSCLC). However, drug resistance mechanisms occur inevitably during the course of treatment leading to disease progression. Activation of epidermal growth factor receptor (EGFR) bypass signaling pathway is an uncommon cause of acquired resistance to ALK TKIs.MethodWe present two patients with EML4-ALK rearranged NSCLC, developing an acquired EGFR resistance mutation after receiving multiple lines of ALK TKIs.ResultsWhile preclinical models have showed encouraging data, there is a critical need for clinical studies on treatment strategies to overcome this drug resistance. Three real-life therapeutic approaches were used in this report: i) using brigatinib, an inhibitor targeting both ALK and EGFR tyrosine kinases; ii) combining two ALK TKIs together; and iii) delivering doublet platinum chemotherapy. In case 1, time to treatment failure (TTF) was 9.5 months with brigatinib; in case 2, TTF was 10 months with combined TKIs (osimertinib and brigatinib), whereas TTF with chemotherapy was only 2 months. Tolerability profile TKIs combotherapy was acceptable.ConclusionThese case reports underline the therapeutic complexity of EGFR-acquired resistance mutation in ALK+ NSCLC and offers some leads to solve this real-life clinical challenge.

Non-invasive prenatal diagnosis of single-gene disorders (SGD-NIPD) has been widely accepted, but is mostly limited to the exclusion of either paternal or de novo mutations. Indeed, it is still difficult to infer the inheritance of the maternal allele from cell-free DNA (cfDNA) analysis. Based on the study of maternal haplotype imbalance in cfDNA, relative haplotype dosage (RHDO) was developed to address this challenge. Although RHDO has been shown to be reliable, robust control of statistical error and explicit delineation of critical parameters for assessing the quality of the analysis have not been fully addressed. We present here a universal and adaptable enhanced-RHDO (eRHDO) procedure through an automated bioinformatics pipeline with a didactic visualization of the results, aiming to be applied for any SGD-NIPD in routine care. A training cohort of 43 families carrying CFTR, NF1, DMD, or F8 mutations allowed the characterization and optimal setting of several adjustable data variables, such as minimum sequencing depth, type 1 and type 2 statistical errors, as well as the quality assessment of intermediate steps and final results by block score and concordance score. Validation was successfully performed on a test cohort of 56 pregnancies. Finally, computer simulations were used to estimate the effect of fetal-fraction, sequencing depth and number of informative SNPs on the quality of results. Our workflow proved to be robust, as we obtained conclusive and correctly inferred fetal genotypes in 94.9% of cases, with no false-negative or false-positive results. By standardizing data generation and analysis, we fully describe a turnkey protocol for laboratories wishing to offer eRHDO-based non-invasive prenatal diagnosis for single-gene disorders as an alternative to conventional prenatal diagnosis.

Some patients with Lynch syndrome (LS) have extreme phenotypes, i.e. cancer before the recommended screening age, or cancer for which there are no screening guidelines. We made the hypothesis that additional germline variants in cancer susceptibility genes (CSG) could explain some of these phenotypes. We compared the prevalence of additional CSG variants in LS patients with a cancer diagnosis before age 30 (early-onset, EO group) and after 40 (usual-onset, UO group). While there was no overall difference, we did find an excess of pathogenic variants and variants of unknown significance in EO cases when only gastrointestinal CSG were considered (OR 2.25; 95% CI: 1.01–5.06, p value = 0.04). Four EO cases stood out: two with POLE/POLD1 variants in the key exonuclease domain, one with a BMPR1A duplication and one with an EPCAM deletion. Additional germline variants should be considered in future screening recommendations, as they might influence cancer risk.

Cholangiocarcinomas (CCAs) are rare digestive tumors classified as intrahepatic (iCCA), perihilar (pCCA), and distal (dCCA) CCAs. These tumors are most often diagnosed at an advanced stage, unresectable or metastatic, and associated with a poor prognosis. The identification in recent years of multiple molecular alterations of interest, particularly in iCCA, has nevertheless allowed the development of new targeted therapeutic options for a significant proportion of patients. Gene fusions are among the most frequent alterations, involving FGFR2 in 10-15% of iCCAs in particular, and NTRK genes at a lower frequency (<1%). A dedicated analysis, most often based on RNA sequencing, is required to identify such alterations. Three FGFR inhibitors, pemigatinib, infigratinib and futinatinib, have recently received FDA approval for use in pre-treated patients. These compounds are currently being evaluated as first-line therapy in several phase III trials. Promising results have also been reported with new-generation inhibitors such as RLY-4008, which may soon constitute new therapeutic options. In the case of NTRK fusion, larotrectinib and entrectinib have also demonstrated their efficacy. The objectives of this review are to clarify the specific diagnostic modalities for gene fusions and to summarize the results of the main trials and developments underway for the management of advanced CCA with gene fusions.

Technological advances, in particular the development of high-throughput sequencing, have led to the emergence of a new generation of molecular biomarkers for tumors. These new tools have profoundly changed therapeutic management in oncology, with increasingly precise molecular characterization of tumors leading to increasingly personalized therapeutic targeting. Detection of circulating tumor cells and/or circulating tumor DNA in blood samples -so-called 'liquid biopsies'- can now provide a genetic snapshot of the patient's tumor through an alternative and less invasive procedure than biopsy of the tumor tissue itself. This procedure for characterizing and monitoring the disease in real time facilitates the search for possible relapses, the emergence of resistance, or emergence of a new therapeutic target. In the long term, it might also provide a means of early detection of cancer. These new approaches require the treatment of ever-increasing amounts of clinical data, notably, with the goal of calculating composite clinical-biological predictive scores. The use of artificial intelligence will be unavoidable in this domain, but it raises ethical questions and implications for the health-care system that will have to be addressed.