Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a critical regulator of cell death and inflammation, but its relevance for human disease pathogenesis remains elusive. Studies of ...monogenic disorders might provide critical insights into disease mechanisms and therapeutic targeting of RIPK1 for common diseases. Here, we report on eight patients from six unrelated pedigrees with biallelic loss-of-function mutations in RIPK1 presenting with primary immunodeficiency and/or intestinal inflammation. Mutations in RIPK1 were associated with reduced NF-κB activity, defective differentiation of T and B cells, increased inflammasome activity, and impaired response to TNFR1-mediated cell death in intestinal epithelial cells. The characterization of RIPK1-deficient patients highlights the essential role of RIPK1 in controlling human immune and intestinal homeostasis, and might have critical implications for therapies targeting RIPK1.
Circulating tumor DNA (ctDNA) has become an attractive biomarker in human oncology, and its use may be informative in canine cancer. Thus, we used droplet digital PCR or PCR for antigen receptor ...rearrangement, to explore tumor-specific point mutations, copy number alterations, and chromosomal rearrangements in the plasma of cancer-affected dogs. We detected ctDNA in 21/23 (91.3%) of histiocytic sarcoma (HS), 2/8 (25%) of oral melanoma, and 12/13 (92.3%) of lymphoma cases. The utility of ctDNA in diagnosing HS was explored in 133 dogs, including 49 with HS, and the screening of recurrent PTPN11 mutations in plasma had a specificity of 98.8% and a sensitivity between 42.8 and 77% according to the clinical presentation of HS. Sensitivity was greater in visceral forms and especially related to pulmonary location. Follow-up of four dogs by targeting lymphoma-specific antigen receptor rearrangement in plasma showed that minimal residual disease detection was concordant with clinical evaluation and treatment response. Thus, our study shows that ctDNA is detectable in the plasma of cancer-affected dogs and is a promising biomarker for diagnosis and clinical follow-up. ctDNA detection appears to be useful in comparative oncology research due to growing interest in the study of natural canine tumors and exploration of new therapies.
Introduction
Glioblastoma (GBM) is the most common and aggressive primary brain cancer in adults. Few cytotoxic chemotherapies have been shown to be effective against GBM, due in part to the presence ...of the blood–brain barrier (BBB), which reduces the penetration of chemotherapies from the blood to the brain. Ultrasound-induced BBB opening (US-BBB) has been shown to increase the penetration of multiple chemotherapeutic agents in the brain in animal models. In the current study, the anti-tumor activity of carboplatin chemotherapy with and without US-BBB was investigated in several GBM mouse models.
Methods
First, the IC50 of two commercial (U87 and U251) and six patient-derived GBM cell lines (PDCL) to carboplatin was measured. Next, U87 was subcutaneously grafted to a nude mouse model to test the in vivo response of the tumor to carboplatin in the absence of the BBB. Lastly, nude mice bearing orthotopically xenografted GBM cell lines (U87 or a PDCL) were randomized to four experimental groups: (i) untreated, (ii) US-BBB alone, (iii) carboplatin alone and, (iv) carboplatin + US-BBB. Mice were treated once weekly for 4 weeks and monitored for toxicity, tumor growth, and survival.
Results
Carboplatin plus US-BBB enhanced survival (p = 0.03) and delayed tumor growth (p < 0.05) of GBM-bearing mice compared to carboplatin alone, with a 4.2-fold increase of carboplatin penetration in the brain, without evidence of significant neurological or systemic toxicity.
Conclusions
Carboplatin efficacy was enhanced in GBM mouse models with US-BBB and appears to be a promising chemotherapy for this approach.
Non-Hodgkin lymphomas (NHL) commonly occur in immune-deficient (ID) patients, both HIV-infected and transplanted, and are often EBV-driven with cerebral localization, raising the question of tumor ...immunogenicity, a critical issue for treatment responses. We investigated the immunogenomics of 68 lymphoproliferative disorders from 51 ID (34 posttransplant, 17 HIV+) and 17 immunocompetent patients. Overall, 72% were Large B Cells Lymphoma (LBCL) and 25% were primary central-nervous-system lymphoma (PCNSL) while 40% were EBV-positive. Tumor whole-exome and RNA sequencing, along with a bioinformatics pipeline allowed analysis of tumor mutational burden (TMB), tumor landscape and microenvironment (TME) and prediction of tumor neoepitopes. Both TMB (2.2 vs 3.4/Mb, p=0.001) and neoepitopes numbers (40 vs 200, p=0.00019) were lower in EBVpositive than in EBV-negative NHL, regardless of the immune status. In contrast both EBV and the immune status influenced the tumor mutational profile, with HNRNPF and STAT3 mutations exclusively observed in EBV-positive and ID NHL, respectively. Peripheral blood T-cell responses against tumor neoepitopes were detected in all EBV-negative cases but in only half EBV-positive ones, including responses against IgH-derived MHC-class-II restricted neoepitopes. The TME analysis showed higher CD8 T cell infiltrates in EBVpositive vs EBV-negative NHL, together with a more tolerogenic profile composed of Tregs, type-M2 macrophages and an increased expression of negative immune-regulators. Our results highlight that the immunogenomics of NHL in patients with immunodeficiency primarily relies on the tumor EBV status, while T cell recognition of tumor- and IgH-specific neoepitopes is conserved in EBV-negative patients, offering potential opportunities for future T cell-based immune therapies.
Abstract Circulating tumor cells (CTCs) represent a rare and heterogeneous population of cancer cells that are detached from the tumor site and entered blood or lymphatic circulation. Once ...disseminated in distant tissues, CTCs could remain dormant or create a tumor mass causing serious danger for patients. Many technologies exist to isolate CTCs from patients’ blood samples, mostly based on microfluidic systems or by sorting them according to their surface antigens, notably EpCAM, and/or cytokeratins for carcinoma. ScreenCell has developed an easy-to-use, antigen-independent, rapid, cost-effective, and efficient technology that isolates CTCs according to their bigger size compared to the blood cells. This study provides the technical information necessary to isolate and characterize CTCs from mouse blood. By using blood samples from transgenic mice with breast cancer or from WT mice in which we spiked cancer cells, we showed that ScreenCell technology is compatible with standard EDTA blood collection tubes. Furthermore, the ScreenCell Cyto kit could treat up to 500 µl and the ScreenCell MB kit up to 200 µl of mouse blood. As the ScreenCell MB kit captures unaltered live CTCs, we have shown that their DNA could be efficiently extracted, and the isolated cells could be grown in culture. In conclusion, ScreenCell provides a rapid, easy, antigen-independent, cost-effective, and efficient technology to isolate and characterize CTCs from the blood samples of cancer patients and murine models. Thanks to this technology CTCs could be captured fixed or alive. Murine cancer models are extensively used in pre-clinical studies. Therefore, this study demonstrates the crucial technical points necessary while manipulating mouse blood samples using ScreenCell technology.
Accurate tools for Toxoplasma gondii detection and quantification can be valuable for the early and effective management of toxoplasmosis. Droplet digital PCR (ddPCR) is a next-generation end-point ...PCR technique with high performance. The objective of the study was to evaluate the performance of ddPCR for the detection and absolute quantification of T. gondii. From January 2019 to October 2020, DNA samples collected at the Laboratory of Parasitology and Mycology of Pitié-Salpêtrière Hospital in Paris were retrospectively analyzed by ddPCR and real-time quantitative PCR (qPCR). To detect T. gondii with the best sensitivity possible, the REP-529 multicopy target was used. For absolute quantification of T. gondii, a specific single-copy target of α-tubulin was designed. T. gondii detection by ddPCR and qPCR was strongly correlated (R2 = 0.93), with a total concordance of 96.7% (n = 145/150). Quantification of T. gondii using ddPCR was successful for 15 of 35 samples showing a parasite load ≥170 copies/mL of DNA eluate using the α-tubulin target. The qPCR REP-529 quantification based on a standard curve was approximate and dependent on the strain genotype, which led to an estimate of parasite copy number 14- to 160-fold superior to the ddPCR result. In total, ddPCR is an effective molecular method for T. gondii detection that shows equivalent performance to qPCR. For robust T. gondii quantification, ddPCR is clearly more accurate than semiquantitative qPCR methods.
Non-hodgkin lymphomas (NHL) are among the most common and fatal cancers in HIV-infected patients and transplant recipients. They are frequently associated with the Epstein Barr Virus (EBV) and a ...central nervous system (CNS) localization. Cancers neoantigens derived from the somatic tumor variants are a key factor of anti-tumor activity and response to immunotherapies, but the immunogenomics and tumor microenvironment (TME) characteristics of NHL from immunodeficient (ID) patients are lacking. We hypothesized they might be influenced by the low immunological pressure, the viral stimulation and/or the immune-privileged site. We report the comprehensive and prospective study of the tumor mutational burden (TMB), numbers of tumor neoepitopes, neoepitopes specific T cell responses and TME in ID patients, according to the EBV status, the immune status and the disease localization.Consecutive HIV-infected patients or transplant recipients with treatment-naïve NHL were included and compared with immunocompetent patients with diffuse large B-cell lymphomas (DLBCL). A whole exome sequencing (WES) was carried out in parallel on tumor and on PBMC for TMB assessment and a whole genome RNA sequencing was performed on frozen tumor biopsies. The neoepitopes derived from the somatic tumor variants (called in WES and RNAseq data) were predicted in silico using a bioinformatic pipeline including pVACseq and NetMHC and filtered according to: VAF≥10%, gene expression≥1 TPM and affinity binding score ≤500 nM. Neoepitopes specific T cell responses were assessed by ELISPOT assays after a 10 days PBMC co-culture with peptides from the Top 50 best predicted autologous neoepitopes (positivity defined as spot forming cells >50/106 cells). For the TME study, the profiling of cell type abundance and the T-cell receptor (TCR) sequencing were performed with deconvolution tools (EPIC) and MiXCR respectively. All statistical tests used R.Sixty-seven patients were included so far: 33 post-transplant lymphoproliferative disorders (PTLD) and 16 HIV-positive NHL, 70% of whom having DLBCL, compared to 18 immunocompetent (IC)-DLBCL. Diseases were systemic in 73% and localized in CNS in 27% cases. The median age was 58 years (range 21-85) and 70% were male. Immuno-molecular data are available for 57 patients so far. The TMB was 3.7/Mb for all patients but was lower in EBV-positive NHL (2.7/Mb) compared to the negative ones (4/Mb) (p=0.012, t-test) (Fig 1) without difference according to the ID status. The overall median number of neoepitopes per tumor predicted from the non-immunoglobulin (Ig) variants was 113 (range 11-720) but was lower in EBV-positive NHL (49 vs 243, p= 0.04, t-test), correlating with the TMB (r=0.8, p<0.0001). Most neoepitopes were MHC-class II restricted (ratio MHC-class II/ MHC- class I= 4.1) independently of the EBV status. The median number of neoepitopes predicted from the Ig heavy chain (IgH) genes was 17.5 (range, 6-36) with a MHC-class II/MHC-class I ratio of 1.4. Neoepitopes specific T cell responses were detectable among 71% cases out of 14 patients tested so far, independently of ID status or CNS localization (Fig 2). All 3 positive responses against Ig-derived neoepitopes out of 5 tested cases were directed against the IgH, MHC-class II restricted and immunodominant compared to those against the non-Ig neoepitopes. There was no shared immunogenic neoepitopes between patients. The TME study showed a higher frequency of CD4+ and CD8+ T cells mean frequency in EBV-positive compared to EBV-negative NHL (11% vs 4% and 10% vs 2% respectively, p<0.03).The intra-tumoral TCR repertoire distribution was mostly polyclonal with a median frequency of dominant clones of 8% (range 1-61) and a lower repertoire diversity, assessed on the ratio of clonotypes number/ total TCR-β reads number, in EBV-positive NHL (29) than in EBV-negative ones (37) (p=0.02), without difference according to the immune status.Our data demonstrate that both immunogenomics and TME of NHL from immuno-compromized patients are preferentially influenced by the EBV status, with lower numbers of tumor neoepitopes correlating with lower TMB, and a lower intra-tumoral TCR diversity in EBV-positive versus EBV-negative NHL. We further demonstrated that anti-tumor immune responses can be mounted despite ID status or CNS localization, especially against MHC-class II presented Ig-variants, and might be targeted for therapeutic strategies.
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