Disequilibrium between BRCA1 and BRCA2 Circular and Messenger RNAs Plays a Role in Breast Cancer.

Breast cancer is a frequent disease for which the discovery of markers that enable early detection or prognostic assessment remains challenging. Circular RNAs (circRNAs) are single-stranded structures in closed loops that are produced by backsplicing. CircRNA and messenger RNA (mRNA) are generated co-transcriptionally, and backsplicing and linear splicing compete against each other. As mRNAs are key players in tumorigenesis, we hypothesize that a disruption of the balance between circRNAs and mRNAs could promote breast cancer. Hence, we developed an assay for a simultaneous study of circRNAs and mRNAs, which we have called splice and expression analyses by exon ligation and high-throughput sequencing (SEALigHTS). Following SEALigHTS validation for BRCA1 and BRCA2, our hypothesis was tested using an independent research set of 95 pairs from tumor and adjacent normal breast tissues. In this research set, ratios of BRCA1 and BRCA2 circRNAs/mRNAs were significantly lower in the tumor breast tissue compared to normal tissue (p = 1.6 × 10-9 and p = 4.4 × 10-5 for BRCA1 and BRCA2, respectively). Overall, we developed an innovative method to study linear splicing and backsplicing, described the repertoire of BRCA1 and BRCA2 circRNAs, including 15 novel ones, and showed for the first time that a disequilibrium between BRCA1 and BRCA2 circRNAs and mRNAs plays a role in breast cancer.

Splicing analyses for variants in MMR genes: best practice recommendations from the European Mismatch Repair Working Group.

Over 20% of the DNA mismatch repair (MMR) germline variants in suspected Lynch syndrome patients are classified as variants of uncertain significance (VUS). Well-established functional assays are pivotal for assessing the biological impact of these variants and provide relevant evidence for clinical classification. In our collaborative European Mismatch Repair Working Group (EMMR-WG) we compared three different experimental approaches for evaluating the effect of seven variants on mRNA splicing in MMR genes: (i) RT-PCR of full-length transcripts (FLT), (ii) RT-PCR of targeted transcript sections (TTS), both from patient biological samples and (iii) minigene splicing assays. An overall good concordance was observed between splicing patterns in TTS, FLT and minigene analyses for all variants. The FLT analysis depicted a higher number of different isoforms and mitigated PCR-bias towards shorter isoforms. TTS analyses may miss aberrant isoforms and minigene assays may under/overestimate the severity of certain splicing defects. The interpretation of the experimental findings must be cautious to adequately discriminate abnormal events from physiological complex alternative splicing patterns. A consensus strategy for investigating the impact of MMR variants on splicing was defined. First, RNA should be obtained from patient's cell cultures (such as fresh lymphocyte cultures) incubated with/without a nonsense-mediated decay inhibitor. Second, FLT RT-PCR analysis is recommended to oversee all generated isoforms. Third, TTS analysis and minigene assays are useful independent approaches for verifying and clarifying FLT results. The use of several methodologies is likely to increase the strength of the experimental evidence which contributes to improve variant interpretation.

Generation of Pure Highly Functional Human Anti-Tumor Specific Cytotoxic T Lymphocytes With Stem Cell-Like Memory Features for Melanoma Immunotherapy.

Adoptive immunotherapy based on the transfer of anti-tumor cytotoxic T lymphocytes (CTLs) is a promising strategy to cure cancers. However, rapid expansion of numerous highly functional CTLs with long-lived features remains a challenge. Here, we constructed NIH/3T3 mouse fibroblast-based artificial antigen presenting cells (AAPCs) and precisely evaluated their ability to circumvent this difficulty. These AAPCs stably express the essential molecules involved in CTL activation in the HLA-A*0201 context and an immunogenic HLA-A*0201 restricted analogue peptide derived from MART-1, an auto-antigen overexpressed in melanoma. Using these AAPCs and pentamer-based magnetic bead-sorting, we defined, in a preclinical setting, the optimal conditions to expand pure MART-1-specific CTLs. Numerous highly purified MART-1-specific CTLs were rapidly obtained from healthy donors and melanoma patients. Both TCR repertoire and CDR3 sequence analyses revealed that MART-1-specific CTL responses were similar to those reported in the literature and obtained with autologous or allogeneic presenting cells. These MART-1-specific CTLs were highly cytotoxic against HLA-A*0201+ MART-1+ tumor cells. Moreover, they harbored a suitable phenotype for immunotherapy, with effector memory, central memory and, most importantly, stem cell-like memory T cell features. Notably, the cells harboring stem cell-like memory phenotype features were capable of self-renewal and of differentiation into potent effector anti-tumor T cells. These "off-the-shelf" AAPCs represent a unique tool to rapidly and easily expand large numbers of long-lived highly functional pure specific CTLs with stem cell-like memory T cell properties, for the development of efficient adoptive immunotherapy strategies against cancers.

Calibration of Pathogenicity Due to Variant-Induced Leaky Splicing Defects by Using BRCA2 Exon 3 as a Model System.

BRCA2 is a clinically actionable gene implicated in breast and ovarian cancer predisposition that has become a high priority target for improving the classification of variants of unknown significance (VUS). Among all BRCA2 VUS, those causing partial/leaky splicing defects are the most challenging to classify because the minimal level of full-length (FL) transcripts required for normal function remains to be established. Here, we explored BRCA2 exon 3 (BRCA2e3) as a model for calibrating variant-induced spliceogenicity and estimating thresholds for BRCA2 haploinsufficiency. In silico predictions, minigene splicing assays, patients' RNA analyses, a mouse embryonic stem cell (mESC) complementation assay and retrieval of patient-related information were combined to determine the minimal requirement of FL BRCA2 transcripts. Of 100 BRCA2e3 variants tested in the minigene assay, 64 were found to be spliceogenic, causing mild to severe RNA defects. Splicing defects were also confirmed in patients' RNA when available. Analysis of a neutral leaky variant (c.231T>G) showed that a reduction of approximately 60% of FL BRCA2 transcripts from a mutant allele does not cause any increase in cancer risk. Moreover, data obtained from mESCs suggest that variants causing a decline in FL BRCA2 with approximately 30% of wild-type are not pathogenic, given that mESCs are fully viable and resistant to DNA-damaging agents in those conditions. In contrast, mESCs producing lower relative amounts of FL BRCA2 exhibited either null or hypomorphic phenotypes. Overall, our findings are likely to have broader implications on the interpretation of BRCA2 variants affecting the splicing pattern of other essential exons. SIGNIFICANCE: These findings demonstrate that BRCA2 tumor suppressor function tolerates substantial reduction in full-length transcripts, helping to determine the pathogenicity of BRCA2 leaky splicing variants, some of which may not increase cancer risk.

Skipping Nonsense to Maintain Function: The Paradigm of BRCA2 Exon 12.

Germline nonsense and canonical splice site variants identified in disease-causing genes are generally considered as loss-of-function (LoF) alleles and classified as pathogenic. However, a fraction of such variants could maintain function through their impact on RNA splicing. To test this hypothesis, we used the alternatively spliced BRCA2 exon 12 (E12) as a model system because its in-frame skipping leads to a potentially functional protein. All E12 variants corresponding to putative LoF variants or predicted to alter splicing (n = 40) were selected from human variation databases and characterized for their impact on splicing in minigene assays and, when available, in patient lymphoblastoid cell lines. Moreover, a selection of variants was analyzed in a mouse embryonic stem cell-based functional assay. Using these complementary approaches, we demonstrate that a subset of variants, including nonsense variants, induced in-frame E12 skipping through the modification of splice sites or regulatory elements and, consequently, led to an internally deleted but partially functional protein. These data provide evidence, for the first time in a cancer-predisposition gene, that certain presumed null variants can retain function due to their impact on splicing. Further studies are required to estimate cancer risk associated with these hypomorphic variants. More generally, our findings highlight the need to exercise caution in the interpretation of putative LoF variants susceptible to induce in-frame splicing modifications. SIGNIFICANCE: This study presents evidence that certain presumed loss-of-function variants in a cancer predisposition gene can retain function due to their direct impact on RNA splicing.

Assessment of the InSiGHT Interpretation Criteria for the Clinical Classification of 24 MLH1 and MSH2 Gene Variants.

Pathogenicity assessment of DNA variants in disease genes to explain their clinical consequences is an integral component of diagnostic molecular testing. The International Society for Gastrointestinal Hereditary Tumors (InSiGHT) has developed specific criteria for the interpretation of mismatch repair (MMR) gene variants. Here, we performed a systematic investigation of 24 MLH1 and MSH2 variants. The assessments were done by analyzing population frequency, segregation, tumor molecular characteristics, RNA effects, protein expression levels, and in vitro MMR activity. Classifications were confirmed for 15 variants and changed for three, and for the first time determined for six novel variants. Overall, based on our results, we propose the introduction of some refinements to the InSiGHT classification rules. The proposed changes have the advantage of homogenizing the InSIGHT interpretation criteria with those set out by the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium for the BRCA1/BRCA2 genes. We also observed that the addition of only few clinical data was sufficient to obtain a more stable classification for variants considered as "likely pathogenic" or "likely nonpathogenic." This shows the importance of obtaining as many as possible points of evidence for variant interpretation, especially from the clinical setting.

Artificial antigen-presenting cells expressing HLA class II molecules as an effective tool for amplifying human specific memory CD4(+) T cells.

Owing to their multiple immune functions, CD4(+) T cells are of major interest for immunotherapy in chronic viral infections and cancer, as well as for severe autoimmune diseases and transplantation. Therefore, standardized methods allowing rapid generation of a large number of CD4(+) T cells for adoptive immunotherapy are still awaited. We constructed stable artificial antigen-presenting cells (AAPCs) derived from mouse fibroblasts. They were genetically modified to express human leukocyte antigen (HLA)-DR molecules and the human accessory molecules B7.1, Intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-3 (LFA-3). AAPCs expressing HLA-DR1, HLA-DR15 or HLA-DR51 molecules and loaded with peptides derived from influenza hemagglutinin (HA), myelin basic protein (MBP) or factor VIII, respectively, activated specific CD4(+) T-cell clones more effectively than Epstein-Barr virus (EBV)-transformed B cells. We also showed that AAPCs were able to take up and process whole Ag proteins, and present epitopes to specific T cells. In primary cultures, AAPCs loaded with HA peptide allowed generation of specific Th1 lymphocytes from healthy donors as demonstrated by tetramer and intracellular cytokine staining. Although AAPCs were less effective than autologous peripheral blood mononuclear cells (PBMCs) to stimulate CD4(+) T cells in primary culture, AAPCs were more potent to reactivate and expand memory Th1 cells in a strictly Ag-dependent manner. As the availability of autologous APCs is limited, the AAPC system represents a stable and reliable tool to achieve clinically relevant numbers of CD4(+) T cells for adoptive immunotherapy. For fundamental research in immunology, AAPCs are also useful to decipher mechanisms involved in the development of human CD4 T-cell responses.

Exonic Splicing Mutations Are More Prevalent than Currently Estimated and Can Be Predicted by Using In Silico Tools.

The identification of a causal mutation is essential for molecular diagnosis and clinical management of many genetic disorders. However, even if next-generation exome sequencing has greatly improved the detection of nucleotide changes, the biological interpretation of most exonic variants remains challenging. Moreover, particular attention is typically given to protein-coding changes often neglecting the potential impact of exonic variants on RNA splicing. Here, we used the exon 10 of MLH1, a gene implicated in hereditary cancer, as a model system to assess the prevalence of RNA splicing mutations among all single-nucleotide variants identified in a given exon. We performed comprehensive minigene assays and analyzed patient's RNA when available. Our study revealed a staggering number of splicing mutations in MLH1 exon 10 (77% of the 22 analyzed variants), including mutations directly affecting splice sites and, particularly, mutations altering potential splicing regulatory elements (ESRs). We then used this thoroughly characterized dataset, together with experimental data derived from previous studies on BRCA1, BRCA2, CFTR and NF1, to evaluate the predictive power of 3 in silico approaches recently described as promising tools for pinpointing ESR-mutations. Our results indicate that ΔtESRseq and ΔHZEI-based approaches not only discriminate which variants affect splicing, but also predict the direction and severity of the induced splicing defects. In contrast, the ΔΨ-based approach did not show a compelling predictive power. Our data indicates that exonic splicing mutations are more prevalent than currently appreciated and that they can now be predicted by using bioinformatics methods. These findings have implications for all genetically-caused diseases.

Correlation between Density of CD8+ T-cell Infiltrate in Microsatellite Unstable Colorectal Cancers and Frameshift Mutations: A Rationale for Personalized Immunotherapy.

Colorectal cancers with microsatellite instability (MSI) represent 15% of all colorectal cancers, including Lynch syndrome as the most frequent hereditary form of this disease. Notably, MSI colorectal cancers have a higher density of tumor-infiltrating lymphocytes (TIL) than other colorectal cancers. This feature is thought to reflect the accumulation of frameshift mutations in sequences that are repeated within gene coding regions, thereby leading to the synthesis of neoantigens recognized by CD8(+) T cells. However, there has yet to be a clear link established between CD8(+) TIL density and frameshift mutations in colorectal cancer. In this study, we examined this link in 103 MSI colorectal cancers from two independent cohorts where frameshift mutations in 19 genes were analyzed and CD3(+), CD8(+), and FOXP3(+) TIL densities were quantitated. We found that CD8(+) TIL density correlated positively with the total number of frameshift mutations. TIL densities increased when frameshift mutations were present within the ASTE1, HNF1A, or TCF7L2 genes, increasing even further when at least one of these frameshift mutations was present in all tumor cells. Through in vitro assays using engineered antigen-presenting cells, we were able to stimulate peripheral cytotoxic T cells obtained from colorectal cancer patients with peptides derived from frameshift mutations found in their tumors. Taken together, our results highlight the importance of a CD8(+) T cell immune response against MSI colorectal cancer-specific neoantigens, establishing a preclinical rationale to target them as a personalized cellular immunotherapy strategy, an especially appealing goal for patients with Lynch syndrome.

Direct Toll-Like Receptor 8 signaling increases the functional avidity of human CD8+ T lymphocytes generated for adoptive T cell therapy strategies.

Adoptive transfer of in vitro activated and expanded antigen-specific cytotoxic T lymphocytes (CTLs) is a promising therapeutic strategy for infectious diseases and cancers. Obtaining in vitro a sufficient amount of highly specific cytotoxic cells and capable of retaining cytotoxic activity in vivo remains problematic. We studied the role of Toll-Like Receptor-8 (TLR8) engagement on peripheral CTLs activated with melanoma antigen MART-1-expressing artificial antigen-presenting cells (AAPCs). After a 3-week co-culture, 3-27% of specific CTLs were consistently obtained. CTLs expressed TLR8 in the intracellular compartment and at the cell surface. Specific CTLs activated with a TLR8 agonist (CL075) 24 h before the end of the culture displayed neither any change in their production levels of molecules involved in cytotoxicity (IFN-γ, Granzyme B, and TNF-α) nor major significant change in their cell surface phenotype. However, these TLR8-stimulated lymphocytes displayed increased cytotoxic activity against specific peptide-pulsed target cells related to an increase in specific anti-melanoma CTL functional avidity. TLR8 engagement on CTLs could, therefore, be useful in different immunotherapy strategies.

Germline copy number variation of genes involved in chromatin remodelling in families suggestive of Li-Fraumeni syndrome with brain tumours.

Germline alterations of the tumour suppressor TP53 gene are detected approximately in 25% of the families suggestive of Li-Fraumeni syndrome (LFS), characterised by a genetic predisposition to a wide tumour spectrum, including soft-tissue sarcomas, osteosarcomas, premenopausal breast cancers, brain tumours, adrenocortical tumours, plexus choroid tumours, leukaemia and lung cancer. The aim of this study was to determine the contribution of germline copy number variations (CNVs) to LFS in families without detectable TP53 mutation. Using a custom-designed high-resolution array CGH, we evaluated the presence of rare germline CNVs in 64 patients fulfilling the Chompret criteria for LFS, but without any detectable TP53 alteration. In 15 unrelated patients, we detected 20 new CNVs absent in 600 controls. Remarkably, in four patients who had developed each brain tumour, the detected CNV overlap the KDM1A, MTA3, TRRAP or SIRT3 genes encoding p53 partners involved in histone methylation or acetylation. Focused analysis of SIRT3 showed that the CNV encompassing SIRT3 leads to SIRT3 overexpression, and that in vitro SIRT3 overexpression prevents apoptosis, increases G2/M and results in a hypermethylation of numerous genes. This study supports the causal role of germline alterations of genes involved in chromatin remodelling in genetic predisposition to cancer and, in particular, to brain tumours.

CRTAM receptor engagement by Necl-2 on tumor cells triggers cell death of activated Vγ9Vδ2 T cells.

Human Vγ9Vδ2 T cells exert potent in vitro and in vivo antitumor activities, making them promising candidates for immunotherapy strategies. Recognition of tumor cells by Vγ9Vδ2 T cells requires engagement of the TCR and/or NK receptors. Recently, one of the novel NK receptors, the class I-restricted T cell-associated molecule (CRTAM), has been described to promote cytotoxic function of NK cells and to lead to IFN-γ secretion by CD8(+) T cells through interaction with its ligand, Necl-2. A better understanding of the role of CRTAM in Vγ9Vδ2 T cell functions is highly relevant to optimize innate-like T cell-based cancer immunotherapy. In this article, we report that CRTAM is transiently expressed on activated Vγ9Vδ2 T lymphocytes following TCR engagement. However, CRTAM-Necl-2 interaction does not modify the cytotoxic function or IFN-γ secretion of Vγ9Vδ2 T cells. The expression of CRTAM in activated Vγ9Vδ2 T cells is quickly downregulated following interaction with Necl-2 on tumor cells. Of interest, CRTAM is concurrently acquired at the cell surface of Necl-2(+) tumor cells through Vγ9Vδ2 T cell membrane capture. Finally, we highlight that coculture experiments with tumor cells expressing Necl-2 result in significant cell death of CRTAM(+) Vγ9Vδ2 T cells. CRTAM-mediated cell death is dependent on an autophagic process, but not on apoptosis or necroptosis, as attested by the expression of characteristic markers and blocking experiments with specific inhibitors. On the basis of these findings, we propose that Necl-2 on tumor cells represents a new tumor counterattack mechanism and a potential target to improve efficiency of γδ T cell-based immunotherapy.

HLA-A*0201-restricted CEA-derived peptide CAP1 is not a suitable target for T-cell-based immunotherapy.

Carcinoembryonic antigen (CEA) is a potential target for antigen-specific immunotherapy, as it is frequently overexpressed in human carcinomas. Moreover, an epitope derived from CEA, designated CAP1 (YLSGANLNL), has been proposed as naturally processed and presented by tumors in the human leukocyte antigen (HLA)-A*0201 context. Our aim was to fully characterize and assess the clinical relevance of the HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) response against CEA. Stable and potent artificial antigen presenting cells (AAPCs) were used to evaluate T-cell response against CEA. These cells efficiently activate CTLs against tumor-derived epitopes after transduction with the antigenic peptides or full-length proteins. We found that AAPCs genetically modified to express CAP1, the agonist peptide CAP1-6D, or the whole CEA protein were not able to activate CAP1-specific CTLs from HLA-A*0201+ healthy donors or patients with colorectal carcinoma, even after multiple stimulations. In addition, we showed that a CAP1-specific T-cell clone, obtained after multiple stimulations of T cells of a HLA-A*0201+ healthy donor in vitro with autologous antigen presenting cells, recognized CEA(-) HLA-A*0201+ tumors transduced with a minigene encoding CAP1 but failed to react against HLA-A*0201+ tumor cells expressing CEA. Finally, AAPCs expressing the whole CEA protein did not induce any specific CTL response against CEA+ HLA-A*0201+ tumor cells highlighting the potential difficulty of mounting an efficacious T-cell response against this autoantigen. Altogether, our data indicate that CAP1 is not efficiently processed and presented by CEA+ tumor cells, and therefore, is not an appropriate target for T-cell-based immunotherapy.

Gradual reduction of BUBR1 protein levels results in premature sister-chromatid separation then in aneuploidy.

Biallelic and heterozygous mutations of the BUB1B gene have been reported in mosaic variegated aneuploidy (MVA), a rare disorder characterized by constitutional mosaic aneuploidies associated to severe intrauterine growth retardation, microcephaly and, in most cases, to premature chromatid separation (PCS), highlighting the key role of human BUBR1 in chromosome segregation. To study the consequences of gradual reduction of the BUBR1 protein levels, inhibition of BUB1B expression in model cells was induced using short hairpin RNAs (shRNAs). We obtained stable shRNA-transduced HeLa cells displaying a gradient of residual BUBR1 protein (8.5, 10, 14, 58, and 77%), mimicking the situation of patients' cells harboring one or two BUB1B mutations. Induction of PCS was detected in all transduced cells and its level was correlated to the decrease of BUBR1. Aneuploidy was clearly detected in cells with residual BUBR1 below 50%. Our data demonstrate that the function of the human BUBR1 protein in the spindle checkpoint is remarkably dosage-dependent and that the biological consequences of BUB1B expression reduction on premature chromatid separation and aneuploidy depend on the residual amount of BUBR1. This provides a biological explanation for the mode of inheritance of PCS, which is dominant, and of MVA, which can be recessive in some families and result from the combination of a null allele associated to a common hypomorphic allele in others.