MET variants with activating N-lobe mutations identified in hereditary papillary renal cell carcinomas still require ligand stimulation.

In hereditary papillary renal cell carcinoma (HPRCC), the MET receptor tyrosine kinase (RTK) mutations recorded to date are located in the kinase domain and lead to constitutive MET activation. This contrasts with MET mutations recently identified in non-small cell lung cancer (NSCLC), which lead to exon 14 skipping and deletion of a regulatory domain: in this latter case, the mutated receptor still requires ligand stimulation. Sequencing of MET in samples from 158 HPRCC and 2808 NSCLC patients revealed ten uncharacterized mutations. Four of these, all found in HPRCC and leading to amino acid substitutions in the N-lobe of the MET kinase, proved able to induce cell transformation, further enhanced by HGF stimulation: His1086Leu, Ile1102Thr, Leu1130Ser, and Cis1125Gly. Similar to the variant resulting in MET exon14 skipping, the two N-lobe MET variants His1086Leu, Ile1102Thr further characterized were found to require stimulation by HGF in order to strongly activate downstream signaling pathways and epithelial cell motility. The Ile1102Thr mutation displayed also transforming potential, promoting tumor growth in a xenograft model. In addition, the N-lobe-mutated MET variants were found to trigger a common HGF-stimulation-dependent transcriptional program, consistent with an observed increase in cell motility and invasion. Altogether, this functional characterization revealed that N-lobe variants still require ligand stimulation, in contrast to other RTK variants. This suggests that HGF expression in the tumor microenvironment is important for tumor growth. The sensitivity of these variants to MET TKIs opens the way for use of targeted therapies for patients harboring the corresponding mutations.

Comparative analysis of response to treatments and molecular features of tumor-derived organoids versus cell lines and PDX derived from the same ovarian clear cell carcinoma.

BACKGROUND: In the era of personalized medicine, the establishment of preclinical models of cancer that faithfully recapitulate original tumors is essential to potentially guide clinical decisions. METHODS: We established 7 models [4 cell lines, 2 Patient-Derived Tumor Organoids (PDTO) and 1 Patient-Derived Xenograft (PDX)], all derived from the same Ovarian Clear Cell Carcinoma (OCCC). To determine the relevance of each of these models, comprehensive characterization was performed based on morphological, histological, and transcriptomic analyses as well as on the evaluation of their response to the treatments received by the patient. These results were compared to the clinical data. RESULTS: Only the PDX and PDTO models derived from the patient tumor were able to recapitulate the patient tumor heterogeneity. The patient was refractory to carboplatin, doxorubicin and gemcitabine, while tumor cell lines were sensitive to these treatments. In contrast, PDX and PDTO models displayed resistance to the 3 drugs. The transcriptomic analysis was consistent with these results since the models recapitulating faithfully the clinical response grouped together away from the other classical 2D cell culture models. We next investigated the potential of drugs that have not been used in the patient clinical management and we identified the HDAC inhibitor belinostat as a potential effective treatment based on PDTO response. CONCLUSIONS: PDX and PDTO appear to be the most relevant models, but only PDTO seem to present all the necessary prerequisites for predictive purposes and could constitute relevant tools for therapeutic decision support in the context of these particularly aggressive cancers refractory to conventional treatments.

LSC17 score complements genetics and measurable residual disease in acute myeloid leukemia: an ALFA study.

Whether the LSC17 gene expression can improve risk stratification in the context of next generation sequencing-based risk stratification and measurable residual disease (MRD) in patients with intensively treated AML has not been explored. We analyzed LSC17 in 504 adult patients prospectively treated in the ALFA-0702 trial. RUNX1 or TP53 mutations were associated with higher LSC1 scores while CEBPA and NPM1 mutations were associated with lower scores. Patients with high LSC17 scores had a lower rate of complete response (CR) in a multivariable analysis (odds ratio, 0.41; P = .0007), accounting for European LeukemiaNet 2022 (ELN22), age, and white blood cell count (WBC). LSC17-high status was associated with shorter overall survival (OS) (3-year OS: 70.0% vs 52.7% in patients with LSC17-low status; P < .0001). In a multivariable analysis considering ELN22, age, and WBC, patients with LSC17-high status had shorter disease-free survival (DFS) (hazard ratio [HR], 1.36; P = .048) than those with LSC17-low status. In 123 patients with NPM1-mutated AML in CR, LSC17-high status predicted poorer DFS (HR, 2.34; P = .01), independent of age, WBC, ELN22 risk, and NPM1-MRD. LSC-low status and negative NPM1-MRD identified a subset comprising 48% of patients with mutated NPM1 with a 3-year OS from CR of 93.1% compared with 60.7% in those with LSC17-high status and/or positive NPM1-MRD (P = .0001). Overall, LSC17 assessment refines genetic risk stratification in adult patients with AML treated intensively. Combined with MRD, LSC17 identifies a subset of patients with NPM1-mutated AML with excellent clinical outcome.

Detection of residual and chemoresistant leukemic cells in an immune-competent mouse model of acute myeloid leukemia: Potential for unravelling their interactions with immunity.

Acute myeloid leukemia (AML) is characterized by blocked differentiation and extensive proliferation of hematopoietic progenitors/precursors. Relapse is often observed after chemotherapy due to the presence of residual leukemic cells, which is also called minimal residual disease (MRD). Subclonal heterogeneity at diagnosis was found to be responsible for MRD after treatment. Patient xenograft mouse models are valuable tools for studying MRD after chemotherapy; however, the contribution of the immune system in these models is usually missing. To evaluate its role in leukemic persistence, we generated an immune-competent AML mouse model of persistence after chemotherapy treatment. We used well-characterized (phenotypically and genetically) subclones of the murine C1498 cell line stably expressing the ZsGreen reporter gene and the WT1 protein, a valuable antigen. Accordingly, these subclones were also selected due to their in vitro aracytidine (Ara-c) sensitivity. A combination of 3 subclones (expressing or not expressing WT1) was found to lead to prolonged mouse survival after Ara-c treatment (as long as 150 days). The presence of residual leukemic cells in the blood and BM of surviving mice indicated their persistence. Thus, a new mouse model that may offer insights into immune contributions to leukemic persistence was developed.

Galectin-3 modulates epithelial cell adaptation to stress at the ER-mitochondria interface.

Cellular stress response contributes to epithelial defense in adaptation to environment changes. Galectins play a pivotal role in the regulation of this response in malignant cells. However, precise underlying mechanisms are largely unknown. Here we demonstrate that Galectin-3, a pro and anti-apoptotic lectin, is required for setting up a correct cellular response to stress by orchestrating several effects. First, Galectin-3 constitutes a key post-transcriptional regulator of stress-related mRNA regulons coordinating the cell metabolism, the mTORC1 complex or the unfolded protein response (UPR). Moreover, we demonstrated the presence of Galectin-3 with mitochondria-associated membranes (MAM), and its interaction with proteins located at the ER or mitochondrial membranes. There Galectin-3 prevents the activation and recruitment at the mitochondria of the regulator of mitochondria fission DRP-1. Accordingly, loss of Galectin-3 impairs mitochondrial morphology, with more fragmented and round mitochondria, and dynamics both in normal and cancer epithelial cells in basal conditions. Importantly, Galectin-3 deficient cells also display changes of the activity of the mitochondrial respiratory chain complexes, of the mTORC1/S6RP/4EBP1 translation pathway and reactive oxygen species levels. Regarding the ER, Galectin-3 did not modify the activities of the 3 branches of the UPR in basal conditions. However, Galectin-3 favours an adaptative UPR following ER stress induction by Thapsigargin treatment. Altogether, at the ER-mitochondria interface, Galectin-3 coordinates the functioning of the ER and mitochondria, preserves the integrity of mitochondrial network and modulates the ER stress response.

Transcriptomic signatures of brain regional vulnerability to Parkinson's disease.

The molecular mechanisms underlying caudal-to-rostral progression of Lewy body pathology in Parkinson's disease remain poorly understood. Here, we identified transcriptomic signatures across brain regions involved in Braak Lewy body stages in non-neurological adults from the Allen Human Brain Atlas. Among the genes that are indicative of regional vulnerability, we found known genetic risk factors for Parkinson's disease: SCARB2, ELOVL7, SH3GL2, SNCA, BAP1, and ZNF184. Results were confirmed in two datasets of non-neurological subjects, while in two datasets of Parkinson's disease patients we found altered expression patterns. Co-expression analysis across vulnerable regions identified a module enriched for genes associated with dopamine synthesis and microglia, and another module related to the immune system, blood-oxygen transport, and endothelial cells. Both were highly expressed in regions involved in the preclinical stages of the disease. Finally, alterations in genes underlying these region-specific functions may contribute to the selective regional vulnerability in Parkinson's disease brains.

Functional Analysis of Somatic Mutations Affecting Receptor Tyrosine Kinase Family in Metastatic Colorectal Cancer.

Besides the detection of somatic receptor tyrosine kinases (RTK) mutations in tumor samples, the current challenge is to interpret their biological relevance to give patients effective targeted treatment. By high-throughput sequencing of the 58 RTK exons of healthy tissues, colorectal tumors, and hepatic metastases from 30 patients, 38 different somatic mutations in RTKs were identified. The mutations in the kinase domains and present in both tumors and metastases were reconstituted to perform an unbiased functional study. Among eight variants found in seven RTKs (EPHA4-Met726Ile, EPHB2-Val621Ile, ERBB4-Thr731Met, FGFR4-Ala585Thr, VEGFR3-Leu1014Phe, KIT-Pro875Leu, TRKB-Leu584Val, and NTRK2-Lys618Thr), none displayed significantly increased tyrosine kinase activity. Consistently, none of them induced transformation of NIH3T3 fibroblasts. On the contrary, two RTK variants (FGFR4-Ala585Thr and FLT4-Leu1014Phe) caused drastic inhibition of their kinase activity. These findings indicate that these RTK variants are not suitable targets and highlight the importance of functional studies to validate RTK mutations as potential therapeutic targets.

The stem cell-associated gene expression signature allows risk stratification in pediatric acute myeloid leukemia.

Despite constant progress in prognostic risk stratification, children with acute myeloid leukemia (AML) still relapse. Treatment failure and subsequent relapse have been attributed to acute myeloid leukemia-initiating cells (LSC), which harbor stem cell properties and are inherently chemoresistant. Although pediatric and adult AML represent two genetically very distinct diseases, we reasoned that common LSC gene expression programs are shared and consequently, the highly prognostic LSC17 signature score recently developed in adults may also be of clinical interest in childhood AML. Here, we demonstrated prognostic relevance of the LSC17 score in pediatric non-core-binding factor AML using Nanostring technology (ELAM02) and RNA-seq data from the NCI (TARGET-AML). AML were stratified by LSC17 quartile groups (lowest 25%, intermediate 50% and highest 25%) and children with low LSC17 score had significantly better event-free survival (EFS: HR = 3.35 (95%CI = 1.64-6.82), P < 0.001) and overall survival (OS: HR = 3.51 (95%CI = 1.38-8.92), P = 0.008) compared with patients with high LSC17 scores. More importantly, the high LSC17 score was an independent negative EFS and OS prognosticator determined by multivariate Cox model analysis (EFS: HR = 3.42 (95% CI = 1.63-7.16), P = 0.001; OS HR = 3.02 (95%CI = 1.16-7.85), P = 0.026). In conclusion, we have demonstrated the broad applicability of the LSC17 score in the clinical management of AML by extending its prognostic relevance to pediatric AML.

Sub-clonal analysis of the murine C1498 acute myeloid leukaemia cell line reveals genomic and immunogenic diversity.

BACKGROUND: In acute myeloid leukaemia (AML)-affected patients, the presence of heterogeneous sub-clones at diagnosis has been shown to be responsible for minimal residual disease and relapses. The role played by the immune system in this leukaemic sub-clonal hierarchy and maintenance remains unknown. As leukaemic sub-clone immunogenicity could not be evaluated in human AML xenograft models, we assessed the sub-clonal diversity of the murine C1498 AML cell line and the immunogenicity of its sub-clones in immune-competent syngeneic mice. METHODOLOGY: The murine C1498 cell line was cultured in vitro and sub-clonal cells were generated after limiting dilution. The genomic profiles of 6 different sub-clones were analysed by comparative genomic hybridization arrays (CGH). The sub-clones were then injected into immune-deficient and - competent syngeneic mice. The immunogenicities of the sub-clones was evaluated through 1) assessment of mouse survival, 2) determination of leukaemic cell infiltration into organs by flow cytometry and the expression of a fluorescent reporter gene, 3) assessment of the CTL response ex vivo and 4) detection of residual leukaemic cells in the organs via amplification of the genomic reporter gene by real-time PCR (qPCR). RESULTS: Genomic analyses revealed heterogeneity among the parental cell line and its derived sub-clones. When injected individually into immune-deficient mice, all sub-clones induced cases of AML with different kinetics. However, when administered into immune-competent animals, some sub-clones triggered AML in which no mice survived, whereas others elicited reduced lethality rates. The AML-surviving mice presented efficient anti-leukaemia CTL activity ex vivo and eliminated the leukaemic cells in vivo. CONCLUSION: We showed that C1498 cell sub-clones presented genomic heterogeneity and differential immunogenicity resulting either in immune escape or elimination. Such findings could have potent implications for new immunotherapeutic strategies in patients with AML.

Persistent coxsackievirus B4 infection induces microRNA dysregulation in human pancreatic cells.

Enterovirus infections are implicated in the development of type 1 diabetes (T1D). MicroRNAs as regulators of gene expression are involved in many physiological and pathological processes. Given that viral infections dysregulate cellular microRNAs, we investigated the impact of persistent coxsackievirus B4 infection on microRNA expression of human pancreatic cells. Next-generation sequencing was used to determine microRNA expression in PANC-1 cells persistently infected (for several weeks) with coxsackievirus B4 and uninfected control cells. Target prediction restricted to T1D risk genes was performed with miRWalk2.0. Functional annotation analysis was performed with DAVID6.7. Expression of selected microRNAs and T1D risk genes was measured by quantitative reverse-transcription polymerase chain reaction. Eighty-one microRNAs were dysregulated in persistently infected PANC-1 cells. Forty-nine of the known fifty-five T1D risk genes were predicted as putative targets of at least one of the dysregulated microRNAs. Most functional annotation terms that were enriched in these 49 putative target genes were related to the immune response or autoimmunity. mRNA levels of AFF3, BACH2, and IL7R differed significantly between persistently infected cells and uninfected cells. This is the first characterization of the microRNA expression profile changes induced by persistent coxsackievirus B4 infection in pancreatic cells. The predicted targeting of genes involved in the immune response and autoimmunity by the dysregulated microRNAs as well as the dysregulated expression of diabetes risk genes shows that persistent coxsackievirus B4 infection profoundly impacts the host cell. These data support the hypothesis of a possible link between persistent coxsackievirus B4 infection and the development of T1D.

Application of the cghRA framework to the genomic characterization of Diffuse Large B-Cell Lymphoma.

MOTIVATION: Although sequencing-based technologies are becoming the new reference in genome analysis, comparative genomic hybridization arrays (aCGH) still constitute a simple and reliable approach for copy number analysis. The most powerful algorithms to analyze such data have been freely provided by the scientific community for many years, but combining them is a complex scripting task. RESULTS: The cghRA framework combines a user-friendly graphical interface and a powerful object-oriented command-line interface to handle a full aCGH analysis, as is illustrated in an original series of 107 Diffuse Large B-Cell Lymphomas. New algorithms for copy-number calling, polymorphism detection and minimal common region prioritization were also developed and validated. While their performances will only be demonstrated with aCGH, these algorithms could actually prove useful to any copy-number analysis, whatever the technique used. AVAILABILITY AND IMPLEMENTATION: R package and source for Linux, MS Windows and MacOS are freely available at http://bioinformatics.ovsa.fr/cghRA. CONTACT: mareschal@ovsa.fr or fabrice.jardin@chb.unicancer.fr. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Galectin-3 is a non-classic RNA binding protein that stabilizes the mucin MUC4 mRNA in the cytoplasm of cancer cells.

Pancreatic cancer cells express high levels of MUC1, MUC4 and MUC16 mRNAs that encode membrane-bound mucins. These mRNAs share unusual features such as a long half-life. However, it remains unknown how mucin mRNA stability is regulated. Galectin-3 (Gal-3) is an endogenous lectin playing important biological functions in epithelial cells. Gal-3 is encoded by LGALS3 which is up-regulated in pancreatic cancer. Despite the absence of a RNA-recognition motif, Gal-3 interacts indirectly with pre-mRNAs in the nucleus and promotes constitutive splicing. However a broader role of Gal-3 in mRNA fate is unexplored. We report herein that Gal-3 increases MUC4 mRNA stability through an intermediate, hnRNP-L which binds to a conserved CA repeat element in the 3'UTR in a Gal-3 dependent manner and also controls Muc4 mRNA levels in epithelial tissues of Gal3-/- mice. Gal-3 interacts with hnRNP-L in the cytoplasm, especially during cell mitosis, but only partly associates with protein markers of P-Bodies or Stress Granules. By RNA-IP plus RNA-seq analysis and imaging, we demonstrate that Gal-3 binds to mature spliced MUC4 mRNA in the perinuclear region, probably in hnRNP-L-containing RNA granules. Our findings highlight a new role for Gal-3 as a non-classic RNA-binding protein that regulates MUC4 mRNA post-transcriptionally.

High-throughput sequencing in acute lymphoblastic leukemia: Follow-up of minimal residual disease and emergence of new clones.

Minimal residual disease (MRD) is known to be an independent prognostic factor in patients with acute lymphoblastic leukemia (ALL). High-throughput sequencing (HTS) is currently used in routine practice for the diagnosis and follow-up of patients with hematological neoplasms. In this retrospective study, we examined the role of immunoglobulin/T-cell receptor-based MRD in patients with ALL by HTS analysis of immunoglobulin H and/or T-cell receptor gamma chain loci in bone marrow samples from 11 patients with ALL, at diagnosis and during follow-up. We assessed the clinical feasibility of using combined HTS and bioinformatics analysis with interactive visualization using Vidjil software. We discuss the advantages and drawbacks of HTS for monitoring MRD. HTS gives a more complete insight of the leukemic population than conventional real-time quantitative PCR (qPCR), and allows identification of new emerging clones at each time point of the monitoring. Thus, HTS monitoring of Ig/TR based MRD is expected to improve the management of patients with ALL.

BAP1 Is Altered by Copy Number Loss, Mutation, and/or Loss of Protein Expression in More Than 70% of Malignant Peritoneal Mesotheliomas.

INTRODUCTION: Malignant mesothelioma is a deadly disease that is strongly associated with asbestos exposure. Peritoneal mesotheliomas account for 10% of all the cases. BRCA1 associated protein 1 (BAP1) is a deubiquitinating hydrolase that plays a key role in various cellular processes. Germline and somatic inactivation of BRCA1 associated protein 1 gene (BAP1) is frequent in pleural mesothelioma; however, little is known about its status in peritoneal mesothelioma. METHODS: Taking advantage of the extensive French National Network for the Diagnosis of Malignant Pleural Mesothelioma and Rare Peritoneal Tumors and the French National Network for the Treatment of Rare Peritoneal Surface Malignancies, we collected biological material and clinical and epidemiological data for 46 patients with peritoneal mesothelioma. The status of BAP1 was evaluated at the mutational and protein expression levels and combined with our previous data on copy number alterations assessed in the same samples. RESULTS: We detected mutations in 32% of the malignant peritoneal mesotheliomas analyzed. In addition, we have previously reported that copy number losses occurred in 42% of the samples included in this series. Overall, 73% of the malignant peritoneal mesotheliomas analyzed carried at least one inactivated BAP1 allele, but only 57% had a complete loss of its protein nuclear expression. Better overall survival was observed for patients with BAP1 mutations (p = 0.04), protein expression loss (p = 0.016), or at least one of these alterations (p = 0.007) independently of tumor histological subtype, age, and sex. CONCLUSIONS: As in pleural mesothelioma, inactivation of BAP1 is frequent in peritoneal mesotheliomas. We found that BAP1 protein nuclear expression is a good prognostic factor and a more reliable marker for the complete loss of BAP1 activity than mutation or copy number loss.

RNA-binding disturbances as a continuum from spinocerebellar ataxia type 2 to Parkinson disease.

CAG triplet expansions in Ataxin-2 gene (ATXN2) cause spinocerebellar ataxia type 2 and have a role that remains to be clarified in Parkinson's disease (PD). To study the molecular events associated with these expansions, we sequenced them and analyzed the transcriptome from blood cells of controls and three patient groups diagnosed with spinocerebellar ataxia type 2 (herein referred to as SCA2c) or PD with or without ATXN2 triplet expansions (named SCA2p). The transcriptome profiles of these 40 patients revealed three main observations: i) a specific pattern of pathways related to cellular contacts, proliferation and differentiation associated with SCA2p group, ii) similarities between the SCA2p and sporadic PD groups in genes and pathways known to be altered in PD such as Wnt, Ephrin and Leukocyte extravasation signaling iii) RNA metabolism disturbances with "RNA-binding" and "poly(A) RNA-binding" as a common feature in all groups. Remarkably, disturbances of ALS signaling were shared between SCA2p and sporadic PD suggesting common molecular dysfunctions in PD and ALS including CACNA1, hnRNP, DDX and PABPC gene family perturbations. Interestingly, the transcriptome profiles of patients with parkinsonian phenotypes were prevalently associated with alterations of translation while SCA2c and PD patients presented perturbations of splicing. While ATXN2 RNA expression was not perturbed, its protein expression in immortalized lymphoblastoid cells was significantly decreased in SCA2c and SCA2p versus control groups assuming post-transcriptional biological perturbations. In conclusion, the transcriptome data do not exclude the role of ATXN2 mutated alleles in PD but its decrease protein expression in both SCA2c and SCA2p patients suggest a potential involvement of this gene in PD. The perturbations of "RNA-binding" and "poly(A) RNA-binding" molecular functions in the three patient groups as well as gene deregulations of factors not yet described in PD but known to be deleterious in other neurological conditions, suggest the existence of RNA-binding disturbances as a continuum between spinocerebellar ataxia type 2 and Parkinson's disease.

Multi-loci diagnosis of acute lymphoblastic leukaemia with high-throughput sequencing and bioinformatics analysis.

High-throughput sequencing (HTS) is considered a technical revolution that has improved our knowledge of lymphoid and autoimmune diseases, changing our approach to leukaemia both at diagnosis and during follow-up. As part of an immunoglobulin/T cell receptor-based minimal residual disease (MRD) assessment of acute lymphoblastic leukaemia patients, we assessed the performance and feasibility of the replacement of the first steps of the approach based on DNA isolation and Sanger sequencing, using a HTS protocol combined with bioinformatics analysis and visualization using the Vidjil software. We prospectively analysed the diagnostic and relapse samples of 34 paediatric patients, thus identifying 125 leukaemic clones with recombinations on multiple loci (TRG, TRD, IGH and IGK), including Dd2/Dd3 and Intron/KDE rearrangements. Sequencing failures were halved (14% vs. 34%, P = 0.0007), enabling more patients to be monitored. Furthermore, more markers per patient could be monitored, reducing the probability of false negative MRD results. The whole analysis, from sample receipt to clinical validation, was shorter than our current diagnostic protocol, with equal resources. V(D)J recombination was successfully assigned by the software, even for unusual recombinations. This study emphasizes the progress that HTS with adapted bioinformatics tools can bring to the diagnosis of leukaemia patients.

Vaginal Mucosal Homeostatic Response May Determine Pregnancy Outcome in Women With Bacterial Vaginosis: A Pilot Study.

Bacterial vaginosis (BV) is considered as a trigger for an inflammatory response that could promote adverse pregnancy outcome (APO). We hypothesized that BV-related inflammation could be counterbalanced by anti-inflammatory and mucosal homeostatic responses that could participate in pregnancy outcomes.A total of 402 vaginal self-samples from pregnant women in their first trimester were screened by Nugent score. In this population, we enrolled 23 pregnant women with BV but without APO, 5 pregnant women with BV and developing APO, 21 pregnant women with intermediate flora, and 28 random control samples from pregnant women without BV or APO.BV without APO in pregnant women was associated with 28-fold interleukin-8, 5-fold interleukin-10, and 40-fold interleukin-22 increases in expression compared to controls. BV associated with APO in pregnant women shared 4-fold increase in tumor necrosis factor, 100-fold decrease in interleukin-10, and no variation in interleukin-22 expressions compared to controls. Next-generation sequencing of vaginal microbiota revealed a shift from obligate anaerobic bacteria dominance in BV without APO pregnant women to Lactobacillus dominance microbiota in BV with APO.Our results show that the anti-inflammatory and mucosal homeostatic responses to BV may determine outcome of pregnancy in the setting of BV possibly through effects on the vaginal microbiota.

Fast multiclonal clusterization of V(D)J recombinations from high-throughput sequencing.

BACKGROUND: V(D)J recombinations in lymphocytes are essential for immunological diversity. They are also useful markers of pathologies. In leukemia, they are used to quantify the minimal residual disease during patient follow-up. However, the full breadth of lymphocyte diversity is not fully understood. RESULTS: We propose new algorithms that process high-throughput sequencing (HTS) data to extract unnamed V(D)J junctions and gather them into clones for quantification. This analysis is based on a seed heuristic and is fast and scalable because in the first phase, no alignment is performed with germline database sequences. The algorithms were applied to TR γ HTS data from a patient with acute lymphoblastic leukemia, and also on data simulating hypermutations. Our methods identified the main clone, as well as additional clones that were not identified with standard protocols. CONCLUSIONS: The proposed algorithms provide new insight into the analysis of high-throughput sequencing data for leukemia, and also to the quantitative assessment of any immunological profile. The methods described here are implemented in a C++ open-source program called Vidjil.

Minimal residual disease monitoring in t(8;21) acute myeloid leukemia based on RUNX1-RUNX1T1 fusion quantification on genomic DNA.

Although acute myeloid leukemia (AML) with t(8;21) belongs to the favorable risk AML subset, relapse incidence may reach 30% in those patients. RUNX1-RUNX1T1 fusion transcript is a well-established marker for minimal residual disease (MRD) monitoring. In this study, we investigated the feasibility and performances of RUNX1-RUNX1T1 DNA as MRD marker in AML with t(8;21). In 17/22 patients with t(8;21)-positive AML treated in the French CBF-2006 trial, breakpoints in RUNX1 and RUNX1T1 were identified using long-range PCR followed by next-generation sequencing. RUNX1-RUNX1T1 DNA quantification was performed by real-time quantitative PCR using patient-specific primers and probe. MRD levels were evaluated in 71 follow-up samples from 16 patients, with a median of four samples [range 2-7] per patient. RUNX1 breakpoints were located in intron 5 in all cases. RUNX1T1 breakpoints were located in intron 1b in 15 cases and in intron 1a in two cases. RUNX1-RUNX1T1 MRD levels measured on DNA and RNA were strongly correlated (r = 0.8, P < 0.0001). Discordant MRD results were observed in 10/71 (14%) of the samples: in three samples from two patients who relapsed, RUNX1-RUNX1T1 was detectable only on DNA, while RUNX1-RUNX1T1 was detectable only on RNA in seven samples. MRD monitoring on genomic DNA is feasible, but with sensitivity variations depending on the patient breakpoint sequence and the qPCR assay efficiency. Although interpretation of the results is easier because it is closely related to the number of leukemic cells, this method greatly increases time, cost and complexity, which limits its interest in routine practice.