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.

Cryptic and partial deletions of PRDM16 and RUNX1 without t(1;21)(p36;q22) and/or RUNX1-PRDM16 fusion in a case of progressive chronic myeloid leukemia: a complex chromosomal rearrangement of underestimated frequency in disease progression?

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the presence in leukemic stem cells of the Philadelphia chromosome (Ph) and the formation of the BCR-ABL1 fusion. Untreated, the disease progresses to accelerate phase and blast crisis in which hematopoietic differentiation has become arrested. CML progression is frequently associated with cytogenetic evidence of clonal evolution, defined as additional chromosomal aberrations. We here report a CML resistant to tyrosine kinase inhibitors that rapidly progressed to blastic phase. At this time, array CGH performed on CD34(+) cells revealed cryptic partial deletions of both PRDM16 and RUNX1 and duplication of the der(21) chromosome. These genomic rearrangements were confirmed by FISH with probes targeting the deletion on chromosome 21 (24 kb), and with BAC probes flanking the deletion on 1p36 (220 kb). However, no cryptic t(1;21)(p36;q22) and/or RUNX1-PRDM16 were detected, suggesting that these deletions are the residual hallmarks of a more complex mechanism of chromosomal rearrangement, as indicated by the additional inversion of the region bounded by 1p36.32 and 1p36.12 breaks. At the molecular level, these abnormalities lead to the overexpression of the PR-domain negative oncogenic isoform of PRDM16, associated with two deleted copies within the runt domain of C-teminal aberrant RUNX1. These events are not detectable by conventional cytogenetic and molecular strategies, and may be of underestimated frequency in disease progression.

Detection of somatic quantitative genetic alterations by multiplex polymerase chain reaction for the prediction of outcome in diffuse large B-cell lymphomas.

BACKGROUND: Genomic gains and losses play a crucial role in the development of diffuse large B-cell lymphomas. High resolution array comparative genomic hybridization provides a comprehensive view of these genomic imbalances but is not routinely applicable. We developed a polymerase chain reaction assay to provide information regarding gains or losses of relevant genes and prognosis in diffuse large B-cell lymphomas. DESIGN AND METHODS: Two polymerase chain reaction assays (multiplex polymerase chain reaction of short fluorescent fragments, QMPSF) were designed to detect gains or losses of c-REL, BCL6, SIM1, PTPRK, MYC, CDKN2A, MDM2, CDKN1B, TP53 and BCL2. Array comparative genomic hybridization was simultaneously performed to evaluate the sensitivity and predictive value of the QMPSF assay. The biological and clinical relevance of this assay were assessed. RESULTS: The predictive value of the QMPSF assay for detecting abnormal DNA copy numbers ranged between 88-97%, giving an overall concordance rate of 92% with comparative genomic hybridization results. In 77 cases of diffuse large B-cell lymphomas, gains of MYC, CDKN1B, c-REL and BCL2 were detected in 12%, 40%, 27% and 29%, respectively. TP53 and CDKN2A deletions were observed in 22% and 36% respectively. BCL2 and CDKN2A allelic status correlated with protein expression. TP53 mutations were associated with allelic deletions in 45% of cases. The prognostic value of a single QMPSF assay including TP53, MYC, CDKN2A, SIM1 and CDKN1B was predictive of the outcome independently of the germinal center B-cell like/non-germinal center B-cell like subtype or the International Prognostic Index. CONCLUSIONS: QMPSF is a reliable and flexible method for detecting somatic quantitative genetic alterations in diffuse large B-cell lymphomas and could be integrated in future prognostic predictive models.

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.

HGF/SF regulates expression of apoptotic genes in MCF-10A human mammary epithelial cells.

Hepatocyte growth factor/scatter factor (HGF/SF) induces scattering, morphogenesis, and survival of epithelial cells through activation of the MET tyrosine kinase receptor. HGF/SF and MET are involved in normal development and tumor progression of many tissues and organs, including the mammary gland. In order to find target genes of HGF/SF involved in its survival function, we used an oligonucleotide microarray representing 1,920 genes known to be involved in apoptosis, transcriptional regulation, and signal transduction. MCF-10A human mammary epithelial cells were grown in the absence of serum and treated or not with HGF/SF for 2 h. Total RNA was reverse-transcribed to cDNA in the presence of fluorescent Cy3-dUTP or Cy5-dUTP to generate fluorescently labeled cDNA probes. Microarrays were performed and the ratios of Cy5/Cy3 fluorescence were determined. The expression of three apoptotic genes was modified by HGF/SF, with A20 being upregulated, and DAXX and SMAC being downregulated. These changes of expression were confirmed by real-time quantitative PCR. According to current-knowledge, A20 is antiapoptotic and SMAC is proapoptotic, while a pro- or antiapoptotic function of DAXX is controversial. The fact that HGF/SF upregulates an antiapoptotic gene (A20) and downregulates a proapoptotic gene (SMAC) is in agreement with its survival effect in MCF-10A cells. This study identified novel apoptotic genes regulated by HGF/SF, which can contribute to its survival effect.

Next-generation sequencing of FLT3 internal tandem duplications for minimal residual disease monitoring in acute myeloid leukemia.

Minimal Residual Disease (MRD) detection can be used for early intervention in relapse, risk stratification, and treatment guidance. FLT3 ITD is the most common mutation found in AML patients with normal karyotype. We evaluated the feasibility of NGS with high coverage (up to 2.4.10(6) PE fragments) for MRD monitoring on FLT3 ITD. We sequenced 37 adult patients at diagnosis and various times of their disease (64 samples) and compared the results with FLT3 ITD ratios measured by fragment analysis. We found that NGS could detect variable insertion sites and lengths in a single test for several patients. We also showed mutational shifts between diagnosis and relapse, with the outgrowth of a clone at relapse different from that dominant at diagnosis. Since NGS is scalable, we were able to adapt sensitivity by increasing the number of reads obtained for follow-up samples, compared to diagnosis samples. This technique could be applied to detect biological relapse before its clinical consequences and to better tailor treatments through the use of FLT3 inhibitors. Larger cohorts should be assessed in order to validate this approach.

Dysregulation and overexpression of HMGA2 in myelofibrosis with myeloid metaplasia.

Among cytogenetic studies of patients affected with myelofibrosis with myeloid metaplasia (MMM), a rare chronic myeloproliferative disorder, we found several reports of structural abnormalities of the long arm of chromosome 12. Two MMM patients had a balanced translocation involving 12q: t(4;12)(q32;q15) and t(5;12)(p14;q15), respectively. FISH (fluorescence in situ hybridization) analysis showed that BAC (bacterial artificial chromosome) RP11-366L20 overlaps the breakpoint in both cases. A gene, HMGA2, most of which is included in that BAC, thus was identified as a potential candidate. Using reserves transcriptase-polymerase chain reaction (RT-PCR), we looked for expression of HMGA2 in blood mononuclear cells from these 2 patients and demonstrated a transcript in both. Moreover, we found the gene expressed in the hematopoietic cells of 10 of 10 additional patients bearing no 12q anomalies. HMGA2, not expressed in normal subjects, is implicated in benign solid tumors such as lipomas, leiomyomas, and other rare tumors of mesenchymal origin. We postulate that its dysregulation and overexpression in myeloid progenitors contribute also to the pathogenesis of MMM.