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.

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.

Gene expression signatures predict outcome in non-muscle-invasive bladder carcinoma: a multicenter validation study.

PURPOSE: Clinically useful molecular markers predicting the clinical course of patients diagnosed with non-muscle-invasive bladder cancer are needed to improve treatment outcome. Here, we validated four previously reported gene expression signatures for molecular diagnosis of disease stage and carcinoma in situ (CIS) and for predicting disease recurrence and progression. EXPERIMENTAL DESIGN: We analyzed tumors from 404 patients diagnosed with bladder cancer in hospitals in Denmark, Sweden, England, Spain, and France using custom microarrays. Molecular classifications were compared with pathologic diagnosis and clinical outcome. RESULTS: Classification of disease stage using a 52-gene classifier was found to be highly significantly correlated with pathologic stage (P < 0.001). Furthermore, the classifier added information regarding disease progression of T(a) or T(1) tumors (P < 0.001). The molecular 88-gene progression classifier was highly significantly correlated with progression-free survival (P < 0.001) and cancer-specific survival (P = 0.001). Multivariate Cox regression analysis showed the progression classifier to be an independently significant variable associated with disease progression after adjustment for age, sex, stage, grade, and treatment (hazard ratio, 2.3; P = 0.007). The diagnosis of CIS using a 68-gene classifier showed a highly significant correlation with histopathologic CIS diagnosis (odds ratio, 5.8; P < 0.001) in multivariate logistic regression analysis. CONCLUSION: This multicenter validation study confirms in an independent series the clinical utility of molecular classifiers to predict the outcome of patients initially diagnosed with non-muscle-invasive bladder cancer. This information may be useful to better guide patient treatment.

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.

Genomic Landscape of CXCR4 Mutations in Waldenström Macroglobulinemia.

PURPOSE: Whole-genome sequencing has revealed MYD88 L265P and CXCR4 mutations (CXCR4(mut)) as the most prevalent somatic mutations in Waldenström macroglobulinemia. CXCR4 mutation has proved to be of critical importance in Waldenström macroglobulinemia, in part due to its role as a mechanism of resistance to several agents. We have therefore sought to unravel the different aspects of CXCR4 mutations in Waldenström macroglobulinemia. EXPERIMENTAL DESIGN: We have scanned the two coding exons of CXCR4 in Waldenström macroglobulinemia using deep next-generation sequencing and Sanger sequencing in 98 patients with Waldenström macroglobulinemia and correlated with SNP array landscape and mutational spectrum of eight candidate genes involved in TLR, RAS, and BCR pathway in an integrative study. RESULTS: We found all mutations to be heterozygous, somatic, and located in the C-terminal domain of CXCR4 in 25% of the Waldenström macroglobulinemia. CXCR4 mutations led to a truncated receptor protein associated with a higher expression of CXCR4. CXCR4 mutations pertain to the same clone as to MYD88 L265P mutations but were mutually exclusive to CD79A/CD79B mutations (BCR pathway). We identified a genomic signature in CXCR4(mut) Waldenström macroglobulinemia traducing a more complex genome. CXCR4 mutations were also associated with gain of chromosome 4, gain of Xq, and deletion 6q. CONCLUSIONS: Our study panned out new CXCR4 mutations in Waldenström macroglobulinemia and identified a specific signature associated to CXCR4(mut), characterized with complex genomic aberrations among MYD88L265P Waldenström macroglobulinemia. Our results suggest the existence of various genomic subgroups in Waldenström macroglobulinemia.

Inhibition of human bladder tumour cell growth by fibroblast growth factor receptor 2b is independent of its kinase activity. Involvement of the carboxy-terminal region of the receptor.

The b isoform of fibroblast growth factor receptor 2, FGFR2b/FGFR2-IIIb/Ksam-IIC1/KGFR, a tyrosine kinase receptor, is expressed in a wide variety of epithelia and is downregulated in several human carcinomas including prostate, salivary and urothelial cell carcinomas. FGFR2b has been shown to inhibit growth in tumour cell lines derived from these carcinomas. Here, we investigated the molecular mechanisms underlying the inhibition of human urothelial carcinoma cell growth following FGFR2b expression. Using a nylon DNA array, we analysed the gene expression profile of the T24 bladder tumour cell line, transfected or not with a construct encoding FGFR2b. The expression of FGFR2b in T24 cells decreased insulin-like growth factor (IGF)-II mRNA levels. This decrease was correlated with a decrease in IGF-II secretion and may have been responsible for the observed inhibition of cell growth because the addition of exogenous IGF-II restored growth rates to normal levels. Using SU5402, an inhibitor of FGFR tyrosine kinase activity, and a kinase dead mutant of the receptor, FGFR2b Y659F/Y660F, we also demonstrated that the growth inhibition and decrease in IGF-II secretion induced by FGFR2b did not require tyrosine kinase activity. Finally, we demonstrated the involvement of the distal carboxy-terminal domain of the receptor in decreasing IGF-II expression and inhibiting T24 cell growth, as Ksam-IIC3, a variant of FGFR2b carrying a short carboxy-terminus, neither downregulated IGF-II nor inhibited cell proliferation. Our data suggest that FGFR2b inhibits the growth of bladder carcinoma cells by reducing IGF-II levels via its carboxy-terminal domain, independent of its tyrosine kinase activity.

EGFR as a potential therapeutic target for a subset of muscle-invasive bladder cancers presenting a basal-like phenotype.

Muscle-invasive bladder carcinoma (MIBC) constitutes a heterogeneous group of tumors with a poor outcome. Molecular stratification of MIBC may identify clinically relevant tumor subgroups and help to provide effective targeted therapies. From seven series of large-scale transcriptomic data (383 tumors), we identified an MIBC subgroup accounting for 23.5% of MIBC, associated with shorter survival and displaying a basal-like phenotype, as shown by the expression of epithelial basal cell markers. Basal-like tumors presented an activation of the epidermal growth factor receptor (EGFR) pathway linked to frequent EGFR gains and activation of an EGFR autocrine loop. We used a 40-gene expression classifier derived from human tumors to identify human bladder cancer cell lines and a chemically induced mouse model of bladder cancer corresponding to human basal-like bladder cancer. We showed, in both models, that tumor cells were sensitive to anti-EGFR therapy. Our findings provide preclinical proof of concept that anti-EGFR therapy can be used to target a subset of particularly aggressive MIBC tumors expressing basal cell markers and provide diagnostic tools for identifying these tumors.

Oncogenic properties of the mutated forms of fibroblast growth factor receptor 3b.

Germinal activating mutations of FGFR3 are responsible for several forms of dwarfism due to the inhibitory effect of FGFR3 on bone growth. Surprisingly, identical somatic activating mutations have been found at the somatic level in tumours: at high frequency in benign epithelial tumours (seborrheic keratosis, urothelial papilloma) and in low-grade, low-stage urothelial carcinomas, and at a lower frequency in other types of urothelial carcinoma, in cervix carcinoma, and in haematological cancer, multiple myeloma. FGFR3 exists as two isoforms, FGFR3b and FGFR3c, differs in ligand specificity and tissue expression. FGFR3b is the main form in epithelial cells and derived tumours, whereas FGFR3c is the main form in mesenchyme-derived cells and multiple myeloma. Several lines of evidence suggest that mutated FGFR3c has transforming properties. Although mutated FGFR3b is mostly found in benign epithelial tumours or carcinomas of low malignant potential, we present evidence here that mutated FGFR3b is oncogenic. All bladder tumours presenting FGFR3 mutations expressed this receptor more strongly than normal urothelium or non-mutated tumours. NIH-3T3 cells transfected with a mutated form of FGFR3b--FGFR3b-S249C, the most common mutation in bladder tumours--presented a spindle-cell morphology, grew in soft agar and gave rise to tumours when xenografted into nude mice. We identified one line of 17 bladder cell lines tested (MGH-U3) that expressed a mutated form of FGFR3b, FGFR3b-Y375C. We showed using siRNA and SU5402, an FGFR inhibitor, that the tumour properties of MGH-U3 depended on mutated receptor activity. Thus, in two different models, mutated FGFR3b presents oncogenic properties.