Mutational and transcriptional landscape of pediatric B-cell precursor lymphoblastic lymphoma.

Pediatric B-cell precursor (BCP) lymphoblastic malignancies are neoplasms with manifestation either in bone marrow/blood (BCP acute lymphoblastic leukemia, BCP-ALL) or less common in extramedullary tissue (BCP lymphoblastic lymphoma, BCP-LBL). Although both presentations are similar in morphology and immunophenotype, molecular studies are virtually restricted to BCP-ALL so far. The lack of molecular studies on BCP-LBL is due to its rarity and the restriction to small, mostly formalin-fixed paraffin embedded (FFPE) tissues. Here we present the first comprehensive mutational and transcriptional analysis of what we consider the largest BCP-LBL cohort described to date (n=97). Whole exome sequencing indicates a mutational spectrum of BCP-LBL strikingly similar to that found in BCP-ALL. However, epigenetic modifiers were more frequently mutated in BCP-LBL, whereas BCP-ALL was more frequently affected by mutation in genes involved in B-cell development. Integrating copy number alterations, somatic mutations and gene expression by RNA-sequencing revealed that virtually all molecular subtypes originally defined in BCP-ALL are present in BCP-LBL too, with only 7% of lymphomas that were not assigned to a subtype. Similar to BCP-ALL, the most frequent subtypes of BCP-LBL were high hyperdiploidy and ETV6::RUNX1. Tyrosine kinase/cytokine-receptor rearrangements were detected in 7% of BCP-LBL. These results indicate that genetic subtypes can be identified in BCP-LBL using next-generation sequencing, even on FFPE tissue, and may be relevant to guide treatment.

Genomic imbalances analysis provides new insight into prognostic factors in adult and pediatric T-ALL.

Given the poor outcome of refractory and relapsing T-ALL, identifying prognostic markers is still challenging. Using SNP-array analysis, we provide a comprehensive analysis of genomic imbalances in a cohort of 317 newly-diagnosed T-ALL patients including 135 children and 182 adults with respect to clinical and biological features and outcomes. SNP-array results identified at least one somatic genomic imbalance in virtually all T-ALL patients (~96%). Del(9)(p21) (~70%) and UPD(9)p21)/CDKN2A/B (~28%) were the most frequent genomic imbalances. Unexpectedly del(13q14)/RB1/DLEU1 (~14%) was the second more frequent CNV followed by del(6)(q15)/CASP8AP2 (~11%), del(1)(p33)/SIL-TAL1 (~11%), del(12)(p13)ETV6/CDKN1B (~9%), del(18)(p11)/PTPN2 (~9%), del(1)(p36)/RPL22 (~9%), and del(17)(q11)/NF1/SUZ12 (~8%). SNP-array also revealed distinct profiles of genomic imbalances according to age, immunophenotype, and oncogenetic subgroups. In particular, adult T-ALL patients demonstrated a significantly higher incidence of del(1)(p36)/RPL22, and del(13)(q14)/RB1/DLEU1, and lower incidence of del(9)(p21) and UPD(9p21)/CDKN2A/B. We determined a threshold of 15 genomic imbalances to stratify patients into high- and low-risk groups of relapse. Survival analysis also revealed the poor outcome, despite the low number of affected cases, conferred by the presence of chromothripsis (n=6, ~2%), del(16)(p13)/CREBBP (n=15, ~5%) as well as the newly identified recurrent gain at 6q27 involving MLLT4 (n=10, ~3%). Genomic complexity, del(16)(p13)/CREBBP and gain at 6q27 involving MLLT4 maintained their significance in multivariate analysis for survival outcome. Our study thus demonstrated that whole genome analysis of imbalances provides new insights to refine risk stratification in T-ALL.

PHF6-altered T-ALL Harbor Epigenetic Repressive Switch at Bivalent Promoters and Respond to 5-Azacitidine and Venetoclax.

PURPOSE: To assess the impact of PHF6 alterations on clinical outcome and therapeutical actionability in T-cell acute lymphoblastic leukemia (T-ALL). EXPERIMENTAL DESIGN: We described PHF6 alterations in an adult cohort of T-ALL from the French trial Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL)-2003/2005 and retrospectively analyzed clinical outcomes between PHF6-altered (PHF6ALT) and wild-type patients. We also used EPIC and chromatin immunoprecipitation sequencing data of patient samples to analyze the epigenetic landscape of PHF6ALT T-ALLs. We consecutively evaluated 5-azacitidine efficacy, alone or combined with venetoclax, in PHF6ALT T-ALL. RESULTS: We show that PHF6 alterations account for 47% of cases in our cohort and demonstrate that PHF6ALT T-ALL presented significantly better clinical outcomes. Integrative analysis of DNA methylation and histone marks shows that PHF6ALT are characterized by DNA hypermethylation and H3K27me3 loss at promoters physiologically bivalent in thymocytes. Using patient-derived xenografts, we show that PHF6ALT T-ALL respond to the 5-azacytidine alone. Finally, synergism with the BCL2-inhibitor venetoclax was demonstrated in refractory/relapsing (R/R) PHF6ALT T-ALL using fresh samples. Importantly, we report three cases of R/R PHF6ALT patients who were successfully treated with this combination. CONCLUSIONS: Overall, our study supports the use of PHF6 alterations as a biomarker of sensitivity to 5-azacytidine and venetoclax combination in R/R T-ALL.

Predictive Value of Minimal Residual Disease for Efficacy of Rituximab Maintenance in Mantle Cell Lymphoma: Results From the European Mantle Cell Lymphoma Elderly Trial.

PURPOSE: The outcome of older patients with mantle cell lymphoma (MCL) has improved by the introduction of immunochemotherapy, followed by rituximab (R)-maintenance. Assessment of minimal residual disease (MRD) represents a promising tool for individualized treatment decisions and was a prospectively planned part of the European MCL Elderly trial. We investigated how MRD status influenced the efficacy of R-maintenance and how MRD can enable tailored consolidation strategies. PATIENTS AND METHODS: Previously untreated patients with MCL age 60 years or older have been randomly assigned to R versus interferon-alpha maintenance after response to rituximab, fludarabine, cyclophosphamide (R-FC) versus rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP). MRD monitoring was performed by real-time quantitative polymerase chain reaction (qPCR) following EuroMRD guidelines. RESULTS: A qPCR assay with a median sensitivity of 1 × 10-5 could be generated in 80% of 288 patients in an international, multicenter, multilaboratory setting. More extensive tumor dissemination facilitated the identification of a molecular marker. The efficacy of R-maintenance in clinical remission was confirmed for MRD-negative patients at the end of induction in terms of progression-free survival (PFS; hazard ratio [HR], 0.38 [95% CI, 0.21 to 0.63]) and overall survival (OS; HR, 0.37 [95% CI, 0.20 to 0.68]), particularly in R-CHOP-treated patients (PFS-HR, 0.23 [95% CI, 0.10 to 0.52]; OS-HR, 0.19 [95% CI, 0.07 to 0.52]). R-maintenance appeared less effective in MRD-positive patients (PFS-HR, 0.51 [95% CI, 0.26 to 1.02]) overall and after R-CHOP induction (PFS-HR, 0.59 [95% CI, 0.28 to 1.26]). R-FC achieved more frequent and faster MRD clearance compared with R-CHOP. MRD positivity in clinical remission after induction was associated with a short median time to clinical progression of approximately 1-1.7 years. CONCLUSION: The results confirm the strong efficacy of R-maintenance in patients who are MRD-negative after induction. Treatment de-escalation for MRD-negative patients is discouraged by our results. More effective consolidation strategies should be explored in MRD-positive patients to improve their long-term prognosis.

Performance of baseline FDG-PET/CT radiomics for prediction of bone marrow minimal residual disease status in the LyMa-101 trial.

The prognostic value of 18F-Fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) at baseline or the predictive value of minimal residual disease (MRD) detection appear as potential tools to improve mantle cell lymphoma (MCL) patients' management. The LyMa-101, a phase 2 trial of the LYSA group (ClinicalTrials.gov:NCT02896582) reported induction therapy with obinutuzumab, a CD20 monoclonal antibody. Herein, we investigated the added prognostic value of radiomic features (RF) derived from FDG-PET/CT at diagnosis for MRD value prediction. FDG-PET/CT of 59 MCL patients included in the LyMa-101 trial have been independently, blindly and centrally reviewed. RF were extracted from the disease area with the highest uptake and from the total metabolic tumor volume (TMTV). Two models of machine learning were used to compare several combinations for prediction of MRD before autologous stem cell transplant consolidation (ASCT). Each algorithm was generated with or without constrained feature selections for clinical and laboratory parameters. Both algorithms showed better discrimination performances for negative vs positive MRD in the lesion with the highest uptake than in the TMTV. The constrained use of clinical and biological features showed a clear loss in sensitivity for the prediction of MRD status before ASCT, regardless of the machine learning model. These data plead for the importance of FDG-PET/CT RF compared to clinical and laboratory parameters and also reinforced the previously made hypothesis that the prognosis of the disease in MCL patients is linked to the most aggressive contingent, within the lesion with the highest uptake.

Multimodal cartography of human lymphopoiesis reveals B and T/NK/ILC lineages are subjected to differential regulation.

The developmental cartography of human lymphopoiesis remains incompletely understood. Here, we establish a multimodal map demonstrating that lymphoid specification follows independent direct or stepwise hierarchic routes converging toward the emergence of newly characterized CD117lo multi-lymphoid progenitors (MLPs) that undergo a proliferation arrest before entering the CD127- (NK/ILC/T) or CD127+ (B) lymphoid pathways. While the differentiation of CD127- early lymphoid progenitors is mainly driven by Flt3 signaling, emergence of their CD127+ counterparts is regulated cell-intrinsically and depends exclusively on the divisional history of their upstream precursors, including hematopoietic stem cells. Further, transcriptional mapping of differentiation trajectories reveals that whereas myeloid granulomonocytic lineages follow continuous differentiation pathways, lymphoid trajectories are intrinsically discontinuous and characterized by sequential waves of cell proliferation allowing pre-commitment amplification of lymphoid progenitor pools. Besides identifying new lymphoid specification pathways and regulatory checkpoints, our results demonstrate that NK/ILC/T and B lineages are under fundamentally distinct modes of regulation. (149 words).

Targeted High-throughput Sequencing for Hematological Malignancies: A GBMHM Survey of Practice and Cost Evaluation in France.

The objective of this study was to assess the clinical impact and financial costs of next-generation sequencing (NGS) in 5 categories of pediatric and adult hematological cancers. NGS prescriptions were prospectively collected from 26 laboratories, with varied technical and reporting practice (all or only significant targets). Impact was defined by the identification of (1) an actionable mutation, (2) a mutation with prognostic and/or theranostic value, and/or (3) a mutation allowing nosological refinement, reported by local investigators. A microcosting study was undertaken in 4 laboratories, identifying the types and volumes of resources required for each procedural step. Individual index prescriptions for 3961 patients were available for impact analysis on the management of myeloid disorders (two thirds) and, mainly mature B, lymphoid disorders (one third). NGS results were considered to impact the management for 73.4% of prescriptions: useful for evaluation of prognostic risk in 34.9% and necessary for treatment adaptation (actionable) in 19.6%, but having no immediate individual therapeutic impact in 18.9%. The average overall cost per sample was 191 € for the restricted mature lymphoid amplicon panel. Capture panel costs varied from 369 € to 513 €. Unit costs varied from 0.5 € to 5.7 € per kb sequenced, from 3.6 € to 11.3 € per target gene/hot-spot sequenced and from 4.3 € to 73.8 € per target gene/hot-spot reported. Comparable costs for the Amplicon panels were 5-8 € per kb and 10.5-14.7 € per target gene/hot-spot sequenced and reported, demonstrating comparable costs with greater informativity/flexibility for capture strategies. Sustainable funding of precision medicine requires a transparent discussion of its impact on care pathways and its financial aspects.

TREC mediated oncogenesis in human immature T lymphoid malignancies preferentially involves ZFP36L2.

The reintegration of excised signal joints resulting from human V(D)J recombination was described as a potent source of genomic instability in human lymphoid cancers. However, such molecular events have not been recurrently reported in clinical patient lymphoma/leukemia samples. Using a specifically designed NGS-capture pipeline, we here demonstrated the reintegration of T-cell receptor excision circles (TRECs) in 20/1533 (1.3%) patients with T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL). Remarkably, the reintegration of TREC recurrently targeted the tumor suppressor gene, ZFP36L2, in 17/20 samples. Thus, our data identified a new and hardly detectable mechanism of gene deregulation in lymphoid cancers providing new insights in human oncogenesis.

Distinct subsets of multi-lymphoid progenitors support ontogeny-related changes in human lymphopoiesis.

Changes in lymphocyte production patterns occurring across human ontogeny remain poorly defined. In this study, we demonstrate that human lymphopoiesis is supported by three waves of embryonic, fetal, and postnatal multi-lymphoid progenitors (MLPs) differing in CD7 and CD10 expression and their output of CD127-/+ early lymphoid progenitors (ELPs). In addition, our results reveal that, like the fetal-to-adult switch in erythropoiesis, transition to postnatal life coincides with a shift from multilineage to B lineage-biased lymphopoiesis and an increase in production of CD127+ ELPs, which persists until puberty. A further developmental transition is observed in elderly individuals whereby B cell differentiation bypasses the CD127+ compartment and branches directly from CD10+ MLPs. Functional analyses indicate that these changes are determined at the level of hematopoietic stem cells. These findings provide insights for understanding identity and function of human MLPs and the establishment and maintenance of adaptative immunity.

Improving outcomes of childhood and young adult non-Hodgkin lymphoma: 25 years of research and collaboration within the framework of the European Intergroup for Childhood Non-Hodgkin Lymphoma.

The European Intergroup for Childhood Non-Hodgkin Lymphoma (EICNHL) was established 25 years ago with the goal to facilitate clinical trials and research collaborations in the field both within Europe and worldwide. Since its inception, much progress has been made whereby major improvements in outcomes have been achieved. In this Review, we describe the different diagnostic entities of non-Hodgkin lymphoma in children and young adults describing key features of each entity and outlining clinical achievements made in the context of the EICNHL framework. Furthermore, we provide an overview of advances in biopathology with an emphasis on the role of biological studies and how they have shaped available treatments. Finally, for each entity, we describe future goals, upcoming clinical trials, and highlight areas of research that require our focus going forward.

Harnessing the MYB-dependent TAL1 5'super-enhancer for targeted therapy in T-ALL.

The acquisition of genetic abnormalities engendering oncogene dysregulation underpins cancer development. Certain proto-oncogenes possess several dysregulation mechanisms, yet how each mechanism impacts clinical outcome is unclear. Using T-cell acute lymphoblastic leukemia (T-ALL) as an example, we show that patients harboring 5'super-enhancer (5'SE) mutations of the TAL1 oncogene identifies a specific patient subgroup with poor prognosis irrespective of the level of oncogene dysregulation. Remarkably, the MYB dependent oncogenic 5'SE can be targeted using Mebendazole to induce MYB protein degradation and T-ALL cell death. Of note Mebendazole treatment demonstrated efficacy in vivo in T-ALL preclinical models. Our work provides proof of concept that within a specific oncogene driven cancer, the mechanism of oncogene dysregulation rather than the oncogene itself can identify clinically distinct patient subgroups and pave the way for future super-enhancer targeting therapy.

Human kidney-derived hematopoietic stem cells can support long-term multilineage hematopoiesis.

Long-term multilineage hematopoietic donor chimerism occurs sporadically in patients who receive a transplanted solid organ enriched in lymphoid tissues such as the intestine or liver. There is currently no evidence for the presence of kidney-resident hematopoietic stem cells in any mammal species. Graft-versus-host-reactive donor T cells promote engraftment of graft-derived hematopoietic stem cells by making space in the bone marrow. Here, we report full (over 99%) multilineage, donor-derived hematopoietic chimerism in a pediatric kidney transplant recipient with syndromic combined immune deficiency that leads to transplant tolerance. Interestingly, we found that the human kidney-derived hematopoietic stem cells took up long-term residence in the recipient's bone marrow and gradually replaced their host counterparts, leading to blood type conversion and full donor chimerism of both lymphoid and myeloid lineages. Thus, our findings highlight the existence of human kidney-derived hematopoietic stem cells with a self-renewal ability able to support multilineage hematopoiesis.

The gaps between the new EU legislation on in vitro diagnostics and the on-the-ground reality.

The background to this debate is now well-known: an EU policy decision to tighten controls on the devices and diagnostics sector led to the adoption of a regulation in 2017 with a schedule for implementation over coming years - a timetable extended still further by last-minute legislation in early 2022, to provide the sector and regulators with more time to adapt to the changes. Discussions among experts organised in April by the European Alliance for Personalized Medicine (EAPM) exposed continuing challenges that cannot be fully resolved by the recent deferral of implementation deadlines. One salient problem is that there is little awareness of the In Vitro Diagnostic Regulation (IVDR) across Europe, and only limited awareness of the different structures of national systems involved in implementing IVDR, with consequent risks for patient and consumer access to in vitro diagnostics (IVDs). The tentative conclusion from these consultations is that despite a will across the sector to seek workable solutions, the obstacles remain formidable, and the potential solutions so far proposed remain more a matter of aspirations than of clear pathways.