Frequency and clinical impact of CDKN2A/ARF/CDKN2B gene deletions as assessed by in-depth genetic analyses in adult T cell acute lymphoblastic leukemia

© The Author(s).Recurrent deletions of the CDKN2A/ARF/CDKN2B genes encoded at chromosome 9p21 have been described in both pediatric and adult acute lymphoblastic leukemia (ALL), but their prognostic value remains controversial, with limited data on adult T-ALL. Here, we investigated the presence of homozygous and heterozygous deletions of the CDKN2A/ARF and CDKN2B genes in 64 adult T-ALL patients enrolled in two consecutive trials from the Spanish PETHEMA group. Alterations in CDKN2A/ARF/CDKN2B were detected in 35/64 patients (55%). Most of them consisted of 9p21 losses involving homozygous deletions of the CDKNA/ARF gene (26/64), as confirmed by single nucleotide polymorphism (SNP) arrays and interphase fluorescence in situ hybridization (iFISH). Deletions involving the CDKN2A/ARF/CDKN2B locus correlated with a higher frequency of cortical T cell phenotype and a better clearance of minimal residual disease (MRD) after induction therapy. Moreover, the combination of an altered copy-number-value (CNV) involving the CDKN2A/ARF/CDKN2B gene locus and undetectable MRD (≤ 0.01%) values allowed the identification of a subset of T-ALL with better overall survival in the absence of hematopoietic stem cell transplantation.This project was supported by the Asociación Española Contra el Cáncer, AECC (project ref.: GC16173697BIGA), by CERCA Program/Generalitat de Catalunya, the Catalan Government: 2014-SGR225 (GRE), Obra Social “La Caixa” and by Celgene Spain. E. Genescà is the recipient of agrant from the Spanish Health Ministry (ISCIII, CA12/00468) and an unrestricted grant from Gilead.A. Gonzalez-Perez is supported by a Ramon y Cajal fellowship (RYC-2013-14554) of the Educational Ministry (Madrid, Spain). This work was also partially supported by FEDER funds from the ISCIII (PT13/0010/0026, CIBERONC (CB16/12/00284 and CB16/12/00400), Madrid, Spain)

Caracterització i valor pronòstic de l'estudi citogenètic de les leucèmies mieloides agudes incloses en protocols CETLAM

Les leucèmies mieloides agudes (LMA) són un grup heterogeni de neoplàsies hematològiques amb diferents característiques clíniques i genètiques. Les alteracions citogenètiques de la LMA s'associen tant a característiques clíniques específiques com a la resposta al tractament; i defineixen els esquemes d'estratificació del risc en la LMA. Tanmateix, la importància pronòstica d'algunes anomalies citogenètiques recurrents segueix sent incerta. En aquest treball s'han analitzat les alteracions citogenètiques i el seu impacte sobre el pronòstic de 1417 pacients adults diagnosticats de LMA de novo (excloent LMA amb t(15;17) o reordenació PML-RARA) i inclosos en protocols CETLAM de quimioteràpia intensiva entre el 1994 i el 2012. En l'anàlisi multivariant, la t(8;21)(q22;q22) i la inv(16)(p13q22)/t(16;16)(p13;q22) han estat les úniques anomalies que prediuen un pronòstic relativament favorable. A més, en pacients amb t(8;21) o inv(16)/t(16;16), les anomalies citogenètiques addicionals no tenen un efecte advers significatiu en el pronòstic. Per altra banda, diverses anomalies han predit un resultat significativament més dolent, concretament la monosomia del cromosoma 7, les alteracions estructurals del cromosoma 1, el cariotip complex de més de 4 anomalies (CK) i el cariotip monosòmic (MK). Finalment, els pacients d'aquesta sèrie s'han distribuït en tres grups pronòstic segons les alteracions citogenètiques que presentaven i d'acord amb tres sistemes d'estratificació del risc (MRC, CALGB i el proposat per CETLAM en aquest treball). S'ha demostrat que els tres sistemes són vàlids per classificar els pacients en tres grups pronòstic ben definits. Aquest treball consolida la importància pronòstica de les alteracions citogenètiques en la LMA i obre algunes vies per seguir investigant en el camp de la citogenètica de la LMA.Las leucemias mieloides agudas (LMA) son un grupo heterogéneo de neoplasias hematológicas con distintas características clínicas y genéticas. Las alteraciones citogenéticas de la LMA se asocian tanto a características clínicas específicas como a la respuesta al tratamiento; y definen los esquemas de estratificación del riesgo en la LMA. Sin embargo, la importancia pronóstica de algunas anomalías citogenéticas recurrentes sigue siendo incierta. En este trabajo se han analizado las alteraciones citogenéticas y su impacto sobre el pronóstico de 1417 pacientes adultos diagnosticados de LMA de novo (excluyendo LMA con t(15;17) o reordenación PML-RARA) e incluidos en protocolos CETLAM de quimioterapia intensiva entre el 1994 y 2012. En el análisis multivariante, la t(8;21)(q22;q22) y la inv(16)(p13q22)/t(16;16)(p13;q22) han sido las únicas anomalías que predicen un pronóstico relativamente favorable. Además, en pacientes con t(8;21) o inv(16)/t(16;16), las anomalías citogenéticas adicionales no tienen un efecto adverso significativo en el pronóstico. Por otra parte, varias anomalías han predicho un resultado significativamente peor, concretamente la monosomía del cromosoma 7, las alteraciones estructurales del cromosoma 1, el cariotipo complejo de más de 4 anomalías (CK) y el cariotipo monosómico (MK). Por último, los pacientes de esta serie se han distribuido en tres grupos pronóstico según las alteraciones citogenéticas que presentaban y de acuerdo con tres sistemas de estratificación del riesgo (MRC, CALGB y el propuesto por CETLAM en este trabajo). Se ha demostrado que los tres sistemas son válidos para clasificar a los pacientes en tres grupos pronóstico bien definidos. Este trabajo consolida la importancia pronóstica de las alteraciones citogenéticas en la LMA y abre algunas vías para seguir investigando en el campo de la citogenética de la LMA.Acute myeloid leukemias (AML) are a heterogeneous group of hematological neoplasms with distinct clinical and genetic features. Cytogenetic abnormalities in AML are strongly associated with both clinical features and therapeutic outcome and provide the framework for risk-stratification schemes in AML. However, the prognostic significance of some recurring cytogenetic abnormalities remains uncertain. This study analyzes the cytogenetic alterations and their impact on the prognosis of 1417 adult patients diagnosed with de novo AML (excluding AML with t(15;17) or PML::RARA rearrangement) and included in CETLAM protocols for intensive chemotherapy between 1994 and 2012. In multivariable analysis, t(8;21)(q22;q22) and inv(16)(p13q22)/t(16;16)(p13;q22) were the only abnormalities predicting a relatively favorable prognosis. In patients with t(8;21) or inv(16)/t(16;16), additional cytogenetic abnormalities did not have a significant adverse effect. On the other hand, several abnormalities predicted a significantly worse outcome, namely monosomy of chromosome 7, structural alterations of chromosome 1, complex karyotype with more than 4 abnormalities (CK) and monosomal karyotype (MK). Finally, the patients in this series were distributed in three prognostic groups according to their cytogenetic alterations and according to three risk stratification systems (MRC, CALGB and the one proposed by CETLAM in this work). All three systems have been shown to be valid for classifying patients into three well-defined prognostic groups. This work consolidates the prognostic importance of cytogenetic alterations in AML and opens some paths for further research in the field of AML cytogenetics

Extramedullary multiple myeloma patient-derived orthotopic xenograft with a highly altered genome: combined molecular and therapeutic studies

Extramedullary multiple myeloma (EMM) has an overall survival of 6 months and occurs in 20% of multiple myeloma (MM) patients. Genetic and epigenetic mechanisms involved in EMM and the therapeutic role of new agents for MM are not well established. Besides, well-characterized preclinical models for EMM are not available. Herein, a patient-derived orthotopic xenograft (PDOX) was generated from a patient with an aggressive EMM to study in-depth genetic and epigenetic events, and drug responses related to extramedullary disease. A fresh punch of an extramedullary cutaneous lesion was orthotopically implanted in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ(NSG) mouse. The PDOX mimicked histologic and phenotypic features of the tumor of the patient. Cytogenetic studies revealed a hyperploid genome with multiple genetic poor-prognosis alterations. Copy number alterations (CNAs) were detected in all chromosomes. The IGH translocation t(14;16)(q32;q23)IGH/MAF was already observed at the medullary stage and a new one, t(10;14)(p?11-12;q32), was observed only with extramedullary disease and could be eventually related to EMM progression in this case. Exome sequencing showed 24 high impact single nucleotide variants and 180 indels. From the genes involved, only TP53 was previously described as a driver in MM. A rather balanced proportion of hyper/hypomethylated sites different to previously reported widespread hypomethylation in MM was also observed. Treatment with lenalidomide, dexamethasone and carfilzomib showed a tumor weight reduction of 90% versus non-treated tumors, whereas treatment with the anti-CD38 antibody daratumumab showed a reduction of 46%. The generation of PDOX from a small EMM biopsy allowed us to investigate in depth the molecular events associated with extramedullary disease in combination with drug testing

Characterizing patients with multiple chromosomal aberrations detected by FISH in chronic lymphocytic leukemia

On behalf of Grupo Español de Leucemia Linfática Crónica (GELLC) and Grupo Cooperativo Español de Citogenética Hematológica (GCECGH).We analyzed the features of chronic lymphocytic leukemia (CLL) with multiple abnormalities (MA) detected by FISH. A local database including 2095 CLL cases was used and 323 with MA (15.4%) were selected. MA was defined by the presence of two or more alterations (deletions of 13q14 (13q-), 11q22 (11q-), 17p13 (17p-) or trisomy 12 (+12)). The combination of 13q- with 11q- and 13q- with 17p-, accounted for 58.2% of the series, in contrast to 11q- with 17p- (3.7%). Patients carrying MA since diagnosis presented a short time to first therapy(TTFT) (27 months) and overall survival (OS) (76 months). The combinations including 17p- had a shorter OS (58 months) than the ones without 17p- (not reached, p =.002). Patients with a complex-FISH were the ones with worse OS (34 months). MA imply poor prognosis when they emerge at diagnosis, probably due to the high incidence of bad prognosis markers, which may be a reflection of a more complex karyotype.Peer Reviewe

Development Refractoriness of MLL-Rearranged Human B Cell Acute Leukemias to Reprogramming into Pluripotency

Altres ajuts: Asociación Española Contra el Cáncer, Obra Social La Caixa i Fundació Josep CarrerasInduced pluripotent stem cells (iPSCs) are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL) has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L)-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming "boosters" also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency

Patients with chronic lymphocytic leukemia and complex karyotype show an adverse outcome even in absence of TP53/ATM FISH deletions

Genomic complexity identified by chromosome banding analysis (CBA) predicts a worse clinical outcome in CLL patients treated either with standard or new treatments. Herein, we analyzed the clinical impact of complex karyotypes (CK) with or without high-risk FISH deletions (ATM and/or TP53, HR-FISH) in a cohort of 1045 untreated MBL/CLL patients. In all, 99/1045 (9.5%) patients displayed a CK. Despite ATM and TP53 deletions were more common in CK (25% vs 7%; P < 0.001; 40% vs 5%; P < 0.001, respectively), only 44% (40/90) patients with TP53 deletions showed a CK. CK group showed a significant higher two-year cumulative incidence of treatment (48% vs 20%; P < 0.001), as well as a shorter overall survival (OS) (79 mo vs not reached; P < 0.001). When patients were categorized regarding CK and HR-FISH, those with both characteristics showed the worst median OS (52 mo) being clearly distinct from those non-CK and non-HR-FISH (median not reached), but no significant differences were detected between cases with only CK or HR-FISH. Both CK and TP53 deletion remained statistically significant in the multivariate analysis for OS. In conclusion, CK group is globally associated with advanced disease and poor prognostic markers. Further investigation in larger cohorts with CK lacking HR-FISH is needed to elucidate which mechanisms underlie the poor outcome of this subgrou

Patients with chronic lymphocytic leukemia and complex karyotype show an adverse outcome even in absence of TP53/ATM FISH deletions

Genomic complexity identified by chromosome banding analysis (CBA) predicts a worse clinical outcome in CLL patients treated either with standard or new treatments. Herein, we analyzed the clinical impact of complex karyotypes (CK) with or without high-risk FISH deletions (ATM and/or TP53, HR-FISH) in a cohort of 1045 untreated MBL/CLL patients. In all, 99/1045 (9.5%) patients displayed a CK. Despite ATM and TP53 deletions were more common in CK (25% vs 7%; P < 0.001; 40% vs 5%; P < 0.001, respectively), only 44% (40/90) patients with TP53 deletions showed a CK. CK group showed a significant higher two-year cumulative incidence of treatment (48% vs 20%; P < 0.001), as well as a shorter overall survival (OS) (79 mo vs not reached; P < 0.001). When patients were categorized regarding CK and HR-FISH, those with both characteristics showed the worst median OS (52 mo) being clearly distinct from those non-CK and non-HR-FISH (median not reached), but no significant differences were detected between cases with only CK or HR-FISH. Both CK and TP53 deletion remained statistically significant in the multivariate analysis for OS. In conclusion, CK group is globally associated with advanced disease and poor prognostic markers. Further investigation in larger cohorts with CK lacking HR-FISH is needed to elucidate which mechanisms underlie the poor outcome of this subgroup