Caracterització biològica de la trombocitèmia essencial i la policitèmia vera

Consultable des del TDXTítol obtingut de la portada digitalitzadaEl concepte de síndromes mieloproliferatives cròniques (SMPC) engloba un conjunt d'entitats hematològiques amb característiques clíniques i evolutives molt similars i d'etiopatogènia probablement comú. Es tracta de processos caracteritzats per l'expansió clonal d'una cèl·lula mare (stem cell) pluripotent, que dóna com a resultat una hipercel·lularitat medul·lar amb predomini d'una o més línies que arriben a diferenciar-se en elements madurs. Totes les SMPC es troben subjectes a evolució clonal però en un grau força variable: un 90% d'evolució a leucèmia aguda en la leucèmia mieloide crònica front un 1-2% a la trombocitèmia essencial. Les SMPC clàssiques comprenen quatre entitats: la leucèmia mieloide crònica (LMC), la mielofibrosi idiopàtica (MI), la policitèmia vera (PV) i la trombocitèmia essencial (TE). Aquesta tesi pretén caracteritzar biologicament millor la PV i la TE. La PV és el resultat de la proliferació anormal d'una cèl·lula mare pluripotent, que dóna lloc a una hemopoesi clonal d'hematies, granulòcits i plaquetes, amb predomini d'hiperplàsia eritroide sobre la resta de línies hemopoètiques. La TE és una SMPC caracteritzada per un increment persistent de la xifra de plaquetes i per una hiperplàsia megacariocítica en la medul·la òssia. Un dels criteris diagnòstics majors de la PV i majoria dels criteris diagnòstics de la TE són criteris d'exclusió. Aquest fet fa que constantment s'estiguin buscant nous biomarcadors que ajudin al diagnòstics d'aquestes patologies. Les tècniques que s'han utilitzat en aquestes tesi amb aquest propòsit han sigut: 1. Citogenètica convencional: - PV: Al moment del diagnòstic entre el 13-18% dels pacients presenten un cariotip alterat mitjançant tècniques de citogenètica convencional. Les alteracions més freqüents són: del(20q) (25%), trisomia 8 (16%), trisomia 9 (16%) (tampoc era rar trobar les dues trisomies 8 i 9 juntes en el mateix pacient), duplicacions de bandes inespecífiques d'1q o trisomies d'1q (10%), seguit per del(13q), del(11q), del(7q), del(5q) i trisomia 21. - TE: Al moment del diagnòstic entre el 5% i 10% dels casos presenten alteracions cromosòmiques. Entre les alteracions citogenètiques més descrites en la TE trobem les delecions dels braços llargs dels cromosomes 20 (del(20q)) i 13 (del(13q)) i les trisomies 8 i 9. 2. Hibridació in situ fluorescent: - PV: El percentatge de pacients amb alteracions cromosòmiques que es detecten mitjançant la tècnica de FISH varia entre 14.3% i 71.5% en funció de cada sèrie. - TE: El percentatge de pacients amb alteracions cromosòmiques que es detecten mitjançant la tècnica de FISH varia entre 15% i 55% en funció de cada sèrie. 3. Estudi de clonalitat mitjançant el gen HUMARA: - PV: El percentatge de clonalitat entre les pacients afectes de PV oscil·la entre el 41% i el 73%. - TE: El percentatge de clonalitat entre les pacients afectes de TE oscil·la entre el 18.7% i el 68%. La tècnica més útil com a nou biomarcador ha estat l'estudi de clonalitat mitjançant el gen HUMARA. Ara bé, la recerca constant de possibles biomarcadors (ex. PRV-1, JAK2...) poden superar la eficacia d'aquesta tècnica.Essential thrombocythemia (ET) and polycythemia vera (PV) are chronic myeloproliferative disorders (MPD) arising from the clonal expansion of a pluripotential stem cell. Specific genetic lesions have not been recognized for both disorders, so, diagnostic criteria still are based on the presence or absence of particular clinical and laboratory features In recent years, there have been attempts to find new positive criteria that could help in the diagnosis of ET and PV, such as spontaneous in vitro colony formation of erythroid and megakaryocytic progenitors, conventional cytogenetics, fluorescence in situ hybridization techniques and analysis of X-chromosome inactivation patterns (XCIPs). This thesis wants to better characterize PV and ET. For this propose it has been used the following methodologies: 1. Conventional Cytogenetics: - PV: At diagnosis between 13-18% of patients has an abnormal karyotype with conventional cytogenetics methodologies. The most frequent abnormalities are del(20q) (25%), trisomy 8 (16%), trisomy 9 (16%) (we can also find both alterations together in the same patient), duplications of unspecific bands from 1q or trisomy 1q (10%), del(13q), del(11q), del(7q), del(5q) and trisomy 21. - TE: At diagnosis between 5-10% of patients has an abnormal karyotype with conventional cytogenetics methodology. The most frequent abnormalities are del(20q)), del(13q) and trisomy 8 and 9. 2. Fluorescent in situ hybridization: - PV: The percentage of patients with abnormalities detected by FISH goes from 14.3% to 71.5% depending on the series. - TE: The percentage of patients with abnormalities detected by FISH goes from 15% to 55% depending on the series. 3. Clonality study with HUMARA gene: - PV: The percentage of clonality between patients goes from 41% to 73%. - TE: The percentage of clonality between patients goes from 18.7% to 68%. The most useful technique as a biomarker has been the clonality study with the HUMARA gene. But the constant investigation of new possible biomarkers (ex. PRV-1, JAK2...) could be even more useful than this one

Association between Germline Single-Nucleotide Variants in ADME Genes and Major Molecular Response to Imatinib in Chronic Myeloid Leukemia Patients

Chronic myeloid leukemia; Imatinib; Single-nucleotide polymorphismsLeucèmia mieloide crònica; Imatinib; Polimorfismes d'un sol nucleòtidLeucemia mieloide crónica; Imatinib; Polimorfismos de un sólo nucleótidoImatinib is the most common first-line tyrosine kinase inhibitor (TKI) used to treat chronic-phase chronic myeloid leukemia (CP-CML). However, only a proportion of patients achieve major molecular response (MMR), so there is a need to find biological factors that aid the selection of the optimal therapeutic strategy (imatinib vs. more potent second-generation TKIs). The aim of this retrospective study was to understand the contribution of germline single-nucleotide variants (gSNVs) in the achievement of MMR with imatinib. In particular, a discovery cohort including 45 CP-CML patients was analyzed through the DMET array, which interrogates 1936 variants in 231 genes related to the absorption, distribution, metabolism and excretion (ADME) process. Variants statistically significant in the discovery cohort were then tested in an extended and independent cohort of 137 CP-CML patients. Finally, a total of 7 gSNVs (ABCG1-rs492338, ABCB11-rs496550, ABCB11-rs497692, CYP2D6-rs1135840, CYP11B1-rs7003319, MAT1A-rs4934027 and SLC22A1-rs628031) and one haplotype in the ABCB11 gene were significantly associated with the achievement of MMR with first-line imatinibtreatment. In conclusion, we identified a genetic signature of response to imatinib in CP-CML patients that could be useful in selecting those patients that may benefit from starting imatinib as first-line therapy, therefore avoiding the toxicity related to second-generation TKIs.This work was supported by Novartis Oncology (NF.5.1.2-3d.018/2014) and the Fundación Española de Hematología y Hemoterapia (FEHH-SEHH)

Association between Germline Single-Nucleotide Variants in ADME Genes and Major Molecular Response to Imatinib in Chronic Myeloid Leukemia Patients

Leucèmia mieloide crònica; Imatinib; Inhibidor de la tirosina quinasa:Chronic myeloid leukemia; Imatinib; Tyrosine kinase inhibitorLeucemia mieloide crónica; Imatinib; Inhibidor de la tirosina quinasaImatinib is the most common first-line tyrosine kinase inhibitor (TKI) used to treat chronic-phase chronic myeloid leukemia (CP-CML). However, only a proportion of patients achieve major molecular response (MMR), so there is a need to find biological factors that aid the selection of the optimal therapeutic strategy (imatinib vs. more potent second-generation TKIs). The aim of this retrospective study was to understand the contribution of germline single-nucleotide variants (gSNVs) in the achievement of MMR with imatinib. In particular, a discovery cohort including 45 CP-CML patients was analyzed through the DMET array, which interrogates 1936 variants in 231 genes related to the absorption, distribution, metabolism and excretion (ADME) process. Variants statistically significant in the discovery cohort were then tested in an extended and independent cohort of 137 CP-CML patients. Finally, a total of 7 gSNVs (ABCG1-rs492338, ABCB11-rs496550, ABCB11-rs497692, CYP2D6-rs1135840, CYP11B1-rs7003319, MAT1A-rs4934027 and SLC22A1-rs628031) and one haplotype in the ABCB11 gene were significantly associated with the achievement of MMR with first-line imatinibtreatment. In conclusion, we identified a genetic signature of response to imatinib in CP-CML patients that could be useful in selecting those patients that may benefit from starting imatinib as first-line therapy, therefore avoiding the toxicity related to second-generation TKIs

Analysis of Intratumoral Heterogeneity in Myelodysplastic Syndromes with Isolated del(5q) Using a Single Cell Approach

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological diseases. Among them, the most well characterized subtype is MDS with isolated chromosome 5q deletion (MDS del(5q)), which is the only one defined by a cytogenetic abnormality that makes these patients candidates to be treated with lenalidomide. During the last decade, single cell (SC) analysis has emerged as a powerful tool to decipher clonal architecture and to further understand cancer and other diseases at higher resolution level compared to bulk sequencing techniques. In this study, a SC approach was used to analyze intratumoral heterogeneity in four patients with MDS del(5q). Single CD34+CD117+CD45+CD19- bone marrow hematopoietic stem progenitor cells were isolated using the C1 system (Fluidigm) from diagnosis or before receiving any treatment and from available follow-up samples. Selected somatic alterations were further analyzed in SC by high-throughput qPCR (Biomark HD, Fluidigm) using specific TaqMan assays. A median of 175 cells per sample were analyzed. Inferred clonal architectures were relatively simple and either linear or branching. Similar to previous studies based on bulk sequencing to infer clonal architecture, we were able to observe that an ancestral event in one patient can appear as a secondary hit in another one, thus reflecting the high intratumoral heterogeneity in MDS del(5q) and the importance of patient-specific molecular characterization

Divergent leukaemia subclones as cellular models for testing vulnerabilities associated with gains in chromosomes 7, 8 or 18

Haematopoietic malignancies are frequently characterized by karyotypic abnormalities. The development of targeted drugs has been pioneered with compounds against gene products of fusion genes caused by chromosomal translocations. While polysomies are equally frequent as translocations, for many of them we are lacking therapeutic approaches aimed at synthetic lethality. Here, we report two new cell lines, named MBU-7 and MBU-8, that differ in complete trisomy of chromosome18, a partial trisomy of chromosome 7 and a tetrasomy of the p-arm of chromosome 8, but otherwise share the same mutational pattern and complex karyotype. Both cell lines are divergent clones of U-937 cells and have the morphology and immunoprofile of monocytic cells. The distinct karyotypic differences between MBU-7 and MBU-8 are associated with a difference in the specific response to nucleoside analogues. Taken together, we propose the MBU-7 and MBU-8 cell lines described here as suitable in vitro models for screening and testing vulnerabilities that are associated with the disease-relevant polysomies of chromosome 7, 8 and 18

Association between Germline Single-Nucleotide Variants in ADME Genes and Major Molecular Response to Imatinib in Chronic Myeloid Leukemia Patients

Imatinib is the most common first-line tyrosine kinase inhibitor (TKI) used to treat chronic-phase chronic myeloid leukemia (CP-CML). However, only a proportion of patients achieve major molecular response (MMR), so there is a need to find biological factors that aid the selection of the optimal therapeutic strategy (imatinib vs. more potent second-generation TKIs). The aim of this retrospective study was to understand the contribution of germline single-nucleotide variants (gSNVs) in the achievement of MMR with imatinib. In particular, a discovery cohort including 45 CP-CML patients was analyzed through the DMET array, which interrogates 1936 variants in 231 genes related to the absorption, distribution, metabolism and excretion (ADME) process. Variants statistically significant in the discovery cohort were then tested in an extended and independent cohort of 137 CP-CML patients. Finally, a total of 7 gSNVs (ABCG1-rs492338, ABCB11-rs496550, ABCB11-rs497692, CYP2D6-rs1135840, CYP11B1-rs7003319, MAT1A-rs4934027 and SLC22A1-rs628031) and one haplotype in the ABCB11 gene were significantly associated with the achievement of MMR with first-line imatinibtreatment. In conclusion, we identified a genetic signature of response to imatinib in CP-CML patients that could be useful in selecting those patients that may benefit from starting imatinib as first-line therapy, therefore avoiding the toxicity related to second-generation TKIs

A novel targeted RNA-Seq panel identifies a subset of adult patients with acute lymphoblastic leukemia with BCR-ABL1-like characteristics

BCR-ABL1-like B-cell precursor acute lymphoblastic leukemia (BCP-ALL) remains poorly characterized in adults. We sought to establish the frequency and outcome of adolescent and adult BCR-ABL1-like ALL using a novel RNA-Seq signature in a series of patients with BCP-ALL. To this end, we developed and tested an RNA-Seq custom panel of 42 genes related to a BCR-ABL1-like signature in a cohort of 100 patients with BCP-ALL and treated with risk-adapted ALL trials. Mutations related to BCR-ABL1-like ALL were studied in a panel of 33 genes by next-generation sequencing (NGS). Also, CRLF2 overexpression and IKZF1/CDKN2A/B deletions were analyzed. Twenty out of 79 patients (12-84 years) were classified as BCR-ABL1-like (25%) based on heatmap clustering, with significant overexpression of ENAM, IGJ, and CRLF2 (P ≤ 0.001). The BCR-ABL1-like subgroup accounted for 29% of 15-60-year-old patients, with the following molecular characteristics: CRLF2 overexpression (75% of cases), IKZF1 deletions (64%), CDKN2A/B deletions (57%), and JAK2 mutations (57%). Among patients with postinduction negative minimal residual disease, those with the BCR-ABL1-like ALL signature had a higher rate of relapse and lower complete response duration than non-BCR-ABL1-like patients (P = 0.007). Thus, we have identified a new molecular signature of BCR-ABL1-like ALL that correlates with adverse prognosis in adult patients with ALL

ALL-268 genetic classification of B-Cell precursor adult acute lymphoblastic leukemia patients enrolled in LAL19 trial from the pethema group: response to treatment and survival

Context: B-cell precursor acute lymphoblastic leukemia (BCP ALL) is a genetically heterogeneous neoplasm with >20 biologic subtypes. Each subtype shows specific genetic traits that determine relapse risk and patients' survival. Objectives: To establish the genetic subtype (primary alteration) of adult BCP ALL patients enrolled in the PETHEMA LAL19 trial (NCT 04179929) and to correlate them with measurable residual disease (MRD) level and survival. Patients and Methods: In the LAL19 trial (NCT04179929), Ph-negative patients (18–65 y) with MRD≥0.01% at day+35 or high-risk genetics receive alloHSCT and MRD<0.01% patients with standard-risk genetics receive maintenance chemotherapy. The genetic analyses are centralized: FISH and NGS DNA panel (Hospital de Salamanca), RNAseq panel (Hospital 12 de Octubre), FISH panel (Hospital La Fe), and SNP array (Josep Carreras Institute/ICO-Hospital Germans Trias i Pujol). MRD determinations are centrally done by next-generation flow cytometry in the Cytometry Service, NUCLEUS, University of Salamanca. Results: The genetic subtype was identified in 54% (82/152) of patients. The most recurrent subtypes were KMT2Ar (11%), Ph-like (mostly CRLF2::IGH, 11%), low-hypodiploid (7%), PAX5 P80R (7%), high-hyperdiploid (6%), and t(1;19)/TCF3::PBX1 (6%). In addition, t(12;21)/ETV6::RUNX1, ZNF384r, and iAMP21 subtypes (1.5% each) and MEF2Dr, MYCr, IDH1 R132 subtypes (<1% each) were found. Regarding secondary alterations, NRAS (15%), TP53 (13%), PAX5 (13%), and KRAS (10%) mutations were the most frequently observed. Twelve patients were refractory (mainly low-hypodiploid, Ph-like, MYCr, and B-other/unclassified patients). Statistically significant differences were observed for day+35 MRD levels between genetic subtypes. Ph-like, low-hypodiploid, and KMT2Ar showed lower frequencies of MRD<0.01% (17%, 33%, and 57%, respectively) than patients with PAX5P80R (100%), t(1;19)/TCF3::PBX1 (83%), and high-hyperdiploid (75%) (P=0.006). Despite the short median follow-up (11 months), differences in response to treatment were reflected in patients' survival. Significant differences in survival were observed between poor-response subtypes (Ph-like, KMT2Ar, and low-hypodiploid) and good-response subtypes (PAX5 P80R, t(1;19)/TCF3::PBX1, and high-hyperdiploid). Conclusions: Knowing the genetic subtype of each ALL is crucial to better predict relapse risk and offer the best (personalized) treatment for each patient

A novel targeted RNA-Seq panel identifies a subset of adult patients with acute lymphoblastic leukemia with BCR-ABL1-like characteristics

BCR-ABL1-like B-cell precursor acute lymphoblastic leukemia (BCP-ALL) remains poorly characterized in adults. We sought to establish the frequency and outcome of adolescent and adult BCR-ABL1-like ALL using a novel RNA-Seq signature in a series of patients with BCP-ALL. To this end, we developed and tested an RNA-Seq custom panel of 42 genes related to a BCR-ABL1-like signature in a cohort of 100 patients with BCP-ALL and treated with risk-adapted ALL trials. Mutations related to BCR-ABL1-like ALL were studied in a panel of 33 genes by next-generation sequencing (NGS). Also, CRLF2 overexpression and IKZF1/CDKN2A/B deletions were analyzed. Twenty out of 79 patients (12-84 years) were classified as BCR-ABL1-like (25%) based on heatmap clustering, with significant overexpression of ENAM, IGJ, and CRLF2 (P <= 0.001). The BCR-ABL1-like subgroup accounted for 29% of 15-60-year-old patients, with the following molecular characteristics: CRLF2 overexpression (75% of cases), IKZF1 deletions (64%), CDKN2A/B deletions (57%), and JAK2 mutations (57%). Among patients with postinduction negative minimal residual disease, those with the BCR-ABL1-like ALL signature had a higher rate of relapse and lower complete response duration than non-BCR-ABL1-like patients (P = 0.007). Thus, we have identified a new molecular signature of BCR-ABL1-like ALL that correlates with adverse prognosis in adult patients with ALL