Publications by authors named "Miguel Alcoceba"

68 Publications

Revised International Prognostic Index and genetic alterations are associated with early failure to R-CHOP in patients with diffuse large B-cell lymphoma.

Br J Haematol 2021 Oct 10. Epub 2021 Oct 10.

Department of Hematology, Hospital Clínic, Barcelona, Spain.

Relapsed or refractory diffuse large B-cell lymphoma (DLBCL) cases have a poor outcome. Here we analysed clinico-biological features in 373 DLBCL patients homogeneously treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP), in order to identify variables associated with early failure to treatment (EF), defined as primary refractoriness or relapse within 12 months from diagnosis. In addition to clinical features, mutational status of 106 genes was studied by targeted next-generation sequencing in 111 cases, copy number alterations in 87, and gene expression profile (GEP) in 39. Ninety-seven cases (26%) were identified as EF and showed significantly shorter overall survival (OS). Patients with B symptoms, advanced stage, high levels of serum lactate dehydrogenase (LDH) or β2-microglobulin, low lymphocyte/monocyte ratio and higher Revised International Prognostic Index (R-IPI) scores, as well as those with BCL2 rearrangements more frequently showed EF, with R-IPI being the most important in logistic regression. Mutations in NOTCH2, gains in 5p15·33 (TERT), 12q13 (CDK2), 12q14·1 (CDK4) and 12q15 (MDM2) showed predictive importance for EF independently from R-IPI. GEP studies showed that EF cases were significantly enriched in sets related to cell cycle regulation and inflammatory response, while cases in response showed over-representation of gene sets related to extra-cellular matrix and tumour microenvironment.
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http://dx.doi.org/10.1111/bjh.17858DOI Listing
October 2021

Interlaboratory Analytical Validation of a Next-Generation Sequencing Strategy for Clonotypic Assessment and Minimal Residual Disease Monitoring in Multiple Myeloma.

Arch Pathol Lab Med 2021 Oct 7. Epub 2021 Oct 7.

From Departamento de Hematología, Hospital Universitario de Salamanca (HUSAL/IBSAL), Centro de Investigación del Cáncer - IBMCC (USAL-CSIC), CIBERONC-CB16/12/00233, Salamanca, Spain (Medina, Jiménez, Puig, Sarasquete, García-Álvarez, Prieto-Conde, Chillón, Alcoceba, González-Calle, Gutiérrez, González, García-Sanz).

Context.—: Minimal residual disease (MRD) is a major prognostic factor in multiple myeloma, although validated technologies are limited.

Objective.—: To standardize the performance of the LymphoTrack next-generation sequencing (NGS) assays (Invivoscribe), targeting clonal immunoglobulin rearrangements, in order to reproduce the detection of tumor clonotypes and MRD quantitation in myeloma.

Design.—: The quantification ability of the assay was evaluated through serial dilution experiments. Paired samples from 101 patients were tested by LymphoTrack, using Sanger sequencing and EuroFlow's next-generation flow (NGF) assay as validated references for diagnostic and follow-up evaluation, respectively. MRD studies using LymphoTrack were performed in parallel at 2 laboratories to evaluate reproducibility.

Results.—: Sensitivity was set as 1.3 tumor cells per total number of input cells. Clonality was confirmed in 99% and 100% of cases with Sanger and NGS, respectively, showing great concordance (97.9%), although several samples had minor discordances in the nucleotide sequence of rearrangements. Parallel NGS was performed in 82 follow-up cases, achieving a median sensitivity of 0.001%, while for NGF, median sensitivity was 0.0002%. Reproducibility of LymphoTrack-based MRD studies (85.4%) and correlation with NGF (R2 > 0.800) were high. Bland-Altman tests showed highly significant levels of agreement between flow and sequencing.

Conclusions.—: Taken together, we have shown that LymphoTrack is a suitable strategy for clonality detection and MRD evaluation, with results comparable to gold standard procedures.
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http://dx.doi.org/10.5858/arpa.2021-0088-OADOI Listing
October 2021

Clinico-biological features and outcome of patients with splenic marginal zone lymphoma with histological transformation.

Br J Haematol 2021 Sep 14. Epub 2021 Sep 14.

Hematology Department and Hematopathology Section, Pathology Department, Hospital Clínic of Barcelona, Barcelona, Spain.

We describe 36 patients with splenic marginal zone lymphoma (SMZL) with transformation (SMZL-T), including 15 from a series of 84 patients with SMZL diagnosed at the Hospital Clinic of Barcelona (HCB) and 21 diagnosed with SMZL-T in other centres. In the HCB cohort, the cumulative incidence of transformation at 5 years was 15%. Predictors for transformation were cytopenias, hypoalbuminaemia, complex karyotype (CK) and both the Intergruppo Italiano Linfomi (ILL) and simplified Haemoglobin, Platelet count, lactate dehydrogenase (LDH) and extrahilar Lymphadenopathy (HPLL)/ABC scores (P < 0·05). The only independent predictor for transformation in multivariate analysis was CK [hazard ratio (HR) 4·025, P = 0·05]. Patients with SMZL-T had a significantly higher risk of death than the remainder (HR 3·89, P < 0·001). Of the 36 patients with SMZL-T, one developed Hodgkin lymphoma and 35 a diffuse large B-cell lymphoma, 71% with a non-germinal centre phenotype. The main features were B symptoms, lymphadenopathy, and high serum LDH. CK was observed in 12/22 (55%) SMZL-T and fluorescence in situ hybridisation detected abnormalities of MYC proto-oncogene, basic helix-loop-helix transcription factor (MYC), B-cell leukaemia/lymphoma 2 (BCL2) and/or BCL6 in six of 14 (43%). In all, 21 patients received immunochemotherapy, six chemotherapy, one radiotherapy and three splenectomy. The complete response (CR) rate was 61% and the median survival from transformation was 4·92 years. Predictors for a worse survival in multivariate analysis were high-risk International Prognostic Index (HR 5·294, P = 0·016) and lack of CR (HR 2·67, P < 0·001).
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http://dx.doi.org/10.1111/bjh.17815DOI Listing
September 2021

Primary refractory follicular lymphoma: a poor outcome entity with high risk of transformation to aggressive B cell lymphoma.

Eur J Cancer 2021 Sep 8;157:132-139. Epub 2021 Sep 8.

CHIMOMODepartment, University of Modena and Reggio Emilia, Modena, Italy.

Background: Primary refractory (PREF) follicular lymphoma (FL) has a completely different clinical course from that of FL that responds to front-line treatments. In addition to having poor responses to salvage therapies, it seems that patients with PREF are at increased risk of histological transformation (HT). The Aristotle consortium presented the opportunity of investigating the risk of HT in a very large series of cases. Thus, we investigated the risk of HT in patients with PREF FL compared with that of responding patients or in stable disease and ultimately their outcome.

Methods: Six thousand three hundred thirty-nine patients from the Aristotle database were included in the analysis. These patients had a histologically confirmed grade 1, 2 or 3a FL diagnosed between 1997 and 2013. The primary end-points were the cumulative incidence (CI) of HT at the first progression or relapse and the survival after transformation.

Findings: The 5-year CI of HT among patients with PREF was 34% (95% confidence interval (CI): 27-43), whilst it was 7.1% (95% CI: 6.0-8.5) in the group of patients with partial response (PR) or stable disease (SD) (PR + SD) and 3.5% (95% CI: 3.0-4.2) in the group of patients achieving complete response (CR). The 5-year survival after relapse (SAR) was 33% (95% CI: 28-39) for the PREF group, 57% (95% CI 54-61) in patients with PR, 51% (95% CI 43-58) in the SD group after first-line therapy and 63% (95% CI: 66-72) in patients with CR after initial treatment (p-value <0.001). The 5-year SAR for those patients with PREF who developed HT was 21% (95% CI: 12-31), clearly diminished when compared with those patients with PREF who did not experience HT (38% [95% CI: 31-44]) (p-value = 0.001).

Interpretation: Patients with PREF FL have a dismal outcome and an associated very high rate of HT that further worsens their poor prognosis.
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http://dx.doi.org/10.1016/j.ejca.2021.08.005DOI Listing
September 2021

Genomic mutation profile in progressive chronic lymphocytic leukemia patients prior to first-line chemoimmunotherapy with FCR and rituximab maintenance (REM).

PLoS One 2021 10;16(9):e0257353. Epub 2021 Sep 10.

Hematology Department, Hospital Universitario Puerta de Hierro-Majadahonda and Instituto de Investigación Sanitaria Puerta de Hierro-Segovia de Arana (IDIPHISA), Madrid, Spain.

Chronic Lymphocytic Leukemia (CLL) is the most prevalent leukemia in Western countries and is notable for its variable clinical course. This variability is partly reflected by the mutational status of IGHV genes. Many CLL samples have been studied in recent years by next-generation sequencing. These studies have identified recurrent somatic mutations in NOTCH1, SF3B1, ATM, TP53, BIRC3 and others genes that play roles in cell cycle, DNA repair, RNA metabolism and splicing. In this study, we have taken a deep-targeted massive sequencing approach to analyze the impact of mutations in the most frequently mutated genes in patients with CLL enrolled in the REM (rituximab en mantenimiento) clinical trial. The mutational status of our patients with CLL, except for the TP53 gene, does not seem to affect the good results obtained with maintenance therapy with rituximab after front-line FCR treatment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0257353PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8432772PMC
September 2021

Anti-TRBC1 Antibody-Based Flow Cytometric Detection of T-Cell Clonality: Standardization of Sample Preparation and Diagnostic Implementation.

Cancers (Basel) 2021 Aug 30;13(17). Epub 2021 Aug 30.

Translational and Clinical Research Program, Centro de Investigación del Cáncer and IBMCC (CSIC-University of Salamanca), Cytometry Service, NUCLEUS, Department of Medicine, University of Salamanca (USAL) and Institute of Biomedical Research of Salamanca (IBSAL), 37007 Salamanca, Spain.

A single antibody (anti-TRBC1; JOVI-1 antibody clone) against one of the two mutually exclusive T-cell receptor β-chain constant domains was identified as a potentially useful flow-cytometry (FCM) marker to assess Tαβ-cell clonality. We optimized the TRBC1-FCM approach for detecting clonal Tαβ-cells and validated the method in 211 normal, reactive and pathological samples. TRBC1 labeling significantly improved in the presence of CD3. Purified TRBC1 and TRBC1 monoclonal and polyclonal Tαβ-cells rearranged TRBJ1 in 44/47 (94%) and TRBJ1+TRBJ2 in 48 of 48 (100%) populations, respectively, which confirmed the high specificity of this assay. Additionally, TRBC1/TRBC1 ratios within different Tαβ-cell subsets are provided as reference for polyclonal cells, among which a bimodal pattern of TRBC1-expression profile was found for all TCRVβ families, whereas highly-variable TRBC1/TRBC1 ratios were observed in more mature vs. naïve Tαβ-cell subsets (vs. total T-cells). In 112/117 (96%) samples containing clonal Tαβ-cells in which the approach was validated, monotypic expression of TRBC1 was confirmed. Dilutional experiments showed a level of detection for detecting clonal Tαβ-cells of ≤10 in seven out of eight pathological samples. These results support implementation of the optimized TRBC1-FCM approach as a fast, specific and accurate method for assessing T-cell clonality in diagnostic-FCM panels, and for minimal (residual) disease detection in mature Tαβ leukemia/lymphoma patients.
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http://dx.doi.org/10.3390/cancers13174379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8430560PMC
August 2021

Validation of the EuroClonality-NGS DNA capture panel as an integrated genomic tool for lymphoproliferative disorders.

Blood Adv 2021 08;5(16):3188-3198

Laboratory of Onco-Hematology, Necker-Enfants Malades Hospital, Assistance Publique-Hôpitaux de Paris, University de Paris, Paris, France.

Current diagnostic standards for lymphoproliferative disorders include multiple tests for detection of clonal immunoglobulin (IG) and/or T-cell receptor (TCR) rearrangements, translocations, copy-number alterations (CNAs), and somatic mutations. The EuroClonality-NGS DNA Capture (EuroClonality-NDC) assay was designed as an integrated tool to characterize these alterations by capturing IGH switch regions along with variable, diversity, and joining genes of all IG and TCR loci in addition to clinically relevant genes for CNA and mutation analysis. Diagnostic performance against standard-of-care clinical testing was assessed in a cohort of 280 B- and T-cell malignancies from 10 European laboratories, including 88 formalin-fixed paraffin-embedded samples and 21 reactive lesions. DNA samples were subjected to the EuroClonality-NDC protocol in 7 EuroClonality-NGS laboratories and analyzed using a bespoke bioinformatic pipeline. The EuroClonality-NDC assay detected B-cell clonality in 191 (97%) of 197 B-cell malignancies and T-cell clonality in 71 (97%) of 73 T-cell malignancies. Limit of detection (LOD) for IG/TCR rearrangements was established at 5% using cell line blends. Chromosomal translocations were detected in 145 (95%) of 152 cases known to be positive. CNAs were validated for immunogenetic and oncogenetic regions, highlighting their novel role in confirming clonality in somatically hypermutated cases. Single-nucleotide variant LOD was determined as 4% allele frequency, and an orthogonal validation using 32 samples resulted in 98% concordance. The EuroClonality-NDC assay is a robust tool providing a single end-to-end workflow for simultaneous detection of B- and T-cell clonality, translocations, CNAs, and sequence variants.
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http://dx.doi.org/10.1182/bloodadvances.2020004056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8405189PMC
August 2021

Liquid biopsy: a non-invasive approach for Hodgkin lymphoma genotyping.

Br J Haematol 2021 Jul 27. Epub 2021 Jul 27.

Department of Hematology, University Hospital of Salamanca (HUS/IBSAL), CIBERONC and Cancer Research Institute of Salamanca-IBMCC (USAL-CSIC), Spain.

The Hodgkin lymphoma (HL) genomic landscape is hardly known due to the scarcity of tumour cells in the tissue. Liquid biopsy employing circulating tumour DNA (ctDNA) can emerge as an alternative tool for non-invasive genotyping. By using a custom next generation sequencing (NGS) panel in combination with unique molecule identifiers, we aimed to identify somatic variants in the ctDNA of 60 HL at diagnosis. A total of 277 variants were detected in 36 of the 49 samples (73·5%) with a good quality ctDNA sample. The median number of variants detected per patient was five (range 1-23) with a median variant allele frequency of 4·2% (0·84-28%). Genotyping revealed somatic variants in the following genes: SOCS1 (28%), IGLL5 (26%), TNFAIP3 (23%), GNA13 (23%), STAT6 (21%) and B2M (19%). Moreover, several poor prognosis features (high LDH, low serum albumin, B-symptoms, IPI ≥ 3 or at an advanced stage) were related to significantly higher amounts of ctDNA. Variant detection in ctDNA by NGS is a feasible approach to depict the genetic features of HL patients at diagnosis. Our data favour the implementation of liquid biopsy genotyping for the routine evaluation of HL patients.
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http://dx.doi.org/10.1111/bjh.17719DOI Listing
July 2021

The novel HLA-DQB1*06:03:27 allele characterised by sequence-based typing in a European bone marrow donor.

HLA 2021 Nov 14;98(5):498-500. Epub 2021 Apr 14.

Department of Clinical Analysis, Immunology Unit, Albacete University Hospital Complex, Albacete, Spain.

A single nucleotide substitution in exon 3 of HLA-DQB1*06:03:01 results in a new allele, HLA-DQB1*06:03:27.
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http://dx.doi.org/10.1111/tan.14271DOI Listing
November 2021

Building a network of TP53 and IGHV testing reference centers across Spain: the Red53 initiative.

Ann Hematol 2021 Mar 6;100(3):825-830. Epub 2021 Jan 6.

Department of Hematology, University Clinic Hospital of Valencia, INCLIVA Institute, Valencia, Spain.

Among the different biomarkers predicting response in chronic lymphocytic leukemia (CLL), the most influential parameters are the mutational status of the IGHV genes and the presence of TP53 gene disruptions. Nevertheless, these important assessments are not readily available in most centers dealing with CLL patients. To provide this molecular testing across the country, the Spanish Cooperative Group on CLL (GELLC) established a network of four analytical reference centers. A total of 2153 samples from 256 centers were analyzed over a period of 30 months. In 9% of the patients, we found pathological mutations in the TP53 gene, whereas 48.96% were classified as IGHV unmutated. Results of the satisfaction survey of the program showed a Net Promoter Score of 85.15. Building a national network for molecular testing in CLL allowed the CLL population a broad access to complex biomarkers analysis that should translate into a more accurate and informed therapeutic decision-making.
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http://dx.doi.org/10.1007/s00277-020-04331-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7914181PMC
March 2021

and Mutations in T/NK-Cell Chronic Lymphoproliferative Disorders of Large Granular Lymphocytes (LGL): Association with Disease Features.

Cancers (Basel) 2020 Nov 25;12(12). Epub 2020 Nov 25.

Translational and Clinical Research Program, Centro de Investigación del Cáncer and IBMCC (CSIC-University of Salamanca), Cytometry Service, NUCLEUS, Department of Medicine, University of Salamanca (USAL) and Institute of Biomedical Research of Salamanca (IBSAL), 37007 Salamanca, Spain.

and () mutations are the most common mutations in T-cell large granular lymphocytic leukemia (T-LGLL) and chronic lymphoproliferative disorders of NK cells (CLPD-NK), but their clinical impact remains unknown. We investigated the frequency and type of mutations in FACS-sorted populations of expanded T/NK-LGL from 100 (82 clonal; 6 oligoclonal; 12 polyclonal) patients, and its relationship with disease features. Seventeen non-LGL T-CLPD patients and 628 age-matched healthy donors were analyzed as controls. ( = 30) and ( = 1) mutations were detected in 28/82 clonal T/NK-LGLL patients (34%), while absent (0/18, 0%) among oligoclonal/polyclonal LGL-lymphocytosis. Mutations were found across all diagnostic subgroups: TCD8-LGLL, 36%; CLPD-NK, 38%; TCD4-LGLL, 7%; TαβDP-LGLL, 100%; TαβDN-LGLL, 50%; Tγδ-LGLL, 44%. -mutated T-LGLL/CLPD-NK showed overall reduced ( < 0.05) blood counts of most normal leukocyte subsets, with a higher rate (vs. nonmutated LGLL) of neutropenia ( = 0.04), severe neutropenia ( = 0.02), and cases requiring treatment ( = 0.0001), together with a shorter time-to-therapy ( = 0.0001), particularly in non-Y640F mutated patients. These findings confirm and extend on previous observations about the high prevalence of mutations across different subtypes of LGLL, and its association with a more marked decrease of all major blood-cell subsets and a shortened time-to-therapy.
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http://dx.doi.org/10.3390/cancers12123508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760806PMC
November 2020

Comparison of next-generation sequencing (NGS) and next-generation flow (NGF) for minimal residual disease (MRD) assessment in multiple myeloma.

Blood Cancer J 2020 10 30;10(10):108. Epub 2020 Oct 30.

Departamento de Hematología, Hospital Universitario de Salamanca (HUSA/IBSAL), CIBERONC, CIC-IBMCC (USAL-CSIC), Salamanca, Spain.

Detecting persistent minimal residual disease (MRD) allows the identification of patients with an increased risk of relapse and death. In this study, we have evaluated MRD 3 months after transplantation in 106 myeloma patients using a commercial next-generation sequencing (NGS) strategy (LymphoTrack®), and compared the results with next-generation flow (NGF, EuroFlow). The use of different marrow pulls and the need of concentrating samples for NGS biased the applicability for MRD evaluation and favored NGF. Despite that, correlation between NGS and NGF was high (R = 0.905). The 3-year progression-free survival (PFS) rates by NGS and NGF were longer for undetectable vs. positive patients (NGS: 88.7% vs. 56.6%; NGF: 91.4% vs. 50%; p < 0.001 for both comparisons), which resulted in a 3-year overall survival (OS) advantage (NGS: 96.2% vs. 77.3%; NGF: 96.6% vs. 74.9%, p < 0.01 for both comparisons). In the Cox regression model, NGS and NGF negativity had similar results but favoring the latter in PFS (HR: 0.20, 95% CI: 0.09-0.45, p < 0.001) and OS (HR: 0.21, 95% CI: 0.06-0.75, p = 0.02). All these results reinforce the role of MRD detection by different strategies in patient prognosis and highlight the use of MRD as an endpoint for multiple myeloma treatment.
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http://dx.doi.org/10.1038/s41408-020-00377-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7603393PMC
October 2020

6q deletion in Waldenström macroglobulinaemia negatively affects time to transformation and survival.

Br J Haematol 2021 03 11;192(5):843-852. Epub 2020 Aug 11.

Haematology Department, University Hospital of Salamanca, Research Biomedical Institute of Salamanca (IBSAL), CIBERONC and Center for Cancer Research-IBMCC (USAL-CSIC), Salamanca, Spain.

Deletion of the long arm of chromosome 6 (del6q) is the most frequent cytogenetic abnormality in Waldenström macroglobulinaemia (WM), occurring in approximately 50% of patients. Its effect on patient outcome has not been completely established. We used fluorescence in situ hybridisation to analyse the prevalence of del6q in selected CD19+ bone marrow cells of 225 patients with newly diagnosed immunoglobulin M (IgM) monoclonal gammopathies. Del6q was identified in one of 27 (4%) cases of IgM-monoclonal gammopathy of undetermined significance, nine of 105 (9%) of asymptomatic WM (aWM), and 28/93 (30%) of symptomatic WM (sWM), and was associated with adverse prognostic features and higher International Prognostic Scoring System for WM (IPSSWM) score. Asymptomatic patients with del6q ultimately required therapy more often and had a shorter time to transformation (TT) to symptomatic disease (median TT, 30 months vs. 199 months, respectively, P < 0·001). When treatment was required, 6q-deleted patients had shorter progression-free survival (median 20 vs. 47 months, P < 0·001). The presence of del6q translated into shorter overall survival (OS), irrespective of the initial diagnosis, with a median OS of 90 compared with 131 months in non-del6q patients (P = 0·01). In summary, our study shows that del6q in IgM gammopathy is associated with symptomatic disease, need for treatment and poorer clinical outcomes.
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http://dx.doi.org/10.1111/bjh.17028DOI Listing
March 2021

Clinical outcome and prognostic factors of patients with Richter syndrome: real-world study of the Spanish Chronic Lymphocytic Leukemia Study Group (GELLC).

Br J Haematol 2020 09 9;190(6):854-863. Epub 2020 Jun 9.

Department of Hematology, Hospital Vall d'Hebron, Barcelona, Spain.

Richter syndrome (RS) is an uncommon evolution of chronic lymphocytic leukaemia (CLL) with a dismal prognosis. Clinical-biological features predicting outcome and best therapeutic approach for these patients remain to be established. In this study, 128 patients with RS, including 112 diffuse large B-cell lymphoma (DLBCL)-type RS, 15 Hodgkin lymphoma (HL)-type RS, and one plasmablastic lymphoma, were identified in 11 centres of the Spanish CLL Study Group (GELLC). The median overall survival (OS) was 5·9 months for DLBCL-type RS and 30·8 months for HL-type RS. Eastern Cooperative Oncology Group Performance Status, haemoglobin level, platelet count, serum lactate dehydrogenase and β2-microglobulin levels, tumour protein p53 (TP53) abnormalities in the CLL clone concomitant to RS, number of prior therapies, and clonal relationship between CLL and RS, were associated with OS in patients with DLBCL-type RS. A platelet count of <100 × 10 /l, prior CLL therapy (0 vs. ≥1), and presence of TP53 alterations maintained an independent prognostic impact in the multivariate analysis. Patients without any of these factors had a better clinical outcome, with a median OS of 75·3 months, while patients with one or two or more of these factors presented a median OS of 25·5 and 3 months, respectively. Although OS of patients with RS is generally poor, a proportion of patients achieved prolonged survival. Treatment of RS remains a medical need, and further therapeutic approaches with novel therapies are warranted.
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http://dx.doi.org/10.1111/bjh.16748DOI Listing
September 2020

Identification of relapse-associated gene mutations by next-generation sequencing in low-risk acute myeloid leukaemia patients.

Br J Haematol 2020 05 2;189(4):718-730. Epub 2020 Mar 2.

Department of Hematology, IBSAL, CIBERONC and Center for Cancer Research-IBMCC (USAL-CSIC), University Hospital of Salamanca, Salamanca, Spain.

Recommended genetic categorization of acute myeloid leukaemias (AML) includes a favourable-risk category, but not all these patients have good prognosis. Here, we used next-generation sequencing to evaluate the mutational profile of 166 low-risk AML patients: 30 core-binding factor (CBF)-AMLs, 33 nucleophosmin (NPM1)-AMLs, 4 biCEBPα-AMLs and 101 acute promyelocytic leukaemias (APLs). Functional categories of mutated genes differed among subgroups. NPM1-AMLs showed frequent variations in DNA-methylation genes (DNMT3A, TET2, IDH1/2) (79%), although without prognostic impact. Within this group, splicing-gene mutations were an independent factor for relapse-free (RFS) and overall survival (OS). In CBF-AML, poor independent factors for RFS and OS were mutations in RAS pathway and cohesin genes, respectively. In APL, the mutational profile differed according to the risk groups. High-risk APLs showed a high mutation rate in cell-signalling genes (P = 0·002), highlighting an increased incidence of FLT3 internal tandem duplication (ITD) (65%, P < 0·0001). Remarkably, in low-risk APLs (n = 28), NRAS mutations were strongly correlated with a shorter five-year RFS (25% vs. 100%, P < 0·0001). Overall, a high number of mutations (≥3) was the worst prognostic factor RFS (HR = 2·6, P = 0·003). These results suggest that gene mutations may identify conventional low-risk AML patients with poor prognosis and might be useful for better risk stratification and treatment decisions.
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http://dx.doi.org/10.1111/bjh.16420DOI Listing
May 2020

Biological Features and Prognostic Impact of Bone Marrow Infiltration in Patients with Diffuse Large B-Cell Lymphoma.

Cancers (Basel) 2020 Feb 18;12(2). Epub 2020 Feb 18.

Department of Hematology, University Hospital of Salamanca (HUS-IBSAL), CIBERONC, and Cancer Research Institute of Salamanca-IBMCC (CSIC-USAL University), 37007 Salamanca, Spain.

The biology and clinical impact of bone marrow (BM) infiltration in patients with diffuse large B-cell lymphoma (DLBCL) remains unclear in the rituximab era. We retrospectively analyzed 232 patients diagnosed with DLBCL at our center between 1999 and 2014. Concordant-presence of large cells similar to those of the lymph node biopsy- and discordant-infiltration by small cells forming lymphoid aggregates, lacking cytological atypia-BM infiltration was defined by histological criteria and further characterized by flow cytometry (FCM). Cell of origin (COO) was determined using Hans' algorithm. For the clonal relationship between tumor and discordant BM, the VDJH rearrangement was analyzed. Survival analyses were restricted to 189 patients treated with rituximab and chemotherapy. Thirty-six (16%) had concordant, and 37 (16%) discordant BM infiltration. FCM described different indolent lymphomas among discordant cases, clonally related with DLBCL in 10/13 available samples. Median follow-up was 58 months. 5-year-progression-free survival (PFS) for non-infiltrated, discordant and concordant groups was 68%, 65% and 30%, respectively ( < 0.001). Combining COO and BM infiltration, patients with discordant BM and non-germinal center B-cell COO also had decreased 5-year-PFS (41.9%). In multivariate analysis, concordant BM had an independent effect on PFS (HR 2.5, p = 0.01). Five-year cumulative incidence of central nervous system (CNS) relapse was 21%, 4% and 1% in concordant, discordant and non-infiltrated groups, respectively ( < 0.001). In conclusion, concordant BM infiltration represents a subset with poor prognosis, whereas the prognostic impact of discordant BM infiltration could be limited to non-CGB cases.
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http://dx.doi.org/10.3390/cancers12020474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072385PMC
February 2020

Molecular profiling of immunoglobulin heavy-chain gene rearrangements unveils new potential prognostic markers for multiple myeloma patients.

Blood Cancer J 2020 02 6;10(2):14. Epub 2020 Feb 6.

Hospital Universitario de Salamanca (HUSAL), IBSAL, IBMCC (USAL-CSIC), CIBERONC, Salamanca, Spain.

Multiple myeloma is a heterogeneous disease whose pathogenesis has not been completely elucidated. Although B-cell receptors play a crucial role in myeloma pathogenesis, the impact of clonal immunoglobulin heavy-chain features in the outcome has not been extensively explored. Here we present the characterization of complete heavy-chain gene rearrangements in 413 myeloma patients treated in Spanish trials, including 113 patients characterized by next-generation sequencing. Compared to the normal B-cell repertoire, gene selection was biased in myeloma, with significant overrepresentation of IGHV3, IGHD2 and IGHD3, as well as IGHJ4 gene groups. Hypermutation was high in our patients (median: 8.8%). Interestingly, regarding patients who are not candidates for transplantation, a high hypermutation rate (≥7%) and the use of IGHD2 and IGHD3 groups were associated with improved prognostic features and longer survival rates in the univariate analyses. Multivariate analysis revealed prolonged progression-free survival rates for patients using IGHD2/IGHD3 groups (HR: 0.552, 95% CI: 0.361-0.845, p = 0.006), as well as prolonged overall survival rates for patients with hypermutation ≥7% (HR: 0.291, 95% CI: 0.137-0.618, p = 0.001). Our results provide new insights into the molecular characterization of multiple myeloma, highlighting the need to evaluate some of these clonal rearrangement characteristics as new potential prognostic markers.
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http://dx.doi.org/10.1038/s41408-020-0283-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7004993PMC
February 2020

A New Next-Generation Sequencing Strategy for the Simultaneous Analysis of Mutations and Chromosomal Rearrangements at DNA Level in Acute Myeloid Leukemia Patients.

J Mol Diagn 2020 01 9;22(1):60-71. Epub 2019 Oct 9.

Department of Hematology, University Hospital of Salamanca (HUS/IBSAL), CIBERONC and Center for Cancer Research-IBMCC (USAL-CSIC), Salamanca, Spain.

Acute myeloid leukemias (AMLs) are currently genomically characterized by karyotype, fluorescence in situ hybridization (FISH), real-time quantitative PCR, and DNA sequencing. Next-generation sequencing offers the promise of detecting all genomic lesions in a single run. However, technical limitations have hampered the detection of chromosomal rearrangements, so most studies are limited to somatic mutation assessment or require the use of RNA-based strategies. To overcome these limitations, we designed a targeted-DNA capture next-generation sequencing approach associated with easy-to-perform public bioinformatic tools for one-step identification of translocations, inversions, and somatic mutations in AML. Thirty well-characterized newly diagnosed myeloid leukemia patients (27 AML and 3 chronic myeloid leukemia) were tested with the panel. Twenty-three of 24 known rearrangements, as well as one novel fusion gene that could not be detected by karyotype/fluorescence in situ hybridization/real-time quantitative PCR, were detected. This strategy also identified all chromosomal breakpoints as potential targets for future high-sensitive minimal residual disease studies. In addition, mutation analysis revealed the presence of missense protein-coding alterations in at least 1 of the 32 genes evaluated in 21 of 30 patients (70%). This strategy may represent a time- and cost-effective diagnostic method for molecular characterization in AML.
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http://dx.doi.org/10.1016/j.jmoldx.2019.08.002DOI Listing
January 2020

Immunoglobulin gene rearrangement IGHV3-48 is a predictive marker of histological transformation into aggressive lymphoma in follicular lymphomas.

Blood Cancer J 2019 06 17;9(7):52. Epub 2019 Jun 17.

Department of Hematology, University Hospital of Salamanca (HUS/IBSAL), Salamanca, Spain.

Follicular lymphoma (FL) is a heterogeneous disease whose pathogenesis remains partially unknown. Around 20% of FL patients experience early progression or treatment-refractory disease and 2-3% of patients per year experience histological transformation (HT) into a more aggressive lymphoma (tFL). Here, we evaluate the immunoglobulin heavy chain variable (IGHV) gene usage and mutational status in 187 FL cases to assess its impact on clinical outcome and histological transformation. The IGHV gene repertoire was remarkably biased in FL. The IGHV4-34 (14%), IGHV3-23 (14%), IGHV3-48 (10%), IGHV3-30 (9%) and IGHV3-21 (7%) genes accounted for more than half of the whole cohort. IGHV3-48 was overrepresented in cases of tFL (19%) compared with non-transformed FL at 5 years (5%, P = 0.05). Patients with the IGHV3-48 gene were significantly more likely to have had HT after 10 years than those who used other genes (71% vs. 25%, P < 0.05), irrespective of the therapy they received. Moreover, IGHV3-30 was also overrepresented in cases of FL (9%) and tFL (13%) compared with diffuse large B-cell lymphoma in which it was nearly absent. In conclusion, our results indicate a role for antigen selection in the development of FL, while the use of IGHV3-48 could help predict histological transformation.
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http://dx.doi.org/10.1038/s41408-019-0213-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6579759PMC
June 2019

Vitamin D Modifies the Incidence of Graft-versus-Host Disease after Allogeneic Stem Cell Transplantation Depending on the Vitamin D Receptor (VDR) Polymorphisms.

Clin Cancer Res 2019 08 1;25(15):4616-4623. Epub 2019 May 1.

Department of Hematology, Hospital Universitario Virgen del Rocío/Instituto de Biomedicina de Sevilla (IBIS)/CSIC/CIBERONC, Universidad de Sevilla, Seville, Spain.

Purpose: The biologically active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (vit D), has immunoregulatory properties via binding vitamin D receptor (VDR). In a prospective trial, we previously reported a reduction in the incidence of chronic GvHD (cGvHD) among patients who received vit D after allogeneic stem cell transplantation (allo-HSCT; Clinical Trials.gov: NCT02600988). Here we analyze the role of patients and donors' VDR SNPs on the immunomodulatory effect of vit D.

Patients And Methods: Patients undergoing allo-HSCT were included in a prospective phase I/II clinical trial (Alovita) in three consecutive cohorts: control (without vit D), low-dose (1,000 IU/day), and high-dose (5,000 IU/day) groups. Vit D was given from day -5 until +100 after transplant. Genotyping of four SNPs of the VDR gene, FokI, BsmI, ApaI, and TaqI, were performed using TaqMan SNP genotyping assays.

Results: We observed a decrease in the incidence of overall cGvHD at 1 year after allo-HSCT depending on the use or not of vit D among patients with FokI CT genotype (22.5% vs 80%, = 0.0004) and among those patients without BsmI/ApaI/TaqI ATC haplotype (22.2% vs 68.8%, = 0.0005). In a multivariate analysis, FokI CT genotype significantly influenced the risk of cGvHD in patients treated with vit D as compared with the control group (HR 0.143, < 0.001).

Conclusions: Our results show that the immunomodulatory effect of vit D depends on the VDR SNPs, and patients carrying the FokI CT genotype display the highest benefit from receiving vit D after allo-HSCT.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-3875DOI Listing
August 2019

Life expectancy of follicular lymphoma patients in complete response at 30 months is similar to that of the Spanish general population.

Br J Haematol 2019 05 22;185(3):480-491. Epub 2019 Feb 22.

Haematology Department, Hospital Virgen del Puerto, Plasencia, Spain.

The use of immunochemotherapy has improved the outcome of follicular lymphoma (FL). Recently, complete response at 30 months (CR30) has been suggested as a surrogate for progression-free survival. This study aimed to analyse the life expectancy of FL patients according to their status at 30 months from the start of treatment in comparison with the sex and age-matched Spanish general population (relative survival; RS). The training series comprised 263 patients consecutively diagnosed with FL in a 10-year period who needed therapy and were treated with rituximab-containing regimens. An independent cohort of 693 FL patients from the Grupo Español de Linfomas y Trasplante Autólogo de Médula Ósea (GELTAMO) group was used for validation. In the training cohort, 188 patients were in CR30, with a 10-year overall survival (OS) of 53% and 87% for non-CR30 and CR30 patients, respectively. Ten-year RS was 73% and 100%, showing no decrease in life expectancy for CR30 patients. Multivariate analysis indicated that the FL International Prognostic Index was the most important variable predicting OS in the CR30 group. The impact of CR30 status on RS was validated in the independent GELTAMO series. In conclusion, FL patients treated with immunochemotherapy who were in CR at 30 months showed similar survival to a sex- and age-matched Spanish general population.
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http://dx.doi.org/10.1111/bjh.15805DOI Listing
May 2019

Exportin-1 E571K mutation is a common finding in patients with classical Hodgkin lymphoma.

Hematol Oncol 2019 Apr 8;37(2):215-218. Epub 2019 Jan 8.

Centro de Investigación del Cáncer de Salamanca; Servicio de Hematología, Hospital Universitario de Salamanca; Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, Spain.

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http://dx.doi.org/10.1002/hon.2570DOI Listing
April 2019

Mutations in the RAS-BRAF-MAPK-ERK pathway define a specific subgroup of patients with adverse clinical features and provide new therapeutic options in chronic lymphocytic leukemia.

Haematologica 2019 03 27;104(3):576-586. Epub 2018 Sep 27.

Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CIBERONC, Barcelona.

Mutations in genes of the RAS-BRAF-MAPK-ERK pathway have not been fully explored in patients with chronic lymphocytic leukemia. We, therefore, analyzed the clinical and biological characteristics of chronic lymphocytic leukemia patients with mutations in this pathway and investigated the response of primary cells to BRAF and ERK inhibitors. Putative damaging mutations were found in 25 of 452 patients (5.5%). Among these, was mutated in nine patients (2.0%), genes upstream of (, , , , and ) were mutated in 12 patients (2.6%), and genes downstream of (, , and ) were mutated in five patients (1.1%). The most frequent mutations were missense, subclonal and mutually exclusive. Patients with these mutations more frequently had increased lactate dehydrogenase levels, high expression of ZAP-70, CD49d, CD38, trisomy 12 and unmutated immunoglobulin heavy-chain variable region genes and had a worse 5-year time to first treatment (hazard ratio 1.8, =0.025). Gene expression analysis showed upregulation of genes of the MAPK pathway in the group carrying RAS-BRAF-MAPK-ERK pathway mutations. The BRAF inhibitors vemurafenib and dabrafenib were not able to inhibit phosphorylation of ERK, the downstream effector of the pathway, in primary cells. In contrast, ulixertinib, a pan-ERK inhibitor, decreased phospho-ERK levels. In conclusion, although larger series of patients are needed to corroborate these findings, our results suggest that the RAS-BRAF-MAPK-ERK pathway is one of the core cellular processes affected by novel mutations in chronic lymphocytic leukemia, is associated with adverse clinical features and could be pharmacologically inhibited.
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http://dx.doi.org/10.3324/haematol.2018.196931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395334PMC
March 2019

Rituximab and the risk of transformation of follicular lymphoma: a retrospective pooled analysis.

Lancet Haematol 2018 Aug 4;5(8):e359-e367. Epub 2018 Jul 4.

Hospices Civils de Lyon, Universite Claude Bernard Lyon 1, Department of Haematology, Pierre Benite, France.

Background: Histological transformation of follicular lymphoma to aggressive lymphoma is a serious event with a substantial effect on patient outcome. The aim of the Aristotle study was to assess the effect of rituximab on the risk of histological transformation and its outcome.

Methods: 11 cooperative groups or institutions across Europe contributed data to this study. Eligible patients (≥18 years) had histologically confirmed follicular lymphoma grade 1, 2, or 3a, diagnosed between Jan 2, 1997, and Dec 20, 2013. Histological transformation was defined as a biopsy-proven aggressive lymphoma that occurred as a first event after first-line therapy. The primary endpoints were the cumulative hazard of histological transformation and survival after transformation.

Findings: Information was available for 10 001 patients with follicular lymphoma, 8116 of whom were eligible for analysis. 509 histological transformations were reported. After a median follow-up of 87 months (range 1-221; 2·5-97·5th percentile 5-160), the 10-year cumulative hazard of histological transformation was 7·7% (95% CI 6·9-8·5). The 10-year cumulative hazard of histological transformation was 5·2% (95% CI 4·5-6·2) in patients who received rituximab and 8·7% (7·2-10·6) in those who did not (hazard ratio [HR] 0·73, 95% CI 0·58-0·90; p=0·004). The 10-year cumulative hazard of histological transformation was 5·9% (95% CI 5·0-7·0) for patients who received induction rituximab only and 3·6% (95% CI 2·3-5·5) for those treated with induction and maintenance rituximab (HR 0·55, 95% CI 0·37-0·81; p=0·003). This finding was confirmed in a multivariate analysis (p=0·016). 287 deaths were recorded in 509 patients with histological transformation, resulting in a 10-year survival after transformation of 32% (95% CI 26-38). Survival after transformation did not differ between patients not exposed to rituximab and those who received rituximab in induction only (HR 0·94, 95% CI 0·69-1·28; p=0·70), and those who received rituximab in induction and maintenance (0·96, 0·58-1·61; p=0·88).

Interpretation: The risk of histological transformation as a first event can be significantly reduced by the use of rituximab. These findings support the need to inform patients using rituximab nowadays that the risk of transformation is lower than it was before the introduction of rituxumab.

Funding: Associazione Angela Serra per la Ricerca sul Cancro, European Lymphoma Institute, European Hematology Association Lymphoma Group, Fondazione Italiana Linfomi, Spanish Group of Lymphoma and Bone Marrow Transplantation.
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http://dx.doi.org/10.1016/S2352-3026(18)30090-5DOI Listing
August 2018
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