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

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.

Chronic lymphocytic leukemia patients with IGH translocations are characterized by a distinct genetic landscape with prognostic implications.

Chromosome 14q32 rearrangements/translocations involving the immunoglobulin heavy chain (IGH) are rarely detected in chronic lymphocytic leukemia (CLL). The prognostic significance of the IGH translocation is controversial and its mutational profile remains unknown. Here, we present for the first time a comprehensive next-generation sequencing (NGS) analysis of 46 CLL patients with IGH rearrangement (IGHR-CLLs) and we demonstrate that IGHR-CLLs have a distinct mutational profile with recurrent mutations in NOTCH1, IGLL5, POT1, BCL2, FBXW7, ZMYM3, MGA, BRAF and HIST1H1E genes. Interestingly, BCL2 and FBXW7 mutations were significantly associated with this subgroup and almost half of BCL2, IGLL5 and HISTH1E mutations reported were previously identified in non-Hodgkin lymphomas. Notably, IGH/BCL2 rearrangements were associated with a lower mutation frequency and carried BCL2 and IGLL5 mutations, while the other IGHR-CLLs had mutations in genes related to poor prognosis (NOTCH1, SF3B1 and TP53) and shorter time to first treatment (TFT). Moreover, IGHR-CLLs patients showed a shorter TFT than CLL patients carrying 13q-, normal fluorescence in situ hybridization (FISH) and +12 CLL, being this prognosis particularly poor when NOTCH1, SF3B1, TP53, BIRC3 and BRAF were also mutated. The presence of these mutations not only was an independent risk factor within IGHR-CLLs, but also refined the prognosis of low-risk cytogenetic patients (13q-/normal FISH). Hence, our study demonstrates that IGHR-CLLs have a distinct mutational profile from the majority of CLLs and highlights the relevance of incorporating NGS and the status of IGH by FISH analysis to refine the risk-stratification CLL model.

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

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.

Evaluation of the MD Anderson tumor score for diffuse large B-cell lymphoma in the rituximab era.

OBJECTIVES: Diffuse large B-cell lymphoma (DLBCL) is an aggressive heterogeneous lymphoma with standard treatment. However, 30%-40% of patients still fail, so we should know which patients are candidates for alternative therapies. IPI is the main prognostic score but, in the rituximab era, it cannot identify a very high-risk (HR) subset. The MD Anderson Cancer Center reported a score in the prerituximab era exclusively considering tumor-related variables: Tumor Score (TS). We aim to validate TS in the rituximab era and to analyze its current potential role. METHODS: From GELTAMO DLBCL registry, we selected those patients homogeneously treated with R-CHOP (n = 1327). RESULTS: Five-years PFS and OS were 62% and 74%. All variables retained an independent prognostic role in the revised TS (R-TS), identifying four different risk groups, with 5-years PFS of 86%, 71%, 50%, and very HR (28%). With a further categorization of three variables of the original TS (Ann Arbor Stage, LDH and B2M), we generated a new index that allowed an improvement in HR assessment. CONCLUSIONS: (a) All variables of the original TS retain an independent prognostic role, and R-TS remains predictive in the rituximab era; (b) R-TS and additional categorization of LDH, B2M, and AA stage (enhanced TS) increased the ability to identify HR subsets.

Biological significance of monoallelic and biallelic BIRC3 loss in del(11q) chronic lymphocytic leukemia progression.

BIRC3 is monoallelically deleted in up to 80% of chronic lymphocytic leukemia (CLL) cases harboring del(11q). In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which has been shown to be a marker for reduced survival in CLL. Nevertheless, the biological mechanisms by which these lesions could contribute to del(11q) CLL pathogenesis and progression are partially unexplored. We implemented the CRISPR/Cas9-editing system to generate isogenic CLL cell lines harboring del(11q) and/or BIRC3 mutations, modeling monoallelic and biallelic BIRC3 loss. Our results reveal that monoallelic BIRC3 deletion in del(11q) cells promotes non-canonical NF-κB signaling activation via RelB-p52 nuclear translocation, being these effects allelic dose-dependent and therefore further enhanced in del(11q) cells with biallelic BIRC3 loss. Moreover, we demonstrate ex vivo in primary cells that del(11q) cases including BIRC3 within their deleted region show evidence of non-canonical NF-κB activation which correlates with high BCL2 levels and enhanced sensitivity to venetoclax. Furthermore, our results show that BIRC3 mutations in del(11q) cells promote clonal advantage in vitro and accelerate leukemic progression in an in vivo xenograft model. Altogether, this work highlights the biological bases underlying disease progression of del(11q) CLL patients harboring BIRC3 deletion and mutation.

Long FLT3 internal tandem duplications and reduced PML-RARα expression at diagnosis characterize a high-risk subgroup of acute promyelocytic leukemia patients.

BACKGROUND: Internal tandem duplications of the FLT3 gene (FLT3-ITDs) are frequent in patients with acute promyelocytic leukemia (APL), however its clinical impact remains controversial. DESIGN AND METHODS: We analyzed the prognostic significance of FLT3-ITD mutant level and size, as well as FLT3-D835 point mutations, PML-RARalpha expression and other predictive factors in 129 APL patients at diagnosis enrolled on the Spanish LPA96 (n=43) or LPA99 (n=86) PETHEMA trials. RESULTS: FLT3-ITDs and D835 mutations were detected in 21% and 9% of patients, respectively. Patients with increased ITD mutant/wild-type ratio or longer ITD size displayed shorter 5-year relapse-free survival (RFS) (P=0.048 and P<0.0001, respectively). However, patients with D835 mutations did not show differences in RFS or overall survival (OS). Moreover, patients with initial normalized copy number (NCN) of PML-RARalpha transcripts less than the 25(th) percentile had adverse clinical features and shorter 5-year RFS (P<0.0001) and OS (P=0.004) compared to patients with higher NCN. Patients with low NCN showed increased incidence of ITDs (P=0.001), with higher ratios (P<0.0001) and/or longer sizes (P=0.007). Multivariate analysis showed that long FLT3-ITD (P=0.001), low PML-RARalpha levels (P=0.004) and elevated WBC counts (>10x10(9)/L) (P=0.018) were independent predictors for shorter RFS. We identified a subgroup of patients with high WBC, long FLT3-ITD and low NCN of transcripts that showed an extremely bad prognosis (5-year RFS 23.4%, P<0.0001). CONCLUSIONS: In conclusion, FLT3-ITD size and PML-RARalpha transcript levels at diagnosis could contribute to improve the risk stratification in APL.

Integrated Genomic Analysis of Chromosomal Alterations and Mutations in B-Cell Acute Lymphoblastic Leukemia Reveals Distinct Genetic Profiles at Relapse.

The clonal basis of relapse in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is complex and not fully understood. Next-generation sequencing (NGS), array comparative genomic hybridization (aCGH), and multiplex ligation-dependent probe amplification (MLPA) were carried out in matched diagnosis-relapse samples from 13 BCP-ALL patients to identify patterns of genetic evolution that could account for the phenotypic changes associated with disease relapse. The integrative genomic analysis of aCGH, MLPA and NGS revealed that 100% of the BCP-ALL patients showed at least one genetic alteration at diagnosis and relapse. In addition, there was a significant increase in the frequency of chromosomal lesions at the time of relapse (p = 0.019). MLPA and aCGH techniques showed that IKZF1 was the most frequently deleted gene. TP53 was the most frequently mutated gene at relapse. Two TP53 mutations were detected only at relapse, whereas the three others showed an increase in their mutational burden at relapse. Clonal evolution patterns were heterogeneous, involving the acquisition, loss and maintenance of lesions at relapse. Therefore, this study provides additional evidence that BCP-ALL is a genetically dynamic disease with distinct genetic profiles at diagnosis and relapse. Integrative NGS, aCGH and MLPA analysis enables better molecular characterization of the genetic profile in BCP-ALL patients during the evolution from diagnosis to relapse.

The relevance of preferentially expressed antigen of melanoma (PRAME) as a marker of disease activity and prognosis in acute promyelocytic leukemia.

BACKGROUND: The gene for preferentially expressed antigen of melanoma (PRAME) has been shown to be over-expressed in acute promyelocytic leukemia, but its actual incidence and clinical impact are still unknown. DESIGN AND METHODS: We studied PRAME expression at diagnosis using real-time quantitative polymerase chain reaction in 125 patients with acute promyelocytic leukemia enrolled in the Spanish PETHEMA-96 (n=45) and PETHEMA-99 (n=80) clinical trials. In addition, PRAME expression was evaluated as a marker of disease activity in 225 follow-up samples from 67 patients with acute promyelocytic leukemia. RESULTS: At diagnosis, PRAME expression in patients with acute promyelocytic leukemia was significantly higher (p<0.001) than in patients with non-M3 acute myeloid leukemia (n=213) and in healthy controls (n=10). Furthermore, patients with acute promyelocytic leukemia with high PRAME expression had a favorable outcome. Thus, the 5-year relapse-free survival was better in patients with >100-fold PRAME expression (86% vs. 74%; p=0.03), and this cut-off established two sub-groups with different relapse-free survival rates among patients with a white cell count <10(9)/L (5-year relapse-free survival 94% vs. 80%, p=0.01). This effect was similar in patients with a white cell count >10(9)/L, although differences were not statistically significant. In multivariate analysis, white cell count >10(9)/L (p<0.001), bone marrow blasts >90% (p=0.001), and PRAME expression <100-fold (p=0.009) were associated with short relapse-free survival. Samples at remission showed PRAME levels similar to those in normal controls while samples at relapse over-expressed PRAME again. Furthermore, 12/13 samples collected within the 6-month period preceding relapse showed a >10-fold increase in PRAME expression levels. CONCLUSIONS: Low PRAME expression defines a subgroup of patients with acute promyelocytic leukemia with a short relapse-free survival. This marker could be useful as a secondary marker for monitoring patients with acute promyelocytic leukemia.

Nephrotic syndrome resulting from focal segmental glomerulosclerosis in a peripheral blood stem cell transplant patient.

We describe the case of a male patient who was diagnosed with acute monoblastic leukemia and received a peripheral stem cell transplantation (PSCT) with peripheral blood hematopoietic progenitors. Because he was in clinical remission with no evidence of chronic graft-versus-host disease (GVHD), immunosuppression was withdrawn, and he developed nephrotic syndrome (NS) months later. A kidney biopsy showed focal segmental glomerulosclerosis (FSGS) as part of the GVHD. Soon after the reintroduction of previous immunosuppressive therapy, we observed a complete remission of the NS.

HLA specificities are associated with prognosis in IGHV-mutated CLL-like high-count monoclonal B cell lymphocytosis.

INTRODUCTION: Molecular alterations leading progression of asymptomatic CLL-like high-count monoclonal B lymphocytosis (hiMBL) to chronic lymphocytic leukemia (CLL) remain poorly understood. Recently, genome-wide association studies have found 6p21.3, where the human leukocyte antigen (HLA) system is coded, to be a susceptibility risk region for CLL. Previous studies have produced discrepant results regarding the association between HLA and CLL development and outcome, but no studies have been performed on hiMBL. AIMS: We evaluated the role of HLA class I (-A, -B and -C) and class II (-DRB1 and -DQB1) in hiMBL/CLL susceptibility, hiMBL progression to CLL, and treatment requirement in a large series of 263 patients diagnosed in our center with hiMBL (n = 156) or Binet A CLL (n = 107). RESULTS: No consistent association between HLA specificities and hiMBL or CLL susceptibility was found. With a median follow-up of 7.7 years, 48/156 hiMBLs (33%) evolved to asymptomatic CLLs, while 16 hiMBLs (10%) and 44 CLLs (41%) required treatment. No HLA specificities were found to be significantly associated with hiMBL progression or treatment in the whole cohort. However, within antigen-experienced immunoglobulin heavy-chain (IGHV)-mutated hiMBLs, which represents the highest proportion of hiMBL cases (81%), the presence of HLA-DQB1*03 showed a trend to a higher risk of progression to CLL (60% vs. 26%, P = 0.062). Moreover, HLA-DQB1*02 specificity was associated with a lesser requirement for 15-year treatment (10% vs. 36%, P = 0.012). CONCLUSION: In conclusion, our results suggest a role for HLA in IGHV-mutated hiMBL prognosis, and are consistent with the growing evidence of the influence of 6p21 on predisposition to CLL. Larger non-biased series are required to enable definitive conclusions to be drawn.

Blastic plasmacytoid dendritic cell neoplasm frequently shows occult central nervous system involvement at diagnosis and benefits from intrathecal therapy.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare aggressive myeloid neoplasm which shows a high rate of central nervous system (CNS) recurrence and overall survival (OS) of <1 year. Despite this, screening for CNS involvement is not routinely performed at diagnosis and intrathecal (IT) prophylaxis is not regularly administered in BPDCN. Here, we prospectively evaluated 13 consecutive BPDCN patients for the presence of CNS involvement by flow cytometry. Despite none of the patients presented with neurological symptoms, occult CNS involvement was detected in 6/10 cases evaluated at diagnosis and 3/3 studied at relapse/progression. BPDCN patients evaluated at diagnosis received IT treatment -either CNS prophylaxis (n = 4) or active therapy (n = 6)- and all but one remain alive (median follow-up of 20 months). In contrast, all three patients assessed at relapse/progression died. The potential benefit of IT treatment administered early at diagnosis on OS and CNS recurrence-free survival of BPDCN was further confirmed in a retrospective cohort of another 23 BPDCN patients. Our results show that BPDCN patients studied at diagnosis frequently display occult CNS involvement; moreover, they also indicate that treatment of occult CNS disease might lead to a dramatically improved outcome of BPDCN.

A Low Frequency of Losses in 11q Chromosome Is Associated with Better Outcome and Lower Rate of Genomic Mutations in Patients with Chronic Lymphocytic Leukemia.

To analyze the impact of the 11q deleted (11q-) cells in CLL patients on the time to first therapy (TFT) and overall survival (OS), 2,493 patients with CLL were studied. 242 patients (9.7%) had 11q-. Fluorescence in situ hybridization (FISH) studies showed a threshold of 40% of deleted cells to be optimal for showing that clinical differences in terms of TFT and OS within 11q- CLLs. In patients with ≥40% of losses in 11q (11q-H) (74%), the median TFT was 19 months compared with 44 months in CLL patients with <40% del(11q) (11q-L) (P<0.0001). In the multivariate analysis, only the presence of 11q-L, mutated IGHV status, early Binet stage and absence of extended lymphadenopathy were associated with longer TFT. Patients with 11q-H had an OS of 90 months, while in the 11q-L group the OS was not reached (P = 0.008). The absence of splenomegaly (P = 0.02), low LDH (P = 0.018) or ß2M (P = 0.006), and the presence of 11q-L (P = 0.003) were associated with a longer OS. In addition, to detect the presence of mutations in the ATM, TP53, NOTCH1, SF3B1, MYD88, FBXW7, XPO1 and BIRC3 genes, a select cohort of CLL patients with losses in 11q was sequenced by next-generation sequencing of amplicons. Eighty % of CLLs with 11q- showed mutations and fewer patients with low frequencies of 11q- had mutations among genes examined (50% vs 94.1%, P = 0.023). In summary, CLL patients with <40% of 11q- had a long TFT and OS that could be associated with the presence of fewer mutated genes.

Frontline treatment of follicular lymphoma with fludarabine, cyclophosphamide, and rituximab followed by rituximab maintenance: toxicities overcome its high antilymphoma effect. Results from a Spanish Cooperative Trial (LNHF-03).

We assessed the efficacy of fludarabine, cyclophosphamide, and rituximab in combination (FCR) as frontline treatment in patients with follicular lymphoma (FL) followed by rituximab maintenance. Seventy-five untreated patients with FL received FCR followed by maintenance with rituximab 375 mg/m(2) weekly during 4 weeks and every 6 months for 2 years. The overall response rate was 100%, with 89% complete remission (CR) and 11% partial remission (PR). Molecular remission was observed in all but one patient. Only eight patients completed all therapy planned. With a median follow-up of 47 months, the 5-year overall survival (OS), progression-free survival (PFS), and event-free survival (EFS) were 77%, 93%, and 72%, respectively. Age below 60 and low Follicular Lymphoma International Prognostic Index (FLIPI) correlated with a better EFS. Ten patients died due to toxic complications. The FCR regimen is highly effective in untreated patients with FL, with 89% CR, including molecular responses, and a low progression rate. However, the high incidence of treatment-related mortality makes this regimen unsafe and it cannot be recommended as an upfront therapy in FL.