The pathogenesis of diffuse large B-cell lymphoma (DLBCL) remains partially unknown. The analysis of the B-cell receptor of the malignant cells could contribute to a better understanding of the DLBCL ...biology. We studied the molecular features of the immunoglobulin heavy chain (IGH) rearrangements in 165 patients diagnosed with DLBCL not otherwise specified. Clonal IGH rearrangements were amplified according to the BIOMED-2 protocol and PCR products were sequenced directly. We also analyzed the criteria for stereotyped patterns in all complete IGHV-IGHD-IGHJ (V-D-J) sequences. Complete V-D-J rearrangements were identified in 130 of 165 patients. Most cases (89%) were highly mutated, but 12 sequences were truly unmutated or minimally mutated. Three genes, IGHV4-34, IGHV3-23, and IGHV4-39, accounted for one third of the whole cohort, including an overrepresentation of IGHV4-34 (15.5% overall). Interestingly, all IGHV4-34 rearrangements and all unmutated sequences belonged to the nongerminal center B-cell-like (non-GCB) subtype. Overall, we found three cases following the current criteria for stereotyped heavy chain VH CDR3 sequences, two of them belonging to subsets previously described in CLL. IGHV gene repertoire is remarkably biased, implying an antigen-driven origin in DLBCL. The particular features in the sequence of the immunoglobulins suggest the existence of particular subgroups within the non-GCB subtype.
Plasma cell disorders (PCDs) are identified in the clinical lab by detecting the monoclonal immunoglobulin (M-protein) which they produce. Traditionally, serum protein electrophoresis methods have ...been utilized to detect and isotype M-proteins. Increasing demands to detect low-level disease and new therapeutic monoclonal immunoglobulin treatments have stretched the electrophoretic methods to their analytical limits. Newer techniques based on mass spectrometry (MS) are emerging which have improved clinical and analytical performance. MS is gaining traction into clinical laboratories, and has replaced immunofixation electrophoresis (IFE) in routine practice at one institution. The International Myeloma Working Group (IMWG) Mass Spectrometry Committee reviewed the literature in order to summarize current data and to make recommendations regarding the role of mass spectrometric methods in diagnosing and monitoring patients with myeloma and related disorders. Current literature demonstrates that immune-enrichment of immunoglobulins coupled to intact light chain MALDI-TOF MS has clinical characteristics equivalent in performance to IFE with added benefits of detecting additional risk factors for PCDs, differentiating M-protein from therapeutic antibodies, and is a suitable replacement for IFE for diagnosing and monitoring multiple myeloma and related PCDs. In this paper we discuss the IMWG recommendations for the use of MS in PCDs.
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.
We have analyzed brain and acute leukemia, cytoplasmic (BAALC) gene expression and other genetic markers (ERG, EVI1, MN1, PRAME, WT1, FLT3, and NPM1 mutations) in 127 intermediate-risk acute myeloid ...leukemia (AML) patients: 98 cytogenetically normal and 29 with intermediate-risk cytogenetic alterations. High versus low BAALC expressers showed a higher refractoriness to induction treatment (31% vs 10%; p = .005), lower complete remission rate after salvage therapy (82% vs 97%; p = .010), and lower 3-year overall (23% vs 58%, p < .001) and relapse-free survival (26% vs 52%, p = .006). Similar results were found when cytogenetic subgroups were analyzed separately. Multivariate models confirmed the unfavorable prognosis of this marker. In conclusion, BAALC is a relevant prognostic marker in intermediate-risk AML.
Purpose To perform a critical analysis on the impact of depth of response in newly diagnosed multiple myeloma (MM). Patients and Methods Data were analyzed from 609 patients who were enrolled in the ...GEM (Grupo Español de Mieloma) 2000 and GEM2005MENOS65 studies for transplant-eligible MM and the GEM2010MAS65 clinical trial for elderly patients with MM who had minimal residual disease (MRD) assessments 9 months after study enrollment. Median follow-up of the series was 71 months. Results Achievement of complete remission (CR) in the absence of MRD negativity was not associated with prolonged progression-free survival (PFS) and overall survival (OS) compared with near-CR or partial response (median PFS, 27, 27, and 29 months, respectively; median OS, 59, 64, and 65 months, respectively). MRD-negative status was strongly associated with prolonged PFS (median, 63 months; P < .001) and OS (median not reached; P < .001) overall and in subgroups defined by prior transplantation, disease stage, and cytogenetics, with prognostic superiority of MRD negativity versus CR particularly evident in patients with high-risk cytogenetics. Accordingly, Harrell C statistics showed higher discrimination for both PFS and OS in Cox models that included MRD (as opposed to CR) for response assessment. Superior MRD-negative rates after different induction regimens anticipated prolonged PFS. Among 34 MRD-negative patients with MM and a phenotypic pattern of bone marrow involvement similar to monoclonal gammopathy of undetermined significance at diagnosis, the probability of "operational cure" was high; median PFS was 12 years, and the 10-year OS rate was 94%. Conclusion Our results demonstrate that MRD-negative status surpasses the prognostic value of CR achievement for PFS and OS across the disease spectrum, regardless of the type of treatment or patient risk group. MRD negativity should be considered as one of the most relevant end points for transplant-eligible and elderly fit patients with MM.
Abstract 1589
Acquired potentially N-glycosylation sites are produced by somatic hypermutation (SHM) in the immunoglobulin (Ig) variable region. This phenomenon is produced in ∼9% of normal B-cells ...and seems to be related to certain B-cell lymphoproliferative disorders (B-LPDs) such as follicular lymphoma (FL, 79%), endemic Burkitt lymphoma (BL, 82%) and diffuse large B-cell lymphoma (DLBCL, 41%). These data suggest that new potential N-glycosylation sites could be related to germinal center B (GCB)-LPDs. By contrast, in other B-LPDs, such as chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), MALT lymphoma, Waldenström macroglobulinemia (WM) or multiple myeloma (MM), these modifications have not been analyzed in deep.
To evaluate the acquisition of potential N-glycosylation sites in B-LPDs, including immunohystochemical DLBCL subtypes (GCB and non-GCB) and specific non-GCB-LPDs, such as hairy cell leukemia (HCL), splenic marginal-zone lymphoma (SMZL), CLL, MCL, ocular extranodal marginal zone lymphoma (OAEMZL), MM and WM.
A total of 953 sequences (203 from our group and 750 previously published sequences) of B-LPDs were included. Diagnosis distribution was as follows: DLBCL (n=235), MCL (n=235), CLL (n=166), MM (n=96), OAEMZL (n=82), SMZL (n=68), WM (n=38) and HCL (n=33).
Acquired N-glycosylation sites were counted according to the sequence Asn-X-Ser/Thr, where X could be any amino acid except Pro. Natural motifs in germline sequences of IGHV1–08, IGHV4–34 e IGHV-5a were not considered. Fisher test was used to perform comparisons between groups. To distinguish DLBCL biological subtypes (GCB and non-GCB DLBCL), Hans’ algorithm was used.
A total of 83 out of the 235 DLBCL cases acquired at least a new N-glycosylation site, a higher value than in normal B-cells (35% vs. 9%, p<0.0001). Higher incidence of these motifs in the group of GCB as compared to non-GCB DLBCL were observed (52% vs. 20%, p<0.0001). Those cases diagnosed of HCL, CLL, MCL, MM, WM, OAEMZL and SMZL presented a reduced number of new N-glycosylation sites, showing similar values than normal B-cells (range 3–18%, p=ns).
We described for the first time the pattern of N-glycosylation in HCL, SMZL, OAEMZL and in the immunohystochemical DLBCL subtypes, where the GCB-DLBCL showed a higher number of new N-glycosylation sites with respect to non-GCB DLBCL and other non-GCB-LPDs. The presence of novel N-glycosylation sites in FL, BL and in GCB-DLBCL strongly suggests that these motifs are characteristic of the germinal center B-LPDs.
No relevant conflicts of interest to declare.
We have evaluated the use of CD138+ positively selected bone marrow samples to identify a molecular target for minimal residual disease assessment by polymerase chain reaction (PCR) in 25 untreated ...patients with multiple myeloma. A fraction of each sample was used for CD138+ selection, and the rest served as a reference control. VDJH, DJH, and Kde gene rearrangements were tested for amplification according to the BIOMED-2 Concerted Action. PCR products were directly sequenced in an automated ABI 3130 DNA sequencer using Big-Dye terminators. Within the CD138+ selected group, VDJH rearrangements were detected in all cases (100 %), DJH in 16 (64 %), and Kde in 18 (72 %) cases; whereas in the control samples, VDJH, DJH, and Kde rearrangements were detected in 19 (76 %), 11 (44 %), and 12 (48 %) cases, respectively. After sequencing, 24 (96 %) cases within the CD138+ group had a PCR target for MRD detection compared with 15 (60 %) cases in the control group. We conclude that the use of CD138+ positively selected bone marrow samples increases the applicability of minimal residual disease studies by PCR in patients with multiple myeloma.
Abstract 2680
The Human Leukocyte Antigen (HLA) system plays an important role in antitumor immune response. In the last years, different genome-wide association studies have suggested that 6p21.3 ...(which include HLA system), is a risk region for lymphoma susceptibility. It has been also reported that specific alleles could modify the prognosis in patients diagnosed of lymphoma, including diffuse large B cell lymphoma (DLBCL), treated without Rituximab.
Our hypothesis is that HLA system could play an essential role in disease control of DLBCL. Here, we have evaluated the effect of HLA Class I (A, B and C) and II (DRB1 and DQB1) alleles in DLBCL incidence and survival.
A total of 251 patients diagnosed of DLBCL according to the 2008 WHO classification were analyzed (68% of them received Rituximab-based regimens). Control population consisted in 1940 healthy donor individuals analyzed for HLA-A, B, and DRB1 alleles and 200 for HLA-C and HLA-DQB1.
Overall survival (OS) was calculated from diagnosis until death for any cause. Event-free survival (EFS) was calculated from diagnosis until event defined as death for any cause, relapse or progression of the disease.
Allele frequencies and Hardy-Weinberg equilibrium were estimated using the Arlequin software package, version 3.5.1.2. Comparison of allele and phenotype frequencies between populations was performed with the two-sided Fisher’s exact test or χ2 test using GraphPad Prism 4.0 (GraphPad Software, Inc. San Diego, CA, USA) or SPSS software (SPSS 15.0, Inc. Chicago, IL, USA). P-value was adjusted applying a Bonferroni correction (Pc).
The effect of biological and clinical variables on OS and EFS at five years was analyzed by univariate (two-sided log-rank test) and multivariate (Cox multivariate analysis) methods. Differences were considered to be statistically significant when P< 0.05.
HLA specificities frequencies in patients and controls (Alcoceba et al, Tissue Antigens 2011) were consistent in all cases with the Hardy-Weinberg equilibrium.
Phenotypic frequencies showed significant differences in DLBCL patients compared to control population. DRB1*01 frequency was increased in DLBCL patients (28.9% vs. 19.5%, p=0.0009, Pc=0.0117, OR: 1.68; 95% CI: 1.24–2.26). By contrast, DLBCL patients showed a lower frequency in C*03 allele as compared to the control population (6.4% vs. 18.3, p=0.0005, Pc=0.007, OR: 3.2, 95% CI: 1.62–6.3).
As far as the outcome of patients was concerned, the presence of B*18 and/or B*44 was significantly associated with a worse EFS (32% vs. 60%, p<0.001 and 53% vs. 72%, p=0.025, respectively) and OS (41% vs. 78%, p<0.001, and 53% vs. 86%, p=0.006, respectively). These statistical differences were also observed in the group of patients receiving Rituximab-based regimens. The Cox multivariate analysis identified high International Prognostic Index and the presence of any B*18 or B*44 as independent adverse prognostic factors for EFS and OS.
Our data revealed that HLA specificities could influence the incidence (DRB1*01 and C*03) and outcome (B*18 and B*44) of DLBCL. These results suggest a role for HLA in the immune-surveillance of DLBCL and, also open possibilities for future investigations to answer how a specific HLA allele could affect in DLBCL.
No relevant conflicts of interest to declare.
Multiple available combinations of proteasome inhibitors, immunomodulators (IMIDs), and monoclonal antibodies are shifting the relapsed/refractory multiple myeloma (RRMM) treatment landscape. Lack of ...head-to-head trials of triplet regimens highlights the need for real-world (RW) evidence. We conducted an RW comparative effectiveness analysis of bortezomib (V), carfilzomib (K), ixazomib (I), and daratumumab (D) combined with either lenalidomide or pomalidomide plus dexamethasone (Rd or Pd) in RRMM. A retrospective cohort of patients initiating triplet regimens in line of therapy (LOT) ≥ 2 on/after 1/1/2014 was followed between 1/2007 and 3/2018 in Optum’s deidentified US electronic health records database. Time to next treatment (TTNT) was estimated using Kaplan-Meier methods; regimens were compared using covariate-adjusted Cox proportional hazard models. Seven hundred forty-one patients (820 patient LOTs) with an Rd backbone (VRd,
n
= 349; KRd,
n
= 218; DRd,
n
= 99; IRd,
n
= 154) and 348 patients (392 patient LOTs) with a Pd backbone (VPd,
n
= 52; KPd,
n
= 146; DPd,
n
= 149; IPd,
n
= 45) in LOTs ≥2 were identified. More patients ≥75 years received IRd (39.6%), IPd (37.8%), and VRd (36.7%) than other triplets. More patients receiving VRd/VPd were in LOT2 vs other triplets. Unadjusted median TTNT in LOT ≥ 2: VRd, 13.9; KRd, 8.7; IRd, 11.4; DRd, not estimable (NE); and VPd, 12.0; KPd, 6.7; IPd, 9.5 months; DPd, NE. In covariate-adjusted analysis, only KRd vs DRd was associated with a significantly higher risk of next LOT initiation/death (HR 1.72;
P
= 0.0142); no Pd triplet was significantly different vs DPd in LOT ≥ 2. Our data highlight important efficacy/effectiveness gaps between results observed in phase 3 clinical trials and those realized in the RW.
Light chain (AL) amyloidosis is caused by a small B-cell clone producing light chains that form amyloid deposits and cause organ dysfunction. Chemotherapy aims at suppressing the production of the ...toxic light chain (LC) and restore organ function. However, even complete hematologic response (CR), defined as negative serum and urine immunofixation and normalized free LC ratio, does not always translate into organ response. Next-generation flow (NGF) cytometry is used to detect minimal residual disease (MRD) in multiple myeloma. We evaluated MRD by NGF in 92 AL amyloidosis patients in CR. Fifty-four percent had persistent MRD (median 0.03% abnormal plasma cells). There were no differences in baseline clinical variables in patients with or without detectable MRD. Undetectable MRD was associated with higher rates of renal (90% vs 62%, p = 0.006) and cardiac response (95% vs 75%, p = 0.023). Hematologic progression was more frequent in MRD positive (0 vs 25% at 1 year, p = 0.001). Altogether, NGF can detect MRD in approximately half the AL amyloidosis patients in CR, and persistent MRD can explain persistent organ dysfunction. Thus, this study supports testing MRD in CR patients, especially if not accompanied by organ response. In case MRD persists, further treatment could be considered, carefully balancing residual organ damage, patient frailty, and possible toxicity.
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