Medina et al. Blood Cancer Journal (2020)10:14 https://doi.org/10.1038/s41408-020-0283-8 Blood Cancer Journal ARTICLE Open Access Molecular profiling of immunoglobulin heavy- chain gene rearrangements unveils new potential prognostic markers for multiple myeloma patients Alejandro Medina 1, Cristina Jiménez1, M. Eugenia Sarasquete1, Marcos González1, M. Carmen Chillón1, Ana Balanzategui1, Isabel Prieto-Conde 1, María García-Álvarez1, Noemí Puig1, Verónica González-Calle1, Miguel Alcoceba 1, Isabel Cuenca2, Santiago Barrio2, Fernando Escalante3, Norma C. Gutiérrez1, Mercedes Gironella4, Miguel T. Hernández5, Anna Sureda6, Albert Oriol7, Joan Bladé8, Juan-José Lahuerta2, Jesús F. San Miguel9, María-Victoria Mateos 1, Joaquín Martínez-López2, María-José Calasanz 9 and Ramón García-Sanz 1 Abstract 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 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; 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. Introduction and IGHJ immunoglobulin genes go through recombi- B-cell lymphoproliferative disorders are caused by the nation during early B-cell ontogeny that takes place in the expansion of a pathological clone at a specific stage of bone marrow (BM), an entirely random, antigen- differentiation reflected in the B-cell receptor (BCR). independent process driven by the Recombination- Through multiple, hierarchically structured events, BCR Activating Genes (RAG1 and RAG2)2 and Terminal genes from each B cell are assembled in a particular shape deoxynucleotidyl Transferase (TdT)3. In humans, there that confers an extremely high diversity to the B-cell are more than 50 IGHV genes, 27 IGHD genes and 6 IGHJ repertoire to recognize different antigens1. IGHV, IGHD genes. IGHV and IGHD genes are clustered into seven different groups4. During B-cell differentiation, one allele is rearranged to produce a complete heavy-chain rear- Correspondence: M. Eugenia Sarasquete (mealonsos@saludcastillayleon.es) 1 rangement (IGH) with or without the rearrangement of Hospital Universitario de Salamanca (HUSAL), IBSAL, IBMCC (USAL-CSIC), the second IGH allele5. This means that one or two IGH CIBERONC, Salamanca, Spain 2 Hospital 12 de Octubre, CIBERONC, Madrid, Spain rearrangements can be found in each individual B cell, Full list of author information is available at the end of the article © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Blood Cancer Journal Medina et al. Blood Cancer Journal (2020)10:14 Page 2 of 12 although only one of them is expressed as a functional (NCT00480363, n = 7). The 94 remaining patients were protein6. Immunoglobulin-expressing B cells then migrate treated following the same protocols (GEM2000, to germinal centers to undergo two antigen-dependent GEM2005), but they were not formally included in any processes: somatic hypermutation (SHM) and class- clinical trial. switch recombination (CSR)7. According to the International Myeloma Working As a clonal B-cell malignancy, multiple myeloma (MM) Group (IMWG) criteria26, patients with either t(4;14), arises from a single cell, that has been recognized as a t(14;16) or del17p were grouped together as a high-risk post-germinal center, IgA/IgG switched B cell in several (HR) cytogenetics subset for subsequent analysis. Patients studies8. VDJH usage, Complementarity-determining with symptomatic myeloma were also stratified based on Region 3 (CDR3) composition and somatic hypermuta- the Revised International Staging System (R-ISS)27. tion (SHM) levels have been studied for the vast majority This study was approved by the Ethical Committee of of B-cell malignancies, as well as for normal, healthy B the University Hospital of Salamanca in accordance with cells9–12. The most interesting finding to date is the close the Spanish law and the Declaration of Helsinki princi- association between SHM rate and the outcome in ples. Written informed consent was obtained from every chronic lymphocytic leukemia (CLL), since a high SHM patient prior to the inclusion in each trial. rate (>2%) is associated with good prognosis13. Another interesting observation is the presence of stereotyped Sample collection and IGH sequencing immunoglobulin receptors, not only in CLL14 but also in Genomic DNA was isolated from bone marrow aspi- mantle-cell lymphoma (MCL) or marginal zone lym- rates at the time of diagnosis using the automated DNA phoma (MZL), which led researchers to infer that this was Purification kit Maxwell® (Promega, Madison, WI, USA). a common phenomenon for all mature B-cell tumors with DNA quantification and quality assessment were done potential prognostic and therapeutic implications15. Sev- using the NanoDrop2000 Spectrophotometer (Thermo- eral studies performed with MM patients treated with Fisher, Waltham, MA, USA). For the amplification of conventional chemotherapy did not find any correlation complete VDJH rearrangements, we used the BIOMED-2 between prognosis and VDJH gene usage, CDR3 amino (now Euroclonality©) FR1 primers28 in multiplexed PCR acid composition or SHM rates16–18. However, most reactions. All reactions were carried out in a 25 μL mix- series often included an insufficient number of hetero- ture containing ~100 ng DNA and 10 μmol of each pri- geneously treated patients, which makes any conclusion mer. Monoclonal assessment of amplified products was difficult to be inferred. carried out by GeneScanning using 1 μL of PCR reaction. Here, we present the largest-to-date analysis of the PCR products were sequenced in an automated ABI3500 VDJH repertoire in multiple myeloma, consisting in bio- XL DNA sequencer using Big-Dye terminators v3.1 logical and clinical data from 413 patients diagnosed and (Applied Biosystems™, Foster City, CA, USA). treated according to the Spanish Myeloma Group (GEM- One hundred and thirteen samples were also analyzed PETHEMA) protocols. Data were used for a compre- by next-generation sequencing (NGS) using the Lym- hensive investigation of VDJH rearrangement character- phoTrack® methodology (Invivoscribe Technologies, San istics, including IGHV, IGHD and IGHJ gene usage, SHM Diego, CA, USA). We targeted the Framework Region 1 level and distribution, or complementarity-determining to amplify VDJH rearrangements from 113 patients. In region (CDRs) and framework region (FWRs) length and one-step PCR, amplicons were generated and indexed. A composition. We also tried to identify the presence of purification step using Agentcourt AMPure XP potential clusters of stereotyped receptors, assessing microbeads (Beckman Coulter Inc, Brea, CA, USA) was potential relationships between molecular characteristics performed; then, we assessed the quality of our amplicons and clinical outcomes. using the TapeStation 4200 (Agilent, Santa Clara, CA, USA) and Qubit 2.0 (ThermoFisher, Waltham, MA, Methods USA). Libraries were sequenced in an MiSeq platform Patients (Illumina, San Diego, CA, USA) with 2 × 251 of read A total of 413 newly diagnosed MM patients, diag- length and aiming for 1 million reads per sample. nosed from 1995 to 2016, were included in the present study. Most of them, 319 (77%), were enrolled in Immunophenotypic characterization clinical trials from the GEM-PETHEMA Spanish MM Patients were routinely characterized at diagnosis by group: GEM200019 (NCT00560053, n = 27), GEM2005- multiparametric flow cytometry. Bone marrow samples más6520 (NCT00443235, n = 23), GEM2005MENOS6521 were processed within 48 h after collection. Analysis was (NCT00461747, n = 20), GEM2010MAS6522 (NCT01237249, performed using different assays, following the Euro- n = 83), QUIREDEX23 (NCT00480363, n = 29), GEM2012- Clonality/EuroFlow consortium guidelines: six-color MENOS6524 (NCT01916252, n = 130) and GEM-CESAR25 panel for six markers (CD38/CD138/CD45/CD19/CD56/ Blood Cancer Journal Medina et al. Blood Cancer Journal (2020)10:14 Page 3 of 12 CD117) in 78 patients, eight-color panel for eight markers 20.0 software (IBM, Armonk, NY, USA) using Fisher’s (CD38/CD138/CD45/CD19/CD56/CD117/CD27/CD81) exact test for discrete variables and the Mann−Whitney in the remaining 335 patients. test for continuous variables. The Kaplan–Meier method For data analysis, the values of all parameters measured and the log-rank test were used to plot and compare per tube were mathematically calculated for the individual progression-free survival (PFS) and overall survival (OS) plasma-cell events using the merge and calculation curves. Cox regression was used to perform the multi- functions of the INFINICYT™ v2.0 software (Cytognos S. variate analysis. Receiver operating characteristic (ROC) L. Salamanca, Spain). Plasma cells were identified based curves were used to evaluate potential cutoff values for on the characteristic pattern of expression of CD38, some variables. PFS was defined as the time from diag- CD138, CD45 and light scatter features. nosis to either disease progression, death or the last follow-up visit. OS was defined as the time from diagnosis Sequence analysis to the last follow-up visit or decease. All reported p values IGHV, IGHD and IGHJ genes from complete rearran- were obtained by a two-sided exact method, at the con- gements detected using either Sanger or NGS were ventional 5% significance level (p < 0.05). identified comparing them with the IMGT/V-Quest database29, taking into account the option for insertions/ Results deletions. Mutational status was assessed using the closest Clinical characteristics germline gene. Sequences containing more than 2% Baseline characteristics of the 413 patients included in deviation from the germline were considered somatically the study are summarized in Table 1. mutated, following previously described criteria13. Point For transplant-eligible patients (n = 228), R-ISS stages I, mutations were annotated, as well as the length and II and III represented 24.1%, 62.8% and 13.1% of this composition of N-, P- and CDR3 regions. subset, respectively. After induction, before the pre- FastQ files generated by deep sequencing were pro- transplant conditioning regimen started, the overall cessed using the LymphoTrackAnalysis® tool to retrieve response ratio (ORR) was 92.4%: 15.3% were in stringent sequences from virtually every clonal B cell in the sam- complete response (sCR); 24.1% of patients had achieved ples, allowing the identification of tumor clones if the complete response (CR); and 26.5% of patients were in following criteria were met: (i) ≥20,000 total reads; (ii) at very good partial response (VGPR). The remaining least one but not more than 2 merged top reads ≥ 2.5% of patients were in partial response (PR, 26.5%). Median PFS total reads and (iii) top first or second merged reads must was 49.5 months; median OS was 67.9 months. be at least twice more abundant than the third most For transplant-ineligible patients (n = 149), R-ISS stages frequent read to be considered clonotypic. I, II and III represented 13.1%, 77.9% and 9% of this Rearrangements were considered biallelic if the first two subgroup, respectively. After induction, the ORR was reads met the aforementioned criteria, had similar read 89.7%: 7.7% were in sCR, 34.2% achieved CR, 14.5% were count (less than 50% difference) and one was productive in VGPR and 33.3% were in PR. Median PFS was while the other one unproductive. Moreover, rearrange- 26.4 months; median OS was 57.7 months. ments were considered potentially biclonal if the first two As expected, high-risk asymptomatic patients (n = 36) reads met the aforementioned criteria, had similar read were younger (median age: 60 years). Serum albumin count (less than 50% difference) and both were levels were higher (median: 3.76 g/dL) and β2 micro- productive. globulin levels were lower (median: 2.25 mg/L). Conse- quently, 61.9% of smoldering patients were at ISS stage I, CDR3 clustering analysis and 38.1% were at stage II. ClustalX2.0 was used as previously described30,31, comparing CDR3 amino acid sequences obtained from Detection of clonal VDJH gene rearrangements our cohort and 1117 additional sequences obtained from Sanger sequencing allowed the identification of full the IMGT/LIGM-DB database32 corresponding to unique clonal VDJH gene rearrangements in 88% of patients rearrangements from both normal and human tumor (363/413). In 23 cases, the clonal IGH gene rearrangement B cells. was not detected, possibly due to a high polyclonal Primary and secondary standard criteria31,33 were used as background or lack of PCR amplification due to an described before to characterize immunoglobulin clusters. incorrect primer-annealing. In 12 cases, we could only identify the IGHV gene, while in the remaining cases we Statistical analysis could identify two out of three genes (mostly IGHV and Clinical and biological characteristics were analyzed IGHJ, but not IGHD, n = 13). discriminating between transplant-eligible and noneligible Combining Sanger and NGS, the overall VDJH detec- patients, as well as asymptomatic patients, with the SPSS tion rate was 92.5% (382/413). In all, 388 complete Blood Cancer Journal Medina et al. Blood Cancer Journal (2020)10:14 Page 4 of 12 Table 1 Patient characteristics. Variable Global series Transplant-eligible Transplant-ineligible Asymptomatic (N = 413) (N = 228) (N = 149) (N = 36) Sex Men 54.5% 62.1% 45.8% 54.8% Women 45.5% 37.9% 54.2% 45.2% Age 64 years (37−91) 59 years (37−69) 72 years (48−91) 60 years (42−77) IgH IgG 59.4% 55.8% 62.8% 42.3% IgA 32.8% 35.5% 30.4% 50% Bence−Jones 6.9% 7.0% 6.8% 7.7% Nonsecretory 0.9% 1.7% 0% 0% IgL Kappa chain 62.5% 63.5% 60.7% 50% Lambda chain 37.5% 36.5% 39.3% 50% Calcium 9.63 ± 1.72 mg/dL (0.45−17) 9.50 ± 1.99 mg/dL (0.45−17) 9.78 ± 1.37 mg/dL (2.28−14.4) 9.11 ± 0.49 mg/dL (8−10) Creatinine 1.16 ± 0.84 mg/dL (0.20−6.50) 1.11 ± 0.76 mg/dL (0.20−6.50) 1.21 ± 0.93 mg/dL (0.36−5.90) 0.89 ± 0.28 mg/dL (0.50−1.61) Albumin 3.52 ± 0.67 g/dL (1.43−5.50) 3.54 ± 0.73 g/dL (1.70−5.14) 3.51 ± 0.61 g/dL (1.43−5.50) 3.76 ± 0.46 g/dL (2.20−4.60) β2 microglobulin 5.17 ± 5.22 mg/L (0−62) 5.11 ± 5.45 mg/L (0−62) 5.20 ± 4.98 mg/L (0.15−43.40) 2.25 ± 1.13 mg/L (0.30−4.41) Hemoglobin 10.54 ± 1.98 g/dL (4.90−15.60) 10.55 ± 1.99 g/dL (5.50−15.50) 10.56 ± 1.97 g/dL (4.90−15.60) 12.38 ± 1.13 g/dL (10.10−14.70) R-ISSa Stage I 19.1% 24.1% 13.1% 61.9% Stage II 69.7% 62.8% 77.9% 38.1% Stage III 11.2% 13.1% 9.0% 0% ECOG 0 23.8% 27.6% 20.3% 66.7% 1 49.7% 49.3% 50.7% 33.3% 2 20.2% 17.8% 21.6% 0% 3 4.0% 4.6% 3.4% 0% 4 2.3% 0.7% 4.1% 0% Bone lesions None 19.2% 19.7% 18.9% 100% Minor lesions 42.1% 45.4% 38.5% 0% Major lesions 38.7% 34.9% 42.7% 0% Plasmacytoma 11.2% 14.1% 8.3% 0% High LDH 15% 16.6% 13.3% 5.4% t(11;14) 13.2% 7.4% 18.1% 3.4% t(4;14) 11.7% 12.2% 9.4% 19.4% t(14;16) 3% 4.6% 1% 6.9% 17p abnormalities 7% 7.1% 7.2% 4.3% HR cytogenetics 20.3% 21.6% 16.5% 29.6% 1q gain 47.7% 46.4% 50% 26.3% del1p 5.7% 7.2% 3% 20% Clinical features were compared distinguishing between symptomatic (transplant-candidates and noncandidates) and smoldering myeloma patients. IgH immunoglobulin heavy chain, IgL immunoglobulin light chain, R-ISS Revised International Staging System, ECOG Eastern Cooperative Oncology Group Performance Status, LDH lactate dehydrogenase, HR cytogenetics high-risk cytogenetics. a Proportions of each ISS group are shown instead of the R-ISS for asymptomatic myeloma patients. rearrangements were detected, including the identifica- Gene repertoire tion of five cases with a biallelic rearrangement, as well as IGHV, IGHD and IGHJ usage was identified in pro- one case with a potential biclonal myeloma that was later ductive rearrangements. Table 2 shows the frequencies of confirmed by flow cytometry (Fig. S1). Among them, 362 IGHV gene group usage among the 362 productive rear- rearrangements (93.3%) were productive, while 26 rear- rangements, with observed and expected frequencies rangements (6.7%) were unproductive (Fig. 1). Six of these (based on the number of genes per IGHV group), as well unproductive sequences corresponded to light-chain as SHM rates. No major differences were observed com- myeloma patients. paring transplant and nontransplant subgroups. Blood Cancer Journal Medina et al. Blood Cancer Journal (2020)10:14 Page 5 of 12 Fig. 1 Study flowchart. From the starting population (n = 413), 92 patients were excluded. Hence, molecular profiling and survival analyses were performed with 327/413 patients (79.2%). IGHV genes 30-4 (n = 5), IGHV4-38-2 (n = 1) and IGHV5-10-1 (n = 3), According to the IMGT criteria29,32, we detected 50 and one pseudogene (IGHV3-69-1, n = 1). functional genes (Fig. S2 and Table S1) in our cohort. Compared to previous myeloma series, IGHV gene Five canonical IGHV genes were never seen: IGHV1-45, frequencies in our cohort were similar with three excep- IGHV3-20, IGHV4-28, IGHV6-1 and IGHV7-04. Overall, tions (IGHV3-20, IGHV4-30-2 and IGHV6-1) that were IGHV3-30 was the most abundant IGHV gene, with 46/ not found in our patients. We also observed that, while 362 cases (12.7%), followed by IGHV3-33 (5.2%), IGHV3- IGHV3 gene group representation was similar between 23 (5%) and IGHV1-69 (4.7%). On the other hand, some both series, IGHV3-30 was overrepresented and IGHV3- IGHV genes usually rearranged in the normal B-cell 30-3 was underrepresented in our cohort. repertoire, such as IGHV3-20 or IGHV3-74, were underrepresented or totally absent. Another interesting IGHD and IGHJ genes finding was the presence of four cases using the IGHV4- IGHD and IGHJ gene identification was assessed in 349/ 34 gene: two of them had high SHM rates (10 and 18.9%), 362 sequences (96%). both were unproductive, and patients had light-chain IGHD3 and IGHD2 groups were the most abundant myeloma; the other two cases also showed higher SHM (30.4% and 25.4%, respectively), with IGHD3-10 (10%) and rates than the mean (20.8 and 16.7%) and clonal rear- IGHD2-21 (8%) genes significantly overrepresented (p < rangements were productive. One of these patients had 0.05) as compared to the expected value in healthy plasma light-chain myeloma while the other one was IgG cells. Conversely, IGHD4-4 and IGHD6-25 were not found secretory MM. in any sequence, and all members from the IGHD1 gene Finally, we also found noncanonical, haplotype-dependent group but IGHD1-26 seemed to be underrepresented (less IGHV genes in ten patients: IGHV4-30-2 (n = 1), IGHV4- than 1.5% each one, Fig. S3 and Table S2). All these Blood Cancer Journal Medina et al. Blood Cancer Journal (2020)10:14 Page 6 of 12 Table 2 Group usage and SHM rates in IGHV genes. IGHV group Expected frequency Observed frequency Median SHM % (95% CI) IGHV1 10 (18.2%) 55 (15.2%) 10.0 (8.6–10.5) IGHV2 3 (5.4%) 31 (8.6%) 6.5 (6.0–8.0) IGHV3 26 (47.3%) 191 (52.7%) 8.8 (8.7–9.7) IGHV4 12 (21.8%) 67 (18.5%) 9.2 (9.2–11.4) IGHV5 2 (3.6%) 18 (5.0%) 7.2 (6.1–9.0) IGHV6 1 (1.8%) 0 – IGHV7 1 (1.8%) 0 – Total 55 362 8.8 (8.8–9.6) Comparison of SHM % Adjusted p value IGHV1 vs. IGHV2 0.006 IGHV2 vs. IGHV4 0.001 IGHV2 vs. IGHV3 0.012 IGHV1 vs. IGHV5 0.301 IGHV4 vs. IGHV5 0.118 IGHV3 vs. IGHV5 0.790 IGHV1 vs. IGHV4 1 IGHV2 vs. IGHV5 1 IGHV3 vs. IGHV4 1 IGHV1 vs. IGHV3 1 The first two columns show the expected IGHV gene usage if all genes were randomly selected compared with the observed frequency within our cohort, respectively. Observed and expected IGHV distributions were not different when the χ2 test was applied, demonstrating that there is no evidence of selection of specific gene groups in myeloma. Median somatic hypermutation rates per IGHV group are shown on the right side of the table. Below, Kruskal−Wallis-based paired comparisons are listed to determine potential differences in the hypermutation rate; a significantly lower mutation rate was found for patients using IGHV2 compared to those using IGHV1 (p = 0.006), IGHV3 (p = 0.012) and IGHV4 (p = 0.001). 95% CI 95% confidence intervals. features were similar in previous myeloma series and were 0.025) considering only transplant-candidate patients. also seen in other hematologic B-cell malignancies and in IGHD1 gene group was also more frequently used by normal plasma cells, although preference for IGHD2-21 patients harboring high-risk cytogenetics, although this was specially marked only in myeloma. association was restricted to patients not candidates for For IGHJ gene usage, we found a deep bias in the use of transplantation (41.7% vs. 13.2%, p = 0.026). However, certain genes matching previous observations concerning there were not relevant associations between the usage of different hematologic malignancies such as CLL, MCL, these genes and the outcome in terms of PFS or OS. and the normal bone marrow compartment: a significant Transplant-eligible and -ineligible patients showed overrepresentation of IGHJ4 (46.4%) and IGHJ6 (25%) was similar proportions in VDJH gene usage (Table S4). In the estimated (Fig. S4 and Table S3), while IGHJ2 and IGHJ1 same line, high-risk smoldering patients did not show were clearly underrepresented (2.9% and 1.1%, respec- significant differences in the use of VDJH genes compared tively). However, we noticed that, compared to previous to symptomatic patients. series in myeloma, IGHJ4 was underrepresented and IGHJ6 overrepresented. In addition, IGHJ1 and IGHJ6 Somatic hypermutation rates and IGH composition usage in healthy plasma cells looked completely different Somatic mutations could be correctly identified in not only with respect to myeloma, but also with other 349 sequences: mutational load was high (mean: 9.2% ± mature B-cell disorders. 3.8; median: 8.8%; 95% CI: 8.8−9.6%; range: 0−22.7%), We explored associations between VDJH repertoire and with five cases showing more than 98% of homology with risk factors. IGHJ6 gene group was more frequent among germline genes. Comparing SHM rates between IGHV patients with high-risk cytogenetics (37.9% vs. 17.8%, p = groups, we found a significantly lower rate for IGHV2 Blood Cancer Journal Medina et al. Blood Cancer Journal (2020)10:14 Page 7 of 12 compared to IGHV1, IGHV3 and IGHV4 (Table 2). in previous paragraphs were combined with clinical data Median SHM ratios of transplant-eligible and -ineligible to perform statistical comparisons in transplant-eligible patients were similar (9.0% vs. 8.5% respectively, p = and -ineligible patients. 0.869). Median SHM rate in symptomatic and asympto- We first looked for relationships between treatment matic patients was also similar (9.0% vs. 8.8%, p = 0.845). response and molecular variables. No major associations The complete sequence was available in 327/362 cases, so were seen, but two exceptions: (1) SHM ≥ 7% associated we continued the analysis with this set (Fig. 1). with a higher rate of CR/sCR compared to patients with As far as the junction region was concerned, nucleotide SHM < 7% in the nontransplant subset (45.8% vs. 27.3%, addition appeared in 92.5% of cases for the N1 region, and respectively; p = 0.026), and (2) IGHD3-3 usage was in 88.6% of cases for the N2 region. Only three cases did associated with a better response in the transplant sub- not show evidence of nucleotide addition, either in N1 or in group (n = 10), since 80% of MM patients with an IGH N2. Mean Guanine/Cytosine content of the N-regions was rearrangement choosing this particular gene achieved CR/ 58.8%. Evidence of exonuclease activity in at least one of sCR vs. 36.8% for other IGHD genes (p = 0.019). the two junction regions (IGHV-to-IGHD and/or IGHD- In addition, we detected that SHM rates were progres- to-IGHJ) was observed in all rearrangements, although sively higher when symptomatic patients were analyzed 32.2% of cases displayed a fully conserved IGHV-3′ edge. based on the differentiation stage of their pathologic plasma cells: median SHM levels for CD19+/CD81+ CDR3 amino acid composition (immature), CD19−/CD81+ (intermediate) and CD19 According to the IMGT numbering, we identified CDR3 −/CD81− (mature) plasma-cell subsets were 7.0% (95% positions (from amino acid 105 to amino acid 117). CI: 5.2−11.4%), 8.50% (95% CI: 7.9−9.6%), and 9.3% (95% Median CDR3 length was 15 amino acids (range 6−29); CI: 9.1−10.2%), respectively (p = 0.029). Moreover, med- this length was consistent across different myeloma sub- ian PFS increased from intermediate to mature subgroups groups (symptomatic, asymptomatic, transplant-eligible (35.4 vs. 64.0 months respectively, p = 0.024) but not from and transplant-ineligible patients). Amino acid propor- immature to intermediate subsets, probably because we tions in myeloma were not significantly different from the only found 11 CD19+/CD81+ cases in our series. normal B-cell population (Fig. S5), with predominance of Glycine, Alanine, Aspartic acid and Tyrosine. Transplant-ineligible patients In the univariate analysis for PFS, improved survival of Clustering analysis transplant-ineligible patients was associated with 0−1 CDR3 sequences from productive rearrangements (n = ECOG stages (p = 0.003), CR/sCR after the end of induc- 362) were subjected to cluster analysis. We found one pair tion (p = 2 × 10−6), baseline hemoglobin ≥ 9 g/dL (p = from our cohort that showed more than 60% of amino 0.002), no use of the IGHD4 gene group (p = 0.036) acid similarity. Consequently, and given they were clusters (Fig. 2a) or specifically the IGHD4-11 gene (p = 0.0003), use not previously described, we applied the secondary cri- of the IGHD2/IGHD3 gene groups (p = 0.012) (Fig. 2b), and teria. Although CDR3 lengths were similar (13 vs. 15 SHM rate ≥ 7% (p = 0.006) (Fig. 3a). Improved OS rates amino acids) and both rearrangements displayed the same were observed in the univariate analysis for those patients IGHJ4*02 germinal gene, IGHV and IGHD groups, as well with CR/sCR after induction (p = 0.00026), SHM rate ≥ 7% as IGHD reading frames, were not concordant. Thus, we (p = 0.00024) (Fig. 3b), standard-risk cytogenetics (p = could not find any stereotyped immunoglobulin cluster 0.031) and serum hemoglobin ≥ 9 g/dL (p = 0.002). among these myeloma patients. One hundred and eleven transplant-ineligible patients were included in the multivariate analysis. For PFS Impact of molecular variables on clinical outcomes (Table 3), CR/sCR achievement (p = 10−6, HR: 0.311, Considering patients with symptomatic myeloma, after 95% CI: 0.196−0.494) and the use of IGHD2/IGHD3 a median follow-up of 7.6 years (range 0.5−24.1), 120 gene groups (p = 0.006, HR: 0.552, 95% CI: 0.361 patients had died; the majority of them (n = 76) were −0.845) appeared as independent prognostic factors of transplant-ineligible. ROC curves showed that 7 and 8% prolonged survival rates. For OS, CR achievement after were the most optimal cutoffs for prognostic value of induction retained its independent prognostic value for SHM; optimal cutoffs for serum albumin (3.2 g/dL), β2 improved survival probability (p = 0.010, HR: 0.347, 95% microglobulin (3.5 mg/dL), hemoglobin (9 g/dL) and CI: 0.154−0.780) as well as SHM ≥ 7% (p = 0.001, HR: creatinine (1 mg/dL) were also estimated. In addition, 0.291, 95% CI: 0.137−0.618) and standard cytogenetic IGHD2 and IGHD3 users were grouped for subsequent risk (p = 0.016, HR: 0.369, 95% CI: 0.164−0.832). analyses, because they were the most common IGHD Moreover, SHM rate was included in the multivariate groups and this bias has been observed in nearly every B- regression model not only as a discrete variable but also cell malignancy9–18. Finally, all molecular data described as a continuous one to avoid potential bias induced with Blood Cancer Journal Medina et al. Blood Cancer Journal (2020)10:14 Page 8 of 12 Fig. 2 Kaplan−Meier curves comparing PFS for IGHD4 and IGHD2/3 users in transplant-ineligible patients. Survival curves compare progression-free survival rates between IGHD4 (a) or IGHD2/IGHD3 users (b) and other IGHD users. IGHD4 users are represented in red, while IGHD2/ IGHD3 users are represented in black in their respective plots. Patients at risk at each time point appear below each plot. Fig. 3 Kaplan−Meier curves comparing PFS and OS for SHM subsets in transplant-ineligible patients. a Progression-free survival plot. b Overall survival plot. Cases with more than 7% of SHM are represented in black, while cases with SHM below 7% are represented in red. Patients at risk at each time point appear below each plot. the cutoff selection of 7%; in this case, the variable and serum β2 microglobulin < 3.5 mg/dL (p = 0.023). The retained its prognostic value (p = 0.017, HR: 0.909, 95% R-ISS stage (p = 6 × 10−6), high-risk cytogenetics (p = 4 × CI: 0.840−0.983) along with achieving CR/sCR and the 10-6), the ECOG score (p = 0.004), 1q gains (p = 0.016) absence of cytogenetic risk, showing that an increment and serum β2 microglobulin (p = 0.002) also showed sig- of 1% in SHM reduces death risk by 9%. nificant associations with the OS in the same direction as PFS. Interestingly, no molecular variable was significantly Transplant-eligible patients associated with survival in this subset of patients. Univariate analysis for PFS showed improved outcomes One hundred and thirty-seven transplant-eligible for patients in R-ISS stage I vs. II (p = 0.002) and III (p = patients were included in the multivariate analysis. 0.004), standard-risk cytogenetics (p = 3 × 10−6), 0−1 Presence of 1q gains (p = 0.046, HR: 2.187, 95% CI: ECOG stages (p = 0.015), absence of 1q gains (p = 0.019) 1.015−4.714) and high-risk cytogenetics (p = Blood Cancer Journal Medina et al. Blood Cancer Journal (2020)10:14 Page 9 of 12 Table 3 Univariate and multivariate analysis of clinical and biological factors influencing PFS and OS of transplant- ineligible patients in our cohort. Variable Univariate analysis for PFS Multivariate analysis for PFS Univariate analysis for OS Multivariate analysis for OS Median survival p HR [95% CI] p Median survival p HR [95% CI] p ECOG score 0−1 26.94 0.003 NS NS 2−4 21.35 Serum hemoglobin <9 g/dL 14.03 0.001 NS 24.54 0.002 NS ≥9 g/dL 28.32 64.85 Post induction response sCR/CR 49.35 0.008 0.311 [0.196−0.494] 1E−05 Not reached 0.00026 0.347 [0.154−0.780] 0.010 VGPR/PR 23.88 53.19 Cytogenetic risk High risk 36.30 0.196 NS 78.59 0.031 0.369 [0.164−0.832] 0.016 Standard risk 25.03 39.95 IGHD4 usage Yes 14.49 0.036 NS NS No 27.96 IGHD4-11 usage Yes 14.16 0.0003 NS NS No 26.94 IGHD2 and IGHD3 usage Yes 34.46 0.012 0.552 [0.361−0.845] 0.006 NS No 23.46 SHM level <7% 23.46 0.006 NS 34.73 0.00024 0.291 [0.137−0.618] 0.001 ≥7% 26.45 64.85 Univariate analysis was first performed to evaluate the role of each clinical and biological variable described in this study. The table only shows statistically significant variables that were later included in the multivariate analysis. Median survival is shown in months. HR hazard ratio, CI confidence interval, NS nonsignificant. 0.000021, HR: 4.690, 95% CI: 2.267−9.700) were genes34–36, and although these genes are also common in associated with lower PFS rates. High-risk cytogenetics other lymphoproliferative disorders and normal plasma retained its independent prognostic value for shorter cells, we noted that IGHV3-30 was specifically selected OS (p = 0.001, HR: 8.030, 95% CI: 2.406−26.809), only in myeloma. In contrast, IGHV3-23 and IGHV1-69 while serum β2 microglobulin levels < 3.5 mg/dL appeared to be less frequently selected in MM (≥8% of associated with better outcomes (p = 0.039, HR: 0.116, representation in the normal B-cell population35,36 and B- 95% CI: 0.015−0.899). cell pathologies vs. ≤5% in our series). IGHV4-34 representation is high in hematologic Discussion malignancies such as B-ALL, CLL, MCL or diffuse large Here, we analyzed in depth the IGH locus, trying to B-cell lymphoma as well as in normal B cells9,11,31. correlate its characteristics with clinical data in a large Interestingly, this gene is usually absent in the normal cohort of 413 MM patients included in clinical trials. plasma-cell population37, although it shows a wide VDJH usage description provided new insights into the interindividual usage frequency38, and it is rarely selected clonal differentiation and possible clinical implications for in MM17, but in this work it was not completely absent. MM, that could potentially improve patient care. IGHV4-34 has been characterized as an inherently auto- IGHV gene repertoire reflected its normal counterpart, reactive antibody39,40 for it is widely represented in with IGHV3 being the predominant gene group. As in other autoimmune diseases (i.e. systemic lupus erythematosus). series, IGHV3 was overrepresented and IGHV1 under- Here, we found four clonal IGHV4-34 rearrangements; represented, although differences were not significant, with they showed very high SHM rates and were usually a much less pronounced bias than that observed in nonproductive, leading to light-chain myelomas. This CLL12,13,31. Similarly, IGHV3-30, IGHV4-59 and IGHV4- could be explained by the fact that during plasma-cell 39 were overrepresented with respect to other IGHV differentiation, those cells displaying autoreactive-prone Blood Cancer Journal Medina et al. Blood Cancer Journal (2020)10:14 Page 10 of 12 genes within their IGH rearrangements would be specially possible explanation is related to the proliferation ratio of targeted by the somatic hypermutation machinery, trying tumor cells: highly proliferative B cells (and therefore, more to make them able to recognize only foreign antigens. aggressive clones) activate DNA repair pathways that are Moreover, in our series, two out of the three unproductive able to reduce the SHM level, as it has been shown in CLL rearrangements using VH1-69, another gene commonly patients50,51. However, the SHM cutoff is based on FR1 associated with autoimmune events, such as Thrombotic primers; this threshold could differ when using leader Thrombocytopenic Purpura41, and highly represented in primers. CLL, were light-chain myelomas with some of the highest Another striking feature was the correlation between SHM rates in our cohort (mean 12.23%). Interestingly, the use of IGHD2 and IGHD3 gene groups and the out- this gene is commonly unmutated in CLL patients42,43. come. An increased usage of these particular groups has SHM level was high and similar to what has been also been observed in other series suggesting a positive observed in other studies for healthy and pathological selective pressure over them8,17,18, since the bias was plasma and memory B cells. This would be in line with the observed only for complete rearrangements. Nevertheless, post-germinal center origin of myeloma8. A striking whether or not the relationship between IGHD usage and finding was the presence of five MM cases with no survival is a myeloma characteristic remains uncertain. apparent SHM; however, the IGHV region could not be SHM and IGHD usage were associated with the out- completely sequenced (the entire FWR1 and part of the come only in the elderly, transplant-ineligible patients. FWR2 was missing); from our experience, we think the Thereby, the effect of intensive regimens could be the mutational load was most likely underestimated, rather main reason to explain why the negative prognostic than being truly unmutated cases. impact of these molecular characteristics is diluted in CDR3 composition resembled the normal immunoglo- younger, fit patients. bulin repertoire, with similar length, amino acid use, or Regarding the value of clinical variables on the patient evidence of TdT and exonuclease activity44. This compo- outcome, the prognostic role of cytogenetics, hemoglobin, sition diverges from CLL, MCL or MZL patterns, where albumin, β2 microglobulin and ECOG in MM was con- restricted antibody sets account for approximately 30% of firmed52. Cytogenetic risk has to be mentioned as this cases45–47. Accordingly, cluster analysis failed to demon- variable showed significant associations with survival in all strate the presence of stereotyped receptors in our cohort, cases but when applied to PFS in transplant-ineligible as it has been shown before in myeloma patients48. Overall, patients. The absence of FISH studies for most patients the gene repertoire in our series was very close to previous diagnosed from 1995 to 2000 (most of them being older) reports17,18,48, although functional IGHV and IGHD genes, made possible to find only 17 patients with high-risk cyto- whose use has not been reported in myeloma to date, have genetics in this subgroup, which could explain this result. been found in this cohort probably due to its larger size. On In conclusion, we have shown that multiple myeloma the contrary, other genes such as IGHV1-18, IGHV3-20, plasma cells resemble the normal mature B-cell reper- IGHV4-30-2 and IGHV6-1, found in Italian48 or Greek18 toire but, in contrast to CLL, MCL or MZL, stereotyped patients, are absent in Spanish patients, maybe reflecting receptors were absent14,15,53,54. Light-chain and non- differences in the normal repertoire between different secretory myeloma cases could be explained by incor- Mediterranean subpopulations. No differences were rect IGH rearrangements at the genomic DNA level, but observed in the median length of the CDR3 region, and not always, which means that other effects could ham- somatic hypermutation was slightly higher than in previous per the immunoglobulin production, including tran- publications; however, this variation was not statistically scriptional or translational defects. Finally, here, and for significant. the first time, we have reported how some molecular The most remarkable finding was the association between characteristics of the IGH gene rearrangement, such as a higher SHM level and an improved survival rate. The higher SHM rates or IGHD2/IGHD3 gene usage, could explanation for this situation seems to be connected with positively impact on the outcome of patients. A valida- the maturation stage of tumor cells, represented by the tion series is currently planned to elucidate whether expression of CD19 and CD81 biomarkers, which is con- these findings could be applicable in real-life patients; if sistent with previous findings regarding the immune profile validated in independent studies, they could be con- of tumor plasma cells:49 it suggests that early genetic sidered as new molecular markers for PFS and OS in alterations in the ontogeny of germinal-center B cells would multiple myeloma. provide a more aggressive profile in MM, which would be strong enough to overcome SHM and/or CSR earlier. Acknowledgements Conversely, the acquisition of late, more harmless genetic This work was partially supported by the Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness PI15/01956, CIBERONC- events would allow tumor B cells to undergo SHM and CSR CB16/12/00233, and “Una manera de hacer Europa” (Innocampus; CEI-2010- processes and this would lead to better outcomes. Another 1-0010)”. M.G.-A., I.P.-C., and C.J. are supported by the Fundación Española Blood Cancer Journal Medina et al. Blood Cancer Journal (2020)10:14 Page 11 of 12 de Hematología y Hemoterapia (FEHH, co-funded by Fundación Cris in the 2. Oettinger, M. A., Schatz, D. G., Gorka, C. & Baltimore, D. RAG-1 and RAG-2, latter case), A.M. by the European Social Fund and the Spanish Education adjacent genes that synergistically activate V(D)J recombination. Science 248, Council through the University of Salamanca, and M.E.S. by the ISCIII (CPII18/ 1517–1523 (1990). 00028). 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