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Immunogenetic characterization of clonal plasma cells in systemic light-chain amyloidosis

Immunogenetic characterization of clonal plasma cells in systemic light-chain amyloidosis Leukemia (2021) 35:245–249 https://doi.org/10.1038/s41375-020-0800-6 LETTER Multiple myeloma gammopathies Immunogenetic characterization of clonal plasma cells in systemic light-chain amyloidosis 1 2 1 3 2 2 ● ● ● ● ● ● Isabel Cuenca Daniel Alameda Beatriz Sanchez-Vega David Gomez-Sanchez Diego Alignani Marta Lasa 1 2 2 4 5 ● ● ● ● ● Esther Onecha Ramon Lecumberri Felipe Prosper Enrique M. Ocio Maria Esther González 6 7 8 9 10 ● ● ● ● ● Alfonso García de Coca Javier De La Rubia Mercedes Gironella Luis Palomera Albert Oriol 11 12 13 14 15 ● ● ● ● ● Maria Casanova Valentin Cabañas Francisco Taboada Albert Pérez-Montaña Felipe De Arriba 16 1 1 17 ● ● ● ● Noemi Puig Gonzalo Carreño-Tarragona Santiago Barrio Jose Enrique de la Puerta 13 18 19 1 16 ● ● ● ● ● Angel Ramirez-Payer Isabel Krsnik Juan Jose Bargay Juan Jose Lahuerta Maria-Victoria Mateos 2 2 1 ● ● Jesus F. San-Miguel Bruno Paiva Joaquin Martinez-Lopez Received: 19 November 2019 / Revised: 4 March 2020 / Accepted: 9 March 2020 / Published online: 19 March 2020 © The Author(s) 2020. This article is published with open access To the Editor: landscape of AL amyloidosis is required since, for example, potential differences between the genomic profiles of AL Sequence-based analysis has come to play an integral role amyloidosis and multiple myeloma (MM) could help in many hematological malignancies [1], but disorders such identifying patients with monoclonal gammopathies at as systemic light-chain (AL) amyloidosis remain poorly greater risk of developing AL amyloidosis and monitor characterized due to its low incidence and small tumor size presymptomatic organ damage [4, 5]. [2, 3]. Thus, greater knowledge about the immunogenetic To gain further insight into the immunogenetic landscape of AL amyloidosis, we performed whole-exome sequencing (WES) on highly purified bone marrow clonal plasma cells (PCs) isolated by fluorescence activation cell sorting These authors contributed equally: Isabel Cuenca, Daniel Alameda (FACS) based on patient-specific aberrant phenotypes. A These authors jointly supervised this work: Bruno Paiva, Joaquin total of 27 patients with confirmed new diagnosis of AL Martinez-Lopez amyloidosis based on the presence of amyloid-related sys- Supplementary information The online version of this article (https:// temic syndrome, positive amyloid tissue staining with doi.org/10.1038/s41375-020-0800-6) contains supplementary Congo red, restricted light-chain deposition by material, which is available to authorized users. * Joaquin Martinez-Lopez Hospital Clinico Universitario Lozano Blesa, Zaragoza, Spain jmarti01@med.ucm.es Hospital German Trias i Pujol, Badalona, Spain 1 11 Hospital 12 de Octubre, Madrid, CNIO, Universidad Complutese, Hospital Costa del Sol, Marbella, Spain Madrid, Spain Hospital Clínico Universitario Virgen de la Arrixaca, Clinica Universidad de Navarra, Centro de Investigacion Medica Murcia, Spain Aplicada (CIMA), IDISNA, CIBERONC Pamplona, Hospital Universitario Central de Asturias (HUCA), Pamplona, Spain Oviedo, Spain Clinical and Traslational Lung Cancer Research Unit, i+12 Hospital Son Espases, Palma, Spain Research Institute and Biomedical Research Networking Center in Hospital Universitario Morales Meseguer. IMIB-Arrixaca, Oncology (CIBERONC), Madrid, Spain Murcia, Spain Universidad de Cantabria, Hospital Universitario Marqués de Hospital Universitario de Salamanca, Instituto de Investigacion Valdecilla, Santander, Spain Biomedica de Salamanca (IBSAL), Centro de Investigación del Hospital de Cabueñes, Gijon, Spain Cancer (IBMCC-USAL, CSIC), Salamanca, Spain Hospital Clínico Universitario de Valladolid, Valladolid, Spain Hospital de Galdakao, Vizcaya, Spain Hospital Doctor Peset, Valencia, Spain Hospital Puerta de Hierro, Madrid, Spain Hospital Universitari Vall d’Hebron, Barcelona, Spain Hospital Universitario Son Llàtzer, Palma, Spain 1234567890();,: 1234567890();,: 246 I. Cuenca et al. immunohistochemistry or mass spectometry, and evidence of a series of 62 newly diagnosed MM patients. Detailed of PC clonality were investigated. Patients’ demographics methodology is available in the Supplementary methods. and clinical characteristics are described in Supplementary We identified a total of 718 exonic, non-immunoglobu- Table 1. PCs were collected and processed in triplicates lin, nonsynonymous mutations with a variant allelic fraction followed by whole genome amplification of samples with (VAF) >5% (683 SNV and 35 indel). Total number and genomic DNA amounts <50 ng (Supplementary Table 2). type of mutations are described in Supplementary Fig. 1. Afterwards, library construction, exome enrichment, and Interestingly, mutational burden was significantly correlated sequencing were performed individually. An overall aver- with patients’ age (R = 0.51, p < 0.001) (Supplementary age depth of 63× and mean on-target coverage of 84% were Fig. 2) though not tumor burden (data not shown). Only 37 obtained. Data were deposited in the Sequence Read out of 662 (5.5%) mutated genes were altered more than Archive of the NCBI (http://www.ncbi.nlm.nih.gov/sra) once (Fig. 1). That notwithstanding, 23 of the 27 cases under the PRJNA596656 access number. To increase spe- (85%) presented with at least one mutation in one of the 37 cificity, only single-nucleotide variants (SNVs) and indels genes (range, 1–11); being FAT4, IGLL5, MUC16, and detected by both Strelka [6] and Varscan2 [7] variant callers SSH2 the most frequently mutated genes (≥3 patients). With were selected. Moreover, only somatic mutations present in a median of 18 mutations per sample (range, 8–92), patients two of three libraries per patient were considered positive. with AL amyloidosis are closer to monoclonal gammopathy Germline variants were excluded through WES of matched of undetermined significance (MGUS) (median of 19) [10] peripheral blood cells. The mutational profile of patients rather than MM (median of 38 in the CoMMpass IA13c with AL amyloidosis was compared with that of patients dataset, p < 0.0001; Fig. 2a) in terms of mutational load. By with MM enrolled in CoMMpass (n = 930; IA13c dataset). contrast, the presence of CNA was more frequent in The CNVKit [8] was used to determine copy number AL amyloidosis (19/21, 90.5%; Supplementary Fig. 3) than abnormalities (CNA) from WES data (in 21 of the 27 AL MGUS (60.6% in Mikulasova et al.) [10] and similar to patients). Deep sequencing of B-cell receptor immunoglo- MM patients (virtually 100%) [11]. Overall, these results bulin (BcR Ig) gene rearrangements was performed in all underpin recent observations based on the immunopheno- patients as previously described [9], and compared with that typic characterization of clonal PCs [5], and locate AL Nonsense SNV Missense SNV Frame Shift Del In Frame Del Multi Hit 0 0.5 1 1.5 2 2.5 3 11% FAT4 11% IGLL5 11% MUC16 11% SSH2 7% PRK AG3 7% CCIN 7% CFAP74 7% CKAP4 7% CLIC1 7% COL4A5 7% COL6A3 7% CSMD2 DNAH17 7% 7% DNAH2 7% FAM135B 7% FAM170A 7% GABBR1 7% HERC1 7% LAMA1 7% LAMA3 7% MAGEL2 7% MEOX2 7% MORC2 7% MYH13 7% NAV1 7% OPCML 7% PCDH15 7% PCF11 7% PDILT 7% PROB1 7% RIMS2 7% SAFB 7% SMAD1 7% SNAP91 7% SP140 7% VWA7 7% ZFHX3 Patients (n=27) Fig. 1 Genes recurrently mutated in AL. Distribution per patient of the most frequently mutated genes (n = 37). Boxes were colored according to the type of mutation. Top barplots define the total number of mutations per patient. Recurrence (%) 27 Immunogenetic characterization of clonal plasma cells in systemic light-chain amyloidosis 247 AB MM AL 2000 p<0.0001 MM AL IGHV3-30 IGHV3-48 SSH2 FAT4 IGLL5 8 8 MUC16 6 6 TRAF,BRAF NRAS 02468 10 12 14 0 5 10 15 20 25 30 AL IGHV genes recurrence (%) MM recurrent genes (%) Fig. 2 The mutational landscape in AL and MM. a Number of recurrence in AL and MM. d Predominant Ig heavy chain gene rear- mutations in patients with newly diagnosed AL and MM. b Shared and rangements used in AL and MM. private mutations between AL and MM. c Driver genes based in its amyloidosis in the crossroad between MGUS and MM also [13] identified recurrent mutations in KRAS. Furthermore, in genetic grounds. Of note, the only alterations associated IGLL5 emerged as one of the most commonly mutated with inferior progression-free survival were gains in chro- genes in ours and the latter series, but not in that analyzed mosomes 9 and 19 (Supplementary Fig. 4a, b), whereas del by Boyle et al. We have found no association between (13q) was associated with higher NT-proBNP levels (Sup- mutated genes and patients’ outcome, whereas Huang et al. plementary Fig. 5). Furthermore, patients with +1q also identified three mutated genes with prognostic value. displayed greater risk of cardiac involvement (Supplemen- Altogether, further studies in larger series of patients are tary Table 3). warranted to shed more light into the mutational landscape Interestingly, various MM-defined driver mutations [12] and potential clinical correlations in AL amyloidosis. were undetected in patients with AL amyloidosis (e.g., In the absence of a unifying genetic event defining AL NRAS, BRAF, and TRAF) or observed only once (e.g., DIS3 amyloidosis, BcR Ig gene rearrangements emerge as an and DUSP2), most of them being subclonal with a median alternative to understand the propensity for the deposition of VAF of 23% (Supplementary Fig. 6) that was unrelated to monoclonal Ig light-chains in the form of β-sheet fibrils, as the lower tumor burden in AL amyloidosis due to the well as organ tropism. Previous studies based on PCR FACSorting strategy used to isolate clonal PCs, as descri- amplification of individual (typically Lambda) light-chain bed above. Simultaneous analysis of ours and the CoMM- variable gene (VL) families have shown a potential bias in pass datasets unveiled that out of 14,135 mutated genes, germline donor use in patients with AL amyloidosis [14] only 608 (4.4%) were shared between AL amyloidosis and and that VL gene usage may influence clinical presentation, MM (Fig. 2b), with considerable differences in their organ deposition, and outcome [15, 16]. Hence, we per- recurrence (Fig. 2c). Of note, none of the 65 genes exclu- formed next-generation sequencing (NGS) of Ig heavy sively mutated in AL amyloidosis were recurrent. Further- chain gene (IGH) and Kappa light-chain (IGK) rearrange- more, only four genes (XKR5, PRSS45, PKD1L2, and ments both in patients with AL amyloidosis (n = 27) and SRRM5) overlapped with the 105 described by Boyle et al. MM (n = 63) to provide complementary information on [3] as AL restricted. While the results from these two stu- IGH and IGK repertoires, clonal variability, and extent of dies suggest that MM recurrent mutations are unfrequently somatic hypermutations. A total of 39 IGHV-D-J and IGK- detected in patients with AL amyloidosis, recent data based V-J clonotypes were identified and 5 of the 27 (19%) on WES and targeted sequencing reported by Huang et al. patients with AL amyloidosis displayed more than two AL recurrent genes (%) Mutations MM IGHV genes recurrence (%) 248 I. Cuenca et al. clonal rearrangements. This extent of clonal heterogeneity Author contributions JFSM, BP, JML, and JJL conceived the idea and together with IC and BSV designed the study. IC, BSV, DGS, DA, differs (p = 0.024) from that found in our MM series (3.9%). BP, EO, and SB analyzed and interpreted data. IC and GCT performed Using a cut-off of 98% to define homology, 9 of statistical analysis. ML acquired the clinical data. DAlignani per- 39 sequences (23%) in AL amyloidosis were considered as formed cell sorting. RL, FP, EMO, MEG, AGdC, JdlR, MG, LP, AO, mutated, which is slightly less when compared with that MC, VC, FT, AP, FdA, NP, MVM, JJL, and JFSM provided study materials and patients. IC, BSV, DA, JML, and BP wrote the manu- found in MM (36%). The CDR3 length was not significantly script. All authors reviewed and approved the manuscript. different between AL amyloidosis and MM (median of 54 versus 51 amino acids). The most frequent IGH gene Compliance with ethical standards involved in AL amyloidosis was IGHV3-48 (recurrence of 10.3%) and 100% of patients who have this rearrangement Conflict of interest The authors declare that they have no conflict of had kidney involvement (p = 0.025) (Supplementary interest. Table 4), whereas IGHV3-30 was the most recurrent (12%) in MM (Fig. 2d). Thus, expression of IGHV3-48 adds up to Publisher’s note Springer Nature remains neutral with regard to IGLV6-57 as germline Ig genes associated with dominant jurisdictional claims in published maps and institutional affiliations. kidney deposition [14, 15]. Of note, we found no significant differences regarding IGH and IGK repertoires, clonal Open Access This article is licensed under a Creative Commons variability, and extent of somatic hypermutations between Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as patients with light-chain only versus heavy- and light-chain long as you give appropriate credit to the original author(s) and the M-component (data not shown). source, provide a link to the Creative Commons license, and indicate if This study confirms previous observations that AL changes were made. The images or other third party material in this amyloidosis cannot be defined by a singular or a set of well- article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not defined genetic events. In fact, based on combined results included in the article’s Creative Commons license and your intended from WES of 99 patients (27 in this study, 24 in Boyle et al. use is not permitted by statutory regulation or exceeds the permitted [3], and 48 in Huang et al. [13]), 63 genes were found to be use, you will need to obtain permission directly from the copyright recurrently mutated. By contrast, our study further supports holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. the notion that germline Ig gene use is a key determinant in the pathogenesis of AL amyloidosis [16], and unveils novel References associations of cytogenetic abnormalities with organ involvement and outcome. The extent of CNA was similar 1. Merker JD, Valouev A, Gotlib J. Next-generation sequencing in between AL and MM, but MM-driver genes were not hematologic malignancies: what will be the dividends? Ther Adv recurrently mutated in AL, which instead was marked by Hematol. 2012;3:333–9. greater clonal heterogeneity (i.e., similarly to MGUS) [10]. 2. Paiva B, Martinez-Lopez J, Corchete LA, Sanchez-Vega B, Rapado I, Puig N, et al. Phenotypic, transcriptomic, and genomic Taken together with differential predominance of IGH features of clonal plasma cells in light-chain amyloidosis. Blood. rearrangements, our results suggest that amongst other 2016;127:3035–9. factors, Ig germline genes rather than specific cytogenetic 3. Boyle EM, Ashby C, Wardell CP, Rowczenio D, Sachchitha- abnormalities predispose light-chains from a PC clone to nantham S, Wang Y, et al. The genomic landscape of plasma cells in systemic light chain amyloidosis. Blood. 2018;132:2775–7. adopt an aberrant conformation, typically closer to patients’ 4. Merlini G, Comenzo RL, Seldin DC, Wechalekar A, Gertz MA. MGUS stage before the development of higher tumor bur- Immunoglobulin light chain amyloidosis. Expert Rev Hematol. den and accumulation of MM-driver mutations. Given the 2014;7:143–56. high frequency of Lambda light-chain deposition in AL, 5. Puig N, Paiva B, Lasa M, Burgos L, Perez JJ, Merino J, et al. Flow cytometry for fast screening and automated risk further studies are warranted to investigate if clonal muta- assessment in systemic light-chain amyloidosis. Leukemia. tions in IGLL5 (median VAF, 64%) contribute to this pro- 2019;33:1256–67. cess. In such cases, its detection together with other 6. Saunders CT, Wong WSW, Swamy S, Becq J, Murray LJ, candidate genes (e.g., FAT4, MUC16, and SSH2) through Cheetham RK. Strelka: accurate somatic small-variant calling from sequenced tumor–normal sample pairs. Bioinformatics. NGS diagnostics could emerge as novel risk markers for AL 2012;28:1811–7. amyloidosis in patients with monoclonal gammopathies. 7. Koboldt DC, Chen K, Wylie T, Larson DE, McLellan MD, Acknowledgements This study was supported by the Centro de Mardis ER, et al. VarScan: variant detection in massively parallel Investigación Biomédica en Red—Área de Oncología—del Instituto sequencing of individual and pooled samples. Bioinforma Oxf de Salud Carlos III (CIBERONC; CB16/12/00369; and CB16/12/ Engl. 2009;25:2283–5. 00489), Instituto de Salud Carlos III/Subdirección General de Inves- 8. Talevich E, Shain AH, Botton T, Bastian BC. CNVkit: genome- tigación Sanitaria (FIS No. PI13/02196), Asociación Española Contra wide copy number detection and visualization from targeted DNA el Cáncer (GCB120981SAN and the Accelerator Award), CRIS sequencing. PLOS Comput Biol. 2016;12:e1004873. against Cancer foundation grant 2014/0120, and the Black Swan 9. Martinez-Lopez J, Sanchez-Vega B, Barrio S, Cuenca I, Ruiz- Research Initiative of the International Myeloma Foundation. Heredia Y, Alonso R, et al. Analytical and clinical validation of a Immunogenetic characterization of clonal plasma cells in systemic light-chain amyloidosis 249 novel in-house deep-sequencing method for minimal residual 13. Huang X-F, Jian S, Lu J-L, Shen K-N, Feng J, Zhang C-L, et al. disease monitoring in a phase II trial for multiple myeloma. Genomic profiling in amyloid light-chain amyloidosis reveals Leukemia. 2017;31:1446–9. mutation profiles associated with overall survival. Amyloid Int J 10. Mikulasova A, Wardell CP, Murison A, Boyle EM, Jackson GH, Exp Clin Investig J Int Soc Amyloidosis. 2020;27:36–44. Smetana J, et al. The spectrum of somatic mutations in mono- 14. Perfetti V, Casarini S, Palladini G, Vignarelli MC, Klersy C, clonal gammopathy of undetermined significance indicates a less Diegoli M, et al. Analysis of Vλ-Jλ expression in plasma cells complex genomic landscape than that in multiple myeloma. from primary (AL) amyloidosis and normal bone marrow identi- Haematologica. 2017;102:1617–25. fies 3r(λIII) as a new amyloid-associated germline gene segment. 11. López-Corral L, Sarasquete ME, Beà S, García-Sanz R, Mateos Blood. 2002;100:948–53. MV, Corchete LA, et al. SNP-based mapping arrays reveal high 15. Abraham RS. Immunoglobulin light chain variable (V) region genomic complexity in monoclonal gammopathies, from MGUS genes influence clinical presentation and outcome in light chain- to myeloma status. Leukemia. 2012;26:2521–9. associated amyloidosis (AL). Blood. 2003;101:3801–7. 12. Walker BA, Mavrommatis K, Wardell CP, Ashby TC, Bauer M, 16. Perfetti V, Palladini G, Casarini S, Navazza V, Rognoni P, Obici Davies FE, et al. Identification of novel mutational drivers reveals L, et al. The repertoire of λ light chains causing predominant oncogene dependencies in multiple myeloma. Blood. 2018; amyloid heart involvement and identification of a preferentially 132:587–97. involved germline gene, IGLV1-44. Blood. 2012;119:144–50. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Leukemia Springer Journals

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Abstract

Leukemia (2021) 35:245–249 https://doi.org/10.1038/s41375-020-0800-6 LETTER Multiple myeloma gammopathies Immunogenetic characterization of clonal plasma cells in systemic light-chain amyloidosis 1 2 1 3 2 2 ● ● ● ● ● ● Isabel Cuenca Daniel Alameda Beatriz Sanchez-Vega David Gomez-Sanchez Diego Alignani Marta Lasa 1 2 2 4 5 ● ● ● ● ● Esther Onecha Ramon Lecumberri Felipe Prosper Enrique M. Ocio Maria Esther González 6 7 8 9 10 ● ● ● ● ● Alfonso García de Coca Javier De La Rubia Mercedes Gironella Luis Palomera Albert Oriol 11 12 13 14 15 ● ● ● ● ● Maria Casanova Valentin Cabañas Francisco Taboada Albert Pérez-Montaña Felipe De Arriba 16 1 1 17 ● ● ● ● Noemi Puig Gonzalo Carreño-Tarragona Santiago Barrio Jose Enrique de la Puerta 13 18 19 1 16 ● ● ● ● ● Angel Ramirez-Payer Isabel Krsnik Juan Jose Bargay Juan Jose Lahuerta Maria-Victoria Mateos 2 2 1 ● ● Jesus F. San-Miguel Bruno Paiva Joaquin Martinez-Lopez Received: 19 November 2019 / Revised: 4 March 2020 / Accepted: 9 March 2020 / Published online: 19 March 2020 © The Author(s) 2020. This article is published with open access To the Editor: landscape of AL amyloidosis is required since, for example, potential differences between the genomic profiles of AL Sequence-based analysis has come to play an integral role amyloidosis and multiple myeloma (MM) could help in many hematological malignancies [1], but disorders such identifying patients with monoclonal gammopathies at as systemic light-chain (AL) amyloidosis remain poorly greater risk of developing AL amyloidosis and monitor characterized due to its low incidence and small tumor size presymptomatic organ damage [4, 5]. [2, 3]. Thus, greater knowledge about the immunogenetic To gain further insight into the immunogenetic landscape of AL amyloidosis, we performed whole-exome sequencing (WES) on highly purified bone marrow clonal plasma cells (PCs) isolated by fluorescence activation cell sorting These authors contributed equally: Isabel Cuenca, Daniel Alameda (FACS) based on patient-specific aberrant phenotypes. A These authors jointly supervised this work: Bruno Paiva, Joaquin total of 27 patients with confirmed new diagnosis of AL Martinez-Lopez amyloidosis based on the presence of amyloid-related sys- Supplementary information The online version of this article (https:// temic syndrome, positive amyloid tissue staining with doi.org/10.1038/s41375-020-0800-6) contains supplementary Congo red, restricted light-chain deposition by material, which is available to authorized users. * Joaquin Martinez-Lopez Hospital Clinico Universitario Lozano Blesa, Zaragoza, Spain jmarti01@med.ucm.es Hospital German Trias i Pujol, Badalona, Spain 1 11 Hospital 12 de Octubre, Madrid, CNIO, Universidad Complutese, Hospital Costa del Sol, Marbella, Spain Madrid, Spain Hospital Clínico Universitario Virgen de la Arrixaca, Clinica Universidad de Navarra, Centro de Investigacion Medica Murcia, Spain Aplicada (CIMA), IDISNA, CIBERONC Pamplona, Hospital Universitario Central de Asturias (HUCA), Pamplona, Spain Oviedo, Spain Clinical and Traslational Lung Cancer Research Unit, i+12 Hospital Son Espases, Palma, Spain Research Institute and Biomedical Research Networking Center in Hospital Universitario Morales Meseguer. IMIB-Arrixaca, Oncology (CIBERONC), Madrid, Spain Murcia, Spain Universidad de Cantabria, Hospital Universitario Marqués de Hospital Universitario de Salamanca, Instituto de Investigacion Valdecilla, Santander, Spain Biomedica de Salamanca (IBSAL), Centro de Investigación del Hospital de Cabueñes, Gijon, Spain Cancer (IBMCC-USAL, CSIC), Salamanca, Spain Hospital Clínico Universitario de Valladolid, Valladolid, Spain Hospital de Galdakao, Vizcaya, Spain Hospital Doctor Peset, Valencia, Spain Hospital Puerta de Hierro, Madrid, Spain Hospital Universitari Vall d’Hebron, Barcelona, Spain Hospital Universitario Son Llàtzer, Palma, Spain 1234567890();,: 1234567890();,: 246 I. Cuenca et al. immunohistochemistry or mass spectometry, and evidence of a series of 62 newly diagnosed MM patients. Detailed of PC clonality were investigated. Patients’ demographics methodology is available in the Supplementary methods. and clinical characteristics are described in Supplementary We identified a total of 718 exonic, non-immunoglobu- Table 1. PCs were collected and processed in triplicates lin, nonsynonymous mutations with a variant allelic fraction followed by whole genome amplification of samples with (VAF) >5% (683 SNV and 35 indel). Total number and genomic DNA amounts <50 ng (Supplementary Table 2). type of mutations are described in Supplementary Fig. 1. Afterwards, library construction, exome enrichment, and Interestingly, mutational burden was significantly correlated sequencing were performed individually. An overall aver- with patients’ age (R = 0.51, p < 0.001) (Supplementary age depth of 63× and mean on-target coverage of 84% were Fig. 2) though not tumor burden (data not shown). Only 37 obtained. Data were deposited in the Sequence Read out of 662 (5.5%) mutated genes were altered more than Archive of the NCBI (http://www.ncbi.nlm.nih.gov/sra) once (Fig. 1). That notwithstanding, 23 of the 27 cases under the PRJNA596656 access number. To increase spe- (85%) presented with at least one mutation in one of the 37 cificity, only single-nucleotide variants (SNVs) and indels genes (range, 1–11); being FAT4, IGLL5, MUC16, and detected by both Strelka [6] and Varscan2 [7] variant callers SSH2 the most frequently mutated genes (≥3 patients). With were selected. Moreover, only somatic mutations present in a median of 18 mutations per sample (range, 8–92), patients two of three libraries per patient were considered positive. with AL amyloidosis are closer to monoclonal gammopathy Germline variants were excluded through WES of matched of undetermined significance (MGUS) (median of 19) [10] peripheral blood cells. The mutational profile of patients rather than MM (median of 38 in the CoMMpass IA13c with AL amyloidosis was compared with that of patients dataset, p < 0.0001; Fig. 2a) in terms of mutational load. By with MM enrolled in CoMMpass (n = 930; IA13c dataset). contrast, the presence of CNA was more frequent in The CNVKit [8] was used to determine copy number AL amyloidosis (19/21, 90.5%; Supplementary Fig. 3) than abnormalities (CNA) from WES data (in 21 of the 27 AL MGUS (60.6% in Mikulasova et al.) [10] and similar to patients). Deep sequencing of B-cell receptor immunoglo- MM patients (virtually 100%) [11]. Overall, these results bulin (BcR Ig) gene rearrangements was performed in all underpin recent observations based on the immunopheno- patients as previously described [9], and compared with that typic characterization of clonal PCs [5], and locate AL Nonsense SNV Missense SNV Frame Shift Del In Frame Del Multi Hit 0 0.5 1 1.5 2 2.5 3 11% FAT4 11% IGLL5 11% MUC16 11% SSH2 7% PRK AG3 7% CCIN 7% CFAP74 7% CKAP4 7% CLIC1 7% COL4A5 7% COL6A3 7% CSMD2 DNAH17 7% 7% DNAH2 7% FAM135B 7% FAM170A 7% GABBR1 7% HERC1 7% LAMA1 7% LAMA3 7% MAGEL2 7% MEOX2 7% MORC2 7% MYH13 7% NAV1 7% OPCML 7% PCDH15 7% PCF11 7% PDILT 7% PROB1 7% RIMS2 7% SAFB 7% SMAD1 7% SNAP91 7% SP140 7% VWA7 7% ZFHX3 Patients (n=27) Fig. 1 Genes recurrently mutated in AL. Distribution per patient of the most frequently mutated genes (n = 37). Boxes were colored according to the type of mutation. Top barplots define the total number of mutations per patient. Recurrence (%) 27 Immunogenetic characterization of clonal plasma cells in systemic light-chain amyloidosis 247 AB MM AL 2000 p<0.0001 MM AL IGHV3-30 IGHV3-48 SSH2 FAT4 IGLL5 8 8 MUC16 6 6 TRAF,BRAF NRAS 02468 10 12 14 0 5 10 15 20 25 30 AL IGHV genes recurrence (%) MM recurrent genes (%) Fig. 2 The mutational landscape in AL and MM. a Number of recurrence in AL and MM. d Predominant Ig heavy chain gene rear- mutations in patients with newly diagnosed AL and MM. b Shared and rangements used in AL and MM. private mutations between AL and MM. c Driver genes based in its amyloidosis in the crossroad between MGUS and MM also [13] identified recurrent mutations in KRAS. Furthermore, in genetic grounds. Of note, the only alterations associated IGLL5 emerged as one of the most commonly mutated with inferior progression-free survival were gains in chro- genes in ours and the latter series, but not in that analyzed mosomes 9 and 19 (Supplementary Fig. 4a, b), whereas del by Boyle et al. We have found no association between (13q) was associated with higher NT-proBNP levels (Sup- mutated genes and patients’ outcome, whereas Huang et al. plementary Fig. 5). Furthermore, patients with +1q also identified three mutated genes with prognostic value. displayed greater risk of cardiac involvement (Supplemen- Altogether, further studies in larger series of patients are tary Table 3). warranted to shed more light into the mutational landscape Interestingly, various MM-defined driver mutations [12] and potential clinical correlations in AL amyloidosis. were undetected in patients with AL amyloidosis (e.g., In the absence of a unifying genetic event defining AL NRAS, BRAF, and TRAF) or observed only once (e.g., DIS3 amyloidosis, BcR Ig gene rearrangements emerge as an and DUSP2), most of them being subclonal with a median alternative to understand the propensity for the deposition of VAF of 23% (Supplementary Fig. 6) that was unrelated to monoclonal Ig light-chains in the form of β-sheet fibrils, as the lower tumor burden in AL amyloidosis due to the well as organ tropism. Previous studies based on PCR FACSorting strategy used to isolate clonal PCs, as descri- amplification of individual (typically Lambda) light-chain bed above. Simultaneous analysis of ours and the CoMM- variable gene (VL) families have shown a potential bias in pass datasets unveiled that out of 14,135 mutated genes, germline donor use in patients with AL amyloidosis [14] only 608 (4.4%) were shared between AL amyloidosis and and that VL gene usage may influence clinical presentation, MM (Fig. 2b), with considerable differences in their organ deposition, and outcome [15, 16]. Hence, we per- recurrence (Fig. 2c). Of note, none of the 65 genes exclu- formed next-generation sequencing (NGS) of Ig heavy sively mutated in AL amyloidosis were recurrent. Further- chain gene (IGH) and Kappa light-chain (IGK) rearrange- more, only four genes (XKR5, PRSS45, PKD1L2, and ments both in patients with AL amyloidosis (n = 27) and SRRM5) overlapped with the 105 described by Boyle et al. MM (n = 63) to provide complementary information on [3] as AL restricted. While the results from these two stu- IGH and IGK repertoires, clonal variability, and extent of dies suggest that MM recurrent mutations are unfrequently somatic hypermutations. A total of 39 IGHV-D-J and IGK- detected in patients with AL amyloidosis, recent data based V-J clonotypes were identified and 5 of the 27 (19%) on WES and targeted sequencing reported by Huang et al. patients with AL amyloidosis displayed more than two AL recurrent genes (%) Mutations MM IGHV genes recurrence (%) 248 I. Cuenca et al. clonal rearrangements. This extent of clonal heterogeneity Author contributions JFSM, BP, JML, and JJL conceived the idea and together with IC and BSV designed the study. IC, BSV, DGS, DA, differs (p = 0.024) from that found in our MM series (3.9%). BP, EO, and SB analyzed and interpreted data. IC and GCT performed Using a cut-off of 98% to define homology, 9 of statistical analysis. ML acquired the clinical data. DAlignani per- 39 sequences (23%) in AL amyloidosis were considered as formed cell sorting. RL, FP, EMO, MEG, AGdC, JdlR, MG, LP, AO, mutated, which is slightly less when compared with that MC, VC, FT, AP, FdA, NP, MVM, JJL, and JFSM provided study materials and patients. IC, BSV, DA, JML, and BP wrote the manu- found in MM (36%). The CDR3 length was not significantly script. All authors reviewed and approved the manuscript. different between AL amyloidosis and MM (median of 54 versus 51 amino acids). The most frequent IGH gene Compliance with ethical standards involved in AL amyloidosis was IGHV3-48 (recurrence of 10.3%) and 100% of patients who have this rearrangement Conflict of interest The authors declare that they have no conflict of had kidney involvement (p = 0.025) (Supplementary interest. Table 4), whereas IGHV3-30 was the most recurrent (12%) in MM (Fig. 2d). Thus, expression of IGHV3-48 adds up to Publisher’s note Springer Nature remains neutral with regard to IGLV6-57 as germline Ig genes associated with dominant jurisdictional claims in published maps and institutional affiliations. kidney deposition [14, 15]. Of note, we found no significant differences regarding IGH and IGK repertoires, clonal Open Access This article is licensed under a Creative Commons variability, and extent of somatic hypermutations between Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as patients with light-chain only versus heavy- and light-chain long as you give appropriate credit to the original author(s) and the M-component (data not shown). source, provide a link to the Creative Commons license, and indicate if This study confirms previous observations that AL changes were made. The images or other third party material in this amyloidosis cannot be defined by a singular or a set of well- article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not defined genetic events. In fact, based on combined results included in the article’s Creative Commons license and your intended from WES of 99 patients (27 in this study, 24 in Boyle et al. use is not permitted by statutory regulation or exceeds the permitted [3], and 48 in Huang et al. [13]), 63 genes were found to be use, you will need to obtain permission directly from the copyright recurrently mutated. By contrast, our study further supports holder. 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LeukemiaSpringer Journals

Published: Mar 19, 2020

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