Tumor cells in light-chain amyloidosis and myeloma show distinct transcriptional rewiring of normal plasma cell development

Although light-chain amyloidosis (AL) and multiple myeloma (MM) are characterized by tumor plasma cell (PC) expansion in bone marrow (BM), their clinical presentation differs. Previous attempts to identify unique pathogenic mechanisms behind such differences were unsuccessful, and no studies have investigated the differentiation stage of tumor PCs in patients with AL and MM. We sought to define a transcriptional atlas of normal PC development in secondary lymphoid organs (SLOs), peripheral blood (PB), and BM for comparison with the transcriptional programs (TPs) of tumor PCs in AL, MM, and monoclonal gammopathy of undetermined significance (MGUS). Based on bulk and single-cell RNA sequencing, we observed 13 TPs during transition of normal PCs throughout SLOs, PB, and BM. We further noted the following: CD39 outperforms CD19 to discriminate newborn from long-lived BM-PCs; tumor PCs expressed the most advantageous TPs of normal PC differentiation; AL shares greater similarity to SLO-PCs whereas MM is transcriptionally closer to PB-PCs and newborn BM-PCs; patients with AL and MM enriched in immature TPs had inferior survival; and protein N-linked glycosylation–related TPs are upregulated in AL. Collectively, we provide a novel resource to understand normal PC development and the transcriptional reorganization of AL and other monoclonal gammopathies

Circulating tumor cells for comprehensive and multiregional non-invasive genetic characterization of multiple myeloma

Multiple myeloma (MM) patients undergo repetitive bone marrow (BM) aspirates for genetic characterization. Circulating tumor cells (CTCs) are detectable in peripheral blood (PB) of virtually all MM cases and are prognostic, but their applicability for noninvasive screening has been poorly investigated. Here, we used next-generation flow (NGF) cytometry to isolate matched CTCs and BM tumor cells from 53 patients and compared their genetic profile. In eight cases, tumor cells from extramedullary (EM) plasmacytomas were also sorted and whole-exome sequencing was performed in the three spatially distributed tumor samples. CTCs were detectable by NGF in the PB of all patients with MM. Based on the cancer cell fraction of clonal and subclonal mutations, we found that ~22% of CTCs egressed from a BM (or EM) site distant from the matched BM aspirate. Concordance between BM tumor cells and CTCs was high for chromosome arm-level copy number alterations (≥95%) though not for translocations (39%). All high-risk genetic abnormalities except one t(4;14) were detected in CTCs whenever present in BM tumor cells. Noteworthy, ≥82% mutations present in BM and EM clones were detectable in CTCs. Altogether, these results support CTCs for noninvasive risk-stratification of MM patients based on their numbers and genetic profile.This study was supported by the Centro de Investigación Biomédica en Red—Área de Oncología—del Instituto de Salud Carlos III (CIBERONC; CB16/12/00236, CB16/12/00369, CB16/12/00489, and CB16/12/00400); by Cancer Research UK [C355/A26819] and FC AECC and AIRC under the Accelerator Award Program; by the Instituto de Salud Carlos III, FCAECC and co-financed by FEDER (ERANET-TRANSCAN-2 iMMunocell AC17/00101); the Spanish Ministry of Science and Innovation and co-financed by FSE (Torres Quevedo fellowship, PTQ-16-08623); the Black Swan Research Initiative of the International Myeloma Foundation; European Research Council (ERC) under the European Commission’s H2020 Framework Programme (MYELOMANEXT, 680200); the Qatar National Research Fund (QNRF) Award No. 7-916-3-237; the AACR-Millennium Fellowship in Multiple Myeloma Research (15-40-38-PAIV); the Leukemia Research Foundation; and the Multiple Myeloma Research Foundation (MMRF) under the 2019 Research Fellowship Award

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

This study was supported by the Centro de Investigación Biomédica en Red—Área de Oncología—del Instituto de Salud Carlos III (CIBERONC; CB16/12/00369; and CB16/12/00489), Instituto de Salud Carlos III/Subdirección General de Investigación Sanitaria (FIS No. PI13/02196), Asociación Española Contra el Cáncer (GCB120981SAN and the Accelerator Award), CRIS against Cancer foundation grant 2014/0120, and the Black Swan Research Initiative of the International Myeloma Foundation.Peer reviewe

A mouse model of ATR-Seckel shows embryonic replicative stress and accelerated aging.

Although DNA damage is considered a driving force for aging, the nature of the damage that arises endogenously remains unclear. Replicative stress, a source of endogenous DNA damage, is prevented primarily by the ATR kinase. We have developed a mouse model of Seckel syndrome characterized by a severe deficiency in ATR. Seckel mice show high levels of replicative stress during embryogenesis, when proliferation is widespread, but this is reduced to marginal amounts in postnatal life. In spite of this decrease, adult Seckel mice show accelerated aging, which is further aggravated in the absence of p53. Together, these results support a model whereby replicative stress, particularly in utero, contributes to the onset of aging in postnatal life, and this is balanced by the replicative stress-limiting role of the checkpoint proteins ATR and p53.We thank M. Serrano and A. Ramiro for critical comments on the manuscript. We also thank S. P. Jackson for his help with the PIKK inhibitors and A. Garcia for cytometry. M. M. is supported by a Ramon y Cajal contract from the Spanish Ministry of Science (RYC-2003-002731) and from a grant from Fondo de Investigaciones Sanitarias (PI080220). Work in O. F.-C.' s laboratory is supported by grants from the Spanish Ministry of Science (RYC-2003-002731, CSD200700017 and SAF2008-01596), European Molecular Biology Organization Young Investigator Programme, European Research Council (ERC-210520) and Epigenome Network of Excellence.S

Exploiting oncogene-induced replicative stress for the selective killing of Myc-driven tumors.

Oncogene-induced replicative stress activates an Atr- and Chk1-dependent response, which has been proposed to be widespread in tumors. We explored whether the presence of replicative stress could be exploited for the selective elimination of cancer cells. To this end, we evaluated the impact of targeting the replicative stress-response on cancer development. In mice (Mus musculus), the reduced levels of Atr found on a mouse model of the Atr-Seckel syndrome completely prevented the development of Myc-induced lymphomas or pancreatic tumors, both of which showed abundant levels of replicative stress. Moreover, Chk1 inhibitors were highly effective in killing Myc-driven lymphomas. By contrast, pancreatic adenocarcinomas initiated by K-Ras(G12V) showed no detectable evidence of replicative stress and were nonresponsive to this therapy. Besides its impact on cancer, Myc overexpression aggravated the phenotypes of Atr-Seckel mice, revealing that oncogenes can modulate the severity of replicative stress-associated diseases.We thank M. Serrano for critical comments on the manuscript and F.X. Real for advice on the Ela-myc model. M.M. is supported a grant from Fondo de Investigaciones Sanitarias (PI080220). T.S. is supported by German Research Foundation (DFG) Research Fellowship (SCHL 1945/1-1). Work in O.F.-C.'s laboratory is supported by grants from the Spanish Ministry of Science (CSD2007-00017 and SAF2008-01596), Miguel Catalan Award from the Community of Madrid, European Molecular Biology Organization (EMBO) Young Investigator Programme and the European Research Council (ERC-210520).S

Flow cytometry for fast screening and automated risk assessment in systemic light-chain amyloidosis

Early diagnosis and risk stratification are key to improve outcomes in light-chain (AL) amyloidosis. Here we used multidimensional-flow-cytometry (MFC) to characterize bone marrow (BM) plasma cells (PCs) from a series of 166 patients including newly-diagnosed AL amyloidosis (N = 94), MGUS (N = 20) and multiple myeloma (MM, N = 52) vs. healthy adults (N = 30). MFC detected clonality in virtually all AL amyloidosis (99%) patients. Furthermore, we developed an automated risk-stratification system based on BMPCs features, with independent prognostic impact on progression-free and overall survival of AL amyloidosis patients (hazard ratio: ≥ 2.9;P ≤.03). Simultaneous assessment of the clonal PCs immunophenotypic protein expression profile and the BM cellular composition, mapped AL amyloidosis in the crossroad between MGUS and MM; however, lack of homogenously-positive CD56 expression, reduction of B-cell precursors and a predominantly-clonal PC compartment in the absence of an MM-like tumor PC expansion, emerged as hallmarks of AL amyloidosis (ROC-AUC = 0.74;P <.001), and might potentially be used as biomarkers for the identification of MGUS and MM patients, who are candidates for monitoring pre-symptomatic organ damage related to AL amyloidosis. Altogether, this study addressed the need for consensus on how to use flow cytometry in AL amyloidosis, and proposes a standardized MFC-based automated risk classification ready for implementation in clinical practice.This study was supported by the Centro de Investigación Biomédica en Red – Área de Oncología—del Instituto de Salud Carlos III (CIBERONC; CB16/12/00369, CB16/12/00400 and CB16/12/00489), Instituto de Salud Carlos III/Subdirección General de Investigación Sanitaria (FIS No. PI13/02196), Asociación Española Contra el Cáncer (GCB120981SAN and Accelerator Award), the Black Swan Research Initiative of the International Myeloma Foundation, and the European Research Council (ERC) 2015 Starting Grant (MYELOMANEXT)

Tumor cells in light-chain amyloidosis and myeloma show distinct transcriptional rewiring of normal plasma cell development

Although light-chain amyloidosis (AL) and multiple myeloma (MM) are characterized by tumor plasma cell (PC) expansion in bone marrow (BM), their clinical presentation differs. Previous attempts to identify unique pathogenic mechanisms behind such differences were unsuccessful, and no studies have investigated the differentiation stage of tumor PCs in patients with AL and MM. We sought to define a transcriptional atlas of normal PC development in secondary lymphoid organs (SLOs), peripheral blood (PB), and BM for comparison with the transcriptional programs (TPs) of tumor PCs in AL, MM, and monoclonal gammopathy of undetermined significance (MGUS). Based on bulk and single-cell RNA sequencing, we observed 13 TPs during transition of normal PCs throughout SLOs, PB, and BM. We further noted the following: CD39 outperforms CD19 to discriminate newborn from long-lived BM-PCs; tumor PCs expressed the most advantageous TPs of normal PC differentiation; AL shares greater similarity to SLO-PCs whereas MM is transcriptionally closer to PB-PCs and newborn BM-PCs; patients with AL and MM enriched in immature TPs had inferior survival; and protein N-linked glycosylation-related TPs are upregulated in AL. Collectively, we provide a novel resource to understand normal PC development and the transcriptional reorganization of AL and other monoclonal gammopathies

Large T cell clones expressing immune checkpoints increase during multiple myeloma evolution and predict treatment resistance

Abstract Tumor recognition by T cells is essential for antitumor immunity. A comprehensive characterization of T cell diversity may be key to understanding the success of immunomodulatory drugs and failure of PD-1 blockade in tumors such as multiple myeloma (MM). Here, we use single-cell RNA and T cell receptor sequencing to characterize bone marrow T cells from healthy adults (n = 4) and patients with precursor (n = 8) and full-blown MM (n = 10). Large T cell clones from patients with MM expressed multiple immune checkpoints, suggesting a potentially dysfunctional phenotype. Dual targeting of PD-1 + LAG3 or PD-1 + TIGIT partially restored their function in mice with MM. We identify phenotypic hallmarks of large intratumoral T cell clones, and demonstrate that the CD27− and CD27+ T cell ratio, measured by flow cytometry, may serve as a surrogate of clonal T cell expansions and an independent prognostic factor in 543 patients with MM treated with lenalidomide-based treatment combinations

Circulating tumor and immune cells for minimally invasive risk stratification of smoldering multiple myeloma

Early intervention in smoldering multiple myeloma (SMM) requires optimal risk stratification to avoid under and over-treatment. We hypothesized that replacing bone marrow (BM) plasma cells (PCs) for circulating tumor cells (CTCs), and adding immune biomarkers in peripheral blood (PB) for the identification of patients at risk of progression due to lost immune surveillance, could improve the International Myeloma Working Group 20/2/20 model