Cytogenetic profiles in multiple myeloma and monoclonal gammopathy of undetermined significance: a study in highly purified aberrant plasma cells

This is an open-access paper.Cytogenetic studies in clonal plasma cell disorders have mainly been done in whole bone marrow or CD138+ microbead-enriched plasma cells and suggest that recurrent immunoglobulin heavy chain translocations - e.g. t(4;14) - are primary oncogenetic events. The aim of this study was to determine cytogenetic patterns of highly purified aberrant plasma cells (median purity ≥98%) in different clonal plasma cell disorders. We analyzed aberrant plasma cells from 208 patients with multiple myeloma (n=148) and monoclonal gammopathy of undetermined significance (n=60) for the presence of del(13q14), del(17p13) and t(14q32) using multicolor interphase fluorescence in situ hybridization. Additionally, immunoglobulin heavy chain gene arrangements were analyzed and complementarity determining region 3 was sequenced in a subset of patients and combined multicolor interphase fluorescence in situ hybridization/immunofluorescent protein staining analyses were performed in selected cases to confirm clonality and cytogenetic findings. At diagnosis, 96% of cases with multiple myeloma versus 77% of monoclonal gammopathy of undetermined significance cases showed at least one cytogenetic alteration and/or hyperdiploidy. The cytogenetic heterogeneity of individual cases reflected coexistence of cytogenetically-defined aberrant plasma cell clones, and led to the assumption that karyotypic alterations were acquired stepwise. Cases of multiple myeloma and monoclonal gammopathy of undetermined significance frequently showed different but related cytogenetic profiles when other cytogenetic alterations such as deletions/gains of the immunoglobulin heavy chain or the fibroblast growth factor receptor 3 were additionally considered. Interestingly, in 24% of multiple myeloma versus 62% of monoclonal gammopathy of undetermined significance patients with an immunoglobulin heavy chain translocation, aberrant plasma cells with and without t(14q32) coexisted in the same patient. Our data suggest that recurrent immunoglobulin heavy chain translocations might be absent in the primordial plasma cell clone in a significant proportion of patients with clonal plasma cell disorders carrying these cytogenetic alterations.This work was partially supported by grants from the Fundacion Memoria de Don Samuel Solorzano Barruso, Salamanca, Spain (FS/4-2010). The authors would also like to thank the Dr. Werner Jackstädt Foundation (Wuppertal, Germany) for grant supporting the work of Martin Schmidt-Hieber. The authors would like to thank the Cooperative Research Thematic Network (RTICs; RTICC RD06/0020/0035, RD06/0020/0006 and G03/136), MM Jevitt, SL firm, Instituto de Salud Carlos III/Subdirección General de Investigación Sanitaria (FIS: PI060339; 02/0905; 01/0089/01-02; PS09/01897) and Gerencia Regional de Salud de Castilla y León; Ayuda de Excelencia de Castilla y León, Consejeria de Educación (EDU/894/2009, GR37), and Consejería de Sanidad (557/A/10), Junta de Castilla y León, Valladolid, Spain for supporting this study. JMS is supported by a grant (CP05/00321) from the ISCIII, Ministerio de Ciencia e Innovacion, Madrid, Spain.Peer Reviewe

Circulating clonotypic B cells in multiple myeloma and monoclonal gammopathy of undetermined significance

This is an open-access paper.The B-cell compartment in which multiple myeloma stem cells reside remains unclear. We investigated the potential presence of mature, surface-membrane immunoglobulin-positive B lymphocytes clonally related to the tumor bone marrow plasma cells among different subsets of peripheral blood B cells from ten patients (7 with multiple myeloma and 3 with monoclonal gammopathies of undetermined significance). The presence of clonotypic immunoglobulin heavy chain gene rearrangements was determined in multiple highly-purified fractions of peripheral blood B-lymphocytes including surface-membrane IgM+ CD27- naïve B-lymphocytes, plus surface-membrane IgG+, IgA+ and IgM+ memory CD27+ B cells, and normal circulating plasma cells, in addition to (mono)clonal plasma cells, by a highly-specific and sensitive allele-specific oligonucleotide polymerase chain reaction directed to the CDR3 sequence of the rearranged IGH gene of tumor plasma cells from individual patients. Our results showed systematic absence of clonotypic rearrangements in all the different B-cell subsets analyzed, including M-compo-nent isotype-matched memory B-lymphocytes, at frequencies <0.03 cells/mL (range: 0.0003-0.08 cells/mL); the only exception were the myeloma plasma cells detected and purified from the peripheral blood of four of the seven myeloma patients. These results indicate that circulating B cells from patients with multiple myeloma and monoclonal gammopathies of undetermined significance are usually devoid of clonotypic B cells while the presence of immunophenotypically aberrant myeloma plasma cells in peripheral blood of myeloma patients is a relatively frequent finding.This work was supported by grants from European Union FP6 STREP MSCNet (N. E06005FF), Cooperative Research Thematic Network on Cancer (RTICs; RTICC RD06/0020/0035-FEDER, RD06/0020/0006, RD12/0036/0048, RD12/0036/0069 and G03/136), Instituto de Salud Carlos III/Subdirección General de Investigación Sanitaria Ministerio de Sanidad y Consumo (FIS: PI060339; 02/0905; 01/0089/01-02;PS09/01897, and PI06/0824-FEDER), Asociacion Española Contra el Cancer AECC (GCB120981SAN) and Gerencia Regional de Salud de Castilla y León; Ayuda de Excelencia de Castilla y León, Consejería de Educación (EDU/894/2009, GR37) Junta de Castilla y León, Valladolid, Spain. LST received a CAPES/Ministério da Educação scholarship from the Brazilian Government.Peer Reviewe

The cellular origin and malignant transformation of Waldenström macroglobulinemia

Although information about the molecular pathogenesis of Waldenström macroglobulinemia (WM) has significantly advanced, the precise cell of origin and the mechanisms behind WM transformation from immunoglobulin-M (IgM) monoclonal gammopathy of undetermined significance (MGUS) remain undetermined. Here, we undertook an integrative phenotypic, molecular, and genomic approach to study clonal B cells from newly diagnosed patients with IgM MGUS (n = 22), smoldering (n = 16), and symptomatic WM (n = 11). Through principal component analysis of multidimensional flow cytometry data, we demonstrated highly overlapping phenotypic profiles for clonal B cells from IgM MGUS, smoldering, and symptomatic WM patients. Similarly, virtually no genes were significantly deregulated between fluorescence-activated cell sorter-sorted clonal B cells from the 3 disease groups. Interestingly, the transcriptome of the Waldenström B-cell clone was highly different than that of normal CD25-CD22+ B cells, whereas significantly less genes were differentially expressed and specific WM pathways normalized once the transcriptome of the Waldenström B-cell clone was compared with its normal phenotypic (CD25+CD22+low) B-cell counterpart. The frequency of specific copy number abnormalities [+4, del(6q23.3-6q25.3), +12, and +18q11-18q23] progressively increased from IgM MGUS and smoldering WM vs symptomatic WM (18% vs 20% and 73%, respectively; P = .008), suggesting a multistep transformation of clonal B cells that, albeit benign (ie, IgM MGUS and smoldering WM), already harbor the phenotypic and molecular signatures of the malignant Waldenström clone.This study was supported by Cooperative Research Thematic Network grants RD12/0036/0058 and RD12/0036/0048 of the Red de Cancer (Cancer Network of Excellence), Consejería de Sanidad, Junta de Castilla y Leon, Valladolid, Spain (557/A/10).Peer Reviewe

Panorámica actual de las especies no indígenas marinas en el mar Cantábrico y Atlántico adyacente (NO-N de la península Ibérica): primera aproximación a la Directiva Marco de Estrategias Marinas en la demarcación noroeste

Simposio Ibérico de Estudios de Biología Marina (18º. 2014. Gijón (España))La introducción de especies invasoras no indígenas es un problema cada vez más común a escala mundial, siendo considerado una de mayores amenazas para la conservación de la biodiversidad de todo el planeta. Estas invasiones biológicas causan graves problemas ecológicos y en muchos casos ocasionan también importantes pérdidas a la economía local. La gravedad y el alcance de los impactos generados por las especies invasoras es tal, que en los últimos años ha trascendido del ámbito científico al político-social, siendo considerado como uno de los descriptores de la calidad ambiental en la Directiva Marco de la Estrategia Marina (MSFD 2008/56/EC). A pesar de la envergadura y repercusión de este problema, el estudio de las especies invasoras en aguas de la península Ibérica no ha sido abordado hasta fechas muy recientes, estando en su mayoría centrado en el ámbito continental de los ecosistemas dulceacuícolas y en el ámbito marino de la cuenca mediterránea de la península. Para las costas noratlánticas españolas(las cuales constituyen la demarcación noroeste de la Directiva Marco de la Estrategia Marina) los datos existentes son bastante escasos y sobre todo dispersos, siendo también en muchos casos de afiliación taxonómica dudosa. En este trabajo se presenta la primera revisión actualizada de las especies marinas reportadas como no indígenas (exóticas, no autóctonas, alóctonas o “alien”) ocriptogénicas para el mar Cantábrico y el Atlántico adyacente (costas de Galicia) hasta la actualidad; incluyendo también nuevas adiciones procedentes de los muestreos realizadosen las costas cantábricas y atlántico-gallegas entre los años 2010 y 2014. El área de estudio fue dividida en cuatro zonas (A-D) de Oeste a Este. En esta revisión no se reconocen como alóctonosy/o criptogénicosalgunos taxones previamente reportados como tales, ya que según nuestro criterio se tratan de especies nativas y/o especies que están extendiendo sus rangos de distribución de forma natural, en muchos casos como consecuencia de los fenómenos de calentamiento global. Se listan un total de 287 taxones considerados como no indígenas y/o criptogénicos para el área de estudio, constituyendo estos últimos aproximadamente un tercio del total (92 taxones). Para las taxones alóctonos(195), se ha seguido la clasificación de Zenetoset al. basada en el grado de invasión de las mismas. Las especies consideradas como “casuales” representaron aproximadamente una quinta parte del total (17%), mientras que las especies “establecidas” constituyeron algo menos de la mitad (41%). Finalmente, alrededor de un 6% de los taxones recogidos (17) se categorizaron como “invasores” para las aguas objeto de estudio. En función de las zonas geográficas en las que fue dividida el área de estudio, la gran mayoría de las especies (78%) fueron detectadas para una sola zona; un 21% lo fueron para al menos dos zonas; un 11% para tres y sólo un 6% de las especies fueron detectadas en las cuatro zonas (formando parte de éstas, 10 de las especies consideradas “invasoras”). Taxonómicamente, los filos con un mayor número de especies introducidas son los moluscos y los anélidos, agrupando, cada uno de ellos, un 21% del total de especies; seguidos del grupo de las algas rojas (16%) y de los artrópodos (14%). Otros filos que representan porcentajes mucho menores son los cnidarios (7%), los briozoos (5%), las algas pardas (4%), las ascidias (4%), los poríferos (3%), las algasverdes (2%), los gusanos nemertinos (<1%) y los equinodermos (<1%). El filo con mayor número de especies consideradas como “invasoras” es el grupo las algas rojas con 7 especies

Minimal residual disease evaluation by flow cytometry is a complementary tool to cytogenetics for treatment decisions in acute myeloid leukaemia

For PETHEMA Programa para el Estudio de la Terapéutica en Hemopatías Malignas Cooperative Study Group.The clinical utility of minimal residual disease (MRD) analysis in acute myeloid leukaemia (AML) is not yet defined. We analysed the prognostic impact of MRD level at complete remision after induction therapy using multiparameter flow cytometry in 306 non-APL AML patients. First, we validated the prognostic value of MRD-thresholds we have previously proposed (≥0.1%; ≥0.01-0.1%; and <0.01), with a 5-year RFS of 38%, 50% and 71%, respectively (p = 0.002). Cytogenetics is the most relevant prognosis factor in AML, however intermediate risk cytogenetics represent a grey zone that require other biomarkers for risk stratification, and we show that MRD evaluation discriminate three prognostic subgroups (p = 0.03). Also, MRD assessments yielded relevant information on favourable and adverse cytogenetics, since patients with favourable cytogenetics and high MRD levels have poor prognosis and patients with adverse cytogenetics but undetectable MRD overcomes the adverse prognosis. Interestingly, in patients with intermediate or high MRD levels, intensification with transplant improved the outcome as compared with chemotherapy, while the type of intensification therapy did not influenced the outcome of patients with low MRD levels. Multivariate analysis revealed age, MRD and cytogenetics as independent variables. Moreover, a scoring system, easy in clinical practice, was generated based on MRD level and cytogenetics.This work was supported in part by Spanish grants from Fondo de Investigación Sanitaria-ISCIII (FIS 00/0023-03, PI12/02321), DGCYT (SAF 94- 0308, SAF2001-1687), Conserjería de Educación de Castilla y León (HUS416A12), and Red Temática de Investigación Cooperativa en Cáncer (RTICC-ISCIII) (RD12/0036/0069).Peer Reviewe

Impact of measurable residual disease by decentralized flow cytometry: a PETHEMA real-world study in 1076 patients with acute myeloid leukemia

The role of decentralized assessment of measurable residual disease (MRD) for risk stratification in acute myeloid leukemia (AML) remains largely unknown, and so it does which methodological aspects are critical to empower the evaluation of MRD with prognostic significance, particularly if using multiparameter flow cytometry (MFC). We analyzed 1076 AML patients in first remission after induction chemotherapy, in whom MRD was evaluated by MFC in local laboratories of 60 Hospitals participating in the PETHEMA registry. We also conducted a survey on technical aspects of MRD testing to determine the impact of methodological heterogeneity in the prognostic value of MFC. Our results confirmed the recommended cutoff of 0.1% to discriminate patients with significantly different cumulative-incidence of relapse (-CIR- HR:0.71, P < 0.001) and overall survival (HR: 0.73, P = 0.001), but uncovered the limited prognostic value of MFC based MRD in multivariate and recursive partitioning models including other clinical, genetic and treatment related factors. Virtually all aspects related with methodological, interpretation, and reporting of MFC based MRD testing impacted in its ability to discriminate patients with different CIR. Thus, this study demonstrated that “real-world” assessment of MRD using MFC is prognostic in patients at first remission, and urges greater standardization for improved risk-stratification toward clinical decisions in AML.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, CB16/12/00233, CB16/12/00284 and CB16/12/00400), Instituto de Salud Carlos III/Subdirección General de Investigación Sanitaria (FIS No. PI16/01661, PI16/00517 and PI18/01946), Gerencia Regional de Salud de CyL (GRS 1346/A/16) and the Plan de Investigación de la Universidad de Navarra (PIUNA 2014-18). This study was supported internationally by the Cancer Research UK, FCAECC and AIRC under the Accelerator Award Program EDITOR

Pembrolizumab as consolidation strategy in patients with multiple myeloma: Results of the GEM-Pembresid clinical trial

PD1 expression in CD4+ and CD8+ T cells is increased after treatment in multiple myeloma patients with persistent disease. The GEM-Pembresid trial analyzed the efficacy and safety of pembrolizumab as consolidation in patients achieving at least very good partial response but with persistent measurable disease after first- or second-line treatment. Moreover, the characteristics of the immune system were investigated to identify potential biomarkers of response to pembrolizumab. One out of the 17 evaluable patients showed a decrease in the amount of M-protein, although a potential late effect of high-dose melphalan could not be ruled out. Fourteen adverse events were considered related to pembrolizumab, two of which (G3 diarrhea and G2 pneumonitis) prompted treatment discontinuation and all resolving without sequelae. Interestingly, pembrolizumab induced a decrease in the percentage of NK cells at cycle 3, due to the reduction of the circulating and adaptive subsets (0.615 vs. 0.43, p = 0.007; 1.12 vs. 0.86, p = 0.02). In the early progressors, a significantly lower expression of PD1 in CD8+ effector memory T cells (MFI 1327 vs. 926, p = 0.03) was observed. In conclusion, pembrolizumab used as consolidation monotherapy shows an acceptable toxicity profile but did not improve responses in this MM patient population. The trial was registered at clinicaltrials.gov with identifier NCT02636010 and with EUDRACT number 2015-003359-23

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

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

Integrated flow cytometry and sequencing to reconstruct evolutionary patterns from dysplasia to acute myeloid leukemia

Clonal evolution in acute myeloid leukemia (AML) originates long before diagnosis and is a dynamic process that may affect survival. However, it remains uninvestigated during routine diagnostic workups. We hypothesized that the mutational status of bone marrow dysplastic cells and leukemic blasts, analyzed at the onset of AML using integrated multidimensional flow cytometry (MFC) immunophenotyping and fluorescence-activated cell sorting (FACS) with next-generation sequencing (NGS), could reconstruct leukemogenesis. Dysplastic cells were detected by MFC in 285 of 348 (82%) newly diagnosed patients with AML. Presence of dysplasia according to MFC and World Health Organization criteria had no prognostic value in older adults. NGS of dysplastic cells and blasts isolated at diagnosis identified 3 evolutionary patterns: stable (n = 12 of 21), branching (n = 4 of 21), and clonal evolution (n = 5 of 21). In patients achieving complete response (CR), integrated MFC and FACS with NGS showed persistent measurable residual disease (MRD) in phenotypically normal cell types, as well as the acquisition of genetic traits associated with treatment resistance. Furthermore, whole-exome sequencing of dysplastic and leukemic cells at diagnosis and of MRD uncovered different clonal involvement in dysplastic myelo-erythropoiesis, leukemic transformation, and chemoresistance. Altogether, we showed that it is possible to reconstruct leukemogenesis in ∼80% of patients with newly diagnosed AML, using techniques other than single-cell multiomics.This work was supported by grants from the Área de Oncología del Instituto de Salud Carlos III, Centro de Investigación Biomédica en Red (CIBER-ONC) (CB16/12/00369, CB16/12/00233, CB16/12/00489, and CB16/12/00284), Instituto de Salud Carlos III/Subdirección General de Investigación Sanitaria (FIS numbers PI16/01661, PI16/00517, and PI19/01518), and the Plan de Investigación de la Universidad de Navarra (PIUNA 2014-18). This work was supported internationally by the Cancer Research UK, FCAECC, and AIRC under the Accelerator Award Program (EDITOR)