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Trabajo presentado al "13th Congress of the European Hematology Association" celebrado en Copenhague en Junio del 2008.-- et al., Limited knowledge exists about the impact of specific genetic abnormalities on the proliferation of neoplastic B cells from chronic lymphoproliferative disorders (B- CLPDs). Here we analyze the impact of cytogenetic abnormalities on the proliferation of neoplastic B cells in 432 B-CLPD patients, grouped according to diagnosis and site of sampling, versus their normal counterparts. Overall, proliferation of neoplastic B cells highly varied among the different B-CLPD subtypes, the greatest numbers of proliferating cells being identified in diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL). Compared with normal B cells, neoplastic B-CLPD cells showed significantly increased S + G2/M-phase values in mantle cell lymphoma (MCL), B-chronic lymphocytic leukemia (B-CLL), BL, and some DLBCL cases. Conversely, decreased proliferation was observed in follicular lymphoma, lymphoplasmacytic lymphoma/ Waldenstrom macroglobulinemia (LPL/ WM), and some DLBCL patients; hairy cell leukemia, splenic marginal zone, and MALT-lymphoma patients showed S + G 2/ M phase values similar to normal mature B lymphocytes from LN. Interestingly, in B-CLL and MCL significantly higher percentages of S + G 2/M cells were detected in BM versus PB and in LN versus BM and PB samples, respectively. In turn, presence of 14q32.3 gene rearrangements and DNA aneuploidy, was associated with a higher percentage of S + G2/M-phase cells among LPL/WM and B-CLL cases, respectively. © 2008 by The American Society of Hematology., This work has been partially supported by the following grants: FIS 06/0824, from the Ministerio de Sanidad y Consumo (Madrid, Spain) and RETICC RD06/0020/0035 from the Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo (Madrid, Spain). S.Q. is supported by a grant from COLCIENCIAS (Bogotá, Colombia), J.M.S. is supported by a grant from the Ministerio de Sanidad y Consumo (Madrid, Spain; CP05/ 00321), A.R. is supported by a grant from the Ministerio de Ciencia y Tecnología (Madrid, Spain) y Fondo Social Europeo, and C.F. is supported by a grant from the Instituto de Salud Carlos III (Madrid, Spain; CM05/00250).

The identification of subclones in multiple myeloma (MM) could have clinical implications since the complete eradication of all clones is required to prolong patients’ survival. Hence, there is an unmet need to fully characterize such subclones. In the present study, we started by investigating using multidimensional (23-color) flow cytometry (MFC) the presence of ≥2 distinct bone marrow PC subclones in 116 newly-diagnosed (MM) patients included in the Spanish GEM2010 trial: sequential VMP (9 cycles) followed by Rd (9 cycles) vs alternating VMP with Rd (18 cycles). By principal component analysis (PCA) using the Infinicyt software, the immunophenotypic expression profile (based on the simultaneous evaluation of 23 antigens in single-clonal-PCs) showed the presence of two or more subclones in 35/116 (30%) newly-diagnosed MM patients. The most powerful antigens for the identification of subclonal heterogeneity were CD56, CD27, CD81, CD20, CD33, β7, CD138, CD49e, CD54, HLADR, CD19, CD44 and CXCR4. Then, we investigated the genetic profile of distinct phenotypic subclones (sorted by FACS with purity ≥97%) through high-density Cytoscan750K (n=5) arrays and FISH (n=8). In 2/5 (40%) patients, specific copy number abnormalities (CNA; only those with minimum of 25 consecutive imbalanced markers per segment and minimum 100 Kb length were considered) were present in one clone [del(13q), del(14q), del(20p), +(20q), +(21q), or del(22q)] but absent in the other. Similar results were observed by FISH while investigating those probes used as part of the clinical workout. Noteworthy, in three cases del(17p13) was detected in only one subclone, whereas del(13q14) was restricted to one subclone in two patients. To assess the clonogenic potential of distinct phenotypic subclones detected in the whole malignant PC compartment, these were sorted by MFC and seeded in coculture with the human mesenchymal cell line hTERT (with Methocult® supplemented with lymphocyte conditioned medium (10%) plus 20 ng/ml IL-6 plus 20 ng/ml IGF-1). In 2 out of 7 patients (29%) we observed different clonogenic potential between two patient-specific subclones (patient 1, 208 vs 0 colonies; patient 2, 5 vs 1 colony). Finally, we aimed to investigate if distinct phenotypic subclones display different chemoresistance. To address this question, we compared the immunophenotypic expression profile of paired baseline vs MRD clonal PC in 9 patients enrolled in the Spanish GEM2010 trial in which ≥2 subclones were identified at diagnosis. Our results show a phenomenon of clonal restriction in 7/9 (78%) patients after chemotherapy, indicating that among the initial tumor bulk, only specific subclones are primary chemoresistant (i.e.: MRD) in patients otherwise achieving a serological response. As an example, in one patient showing at diagnosis four clonal PC subsets (CD33-/CD117-, CD33+/CD117-, CD33+/CD117+ and CD33+/CD117+), only one subclone (CD33-/CD117-) persisted after 9 cycles of alterning Rd-VMP. In conclusion, our results show that distinct phenotypic subclones translate into a different cytogenetic profile and clonogenic potential. Since the balance between different subclones may change over the course of treatment, so may change the extent of specific cytogenetic abnormalities and the patient risk. Thus, careful monitoring of specific subclones may have implications towards an adapted therapy to overcome chemoresistant cells. This work was supported by funding from the RTICC-Hematology Group (RD12/0036/0058), the Asociación Española Contra el Cáncer (AECC) (GCB120981SAN) and Multiple Myeloma Research Foundation (MMRF) 2012 Research Fellow Award (B Paiva) Disclosures: No relevant conflicts of interest to declare.

Abstract 3945 Background: Recurrent immunoglobulin heavy chain (IGH) translocations - t(14q32) - such as t(4;14) or t(11;14) are considered to be primary cytogenetic events which play an important role in the pathogenesis of clonal plasma cell (PC) disorders. However, previous cytogenetic studies in these diseases have mainly been done in whole bone marrow or CD138+ microbead-enriched PC; the latter technique has several pitfalls such as coexistence of CD138+ and CD138− aberrant PC or apoptotic loss of CD138. Design and Methods: Overall, highly-purified aberrant PC (purity ≥98%) from 217 patients with either multiple myeloma (MM; n=155) or monoclonal gammopathy of undetermined significance (MGUS; n=62) were analyzed for the presence of different cytogenetic alterations such as del(13q14), del(17p13) and t(14q32) by multicolor interphase fluorescence in situ hybridization (iFISH). Purity of sorted PC populations was further confirmed by iFISH studies of residual normal - i.e. polyclonal - PC fractions in a subgroup of 10 exemplarily cases with clonal PC disorder. Additionally, analysis of IGH gene arrangements and complementarity determining region 3 (CDR3) sequencing was carried out in a subset of patients (n=9) and simultaneous application of iFISH and immunofluorescent protein staining (FICTION) was performed in selected cases to confirm the clonal relationship between different fractions of tumor PC from individual patients and specific cytogenetic findings. Results: At diagnosis, 96% of all MM vs. 77% of MGUS cases (p Conclusions: Our findings using highly-purified aberrant PC suggest that also recurrent IGH translocations might evolve secondarily during disease evolution in a significant fraction of MGUS and MM cases, which raises the question about their precise role in the pathogenesis of clonal PC disorders. Our data further supports the existence of a high intratumoral cytogenetic heterogeneity in MM and MGUS, challenging individualized treatment approaches in these diseases. Disclosures: No relevant conflicts of interest to declare.