Search

Interstitial 14q32 deletions involving IGH gene are infrequent events in chronic lymphocytic leukemia (CLL), affecting less than 5% of patients. To date, little is known about their clinical impact and molecular underpinnings, and its mutational landscape is currently unknown. In this work, a total of 871 CLLs were tested for the IGH break-apart probe, and 54 (6.2%) had a 300 kb deletion of 3′IGH (del-3′IGH CLLs), which contributed to a shorter time to first treatment (TFT). The mutational analysis by next-generation sequencing of 317 untreated CLLs (54 del-3′IGH and 263 as the control group) showed high mutational frequencies of NOTCH1 (30%), ATM (20%), genes involved in the RAS signaling pathway (BRAF, KRAS, NRAS, and MAP2K1) (15%), and TRAF3 (13%) within del-3′IGH CLLs. Notably, the incidence of TRAF3 mutations was significantly higher in del-3′IGH CLLs than in the control group (p, Funding information: Universidad de Salamanca; Fundación Española de Hematología y Hemoterapia (FEHH); Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Grant/Award Number: CB16/12/00233; Red Temática de Investigación Cooperativa en Cáncer (RTICC); “Fundación Memoria Don Samuel Solórzano Barruso”: FS/33–2020, Grant/Award Number: RD12/0036/0069; “Gerencia Regional de Salud, SACYL”:, Grant/Award Numbers: GRS2385/A/21, GRS2140/A/20; Consejería de Educación, Junta de Castilla y León, Grant/Award Number: SA118P20; European Regional Development Fund and Instituto de Salud Carlos III, Grant/Award Numbers: CD19/00222, FI19/00191; Spanish Fondo de Investigaciones Sanitarias, Grant/Award Numbers: PI21/00983, PI18/01500

© The Author(s) 2021., BIRC3 is monoallelically deleted in up to 80% of chronic lymphocytic leukemia (CLL) cases harboring del(11q). In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which has been shown to be a marker for reduced survival in CLL. Nevertheless, the biological mechanisms by which these lesions could contribute to del(11q) CLL pathogenesis and progression are partially unexplored. We implemented the CRISPR/Cas9-editing system to generate isogenic CLL cell lines harboring del(11q) and/or BIRC3 mutations, modeling monoallelic and biallelic BIRC3 loss. Our results reveal that monoallelic BIRC3 deletion in del(11q) cells promotes non-canonical NF-κB signaling activation via RelB-p52 nuclear translocation, being these effects allelic dose-dependent and therefore further enhanced in del(11q) cells with biallelic BIRC3 loss. Moreover, we demonstrate ex vivo in primary cells that del(11q) cases including BIRC3 within their deleted region show evidence of non-canonical NF-κB activation which correlates with high BCL2 levels and enhanced sensitivity to venetoclax. Furthermore, our results show that BIRC3 mutations in del(11q) cells promote clonal advantage in vitro and accelerate leukemic progression in an in vivo xenograft model. Altogether, this work highlights the biological bases underlying disease progression of del(11q) CLL patients harboring BIRC3 deletion and mutation., This work was supported by grants from the Spanish Fondo de Investigaciones Sanitarias PI15/01471, PI18/01500, Instituto de Salud Carlos III (ISCIII), European Regional Development Fund (ERDF) “Una manera de hacer Europa”, “Consejería de Educación, Junta de Castilla y León” (SA271P18), “Proyectos de Investigación del SACYL”, Spain GRS 2062/A/19, GRS 1847/A/18, GRS1653/A17,“Fundación Memoria Don Samuel Solórzano Barruso” (FS/23-2018), by grants (RD12/0036/0069) from Red Temática de Investigación Cooperativa en Cáncer (RTICC), Universidad de Salamanca (Programa XIII), Centro de Investigación Biomédica en Red de Cáncer (CIBERONC CB16/12/00233) and SYNtherapy “Synthetic Lethality for Personalized Therapy-based Stratification In Acute Leukemia” (ERAPERMED2018-275); ISCIII (AC18/00093), co-funded by ERDF/ESF, “Investing in your future”. M.Q.Á. and A.E.R.V. are supported with a research grant by FEHH (“Fundación Española de Hematología y Hemoterapia”); M.H.S. holds a Sara Borrell postdoctoral contract (CD19/00222) from the Instituto de Salud Carlos III (ISCIII). C.P.C. was supported by an “Ayuda predoctoral en Oncología” (AECC) and is a recipient of a PFIS grant (FI19/00191) from Instituto de Salud Carlos III; PFIS grant and Sara Borrell postdoctoral contrat are co-founded by Fondo Social Europeo (FSE) “El Fondo Social Europeo invierte en tu futuro”; J.L.O. and R.B.S. are supported by a grant from the University of Salamanca (“Contrato postdoctoral programa II”).

Chronic lymphocytic leukemia (CLL) patients harboring 11q22.3 deletion, del(11q), are characterized by a rapid disease progression. One of the suggested genes to be involved in the pathogenesis of this deletion is BIRC3, a negative regulator of NF-κB, which is monoallelically deleted in ~80% of del(11q) CLL cases. In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which accounts for marked reduced survival in CLL. Nevertheless, the biological mechanisms by which monoallelic or biallelic BIRC3 lesions could contribute to del(11q) CLL pathogenesis, progression and therapy response are partially unexplored. We used the CRISPR/Cas9 system to model monoallelic and biallelic BIRC3 loss in vitro. First, we generated an isogenic HG3 CLL cell line harboring monoallelic del(11q) - HG3-del(11q) - by the introduction of 2 guide RNAs targeting 11q22.1 and 11q23.3 (~17 Mb). Loss-of-function BIRC3 mutations (MUT) were introduced in the remaining allele, generating 3 HG3-del(11q) BIRC3MUT clones. In addition, single BIRC3MUT were introduced in HG3 and MEC1 CLL-derived cells for experimental validation (n = 3 clones/cell line). We first questioned whether monoallelic and biallelic BIRC3 loss had an impact in the DNA-binding activity of NF-κB transcription factors. Interestingly, HG3-del(11q) had higher p52 and RelB (non-canonical NF-κB signaling) activity than HG3WT cells (P = 0.005; P = 0.007), being this activity further increased in HG3-del(11q) BIRC3MUT cells (P < 0.001; P < 0.001). In depth analysis of the non-canonical signaling components by immunoblot revealed that HG3-del(11q) and, to a greater extent, HG3-del(11q) BIRC3MUT cells presented NF-κB-inducing kinase (NIK) cytoplasmic stabilization, high p-IKKα levels and p52-RelB nuclear translocation. Besides, HG3-del(11q) BIRC3MUT cells showed increased levels of the anti-apoptotic proteins BCL2 and BCL-xL. We next assessed this pathway ex vivo in stroma and CpG-stimulated primary CLL cells with or without BIRC3 deletion (n = 22; 11 each group). Remarkably, stimulated BIRC3-deleted primary cells showed higher p52 and RelB activity than BIRC3WT cases (P = 0.01; P = 0.07), and the percentage of BIRC3-deleted cells correlated with p52 activity in del(11q) cases (P = 0.04). We further performed western blot analyses in a homogenous cohort of del(11q) cases including (n = 4) or not including (n = 3) BIRC3 within the deleted region. Interestingly, del(11q)/BIRC3 deleted cases presented high levels of stabilized NIK, which correlated with higher p52 processing (P = 0.003). These patients also showed higher BCL2 levels than those del(11q)/BIRC3 undeleted, and we could further observe a correlation between p52 and BCL2 levels (P = 0.01). Given this p52-dependent BCL2 upregulation, we treated the CRISPR/Cas9 edited clones with venetoclax, demonstrating that HG3-del(11q) BIRC3MUT cells were more sensitive upon BCL2 inhibition than HG3WT clones (mean IC50 3.5 vs. 5.75 μM; P = 0.005). In vitro proliferation assays were performed to interrogate the impact of BIRC3 loss in CLL cell growth, revealing that HG3 BIRC3MUT cell lines had higher growth rates than BIRC3WT cells (P = 0.001). HG3-del(11q) BIRC3MUT cells also showed enhanced proliferation in comparison to HG3-del(11q) clones (P = 0.009). We further determined the clonal dynamics of del(11q) and/or BIRC3MUT cell lines in clonal competition experiments, showing that HG3 BIRC3MUT and HG3-del(11q) BIRC3MUT cells progressively outgrew HG3WT and HG3-del(11q) cells, respectively, overtime (P = 0.02; P = 0.006). Furthermore, we injected these edited cell lines into NSG mice (n = 20) in vivo, showing that mice xenografted with HG3 BIRC3MUT and HG3-del(11q) BIRC3MUT cells presented, by flow cytometry, an increase of human CD45+ cells in spleen 14 days after injection, compared to HG3WT and HG3-del(11q) cells (P = 0.02; P = 0.015). In summary, this work demonstrates that biallelic BIRC3 deletion through del(11q) and mutation triggers non-canonical NF-κB signaling, driving BCL2 overexpression and conferring clonal advantage, which could account for the negative predictive impact of BIRC3 biallelic inactivation in CLL. Taken together, our results suggest that del(11q) CLL patients harboring BIRC3 mutations should be considered as a CLL subgroup at a high risk of progression that might benefit from venetoclax-based therapies. Funding: PI18/01500 Disclosures No relevant conflicts of interest to declare.

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).

[Background]: Chronic lymphocytic leukemia (CLL) is a highly genetically heterogeneous disease. Although CLL has been traditionally considered as a mature B cell leukemia, few independent studies have shown that the genetic alterations may appear in CD34+ hematopoietic progenitors. However, the presence of both chromosomal aberrations and gene mutations in CD34+ cells from the same patients has not been explored. [Methods]: Amplicon-based deep next-generation sequencing (NGS) studies were carried out in magnetically activated-cell-sorting separated CD19+ mature B lymphocytes and CD34+ hematopoietic progenitors (n = 56) to study the mutational status of TP53, NOTCH1, SF3B1, FBXW7, MYD88, and XPO1 genes. In addition, ultra-deep NGS was performed in a subset of seven patients to determine the presence of mutations in flow-sorted CD34+CD19− early hematopoietic progenitors. Fluorescence in situ hybridization (FISH) studies were performed in the CD34+ cells from nine patients of the cohort to examine the presence of cytogenetic abnormalities. [Results]: NGS studies revealed a total of 28 mutations in 24 CLL patients. Interestingly, 15 of them also showed the same mutations in their corresponding whole population of CD34+ progenitors. The majority of NOTCH1 (7/9) and XPO1 (4/4) mutations presented a similar mutational burden in both cell fractions; by contrast, mutations of TP53 (2/2), FBXW7 (2/2), and SF3B1 (3/4) showed lower mutational allele frequencies, or even none, in the CD34+ cells compared with the CD19+ population. Ultra-deep NGS confirmed the presence of FBXW7, MYD88, NOTCH1, and XPO1 mutations in the subpopulation of CD34+CD19− early hematopoietic progenitors (6/7). Furthermore, FISH studies showed the presence of 11q and 13q deletions (2/2 and 3/5, respectively) in CD34+ progenitors but the absence of IGH cytogenetic alterations (0/2) in the CD34+ cells. Combining all the results from NGS and FISH, a model of the appearance and expansion of genetic alterations in CLL was derived, suggesting that most of the genetic events appear on the hematopoietic progenitors, although these mutations could induce the beginning of tumoral cell expansion at different stage of B cell differentiation. [Conclusions]: Our study showed the presence of both gene mutations and chromosomal abnormalities in early hematopoietic progenitor cells from CLL patients., This work was partially supported by grants from the Spanish Fondo de Investigaciones Sanitarias PI12/00281, PI15/01471, Instituto de Salud Carlos III (ISCIII), European Regional Development Fund (ERDF) “Una manera de hacer Europa,” Consejería de Educación, Junta de Castilla y León (SA085U16), Proyectos de Investigación del SACYL, Spain: GRS 1172/A/15, BIO/SA10/14, GRS 1343/A/16, and Fundación Española de Hematología y Hemoterapia (FEHH) and by a grant (RD12/0036/0069) from the Red Temática de Investigación Cooperativa en Cáncer (RTICC), Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness & European Regional Development Fund (ERDF) “Una manera de hacer Europa” (Innocampus; CEI-2010-1-0010), Fundación “Memoria Don Samuel Solórzano Barruso” 2016, and the European Union Seventh Framework Programme [FP7/2007–2013] under Grant Agreement no. 306242-NGS-PTL. MHS is fully supported by an “Ayuda predoctoral de la Junta de Castilla y León” by the Fondo Social Europeo (JCYL-EDU/346/2013 PhD scholarship).

The presence of chromosomal gains other than trisomy 12 in chronic lymphocytic leukaemia (CLL) is unusual. However, some patients may show gains on several chromosomes simultaneously suggesting a hyperdiploid karyotype. Objective: The objective of this study was to analyse by FISH the frequency and prognostic impact of hyperdiploidy in CLL. Method: A review of 1359 consecutive cases diagnosed with CLL referred for FISH analysis to a unique institution was carried out. Hyperdiploidy was considered when a gain of at least three of the five FISH probes used was observed. Results: Seven cases (0.51%) with hyperdiploidy were found, confirming that it is a rare event in this disease. Although most patients presented with early Binet stages at diagnosis, six of seven (86%) shortly progressed. The median of time to the first therapy (TTFT) and overall survival (OS) for the patients with hyperdiploidy were short (1.4 months and 20 months, respectively). Moreover, comparing them with a control group of patients (non-hyperdiploid) with completed follow-up data, TTFT and OS of the patients with hyperdiploidy were significantly shorter than the control group. Conclusion: The presence of hyperdiploidy is uncommon and probably associated with poor prognostic markers in CLL., This work was supported by grants from the Spanish Fondo de Investigaciones Sanitarias FIS 09/01543, PI12/00281 and PI15/01471, Instituto de Salud Carlos III (ISCIII), European Regional Development Fund (ERDF) ‘Una manera de hacer Europa’, Proyectos de Investigación del SACYL 355/A/09, GRS/1172/A15, COST Action EuGESMA (BM0801), Fundación Manuel Solórzano, Obra Social Banca Cívica (Caja Burgos), Fundación Española de Hematología y Hemoterapia (FEHH), by grants (RD12/0036/0069 and RD12/0036/0044) from Red Temática de Investigación Cooperativa en Cancer (RTICC), Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness and European Regional Development Fund (ERDF) ‘Una manera de hacer Europa’ (CEI 2010-1-0010) and the IRON-II collaborative network. The research leading to these results has received funding from the European Union Seventh Framework Programme [FP7/2007-2013] under grant agreement n°306242-NGS-PTL. Maria Hernandez-Sanchez is fully supported by an Ayuda Predoctoral de la Junta de Castilla y Leon from the Fondo Social Europeo (JCYL-EDU/346/2013 Ph.D. scholarship). Ana Eugenia Rodriıguez-Vicente is supported by a grant from Fundacion Ramon Areces.

Prognostic factors in Hodgkin lymphoma (HL) still fail to accurately identify high-risk patients. Tumor microenvironment in HL is a current focus of research for risk definition but few studies have focused on infiltrating lymphocytes. Here, we analyzed the number of tumor infiltrating lymphocytes by flow cytometry in diagnostic biopsies from 96 HL homogeneously treated patients with ABVD with or without radiotherapy. Most lymph node cells were lymphocytes (90 ± 17), with a median T/B/NK distribution of 74%/26%/0.7%, and CD4 T-cell predominance. The amount of CD19 B cells, and NK cells did not show association with disease features. However, high numbers of CD8 and CD4 cells were associated with better and poorer outcomes, respectively. Patients with ≥15% cytotoxic CD8 cells among the total cell population had a longer 10-year freedom from treatment failure (FFTF) (93% vs. 73%, p=.04). In turn, cases with ≥75% of CD4 infiltrating cells showed a significantly decreased FFTF (73% vs. 96%, p=.021). Consequently, CD4/CD8 ratio ≥5 associated with a poorer 10-year FFTF (69.5% vs. 94%, p=.02). This deleterious effect was particularly prominent in advanced disease (n = 58, p=.01). In multivariate analysis, a CD4/CD8 ratio ≥5 was the only independent variable to predict for treatment failure (HR = 4.5, 95% confidence interval, 1.2–16.8). In conclusion, our study shows that high CD4 and low CD8 T-cells infiltrates of tumor specimens associate with poor prognosis in HL patients, and CD4/CD8 ratio might be potentially useful for tailoring therapy.

Imatinib mesylate (IM) is the treatment of choice in patients with newly diagnosed chronic myeloid leukemia (CML), irrespectively of their age. Nevertheless, information regarding tolerability and responses in advanced-age patients, a subgroup in which co-morbidities and other factors may influence outcome, is scarce, since they were excluded from most clinical trials. In this observational study (ELDERGLI), information regarding demographics, concomitant medication, physical examination, performance status, hemogram, biochemistry, hematologic, cytogenetic and molecular responses, time to progression, adverse events (AE) and severe adverse events (SAE) were prospectively recorded in a series of 36 elderly patients with CML, with a median age of 76.6 years. Most patients had cardiovascular co-morbidities, especially hypertension. Regarding IM toxicity, around one third of patients required treatment interruptions because of adverse events, especially hematologic toxicity (66% of cases that needed dose interruptions). When analyzing non hematologic adverse events, the most frequent ones were superficial edemas and GI symptoms. Of note, 9 of patients experienced an infection episode during the follow-up, and 4 were diagnosed during the study period of another type of cancer. Finally, cardiovascular events were reported in 7 patients, most of them with prior cardiovascular risk factors. Regarding responses, after 12 months of imatinib therapy, the rate of complete hematologic response (CHR), complete cytogenetic response (CCyR) and major molecular response (MMolR) were 89%, 72% and 55% respectively. In summary, IM display, in advanced-age patients with chronic phase CML, an efficacy and safety profile comparable to younger patients.

The clinical value of multiparameter flow cytometry (MFC) immunophenotyping in primary or light chain amyloidosis (AL) remains unknown. We studied 44 consecutive bone marrow samples from newly diagnosed patients with amyloidosis; 35 patients with AL and 9 with other forms of amyloidosis. Monoclonal plasma cells (PCs) were identifiable by MFC immunophenotyping in 34 of 35 (97%) patients with AL, whereas it was absent from all but 1 of the 9 (11%) patients with other forms of amyloidosis. Quantification of bone marrow plasma cells (BMPCs) by MFC immunophenotyping was a significant prognostic factor for overall survival (OS) (≤ 1% vs > 1% BMPC cutoff; 2-year OS rates of 90% vs 44%, P = .02). Moreover, detecting persistent normal PCs at diagnosis identifies a subgroup of patients withAL with prolonged OS (> 5% vs ≤ 5% normal PC within all BMPC cutoff, 2-year rates of 88% vs 37%, P = .01). MFC immunophenotyping could be clinically useful for the demonstration of PC clonality in AL and for the prognostication of patients with AL. © 2011 by The American Society of Hematology., This work was supported by the Cooperative Research Thematic Cancer Network (RTIC) grants RD06/0020/0006 and G03/136, MM Jevitt, SL firm, Instituto de Salud Carlos III/Subdirección General de Investigación Sanitaria (FIS) grants PI060339, 02/0905, 01/0089/01-02, PS09/01897, and Consejería de Sanidad, Junta de Castilla y León, Valladolid, Spain grant 557/A/10.

Multiparameter flow cytometry immunophenotyping allows discrimination between normal (N-) and myelomatous (MM-) plasma cells (PCs) within the bone marrow plasma cell compartment (BMPCs). Here we report on the prognostic relevance of detecting more than 5% residual normal plasma cells from all bone marrow plasma cells (N-PCs/BMPCs) by multiparameter flow cytometry in a series of 594 newly diagnosed symptomatic MM patients, uniformly treated according to the Grupo Español de MM 2000 (GEM2000) protocol. Our results show that symptomatic MM patients with more than 5% N-PCs/BMPCs (n = 80 of 594; 14%) have a favorable baseline clinical prospect, together with a significantly lower frequency of high-risk cytogenetic abnormalities and higher response rates. Moreover, this group of patients had a significantly longer progression-free survival (median, 54 vs 42 months, P = .001) and overall survival (median, not reached vs 89 months, P = .04) than patients with less than or equal to 5% N-PCs/BMPCs. Our findings support the clinical value of detecting residual normal PCs in MM patients at diagnosis because this reveals a good prognostic category that could benefit from specific therapeutic approaches. This trial was registered at www.clinicaltrials.gov as NCT00560053.

Background Among patients with B-cell chronic lymphoid leukemia, those with 13q14 deletion have a favorable outcome. However, whether the percentage of cells with 13q- influences the prognosis or the biological characteristics of this disease is unknown. We analyzed the clinico-biological characteristics and outcome of patients with B-cell chronic lymphoid leukemia with loss of 13q as the sole cytogenetic aberration. Design and Methods Three hundred and fifty patients with B-cell chronic lymphoid leukemia were studied. Clinical data were collected and fluorescence in situ hybridization and molecular studies were carried out. In addition, a gene expression profile was obtained by microarray-based analysis. Results In 109 out of the 350 cases (31.1%) loss of 13q was the sole cytogenetic aberration at diagnosis. In the subgroup of patients with 80% or more of cells with loss of 13q (18 cases), the overall survival was 56 months compared with not reached in the 91 cases in whom less than 80% of cells had loss of 13q ( p < 0.0001). The variables included in the multivariate analysis for overall survival were the percentage of losses of 13q14 ( p =0.001) and B symptoms ( p =0.007). The time to first therapy in the group with 80% or more vs. less than 80% of losses was 38 months vs. 87 months, respectively ( p =0.05). In the multivariate analysis the variables selected were unmutated status of IgVH ( p =0.001) and a high level of β2microglobulin ( p =0.003). Interestingly, these differences regarding overall survival and time to first therapy were also present when other cut-offs were considered. The gene expression profile of patients with a high number of losses in 13q14 showed a high proliferation rate, downregulation of apoptosis-related genes, and dysregulation of genes related to mitochondrial functions. Conclusions Patients with B-cell chronic lymphoid leukemia with a high number of losses in 13q14 as the sole cytogenetic aberration at diagnosis display different clinical and biological features: short overall survival and time to first therapy as well as more proliferation and less apoptosis. A quantification of the number of cells showing a genetic abnormality should, therefore, be included in the study of the prognostic factors of B-cell chronic lymphoid leukemia.

Clonal plasma cells (PC) from different types of monoclonal gammopathies (MG) display distinct phenotypes consistent with an increased antigen-presentation and T-cell costimulation in MG of undetermined significance that deteriorates in malignant conditions. Expression of other cell surface and soluble molecules (e.g. adhesion/proliferation molecules) involved in the interaction between clonal PC and the bone marrow (BM) microenvironment has also been related to malignant PC, although the exact clinical significance of their expression remains largely unknown. Analysis of cell surface levels of several of these molecules in multiple myeloma (MM) patients shows an association between lower expression on BMPC of the HLA-I and β2-microglobulin antigen-presenting molecules, the CD126 and CD130 IL6 receptor (IL6R) chains, and CD38, and adverse prognostic features of the disease. Likewise, patients showing higher soluble levels of antigen-presenting molecules (HLA-I and β2-microglobulin), IL6R and CD95 tended to be associated with more aggressive disease behavior. In contrast, CD40, CD86, CD56, CD19, and CD45 were not associated with patients' outcome. Interestingly, upon considering the ratio between the soluble and PC membrane expression of each molecule, an increased adverse prognostic impact was observed for both HLA-I and β2-microglobulin, but not for the other molecules. Multivariate analysis confirmed the independent prognostic value of cell surface expression of CD126 on BMPC together with serum β2-microglobulin and LDH. In summary, our results show an abnormal distribution of the cellular and soluble compartments of the HLA-I, IL6R, and to a lower extent, CD95 molecules, in MM, associated with the clinical characteristics and behavior of the disease., Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo (Madrid, Spain); Grant numbers: Spanish Network on Multiple Myeloma G03/136, RETICC RD06/0020/0035-FEDER.

[Objectives]: The information currently available about dendritic cells (DCs) in patients with different types of monoclonal gammopathy (MG) is limited and frequently controversial. In the present study, we analyzed the ex vivo distribution as well as the phenotypic and functional characteristics of peripheral blood (PB) DCs from different types of MG. [Methods]: For this purpose, 61 untreated patients in total with MG were analyzed - MG of undetermined significance (MGUS), 29 cases; multiple myeloma (MM), 28 cases; and plasma cell leukemia (PCL), 4 cases - in comparison with a group of 10 healthy controls. [Results]: Our results show an absolute overall higher number of all subsets of PB DCs in PCL, together with lower numbers of myeloid DCs in MM patients. From a phenotypic point of view, PB DC subsets from all types of MG expressed significantly higher levels of HLA molecules and altered patterns of expression of the CD2, CD11c, CD16, CD22, CD62L, and CD86 molecules, in association with altered patterns of secretion of inflammatory cytokines. [Conclusion]: In summary, we show the existence of significant abnormalities in the distribution, phenotype, and pattern of secretion of inflammatory cytokines by different subsets of PB DCs from patients with MGs, which could reflect a potentially altered homing of DCs, together with a greater in vivo activation and lower responsiveness of PB DCs, which are already detectable in MGUS patients. ©AlphaMed Press., This work was partially supported by the Spanish Red de mieloma multiple y otras gammapatías (Contract Grant RTIC G03/136), the Spanish Red de Centros de Cáncer

We analyzed patients with hairy cell leukemia (HCL) to achieve a better understanding of the differentiation stage reached by HCL cells and to define the key role of the diversification of cell surface makers, especially CD25 expression.We analyzed 38 previously untreated patients with HCL to characterize their complete (VDJ(H)) and incomplete (DJ(H)) immunoglobulin (Ig) heavy chain (IgH) rearrangements, including somatic hypermutation pattern and gene segment use.A correlation between immunophenotypic profile and molecular data was seen. All 38 cases showed monoclonal amplifications: VDJ(H) in 97%, DJ(H) in 42%, and both in 39%. Segments from the D(H)3 family were used more in complete compared with incomplete rearrangements (45% vs. 12%; P.005). Furthermore, comparison between molecular and immunophenotypic characteristics disclosed differences in the expression of CD25 antigen; CD25(-) cases, a phenotype associated with HCL variant, showed complete homology to the germline in 3 of 5 cases (60%), whereas this characteristic was never observed in CD25(+) cases (P.005). Moreover, V(H)4-34, V(H)1-08, and J(H)3 segments appeared in 2, 1, and 2 CD25(-) cases, respectively, whereas they were absent in all CD25(+) cases.These results support that HCL is a heterogeneous entity including subgroups with different molecular characteristics, which reinforces the need for additional studies with a larger number of patients to clarify the real role of gene rearrangements in HCL.

[BACKGROUND]: The majority of studies published to date regarding the role of the bone marrow (BM) microenvironment in the pathogenesis of monoclonal gammopathies (MG) have focused on the interaction between stroma cells and plasma cells, whereas information concerning the lymphocytes infiltrating the tumor microenvironment is scanty. [METHODS]: The authors measured the distribution, TCR-Vβ repertoire, immunophenotype, and functional characteristics of different subsets of BM T lymphocytes from 61 nontreated patients with MG (30 patients with MG of undetermined significance [MGUS], 27 patients with multiple myeloma [MM], and 4 patients with plasma cell leukemia [PCL]). [RESULTS]: The authors found a significantly increased rate of BM infiltration by T cells in all patient groups, at the expense of CD4+CD8- and CD4-CD8- T lymphocytes and both CD4+CD28- and CD8+CD28- cytotoxic/effector T cell subsets, and associated with TCR-Vβ expansions in both CD4+ and CD8+ BM T cells in the majority of patients with MGUS, MM, and PCL. Moreover, the percentage of T cells secreting interferon (IFN)-γ was found to be increased (P ≤ 0.05) both in CD4+ and CD8+ T cells in MGUS and MM patients, and a higher plasma concentration of IFN-γ was found in patients with MM. It is interesting to note that a positive correlation was noted between the proportion of CD28- and both the percentage of IFN-γ-secreting cells and the proportion of expanded TCR-Vβ lymphocytes within the total BM CD4+ T cells. [CONCLUSIONS]: The results of the current study demonstrated an increased infiltration of BM by T cells associated with frequent TCR-Vβ expansions and a more prominent cytotoxic/Th1 phenotype in all the patient groups studied. © 2006 American Cancer Society.

We evaluated the incidence of trisomy 11 in acute myeloblastic leukemia (AML) and its correlation with the most relevant clinical, biological, and immunophenotypic disease characteristics in a total of 399 consecutive AML patients. Trisomy 11 was found in 15 patients (3.8%), in 3 of them as the sole abnormality. Median age was 68 years (range 48-87); 87% of patients were older than 60 years. Seven patients displayed multilineage dysplasia. Cytogenetics showed an association with trisomy 8 in six patients, and in five cases with a del(5q); nine patients had complex karyotypes. In all cases, in situ hybridization studies revealed three copies of the MLL gene, but no rearrangements or tandem duplications of MLL. Immunophenotypic analysis of blast cells showed a constant immature immunophenotypic profile with CD34 or CD117 expression (or both) in all cases analyzed. The complete remission rate was 43%; median survival time was only 2 months.

Although multiple myeloma (MM) is predominantly a disease of the elderly, few studies have focused on the identification of prognostic factors in this group of patients. Four hundred twenty five MM patients >65 years were uniformly treated with chemotherapy (MP or VCMP/VBAD). Multivariate analysis identified 4 factors with independent unfavorable prognostic influence: high percentage of S-phase bone marrow plasma cells (>2.5%); elevated beta(2) microglobulin (B2M) (>4 mg/L); age >80 years old; and LDH serum levels (above normal limit). The S-phase value was the most powerful independent prognostic factor to discriminate subgroups of patients with different prognosis. Thus, 3 main risk categories could be identified according to S-phase values: 3%, with median survivals of 34, 22 and 12 months, respectively (p < 0.0001). Our study also proved the value for elderly patients of the recently developed International Score System (ISS) based on B2M and albumin. Furthermore, the number of S-phase cells helped to subdivide the ISS III Group identifying a subset of patients with very poor prognosis defined by an additional high S-phase, who displayed a median survival of only 8 months. These results demonstrate that elderly patients can be accurately classified according to prognosis, which may be particularly valuable when comparing the efficacy of new treatment strategies. Moreover, our results underline the high prognostic value of proliferative activity of PC, a parameter that should be considered in routine laboratory investigations of MM.

Recombinant® interferon α (r-IFN-α) has been shown to be an effective drug for chronic myeloid leukaemia (CML). However, higher response rates can be achieved using cytarabine along with r-IFN-α. YNK01 is a derivative of cytosine arabinoside for oral administration. So far, the only published experience with continuous YNK01 was in advanced CML (10 cases). We have performed a pilot study to evaluate the efficacy and toxicity of the combined therapy r-IFN-α and daily oral YNK01 in patients with newly diagnosed Ph+ CML. Ten previously untreated patients were included in the study. Among those patients evaluable for cytogenetic response, 87% (seven out of eight) reached a major cytogenetic response with four reaching complete cytogenetic response (50%). The most significant side-effects were gastrointestinal. Macrocytic anaemia was observed in three patients. In conclusion, continuous oral administration of YNK01 in combination with IFN-α is safe and can result in high-cytogenetic response rates.

The aim of the present study was to analyze whether or not leukemic clonogenic cells are restricted to the CD34+ cell fraction and to investigate the effect of IL-3 and G-CSF on blast cell populations dissected according to their CD34 reactivity. For this purpose 34 patients were studied. Patients were classified into three groups according to CD34 antigen expression: (1) cases in which all blast cells (100%) were positive for the CD34 Ag (n = 9); (2) cases in which all blast cells lacked the expression of this antigen (n = 10); and (3) patients in whom both, CD34 positive and negative blast cell subsets coexisted (n = 15). In 15 cases immunomagnetic cell selection was performed and two subpopulations were separated: one, phenotypically more immature (CD34+), and another, theoretically more differentiated (CD34−/33+). In addition, in three cases both CD34+ and CD34− blast cell subpopulations were sorted using a FACStar flow cytometer. Blast colony assays were performed using 0.9% methylcellulose and two different recombinant human hematopoietic growth factors (HGFs), IL-3 and G-CSF, were used as growth stimulants. Either, a single or a combination of the growth factors was added to cultures. Colony formation was observed in both 100% positive or 100% negative cases for the CD34 antigen as well as in the CD34+ and CD34- cell fractions separated by immunomagnetic selection or flow cytometry. The effect of G-CSF and IL-3 on both cell fractions was as follows: cases with a uniform population according to CD34 expression (100% positive or negative) showed a better growth response with IL-3 especially for the CD34+ cases (87% vs 40% of CD34+ and CD34− cases, respectively). Within the CD34−/33+ selected fractions, IL-3 tended to induce a higher proliferative response than G-CSF while the opposite was found within the CD34+ cell selected fractions. In contrast it was observed that both IL-3 and G-CSF induced a higher PE on the CD34− blast cells (both selected and 100% negative), although the difference was not statistically significant. The existence of a possible synergistic effect (SE) between HGFs was also explored. Overall, a synergistic growth was observed in nine out of the 13 selected cases studied and this effect could be seen in both CD34− or CD34+ blast cell fractions. The analysis of the complete phenotypic characteristics of these cells revealed that cell fractions showing SE were more immature according to the expression of CD15 and HLA-DR antigens. We can conclude that in leukemic hematopoiesis, CD34 antigen expression does not have the same significance as it does in normal hematopoiesis since clonogenic cells are not restricted to the CD34+ acute myeloid leukemia (AMD blast cell fraction. Moreover, our study shows that the heterogeneous response to HGFs observed in AML patients may be associated with the existence of immunophenotypically different blast cell subsets.

Although multiparameter flow cytometry (MFC) has demonstrated clinical relevance in monoclonal gammopathy of undetermined significance (MGUS)/myeloma, immunophenotypic studies on the full spectrum of Waldenström's Macroglobulinemia (WM) remain scanty. Herein, a comprehensive MFC analysis on bone marrow samples from 244 newly diagnosed patients with an immunoglobulin M (IgM) monoclonal protein was performed, including 67 IgM-MGUS, 77 smoldering and 100 symptomatic WM. Our results show a progressive increase on the number and light-chain-isotype-positive B-cells from IgM-MGUS to smoldering and symptomatic WM (P10% B cells or 100% light-chain-isotype-positive B-cells (P, This work was supported by the Cooperative Research Thematic Network (RTICC) RD06/0020/0006, Instituto de Salud Carlos III/Subdirección General de Investigación Sanitaria (FIS: PI060339; 06/1354; 02/0905; 01/0089/01-02; PS09/01897/01370) and Consejería de Sanidad, Junta de Castilla y León, Valladolid, Spain (557/A/10).

AIMS--To analyse the forward scatter/side scatter (FSC/SSC) distribution of acute myeloblastic leukaemia (AML) blast cells in order to assess whether it correlates with their morphology, immunophenotype, and clinical and biological disease characteristics. METHODS--FSC/SSC patterns were established upon taking into account the localisation of the residual T lymphocytes in the FSC/SSC dot plot as an internal biological standard. One hundred and seventy one newly diagnosed AML patients were analysed and five different FSC/SSC patterns were established. These five patterns could be grouped into two major categories taking into account the FSC/SSC distribution of normal cells in a bone marrow aspirate: immature patterns (1 and 2) and mature patterns (3, 4, and 5). These FSC/SSC patterns were correlated with different clinical and biological characteristics of AML patients. RESULTS--No significant associations were detected in relation to the clinical and haematological disease characteristics and the prognosis of these patients. By contrast there was a significant correlation between the FSC/SSC pattern of the AML blast cells and the FAB classification. An increased reactivity for the antigens associated with myeloid differentiation such as CD13, CD33, CD11b, CD15, CD14, CD4, CD56, and/or CD16 was detected among cases showing a mature FSC/SSC pattern (3, 4, and 5), both in the whole series and even within each of the FAB AML subtypes. By contrast, the reactivity for the CD34 precursor cell associated antigen was higher among those cases displaying an immature FSC/SSC pattern, this being observed even within each FAB subgroup. CONCLUSIONS--The FSC/SSC pattern distribution of AML blast cells not only provides an additional objective and reproductible system for the classification of these leukaemias but it may also represent a connection between the FAB morphological groups and the immunophenotypic classification of AML patients.

Introduction: Although multiple myeloma (MM) is typically described as an incurable disease, it has been shown in recent years that a small fraction of patients may reach more than 10-years progression-free survival (PFS), which is considered as the minimum threshold to identify patients in "operational cure". However, because of the scarcity of available data there is significant lack of knowledge in MM regarding the frequency of cases attaining operational cure, nor the existence of biomarkers that could prospectively predict such curability. Methods: Herein, we sought to define the frequency as well as the biomarkers predictive of operational cure in a large series of uniformly-treated transplant-eligible patients enrolled in the PETHEMA/GEM2000 protocol (VBMCP/VBAD followed by HDT/ASCT and 2 years of maintenance with interferon and prednisone). Patients' follow-up was updated at the time of abstract submission, and the median follow-up of the series is now of 12-years. We used an automated multiparameter flow cytometric (MFC) classification model focused on the analysis of the bone marrow plasma-cell compartment to identify among newly diagnosed symptomatic MM those with MGUS-like vs. MM-like phenotypic signatures. Minimal residual disease (MRD) was monitored using a first-generation 4-color MFC assay (CD38-FITC / CD56-PE / CD19-PerCPCy5.5 / CD45-APC) with a limit of detection of 10-4. PFS and overall survival (OS) were measured from the time of diagnosis. Results: From a total of 1075 patients enrolled in the GEM2000 protocol, 763 were eligible for this analysis because they either relapsed or died during the first 10-years from diagnosis (n=666; 87%), or remained progression-free and alive for more than 10-yers (n=97; 13%); accordingly, all patients remaining progression-free and alive but for which the follow-up was inferior to 10-years were excluded from the analysis. We then investigated the biomarkers that could help to identify patients reaching operational cure after HDT/ASCT. As compared to the vast majority of cases, patients reaching >10-years PFS (13%) had significantly less frequent anemia (76% vs. 60%, respectively; P=.002), as well as more frequent Durie-Salmon stage IA (14% vs. 6%; P=.004), MGUS-like signature as determined by the automated MFC algorithm (28% vs. 6%; P10-years PFS vs. those who relapsed earlier. On multivariate analysis, only the presence of an MGUS-like signature at baseline (P=.04; HR: 3.9) and MRD-negativity at day+100 after HDT/ASCT (P=.006; HR: 6.3) emerged as independent predictive markers for >10-years PFS; anemia, Durie-Salmon and CR status were not retained in the logistic regression model. Accordingly, patients with an MFC-defined baseline MGUS-like signature reaching MRD-negativity after HDT/ASCT (n=14) had a median PFS of 10-years and a 10-year OS rate of 79%, which were significantly superior to those observed among cases with MM-like signatures being MRD-negative (n=54) or positive (n=99) after therapy [median PFS of 6 and 3 years (P Conclusions: We demonstrated that operational cure (i.e.: >10-years PFS) was possible for 13% of transplant-eligible MM patients before the era of novel agents. Curability rates were particularly frequent among patients with a benign phenotypic signature at diagnosis and MRD negativity after HDT/ASCT, suggesting a remarkable clinical benefit of attaining deep remissions after intensive treatment for patients with early MM. Disclosures Paiva: Sanofi: Consultancy; Janssen: Consultancy; Millenium: Consultancy; Onyx: Consultancy; BD Bioscience: Consultancy; EngMab AG: Research Funding; Binding Site: Consultancy; Celgene: Consultancy. Puig:The Binding Site: Consultancy; Janssen: Consultancy. Mateos:Takeda: Consultancy; Celgene: Consultancy, Honoraria; Onyx: Consultancy; Janssen-Cilag: Consultancy, Honoraria. San Miguel:Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Onyx: Honoraria; Sanofi-Aventis: Honoraria; Novartis: Honoraria; Millennium: Honoraria; Janssen-Cilag: Honoraria.

The present study explored the impact of two novel criteria; having >95% abnormal plasma cells by flow cytometry at diagnosis and the evolving subtype of the disease, as predictors of progression in 61 smouldering multiple myeloma (SMM) and 311 monoclonal gammopathy of unknown significance (MGUS) patients. Although both criteria were of prognostic value, the risk of progression was better identified by immunophenotyping [Hazard Ratio (HR) 6·2 and 17·2 for SMM and MGUS, respectively] than evolving subtype, which had independent prognostic value only in MGUS (HR 3·6). Immunophenotyping discriminated the different risk of progression within the evolving and non-evolving subgroups of SMM (P = 0·01) and MGUS (P < 0·001). © 2009 Blackwell Publishing Ltd., This work was supported in part by a national Grant from the Spanish ‘Instituto de Salud Carlos III’ (Ref. PI50575) and ‘Ministerio de Educación y Ciencia’ (Ref. SAF: 2004-06587). This work was supported by the Cooperative Research Thematic Network (RTICs; RD06/0020/0006), MM Jevitt, SL firm, and Instituto de Salud Carlos III/ Subdirección General de Investigación Sanitaria (FIS: PI060339; 02/0905; 01/0089/01-02; and PI060339).

8 páginas, 3 figuras, 2 tablas.-- GEM (Grupo Español de MM)/PETHEMA (Programa para el Estudio de la Terapéutica en Hemopatías Malignas) cooperative study groups: et al., Minimal residual disease (MRD) assessment is standard in many hematologic malignancies but is considered investigational in multiple myeloma (MM). We report a prospective analysis of the prognostic importance of MRD detection by multiparameter flow cytometry (MFC) in 295 newly diagnosed MM patients uniformly treated in the GEM2000 protocol VBMCP/VBAD induction plus autologous stem cell transplantation [ASCT]). MRD status by MFC was determined at day 100 after ASCT. Progression-free survival (PFS; median 71 vs 37 months, P < .001) and overall survival (OS; median not reached vs 89 months, P = .002) were longer in patients who were MRD negative versus MRD positive at day 100 after ASCT. Similar prognostic differentiation was seen in 147 patients who achieved immunofixation-negative complete response after ASCT. Moreover, MRD- immunofixation-negative (IFx-) patients and MRD- IFx + patients had significantly longer PFS than MRD- IFx + patients. Multivariate analysis identified MRDstatus byMFCat day 100 afterASCTas the most important independent prognostic factor for PFS (HR = 3.64, P = .002) and OS (HR = 2.02, P = .02). Our findings demonstrate the clinical importance of MRD evaluation by MFC, and illustrate the need for further refinement of MM response criteria. This trial is registered at http://clinicaltrials.gov under identifier NCT00560053., This work was supported by the Cooperative Research Thematic Network (RTICs; RD06/0020/0006), MM Jevitt, SL firm, and Instituto de Salud Carlos III/Subdirección General de Investigación Sanitaria (FIS: PI060339; 02/0905; 01/0089/01-02; and PI060339; Madrid, Spain).

Monoclonal gammopathy of uncertain significance (MGUS) and smoldering multiple myeloma (SMM) are plasma cell disorders with a risk of progression of approximately 1% and 10% per year, respectively. We have previously shown that the proportion of bone marrow (BM) aberrant plasma cells (aPCs) within the BMPC compartment (aPC/BMPC) as assessed by flow cytometry (FC) contributes to differential diagnosis between MGUS and multiple myloma (MM). The goal of the present study was to investigate this parameter as a marker for risk of progression in MGUS (n = 407) and SMM (n = 93). Patients with a marked predominance of aPCs/BMPC (≥ 95%) at diagnosis displayed a significantly higher risk of progression both in MGUS and SMM (P< .001). Multivariate analysis for progression-free survival (PFS) selected the percentage aPC/BMPC (≥ 95%) as the most important independent variable, together with DNA aneuploidy and immunoparesis, for MGUS and SMM, respectively. Using these independent variables, we have identified 3 risk categories in MGUS (PFS at 5 years of 2%, 10%, and 46%, respectively; P< .001) and SMM patients (PFS at 5 years of 4%, 46%, and 72%, respectively; P < .001). Our results show that multiparameter FC evaluation of BMPC at diagnosis is a valuable tool that could help to individualize the follow-up strategy for MGUS and SMM patients. © 2007 by The American Society of Hematology., This work was supported in part by a national grant from the Spanish Instituto de Salud Carlos III (Ref. PI50575 and FIS PI-02/0905) and Ministerio de Educación y Ciencia (Ref. SAF: 2004-06587).

Background: Progressive transformation of germinal centers (PTGC) is a disease of mostly young adults that generally presents as asymptomatic, localized or generalized lymphadenopathy. Although PTGC usually seen as nodal disease, it can occur at extranodal sites. In the differential diagnosis of chronic lymphadenopathies, infection, lymphoproliferative disorders and progressive transformation of germinal centers should be considered. The most significant entities in the differential diagnosis with PTGC are nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) and lymphocyte rich classical Hodgkin lymphoma. PTGC may precede, follow, or be concurrent with lymphomas. Objectives: To review the clinical and pathological features of PTGC, and PTGC’s relationship with lymphoid neoplasia in adult population. Material and Methods: The data of 33 patients who was diagnosed with PTCG at Hospital of Ege University School of Medicine, Pathology Department, between January 2000 and December 2014 were analyzed retrospectively. Clinical and Pathological Features: The median age of the patients was 43.8 years (26-66 years), and of 16 (48.5%) males and 17 (51.5%) females. Biopsy regions were; cervical lymph nodes (LN) 9 (27.3%), axillary LN 9 (27.3%), inguinal LN 6 (18.2%), submandibular LN 3 (9.1%), submental LN 2 (6.1%), parapancreatic LN 1 (3.0%), intraparotid LN 1 (3.0%), supraclavicular LN 1 (3.0%) and intramammarian LN 1 (3.0%). Recurrent PTGC was detected in only one (3.0%) patient. Diffuse large B cell lymphoma (DLBCL) and nodular lymphocyte predominant hodgkin lymphoma (NLPHL) was detected concurrent with PTGC in two cases. Also PTGC was detected 3 years after the diagnosis of DLBCL, NLPHL and T cell rich B cell lymphoma in 3 cases. There was no relapse in these 3 cases and no progression to lymphoma during follow-up of other patients. Conclusion: PTGC is not considered a premalignant condition but development of lymphoma have been reported rarely. If PTGC occurs in the follow of lymphoma cases, follow-ups may be intensified. NLPHL is the most important entity in differantial diagnosis of PTGC and patients should be carefully examined in terms of NLPHL.

The immunological bone marrow (BM) microenvironment plays a major role in controlling growth and survival of clonal plasma cells (PC); this might translate into different patterns of expression of molecules involved in immune responses on PC from different types of monoclonal gammopathies (MG). We have studied the expression of a group of nine such molecules on both BMPC and the plasma of 61 newly diagnosed MG patients (30 MG of undetermined significance (MGUS), 27 multiple myeloma (MM) and four plasma cell leukemia (PCL)) and five normal individuals. Clonal PC from all MG displayed significantly increased levels of CD56, CD86 and CD126, and decreased amounts of CD38 (P

AIM: To evaluate the validity of the colony forming unit-granulocyte macrophage (CFU-GM) assay for predicting relapse in patients with acute myeloid leukaemia (AML). METHODS: The study population comprised 32 patients with AML in remission, followed for a median of 18 months. A mean of four studies was carried out per patient. Three patterns of in vitro growth based on the number of CFU-GM in normal bone marrow were defined: 1 = normal (normal number of CFU-GM and a cluster:colony ratio < 2); 2 = hypoplastic (low number of CFU-GM and a cluster:colony ratio < 2); 3 = anomalous (low or normal number of CFU-GM and a cluster:colony ratio > 2). RESULTS: Eleven patients relapsed, all of whom had previously displayed an abnormal CFU-GM pattern: anomalous in nine and hypoplastic in two. The remaining 25 patients were in complete remission at the time of writing, 16 of whom had a normal growth pattern. The other nine had anomalous (eight patients) or hypoplastic (one patient) growth. The latter may be false positive results. The in vitro growth pattern was not constant during follow up analysis. All 15 patients in whom the growth pattern switched from abnormal to normal remain in complete remission. By contrast, of the five cases in whom the pattern changed from normal to abnormal, three have relapsed and the other two had other indicators of relapse. The growth pattern remained unchanged in the remaining 16 patients. CONCLUSION: The present data show that the sequential investigation of the CFU-GM growth pattern may be of value in predicting relapse in patients with AML.

To describe 5 cases of renal lymphoma.A retrospective study was conducted on 5 cases of renal lymphoma that had been diagnosed at our institution in 1999. Of these 5 cases, one was considered to be primary renal lymphoma. The characteristics, treatment and outcome are discussed.Secondary kidney involvement from systemic lymphoma is common, but primary renal lymphoma is a rare and controversial entity.

The objective of this study was to determine the efficacy and safety of the combination ciprofloxacin plus amoxicillin/clavulanic acid as an empirical treatment of infection in hematologic patients without severe neutropenia. These drugs allowed us to carry out a sequential therapy, first intravenously and later orally, so that the patient could be discharged as soon as there was a response. Serum concentrations of ciprofloxacin were monitored in this study. Forty seven of the sixty-six patients included (71%) responded to the treatment with no differences between the dosages of ciprofloxacin employed (600-900 mg daily in two or three divided doses). In the patients who responded, the signs and symptoms of infection lasted only three days, which could allow a short hospital stay (median of six days). In the first pre and post-dose serum samples, ciprofloxacin concentrations were significantly higher when the drug was administered every 8 h. Nevertheless, 72 h after the beginning of treatment, they had leveled out in either 8 and 12 h schemes. The toxicity of the treatment was very light, with only four cases with adverse effects, grades I and II. This data suggests that the employed combination is effective and safe and can considerably decrease costs incurred through the admission of hematologic patients with serious infections but without severe neutropenia.

It is hypothesized that similarly to multiple myeloma, also in WM there may be a continuum between IgM MGUS, smoldering (SWM) and symptomatic WM, rather than these entities being considered as separate. The very low frequency of MYD88 L265P initially reported in IgM MGUS suggested that this could be implicated in disease transformation. However, using sensitive ASO-PCR a significant proportion of patients of patients with IgM MGUS already harbors the MYD88 mutation. Thus, the molecular mechanisms driving the malignant transformation of WM remain largely unknown. Here, we used high-sensitive 8-color multidimensional flow cytometry (MFC) to detect and sort the specific B-cell clone in BM samples (N=31) from a total of 22 newly-diagnosed WM patients (8 symptomatic, 14 SWM) as well as 9 patients with IgM MGUS. The later 9 cases had negative BM biopsy, but light-chain restricted clonal B-cells (typically CD22low, CD25+, sIgM+, LAIR1-) were identified by MFC (median 1.74%, range 0.2%-7.04%). MYD88 L265P was detected on FACS-sorted (purity ≥97%) clonal B-cells from 9/9, 13/14 and 7/8 IgM MGUS, SWM and WM patients, respectively. We first compared the genomic profile of clonal B-cells through high density Cytoscan750K array. Overall, IgM MGUS, SWM and WM patients showed a median of 2, 1.5, and 3 copy number abnormalities (CNA)/case, respectively [defined by >25 consecutive imbalanced markers/segment, >100Kb genomic size and In accordance to the genomic profiles, preliminary analysis of gene expression profiles (GEP; HumanGene 1.0ST) between FACS-sorted clonal B-cells from IgM MGUS, SWM and WM patients showed virtually no deregulated genes (SAM Excel add-in with a FDR q-value In summary, our results show that clonal B-cells from IgM MGUS patients already show a molecular profile that overlaps with that of WM, and suggest that the Waldenström’s clone may arise from normal CD22low/CD25+ BM B-cells. Disclosures: No relevant conflicts of interest to declare.

In AL, a small PC clone synthesizes a misfolded light-chain that forms amyloid fibrils causing organ dysfunction. Significant progress was made regarding the characterization of the amyloid fibrils, but little attention has been paid to the molecular features of clonal PCs; this is most likely explained by the low tumor burden in AL, often masked by a polyclonal PC background. Here, we investigated the phenotypic, transcriptomic and genomic profile of clonal PCs from a total of 22 patients with newly-diagnosed AL. Through multidimensional (12-color) flow cytometry (MFC) combining the evaluation of 10 antigens plus cyκ/cyλ (thus confirming clonality of aberrant phenotypes), we detected clonal PC in all 22 (100%) patients (median 0.76%; range: 0.01% - 30%). Clonal PCs mainly differed from normal PCs by down-regulation of CD19 (100% of cases), CD27 (50%), CD38 (41%), CD45 (50%) and CD81 (50%); CD117, CD28 and CD56 were aberrantly bright positive in 32%, 50% and 64% of patients, respectively. Principal component analysis showed overlapping immunophenotypic expression profiles between clonal PC from AL vs multiple myeloma (MM) and MGUS patients. Using patient-specific aberrant phenotypes, we then sorted clonal PCs (purity ≥97%) by MFC for subsequent molecular studies. Gene expression profiling (GEP) was performed (HumanGene1.0ST) on extracted RNA from clonal PCs of 10 of the 22 AL patients, and compared to FACS-purified PCs from 7 healthy donors. Overall, clonal PCs showed deregulation (SAM Excel add-in with a FDR q-value Afterwards, we investigated the genomic profile of clonal PC from 8/22 AL patients through high density Cytoscan750K array. Interestingly, copy number abnormalities (CNA; defined by >25 consecutive imbalanced markers per segment, >100Kb length and In this integrated phenotypic, molecular and genomic characterization of purified clonal PCs from AL patients, we showed that these display aberrant phenotypes similar to MM, but that the GEP of AL clonal PCs is far less deregulated. Conversely, CNA are present in virtually all patients and with frequencies similar to MM. However, while hyperdiploidy was a less frequent phenomenon in AL as compared to MM, our results show that in highly purified clonal PCs, gains at 1q21.1 can be detected in approximately two-thirds of AL patients. Disclosures: No relevant conflicts of interest to declare.

In acute non-lymphoblastic leukemia (ANLL) progenitor cells frequently display a certain degree of autonomous growth. The aim of the present work was to analyze the autonomous proliferative capacity of leukemic progenitors in both de novo and secondary to myeloproliferative disorders (MPD) and myelodysplastic syndromes (MDS), acute myeloid leukemias and to correlate with clinical and biological characteristics of the disease. Clonogenic assays with and without leukocyte conditioned medium with PHA (LCM-PHA) were performed and the autonomous proliferation index (API) calculated in a series of 50 patients (34 de novo ANLL, eight secondary to MPD and eight secondary to MDS). Patients were divided into two groups according to their API, low (or = 0.4) or high (0.4). Autonomous growth was observed in 84% of cases studied (82% in de novo ANLL, 75% secondary to MDS and 100% secondary to MPD). The group with the highest API (29 patients) had increased levels of hemoglobin (P = 0.006) and platelets (P = 0.01). A high API was also associated with an immature phenotype of blast cells (P = 0.02). Upon analyzing the de novo ANLL separately we observed that a high API correlated with high Hb values (P = 0.02), a lower rate of complete remission (42% vs 61%) and a lower survival rate (medium of 3 vs 10 months). These findings suggest that the capacity for autonomous proliferation can condition the clinical and biological profile of the disease.

Abstract 936 Demonstration of bone marrow (BM) infiltration by lymphoplasmacytic lymphoma is essential to the diagnosis of WM, and a trephine biopsy is considered mandatory for this assessment. Multiparameter flow cytometry (MFC) has demonstrated its clinical relevance in MGUS and myeloma; however, immunophenotypic studies on IgM monoclonal gammopathies are scanty, and focus only in patients with WM. Herein, MFC immunophenotyping was performed on BM samples from 244 patients, including 67 IgM MGUS, 77 smoldering, and 100 symptomatic WM newly diagnosed patients according to the Second International Workshop. A four color panel that systematically allowed the identification of B cells and plasma cells (PC), and their phenotypic characterization for a total of 24 antigens was used. We first analyzed the percentage of B cells and PC in BM and the percentage of light chain restricted cells in both compartments. Our results show a progressive increment of B cells from IgM MGUS to smoldering and symptomatic WM (medians of 2%, 9% and 12%; P10% B cells, in contrast to 34% and 55% of smoldering and symptomatic WM (P10% vs ≤10%: median TTP of 47m vs 145m; P=.016) and light chain restricted B cells (100% vs 10% B cells showed a trend for inferior OS (P=.080), and significant differences emerged when comparing patients with 100% vs Focusing on the antigenic profiles of B cells and PC, we noted that within the B-cell compartment there was a progressive increment of CD22dim (69%, 92% and 88%; P50% of WM patients, which raises the possibility that the WM clone may arise, at least in some cases, before antigenic stimulation; subsequent maturation of the clone into PC would explain the typical presence of somatic hypermutations. On the other hand, B-cells from IgM MGUS and WM patients were negative in ≥90% of cases for CD5, CD10, CD11c and CD103, which can be useful to differentiate between WM and other B-NHL. Finally, the antigenic profile of PC in IgM MGUS and WM was similar to that of normal PC, and different from myeloma PC by consistently showing a CD27+ and CD56- phenotype, in addition to sIgM+ expression in ≥87% of all cases. Similarly to B-cells, we also noted that within the PC compartment there was a progressive increment of CD19+, CD45+ and sIgM+ CD20+ PC from IgM MGUS to smoldering and symptomatic WM. This underlies that this transition is asssociated with an accumulation of light chain restricted clonal PC displaying an immature/plasmablastic phenotype. In summary, our results highlight the potential value of MFC immunophenotyping for the characterization of the Waldenström's clone, as well as for the differential diagnosis, risk of progression and survival in WM. Disclosures: No relevant conflicts of interest to declare.

Abstract 2871 Deletion at 13q14 (13q) is the most common genomic aberration in CLL. It is present in more than 50% of cases, and is the sole documented cytogenetic abnormality in 36% of the patients. These latter cases are known to have a more favorable clinical course. However, recent data from our group and others, suggest that patients with CLL and 13q deletion as the only FISH abnormality could have a different outcome depending on the number of cells displaying this aberration. Thus CLL patients with a high number of 13q cells usually had both shorter overall survival and shorter time to first therapy. However, to the best of our knowledge the molecular characteristics of these patients have not been so far analyzed in detail. A total of 102 samples were selected for the study, 32 of which served as a validation cohort. A complete immunophenotypic analysis by flow cytometry and FISH studies were carried out in all cases. The median age was 68 years (range, 35 to 90 years). For the purpose of the study, only samples with one cytogenetic abnormality were included. For the gene expression profile analysis, according to our previous results, two groups of patients with 13q were compared: those in whom 80% or more of cells showed 13q (13qH) and those in whom fewer than 80% of cells showed 13q losses (13qL). The distribution of cases in the study cohort was: 13qH (n=25; 36%), 13qL (n=27; 39%), normal FISH (nCLL, n=8; 11%) and 17p/11q (n= 10; 14%); and in the validation cohort: 13qH (n=7; 22%), 13qL (n=11; 34%) and nCLL (n=9; 28%). Peripheral blood mononuclear cells (PBMCs) were isolated from fresh peripheral blood samples using Ficoll gradient, snapfrozen and stored at 80°C. For the validation cohort, CD19positive B cells were purified by magnetically activated cell sorting (MACS) CD19 MicroBeads resulting in a >98% purity, as analyzed by flow cytometry. CD19positive normal B cells from peripheral blood of five healthy donors served as controls. All samples were hybridized with the Affymetrix Human Exon arrays 1.0 ST. A total of 3 450 genes significantly distinguished 13qH from 13qL patients, defining the 13qH signature. To determine the biological significance of the deregulated genes, a further analysis was carried out, revealing that apoptosis, BCR and NFkB signaling were the most significant affected pathways in 13qH CLL patients. Moreover, 13qH CLL patients were also characterized by a striking overrepresentation of deregulated miRNAs. A total of 15 miRNAs were deregulated in 13qH relative to 13qL patients. HsamiR155 was the most highly upregulated miRNA (Rfold=3.70), while hsamiR223 was the most significantly downregulated (Rfold=0.10). The posttranscriptional regulatory network of miRNA and genes in CLL patients with more than 80% of 13q cells was carried out by analyzing the miRNAmRNA relationships and the pathway analysis demonstrated that B cell receptor signaling, PI3K signaling and NFkB signaling were among the most strongly affected pathways in 13qH patients, highlighting the importance of miRNA regulation in CLL. The influence of other factors with prognostic relevance in CLL, such as IGVH mutational status, was discarded. We also analyzed the gene signature of CLL high risk cytogenetic subgroups in comparison with 13q patients. Surprisingly, our results suggest that some of the biological characteristics of 13qH CLL patients were similar to those of highrisk cytogenetic subgroups, since they share the deregulation of several key signaling pathways. To validate the differences observed between the subgroups of 13q CLL patients and get a visualization of these, we applied the Principal Component Analysis (PCA) in an independent series of patients. The expression pattern of CD19+ cells from CLL patients was notably different from the gene expression profile of CD19+ cells from healthy donors. Thus, CLL patients with a high number of 13q cells can be differentiated based on their expression profile. By contrast, the gene expression of B lymphocytes from 13qL and normal FISH subgroups was similar. Therefore, this study provides new evidences regarding the heterogeneity of 13q deletion in CLL patients. Thus an overexpression of BCR and NFKB patways and as well as a deregulation of the balance between the proliferative and apoptotic signals and miRNA expression are involved in cases with higher percentages of 13q- cells. Disclosures: No relevant conflicts of interest to declare.

The efficacy of currently available treatments for Hodgkin's disease (HD) has led to a substantial modification in the prognosis of this disease; nevertheless there is still a group of patients that cannot be cured with conventional treatments and who will be candidates for alternative therapy. In the present work we analysed the prognostic influence of the most relevant clinico-biological characteristics of HD in a consecutive series of 137 patients diagnosed and treated in a single institution. Univariate analyses identified six variables with significant prognostic influence, both on achieving complete remission (CR) and overall survival (OS); LDH > 320 U ml-1, age > 45 years, stages IIB, III and IV, extranodal involvement, alkaline phosphatase > 190 UI dl and ESR > 40 mm h. In addition, Hb < 12.5 gr dl-1 and abdominal disease were statistically relevant for CR while a poor performance score (ECOG > or = 2) affected a lower survival. In the multivariate analysis only LDH, age and the clinical stage retained a significant prognostic influence for achieving CR, while the two first factors above, together with performance status were the variables with independent prognostic value with respect to OS. Moreover, only LDH > 320 U ml-1 had prognostic influence in the probability of relapse and disease free survival (DFS), both in the univariate and multivariate analyses. According to the three independent factors obtained in the multivariate analysis for CR (LDH, age and stage) a predictive model was established that allows the stratification of patients into two prognostic groups: one with poor prognosis that includes patients with the three adverse prognostic factors, or two if one of them was elevated LDH, and the other with good prognosis that includes the remaining patients. This model was also able to separate two independent groups of patients with respect to OS and to DFS. In conclusion, the present study shows that LDH is one of the most important prognostic factors in HD.

Abstract 4051 Multiparameter flow cytometry (MFC) immunophenotyping has shown to be of value for differential diagnosis and minimal residual disease assessment in multiple myeloma. However, the clinical value of MFC immunophenotyping in other plasma cell disorders (PCD) remains largely unexplored. Systemic light chain (AL) amyloidosis is a rare PCD characterized by the accumulation of monoclonal light chain fragments leading to end-organ damage and short survival. Bone marrow (BM) plasma cell (PC) infiltration in AL is usually low and thus the identification of clonal PC can be often difficult by immunohistochemistry and/or immunofluorescence. In the present study we focused on 34 BM samples sent to our institution with a suspected diagnosis of AL. MFC immunophenotypic studies were performed using the following 4-color combinations of MoAbs (FITC/PE/PerCP-Cy5.5/APC): CD38/CD56/CD19/CD45 (n=34); in addition cy-Kappa/cy-Lambda/CD19/CD38 staining was add to confirm the clonal or polyclonal nature of BMPC in equivocal cases. Ploidy and cell cycle analysis were additionally performed in a subset of cases (n=12/34). From the total 34 cases included in the present study, 28 had a confirmed diagnosis of AL. The remaining 6 cases were finally diagnosed with localized - amyloidoma - (n=2) and familial (n=1) forms of amyloidosis, multiple myeloma-associated amyloid (n=2) and congestive pericarditis (n=1). Interestingly, the presence of clonal PC was detected by MFC in 27 of the 28 (96%) patients with AL; in turn, clonal PC were undetectable in the BM of all cases with localized and familial forms of amyloidosis. The median overall level of PC (M-PC plus N-PC) seen in MFC immunophenotypic analyses of BM samples of the 28 patients with AL was 1.9% (range: 0.1% - 15%), with a significant positive correlation between PC enumerated by MFC and conventional morphology (r=0.5; p=.01). Within the BMPC compartment, the median proportion of clonal PC was of 94% (mean 81% ± 29%); in 6 cases all BMPC were clonal while in the remaining 22 patients residual normal PC persisted (median of normal PC/BMPC 13% ± 31%). The most common aberrant phenotypes were down-regulation of CD19 (92%) and CD45 (83%), followed by overexpression of CD56 (56%) and infra-expression of CD38 (42%). Aneuploidy was only found in 18% of cases, all of them hyperdiploid. Cell cycle analysis showed a median % of S-phase and G2-Mitosis PC of 0.7% and 3.5%, respectively. Concerning patients' outcome, cases with undetectable normal PC (6/28, 21%) had a significantly decreased overall survival (OS) compared to patients with persistent BM normal PC at diagnosis (22/28, 79%) with 3-year OS rates of 0% vs. 59%, respectively (p=.001). In summary, these preliminary data suggests that MFC immunophenotyping investigations may be clinically relevant in patients with suspected amyloidosis for i) differential diagnosis between AL and other forms of amyloidosis and, ii) prognostication of patients with AL according to the presence or absence of baseline persistent normal PC. Disclosures: No relevant conflicts of interest to declare.

3Dipartimento di Biotecnologie e Ematologia, Universita La Sapienza, Roma, Italy; 4Clinica Ematologica, Ospedale S. Martino, Universita di Genova, Genova, Italy; 5Cattedra e UO Ematologia e Trapianti midollo, Universita degli Studi di Parma, Parma, Italy; 6UO Ematologia 1, Dipartimento di Ematologia e Oncologia, Ospedale Maggiore, I.R.C.C.S,Milano, Italy; 7Divisione di Ematologia dell’Universita di Torino, A.O. San Giovanni Battista, Torino, Italy; 8Dipartimento di

Monoclonal Gammopathy of Uncertain Significance (MGUS) is a monoclonal disorder defined by the presence of a serum monoclonal protein

In the present review we analyse the current knowledge about the growth properties of AML progenitor cells and their relationship with other clinico-biological characteristics of the disease. Leukaemic colony forming unit L-CFU is considered to be the clonogenic cell in AML and more immature than the blast cell population. Our studies have shown that in leukaemic hematopoiesis colony forming cells can exist among both cell fractions CD34+ and CD34-. Optimal "in vitro" proliferation of L-CFU is dependent upon the addition of exogenous growth factors. However, it has been observed that leukaemic progenitor cells frequently display a certain degree of autonomous proliferation. In order to quantify the "in vitro" behaviour of L-CFU, we have explored 3 parameters: 1) plating efficiency (PE); 2) autonomous growth (AG); and 3) autonomous proliferative index (API) which was calculated as AG divided by PE and we have correlated them with other clinico-biological data. According to the FAB classification we could observe that patients with M3 subtype showed an higher PE than other AML subgroups and a significantly lower API. Regarding CD34 expression we observed that AG was enhanced in CD34+ cases and also in those showing a higher rh123 elimination. In order to determine whether PE could condition clinical evolution, we analysed this parameter in a large series of patients but failed to demonstrate any relationship. By contrast, we observed that patients who displayed a higher API showed a shorter survival than patients with lower API (18% vs 48% surviving at 3 years). We have also shown that abnormalities in the CFU-GM growth pattern could be associated with risk the of relapse in AML patients; a switch from normal to abnormal "in vitro" growth should alert us. But for the assessment of the real value of these analyses sequential follow-up studies are mandatory. In summary, cell culture studies contribute not only to a better understanding of leukaemic hematopoiesis but may also contribute to better disease monitoring.

CONTEXT: Microenvironment of Hodgkin and Reed Sternberg(H-RS) cells is a current focus of interest to define risk and predict evolution of Hodgkin′s Lymphoma. OBJECTIVES: To determine the role of infiltrating CD8+ cells, using flow cytometry (FCM), in the evolution of patients with HL. DESIGN: Cell suspensions obtained after mechanical disintegration of ganglion biopsies of patients with newly diagnosed HL were analyzed using FCM. Definitive diagnosis was made by conventional histology and immunohistochemistry. Clinical data were collected from medical records. Statistical analysis was performed using SPSS 20.0 software. SETTING: In the University Hospital of Salamanca, we consecutively analyzed by FCM all lymph nodes with suspected lymphoma at diagnosis. There was no selection bias when collecting patients, except for some cases with inadequate quality(insufficient cells). PATIENTS OR OTHER PARTICIPANTS: From 1996(earliest available FCM data) to 2014, 104 of that samples had a definitive diagnosis of HL. Treatment depended on stage: a)early diagnosis received ABVD(x3) & local radiotherapy(RT) (20-30 Gy), b)advanced: ABVD(x6 to 8), plus RT in selected cases. Median follow-up was 10 years. INTERVENTIONS :This was a retrospective observational study. MAIN OUTCOMES MEASURES: Primary end points were overall survival(OS) and freedom from treatment failure(FFTF), considered from diagnosis to progression or relapse. RESULTS: Most cells obtained were lymphocytes (Median17.1±86.9%) with a T/B/NK distribution of 72%/27%/1.6%(median) and predominance of CD4+ (median CD4:51%-CD8:12%). Median CD8+ cells(12%) was used to divide patients into 2 groups.FFTF was longer in patients with more than 12% of CD8+cells(93% vs. 71%, p=0.01). When analyzed separately patients with early/advanced disease, the clinical benefit remained in the group with advanced disease (p=0.006), whereas the statistical significance was lost in the group with early disease, possibly due to the excellent prognosis for those patients(FFTF 10 years > 95%). No differences were observed in the OS, because second line therapy was highly effective. In the multivariate analysis using Cox regression, advanced stage (HR=9.6 with 95% CI 1.2 to 73.9) and>12% CD8+ tumor infiltrating T-cells were independent variables(HR=0.26, 95% CI 0.07 to 0.9). CONCLUSIONS: Increased number of CD8+ in the H-RSC microenvironment of HL is associated with better FFTF, particularly in the advanced-disease group. This should be considered as a new biomarker to identify high-risk patients. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.