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Supplementary Figure 8 from E2F1 Expression Is Deregulated and Plays an Oncogenic Role in Sporadic Burkitt's Lymphoma

Current treatments of sporadic Burkitt's lymphoma (sBL) are associated with severe toxicities. A better understanding of sBL formation would facilitate development of less toxic therapies. The etiology of sBL remains, however, largely unknown, C-MYC up-regulation being the only lesion known to occur in all sBL cases. Several studies examining the role of C-MYC in the pathogenesis of BL have concluded that C-MYC translocation is not the only critical event and that additional unidentified factors are expected to be involved in the formation of this tumor. We herein report that a gene distinct from C-MYC, E2F1, is involved in the formation of all or most sBL tumors. We found that E2F1 is highly expressed in Burkitt's lymphoma cell lines and sBL lymphoma specimens. Our data indicate that its elevated expression is not merely the consequence of the presence of more cycling cells in this tumor relative to other cell lines or to other neoplasias. In fact, we show that reduction of its expression in sBL cells inhibits tumor formation and decreases their proliferation rate. We also provide data suggesting that E2F1 collaborates with C-MYC in sBL formation. E2F1 expression down-regulation did not affect, however, the proliferation of human primary diploid fibroblasts. Because E2F1 is not needed for cell proliferation of normal cells, our results reveal E2F1 as a promising therapeutic target for sBL. [Cancer Res 2009;69(9):4052–8]

Supplementary Figure 7 from E2F1 Expression Is Deregulated and Plays an Oncogenic Role in Sporadic Burkitt's Lymphoma

Supplementary Figure 6 from E2F1 Expression Is Deregulated and Plays an Oncogenic Role in Sporadic Burkitt's Lymphoma

Supplementary Figure 9 from E2F1 Expression Is Deregulated and Plays an Oncogenic Role in Sporadic Burkitt's Lymphoma

Supplementary Figures 1-5 from E2F1 Expression Is Deregulated and Plays an Oncogenic Role in Sporadic Burkitt's Lymphoma

Although next-generation sequencing (NGS) data on lymphomas require further validation before being implemented in daily practice, the clinical application of NGS can be considered right around the corner. The aim of our study was to validate an NGS lymphoid panel for tissue and liquid biopsy with the most common types of non-Hodgkin’s lymphoma [follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL)]. In this series, 372 somatic alterations were detected in 93.6% (44/47) of the patients through tissue biopsy. In FL, we identified 93 somatic alterations, with a median of 7.4 mutations per sample. In DLBCL, we detected 279 somatic variants with a median of 8.6 mutations (range 0–35). In 92% (24/26) of the cases, we were able to detect some variant in the circulating tumor DNA. We detected a total of 386 variants; 63.7% were detected in both types of samples, 13.2% were detected only in the circulating tumor DNA, and 23% were detected only in the tissue biopsy. We found a correlation between the number of circulating tumor DNA mutations, advanced stage, and bulky disease. The genetic alterations detected in this panel were consistent with those previously described at diagnosis. The liquid biopsy sample is therefore a complementary tool that can provide new genetic information, even in cases where a solid biopsy cannot be performed or an insufficient sample was obtained. In summary, we describe and analyze in this study the findings and difficulties encountered when incorporating liquid biopsy into clinical practice in non-Hodgkin’s lymphoma at diagnosis.

Plasmablastic lymphoma (PBL) represents a rare and aggressive lymphoma subtype frequently associated with immunosuppression. Clinically, patients with PBL are characterized by poor outcome. The current understanding of the molecular pathogenesis is limited. A hallmark of PBL represents its plasmacytic differentiation with loss of B-cell markers and, in 60% of cases, its association with Epstein-Barr virus (EBV). Roughly 50% of PBLs harbor a MYC translocation. Here, we provide a comprehensive integrated genomic analysis using whole exome sequencing (WES) and genome-wide copy number determination in a large cohort of 96 primary PBL samples. We identify alterations activating the RAS-RAF, JAK-STAT, and NOTCH pathways as well as frequent high-level amplifications in MCL1 and IRF4. The functional impact of these alterations is assessed using an unbiased shRNA screen in a PBL model. These analyses identify the IRF4 and JAK-STAT pathways as promising molecular targets to improve outcome of PBL patients., Plasmablastic lymphoma (PBL) is an aggressive lymphoma subtype characterized by poor prognosis but the molecular knowledge of the disease is limited. Here, the authors perform whole exome sequencing and copy number determination of primary samples highlighting IRF4 and JAK-STAT pathways as therapeutic targets for PBL.

Introduction: Chimeric antigen receptor (CAR) T-Cell therapy is approved for relapse/refractory diffuse large B cell lymphoma (DLBCL) patients after two lines of therapy. Although it is an effective treatment, approximately 20-30% of patients have an early relapse. In this context, biomarkers that helps to identify those patients who will be refractory to this therapy are relevant. Cell-free DNA (cfDNA) has emerged as a new tool for non-invasive monitoring of patients with lymphoma, therefore the aim of this study was to evaluate dynamic cfDNA concentration in addition to other biomarkers (LDH, CRP, ferritin) before CAR T-cell infusion to detect early progressors. Methods: We selected 44 r/r DLBCL patients treated with CAR T-cell in our centre. Plasma samples were collected pre-apheresis (PA) and pre-infusion (PI)(∆cfDNA). Other biomarkers (LDH, CRP, Ferritin) were studied PA, pre-lymphodepletion (PL) and PI, tumour volume metabolic (TMV) are also studied. CfDNA were obtained from plasma using QIAamp® Circulating Nucleic Acid (Qiagen) and quantified by QuantiFluor dsDNA System (Promega). The Mann-Whitney test was used to compare differences between two independent quantitative variables . ROC analysis was conducted to determine the cut-off value of biomarkers. Cumulative incidence of progression (1 and 3 months) was calculated from the date of CAR T-cell infusion. Data analysis was performed using Stata. Results: Cumulative incidence of relapse at 1 month and 3 months was 20% and 30% respectively. The correlation between the progression (1 month, 3 months) and the different biomarkers is shown in Table 1. The CRP PL, CRP PI, LDH PI and ∆cfDNA (PI-PA, median time 41.5 days [range:31-107]) was correlated with early progression (1 month). No differences were found with progression at 3 months. The different cut-off for the biomarkers selected was 9 ng/mL for ∆cfDNA, 225 U/L for LDH and 1.35 mg/dL for CRP. Cumulative incidence of progression at 1 month was calculated for these biomarkers (figure 1): ∆cfDNA, HR: 4.3 (1.05-17), p=0.042; CRP PL: HR: 6.9 (1.5-31.1), p=0.012; CRP PI, HR: 11.2 (1.5-83), p=0.019 and LDH PI, HR: 3.4 (1-17) p=0.11. It should be noted that 83.3% (10/12) of the patients who progressed during the first month had at least one of the above variables. Conclusions: Our results highlight that increase in cfDNA higher than 9 ng/mL, CRP PL and PI >1.35 mg/dL and LDH PI > 225 U/L before CAR T-cell therapy may detect early progressors within the first month after treatment. Therefore, ∆cfDNA, CRP and LDH could be useful to identify patients who highly probable will not benefit from the CAR T infusion, in which another prior strategy could be considered in an attempt to control the disease. These results need to be confirmed with a larger cohort. Figure 1 Figure 1. Disclosures Bailén: Pfizer: Honoraria; Kite-Gilead: Honoraria; Gilead: Honoraria. Kwon: Gilead: Honoraria.

Follicular lymphoma (FL) is an indolent but largely incurable disease. Some patients suffer histological transformation to a more aggressive subtype with poorer prognosis. This study aimed to improve our understanding of the genetics underlying FL histological transformation, and to identify genetic drivers or promoters of the transformation by elucidating the differences between FL samples from patients who did and did not transform. We conducted targeted massive parallel sequencing of 22 pre-transformed FL/transformed diffuse large B-cell lymphoma pairs and 20 diagnostic samples from non-transformed FL patients. Additionally, 22 matched samples from 11 transformed FL patients (pre-transformed FL and diffuse large B-cell lymphoma) and 9 non-transformed FLs were studied for copy number variation using SNP arrays. We identified recurrently mutated genes that were enriched at transformation, most notably LRP1B, GNA13 and POU2AF1, which have roles in B-cell differentiation, GC architecture and migration. Mutations in POU2AF1 might be associated with lower levels of expression, were more frequent in transformed FLs, and seemed to be specific to transformed- compared with de novo-diffuse large B-cell lymphomas. Pre-transformed FLs carried more mutations per sample and had greater subclonal heterogeneity than non-transformed FLs. Finally, we identified four mutated genes in FL samples that differed between patients who did and did not transform: NOTCH2, DTX1, UBE2A and HIST1H1E. The presence of mutations in these genes was associated with shorter time to transformation when mutated in the FL biopsies. This information might be useful for identifying patients at higher risk of transformation.

Aim Lymphoplasmacytic lymphoma (LPL) is an indolent mature B-cell-neoplasm with involvement of the bone marrow. At least 90% of LPLs carry MYD88-L265P mutation and some of them (~10%) transform into diffuse large B-cell-lymphoma (DLBCL). Material and methods Over the past 15 years we have collected 7 cases where the both LPL and DLBCL were diagnosed in the same patient. Clinical records, analytical data and histopathological specimens were reviewed. FISH studies on paraffin-embedded tissue for MYC, BCL2 and BCL6 genes were performed, as well as MYD88-L265P mutation and IGH rearrangement analysis by PCR. A mutational study was done by massive next generation sequencing (NGS). Results There were 4 women and 3 men between 36–91 years of age. Diagnoses were made simultaneously in 4 patients. In two cases the LPL appeared before the DLBCL and in the remaining case the high-grade component was discovered 5 years before the LPL. In 6 cases both samples shared the MYD88-L265P mutation. IGH rearrangement analysis showed overlapping features in two of 6 cases tested. Mutational study was evaluable in three cases for both samples showing shared and divergent mutations. Conclusions These data suggest different mechanisms of DLBCL development in LPL patients.

Introduction: Non-Hodgkin B lymphomas (NHL-B) are a large group of heterogeneous diseases, with well-defined, histological and clinical features. Currently, the lymph node biopsy is essential for diagnosis, often obtained from hard to reach areas. Our aims with this study was to analyze genetics variants at diagnosis trying to discriminate between different NHL-B histologic subtypes using formalin fixed paraffin embedded (FFPE) tissue sections and circulating tumor free DNA (ctDNA) samples. Material and Methods: This is a retrospective, cross-sectional, single-center study. Sixty patients were selected with a diagnosis of different NHL-B subtypes: Diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), marginal lymphoma (ML), mantle cell lymphoma (MCL) and plasmablastic lymphoma (PL). Sixty FFPE tissue samples and 23 ctDNA specimens obtained at diagnosis were selected. We performed an enrichment panel of 54 genes recurrently mutated in lymphomas (Lymphoma Solution; Sophia Genetics) by next generation sequencing (NGS; NextSeq; Illumina). The depth of 90% of the readings was greater than 2600x. Quantitative variables were expressed as median and range. Categorical variables were expressed as frequency and percentage. The Fisher exact test was used to compare the distribution of categorical variables. The Mann-Whitney test was used to compare differences between quantitative variables. Statistical significance was set at p < 0.05. Results: Our study shows that 97% (58/60) of patients presented any variant. The most frequently mutated genes were KMT2D, EP300 and SOCS1. Exclusive variants were detected in FL in the genes CCND1, PAX5, CREBBP, BCL2 and REL genes. In the same way, in DLCBL the variants detected in the BRAF, TCF3, XPO1, PIM1, CHD2, ID3 and BCL6 genes were exclusive of this subtype. Furthermore, the genes BCL2 (p=0.0021), CREBBP (p=0.0004), NOTCH2 (p=0.03), PAX5 (p=0.01) and TNFRSF14 (p=0.04) are more frequently altered in patients with FL, ATM (p=0.02) gene in MCL; NFKBIE (p=0.03), CIITA (p=0.02), MYC (p=0.009) and PIM1 (p=0.04) in DLCBL, among which it was found that high-grade lymphomas are more frequently associated with mutations in CHD2 (p=0.02), MYC (p=0.03) and MYD88 (p=0.02; data not shown). In the subgroup of 23 patients with paired samples (FFPE/ctDNA), 95% (22/23) of patients had mutations in any of the genes on the panel in FFPE tissue samples and 82% (19/23) in ctDNA specimens. The number of variants detected was different depending on the type of sample analysed, 52% of the variants were detected in both specimens, 39 only in FFPE tissue and 9% in ctDNA; Figure 1). In variants detected in both samples, the average value of the VAF was higher that when is detected only in one of the samples (FFPE: 28.1 vs 5.8 (p We did not find significant differences in the number of variants depending on the stage, classification, tumour burden or bulky mass (Table 1). Conclusion: In our study, we were able to identify genetic variables that could characterize different histological groups of NHL-B in FFPE tissue and ctDNA samples, by using a commercial gene panel of NGS. The mutational profile obtained from ctDNA and FFPE tissue is comparable in all histological subtypes, regardless tumour burden, aggressiveness or stage, throwing each one of them complemental information. These results support the hypothesis that could be possible to distinguish different histologies through non-invasive strategies, specially oriented to lesions where obtaining a tissue sample is difficult. We are working on the development of clinical-genetic algorithms that allow us to achieve our goal. Disclosures No relevant conflicts of interest to declare.

Mata et al., Defining the mutational landscape of classic Hodgkin lymphoma is still a major research goal. New targeted next-generation sequencing (NGS) techniques may identify pathogenic mechanisms and new therapeutic opportunities related to this disease. We describe the mutational profile of a series of 57 cHL cases, enriched in Hodgkin and Reed-Sternberg (HRS) cells. Overall, the results confirm the presence of strong genomic heterogeneity. However, several variants were consistently detected in genes related to relevant signaling pathways, such as GM-CSF/IL-3, CBP/EP300, JAK/STAT, NF-kappaB, and numerous variants of genes affecting the B-cell receptor (BCR) pathway, such as BTK, CARD11, BCL10, among others. This unexpectedly high prevalence of mutations affecting the BCR pathway suggests some requirement for active BCR signaling for cHL cell viability. Additionally, incubation of a panel of cHL cellular models with selective BTK inhibitors in vitro constrains cell proliferation and causes cell death. Our results indicate new pathogenic mechanisms and therapeutic opportunities in this disease., This work was supported by grants from the Plan Nacional de I+D+I co-financed by the ISCIII-Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER), PI12/1832, RTICC RD06/0020/0107, PI14/00221, CIBER de Cancer, PIE15/0081 and PI16/01294, the Spanish Association for Cancer Research (AECC), and by funds of the U.T M.D. Anderson Cancer Center. MSB currently holds a Miguel Servet contract (CP11/00018 and CPII16/00024) from the ISCIII- MINECO-AES-FEDER (P.N.I+D+I 2008-2011), Spain.

A consensus based on Delphi methodology was developed to produce a guide for the evaluation and reporting of bone marrow biopsies in patients with a clinical suspicion of myeloproliferative neoplasm with fibrosis. Ten expert haematopathologists formulated a questionnaire including: clinical and laboratory data required before regarding a biopsy suspicious for primary myelofibrosis (PMF), descriptive aspects to be reported and the main histopathological differential diagnoses to be considered. It was circulated among 37 hematopathologists and consensus was defined when more than 70% of the experts "strongly agreed" or "agreed" after two rounds. The recommendations gave rise to a proposal for a standardized diagnostic report form to aid in the diagnostic workup and homogeneous reporting of cases with a clinical suspicion of PMF.

Funding: this study was supported by grants from the Asociacion Espanola contra el Cancer (AECC), Ministerio de Economia y Competitividad (MINECO) (SAF2013-47416-R), Instituto Salud Carlos III (ISCIII) – Fondos de Investigacion Sanitaria (RD06/0020/0107, RD012/0036/0060 and FIS11/1759). The Instituto de Investigacion Marques de Valdecilla (IDIVAL) is partly funded by the Sociedad para el Desarrollo Regional de Cantabria (SODERCAN)

Crimean-Congo hemorrhagic fever (CCHF) is a widely distributed, viral, tickborne disease. In Europe, cases have been reported only in the southeastern part of the continent. We report two autochthonous cases in Spain. The index patient acquired the disease through a tick bite in the province of Ávila - 300 km away from the province of Cáceres, where viral RNA from ticks was amplified in 2010. The second patient was a nurse who became infected while caring for the index patient. Both were infected with the African 3 lineage of this virus. (Funded by Red de Investigación Cooperativa en Enfermedades Tropicales [RICET] and Efficient Response to Highly Dangerous and Emerging Pathogens at EU [European Union] Level [EMERGE].). Supported by Red de Investigación Cooperativa en Enferme-dades Tropicales (RICET grant, RD12/0018/0023) and EMERGE (Efficient Response to Highly Dangerous and Emerging Patho-gens at EU [European Union] Level), a joint action funded under the third EU Health Program (677066),.Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.We thank Julio Farias of the Pathology Department at Gregorio Marañón University General Hospital and Maria José Buitrago and the rest of the members of the Grupo de Respuesta Rápida of the Centro Nacional de Microbiología for their help. Sí

Part of this study has been reported as an oral presentation at the EHA Meeting in Vienna 2015., The introduction of Rituximab has improved the outcome and survival rates of Burkitt lymphoma (BL). However, early relapse and refractoriness are current limitations of BL treatment and new biological factors affecting the outcome of these patients have not been explored. This study aimed to identify the presence of genomic changes that could predict the response to new therapies in BL. Forty adolescent and adult BL patients treated with the Dose-Intensive Chemotherapy Including Rituximab (Burkimab) protocol (Spanish Programme for the Study and Treatment of Haematological Malignancies; PETHEMA) were analysed using array-based comparative genomic hybridization (CGH). In addition, the presence of TP53, TCF3 (E2A), ID3 and GNA13 mutations was assessed by next-generation sequencing (NGS). Ninety-seven per cent of the patients harboured genomic imbalances. Losses on 11q, 13q, 15q or 17p were associated with a poor response to Burkimab therapy (P = 0·038), shorter progression-free survival (PFS; P = 0·007) and overall survival (OS; P = 0·009). The integrative analysis of array-CGH and NGS showed that 26·3% (5/19) and 36·8% (7/19) of patients carried alterations in the TP53 and TCF3 genes, respectively. TP53 alterations were associated with shorter PFS (P = 0·011) while TCF3 alterations were associated with shorter OS (P = 0·032). Genetic studies could be used for risk stratification of BL patients treated with the Burkimab protocol., This work was supported in part by: grant no 306242-NGS-PTL from European Union’s Seventh Framework Programme (FP7/2007-2013), Fundacion-Castellano-Leonesa de Hematologia-y-Hemoterapia (FUCALHH 2013), Consejeria de Educacion, Junta de Castilla y León (HUS272U13), SACYL research projects: GRS1172/A/15, GRS 994/A/14, BIO/SA10/14, BIO/SA31/13. Fondo de Investigaciones Sanitarias FISPI09/01543, FIS-PI12/00281, PI14/01971, COST-ActionEuGESMA (BM0801), Fundacion Española de Hematologia y Hemoterapia (FEHH), and by grants RD12/0036/0069, RD12/ 0036/0029 and RD12/0036/0044 from the Red Tematica de Investigacion Cooperativa en Cancer (RTICC), Instituto de Salud Carlos III (ISCIII), the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (ERDF) ‘Una manera de hacer Europa’ (Innocampus). NGS was performed as part of the IRON-II collaborative network of haematological laboratories. MFC was supported by study commission no 223-2011 granted by the UPTC, Colombia. MHS was supported by Junta de Castilla y Leon from the ERDF.

The incidence of non-Hodgkin's lymphoma and Hodgkin's lymphoma is higher in patients with HIV infection than in the general population. Following the introduction of combination antiretroviral therapy (cART), the prognostic significance of HIV-related variables has decreased, and lymphoma-related factors have become more pronounced. Currently, treatments for lymphomas in HIV-infected patients do not differ from those used in the general population. However, differentiating characteristics of seropositive patients, such as the need for cART and specific prophylaxis and treatment of certain opportunistic infections, should be considered. This document updates recommendations on the diagnosis and treatment of lymphomas in HIV infected patients published by GESIDA/PETHEMA in 2008.

Background Plasmablastic lymphoma has recently come to be considered a distinct entity among mature B cell neoplasms, although the limits with diffuse large B-cell lymphoma (DLBCL) need to be more accurately defined.Design and Methods Here we show the results of an immunohistochemical study of 35 cases of plasmablastic lymphoma compared with a set of 111 conventional DLBCLs.Results Our results demonstrate that the use of a limited combination of immunohistochemical markers (PAX5&CD20, PRDM1/BLIMP1 and XBP1s) enables the identification of a plasmablastic immunophenotype highly characteristic of plasmablastic lymphoma cases and associated with an aggressive clinical behavior. Additionally, the study shows that the acquisition of a partial plasmablastic phenotype (PRDM1/BLIMP1 expression) in DLBCL is associated with shorter survival in R-CHOP-treated patients.Conclusions The use of a restricted combination of immunohistochemical markers (PAX5&CD20, PRDM1/BLIMP1 and XBP1s) enables a more accurate definition of terminal differentiation for large B-cell lymphoma.

Current treatments of sporadic Burkitt's lymphoma (sBL) are associated with severe toxicities. A better understanding of sBL formation would facilitate development of less toxic therapies. The etiology of sBL remains, however, largely unknown, C-MYC up-regulation being the only lesion known to occur in all sBL cases. Several studies examining the role of C-MYC in the pathogenesis of BL have concluded that C-MYC translocation is not the only critical event and that additional unidentified factors are expected to be involved in the formation of this tumor. We herein report that a gene distinct from C-MYC, E2F1, is involved in the formation of all or most sBL tumors. We found that E2F1 is highly expressed in Burkitt's lymphoma cell lines and sBL lymphoma specimens. Our data indicate that its elevated expression is not merely the consequence of the presence of more cycling cells in this tumor relative to other cell lines or to other neoplasias. In fact, we show that reduction of its expression in sBL cells inhibits tumor formation and decreases their proliferation rate. We also provide data suggesting that E2F1 collaborates with C-MYC in sBL formation. E2F1 expression down-regulation did not affect, however, the proliferation of human primary diploid fibroblasts. Because E2F1 is not needed for cell proliferation of normal cells, our results reveal E2F1 as a promising therapeutic target for sBL. [Cancer Res 2009;69(9):4052–8]

Follicular lymphomas (FLs) usually carry BCL2 translocations although BCL6 translocations are also present. We explored relationships between translocations status and clinical or histological parameters at diagnosis in 182 patients stratified in four groups: BCL2-/BCL6-, BCL2+/BCL6-, BCL2-/BCL6+ and BCL2+/BCL6+. BCL2-/BCL6- and BCL2+/BCL6-. Double negative cases were ascribed to lower histological grades. In contrast, BCL2-/BCL6+ cases corresponded to higher grades. However, a majority of BCL2+/BCL6+ tumours were classified as lower grades. These results were reinforced by the finding that double positive patients had lower LDH levels and PS than those with solitary BCL6 rearrangements. Bone marrow involvement was more frequent in BCL2+/BCL6+ compared with BCL2-/BCL6+ tumours. Our data confirm the presence of a relationship between histological grade and translocation status, suggesting that FLs carrying BCL6 translocations probably constitute a special biological subtype. Clinical and histological differences between BCL2-/BCL6+ and BCL2+/BCL6+ tumours could reflect an interplay between both translocations.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western world. Pathogenic mechanisms involve multiple external events (such as microenvironmental and antigenic stimuli) and internal events (genetic and epigenetic alterations) that are associated with the transformation, progression and evolution of CLL. CLL is characterized by an accumulation of mature B cells in peripheral blood, bone marrow and lymphoid tissues. Extracellular stimuli play an important role in the development and maintenance of neoplastic cells. B-CLL cells proliferate and activate pathogenic signaling pathways in anatomical structures known as proliferation centers, which are usually more conspicuous in involved lymph nodes.1 Its clinical course is quite heterogeneous, whereby some patients progress rapidly and have short survival, whereas others have a more stable clinical course that may not need treatment for years. This work was supported by grants from the Ministerio de Economía y Competitividad (MINECO) (SAF2013-47416-R) Instituto de Salud Carlos III (ISCIII)- FEDER – MINECO- AES (CP11/00018, PI10/00621, RD012/0036/0060), and Asociación Española contra el Cancer (AECC). MS-B is supported by a Miguel Servet contract from ISCIII-FEDER (CP11/00018). Salary support to SG is provided by CP11/00018, from ISCIII-FEDER. JG-R is supported by a predoctoral grant from the Fundación Investigación Puerta de Hierro. Sí

Around 20% to 30% of patients with Hodgkin lymphoma (HL) do not benefit from standard therapies and finally succumb to their disease. The factors that influence the outcome of HL have not been elucidated, underscoring the demand for the identification of biologic risk factors and new therapeutic targets. We analyzed the gene expression profiles of samples from 29 patients with advanced classic HL treated with standard therapy and compared the expression profiles of patients with favorable and unfavorable clinical outcome. Using supervised methods, we identified 145 genes associated with outcome, which were grouped into 4 signatures representing genes expressed by either the tumoral cells (genes involved in the regulation of mitosis and cell growth/apoptosis) or the tumor microenvironment. The relationship between the expression of 8 representative genes and survival was successfully validated in an independent series of 235 patients by quantification of protein expression levels on tissue microarrays. Analysis of centrosomes and mitotic checkpoint confirmed the existence of an abnormal transition through mitosis in HL cells. Therefore, genes related to tumor microenvironment, cell growth/apoptosis, and regulation of mitosis are associated with treatment response and outcome of patients with HL.

Currently, there is no efficient therapy for patients with peripheral T cell lymphoma (PTCL). The Proviral Integration site of Moloney murine leukemia virus (PIM) kinases are important mediators of cell survival. We aimed to determine the therapeutic value of PIM kinases because they are overexpressed in PTCL patients, T cell lines and primary tumoral T cells. PIM kinases were inhibited genetically (using small interfering and short hairpin RNAs) and pharmacologically (mainly with the pan-PIM inhibitor (PIMi) ETP-39010) in a panel of 8 PTCL cell lines. Effects on cell viability, apoptosis, cell cycle, key proteins and gene expression were evaluated. Individual inhibition of each of the PIM genes did not affect PTCL cell survival, partially because of a compensatory mechanism among the three PIM genes. In contrast, pharmacological inhibition of all PIM kinases strongly induced apoptosis in all PTCL cell lines, without cell cycle arrest, in part through the induction of DNA damage. Therefore, pan-PIMi synergized with Cisplatin. Importantly, pharmacological inhibition of PIM reduced primary tumoral T cell viability without affecting normal T cells ex vivo. Since anaplastic large cell lymphoma (ALK+ ALCL) cell lines were the most sensitive to the pan-PIMi, we tested the simultaneous inhibition of ALK and PIM kinases and found a strong synergistic effect in ALK+ ALCL cell lines. Our findings suggest that PIM kinase inhibition could be of therapeutic value in a subset of PTCL, especially when combined with ALK inhibitors, and might be clinically beneficial in ALK+ ALCL. This work was supported by grants from the Asociación Española Contra el Cáncer, Fondo de Investigaciones Sanitarias (PI051623, PI052800 and FIS11/1759), RTICC (RD06/0020/0107) and Ministerio de Ciencia e Innovación (SAF2008-0387-1). EMS was supported by a grant from the Department of Education, Universities and Research of the Basque Government (BFI08.207). MSB was supported by a Contract Miguel Servet from Fondo de Investigaciones Sanitarias (CP11/00018).

Splenic marginal zone lymphoma (SMZL) is a newly recognized lymphoma type whose precise molecular pathogenesis is still essentially unknown. This hampers differential diagnosis with other small B-cell malignancies. With the aim of characterizing this tumor more comprehensively, and of identifying new diagnostic and prognostic markers, we performed cDNA microarray expression profiling and tissue microarray (TMA) immunohistochemical studies in a relatively large series of 44 SMZLs. The results were related to immunoglobulin heavy chain variable region (IgVH) mutational status and clinical outcome. SMZLs display a largely homogenous signature, implying the existence of a single molecular entity. Of the genes deregulated in SMZLs, special mention may be made of the genes involved in B-cell receptor (BCR) signaling, tumor necrosis factor (TNF) signaling and nuclear factor-κB (NF-κB) activation, such as SYK, BTK, BIRC3, TRAF3, and LTB. Other genes observed were SELL and LPXN, which were highly expressed in spleen, and lymphoma oncogenes, such as ARHH and TCL1. In contrast, the genes CAV1, CAV2, and GNG11 located in 7q31, a commonly deleted area, were down-regulated in the entire series. A comparison with the genes comprising the signature of other small B-cell lymphomas identified 3 genes whose expression distinguishes SMZL, namely ILF1, SENATAXIN, and CD40. Shorter survival was associated with CD38 expression, naive IgVH genes, and the expression of a set of NF-κB pathway genes, including TRAF5, REL, and PKCA. (Blood. 2005;106:1831-1838)

Purpose: Based on previous preliminary observations, we hypothesize that the molecular and clinical variability of chronic lymphocytic leukemia (CLL) reflects differences in the degree of nuclear factor (NF)-κB activation, as determined by the expression of phosphorylated IκBα (p-IκBα).Experimental Design: The expression profile (mRNA and protein expression) was analyzed with the Centro Nacional de Investigaciones Oncológicas Oncochip, a cDNA microarray containing 6386 cancer-related genes, and a tissue microarray (TMA). The results were correlated with the IgVH mutational status, ZAP-70 expression, cytogenetic alterations, and clinical outcome.Results: We found correlations between the presence of p-IκBα, a surrogate marker of NF-κB activation, and changes in the expression profile (mRNA and protein expression) and clinical outcome in a series of CLL cases with lymph node involvement. Activation of NF-κB, as determined by the expression of p-IκBα, was associated with the expression of a set of genes comprising key genes involved in the control of B-cell receptor signaling, signal transduction, and apoptosis, including SYK, LYN, BCL2, CCR7, BTK, PIK3CD, and others. Cases with increased expression of p-IκBα showed longer overall survival than cases with lower expression. A Cox regression model was derived to estimate some parameters of prognostic interest: IgVH mutational status, ZAP-70, and p-IκBα expression. The multivariate analysis disclosed p-IκBα and ZAP-70 expression as independent prognostic factors of survival.Conclusions: A variable degree of activation of NF-κB, as determined by the expression of p-IκBα, is an identifiable event in CLL, and is correlated with changes in the expression profile and overall survival.

Only a few series of splenic large B-cell lymphoma have been previously reported, including limited immunophenotypic studies and clinical data. Here we review the clinical data, morphology, and immunophenotype of series of 33 cases of large B-cell lymphoma presenting in the spleen. Three main groups of tumors are identified. Group A was characterized by macronodular tumors (20 cases), with predominantly stage I disease and a favorable clinical outcome. All cases were bcl6 positive. Group B was characterized by a micronodular pattern (nine cases), including a subset with T-cell-rich B-cell lymphoma features. Most of the patients in this group were diagnosed at advanced clinical stages and died of the disease. All cases were bcl6 positive. Group C was characterized by diffuse red pulp infiltration (four cases) and advanced clinical stages and showed an aggressive behavior. All but one case were bcl6 positive. The results of this series define a characteristic type of large B-cell lymphoma presenting in the spleen as a tumoral mass, associated with a relatively favorable clinical course. Additionally, they provide evidence that clinical presentation as a tumor confined to the spleen and the hilar lymph nodes is associated with lower aggressivity.

Chromosomal aberrations, the consequence of refractory hyperparathyroidism: Its relationship with biochemical parameters.BackgroundIt has been shown that refractory hyperparathyroidism (HPT) correlates biologically with a monoclonal true neoplasm, but the chromosomal changes and their relationship with biochemical variables such as high levels of phosphate, low levels of calcium (Ca), and calcitriol deficiency are still in need of a deeper analysis.MethodsComparative genomic hybridization was used to scan for DNA copy number changes in two groups of samples: 57 glands from refractory secondary HPT and 28 glands from refractory HPT after kidney transplantation. Biochemical HPT-related parameters from these patients were collected and analyzed.ResultsSixty-one percent of the glands from dialysis patients and 53.6% of the glands from transplanted patients suffering severe secondary hyperparathyroidism had clonal chromosomal imbalances. Losses were far more common than gains. The most recurrent changes were losses of 1p (71%), monosomies of chromosomes 19 and 22 (45%), and losses of 20q (44%) and 16p (42%). The most frequent gains were 5q, 6q, and 13q. Biochemical parameters suggested that Ca excess is related to the development of these chromosomal aberrations, although it is not known if it is by playing a role in producing the alterations or merely as a reflection of HPT severity. Phosphate levels, despite their known effect in increasing the proliferation of the parathyroid glands, were not related to the chromosomal aberrations found in severe secondary HPT.ConclusionClonal recurrent chromosomal changes are present in more than half of the glands from patients with refractory HPT, which undergo extreme biochemical levels in hyperparathyroidism effectors. These changes support the idea of the monoclonal neoplastic nature of this disorder.

Comparative study of the expression of proteins involved in the cell cycle in renal secondary hyperparathyroidism. Background In renal hyperparathyroidism, parathyroid cell proliferation seems to play a key role in the progression of the disease. Therefore, G1/S transition, a main cell cycle regulatory step, could be deregulated in these patients. Methods One hundred and one parathyroid glands, taken from parathyroidectomies performed on 41 patients on hemodialysis (HD), and 15 glands, taken from 7 patients with post-transplantation persistent hyperparathyroidism (HPT), were studied. Twelve normal parathyroid (PT) glands were used as the control. Biochemical data, immunohistochemical (IHC) profiles of G1/S transition regulators belonging to the two main pathways (cyclin D1/p16 INK4A /pRb and p14 ARF /p53/MDM2), and proliferation rate (Ki67) were correlated. Results All of the other proteins differed from normal IHC profiles in both groups that showed significant higher proliferating rates, decreases in p27 KIP1 , pRb, and cyclin D1, as well as increases in p16 INK4A , p53, MDM2, and p21 WAF1 levels, in comparison with normal PT glands, with the exception of cyclin D3. Contrary to patients with HPT who were on hemodialysis, in post-transplantation HPT, consistent correlations between biochemical data and IHC profiles were obtained. Conclusion In both groups IHC profiles of proteins involved in G1/S transition regulation significantly differed from normal PT glands. The results support partial reversion to normal IHC profile in post-transplantation HPT.

Tumoral cells in Hodgkin lymphoma (HL) display an increased growth fraction and diminished apoptosis, implying a profound disturbance of the cell cycle and apoptosis regulation. However, limitations of molecular techniques have prevented the analysis of the tumor suppressor pathways and cell-cycle checkpoints. Tissue microarray (TMA) is a powerful tool for analyzing a large number of molecular variables in a large series of tumors, although the feasibility of this technique has not yet been demonstrated in heterogeneous tumors. The expression of 29 genes regulating the cell cycle and apoptosis were analyzed by immunohistochemistry and in situ hybridization in 288 HL biopsies using TMA. The sensitivity of the technique was validated by comparing the results with those obtained in standard tissue sections. The results revealed multiple alterations in different pathways and checkpoints, including G1/S and G2/M transition and apoptosis. Striking findings were the overexpression of cyclin E, CDK2, CDK6, STAT3, Hdm2, Bcl2, Bcl-X(L), survivin, and NF-kappaB proteins. A multiparametric analysis identified proteins associated with increased growth fraction (Hdm2, p53, p21, Rb, cyclins A, B1, D3, and E, CDK2, CDK6, SKP2, Bcl-X(L), survivin, STAT1, and STAT3), and proteins associated with apoptosis (NF-kappaB, STAT1, and RB). The analysis also demonstrated that Epstein-Barr virus (EBV)-positive cases displayed a characteristic profile, confirming the pathogenic role of EBV in HL. Survival probability depends on multiple biologic factors, including overexpression of Bcl2, p53, Bax, Bcl-X(L), MIB1, and apoptotic index. In conclusion, Hodgkin and Reed-Sternberg cells harbor concurrent and overlapping alterations in the major tumor suppressor pathways and cell-cycle checkpoints. This appears to determine the viability of the tumoral cells and the clinical outcome.

Multiple endocrine neoplasia type 2A (MEN 2A) is associated with specific germline missense mutations in the RET proto-oncogene. This locus encodes a receptor tyrosine kinase whose activation requires the formation of a multimeric receptor complex including GDNF as a ligand and GFR alpha 1 as a coreceptor. In order to explore the role of RET, GFR alpha 1 and GDNF genes in the variation of phenotypes observed in MEN2A families, we analysed germline mutations of these genes in 4 unrelated Spanish MEN2A families (23 cases studied). We found 2 novel variants corresponding to a single change in position + 47 (intron 12) of RET and position +22 (intron 7) of GFR alpha 1. Furthermore, we observed strong co-segregation between 2 polymorphisms of RET [G691S (exon 11) and S904S (TCC-TCG, exon 15) (100%, Fisher's exact test, p< 0.001)]. More interestingly, we found that these polymorphisms occurred at a significantly high frequency in patients with age at onset < 20 years old (Kruskal-Wallis's and Fisher's exact test, p = 0.007). These findings suggest that the G691S and S904S variants of RET may somehow play a role on the age of onset of MEN 2A.

Splenic marginal zone lymphoma (SMZL) is a B-cell neoplasm whose molecular pathogenesis remains fundamentally unexplained, requiring more precise diagnostic markers. Previous molecular studies have revealed 7q loss and mutations of nuclear factor κB (NF-κB), B-cell receptor (BCR) and Notch signalling genes. We performed whole-exome sequencing in a series of SMZL cases. Results confirmed that SMZL is an entity distinct from other low-grade B-cell lymphomas, and identified mutations in multiple genes involved in marginal zone development, and others involved in NF-κB, BCR, chromatin remodelling and the cytoskeleton.

Aims The diagnosis of primary myelofibrosis (PMF) strongly relies on the bone marrow biopsy findings, but a report model has not been standardised. Our aim was to establish general recommendations for bone marrow evaluation and standardised reporting in a case suspicious of PMF. Methods The Delphi method was employed to obtain expert consensus. An advisory panel of 10 leading members identifies a total of 37 haematopathology experts to participate. The first Delphi round included a questionnaire with three main groups of items: minimal clinical and laboratory data considered necessary before reporting, minimal descriptive aspects to record and main histological differential diagnosis. The final report content was based on consensus obtained after the second Delphi round. Results The minimal data considered necessary were age, splenomegaly, haemoglobin, leucocyte and platelet counts, differential blood cell count, leucoerythroblastic blood picture, lactate dehydrogenase (LDH) level, BCR-ABL and JAK2 mutational status, reticulin stain and the internal control for the reticulin staining. The minimal descriptive aspects to report were cellularity, osteosclerosis, megakaryocytic morphology and localisation, dense megakaryocytic clusters, quantity of granulocytic precursors, grade of myelofibrosis in a scale of 4, and a proposed final diagnostic approach. The entities to be considered for differential diagnosis were mainly the other classical chronic myeloproliferative neoplasms. Conclusions The Delphi method is a robust tool to determine essential information to be included in a pathology report. A standardised good-quality histopathological report form may help to homogenise PMF diagnosis. A close collaboration between the pathologist and the haematologist is desirable according to our survey.

Parathyroid adenomas (PTAs) are the main cause of primary hyperparathyroidism. Cell cycle regulation in normal parathyroid tissue (NPT) and PTA remains largely unknown. We have systematically explored several components involved in the p53/MDM2/p19ARF pathway in PTA and compared the results were with NPT. Forty-six PTA and 12 NPT were immunostained with anti-p21WAF-1, MDM2, p53, and p27KIP1 antibodies. The slides were processed by cytometry and the results were statistically analyzed using nonparametric methods (Mann-Whitney test). p21WAF-1 and MDM2 expression were significantly higher in PTA compared with NPT (p

Se expone el caso de una paciente de 73 anos que presento cuadro constitucional y dolor en miembros inferiores, con sospecha inicial de mieloma multiple. Durante el proceso diagnostico aparecieron discordancias entre la evolucion clinica y los hallazgos en pruebas complementarias. Tras un curso clinico fulminante en pocos dias, la paciente fallecio; se obtuvo el diagnostico post mortem de linfoma angioinmunoblastico. Se repasan los aspectos fundamentales de esta patologia poco frecuente, de patogenia todavia incierta.