ALBERT

Description: Proton nuclear magnetic resonance spectroscopy (1H MRS) has proven useful in the metabolic characterization of neoplastic tissues both in vitro and in vivo. In vivo 1H MRS can be easily performed in conjunction with conventional MR imaging. Although 1H MRS studies of lymphoma have been performed both in cell lines and animal models, its usefulness as a differential diagnostic tool in lymphoma remains to be explored. T2-filtered high resolution proton magic-angle spinning (HRMAS) MRS of small tissue samples provides sufficient spectral resolution for the identification of chemical cell changes associated with malignancy. HRMAS data from 16 fresh frozen lymph nodes were studied. Histologically, 7 lymph nodes revealed grade I or II follicular lymphoma (FL) and 9 showed diffuse large B cell lymphoma (DLBCL). Each spectrum consisted of the accumulation of 64 scans with a CPMG sequence with an effective T2 delay of 32 ms and an overall experimental time of 6 minutes. Pattern recognition analysis was feasible in all cases except in one FL sample consisting almost exclusively of adipose tissue. Compared with DLBCL, FL cases showed increases in the signal intensity of resonances associted to taurine (3.43 ppm) and decreases in the signal intensity of resonances associated to alanine (1.47 ppm). The mean taurine/alanine ratio was 4.63 (95%SD 1.67) for FL and 1.41 (95%SD 0.69) for DLBCL (P = 0.004). The taurine/alanine ratio proved to be a powerful discriminator between FL and DLBCL at a 2.5 cutpoint, yelding a 90% specificity and an 83% sensitivity to identify DLBCL. Overlapping cases included a case of FL with a diffuse pattern and two cases of DLBCL evolving from indolent lymphomas. The 1H MRS assay proved that FL and DLBCL can be differentiated from a metabolomic standpoint and offers a unique way to explore the underlying biochemistry of malignant transformations. Unlike other techniques, MRS is potentially applicable in vivo and non-invasively in the diagnostic setting. The rapid assessment of nodes or masses before tissue sampling would allow preoperative decisions to be made more efficiently.

Description: Abstract 1948 Poster Board I-971 Survival after treatment of diffuse large B-cell lymphoma (DLBCL) is influenced by differences in the tumor microenvironment. Gene expression profiling (GEP) studies have shown that the angiogenesis-related signature (stromal-2 signature) is prognostically unfavorable. However, the clinical and biological significance of angiogenesis quantified in tumor tissue sections of DLBCL from patients treated with rituximab plus chemotherapy (R-CT) is not yet fully explored. CD31, the platelet adhesion molecule PECAM1, is one of the genes included in the “stromal-2 signature” reported in the GEP studies. The objective of this study was to determine whether the microvessel density (MVD) and microvessel number (MVN) in DLBCL patients treated with R-CT were associated with the clinicopathological features of the tumors and related to the outcome of the patients. The MVD and MVN were assessed in a series of 160 patients with DLBCL from the Leukemia Lymphoma Molecular Profiling Project consortium (LLMPP) 86M /74F; median age 64 yrs. The GEP was investigated in 116 of these including 50 germinal center B (GCB), 55 activated B-cell (ABC) and 11 unclassifiable cases. An independent series of 129 patients from the Catalan Lymphoma-Study Group (GELCAB) (67M/62F; median age 64 yrs) was used to validate the results. Front-line treatment was R-CT in all cases of both series. Tissue Microarrays (TMA) were constructed from pretreatment biopsy specimens of de novo DLBCL. High grade B cell lymphoma otherwise unclassifiable, primary mediastinal B cell lymphoma, T-cell-rich B cell lymphoma, and tumors associated with immunodeficiency were excluded. All cases were stained in an automated immunostainer with an antibody against CD31 (DAKO). The MVD and MVN were quantified using digitalized images of the tumor using Olympus Cell B Basic Imaging Software. Microvessel areas were defined as vascular areas delineated by CD31+ staining. The MVD was calculated as the sum of all microvessel areas divided by the total area analyzed. The MVN was the sum of all identified individual vessels, divided by the total area analyzed. TMAs were independently scored by two observers and discrepancies were resolved over a double-headed microscope. To determine whether the angiogenic values scored using the TMA were representative of the tumor sample, whole tissue sections and TMA cores from the same tumor were evaluated in 40 cases and compared by a linear regression analysis. MVD and MVN were grouped in quartiles when necessary and considered high or low when above or below the 50th percentile, respectively. Linear correlation analysis between the CD31 (+) MVD results on TMA cores and on the corresponding whole tissue sections in 40 cases showed a good correlation (R2=0.81). In the LLMPP cohort, DLBCL with an ABC profile showed higher MVD than those with GCB profile (p=0.05). In addition, higher MVD was observed in patients with advanced stage (p

Description: Background: In the combination antiretroviral therapy (cART) era the prognosis of HIV-infected patients with Hodgkin’s lymphoma (HL) approaches to that of the HIV-negative population. Treatment of advanced stage HL with ABVD has been shown feasible and effective in HIV patients. However, there is scarce information about the impact of cART on clinical and biological characteristics of HIV-related HL as well as on its prognosis. Aim: To compare the clinical and biological characteristics and outcome of patients with advanced stage HL treated with ABVD according to HIV-status in the cART era. Methods: Two groups of patients with advanced stage HL treated with ABVD were studied. The first group consisted of 63 HIV-infected patients diagnosed with HL in the cART era in 15 Spanish hospitals, and the control group included 31 HIV-negative patients diagnosed in the same period in a single institution. Demographic, clinical and biological data were collected, and complete response rate (CRR), disease-free survival (DFS) and overall survival (OS) were calculated. Then, HIV-infected patients were stratified according to whether they started cART therapy before or concomitantly with chemotherapy. Survival analyses were performed using the Kaplan-Meier method and compared using the log-rank test. The Cox regression proportional hazards model was used for multivariate analyses. Significance level was established at p

Description: INTRODUCTION. Mantle cell lymphoma (MCL) is a well defined mature B-cell neoplasm, usually with an aggressive behaviour and a median survival of 3–5 years. It is characterized by the presence of t(11;14)(q13;q32), that leads to overexpression of CCND1. This translocation is detected by conventional cytogenetics (CC) in more than 65% of the cases. Nevertheless, cyclin D1 overexpression alone is not sufficient to give rise to a MCL. Several secondary genetic abnormalities with a potential role in the oncogenic process have been described using different molecular and cytogenetic techniques but no data from large series of MCL cases studied by CC have been correlated with survival and proliferation information. AIMS. 1. To correlate secondary chromosomal abnormalities, histological subtype and proliferative index with survival. 2. To describe the frequency of secondary chromosomal aberrations using CC and a large series of MCL patients. PATIENTS AND METHODS. We selected 145 MCL cases (94M/51F; mean age 65.4 yrs; mean survival 33 mo.), all of them with an abnormal karyotype. Histological subtype, proliferative index (Ki-67) and survival data were collected. All patients were studied by conventional GTG-banding cytogenetics, and FISH with a dual colour dual fusion CCND1/IGH probe was applied in those cases with an abnormal karyotype but without t(11;14) detected by CC. RESULTS. Translocation t(11;14) was detected as a single anomaly in 36% of patients, whereas 57% of cases displayed t(11;14) plus other additional aberrations and 6% of patients showed an abnormal karyotype without t(11;14) which was detected by FISH. We observed a total of 550 abnormalities (147 numerical and 403 structural), being the most frequent deletions of 1p (12%), 13q and 17p (10% each) and gains of 3q (9%). Recurrent breakpoints were identified at 1p31–p32, 1p21–p22, 17p13 and 1p36 (in eight, six, five and four cases, respectively). Blastoid variants of MCL displayed more karyotypic complexity with high number of structural alterations, higher number of 1p and 17p deletions, higher proliferation index and poor survival. CONCLUSIONS. 1. MCL cases associated with 17p losses (overall survival at 4 yrs: 15% vs 50%; P=0.01) and high proliferative index (15% vs 55%; P=0.005), generally corresponding to blastoid variants, display a worse outcome. 2. Our results confirm, by means of a routine technique as CC, previous observations made using more sophisticated molecular techniques in shorter series of patients.

Description: Abstract 4134 Gene expression profile (GEP) allows to distinguish two groups with different origin in patients with diffuse large B-cell lymphoma (DLBCL): germinal-center (GC) and activated (ABC), with the latter having a significantly poorer outcome. However, GEP is a technique not available in current clinical practice. For this reason, attempts to reproduce GEP data by immunophenotyping algorithms have been made. The aim of this study was to apply the most popular algorithms in a series of patients with DLBCL homogeneously treated with immunochemotherapy, in order to assess the correlation with GEP data and their usefulness to predict response and outcome of the patients. One hundred fifty seven patients (80M/77F; median age 65 years) diagnosed with DLBCL in 5 institutions of the Grup per l'Estudi dels Limfomes de Catalunya I Balears (GELCAB) during a 5-year period, treated with Rituximab-containing regimens (in most cases, R-CHOP), in whom histological material to construct a tissue microarrays (TMA) was available, constituted the subjects of the present study. Four algorithms were applied: Colomo (Blood 2003, 101:78) using CD10, bcl-6 and MUM1/IRF4; Hans (Blood 2004, 103:275) using CD10, bcl-6 and MUM1/IRF4; Muris (J Pathol 2006, 208:714) using CD10 and MUM1/IRF4, and Choi (Clin Cancer Res 2009, 15:5494), using CD10, bcl-6, GCET1, FOXP1 and MUM1/IRF4. The thresholds used were those previously described. GEP studies were performed in 62 patients in whom fresh frozen material was available. Main clinical and evolutive data were recorded and analyzed. The proportion of positive cases for the different single antigens was as follows: CD10 26%, bcl-6 64%, GCET1 46%, FOXP1 78% and MUM1/IRF4 28%. The distribution of cases (GC vs. non-GC) according to the algorithms is detailed in the table. In 88 of 110 patients (80%) with all the antigens available, the patients were allocated in the same group (either GC or non-GC). When the immunochemistry was compared with GEP data, the sensitivity in the GC group was 59%, 52%, 70% and 40% for Colomo, Hans, Muris and Choi algorithms, respectively. The sensitivity in the non-GC group was 81%, 85%, 62% and 84%, respectively. On the other hand, the positive predictive value (PPV) in the GC group was 81%, 83%, 72% and 77%, respectively. In non-GC subset the PPV for the different algorithms was 59%, 55%, 72% and 52%, respectively. We observed a higher percentage of misclassified cases in the GC-phenotype subset than in the non-GC subgroup. None of the immunohistochemical algorithms showed a significant superiority as surrogate of GEP information among the others. The ability of GEP groups as well as of groups defined by the algorithms to predict complete response (CR) rate, progression-free survival (PFS) and overall survival (OS) of the patients is showed in the table. Thus, whereas the GEP groups showed significant prognostic value for CR rate, PFS and OS, none of the immunohistochemical algorithms were able to predict the outcome. In conclusion, in a homogeneous series of DLBCL patients treated with immunochemotherapy, the different immunohistochemical algorithms were not able to mimic the GEP information. The prognostic impact of the groups defined by immunohistochemistry (GC vs. non-GC) was particularly low. N (%) CR rate N (%) 5-year PFS (%) 5-year OS (%) Colomo algorithm GC 53 (44) 39 (74) 48 54 Non-GC 68 (56) 53 (78) 55 62 Hans algorithm GC 61 (41) 47 (77) 54 60 Non-GC 88 (59) 67 (76) 52 59 Muris algorithm GC 87 (57) 63 (72) 48 57 Non-GC 65 (43) 51 (78) 56 63 Choi algorithm GC 45 (33) 32 (71) 48 54 Non-GC 90 (67) 70 (78) 52 61 Gene expression profile 30 (58) 25 (83) 76* 80** GC Activated 22 (42) 17 (77) 31* 45** * p=0.005, ** p=0.03. Disclosures: No relevant conflicts of interest to declare.

Description: Aggressive B-cell Lymphomas are the second most frequent AIDS-defining cancers. Few studies have compared the molecular characteristics of aggressive B-cell lymphomas in patients with and without HIV-infection; and to our knowledge, there are no reports comparing the incidence of gene rearrangements between the two groups and their impact on outcome in series treated with RCHOP. We retrospectively studied two series of patients with (N=32) and without HIV-infection (N=43) with diffuse large B-cell lymphomas (DLBCL) NOS (75% and 70%, respectively), T-rich DLCBL (13% and 5%), transformed DLBCL (3% and 14%) and double-hit (DH) DLCBL (9% and 11%) [defined by translocations affecting MYC (TMYC) concomitantly with translocation affecting BCL2 (TBLC2) and/or BCL6 (TBCL6)]. Tissue microarrays were constructed and translocations were studied by fluorescent in situ hybridization. The germinal center (GC) phenotype was defined according to the Hans' algorithm based on the expression of CD10, MUM1 and BCL6. Clinical data was retrieved from records. Continuous and categorical variables are presented using descriptive statistics and compared using Fisher's exact test, χ 2-test, and Mann-Whitney U-test. Survival analyses were performed using the Kaplan-Meier method. P-valuesof less than 0.05 were considered statistically significant. The median follow-up of HIV-infected patients was 6.9 years and of HIV-uninfected was 5 years. HIV-uninfected patients were older than HIV-infected patients, median age (range) 59 years (25-80) and 45 years (37-68) respectively; (P=0.002). There were differences between HIV-infected and HIV-uninfected patients regarding; male gender (81% vs. 54%, P=0.015), ECOG performance status higher than 2 (56% vs. 26%; P=0.018) and elevated ß-2 microglobulin (82% vs. 47%, P=0.005). On the other hand, the percentage of patients with III and IV Ann Arbor stages, two or more extranodal involvement, elevated LDH, 3 to 5 International Prognostic Index (IPI) scores, B-symptoms and bulky disease were similar in both series. The study of molecular features showed that more HIV-infected cases (57%) tended to have a GC phenotype than HIV-uninfected (35%); P=0.093. Regarding gene rearrangements, there was a trend for more HIV-infected patients (30%) to present TBCL2 than HIV-uninfected patients (11%); P=0.056. Of note, only 2 patients of the HIV-infected series were transformed from follicular lymphoma. The frequency of TMYC, TBCL6 and DH was similar in HIV-infected (24%, 28% and 9%, respectively) and HIV-uninfected (15%, 28% and 11%, respectively). HIV-infected patients were treated with RCHOP (N=27), intensive immunochemotherapy for Burkitt Lymphoma (IICT-BL) (N=4), CHOP (N=1) and HIV-uninfected patients with RCHOP (N=39), IICT-BL (N=2), RCOP (N=1), RESHAP (N=1). The complete response rate was not statistically different in HIV-infected and HIV-uninfected patients neither the relapse rate when considered all treatments given. Only patients treated with RCHOP were considered in survival analyses. The overall survival (OS) of HIV-uninfected patients with high IPI scores (3 to 5) (N=15) tended to be shorter than in patients with low IPI scores (0 to 2) (N=24), (5-y OS 60% [35%-85%] vs. 81% [64%-98%], P=0.089) and the progression free survival (PFS) was clearly inferior (5-y PFS 33% [9%-57%] vs. 73% [54%-92%], P=0.004). HIV-infected patients with high IPI scores presented similar OS and PFS than patients with low IPI scores. Contrary to other reports, TMYC had no statistically significant adverse impact on OS and PFS of HIV-uninfected patients, most probably because of small size of our series. The same reasoning could be applied for HIV-infected patients since both OS and PFS of patients with TMYC (N=5) was shorter than in those without TMYC (N=20) (5-year OS 40% [0%-83%] vs. 65% [44%-86%], P=0.365 and 5-year PFS 40% [0%-83%] vs. 60% [38%-82%], P=0.546). TBCL2, TBCL6 and GC phenotype had no impact on OS and on PFS of both HIV-infected and HIV-uninfected patients. Survival analyses of DH were not performed due to the small number of events. In summary, the frequency of TMYC and TBCL6 in aggressive B-cell lymphomas was similar in HIV-infected and HIV-uninfected patients but TBCL2 was less frequent in HIV-infected patients. Supported in part by grants EC11-041 from ISCIII, Spain. Disclosures No relevant conflicts of interest to declare.

Description: In some studies DLBCL germinal center (GC) phenotype has better prognosis than non-germinal center (non-GC) according to IHC expression profile by tissue microarrays (TMA), but the frequency and prognostic significance of ICH expression profile in HIV-related DLBCL is not well known. The objectives of this study were to characterize the IHC expression pattern in 98 pts with DLBCL, of which 34 were HIV positive, and to evaluate its prognostic significance. From August 1991 to February 2006, 98 consecutive pts with DLBCL from a single institution were retrospectively studied. Group A: HIV-negative DLBCL pts, n=64; group B: HIV-related DLBCL pts, n=34. TMA was constructed containing sections of paraffin-embedded tissue samples and cases were assessed for IHC study (expression of CD10, BCL-6, MUM-1). Patients were classified as CG pattern (CD10+ or CD10−/BCL-6+/MUM1−) and non-GC pattern (any other combination of these markers). Clinical and biological characteristics as well as overall survival (OS) and disease-free survival (DFS) were analyzed comparing: 1-GC vs non-GC pattern within groups A and B; 2-GC pattern from groups A vs B; 3-Non-GC pattern from groups A vs B; 4-non-GC pts from group A vs non-GC pts from group B, considering only the pts treated with highly active antiretroviral therapy (HAART) (n=13/32, 41%). There were no differences in clinico-biological parameters according to IHC pattern within each group. The only differences in OS and DFS between subgroups were observed in HIV-related DLBCL with non-GC pattern, whose survival was poorer than non-HIV related pts. Whithin the former group, only those pts not receiving HAART retained their bad prognosis. 5-year OS (95% CI) and 5-year DFS (95%CI) are summarized in the table. In our series the frequency of GC and non-GC subtypes was not significantly different in HIV positive and negative pts with DLBCL. Only those pts with HIV-related DLBCL with non-GC expression pattern not receiving HAART had a significantly inferior survival.

Description: Introduction MYC translocations involving chromosome 8q24 can occur in a wide variety of B-cell lymphomas other than Burkitt’s lymphoma (BL), especially diffuse large B-cell lymphoma (DLBCL) and B-cell lymphoma unclassifiable with intermediate features between DLBCL and BL (BCLU). These lymphomas can harbor additional translocations of BCL2 and/or BCL6, that have been referred to as double and triple-hit lymphomas. MYC positive lymphomas other than BL are not well characterized and the standard treatment has to be established. Aim The objective was to study the clinic-biological characteristics and prognosis of a series of lymphomas with MYC-translocation other than BL. Methods Retrospective study of patients with MYC-translocation other than BL treated in three hospitals of Spain between 2003 and 2012. Cases with diagnosis of BL, Burkitt’s-like lymphoma and any other with MYC-translocation were reviewed and classified according to the WHO 2008 criteria. The status of MYC, BCL2 and BCL6 genes were evaluated in all cases by fluorescent in situ hybridization (FISH) using dual-colour break-apart commercial probes (LSI MYC DC BA, LSI BCL6 DC BA and LSI BCL2 DC BA; Abbot Molecular, Abbot Park, IL, USA) on whole tissue sections of formalin-fixed paraffin-embedded tissue. Main clinical and biological data were collected from the records. Results Between 2003 and 2012, 34 patients with a median follow-up of 1.9 years (range 0.7-9.7) were included. Median age was 59.5 years (range 36-83) and 21 (62%) were male. ECOG score at diagnosis was ≥2 in 14 patients (42%), 16 (49%) had ≥2 extranodal sites involved, serum LDH was elevated in 24 out of 32 (75%), Ann Arbor stage III/IV in 24 (73%) and B symptoms were present in 18 (56%). IPI was high or intermediate/high in 19 out of 32 (59%). Eighteen patients (53%) presented with a mass (13 abdominal location). Twenty-four cases were diagnosed with DLBCL and 10 with BCLU. Conventional cytogenetics showed a complex karyotype in the 9 studied cases. MYC rearrangement alone without BCL2 or BCL6 rearrangements was observed in 13 (38%) cases, 15 were double-hit, and 6 triple-hit lymphomas. There were no differences between patients with MYC translocation alone and patients with double or triple-hit, regarding the clinical and biological characteristics. Moreover, there were no clinical and biological differences between patients with DLBCL and those with BCLU. Twenty-one cases were treated with R-CHOP (19 DLBCL and 2 BCLU), and 13 with a specific treatment for BL (Burkimab) (8 BCLU and 5 DLBCL) (P=0.002). Complete response (CR) was achieved in 14 out of 32 (44%) cases (9 out of 21 [45%] treated with R-CHOP and 5 out of 13 [42%] with Burkimab). Two patients died during chemotherapy. Six out of the 9 patients in CR to R-CHOP relapsed, but none of the 5 patients in CR to Burkimab did it. The overall survival (OS) probability and progression free survival (PFS) at 2 years, (95% CI) for the whole series were 32% (14%, 50%) and 21% (6%, 36%) respectively. The two-year OS and PFS probabilities were not significantly different between patients treated with Burkimab and those treated with R-CHOP: 62% (95% CI: 36%, 88%) versus 28% (95% CI: 8%, 48%) for OS (P=0.67); and 46% (95% CI: 19%, 73%) versus 15% (95% CI: 0%, 31%) for PFS (P=0.549), respectively. Conclusions Lymphomas with MYC translocation other than BL present aggressive characteristics at diagnosis and have poor response to immunochemotherapy. Supported in part by grants EC11-041 and RD12/0036/0029 RTICC from Instituto Carlos III, Spain Disclosures: Lopez-Guillermo: Roche: Membership on an entity’s Board of Directors or advisory committees.

Description: Clinical-pathological sessions are a good method for solving diagnostic and/or therapeutic problems in patients with hemopathies. In these sessions, errors made during health care given to the patient can be detected. By analyzing how and why these errors are made, we can improve patient care and prevent further mistakes. The objective of this study is to describe 34 errors identified in 874 patients with hemopathies at clinical-pathological meetings in two centers, performed with the aim of solving a diagnostic problem and/or a therapeutic decision or due to a great interest of the case, in two institutions, over 22 years (1982–2004). An intererdisciplinary team of hematology specialists gathered every week at interactive sessions of about 45 minutes each, in both institutions. The methodology of sessions was: a description of the medical history of a patient in a one or two-page report and a revision of the different samples (peripheral blood, bone marrow and lymph node morphology, immunocytochemistry, flow-cytometry, cytogenetic and molecular studies) with the aid of a microscope and a TV monitor. A diagnostic and/or treatment were proposed at the end of the session. Eight-hundred and seventy-four reports were analyzed. All the diagnostics were classified: chronic lymphoproliferatives disorders (445), myeloproliferative and myelodysplastic syndromes (136), acute leukemias (136), other haematological diseases (74), non-haematological diseases (31), without a diagnosis after the meeting (52). We identified diagnostic (D) and therapeutic (T) mistakes and considered as the main causes of the medical error (mistake in the diagnosis and/or treatment): lack of expertise (LE), malpractice (MP), impetuosity (IM), bad logistic support (LS), inexplicable (IN). We divided the 22 years into two decades and each error was classified in one of these two groups. Our own mistakes (OM) and the errors made in other institutions (OI) were identified. A comparison between number of errors made in the first 11 years and the second 11 years was made using a Chi-square test. P