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In recent years, the outcome of mantle cell lymphoma (MCL) has improved, especially in younger patients, receiving cytarabine-containing chemoimmunotherapy and autologous stem cell transplantation. Nevertheless, a proportion of MCL patients still experience early failure. To identify biomarkers anticipating failure of intensive chemotherapy in MCL, we performed target resequencing and DNA profiling of purified tumor samples collected from patients enrolled in the prospective FIL-MCL0208 phase 3 trial (high-dose chemoimmunotherapy followed by autologous transplantation and randomized lenalidomide maintenance). Mutations of KMT2D and disruption of TP53 by deletion or mutation associated with an increased risk of progression and death, both in univariate and multivariate analysis. By adding KMT2D mutations and TP53 disruption to the MIPI-c backbone, we derived a new prognostic index, the “MIPI-genetic” (“MIPI- g”). The “MIPI-g” improved the model discrimination ability compared to the MIPI-c alone, defining three risk groups: i) low-risk patients (4-year progression free survival and overall survival of 72.0% and 94.5%); ii) inter-mediate-risk patients (4-year progression free survival and overall survival of 42.2% and 65.8%) and iii) high-risk patients (4-year progression free survival and overall survival of 11.5% and 44.9%). Our results: i) confirm that TP53 disruption identifies a high-risk population characterized by poor sensitivity to conventional or intensified chemotherapy; ii) provide the pivotal evidence that patients harboring KMT2D mutations share the same poor outcome as patients harboring TP53 disruption; and iii) allow to develop a tool for the identification of high-risk MCL patients for whom novel therapeutic strategies need to be investigated. (Trial registered at clinicaltrials.gov identifier: NCT02354313).

An accurate diagnosis of clinically distinct subgroups of aggressive mature B cell lymphomas is crucial for the choice of proper treatment. Presently, precise recognition of these disorders relies on the combination of morphological, immunophenotypical, and cytogenetic/molecular features. The diagnostic workup in such situations implies the application of costly and time-consuming analyses, which are not always required, since an intensified treatment option is reasonably reserved to fit patients. The Italian Group of Haematopathology proposes herein a practical algorithm for the diagnosis of aggressive mature B cell lymphomas based on a stepwise approach, aimed to select cases deserving molecular analysis, in order to optimize time and resources still assuring the optimal management for any patient.

An accurate diagnosis of clinically distinct subgroups of aggressive mature B cell lymphomas is crucial for the choice of proper treatment. Presently, precise recognition of these disorders relies on the combination of morphological, immunophenotypical, and cytogenetic/molecular features. The diagnostic workup in such situations implies the application of costly and time-consuming analyses, which are not always required, since an intensified treatment option is reasonably reserved to fit patients. The Italian Group of Haematopathology proposes herein a practical algorithm for the diagnosis of aggressive mature B cell lymphomas based on a stepwise approach, aimed to select cases deserving molecular analysis, in order to optimize time and resources still assuring the optimal management for any patient.

Background To evaluate the efficacy of the use of flow cytometry (FC) immunophenotyping together with fine-needle aspiration cytology (FNAC) in the diagnosis of thyroid lymphoma. Methods FC was performed in parallel with FNAC in 35 samples of suspected thyroid lymphoma over a 12 years period. Results were correlated with histological or molecular findings and follow-up, when available. Results A final diagnosis of lymphoma was given in 13 of 35 (37.1%) specimens. Among the 22 cases considered negative for lymphoma by FC, 11 were diagnosed as thyroiditis by cytology, 7 as reactive, 2 were anaplastic carcinoma, and 2 cases were considered cytologically suspicious for lymphoma but were not confirmed by further investigations. Histology on core biopsy or molecular analysis was available in 12 of 13 lymphoma cases (92.3%). Data obtained by the combination cytology/FC were confirmed in all cases on histology biopsies. Correlation with histology showed a sensitivity and a specificity of 100% for the combination cytology/FC. Conclusions FC is an important additional test that can contribute with cytology to the identification of lymphomas of the thyroid. FC can detect the presence of small neoplastic lymphocyte populations and may contribute to the diagnosis of cases in which the lymphoid infiltrate is difficult to interpret on cytology alone. © 2014 International Clinical Cytometry Society

JAK/STAT3 signaling pathway is often deregulated in hematopoietic disorders including peripheral T-cell lymphoma. We describe two novel mechanisms leading to the constitutive activation of STAT3 in ALK- ALCL. Oncogenic JAK1 or STAT3 mutations are associated to hyperactive pSTAT3 that regulated canonical STAT3 and ATF3 genes. Moreover, synergizing JAK1 and STAT3 mutants sustain the neoplastic growth, which can be efficiently controlled in vitro and in an ALCL patient derived tumorgraft model by JAK1/2 inhibitors. We have discovered that novel chimera, displaying concomitant transcriptional and kinase activities, are power oncogenes capable to sustain via STAT3 the ALCL phenotype and can be uniquely neutralized by a novel ROS1 inhibitor. The pharmacological inhibition of JAK/STAT3 represents a novel strategy for the treatment of molecular stratified ALCL.

Objective Lymphoid proliferations of the salivary glands can be either reactive or malignant. Diagnosis based solely on fine needle aspiration (FNA) cytology may be troublesome in view of the difficulty in distinguishing low-grade B-cell and mucosa-associated lymphoid tissue (MALT) lymphomas from reactive lymphoid proliferations. We report our experience with FNA cytology combined with flow cytometry (FC) immunophenotyping for the diagnosis of lymphoproliferative processes affecting the salivary glands. Methods Sixty-one FNA specimens, obtained from salivary glands over a 10-year period, were analysed by cytology and FC. The results were correlated with histological follow-up if available. Results A diagnosis of lymphoma was given in 37 of 61 (61%) specimens; 22 of 61 (36%) specimens were considered as benign/reactive or non-lymphomatous processes; two of 61 (3%) specimens were considered as suspicious for lymphoma on cytological analysis and negative on FC. Histological control was available in 23 malignant, four non-lymphomatous and one cytologically suspicious case. Data obtained by the combination of cytology and FC were confirmed in all but one case: the case suspicious on cytology received a histological diagnosis of carcinoma. Four of seven cases with small populations of clonal cells (less than 15%) were histologically confirmed as lymphoma, whereas two remain under surveillance and one was reactive. Correlation with histological data showed a sensitivity of 100% and a specificity of 83% for the combination of cytology and FC. Conclusions FC is fundamental for the diagnosis of lymphoproliferative lesions of the salivary glands. It may solve cytologically suspicious cases and detect the presence of neoplastic B or T cells. This combined approach reduces the time to therapy and may prevent unnecessary surgical biopsies.

A retrospective analysis of 1,792 solid tissues suggestive of lymphoma, submitted over a 12-year period, was carried out and flow cytometry (FC) results were compared with histologic findings. The final histologic diagnosis of cases documented in this report is as follows: 1,270 non-Hodgkin's lymphomas (NHL); 17 composite lymphomas; four NHL plus carcinomas; five post-transplant lymphoproliferative disorders; 105 Hodgkin's lymphomas (HL); eight acute leukemias; 42 tissue cancers; and 341 non-neoplastic diseases. A strong correlation between morphology and FC data was observed among hematological malignancies (1,268/1,304, 97.2%) with the exception of HL. Among B-NHL, FC detection of clonally restricted B-cell allowed the identification of lymphomas that were not histologically clear and the differential diagnosis between follicular lymphoma and reactive hyperplasia. A high correlation level (r = 0.83; P < 0.0001) was obtained in comparing proliferation results obtained by FC and immunohistochemistry. Among T-NHL, FC detection of an aberrant phenotype direct histologic diagnosis in cases having less than 20% of neoplastic cells. In nine cases, FC suggested the need to evaluate a neoplastic population, not morphologically evident. Results show that FC routinely performed on tissue samples suspected of lymphomas is a fundamental adjunct to morphology in the diagnosis of NHL and may enhance the performance of the histologic evaluation so as to achieve the final diagnosis. To the best of our knowledge, this is the first report in the literature of a wide series of tissues also studied by FC.

Objectives: Flow cytometry (FC) has become a useful support for cytomorphologic evaluation (CM) of fine-needle aspirates (FNA) and serous cavity effusions (SCE) in cases of suspected non-Hodgkin lymphoma (NHL). FC results may be hampered by the scarce viability and low cellularity of the specimens. Study Design: We developed a single-tube FC assay (STA) that included 10 antibodies cocktailed in 8-color labeling, a cell viability dye, and a logical gating strategy to detect NHL in hypocellular samples. The results were correlated with CM and confirmed by histologic or molecular data when available. Results: Using the STA, we detected B-type NHL in 31 out of 103 hypocellular samples (81 FNA and 22 SCE). Of these, 8 were not confirmed by CM and 2 were considered to be only suspicious. The FC-negative samples had a final diagnosis of benign/reactive process (42/72), carcinoma (27/72), or Hodgkin lymphoma (3/72). Conclusions: The STA approach allowed obtainment of maximum immunophenotyping data in specimens containing a low number of cells and a large amount of debris. The information obtained by STA can help cytomorphologists not only to recognize but also to exclude malignant lymphomas.

Background Allogeneic hematopoietic stem cell transplantation (HSCT) is potentially curative in a variety of hematological malignancies. Graft-vs.-host disease (GvHD) remains a life-threatening complication. Standard treatment is high-dose (HD) corticosteroids. Steroid-refractory (SR) GvHD is associated with poor prognosis. At present, second-line treatment is ill-defined and includes a number of agents. Novel insights into the pathophysiology of acute GvHD (aGvHD) highlight the relevant role of the host inflammatory response governed by several kinase families, including Janus kinases (JAK)1/2. Ruxolitinib, a JAK1/2 inhibitor approved for intermediate-2/high-risk myelofibrosis, was recently employed in SR-GvHD with encouraging overall response rates. Clinical experience however remains limited. Case presentation A 51-year-old male with refractory anemia with excess blast type-2 underwent a myeloablative allogeneic HSCT from a 9/10 HLA-matched unrelated donor after conditioning with busulfan and cyclophosphamide. GvHD prophylaxis consisted of cyclosporine, methotrexate, and thymoglobulin. CD34+ cells/kg infused were 8.69 × 106 kg. On day 29, the patient developed overall grade IV aGvHD with biopsy proven stage IV gastrointestinal (GI) GvHD refractory to HD corticosteroids. Patient conditions rapidly deteriorated and became critical despite the addition of mycophenolate mofetil and budesonide. On day 33, Ruxolitinib was started, and on day 39 the patient clinical conditions gradually improved. Complete resolution of aGvHD was also confirmed by histology on day 54. Conclusions At 5 months from HSCT, the patient is well and in continuous hematological complete remission without flare of GvHD. Ruxolitinib was discontinued on day 156. Ruxolitinib is feasible and effective in SR-aGvHD though large prospective clinical trials are warranted.

Stacchini A, Barreca A, Demurtas A, Aliberti S, di Celle P F & Novero D (2012) Histopathology 60, 452–459 Flow cytometric detection and quantification of CD56 (neural cell adhesion molecule, NCAM) expression in diffuse large B cell lymphomas and review of the literature Aim: To report unusual CD56 (neural cell adhesion molecule, NCAM) expression on diffuse large B cell lymphoma (DLBCL). Methods and results: CD56 expression was first detected and quantified on tissues obtained from five cases of DLBCL by flow cytometry (FC), then confirmed by immunohistochemistry. The CD56 expression pattern was heterogeneous among the cases [the molecular equivalent of soluble fluorochrome (MESF) level ranged from 2214 to 133 466]. All were CD10 and Bcl-6 positive, suggesting their germinal centre origin; one was also CD5 positive. An extranodal presentation occurred in three of five cases. Conclusions: CD56 expression in B cell lymphoma is a rare occurrence. FC is able to identify aberrant immunophenotypes that can be useful in the identification and monitoring of B cell lymphoma subtypes. The presence of CD56 reported by the literature on certain DLBCL with extranodal presentation might be related to mechanisms involved in growth and expansion.

Composite lymphoma (CL) is a rare occurrence of 2 or more morphologically and immunophenotypically distinct lymphoma clones in a single anatomic site. A retrospective analysis of 1,722 solid tissue samples clinically suggestive of lymphoma was carried out in our institute during a 12-year period to evaluate the efficacy of flow cytometry (FC) in identifying CL. We report 17 CL cases. A strong correlation between morphologic findings and FC was observed in 13 cases (76%). In the 4 cases diagnosed as non-Hodgkin lymphoma plus Hodgkin lymphoma, although FC did not detect Reed-Sternberg cells, it accurately identified the neoplastic B- or T-cell component. In 3 cases, FC indicated the need to evaluate an additional neoplastic component that was not morphologically evident. Our data demonstrate that FC immunophenotyping of tissues may enhance the performance of the diagnostic morphologic evaluation of CL. To the best of our knowledge, this is the first report in the literature of a wide series of CL studied also by FC.

Anaplastic large cell lymphoma (ALCL) is a subtype of peripheral T-cell lymphoma (PTCL) first described in 1985 as a lymphoid malignancy characterized by marked cellular pleomorphism, propensity to grow cohesively, tendency to invade lymph node sinuses and diffuse expression of CD30 1. The discovery of the t(2;5), involving the anaplastic lymphoma kinase (ALK) gene on chromosome 2 and the nucleophosmin (NPM) gene on chromosome 5 in the majority of systemic ALCL, has soon pointed out that ALCL is a clinically and biologically heterogeneous disease. While ALK-positive (ALK+) ALCL is usually characterized by onset in children and young adults and better prognosis, epidemiology, poor outcome and possibly genetic defects of ALK-negative (ALK−) ALCL suggest that this neoplasms should be considered an independent pathological entity. The aim of this review is to illustrate clinical features, histology, immunophenotype, genetics and biology of ALCL and discuss possible relationship(s) among different T-non-Hodgkin lymphoma (T-NHL). Copyright © 2009 John Wiley & Sons, Ltd.

T-cell antigen expression can be observed in B-cell non-Hodgkin lymphoma (B-NHL). Although CD5 is expressed in B-cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma, the presence of other T-cell antigens is less common. This article reports a retrospective multicenter analysis in which flow cytometry was used to evaluate aberrant CD8 expression on the pathologic B cells of 951 bone marrow samples from patients with various types of B-NHL. In a total of 18 patients, CD8 was coexpressed: 10 had B-CLL; 1, small lymphocytic lymphoma (SLL); 1, marginal zone lymphoma; 1, lymphoplasmacytic lymphoma; 2, diffuse large B-cell lymphoma; and 3, follicular lymphoma. There was a 1.89% overall frequency of CD8 coexpression in which B-CLL/SLL had a higher frequency (3.03%) than did the other B-cell neoplasms (1.18%). Most cases were characterized by a favorable outcome.

Mucous membrane plasmacytosis (MMP) is a rare idiopathic condition characterized by infiltration of the mucosa by non neoplastic plasma cells. In this report we describe a case of mucous membrane plasmacytosis of the nose in a 72-year-old woman patient affected by B-cell chronic lymphocytic leukemia (B-CLL). Two different biopsies of the lesion showed diffuse plasma cell, lymphocyte and granulocyte infiltration compatible with granulation tissue. A complete exeresis of the neoplasm was performed endoscopically without complications, allowing the diagnosis of MMP; a monthly follow up was performed with no signs of local relapse 15 months after surgery. Topical steroid therapy with budesonide nasal spray was administered. There is no standardized treatment for MMP: we have reported good result of surgical approach in a unique case of nasal MMP in a patient with B-CLL; the relation between these two diseases deserves more studies.

Although anaplastic large-cell lymphomas (ALCL) carrying anaplastic lymphoma kinase (ALK) have a relatively good prognosis, aggressive forms exist. We have identified a novel translocation, causing the fusion of the TRAF1 and ALK genes, in one patient who presented with a leukemic ALK+ ALCL (ALCL-11). To uncover the mechanisms leading to high-grade ALCL, we developed a human patient-derived tumorgraft (hPDT) line. Molecular characterization of primary and PDT cells demonstrated the activation of ALK and nuclear factor kappa B (NF kappa B) pathways. Genomic studies of ALCL-11 showed the TP53 loss and the in vivo subclonal expansion of lymphoma cells, lacking PRDM1/Blimp1 and carrying c-MYC gene amplification. The treatment with proteasome inhibitors of TRAF1-ALK cells led to the downregulation of p50/p52 and lymphoma growth inhibition. Moreover, a NF kappa B gene set classifier stratified ALCL in distinct subsets with different clinical outcome. Although a selective ALK inhibitor (CEP28122) resulted in a significant clinical response of hPDT mice, nevertheless the disease could not be eradicated. These data indicate that the activation of NF kappa B signaling contributes to the neoplastic phenotype of TRAF1-ALK ALCL. ALCL hPDTs are invaluable tools to validate the role of druggable molecules, predict therapeutic responses and implement patient specific therapies.

OBJECTIVES: The World Health Organization (WHO) thymoma histological classification clinical value remains a controversy. In this study, we evaluated its prognostic significance in patients with thymoma treated with radical intent. METHODS: Six high-volume Italian Thoracic Surgery Institutions collaborated with their own retrospective anonymized datasets. Demographic, clinical, pathological and treatment data were examined. AWHO histological classification (WHO-HC) collapsed scheme (A/AB and B1/B2 types merged) was proposed and compared with the traditional one. Predictors of survival were assessed using a Cox model with shared frailty. Competing-risk regression models were performed to identify the association between individual factors and freedom from recurrence. RESULTS: Between 1990 and 2011, 750 thymomas were operated on in participating centres. Myasthenia gravis was observed in 363 (48%) patients. A complete resection was achieved in 676 (91%) cases. One hundred and nine patients (15%) had a WHO-HC A type, 166 (22%) AB, 179 (24%) B1, 158 (21%) B2 and 135 (18%) B3. The rate of 5-year OS and cumulative incidence of recurrence for all cases was 91% and 0.11, respectively. Five-year survival rates by WHO-HC in the collapsed scheme were A/AB 93%, early-B 90% and advanced-B 85%. Masaoka stage only was demonstrated to be an independent predictor for survival and recurrence. The WHO-collapsed scheme showed a trend in influencing recurrence overall survival development (hazard ratio: 1.32; P=0.16). CONCLUSIONS: Our results show evidence of lack of signifi cance by WHO-HC in influencing prognosis, even though the proposed collapsed scheme revealed a fair stratification of risk to relapses and better correlation with patients’ clinical characteristics.

The histologic classification of thymoma remained controversial since 1999, when the World Health Organization (WHO) Consensus Committee published a histologic typing system for tumours of thymus. Clinical features, postoperative relapsing rates, and survival of patients with thymoma were evaluated with reference to the WHO histologic classification, based on a series of 178 patients, submitted to surgery between 1988 and 2000. There were 21 type A, 49 type AB, 45 type B1, 50 type B2 and 13 type B3 tumours. The invasiveness of tumours was 23.8%, 51%, 73.3%, 82% and 100% for types A, AB, B1, B2 and B3 tumours, respectively. The frequency of invasion of the great vessels increased according to the tumour type in the order A (0%), AB (4%), B1 (6.6%), B2 (22%), and B3 (23%). The 10-year disease-free survival was 95%, 90%, 85%, 71% and 40% for types A, AB, B1, B2 and B3, respectively. According to the Masaoka staging system, the disease-free survival rates were 94%, 88% and 66% for stages I, II and III, respectively, at 10 years. No stage IVA thymomas reached 10 years follow-up. Overall survival at 10 years were 88% and 25% when complete and incomplete resection were considered. By multivariate analysis, Masaoka staging system, WHO histologic classification and complete resection were significant independent prognostic factors, whereas age- and sex-associated myasthenia gravis were not. The present study demonstrated the World Health Organization histologic classification a good prognostic factor, such as completeness of surgical resection and Masaoka staging system.

Most follicular lymphomas (FLs) have a phenotype consistent with the origin from CD5-, CD10+, bcl-6+ follicular center cells and can progress to diffuse large B-cell lymphoma (DLBCL). CD5 is expressed in about 10% of DLBCLs, showing prognostic value, whereas expression is rare in FL. We present 6 cases with coexisting features of CD5+ FL and CD5+ DLBCL, supporting a follicular origin for some CD5+ DLBCLs. The follicular areas showed a meshwork of CD21+ follicular dendritic cells that were lacking in the DLBCL areas. All cases showed a clonal CD19+, CD20+, CD5+, and CD10+ population in both follicular and diffuse areas. Molecularly, 4 of 6 cases demonstrated immunoglobulin heavy chain rearrangements and 1 case, a bcl-2/immunoglobulin heavy chain gene rearrangement. Somatic hypermutations were high in all 4 cases, in keeping with their germinal center origin. Four of five patients died of disease within 42 months, consistent with the proposed prognostic value of CD5 expression in DLBCL. Our data describe an aggressive variant of CD5+ FL suggesting the follicular origin of some CD5+ DLBCLs.

Introduction: MYC and BCL2 overexpression assessed by IHC and rearrangement detected by FISH are important prognostic factor in Diffuse Large B-Cell Lymphoma (DLBCL). Double Hit Lymphoma (DHL) patients have a poor prognosis with conventional therapy and Double expressor Lymphoma (DE) have worse outcome compared with conventional DLBCL although data are controversial. Aimed at a better knowledge of this issue, we performed a retrospective analysis to determine prevalence and outcome of Single Hit Lymphoma (SHL), DHL and DE in patients with de-novo DLBCL treated with Rituximab and CHOP. Methods: de novo DLBCL treated with R-CHOP between January 2003 and December 2013 were included in the study. BCL2 and BCL6 expression were evaluated by IHC at diagnosis while MYC expression was retrospectively investigated with Tissue Micro Arrays (TMA) technique; cases were considered positive for MYC, BCL2 or BCL6 expression by IHC if >40%, >40% or >25% of cells stained positive, respectively. FISH analysis for MYC and BCL2 rearrangements were performed with dual color break apart probes on TMA. PFS and OS were estimated with Kaplan-Meier method and compared between groups with the Cox model. We further evaluated in this series the IHC score proposed by Botto et al. (Blood 2014 124:2964) in DLBCL. The score was based on the assessment in IHC of the expression of MYC, BCL2 and BCL6. The three variable contributed with different risk in the multivariate analysis and an IHC sum additive score of 0-5 was calculated proportionally to the coefficient estimated (coefficient [Log hazard ratio] 0.92 for MYC+, 0.73 for BCL2+ and 0.48 for BCL6-), assigning an individual risk of 2 points for MYC or BCL2 positivity and 1 point for BCL6 negativity. Patients were stratified in three different risk groups; Low risk (0-1 point), Intermediate risk (2 points) and High risk (≥3 points). Results: Of a total of299 DLBCL screened, 267 were evaluable for survival analysis; median age was 65 years (range 20-90). 154 patients had complete immunohystochemical data and 101 were fully investigated by IHC and FISH. No significant differences in clinical presentation or in the outcome were seen between patients with or without available histologic tissue for IHC and FISH. Among 154 patients with complete IHC data we found 12 (8%) DLBCL without expression (DLBCL), 96 (62%) Single expressor (SE), 46 DE (30%). With a median follow up of 60 months, 5-year PFS rates were: DLBCL 90%, SE 60% and DE 43% respectively (fig 1) (HR 8.25 (95% CI: 1.12 -60.99) p 0.039); 5ys OS rates were 91%, 68% and 57% respectively (HR 5.72 (95% CI: 0.77 - 42.82) p 0.08). Applying the prognostic model (adjusted for IPI and age) defined in our previous pilot study we recorded 12/154 patients with low risk score, 61/154 with intermediate and 81/154 patients in high risk group. 5y-PFS rates were 91% vs 67% vs 45% (p=0.014) respectively (HR 1.74 (95% CI: 1.12 -2.69) p 0.014). Among 101 patients investigated by FISH we recorded 10 SHL (10%) and 8 DHL (8%). Clinical characteristics were superimposable among DLBCL, SHL and DHL with a prevalence of non GCB phenotype in DE group (56%). Among 101 patients fully investigated for FISH and IHC, 38 patients had MYC overexpression by IHC; 11 of them had also a MYC translocation. We found 3 cases with MYC rearrangement without protein overexpression. With a median follow up of 60 months PFS in DLBCL, SHL and DHL was 65%, 58% and 25% respectively (fig. 2); 5 ys OS was 70%, 77% and 25% respectively. The worse prognosis of DHL was statistically significant with an HR of 3.3 (95% CI: 1.37- 7.94, p 0.008) in terms of PFS and 3.9 (95% CI:1.59- 9.52 p 0.003) in terms of OS. Conclusion: Our data confirmed that our IHC prognostic score based on MYC, BCL2 and BCL6 expression, is a simple, reproducible and valid prognostic assessment that identify three groups with a different outcome in a large cohort of DLBCL. Moreover these data confirm intermediate prognosis for patients with DE lymphoma and poor prognosis of DHL treated with conventional chemoimmunotherapy. Figure 1 PFS in patients with complete IHC data Figure 1. PFS in patients with complete IHC data Figure 2 PFS in patients with complete FISH data Figure 2. PFS in patients with complete FISH data Disclosures Chiappella: Roche: Speakers Bureau; Celgene: Speakers Bureau; Janssen-Cilag: Speakers Bureau; Teva: Speakers Bureau; Pfizer: Speakers Bureau; Amgen: Speakers Bureau. Cavallo:Celgene: Honoraria; Onyx: Honoraria; Janssen-Cilag: Honoraria. Vitolo:Celgene: Honoraria; Gilead: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Summary Background B-diffuse large-cell lymphomas (DLCL) have been associated with some molecular lesions, but the role of such lesions as prognostic markers is still controversial. This report concerns an investigation of the frequency and clinical correlation of bcl-6, bcl-2, c-myc rearrangements and 6(q) deletions in B-DLCL. Patients and methods The presence of these genetic lesions was analyzed in samples of lymph nodes or bone marrow collected at diagnosis in 71 patients with B-DLCL, all treated with an antracycline-containing chemotherapy regimen. Results Rearrangement of bcl-6 was found in 11 patients (15%), rearranged bcl-2 in 12 (17%), 6(q) deletions in 10 patients (14%) and c-myc rearrangement in four (6%). Patients with rearranged bcl-6 tended to have a more aggressive disease than patients with germ-line bcl-6 (intermediate–high/high risk according to IPI criteria: 73% vs. 43%), but there were no differences in three-year survival rates (62% vs. 42%) between the two groups. The numbers of involved extranodal sites were similar in patients with rearranged and those with germ-line bcl-6. Patients with bcl-2 rearrangement appeared to have a less aggressive disease than those with germ-line bcl-2 (low/low–intermediate risk 75% vs. 47%) and a slightly better three-year survival rate (70% vs. 41%) but again the difference was not significant. Both groups with or without 6(q) deletion had similar clinical characteristics and outcomes. The four patients with c-myc rearrangement had aggressive disease and did poorly. Conclusions The analysis of molecular lesions in B-DLCL may be useful for a better diagnostic definition; however, in this study we were unable to show that the evaluated genetic lesions had a significant impact on clinical outcome.

Objective Thymic carcinoma (TC) is a rare and invasive mediastinal tumor, with poor prognosis. Most of the previous published papers are single-institution based, reporting small series of patient, sometimes including palliative resection. This study collected patients with TC treated in 5 high-volume Italian Thoracic Surgery Institutions. Methods A multicenter retrospective study of patients operated for TC between 2000 and 2011 was conducted. Exclusion criteria were: Neuroendocrine thymic neoplasms, debulking/palliative resection and tumor biopsy. Cause specific survival (CSS) was the primary endpoint. Results Four hundred and seventy-eight patients underwent surgery for thymic malignancies: 40 of them (8.4%) had TC. Eleven (27.5%) received induction chemotherapy because of their radiological invasiveness. A complete resection (R0) was achieved in 36 (90%; 9/11 submitted to induction chemotherapy). Adjuvant radio/chemotherapy was offered to 37 patients, according to the type of surgical resection and tumor invasiveness. Three, 5 and 10-year survival rates were 79%, 75% and 58%. Recurrences developed in 10 patients. R0 resection ( p p =0.03) resulted significant prognostic factors at univariate analysis. Independent CSS predictor was the achievement of a complete resection ( p Conclusions TC is a rare and invasive mediastinal tumor. A multimodal approach is indicated especially in TC invasive forms. The achievement of a complete surgical resection is fundamental to improve survival.

Thymic epithelial tumors include several entities with different biologic behavior. Chemotherapy is indicated in advanced disease, but limited data exist on gene expression correlation with the response to chemotherapeutic agents.A series of 69 thymic neoplasms (7 A-, 6 AB-, 6 B1-, 10 B2-, 14 B3-thymomas, 22 carcinomas and 4 combined tumors) was collected to assess gene expression of thymidylate synthase (TS), excision repair cross complementing-1 (ERCC1), ribonucleotide reductase subunit 1 (RRM1), topoisomerase 2α (TOP2A) and mTOR.A strong linear correlation between TS gene and protein expression was observed (P0.0001, R=0.40). TS expression was significantly lower in pure A-thymomas and thymic carcinomas (P0.0001) and progressively decreasing from B1-type to thymic carcinomas (B1B2B3C; P0.0001). RRM1 and TOP2A mRNA expression levels were significantly correlated with TS levels (both P=0.03) with a similar trend of expression among histotypes. RRM1 and TOP2A high levels were significantly correlated with high TS (P=0.03) and low tumor stages (I-II) (P0.0001 and P0.01, respectively). No relevant changes of ERCC1 and mTOR were detected.Low TS and, to a minor extent, RRM1 and TOP2A expression were detected in aggressive thymic tumors. These findings should be prospectively considered in selecting the most appropriate chemotherapy.

B-lineage diffuse large cell lymphoma (B-DLCL) arising de novo is characterized by a marked degree of clinical heterogeneity. To determine whether or not the clinical heterogeneity of de novo B-DLCL is reflected by heterogeneity in the molecular features of these tumors, we investigated the pattern of distribution of several genetic lesions in 70 cases of de novo B-DLCL at diagnosis. The panel of genetic lesions tested comprised the molecular alterations most frequently detected in B-DLCL, including rearrangements of BCL2, BCL6, and MYC as well as deletions of 6q and mutations of TP53. One or more genetic lesions were detected in 39/70 cases of B-DLCL. Isolated structural alterations of BCL2, BCL6, 6q or TP53 were detected in 8/70, 10/70, 11/70, and 3/70 cases, respectively. No isolated MYC lesions were detected. Six cases carried different combinations of two genetic lesions, including lesions of BCL2 + BCL6 (1 case), BCL2 + MYC (1 case), BCL2 + 6q (2 cases), or BCL6 + 6q (2 cases). One case had accumulated three genetic lesions, namely a rearrangement of BCL2 and BCL6 and a mutation of TP53. Overall, these data show that multiple distinct patterns of genetic lesions may associate with de novo B-DLCL, indicating that the molecular pathogenesis of this group of lymphomas is characterized by a high degree of molecular heterogeneity. Genes Chromosom Cancer 16:21–30 (1996). © 1996 Wiley-Liss, Inc.

Background. Bortezomib, an inhibitor of the proteasome, is effective in relapsed mantle cell lymphoma (MCL) and indolent lymphomas and it is synergistic with Rituximab to enhance apoptosis and NFkB depletion. On these basis, the FIL conducted a phase II multicenter study aimed to evaluate safety and efficacy of Bortezomib in association with Rituximab in relapsed/refractory non-follicular Lymphoma (Linfocytic, LL and Marginal Zone Lymphoma, MZL) and MCL, not eligible to high dose chemotherapy with stem cell transplantation. Patients and methods. The study was a prospective single arm phase II trial, designed on Simon two-stage Optimal Design. Primary end-point was to obtain an Overall Response Rate (ORR) > 40%. The aim of this analysis is to evaluate long term follow-up of Bortezomib and Rituximab combination. A central histological revision was planned in all the patients at the enrollment. Inclusion criteria were: 18-75 years, relapsed/refractory LL, MZL, MCL after 1-4 lines. Treatment schedule was: one course of 1.6 mg/sqm Bortezomib weekly in combination with standard 375 mg/sqm Rituximab on days 1, 8, 15, 22 followed by two courses of four weekly intravenous bolus of Bortezomib alone; patients with complete (CR), partial remission and stable disease at the intermediate evaluation were planned to be given three further courses with the same schedule. Results. From September 2006 to March 2008, 55 patients were enrolled and six were excluded at central histological revision. Clinical characteristics were: median age 68 (50-74); 16 (33%) LL, 8 (16%) MZL, 25 (51%) MCL; 42 (86%) stage III/IV; 33 (67%) bone marrow involvement. Median number of previous treatments was 2 (range 1-7); 34 (69%) were Rituximab pretreated; 21 (43%) had refractory disease. Thirty (61%) patients completed the treatment and 233 courses were delivered (median: 4.7 courses/patient); 19 (39%) patients did not because of no response in 13, adverse events in five, with only one toxic death due to interstitial pneumonia. ORR was 53% (CR 26.5%); no response was seen in 43% and 4% were not evaluable for response. ORRs by clinical subgroup were: LL 37%, MZL 50%, MCL 64%; Rituximab pretreated 62%, Rituximab naïve 33%; relapsed 64% and refractory 38%. With a median follow-up of 85 months, median Overall Survival (OS) was 61.5 months (95%CI: 35.0-81.5), with 5-years OS 51% (95% CI: 36-65) and median Progression Free Survival (PFS) was 8.9 months (95%CI: 5.3-18.3), with 5-years PFS 16% (95% CI: 7-28%). Five-years PFS by histology was: 12% (95% CI: 2-31) for LL, 17% (95% CI: 5-34) for MCL and 19% (95% CI: 11-53) for MZL. PFS rates were not different between Rituximab pretreated versus naïve nor international prognostic index 1-2 versus 3-4-5 nor refractory versus relapsed. By number of previous therapies, 5-years PFS for 1 previous therapy versus 2 versus 3 or more was: 24%, 14% and 13%, respectively, p=0.36. Conclusions. Weekly infusion of Bortezomib in combination with Rituximab is effective in relapsed/refractory indolent and MCL and represents a treatment option in this setting of patients. Disclosures No relevant conflicts of interest to declare.

Introduction: MYC and BCL2 overexpression and low BCL6 expression assessed by immunohistochemistry (IHC) have been reported as negative prognostic factors in DLBCL. We presented in a previous pilot study (Botto B, ASH 2014) a prognostic score based on overexpression of MYC, BCL2 and low expression of BLC6, assessed by IHC, in a retrospective cohort of 69 de-novo DLBCL, with an high proliferation index (MIB1 >70%), treated with R-CHOP between 2010 and 2013 (study cohort). Methods: The aim of the present study was to expand the analysis and validate this score in a larger retrospective cohort of DLBCL, regardless the proliferation index. Planned inclusion criteria were de-novo DLBCL diagnosis and combined modality treatment with Rituximab and CHOP treatment between January 2003 and December 2013 (validation cohort). Patients enrolled in the previous pilot study were considered as control group. Samples were investigated for MYC expression on Tissue Micro Arrays (TMA), while BCL2 and BCL6 expression were evaluated at diagnosis. Cases were considered positive for MYC, BCL2 or BCL6 expression by IHC if >40%, >40% or >25% of cells stained positive, respectively. FISH studies for MYC and BCL2 rearrangements are currently ongoing. PFS and OS were estimated with Kaplan-Meier method and compared between groups with the Cox model. Results: A total of 346 DLBCL were screened for this study; 203/346 had complete data for survival analysis; at the present time, 97/203 cases were fully investigated by histology and IHC. Clinical characteristics were: median age 65 years (IQR 55;73), 66 (69%) stage III-IV, 36 (38%) with LDH upper normal and 37 (39%) with IPI >2. These characteristics are superimposable to those of the pilot study, a part from a lower rate of IPI>2 (39% vs 67%). No differences in clinical presentation or in the outcome were seen between patients with or without histologic tissue for IHC. At the moment of the present analysis, MYC overexpression was detected in 33 (34%); BCL2 overexpression in 84 (87%) and BCL6 low expression in 32 (33%). Overall, 3y-PFS and OS were 68% and 77% respectively. Applying the prognostic model defined in our previous pilot study (adjusted for IPI and age) and based on different coefficient score (2 points for MYC or BCL2 positivity and 1 point for BCL6 negativity, pooled scores 0-1, 2 and >3) 5y-PFS rates were different across the 3 groups: 85% vs 67% vs 54% (HR 2.67 IC 0.85-8.40 p=0,094). Having found similar results in both cohorts, we integrated the two population (validation cohort and study cohort) in a total of 153 patients: 12 had score 0-1, 60 score 2 and 80 >3. 5y-PFS rates were significantly different across the 3 groups (Figure 1): 90% vs 62% vs 44% (HR 2.02 IC 95% 2.02-3.19, p=0.003). Indeed, 5-yrs-OS was significantly worse in high score group with 90% vs 76% vs 61% (HR 1.77 IC 95% 1.03-1.05, p=0,038) (Figure 2). Conclusion: Our data showed that a IHC prognostic score based on MYC, BCL2 and BCL6 expression, is a simple, reproducible and valid prognostic assessment that predicts outcome in a larger cohort of DLBCL, regardless of the proliferation index, forecasting significant different PFS and OS rates. Further extension of the study applying IHC and FISH analysis in the whole population of patients will be available at the time of ASH meeting and will be helpful to better refine the model. Figure 1. 5-yrs PFS in 153 patients (Validation cohort + Study cohort) Figure 1. 5-yrs PFS in 153 patients (Validation cohort + Study cohort) Figure 2. 5-yrs OS in 153 patients (Validation cohort + Study cohort) Figure 2. 5-yrs OS in 153 patients (Validation cohort + Study cohort) Disclosures No relevant conflicts of interest to declare.

Background: Peripheral T-cell lymphomas (PTCL) have an aggressive clinical course with a poor 5-year overall survival with conventional therapy. Autologous stem cell transplantation (autoSCT) and allogeneic stem cell transplantation (alloSCT) can improve long-term disease control in first and second remission, respectively. Prognostic factors are not able to discriminate patients with chemorefractory disease. Preliminary evidence from gene expression profiling (GEP) and immunoistochemical study have suggested that the majority of PTCL-not otherwise specified (PTCL-NOS) can be subdivided in two different groups based on GATA3 and Tbet (transcription regulators of T-helper 2 and T-helper 1 lymphocytes) expression with a poorer prognosis in the former group. Because GEP studies are not feasible in routine clinical practice, we investigated whether expression changes of those proteins could be evaluated by immunohistochemistry (IHC) and used to predict prognosis. Material and Methods: We collected paraffin tissues from 63 consecutive patients (pts) with a diagnosis of PTCL [subtypes: PTCL-NOS (n=32), ALK-negative anaplastic large cell lymphomas (n=15, ALCL ALK-negative), angioimmunoblastic (n=16, AITL)] treated at different Italian Institutions. Sections were analyzed for Ki-67, GATA3, Tbet and CCR4 expression by IHC. Results were expressed as percentages of positive tumor cells. Histology was centrally reviewed. Median age was 58 years (range, 18-76 years); 20 of 63 (32%) pts were characterized by intermediate-high/high IPI; 47 of 63 (74%) pts were candidated to transplantation whereas 16 (25%) were not due to age ≥65 years (n=14) or limited stage/IPI 0 (n=2). Fifty-nine pts (93%) received anthracycline-based induction chemotherapy. Twenty of 47 (43%) pts underwent autoSCT in first (n=11) or subsequent remissions; 23 of 47 (49%) underwent alloSCT in first remission (n=3) or at relapse (n=20). The median follow-up of alive pts was 30 months (range, 8-250). Results: Expression of GATA3 was highly variable, but was significantly higher on average in PTCL-NOS [34% positive tumor cells (range, 1-99%)] than in other subtypes [11% (range,1-97%) in ALK-negative ALCL (p=0.027); 12% (range, 1-82) in AITL (p=0.0025)]; The mean value of Ki67 expression was higher in ALK-negative ALCL [87% (range, 70-95%)] as compared to other subtypes [46% (range,15-90%) in PTCL-NOS (p In PTCL-NOS (n=32), ROC curve analysis identified a cut-off of 27% for GATA3 with 16 pts showing a GATA3 expression higher than the cut-off value. While no significant difference in baseline clinical characteristics was observed among the two groups, pts with GATA3 ≥27% showed a significantly lower CR rate following induction [19% versus 62% (p=0.02)]. Cases with high GATA3 expression were significantly associated with a reduced 5-year PFS and OS as compared to those with low expression [PFS: 6% (95%CI:1%-24%) versus 49% (95%CI:22%-71%), (p=0,004); OS: 39% (95%CI:13%-65%) versus 72% (95%CI:35%-90%) (p=0,04) respectively]. In multivariate analysis including age, IPI score ≥2 and sex, high GATA3 expression retained a strong prognostic value in terms of PFS (p Conclusions: Our analysis identifies GATA3 ICH expression as a strong prognostic and predictive biomarker among PTCL-NOS. Pts with high GATA3 expression values were characterized by chemorefractory disease and poorer outcome even with transplantation strategies. Validation of this biomarker in a larger series of patients is ongoing. Disclosures No relevant conflicts of interest to declare.

AIMS--To verify the correlation between MIB-1, Ki67, and proliferating cell nuclear antigen (PCNA-PC10) scores and S-phase fraction in intermediate grade non-Hodgkin's lymphomas (Working Formulation F); and their reliability in differently processed tissues. METHODS--Forty one non-Hodgkin's lymphomas were classified as (F) intermediate grade malignant lymphomas according to the Working Formulation; mitotic counts and percentage of large cells were assessed for each case. Sections from formalin fixed, paraffin wax embedded tissues were stained with anti MIB-1 monoclonal antibody, after microwave oven processing, and anti-PCNA (PC10) monoclonal antibody using an avidin-biotin immunoperoxidase (ABC) method. One thousand cells from 10 representative fields were scored. Frozen sections from surgical specimens were stained with Ki67 monoclonal antibody using the ABC method; the fraction of Ki67 positive cells was calculated scoring 1000 cells. Flow cytometry analysis (FCM) was performed on cell suspensions from fresh tissues. Correlations between data were estimated using linear regression. RESULTS--A linear correlation was found between MIB-1 and Ki67 scores (r = 0.92; p < 0.00001); between MIB-1 and PCNA scores (r = 0.79; p < 0.00001); and between MIB-1 score and S-phase fraction (r = 0.51; p = 0.0006). A linear correlation was also found between Ki67 and PCNA scores (r = 0.85; p < 0.00001); between Ki67 score and S-phase fraction (r = 0.6; p = 0.0002); and between PCNA score and S-phase fraction (r = 0.74; p < 0.00001). A correlation was found between mitotic counts and MIB-1 (r = 0.56; p = 0.0001), PCNA (r = 0.51; p = 0.0007), or Ki67 scores (r = 0.47; p = 0.002); between the percentage of large cells and MIB-1 (r = 0.49; p = 0.0009), PCNA (r = 0.6; p = 0.00003), and Ki67 scores (r = 0.53; p = 0.0003) and S-phase fraction (r = 0.55; p = 0.0002). CONCLUSION--MIB-1, Ki67, and PCNA (PC10) scores and S-phase fraction are highly correlated and equally well represent the proliferative activity of intermediate grade non-Hodgkin's lymphomas in differently processed material. MIB-1 and PCNA stains can be applied even on small biopsy specimens. MIB-1 produces homogenous staining without background; it also strongly stains mitotic figures. It can be performed on routinely processed tissues, permitting the simultaneous evaluation of the morphology and tumour cell kinetics. The wide standard deviations of the proliferative indices found for intermediate grade NHL suggest that this category probably includes various degrees of malignancy.

The concept of anaplastic large-cell lymphoma (ALCL) has changed over the years because of a stream of new information and novel understanding regarding the cell of origin, biology, genetics, and clinical features of these neoplasms. This new information has led to the current classification proposed by the expert reviewers of the World Health Organization. The objective of this review is to present the most updated information on the cytologic and histologic features of these entities, with a special reference to diagnostic algorithms. A detailed description of the genetic aberrations and the pathogenetic mechanisms leading to transformation is presented. The clinical features of ALCL and novel tailored strategies are briefly illustrated.

We report two cases of diffuse large B cell lymphoma (DLBCL), both occurring in the small bowel, which coexpress PAX5, weak or no CD20 and the CD4 antigen. The CD4 was initially identified by flow cytometry and then confirmed by immunohistochemistry. CD4 is a representative marker for helper T-lymphocytes and is present on a subset of thymocytes, peripheral T cells and monocytes or macrophages. Unlike CD2 and CD5, no B cell fractions are known to express CD4. It might be hypothesized that the deregulated control of gene expression in malignant B cells, in particular PAX5, leads to the activation of some silent or repressed genes of T cell differentiation. Although lineage infidelity is described in some B lymphomas, it remains as an uncommon phenomenon; to our knowledge, cases reported here are the first two cases of DLBCL of the gastrointestinal tract coexpressing the CD4 antigen to be described to date.

Objective: In thymomas, World Health Organization (WHO) histology, Masaoka stage and myasthenia gravis (MG) have long been considered important for patient management and outcome. Their role has been independently investigated in the past. Few studies, however, focussed on the correlations among these variables. The aim of the present study was to retrospectively evaluate, in our patient population of resected thymomas, the inter-relationships among MG, WHO histology and Masaoka stage, and to look at how and to what extent one variable is associated with theothertwo interms of clinicalpresentationand survival.Methods:FromJanuary 1990to October2008, 255patientsreceivedresectionof thymoma. MG was present in 105 cases (41%). Histology by WHO was: 25 A (10%), 72 AB (28%), 65 B1 (25%), 69 B2 (27%) and 24 B (9%). Masaoka staging was stage I, 54 cases (21%), stage II, 86(34%), stage III 79 (31%), and stage IVA 36 (14%). Ordinal and logistic regression models were undertaken to analyse correlations among ordinal (WHO histology and Masaoka stage) and categorical (MG, A vs B WHO types) variables. Univariate and multivariate survival analysis were also performed using the same covariates. Overall survival (OS) and disease-free survival (DFS) were calculated. Results: MG was associated with early Masaoka stages (odds ratio (OR) 0.45, 95% confidence interval (CI) 0.33—0.62) and B-type thymomas (OR 1.59, 95% CI 1.23—2.05). B-type thymomas were associated with high Masaoka stage (OR 0.46, 95% CI 0.36—0.60). High Masaoka stage wasassociatedwith non-MG (OR 3.27;95% CI 2.00—5.34). In univariate survivalanalysis, MG (p = 0.01) and Masaokastage (p = 0.0001)were significant prognostic indicators using OS. Using DFS, WHO histology (A/AB vs B1/B2/B3 types) (p = 0.05) and Masaoka stage (p = 0.0001) had a prognostic significance. In multivariate analysis, only Masaoka stage was an independent prognostic covariate using OS (hazard ratio (HR) 2.57, 95% CI 1.46—4.52, p = 0.001) and DFS (HR 3.18, 95% CI 1.56—6.52, p = 0.001). Conclusions: In thymomas, MG, WHO histology and Masaoka stage are inter-related. MG has an influence on histology and stage at presentation, while two clinical/histologic patterns are more likely: early Masaoka stage A/AB WHO type and high Masaokastage/B WHO type. Among the three factors, only Masaoka stage had a prognostic significance on OS and DFS. Our results suggest that a consistent staging system for thymomas should take into account all three variables. # 2010 European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved.

Summary In a series of 193 patients with advanced stage diffuse large-cell lymphoma (DLCL) treated with MACOP-B, 18 (11%) were defined as having a stage II large B-cell lymphoma with sclerosis of the mediastinum. This type of lymphoma has been reported to have a highly aggressive behaviour and special histological and clinical features. In our series young women were more commonly affected and the most striking clinical feature was the presence of a bulky mediastinal mass in 81%. A comparison was made between stage II patients with DLCL with and without sclerosis. The group of patients with sclerosis had prognostic parameters significantly worse than those of the patients without sclerosis, namely, elevated LDH level and bulky disease. The complete remission rates (89% vs 76%) were similar in the two groups and, with a median follow-up of 23 months, survival and disease-free survival rates were also superimposable. MACOP-B chemotherapy has been proven effective in this subgroup of lymphoma patients with sclerosis that had thus far been reported to have a poor prognosis.

Comparative arteriographic and clinical studies have clearly demonstrated the superior long-term patency of internal mammary artery (IMA) versus eaphenous vein coronary bypass grafts since the IMA was first used for this purpose in 1972.‘-15 Little attention, on the other hand, has been directed to the histology and pathology of the saphenous vei#, l6 and the IMA and coronary arteries. l7 Moreover, the data are primarily confined to patients with coronary diseases and vessels already employed as prostheses. The better patency offered by the IMA, however, has highlighted its almost complete freedom from atherosclerosis on intraoperative inspection and clinical and arteriographic examination, even in patients whose coronary arteries were severely or critically atherosclerotic.18T lg The histologic side to these empiric observations has not been fully investigated through comparison of atherosclerotic lesions in several arterial regions. l8 This report therefore compares the basic anatomic structure and atherosclerotic changes observed in IMA, left coronary artery (LCA), and renal artery (RA) segments obtained postmortem from 27 unselected subjects. Arteries of comparable size were chosen to determine differences in the topography and local extent of atherosclerotic lesions. Methodology. IMA, LCA, and RA segments were removed during autopsy from 17 men aged 19 to 76 years (11 over 65) and from 10 women aged 50 to 72 years (7 over 65). The mean age of the 27 subjects was 59 + 14.3 years. The causes of death are shown in Table I. Sections from three distinct segments of

CD38 rules proliferation signals in chronic lymphocytic leukemia (CLL) cells, suggesting that the molecule is not merely a prognostic marker but also a key element in the pathogenetic network underlying the disease. CD38 has a genetic polymorphism, characterized by a C>G variation in the regulatory region of intron 1. The working hypothesis is that the presence of different alleles in CLL patients marks (or accounts for) some of the clinical heterogeneity. CD38 allele distribution in 248 Italian patients overlapped with that of the controls (n = 232), suggesting that susceptibility to CLL is not influenced by CD38 genotype. Stratification of patients according to markers of unfavorable prognosis constantly resulted in a significantly higher frequency of the rare G allele. Furthermore, analysis of clinical parameters showed that G allele is independently associated with nodal/splenic involvement. The highest G allele frequency was observed in the 16 patients of the cohort that developed Richter syndrome (RS). Five-year cumulative incidence of transformation was significantly higher in G allele carriers than in CC homozygotes. Multivariate analysis on a total of 30 RS patients confirmed that the probability of transformation is strongly associated with G allele, likely representing an independent risk factor for RS development.

The phenotype of the lymphoid cell component of 35 thymomas was investigated by analyzing cryostat sections and lymphocyte suspensions. The morphology in each case was determined by examining multiple tissue samples from different parts of the tumor. Structural heterogeneity was shown in 14 thymomas, and a homogeneous morphology of cortical or medullary or mixed types in the others. To assess whether this heterogeneity was correlated with differences in the lymphoid phenotype, we analyzed both lymphocyte suspensions and frozen sections from the same samples. Phenotypical differences in the suspensions of each thymoma in the heterogeneous group were noted and similar differences were also observed in the cryostat sections. Phenotypical abnormalities were found in some thymomas. They consisted of the simultaneous expression of cortical and medullary markers, which was most marked in the heterogenous mixed-type thymomas invading the lung. Furthermore, the global phenotype was tested on a pool of lymphocyte suspensions in all thymomas. This procedure distinguished cortical, medullary and intermediate cortico-medullary immunophenotype models which closely correlated with the tumor histology. It was concluded that, due to the frequent structural and immunological heterogeneity of thymomas, correct assessment of their lymphoid component requires a two-step analysis. This comprises: 1) individual suspensions from samples taken from different areas of the same thymoma, and 2) a pool of these suspensions. The first step will reveal the different immunological characteristics. In the second, the lymphocyte phenotype, which may vary widely throughout the tumor, will be represented in its totality. These findings may be of great help in predicting clinical patterns, especially possible malignant evolution.

We studied the histologic and multiparameter flow cytometry (MFC) features of 12 cases of angioimmunoblastic T-cell lymphoma (AITL), 13 of mature T-cell lymphoma, and 25 control cases of reactive lymphoid hyperplasia to evaluate the role of CD10 in the differential diagnosis of peripheral T-cell lymphomas (PTCLs). A characteristic immunophenotypic profile (CD2+/CD4+) with recurrent phenotypic aberrancies (eg, CD3 and CD7 loss) was identified in most AITL cases; MFC documented CD10 coexpression on T cells in 10 (83%). Mature T-cell lymphoma showed a more heterogeneous altered immunophenotypic pattern, and 2 cases of PTCL, unspecified, had clear evidence of aberrant CD10 expression on T cells. A small physiologic CD3+/CD4+/CD10+ T-cell population was detected by MFC in all control cases tested (range, 0.28%-4.71%), suggesting that a normal subset of peripheral CD10+ T cells exists. CD10 was a highly sensitive but incompletely specific phenotypic marker for diagnosing AITL; the differential diagnosis of PTCL, unspecified, must be related with traditional histologic features. A small number of CD10+ T cells in reactive lymph nodes suggests that this subpopulation may be the normal counterpart of neoplastic T cells in AITL. The biologic role of CD10+ T cells should be studied further.

Myeloid sarcoma ( MS) is a rare neoplasm whose knowledge is largely based on case reports and/or technically dated contributions. Ninety-two MSs in adulthood with clinical data available were evaluated both morphologically and immunohistochemically. Seventy-four cases were also studied by fluorescent in situ hybridization on tissue sections and/or conventional karyotyping on bone marrow or peripheral blood. Histologically, 50% of the tumors were of the blastic type, 43.5% either monoblastic or myelomonocytic and 6.5% corresponded to different histotypes. CD68/KP1 was the most commonly expressed marker (100%), followed by myeloperoxidase (83.6%), CD117 (80.4%), CD99 (54.3%), CD68/PG-M1 (51%), CD34 (43.4%), terminal-deoxy-nucleotidyl-transferase (31.5%), CD56 (13%), CD61/linker for activation of T cells (2.2%), CD30 ( 2.2%) and CD4 (1.1%). Foci of plasmacytoid monocyte differentiation were observed in intestinal cases carrying inv16. Chromosomal aberrations were detected in about 54% of cases: monosomy 7(10.8%), trisomy 8(10.4%) and mixed lineage leukemia-splitting (8.5%) were the commonest abnormalities, whereas t(8; 21) was rare ( 2.2%). The behavior was dramatic irrespective of presentation, age, sex, phenotype and cytogenetics. Most if not all, long survivors received bone-marrow transplantation. The present report expands the spectrum of our knowledge showing that MS has frequent monoblastic/myelomonocytic differentiation, displays distinctive phenotypic profile, carries chromosomal aberrations other than t( 8; 21), and requires supra-maximal therapy.

Objective: To determine whether or not patients with completely resected Masaoka stage II thymoma benefit from postoperative radiotherapy (RT). Methods: We retrospectively review the case records and compared the long-term outcomes of patients affected by Masaoka stage II thymoma treated by resection alone with same stage thymoma patients submitted to resection and RT. Surgical specimens were reviewed to confirm pathological stage, negative resection margins and histological subtype. Results: Between 1988 and 2000, we performed 197 resections for thymoma; 58 patients resulted to be affected by completely resected tumours with microscopic transcapsular invasion (stage IIA, n = 25) or macroscopic invasion into the surrounding fatty tissue with or without adhesion to the mediastinal pleura (stage IIB, n = 33). Thirty-two patients underwentonlycompletesurgicalresection(14stageIIA and18stageIIB);26patients underwent completeresectionand subsequentmediastinal RT (11 stage IIA and 15 stage IIB). RT dosages were 45—54 grays (Gy), in 25—30 fractions. Histological subtypes were similarly represented in both groups. Median follow-up was 91 months (range 9—170). Five intrathoracic recurrences occurred: three radiated patients (2 stage IIB — 1 AB and 1 B2 thymoma; 1 stage IIA B1 thymoma) and two not-radiated patients (1 stage IIA AB thymoma and 1 stage IIB B1 thymoma). Disease-free survival rate at 5- and 10-year were 94% and 87%, respectively. Log-rank test showed no difference in Kaplan—Meier survival curves (p = 0.432) between radiated and not-radiated patients. Conclusions: These data support the concept that radical surgical resection alone should be considered a sufficient treatment for stage II thymoma.

Introduction : MYC, BCL2 and BCL6 overexpression, assessed by IHC, with the latter conferring a better prognosis, have been reported to be a prognostic factor in DLBCL, but data are not consistent and sometimes contradictory. The aim of the present study was to assess the prognostic impact of overexpression of MYC, BCL2, and BCL6 in a retrospective cohort of de-novo DLBCL, selected for an high proliferation index (MIB1 ≥70%), treated consecutively with R-CHOP regimen. Methods: Patients with de-novo DLBCL diagnosed between January 2010 and December 2013 were included into the study. Inclusion criteria were: high proliferation index MIB1 ≥ 70% and a full course of R-CHOP regimen. Paraffin-embedded tumor samples were collected and investigated using immunohistochemistry (IHC) for MYC, BCL2 and BCL6. Fluorescence in situ hybridization (FISH) is ongoing. MYC/BCL2+ or MYC/BCL6+ double expression cases were identified if they had rearrangements of MYC and BCL2 or BCL6. MYC immunochemistry was done on TMA sections using the antibody clone Y69. BCL2 and BCL6 staining had been evaluated previously at diagnosis. Tumor cells were defined positive for MYC and BCL2 or BCL6 protein expression by immunostaining if >40%, >40% and >25% of cells showed positive expression, respectively. Progression free survival curves (PFS) were estimated using the Kaplan-Meier method and compared between groups using the log-rank test and Cox models. Results : One hundred and sixty seven patients are evaluable for clinical characteristics and 69/167 had paraffin embedded tumor samples available for immunohistochemistry at the time of present analysis. Clinical characteristics of the 69 cases were: median age 66 years (IQR 57;73), 45 (65%) male, 47 (68%) stage III-IV, 35 (54%) with elevated LDH levels and 46 (67%) at International Prognostic Index (IPI) high intermediate or high risk. Overexpression of MYC was detected in 28 cases (41%), 50 (72%) and 38 (55%) showed BCL2 and BCL6 overexpression respectively. Nineteen (28%) cases showed MYC/BCL2+ and 17 (25%) MYC/BCL6+ double expression. With a median follow up of 26 months, the median 2-years PFS was 59%. Overexpression of MYC and BCL2 proteins and low expression of BCL6 were associated with an inferior 2-years PFS in univariate analysis: MYC- vs MYC+ 64% vs 55%; BCL2- vs BCL2+ 71% vs 56%; BCL6+ vs BCL6- 61% vs 54%. In a Cox multivariate regression model adjusted for IPI and age, MYC overexpression, BCL2 positivity and BCL6 negativity showed prognostic relevance as significant independent indicators with different risk (Hazard ratio 2.53 for MYC+, 2.08 for BCL2+ and 1.62 for BCL6-). Established that the three variable contributed with different risk in the multivariate analysis, an IHC sum additive score of 0-5 was calculated proportionally to the coefficient estimated (coefficient [Log hazard ratio] 0.92 for MYC+, 0.73 for BCL2+ and 0.48 for BCL6-), assigning an individual risk of 2 points for MYC or BCL2 positivity and 1 point for BCL6 negativity. Two years-PFS was significantly different between all separate groups (Hazard ratio for unit increase 1.57 95% CI 1.11-2.22, p=0.01). After pooling scores 0-1 (with or without BCL6), 2 (presence of MYC or BCL2 only), and 3-4-5 (MYC+/BCL6-, BCL2+/BCL6-, MYC+/BCL2+, MYC+/BCL2+/BCL6-) 2-yrs PFS rates were different across the three groups: 100% vs 64% vs 50% (log rank p= 0.04) (figure 1). Conclusion: Our data showed, with the limits of a small sample size, that MYC overexpression alone or with high expression of BCL2 and/or low expression of BCL6 correlates with a worse prognosis independently by IPI score in a cohort of DLBCL selected for high proliferation index and treated with R-CHOP. Assessment of MYC, BCL2 and BCL6 expression by IHC represents a rapid, inexpensive, and reproducible technique. These results need to be confirmed in our complete series of 167 patients (analysis ongoing) and validated prospectively in a larger cohort, using standardized staining and scoring methodologies. Thus, MYC and BCL2 represent relevant biomarkers that should be tested in future clinical trials using novel effective and targeted agents in order to improve the prognosis of DLBCL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Peripheral T-cell lymphoma not otherwise specified (PTCL/NOS) is a rare non Hodgkin lymphoma characterized by heterogeneous morphologic and phenotypic features, aggressive clinical behavior, poor response to conventional treatments and dismal prognosis. Its molecular basis and relationships to normal T-cells are still unclear. We performed gene expression profiling of 17 PTCL/NOS cases, collected at diagnosis before any treatment, and 20 samples of normal T-lymphocytes. The comparison of the gene expression profiles of PTCL/NOS and purified normal T-cell subpopulations, including CD4+, CD8+, resting (HLA-DR-), and activated (HLA-DR+) elements, shows that PTCLs/NOS are more related to activated peripheral T-lymphocytes either CD4+ or CD8+, suggesting derivation from these compartments. Notably, immunohistochemistry on routine sections by using anti-CD4 and CD8 antibodies does not vicariate molecular analysis, as there is no correspondence between the global gene expression profile and CD4/CD8 antigen positivity. When compared with normal T-cells, PTCLs/NOS displayed a remarkable deregulation of functional programs often involved in tumorigenesis, such as apoptosis, proliferation, cell adhesion, and matrix remodeling. In addition, our analyses identified several genes that are specifically expressed in PTCLs/NOS. Their expression was confirmed at the protein level by immunohistochemical analysis of tissue micro-arrays including 160 primary PTCL/NOS cases. We applied several different markers that provided significant information as concerns the ectopic, paraphysiologic or stromal expression of the proteins corresponding to the deregulated genes. Among others, PTCLs/NOS aberrantly express CYR61, a molecule involved in drug resistance, and PDGFRA, a tyrosine kinase receptor whose deregulation is often related to a malignant phenotype. Notably, we demonstrated the constitutive activation of PDGFRA in the majority of cases - as indicated by its phosphorylation. Finally, we showed that imatinib mesylate significantly reduces PTCL/NOS primary cells viability after in vitro exposure at 1 mM concentration, independently from their sensitivity to conventional cytotoxic agents. These results are provided with biological implications relevant to the pathogenesis of the tumor, as well as for its clinical management.

Gastric marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT)type can regress after anti-Helicobacter pylori treatment. The International Extranodal Lymphoma Study Group, the United Kingdom Lymphoma Group, and the Groupe d'Etude des Lymphomes de I'Adulte have conducted a trial to ascertain whether the addition of chlorambucil is of benefit after anti-H pylori therapy. At the last interim analysis, 105 (55%) of 189 patients had achieved a complete histologic remission after anti-Helicobacter therapy. To further assess the ability of treatment to eradicate the lymphoma clone, we analyzed the gastric biopsies from a subset of the patients by polymerase chain reaction (PCR) targeted to the immunoglobulin heavy chain genes as a molecular marker for minimal: residual disease. Sixty-two cases were examined at diagnosis. Fifty-four cases were monoclonal by PCR. Forty-two of these patients achieved histologic complete remission (hCR) after anti-Helicobacter treatment: 34 cases! underwent molecular follow-up analysis. Fifteen patients (44%) were in molecular remission with a median follow-up of 2 years after antibiotic treatment and of 1 year after the achievement of hCR. Less than half of the patients with MALT lymphoma can achieve sustained molecular remission after anti-Helicobacter therapy. The presence of molecular disease in the absence of histologic disease does not appear to be associated with histologic relapse, but, given the indolent nature of MALT lymphomas, a longer follow-up is needed. (Blood. 2002; 99:2541-2544) (C) 2002 by The American Society of Hematology.

Long-term outcome, after first line intensified high-dose sequential (i-HDS) chemotherapy, was evaluated in 46 patients, aged

Anaplastic large cell lymphoma (ALCL) is a subtype of peripheral T-cell lymphoma that includes two entities ALK+ and ALK- ALCL based on chromosomal translocations of the anaplastic lymphoma kinase (ALK). ALK- ALCL have very poor clinical outcome with conventional chemotherapy. Lack of representative cell lines and/or models has compromised the development of successful therapy. Toward this end, we report the generation and characterization of a group of novel ALCL tumor grafts. We implanted subcutaneously (s.c.) tumor tissue fragments, fresh or viable cryopreserved, from 11 cases of primary systemic ALCL (3 ALK+ and 8 ALK-) in six- to eight-week old severe immunocombined immunodeficiency NOD Cg-Prkdcscid/Il2rgtm1wjl/SzJ (NSG) mice. A single leukaemic ALCL sample was injected i.v. or s.c. Six cases led to successful lymphoma growth within 6-12 weeks as subcutaneous solid spheroid masses constituted by a uniform population of large pleomorphic cells CD30-positive. All tumor grafts were re-implanted in recipient animals, in same cases up to 21 consecutive implants with an analogous time growth rate. Two cases, in the same way of the original human neoplasia, were early associated with concomitant enlargement of multiple organs. All tumor graft lines eventually showed disseminate disease with involvement of distant lymph nodes and of spleen (with homig in peri-arteriolar spaces), liver (initially observed within the sinusoidal spaces) and kidney. The morphological and immunohistochemical (IHC) evaluation (expression of CD30, cytotoxic proteins and ALK) of the serially propagated ALCL tumor grafts demonstrated a high concordance between primary and derived animal tumors which showed highly stable profile along different passages from the early generation (T1) until the final ones (up to T16). The clonal relationship with primary and corresponding tumor grafts was confirmed by gene TCR rearrangement analyses and by ALK FISH studies. In addition we interrogated the model using gene expression profiling and single nucleotide polymorphism which established a close relationship among primary and derived tumors. Massive parallel sequencing on mRNA transcripts defines the presence of two novel chimeras involving NF1-MAZ and TRAF1-ALK1, which confirmes the evidence of ALK signaling and pinpoint a role of RAS in ALK- ALCL. The tumor grafts displayed, also, therapeutic responses with conventional therapy (CHOP) which reflected that seen in the donor patients: initial partial responses followed invariably by clinical relapses. To overcome this refractory we assayed the sensitivity of two tumorgraft lines, generated from ALK+ ALCL, to a new selective ALK inhibitor (CEP28122) that led a rapid and stable response. This study demonstrated the key role of these libraries of tumor grafts, especially in developing and testing new therapeutic regimens in refractory ALCL patients. Citation Format: Fabrizio Tabbo’, Antonella Barreca, Rodolfo Machiorlatti, Katia Messana, Indira Landra, Francesco Abate, Michela Boi, Maria Todaro, Cristina Abele, Domenico Novero, Alberto Zamo’, Marco Chilosi, Maurilio Ponzoni, Mangeng Cheng, Bruce Ruggeri, Pierpaolo Piccaluga, Stefano Pileri, Enrico Tiacci, Brunangelo Falini, Lenny Shultz, Roberto Piva, Enzo Medico, Francesco Bertoni, Raul Rabadan, Giorgio Inghirami. Humanized NOD/Scid/IL2g-/- tumor grafts recapitulate primary anaplastic large cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3853. doi:10.1158/1538-7445.AM2013-3853

The cell proliferative activity of the clinico-pathologically heterogeneous non-Hodgkin's lymphomas (NHL) included in the intermediate grade F category of the Working Formulation (WF) was investigated. S-phase fraction with flow cytometry on cell suspensions, and Ki67 on frozen tissue sections were performed in 42 F NHL. An avidin-biotin immunocomplex method was used and 1000 cells from 10 representative fields were counted. DNA content, S-phase and Ki67 were also detected in 194 NHL covering the whole spectrum of the WF. DNA content anomalies were found in 52 of 194 NHL. Their incidence, like that of S-phase fraction and Ki67 positive cells, progressively increased from low- to high-grade. A linear correlation was found between Ki67 and S-phase (r = .59). Using the median value of proliferating cells obtained with both procedures as a cut off, two very different groups of lymphomas could be distinguished within a series of 42 F-intermediate NHL: with low and high proliferative cell activity (p < .0001) that were termed F(low) and F(high), respectively. A intermediate group was placed between them. It differed significantly from the others if Ki67 was used but only from the F(high) group if the S-phase fraction analysis was applied. No significant differences were seen when comparing F(low) with the single categories of low-grade NHL and F(high) with H high-grade NHL; no significant differences were found between F(high) and G, and between G and H categories. The existence of distinct groups of NHL in the F category, as defined by biological parameters assessing the cell proliferative activity, indicates that this category includes biologically heterogeneous lymphoma subtypes with different grades of aggressiveness. The results also indicate that the G intermediate category displays proliferation indices similar to those of H high grade category.

BACKGROUND The relationship between chronic lymphocytic leukemia (CLL) and supervening non-Hodgkin lymphoma is debated, as is whether a particular genomic pattern is related to the emergence of the terminal lymphoma. To investigate these features, the molecular organization of the immunoglobulin (Ig) gene region in a case during both the B-CLL and Richter transformation phase was studied. METHODS B-CLL and non-Hodgkin lymphoma cells were processed for Southern blot analysis of Ig heavy- and light-chain gene configuration. RESULTS Molecular studies of B-CLL cells revealed the presence of a single Ig heavy-chain rearrangement with both kappa and lambda light-chain rearranged genes, which was consistent with the occurrence of multiple mutational events during the development of the B-CLL clone. Molecular analysis of the lymphoma DNA showed new Ig heavy- and kappa light-chain rearrangements in addition to the original ones related to the CLL phase, indicating that the lymphoma tissue consisted of two genotypically distinct populations of cells. CONCLUSIONS On the basis of the overall molecular configuration, this heterogeneous pattern of Ig gene rearrangement was interpreted as an inherent genetic instability of the CLL clone, in which multiple mutational events allowed a selective pressure toward more aggressive subclones, resulting in the emergence of the terminal lymphoma.

Abstract 1784 Introduction. The outcome of MCL is unfavourable, with continuous relapses. The MIPI, a clinical score, defined performance status, age, LDH and leucocyte counts as predictors of MCL outcome. Ki-67 as cell proliferation index was evaluated in biological-MIPI (MIPI-b). Aim of the study was to tested MIPI on a retrospective group of MCL patients treated with Rituximab-chemotherapy with or without High Dose Chemotherapy and autologous stem cell transplantation (R-HDC); secondary endpoints were: to evaluate the feasibility of MIPI-b on a retrospective population and to quantify the predictive discrimination of IPI, MIPI, MIPI-b on the outcome of MCL in the Rituximab era. Methods. Between 1999 and 2009, 206 MCL >18 years at diagnosis consecutively treated in seven Italian institutions were included into the study. Histology was centrally reviewed and, if possible, Ki-67 evaluation was performed. Overall survival (OS) and failure-free survival (FFS) curves were estimated both overall and stratified by MIPI, MIPI-b and IPI score. Differences between curves were tested using the 2-tailed log-rank test. In order to quantify the predictive discrimination of MIPI, MIPI-b and IPI scores, a Cox's model analysis and univariate logistic models (with death and failure event as binary outcomes) were fitted and the area under the receiver operating characteristic (ROC) curves (c-index) was estimated in a subgroup of 120 patients that fulfilled MIPI, MIPI-b and IPI scores. Results. Clinical characteristics were: median age 61 (34-85) years, 78% stage IV, 73% with bone marrow involvement, 16% with blastoid variant; median leucocyte counts at diagnosis was 7.53×103 (2.38-175). First-line treatments were: R-HDC in 51%, Rituximab-Fludarabine based chemotherapy in 12%, Rituximab-CHOP in 32% and other Rituximab containing regimens in 5%. Ki-67 evaluation was performed in 135 patients; median Ki-67 value was 30%. Patients at high-risk (HR) were 29% according to MIPI, 18% according to MIPI-b and 33% to IPI. With a median follow-up of 48 months, 4-year OS was 72% (95% CI:65-78) and 4-year FFS was 49% (95%CI: 41–56). Four-year OS according to MIPI by risk groups was: LR 94% (95%CI: 85–97), IR 66% (95%CI: 47–80), HR 41% (95%CI: 26–55) and according to IPI: LR 87% (95%CI: 73–94), IR 88% (95%CI: 74–95), HR 41% (95%CI: 26–55) (Figure 1). In the subgroup of 120 patients that fulfilled MIPI, MIPI-b and IP scores an univariate logistic model and a Cox's model analysis were fitted. The c-index and Cox-index for death event were 76% and 75% for MIPI, 69% and 69% for MIPIb, 76% and 71% for IPI respectively; the c-index and Cox-index for failure event were 61% and 68% for MIPI, 59% and 64% for MIPIb, 68% and 66% for IPI respectively. A sub-analysis was conducted to validate the role of MIPI in R-HDC group; an univariate logistic model and a Cox's model analysis were fitted and the c-index and Cox-index for death event and for failure event were calculated (Table 1). Conclusions. MIPI score was confirmed as a good predictor of death event in MCL retrospective patients treated with Rituximab-chemotherapy regimens. MIPI score should be a good predictor of death event also in patients treated with R-HDC. The impact of MIPI-b score should be tested in prospective trials, with central histology review. New therapeutic strategies are warranted to improve the outcome of MCL namely in MIPI-HR group. Disclosures: No relevant conflicts of interest to declare.

Introduction: A specific molecular signature divides DLBCL into GCB and ABC profiles; in pre-Rituximab era, GCB group had a better prognosis. The addition of Rituximab (R) to chemotherapy improves the outcome in DLBCL. It remains uncertain the prognostic significance of GCB and ABC subgroups in Rituximab era. Patients and methods: from August 2000 to September 2006, 120 previously untreated patients Results: 92 samples were centrally reviewed and studied in immunoistochemistry. Overall 69 were evaluable; 23 were excluded due to inadequate pathological materials in 16 and incorrect inclusion criteria in 7. Thirty-seven patients were classified as GCB, 24 as ABC and 8 as GC-activated. Patient characteristics were well balanced between GCB and ABC subtypes. With a median follow-up of 53 months, OS in all 92 patients was 81% (95% CI: 71% - 88%); at the same timeframe, OS in GCB was 77% vs 86% for ABC (p=.5). With a median follow-up of 46 months, EFS in all 92 patients was 74% (95% CI: 64% - 82%); at that time, EFS in GCB was 65% vs 87% for ABC (p=.2). Conclusions: Rituximab as adjuvant to dose-dense and HDC with ASCT support is effective in high risk DLBCL in both GCB and ABC subgroups. In this R-HDC+ASCT study ABC profile as determined by TMA does not represent a poor prognostic feature. However 25% of patients still experienced progression. Further and more detailed biological analysis are needed to better identify these patients.