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The CTLA4 receptor is a CD28 homologue which induces inhibitory effect on activated T-cells. Peripheral T-cells proliferate spontaneously in CTLA4-deficient mice. These results led to an analysis of CTLA4 expression in human lymphomas (n = 82) including Hodgkin's disease (HD) and non-Hodgkin's lymphomas (NHLs), using immunohistochemistry. CTLA4 was present in neoplastic cells from most (10/11) T-cell malignancies, except for anaplastic and lymphoblastic subtypes (0/4). Malignant B-cells from rare (3/55) B-NHLs (all of follicular subtype) were also CTLA4-positive. Other B-NHLs (52/55) were negative in malignant B-cells and occasionally positive in T-cells. Reactive small lymphocytes, but not Reed-Sternberg cells, from all (12/12) HD cases were strongly CTLA4-positive. The CTLA4 ligands CD80 and CD86 were simultaneously expressed in most CTLA4-negative lymphoma cases. CTLA4 is thus expressed either in the reactive or in the malignant cell populations, depending on the lymphoma subtype. These results provide new insights leading towards therapeutic strategies based either on enhancement of anti-tumour immunity by CTLA4 blockade in reactive lymphocytes or on triggering of a CTLA4-mediated inhibitory pathway in lymphoma cells.

Fas ligand (FasL) is capable of inducing apoptosis of lymphoid cells by cross-linking with its natural receptor, Fas. We aimed to investigate the possible role of the Fas/FasL-mediated apoptosis in the development of human lymphomas. FasL mRNA was detected by reverse transcriptase-polymerase chain reaction in 38 out of 63 lymphoma biopsy specimens representative of various subtypes of non-Hodgkin's lymphoma (NHL) and Hodgkin's disease. FasL was co-expressed with Fas mRNA in most cases. Flow cytometry (FACS) analysis showed a bright FasL staining in 31% to up to 75% of the total cell population from 14 out of 16 samples; the presence of the FasL protein was confirmed by Western blotting. Dual-color FACS analysis showed that FasL was expressed by T cells in B-NHLs and T-NHLs. A significant percentage of B cells in various B-NHLs also stained positively for FasL. Freshly separated neoplastic B cells from three FasL+ and one FasL- B-NHLs displayed a relative resistance to Fas-mediated apoptosis, when compared to reactive T cells isolated from the same tissue samples. In contrast, the sensitivity to Fas-mediated killing of the T cells isolated from two FasL+ T-NHLs was not uniform. These data show that (1) FasL is expressed in both neoplastic and reactive cells from a significant proportion of lymphoma cases, and (2) that the intratumoral FasL+/Fas+ reactive T cells are more sensitive to Fas-induced apoptosis than the neoplastic FasL+/Fas+ malignant B cells. A putative defect in the Fas/FasL pathway may thus favor the development of malignant B cell populations.

Ich-1/Nedd2 and CPP32/YAMA are cysteine proteases related to interleukin 1-β-converting enzyme (ICE), which act as apoptosis effectors. Both molecules are expressed in T- and B-cell lines. The authors investigated their in vivo cellular distribution in normal and neoplastic human lymphoid tissues. Sixty-eight representative non-Hodgkin's lymphomas (NHL) and Hodgkin's disease (HD) samples, normal lymphoid organs, and nonlymphoid tumors were analyzed by immunohistochemistry (IHC). CPP32 expression in benign tissues was restricted to germinal center B cells, plasma cells, and a few interfollicular immunoblasts. All follicular NHLs and most diffuse large cell NHLs were CPP32 positive. Among T-cell NHLs, CPP32 expression was mainly observed in anaplastic large cell NHLs, whereas the other subtypes were less frequently positive. In contrast, lymphoid organs displayed only weak Ichl-L expression, located in sinusal histiocytes and thymic epithelial cells. Lymphomas were Ich1L negative, except for T-cell-rich B-cell NHLs, and about half of the HD samples, in which Reed-Sternberg cells (RSC) were usually Ich1L positive/CPP32 negative. Extralymphoid Ichl-L reactivity was found in particular organs like the kidney and various tumors. Western blot analysis confirmed the specificity of immunostaining. Neither CPP32 nor Ichl-L expression were correlated with intratumoral DNA fragmentation, as determined by the TUNEL assay. Altogether, these results indicate that CPP32 is preferentially expressed in germinal centers and thus could be involved in B-cell maturation. The differential expression of CPP32 and Ich1-L suggests that cysteine proteases differ in substrate specificities and carry out functions unrelated to apoptosis.

Despite the promise of the cancer stem cell (CSC) model, its clinical relevance remains to be proven in breast cancer (BC) and requires a sensitive xenograft assay to define CSC on key functional properties i.e tumorigenicity, self-renewal and differentiation potential. We report the establishment of primary breast tumor-derived xenografts (PDXs) that retain main primary tumors features (histo-phenotypic, molecular and genomic features, hierarchical organization of the tumoral cell population). We demonstrate that success in engraftment is correlated with the presence of CSC in primary tumor and predicts prognosis in patients (HR= 3.61; 95%CI [1.12-11.65], p=3.20E-02). The xenograft assay demonstrated the hierarchical organization of BC. Analysis of gene expression from functionally validated CSC yield to a breast CSC signature (BCSC-GES) is an independent predictor of patient survival. We then identified a core of genes commonly expressed by BCSC and embryonic, hematopoietic, neural stem cells, the CE-4SC. This shared transcriptional program comprised 19 genes encoding mismatch and nucleotide excision repair proteins, proteins implicated in transport through the endoplasmic reticulum, RNA processing, translation and transcriptional regulators, enzymes of detoxification and mitochondrial/oxidative stress, or enzymes of lipid metabolism. This “universal” stem cell program is also an independent predictor of patient survival. Using a screening of an RNA-interference library specially designed to block the expression of these 19 target genes, we validated the effect of single gene knock-down (KD) on the breast CSC population for 14 genes (KD of 5 genes decreased the CSC population and were called “self-renewers”genes, KD of 9 genes increased the CSC, and were designated as“differentiators”). Hence, this study validated the clinical relevance of CSC model in breast cancer for the first time, and the ability of PDXs to accelerate transfer for personalized CSC therapy into clinics. Citation Format: Emmanuelle Charafe-Jauffret, Christophe Ginestier, Francois Bertucci, Olivier Cabaud, Jose Adelaide, Max Chaffanet, Luc Xerri, Patrice Viens, Daniel Birnbaum. Cancer stem cells predict engraftment and poor prognosis of primary breast tumor. [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 244. doi:10.1158/1538-7445.AM2013-244

OBJECTIVE To test the hypothesis that tumor necrosis factor (TNF)-α may mediate the loss and the dedifferentiation of subcutaneous fat tissue in the insulin-induced lipoatrophies of a diabetic patient who presented extensive lesions. RESEARCH DESIGN AND METHODS An in vitro exploration of cytokine production by peripheral blood mononuclear cells (PBMC) from the reported case was performed and compared with the same explorations of PBMC from three nondiabetic subjects and three diabetic patients without lipoatrophic lesions. A proliferation test and an evaluation of TNF-α and interleukin (IL)-6 production from PBMC in presence of insulin were studied. RESULTS The production of TNF-α and IL-6 by the macrophages of the patient in presence of insulin were dramatically increased in comparison with control subjects. This process needed cooperation with other lymphoid cells and was abrogated by dexamethasone. CONCLUSIONS In our reported case, a local hyperproduction of TNF-α from macrophages that was induced by the injected insulin could explain the dedifferentiation of the adipocytes of the subcutaneous tissue and the reversion that was induced by the local injection of dexamethasone.

Breast cancer is a major health problem and heterogeneity of the disease has been considered as a strong limitation to find the best therapies to cure cancer, overcome recurrences and metastases. The establishment of models that reflect tumor biology and metastatic progression is critical to develop successful new therapeutic strategies. In the breast, orthotopic xenografts currently appear as the best models to study tumor growth, metastasis and develop tools for prognosis prediction. Furthermore, mouse transplant assays have been used to assess cancer stem cell (CSC) activity and demonstrate that leukemia and many solid tumors are organized along a hierarchical model. Despite the promise of the CSC model sustained by mouse transplantation assays, the clinical relevance of xenografts studies to identify determinants of stemness able to influence clinical outcome remains challenging. In breast cancer, transcriptional programs from functionally validated CSC populations remain to be deciphered. Here, we report the establishment of a bank of primary breast tumor-derived xenografts (xenobank). We showed that the xenografts retain the main features of primary tumors, that engraftment is correlated with the presence of CSC in tumors, and that engraftment in the mouse is able to predict prognosis in patients. This suggests that CSCs may govern breast cancer prognosis. We established the gene expression profiles of functionally validated ALDEFLUOR-positive CSC populations (breast CSC-GES) and demonstrated their clinical relevance. Among 2609 patients with breast cancers, we validated that he expression of the breast CSC-GES is correlated with poor outcome and metastasis in uni-and multivariate analysis (5-year MFS was 70% CI95 [67–74] in the breast CSC-positive class and 80% (CI95 [77–83]) in the breast CSC-negative class (p = 5.5E−04 with log-rank test). Furthermore, we identified a core of 19 genes commonly expressed in breast CSC, murine embryonic, neural and hematopoietic stem cells programs and demonstrated for each gene its ability to modulate breast CSC population, being implicated in self-renewing or differentiation programs. We found that the core of genes in common between four stem cell gene expression studies (CE-BCSC-3SC) displayed an adverse prognostic impact for patients with breast cancer. The core contained genes implicated in oxidative phosphorylation, detoxification, lipid metabolism, and genomic stability, and these shared determinants of stemness influenced clinical outcome. Thus, we show ed that CSCs from orthotopically engrafted primary breast tumors have clinical and biological relevance. This functionally validated CSC population is highly correlated with survival and express genes governing main stem cell functions, substantiating a major prediction of the CSC model and opening further promises for new CSC therapies using valid preclinical models. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-01.

Background and Design: Polymerase chain reaction (PCR) detection of circulating tumor cells from malignant melanoma (MM) was recently described, but the prognostic value of this method in the treatment of patients with MM remained unclear. In the present prospective study, blood samples (n=193) were collected from 93 patients with MM: 10 stage I patients after primary tumor resection, 18 patients with regional lymph node metastases before node resection, 33 disease-free but high-risk patients (previously treated for node metastases), and 32 patients with distant metastases. Circulating melanocytes were detected using a reverse transcriptase PCR method that analyzes tyrosinase gene expression. All patients were kept under regular surveillance. Results: The PCR assay was always negative in normal individuals and in subjects with non-MM metastatic cancer, while it was positive in 16 of 32 patients with dis- seminated MM. Five of eight patients who were PCR-positive before node dissection vs one of 10 who were PCR-negative relapsed within 6 months after surgery. In high-risk but apparently disease-free patients, the risk of relapse within the next 6 months was 3.8 times higher after a positive test result. In patients with distant metastases, a positive PCR predicted rapid disease progression. Conclusions: These data suggest that PCR detection of circulating melanocytes can be considered as a marker for rapid postoperative relapse after node dissection in patients with MM with regional node metastases, for short-term relapse in high-risk disease-free patients, and for rapid and severe progression in patients with distant metastases. This test may have a crucial interest in the treatment of patients with MM. (Arch Dermatol. 1995;131:443-447)

Development of pre-clinical models that reflect tumor biology and metastatic progression is a critical issue to better develop therapeutic strategies. The xenotransplantation mouse model of human primary tumors is presented as the best model to mimic patient tumor biology in different pathologies. In breast tumors, orthotopic transplantation recently provides strong evidence of maintenance in xenotransplants of main cognate breast tumoral features. As it has recently been reported that primary breast tumor engraftment is a prognostic indicator of disease outcome for women with breast cancer, we aimed to determine parameters that influence engraftment success. Between January 2008 and June 2009, we transplanted 76 fresh primary tumors from 76 different individuals into cleared and humanized fat pad of female NOD-SCID/ gamma (c) null mice. Tumors grew from 20 out of the 76 samples (29%), and we maintained the main molecular (molecular subtypes, genomic profiles, phenotypes) and histological features of matched primary tumors in xenografts. If a high grade, an absence of hormone receptor expression, a basal-like/ERBB2 molecular subtype was correlated with engrafment rate, the presence of ALDH-expressing CSC in primary tumors was the only parameter predicting engraftment in an independent manner. Using this model, we showed that breast cancer xenografts were hierarchically organized with a subpopulation of ALDEFLUOR-positive cells that maintains tumorigenic activity. In order to identify pathways regulating tumorigenic activity of the CSC population, we derived a CSC gene expression signature (GES) from the comparison of expression profiles of the ALDEFLUOR-positive and -negative populations from our xenograft series. This CSC signature presented three mains core transcriptional programs containing DNA repair genes, suggesting a special ability to resist to DNA damage, gene of G1-S transition checkpoint control including pRb and E2F, and gene of G2-M transition checkpoint, suggesting a potential role in self-renewal and differentiation programs. Given that the presence of CSC in primary tumors is an indicator of tumor engraftment and engraftment predict prognosis, we surmised that molecular machinery governing CSC properties is likely to influence clinical outcome. Indeed, our established CSC GES was able to predict bad outcome in primary breast cancer. Thus, we can speculate that the greater retention of cancer stem cell programs observed in patients with shorter survival compared with those with better outcome may reflect greater uncoupling of self-renewal and maturation programs. Overall, our data indicate the importance of developing CSC biomarkers to contribute to personalized cancer therapy and the need to identify therapeutic targets directed toward CSCs using orthotopic xenografts models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5339. doi:1538-7445.AM2012-5339

Hodgkin's disease is histologically characterized by the presence of Reed-Sternberg cells (RSC) and reactive cells, including numerous T lymphocytes. Some forms of B cell-non-Hodgkin's lymphoma also contain a rich T-cell population. Previous studies have suggested that the T-cell receptor repertoire of tumor-infiltrating T lymphocytes (TITL) that act specifically against tumor-related antigens, should be restricted. The authors in this study used the polymerase chain reaction to explore the possible existence of such an immunologic response of TITL against RSC or neoplastic B cells. They studied variable (v) region genes of T-cell receptor alpha and beta chains expressed by infiltrating lymphocytes in biopsy specimens from seven patients with Hodgkin's disease and three with B cell-non-Hodgkin's lymphoma. These latter samples were selected based on a rich T-cell content. Primers specific for 18 different V-alpha and 21 V-beta families were used. In every case, TITL showed an unrestricted pattern of expression similar to the repertoire observed in peripheral blood lymphocytes. Although such experiments are limited, the apparent lack of selection of a single or a limited number of T-cell subsets in the affected tissues did not support the existence of an in vivo immunologic interaction between TITL and antigens related to RSC or neoplastic B cells.

Abstract 1788 The GELA-sponsored intergroup PRIMA Phase III study was designed to investigate the potential benefit of 2-years of rituximab maintenance in high tumour burden follicular lymphoma patients responding to one of three non-randomised first line immunochemotherapy treatments. The results of the pre-planned interim analysis with 24 months median follow-up were positive and demonstrated a significant reduction in the risk of progression for patients randomized to rituximab maintenance (Salles et al., ASCO 2010). We present here the updated efficacy and safety results after an additional year of follow-up, when all randomized patients had completed the observation/maintenance period. From December 2004 until April 2007, 1217 patients were enrolled from 223 centres. Complete data are available for 1193 patients who had the following pre-induction treatment characteristics: median age 56 years [range 22–87]; 52% male; 90% Ann Arbor stage III-IV; 33% B symptoms; 55% bone marrow involvement; 4% ECOG performance status >1; 34% elevated LDH; 32% β2-microglobulin≥3mg/L; FLIPI score 0–1 (21%), FLIPI 2 (36%), FLIPI 3–5 (43%). Most patients (74%) received R-CHOP induction (22% R-CVP, 4% R-FCM). Patients responding to induction therapy were stratified based on their immunochemotherapy regimen and response [CR/CRu versus PR], and randomized to observation or rituximab maintenance, 1 infusion (375 mg/m2) every 8 weeks for 2 years. A total of 1018 randomised patients were analyzed according to the ITT principle (513 observation/505 rituximab maintenance). All initial pre-treatment characteristics were well balanced between arms and the response status at time of randomization was CR=39%; CRu=31% and PR=29% (others 1%). After a median follow-up of 36 months, 3-year progression free survival was 60.3% (95% confidence interval [CI] 55.8 – 64.5%) in the observation arm and 78.6% (95% CI 74.7 – 82%) in the rituximab maintenance arm, respectively (stratified Log-Rank, P The most common adverse events reported were infections, which occurred in 24% and 39% of the patients in the observation and rituximab maintenance arms respectively. Grade 3–4 adverse events were reported in 17% of patients in the observation arm and 24% in the rituximab maintenance arm (neutropenia 1% vs 4%; infections 1% vs 4%, respectively) but these lead to treatment discontinuation in only 8 and 19 patients, respectively. In conclusion, after an additional year of follow-up, these data demonstrate a sustained benefit of 2 years of rituximab maintenance therapy after immunochemotherapy, resulting in improved progression free survival and complete response rates. No additional or unexpected toxicities were observed. All randomized patients have an excellent survival to date and longer follow-up will explore factors affecting overall survival in these patients. The PRIMA results indicate that rituximab maintenance should be considered in all follicular lymphoma patients who have responded to first line immunochemotherapy. Disclosures: Salles: Roche: Consultancy, Honoraria. Off Label Use: The use of rituximab maintenance in follicular lymphoma patients. Catalano:Celgene: Research Funding; Roche: Honoraria, Research Funding, Travel Grants. Feugier:roche: Consultancy, Honoraria. Belada:Roche: Consultancy, Honoraria. Tilly:Amgen: Honoraria. Leppa:Roche: Honoraria. Soubeyran:Roche: Honoraria, Research Funding. Hagenbeek:roche: Consultancy. Lister:Allos: Consultancy; Bioconnections: Consultancy; Astra Zeneca: Consultancy; Tenet: Consultancy; GSK: Chairman of Safety Monitoring Committee. Lopez-Guillermo:Roche: Consultancy, Honoraria. Seymour:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau, Travel support; Bayer-Schering: Honoraria, Travel Support.

8004 Background: The GELA sponsored intergroup PRIMA Phase III study investigated 2 years of rituximab (R) maintenance in follicular lymphoma (FL) patients responding to first-line immunochemotherapy consisting of either 8 cycles of R-CVP, or 6 cycles of R-CHOP or R-FCM (plus 2 additional rituximab infusions). Methods: 1,217 patients were enrolled from 223 centres (25 countries) between Dec 2004 and Apr 2007. Pre-induction characteristics: median age 56 years (range 22–87); 52% male; 90% Ann Arbor stage III-IV; 33% B symptoms; 56% bone marrow involvement; 4% ECOG performance status >1; 34% elevated LDH; 32% β2-microglobulin >3mg/L; 21% FLIPI 0-1: 36% FLIPI 2; 43% FLIPI 3-5. Most patients (75%) received R-CHOP induction (22% R-CVP, 3% R-FCM). 1,018 eligible patients responding to induction therapy were randomized (stratified by regimen and response to induction) to observation or R-maintenance, 375 mg/m2 i.v. every 8 weeks for 2 years. Results: The primary endpoint of PFS was met at the planned interim ana...

Abstract #5068 Background: Breast cancers are traditionally divided into hormone receptor positive and negative cases that guide patient management. The two luminal subtypes in gene expression profiling , A and B, are defined by high expression of ER, GATA3, and FOXA1. There is a little doubt that ER status is a strong predictor of response to endocrine therapy. Prognostic immunohistochemical biomarkers for ER-positive breast cancer include progesterone (PR) and androgen (AR) receptors, proteins related to proliferation or apoptotic resistance. The aim of this study was to identify the best predictors of success of hormonal therapy among immunohistochemical and classical factors. Methods: We studied 10 markers in a consecutive series of 832 cases of breast carcinoma treated at the Paoli-Calmettes Institute from 1990 to 2002 and deposited onto tissue-micro arrays (TMA): luminal-related ER, PR, AR, FOXA1, and GATA3 factors, proliferation-related KI67 and CCND1, ERBB2, anti-apoptotic BCL2, and p53. We also measured the vascular peritumoral invasion (VPI) size, Grade and lymph nodes involvement (N). On these series 145 cases had been profiled on Affymetrix microarrays. The patients had been treated with adjuvant chemotherapy and/or hormonal therapy . The 132 events observed and taken into account were metastases (MF). Results: Of the 67 luminal A cases of this series 63 were ER-positive, 98% were FOXA1-positive; only half of them expressed the five luminal-related markers. In 584 ER-positive patients, 200 did not receive adjuvant hormonal therapy with a highly significant difference in MF with those who received Tamoxifen (p=0.017). For the 240 cases who received hormonal therapy, the factors associated to the MF in multivariate analysis were: A prognostic score can be established summing the impact of each four parameters with a significant Logrank test : p= 0.0003. Discussion: A panel of three antibodies (Ki67, P53 and GATA3), associated with VPI can significantly improve the traditional prognosticators in predicting outcome for ER-positive breast cancer patients receiving hormonal therapy. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5068.

Genotypic analyses were performed in six primary cutaneous lymphomas whose lineage could not be assessed on the basis of histologic and phenotypic data. By immunophenotyping, these neoplasms expressed leukocyte common antigen and HLA-DR but did not show consistent immunostaining for B-cell or T-cell differentiation antigens. Expression of nonspecific histiocytic markers such as lysozyme and alpha 1-antitrypsin was found in three cases. By genotyping, three cases retained a germline configuration and immunoglobulin gene rearrangement was observed in one case, T-cell receptor gene rearrangement was found in one case, and both types of rearrangements in one case. Of the three patients in whom gene rearrangements were noted, two rapidly died and the other patient, with a dual genotype, is still alive 15 years after diagnosis. The three patients without gene rearrangements are alive and well after a mean follow-up of 2.5 years. It appears that cutaneous lymphomas with an uncertain phenotype include at least some cases of authentic B-cell or T-cell lymphomas. The germline configuration that we observed in cases with a chronic course remains difficult to explain. It may be related to a low malignancy form of histiocytic lymphoma, an atypical polyclonal hyperplasia, or even a low-grade lymphoma arising from a primitive cell without established commitment to either B- or T-cell lineage.

Sialomucin and sulphomucin-secreting cells were studied in the normal and pathologic human pancreas with the high iron diamine-alcian blue technique which allows differentiation between the two types of mucin. In six normal autopsy pancreata, only sulphomucin was found. In benign lesions of either calcifying chronic pancreatitis (seven cases) or obstructed chronic pancreatitis (six cases), sulphomucins were widely predominant. In contrast, malignant lesions (pancreatic adenocarcinoma [12 cases], cystadenocarcinoma [two cases]) or premalignant lesions (mucinous cystadenoma [one case], ductectatic mucinous cystadenoma [four cases], villous adenoma of the main pancreatic duct [two cases]) showed a predominant sialomucin secretion, except for three poorly differentiated pancreatic carcinomas that did not show mucin staining. The sialomucin positivity was not observed at distance from the malignant lesions. In one case of benign enteroid cyst, sulphomucins predominated. These findings indicate a preponderance of sialomucin secretion in malignant or premalignant pancreatic lesions.

Background. The FL2000 study evaluated the combination of the anti-CD20 monoclonal antibody rituximab with chemotherapy plus interferon in the first line treatment of follicular lymphoma patients. Methods. Untreated follicular lymphoma patients (n=359) presenting with a high tumor burden were randomly assigned to receive either 12 courses of the chemotherapy regimen CHVP (cyclophosphamide, adriamycin, etoposide and prednisolone) plus interferon-α2a (CHVP+I arm) over 18 months or 6 courses of the same chemotherapy regimen combined with 6 infusions of 375 mg/m2 of rituximab and interferon for the same time period (R-CHVP+I arm). The primary endpoint of the study was event free survival and all results are shown as intent to treat. Results. Six months after treatment initiation, 156 out of 183 (85%) and 164 out of 175 (94%) patients had a response to therapy in the CHVP+I and R-CHVP+I study arm, respectively (P=.009). At the end of the 18 months treatment period, 59% and 75% of the patients were respectively in CR or CRu in the CHVP+I and R-CHVP+I arm (P Conclusions. These results extend our previous interim analyses and confirm that with a five year follow-up, the combination of rituximab with CHVP+I provides superior disease control in follicular lymphoma patients despite a shorter duration of chemotherapy. However, this combination appears to benefit essentially to high risk patients for whom overall survival is also significantly improved.

High amounts of intra-tumoral reactive macrophages have been reported to be associated with a poor prognosis in patients with follicular lymphoma (FL) (Blood2005;106:2169). However, this study has been retrospectively performed in a patients’ population selected before the monoclonal antibody therapeutic area, and has not been confirmed on independent series. To establish whether the intra-tumoral macrophages count (MC) is definitely able to predict the outcome of FL patients, we analysed both immunohistochemical CD68 expression and clinical outcome in patients included in the GELA-GOELAMS FL-2000 trial (ASH-2004; ASCO-2006). Patients between 18 and 75 years of age with high tumor burden FL were randomized to receive either CHVP-I or R-CHVP-I. Among the 358 patients included on the basis of histologically proven FL after reviewing by 3 hematopathologists, 194 had available biopsy specimen. Clinical characteristics of those 194 patients were not different of those of the whole population and median time to event was 43.1 months in 92 pts receiving CHVP-I arm and 54.5 months in the 102 pts receiving R-CHVP-I. Slides were stained with the anti-CD68 KP1 antibody using a standard IHC procedure, then scored on high power field (hpf) (× 400 magnification) by 3 different pathologists. Analysis of the relative risk of event according to the intra-follicular MC (IF-MC) led us to determine a single cut-off point to categorize the covariate, the best cut-off point being estimated to be 10 macrophages/hpf. The IF-MC was equal or less than 10 intra-follicular KP1+ macrophages/hpf in 53 patients, and more than 10 in 141 patients. With this cut-off of 10, a low MC was significantly associated with a better EFS for all patients (p=0.011). However, this effect was predominantly observed in patients that received CHVP-I (p=0,012, OR) but not in those receiving R-CHVP-I (p=0.15).Multivariate analysis adjusting for 10 IF-MC cut-off (p

AITL and PTCL-U, the two most common forms of T-cell lymphomas in western countries, usually present as nodal disease and pursue an aggressive clinical course. AITL is commonly associated with a constellation of clinical symptoms and distinct pathological features. Conversely, PTCL-U lacks precise diagnostic criteria, and by default comprises cases not fulfilling criteria for other entities, including tumors with borderline features to ALCL and AITL. The genetic alterations and pathogenic mechanisms underlying AITL and PTCL-U are largely unknown. To determine whether the molecular signature of AITL and PTCL-U could help in distinguishing both entities and in understanding ther ontogeny, we performed gene expression profile (GEP) analysis of 15 PTCL-U tissue samples (6 CD30+ and 9 CD30−) and 19 AITL samples (including 2 sorted tumor cell suspensions) using Affymetrix HG-U133A Plus2.0 pan-genomic oligonucleotide microarrays, with comparison to that of previously published normal T-cell subsets (J Immunol173:68; J Immunol175: 7837; Blood 104: 1952). Principle component analysis (PCA, accumulated variance 95%) of all 33 tissue samples yielded three groups of tumors: one group of 12 AITLs, one group of 10 PTCLs-U and one mixed group comprising 5 AITLs (some with features borderline to PTCL-U) and 6 PTCLs-U (including 5 of 6 CD30+ tumors). The AITL molecular signature consisted of 442 genes with increased levels of expression in AITL compared to PTCL-U (t test, p

This study aimed to evaluate the role of RIC allo-SCT for relapsed or refractory non-Hodgkin’s lymphoma (NHL). We report here our experience in 25 consecutive patients transplanted in a single center for high grade (n=17) or follicular NHL (FL; n=8). In the high grade NHL group, median age was 46 (range, 24–63) years, and all 17 patients received 2 or more previous chemotherapy regimens prior to RIC allo-SCT. In addition, 12 patients (71%) had failed autologous SCT and 6 patients (35%) had chemoresistant disease at time of allo-SCT. Among the 8 patients transplanted for a heavily pretreated follicular NHL (FL), median age was 52 (range, 34–59) years and median number of prior lines of therapy was 3 (range, 2–5), with 3 patients (38%) having chemoresistant diseases and 4 patients (50%) relapsing after autologous SCT. Among the 17 patients with aggressive high grade NHL, we compared the outcome of T-cell and B-cell aggressive NHL. With a median follow-up of 15.4 (range, 3.4-65.2) months, the cumulative incidence of non-relapse mortality was 6%, (95%CI, 0.3%-31%) and the Kaplan-Meier estimate of progression-free survival (PFS) was significantly higher in the T-cell as compared to the B-cell group (P= 0.03; 100% vs. 40% at 3 years). In the FL group, the cumulative incidence of non-relapse mortality was 25% (95%CI, 3%–65%). Six patients (75%) showed objective disease response with complete remission (CR) occurring concomitantly to graft-versus-host disease, including one CR after donor lymphocytes infusion. With a median follow-up of 19 (range, 7–85) months, 6 patients from the FL group are still alive of whom 5 in CR. We conclude that a potent graft-vs.-lymphoma (GVL) may be achieved in FL patients, even those with chemoresistant disease or who have relapsed after autologous SCT. In the high grade NHL group, strategies aiming to enhance the GVL effect (Rituximab-based RIC and/or Rituximab maintenance therapy) in the B cell subtype are still needed. However, RIC allo-SCT is a feasible and promising strategy for aggressive NHL, with particularly low toxicity, and T-cell aggressive NHL benefiting most from a potent GVL effect, likely overcoming the poor prognosis usually associated with this phenotype.

Background Androgen-independent prostate adenocarcinomas are responsible for about 6% of overall cancer deaths in men. Methods We used DNA microarrays to identify genes related to the transition between androgen-dependent and androgen-independent stages in the LuCaP 23.1 xenograft model of prostate adenocarcinoma. The expression of the proteins encoded by these genes was then assessed by immunohistochemistry on tissue microarrays (TMA) including human prostate carcinoma samples issued from 85 patients who had undergone radical prostatectomy. Results FGFR1, TACC1 and WT1 gene expression levels were associated with the androgen-independent stage in xenografts and human prostate carcinoma samples. MART1 protein expression was correlated with pT2 tumor stages. Conclusion Our results suggest that each of these four genes may play a role, or at least reflect a stage of prostate carcinoma growth/development/progression.

Les lymphomes T peripheriques (LTP) representent environ 10 % des lymphomes non-Hodgkiniens et leur classification est toujours sujet a debat. De nombreux sous-types histologiques de LTP ont ete decrits par le passe, mais en l’absence d’arguments solides, ils n’ont pas ete reconnus comme de veritables entites clinico-pathologiques par la recente classification de l’OMS, qui les regroupe sous l’appellation « unspecified » (UNSP). En dehors de la categorie UNSP, les seuls types de LTP reconnus sont les LTP angio-immunoblastiques (LAI), et les LTP anaplasiques (LAN) et quelques varietes rares comme les LTP gamma-delta (G/D) ou associes aux enteropathies. Dans le but d’identifier une classification coherente des LTP, notamment pour la categorie tres heterogene des « UNSP » nous avons teste 59 echantillons de LTP par la technique de c-DNA micro-array, qui permet d’analyser simultanement le niveau d’expression d’environ 10 000 genes. Les profils d’expression obtenus etaient statistiquement correles aux lesions histopathologiques, permettant de separer les LTP UNSP, les LAI et les LAN, Les 3 cas de LNH T lymphoblastiques montraient un profil moleculaire nettement distinct. Dans le groupe des UNSP, le dendrogramme permet d’identifier 3 groupes moleculaires distincts (UNSP1, UNSP2, UNSP3). Le groupe UNSP2 englobe les LTP dits de Lennert ainsi d’autres LTP assez riches en histiocytes. Les groupes UNSP1 et UNSP3 ne montraient de differences histologiques notables, mais presentaient des profils transcriptionnels distincts, qui suggerent des differences physiopathologiques concernant notamment l’activation des interleukines et de la voie STAT. Les correlations retrospectives preliminaires avec les donnees cliniques semblent indiquer une association des UNSP1 avec un plus mauvais pronostic. Ces resultats suggerent l’existence de sous-groupes moleculaires de LTP, dont certains semblent associes a un pronostic specifique, ce qui pourrait permettre de rationaliser la classification des LTP sur des bases physiopathologiques, et d’ameliorer la prise en charge therapeutique des patients.

Immunocytochemical assay (ICAs) were performed on frozen sections from human endometrial samples ( n = 89) including normal endometrium, decidua, hyperplasia with and without atypia, and carcinomas. Monoclonal antiestrogen receptor (ER), antilaminin (Lam), anti-type IV collagen (Coll IV), and anti-Ki67 were applied with avidin-biotin-peroxidase complex or peroxidaseantiperoxidase complex. The results of the ICAs were evaluated through a computerized system of image analysis referred to as SAMBA. It was shown that this system provided for an accurate reliable and reproducible analysis of ICAs in tissue sections. It is concluded that this multiparametric and standardized method of analysis of ICAs can further be applied in correlations with clinical and biochemical data.

Immunocytochemical assays (ICA) using monoclonal antiestrogen receptors (ER ICA), antiprogesterone receptors (PR ICA), and monoclonal antibody Ki67 (Ki67 ICA) were performed in 127 breast carcinomas. The immunostaining procedures were applied on frozen tissue sections, tumour imprints, and fine-needle aspirates in order to compare the variations in the distribution of the antigens detected in the three different types of preparations. Positive reactions detected with peroxidase-antiperoxidase and avidinbiotin-peroxidase, and alkaline phosphatase-antialkaline phosphatase complexes were evaluated through a computerized system of image analysis referred to as SAMBA 200 (SAMBA TITN, Grenoble, France). Application programs specifically developed for the analysis of tissue sections and of cytologic preparations were applied. This system allowed a multiparametric, accurate, reliable, reproducible and automatized evaluation of the heterogeneity of the antigenic sites in tumors. For each markers positive cell surface (PS), and integrated and mean optical densities (IOD, MOD) and IOD histograms were compared. It was shown that (1) there was no significant variation in optical densities in cell imprints and aspirates whereas PS significantly (P less than 0.01) differed in both preparations; (2) there were significant differences of the optical densities between tissue sections and cytological preparations, either imprints or aspirates, likely due to randomly cut nuclei in tissue sections; and (3) there was a significant difference between the PS of tissue sections and aspirates but no significant difference between tissue sections and imprints. It is concluded that fine-needle aspiration constitutes a convenient method for cell sampling, reliable for the diagnosis of malignancies. However, it may not reflect the heterogeneity of cell subpopulations in tissue.

Type IV collagen immunostaining was performed on tissue sections from a large series of non-malignant and malignant disorders of the breast and endometrium. The results were analysed by means of a computerized system of image analysis referred to as SAMBA. It was shown that this system provided an accurate, reliable, reproducible, automated and multiparameteric analysis of collagen IV immunoprecipitates. It was concluded that this standardized method of analyses can be routinely used for the measurement of collagen IV, thus enabling correlations to be sought with histopathological and clinical data.

A study was carried out to determine whether the minisatellite DNA probes described by Jeffreys and coworkers could be used routinely to analyze engraftment, hematopoietic chimerism, and relapse in recipients of bone marrow transplants. The probes were informative for all of the recipient/donor pairs analyzed. Their limit of sensitivity was determined in reconstruction experiments and was found to vary from 2%, in the best cases, to 10%. We were able to confirm that engraftment and hematopoietic chimerism can, indeed, be sought routinely using this simple molecular approach. Only one set of probes is required for all patients and, unlike cytogenetic analysis, this analysis can be used whether or not blood or marrow cells are dividing.

Abstract 2649 Background. Conflicting results have been published to date concerning the impact of statin use on the prognosis of patients with non-Hodgkin lymphoma (NHL) treated with a rituximab-containing first-line regimen. By impairing protein prenylation and possibly lipid-raft formation, statins have been shown to exert an antitumoral activity both on solid and hematological malignancies in vitro as well as in animal models. However, in vitro experiments have demonstrated an unexpected impairment of anti-CD20 antibody binding due to changes in conformational epitopes of the molecule. As a result, statins were found to decrease anti-CD20 induced complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity. Several groups worldwide attempted to assess the outcome of patients on statins receiving rituximab for NHL. No detrimental effect was observed across studies for patients with diffuse large B-cell lymphoma treated upfront with R-CHOP or R-CHOP-like regimen but a surprising prolonged event-free survival (EFS) was observed for patients with follicular lymphoma (FL) in one study. However, several caveats of the study precluded definitive conclusions to be drawn such as treatment heterogeneity, the enrollment of patients who did not receive rituximab, the retrospective assessment of statin use through medical charts and the consideration of EFS as the primary endpoint without specific evaluation of overall survival (OS). The impact of statin use on the prognosis of patients with FL therefore requires further investigation. Patients and methods. The randomized, open-label PRIMA study enrolled 1217 patients with de novo FL from 223 centers in 25 countries. Patients achieving at least a partial response following frontline therapy with R-CHOP, R-CVP or R-FCM were randomized between 2-years rituximab maintenance (every 8 weeks) or observation. A significant improvement of progression-free survival (PFS) was observed in the rituximab maintenance arm (HR=0.55, 95% CI=0.44–0.68, p Results. As expected, the use of statins was associated to an older age at registration (P No difference in overall response rate to induction regimen as well as in the quality of the response (CR/Cru versus PR versus others) was noted (P=0.53). No further difference was found at the end of the maintenance period either for patients in the observation or in the rituximab treatment arm. Of note, no imbalance was detected concerning the rate of adverse events between the 2 groups of patients as a whole (grade 1–2 versus 3–4, P=0.18) or with regards to each particular toxicity. Finally, outcome in terms of EFS, PFS (see figure above), OS, TTNLT and TTNCT were similar regardless of the use of statins (P=0.76, P=0.43, P=0.21, P=0.83 and P=0.31 respectively). Importantly, further adjustment for potential confounding factors in multivariate Cox models such as age, FLIPI score or LDH (which were imbalanced between the 2 groups) did not reveal a potential beneficial effect. Conclusion. As for other subtypes of NHL, the use of statins in patients with FL in the rituximab era appeared safe and was not associated with an inferior outcome as suggested by in vitro experiments. Disclosures: No relevant conflicts of interest to declare.

Genetic alterations underlying angioimmunoblastic and unspecified peripheral T-cell lymphomas (AITL and PTCL-u) are largely unknown. Seventeen AITL and 16 PTCL-u previously characterized by gene expression profiling, were analyzed by CGH on DNA microarrays comprising 4434 BAC clones with a resolution of about 600 KB. In the PTCL-u group, the mean number of chromosomal aberrations per case was 302 (range, 55 to 892). Gains (n=237, 41 to 587) were more frequent than losses (n=65, 8 to 305). AITL samples had, on average, a lesser number of genomic alterations than PTCL-u cases (n=243, range, 55 to 485), comprising more gains (n=201, 42 to 541) than losses (n=42, 9 to 262). Overall, the most frequent recurrent gains, present in 50% of all samples, were observed at 1p36.1; 1p36.3 ; 1q32 ; 2q37 ; 4p16 ; 5p15.3 ; 6q12 ; 7p22 ; 7p12 ; 7p11.2 ; 7q35-36 ; 8q24.3 ; 9q34 ; 11p15 ; 11q13 ; 16p13.3 ; 16q24 ; 17q12,q21,q25 ; 19p13.3 ; 19q13.2-q13.3 ; 20q11.2-q13.3 ; 22q11.1-q11.2 ; Xp11 ; Xp21-22 ; Xq27-28. The comparison of the genomic profiles of AITL and PTCL-u identified 73 genomic alterations (at clones or zones of clones) significantly associated with either group of tumors (Fisher test, p < 0,05). Six genomic gains mapping at 5p15 and 22q11 were associated with the AITL subtype. Thirty-four gains (mapping at 6p25, 7p1, 7q3, 8q24, 11p14, 14q32, 17q, 22q) and 33 losses (mapping at 6q, 10p and 13q), were overrepresented in PTCL-u. The coordinate analysis of the transcriptomic and CGH array data identified 10 regions with genomic imbalances containing genes differentially expressed in AITL versus PTCL-u. Seven regions amplified in PTCL-u contained genes overexpressed in PTCL-u, mostly related to metabolic pathways. Conversely, loss of genomic material at 13q12 correlated with decreased expression in PTCL-u of a few genes of the AITL signature. In AITL tumors, gain at 22q11 correlated with increased transcription of the LIF gene, previosuly characterized as part of the tumor cell signature in AITL. CD30+ PTCL-u samples had on average a higher number of genomic aberrations than CD30-negative cases (n=408 versus 238). Thirty-three genomic gains and 22 losses were exclusively seen in CD30+ tumors, and regions with chromosomal imbalances at 1q, 6q, 10p contained genes differentially expressed in CD30+ and CD30− tumors. For example, reduced transcription of FYN in CD30+ PTCL-u correlated with deletion of the corresponding chromosomal region. In conclusion, all 33 nodal PTCL analyzed harbor genomic imbalances (gains>losses), of which many are common to both AITL and PTCL-u subgroups; the pattern of genomic aberrations differs between the two subgroups, with certain aberrations being overrepresented in PTCL-u, and only a few specific for AITL; coordinate appraisal of transcriptomic and genomic data highlights correlations between genomic imbalances and gene expression signatures in subgroups of tumors.

Undifferentiated carcinoma of the nasopharyngeal type (UCNT) that histologically mimics Hodgkin's lymphoma (HL) ('Hodgkin's lymphoma-like UCNT' - HL-like UCNT) is known as a diagnostic pitfall. Using immunohistochemistry, Western blot and cDNA array technology, we wanted to document its phenotypical and molecular characteristics. We report herein 5 cases of UCNT that morphologically mimic HL and 3 classical UCNT cases. We compared the expression profiles of a thousand selected genes in HL-like UCNT and in classical UCNT cases. No difference in the profile of EBV infection was noted between the HL-like UCNT and control cases. Significant differences were detected in the expression of genes involved in the matrix modelling, angiogenesis, apoptosis and regulation of the Th-2 interleukins. The eosinophil chemoattractant eotaxin was expressed in the stroma of HL-like UCNT, but not in the control cases. The eotaxin receptor CCR3 was expressed in both stromal and carcinoma cell populations of HL-like UCNT, this pattern being similar to the one observed in HL. These results show that UCNT morphologically resembling HL share also some specific phenotypical and molecular features with HL, and might deserve to be isolated as a particular UCNT subtype.

BackgroundInflammatory breast cancer (IBC) is a distinct and aggressive form of locally-advanced breast cancer with high metastatic potential. In Tunisia, IBC is associated with a high death rate. Among the major molecular subtypes, basal breast carcinomas are poorly differentiated, have metastatic potential and poor prognosis, but respond relatively well to chemotherapy. The aim of this study was to determine the distribution of molecular subtypes in IBC and identify factors that may explain the poor prognosis of IBC.MethodsTo determine breast cancer subtypes we studied by immunohistochemistry the expression of 12 proteins in a series of 91 Tunisian IBC and 541 non-IBC deposited in tissue microarrays.ResultsWe considered infiltrating ductal cases only. We found 33.8% of basal cases in IBC vs 15.9% in non-IBC (p < 0.001), 33.3% of ERBB2-overexpressing cases in IBC vs 14.5% in non-IBC (p < 0.001), and 29.3% of luminal cases in IBC vs 59.9% in non-IBC (p < 0.001). The most differentially-expressed protein between IBCs and non-IBCs was P-cadherin. P-cadherin expression was found in 75.9% of all IBC vs 48.2% of all non-IBC (p < 0.001), 95% of IBC vs 69% of non-IBC (p = 0.02) in basal cases, and 82% of IBC vs 43% of non-IBC (p < 0.001) in luminal cases. Logistic regression determined that the most discriminating markers between IBCs and non-IBCs were P-cadherin (OR = 4.9, p = 0.0019) MIB1 (OR = 3.6, p = 0.001), CK14 (OR = 2.7, p = 0.02), and ERBB2 (OR = 2.3, p = 0.06).ConclusionTunisian IBCs are characterized by frequent basal and ERBB2 phenotypes. Surprisingly, luminal IBC also express the basal marker P-cadherin. This profile suggests a specificity that needs further investigation.

The intergroup PRIMA Phase III study was designed to investigate the potential benefit of 2-years of rituximab maintenance in patients with follicular lymphoma (FL) responding to one of three non-randomised first line immunochemotherapy treatments. The results of the final analysis with 36 months follow-up (Salles et al., Lancet 2011) demonstrated a significant reduction of the risk of progression or death with a hazard ratio (HR) of 0.55 in favour of patients randomized to rituximab maintenance. We present here the updated results with 3 additional years of follow-up. From December 2004 until April 2007, 1217 patients were enrolled from 223 centres and complete data were available for 1193 patients who had the following pre-induction treatment characteristics: median age 56 years [range 22–87]; 52% male; 90% Ann Arbor stage III-IV; 33% B symptoms; 56% bone marrow involvement; 4% ECOG performance status >1; 34% elevated LDH; 32% β2-microglobulin >3mg/L; FLIPI score 0-1 (21%), FLIPI 2 (36%), FLIPI 3-5 (43%). Most patients (75%) received R-CHOP induction (22% R-CVP, 3% R-FCM). Patients responding to induction therapy were stratified based on their immunochemotherapy regimen and response [CR/CRu versus PR] and randomized to observation or rituximab maintenance, 1 infusion (375 mg/m2) every 8 weeks for 2 years. A total of 1018 randomised patients were analyzed according to the ITT principle (513 observation / 505 rituximab maintenance). All initial pre-treatment characteristics were well balanced between arms and the response status at time of randomization was CR=39%; CRu=32% and PR=28% (others 1%). With a median follow-up of 73 months from randomization, 6-year progression free survival estimate was 42.7% (95% CI 38 – 46.9%) in the observation arm (284 events, median=48 months) and 59.2% (95% CI 54.7 – 63.7%) in the rituximab maintenance arm (194 events, median not reached), respectively (stratified Log-Rank, P In pre-planned analyses of patients subgroups categorized by age, sex, FLIPI score category, induction chemotherapy and response to induction, the effect of rituximab maintenance was examined and found to be consistent among these different subgroups. In a Cox regression multivariate analysis, rituximab maintenance (HR=0.57; P The rate of histological transformation did not appear to differ between the 2 treatment arms: in the observation arm, transformation was documented in 24 patients (114 cases with morphological documentation out of 278 progressions) versus 16 patients in the rituximab maintenance arm (80 out of 186) respectively. Overall response rate to second-line therapy was reported by investigators to be 180/227 (79%) in patients from the observation arm (CR/CRu=61%; PR=19%) versus 109/144 (76%) in patients from the rituximab maintenance arm (CR/CRu =51%; PR=22%) (P=NS). At the time of the data cut-off, overall survival (OS) remains favourable in both study arms: 58 patients (11.3%) have died in the observation arm (6-years OS estimate 88.7%) compared to 59 patients (11.7%) in the rituximab maintenance arm (6-year OS estimate 87,4%). Main causes documented for death in the observation and rituximab maintenance arm respectively were lymphoma (28 ; 28), other malignancy (19 ; 5) and infections (4 ; 7). No new significant safety data were captured with this additional follow-up period. In conclusion, with 3 additional years of follow-up, these data demonstrate a sustained and persistent benefit of 2 years of rituximab maintenance therapy after immunochemotherapy, resulting in improved progression free survival. No additional or unexpected long term toxicities were observed and second line therapy efficacy results did not significantly differ between the 2 study arms. Overall survival appears very favourable for these randomized patients. Disclosures: Salles: Roche: Consultancy, Honoraria, Research Funding. Seymour:Roche: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau, Travel support Other; Genetech: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Feugier:Roche: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Offner:Lilly: Membership on an entity’s Board of Directors or advisory committees. Lopez-Guillermo:Roche: Membership on an entity’s Board of Directors or advisory committees. Belada:Roche: Consultancy. Catalano:Roche: Membership on an entity’s Board of Directors or advisory committees. Haioun:Roche: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Simpson:Janssen Research & Development: Honoraria. Leppa:Roche: Consultancy, Honoraria, Research Funding, Travel support Other. Soubeyran:Roche: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Hagenbeek:Takeda/Millennium: Consultancy. Casasnovas:ROCHE: Consultancy, Honoraria, Research Funding. Coiffier:Millennium Pharmaceuticals : Consultancy. Tilly:Roche: Honoraria; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity’s Board of Directors or advisory committees; Pfizer: Honoraria; Janssen: Honoraria; Amgen: Research Funding.

Introduction : PD1 est un antigène décrit dans les cellules du micro-environnement de nombreuses tumeurs solides, notamment dans les lymphomes folliculaires (LF) au sein des lymphocytes TFH, jouant un rôle en temps normal dans la régulation de l’immunité. Il a cependant été mis en évidence, en grande majorité dans des LF riche en lymphocytes T, une expression du PD1 au sein des cellules tumorales. Notre étude a pour but de caractériser ces LF sur le plan clinique, diagnostic et pronostic.Méthode : nous avons analysé 9 prélèvements de 9 patients avec un diagnostic initial de LF. Les critères d’inclusions étaient un pourcentage de lymphocytes T associés supérieur à 60% et une expression du PD1 au sein des cellules tumorales. Un examen morphologique, immunohistochimique et des analyses moléculaires (FISH, clonalités) ont été effectués pour chaque patient.Résultats : au diagnostic 75% des patients avaient un stade de Ann Arbor III/IV et 37,5% étaient atteints de symptômes B. Trois patients présentaient initialement un LF de grade 1-2, quatre un LF de grade 3B et deux un LF de grade 3 non conventionnel. Un des LF de grade 1-2 a évolué en LF de grade 3A, et un autre s’est transformé en Lymphome B diffus à grandes cellules. Deux des LF de grade 3B se sont transformés en LBDGC. Un des deux LF de grade 3 non conventionnel s’est transformé en LBDGC. Sept des neuf lymphomes exprimaient BCL2, six exprimaient CD10 et tous exprimaient BCL6 en immunohistochimie. L’exploration par FISH objectivait un réarrangement de BCL2 pour 3 patients, de BCL6 pour 1 patient et de MYC pour 1 patient. Des études de clonalité B et T ont été réalisées pour tous les patients. Une population clonale B a été retrouvée chez tous les patients. Il n’a été mis en évidence de population clonale T chez aucun patient. La survie sans récidive était comprise entre 8 mois et 173 mois avec une médiane de 39 mois. Conclusion : il est important de ne pas méconnaître ce sous-groupe de lymphome folliculaire en raison de possibles pièges diagnostiques avec le lymphome T folliculaire et de recourir à l’analyse de clonalité au moindre doute. Notre étude suggère aussi un possible pronostic plus péjoratif, avec notamment des signes de gravité fréquents et un grade histologique élevé, qui reste à confirmer sur une plus grande série.

Introduction : au cours des dernières années, l’introduction de l’immunothérapie par CAR T-cells (Chimeric Antigen Receptor T-cells) dans le traitement des lymphomes B diffus à grandes cellules réfractaires au traitement conventionnel a révolutionné le pronostic de ces patients. Cependant les rechutes restent nombreuses et les caractéristiques histophénotypiques et moléculaires ainsi que les mécanismes associés aux récidives post-CART sont mal connus. Méthode : nous avons analysé 19 échantillons provenant de neuf patients, avant et après traitement par des CART de 2ème génération dirigés contre le CD19. Des analyses histologiques, immunohistochimiques et moléculaires par FISH, CGH-array, étude du profil de méthylation de l’ADN, séquençage haut débit et RNAseq ont été réalisées.Résultats : les patients inclus étaient atteints de lymphome B diffus à grandes cellules (LBDGC, n=5), de lymphome B de haut grade double-hit (n=1) et de lymphome de Burkitt (LB, n=3). Sept patients sur 9 ne présentaient pas de modification histophénotypique majeure après traitement, excepté la perte fréquente d’un ou plusieurs marqueurs B dont le CD19. Deux patients ( 1 LBDGC et 1 LB) présentaient à l’inverse des signes de transdifférenciation avec une perte des marqueurs B associée à l’apparition de marqueurs T ou histiocytaires, malgré la persistance d’un clone identique porteur d’un réarrangement du gène des immunoglobulines. Dans le cas de la tumeur post-CART qui exprimait un phénotype T aberrant, on mettait en évidence une diminution importante de l’expression des gènes du programme B au niveau de l’ARNm, associée à une méthylation accrue de leur promoteur. En FISH et CGH-array nous observions une relative stabilité des anomalies en pré- et post-CART, avec notamment des délétions 17p/TP53. Le séquençage haut débit mettait en évidence l’apparition de nouveaux variants notamment au niveau de la voie PI3K ou liés à l’agressivité tumorale (KRAS, INPP4B, SF3B1, SYNE1, TBL1XR1).Conclusion : ces données montrent que les mécanismes de résistance aux CART dans le cadre des LBDGC reposent sur des modifications génétiques, pouvant aller jusqu’à la perte de la différenciation B . L’existence de mutations acquises dans les voies PI3K et KRAS pourrait ouvrir la voie à des thérapies ciblées chez les patients réfractaires aux CART.