ALBERT

Description: Key Points IHC is a valuable clinical tool for assessing CD30+ PTCL patients who may respond to CD30-targeting treatment. CD30 mRNA and protein expression are highly correlated.

Description: Introduction: A number of approaches have been explored to prevent relapse in AML setting, including immune-strategies such as dendritic cells (DC) vaccination. There is no report so far of the use of autologous apoptotic leukemic cells as a source of tumor antigen for DC vaccine. Methods: The main objective of this prospective monocentric Phase I/II study was to explore the feasibility to produce autologous leukemic apoptotic corpse-pulsed DC for elderly AML patients in first or second complete remission (CR) and to report the toxicity of such a vaccine. Inclusion criteria were AML (except promyelocytic) patients older than 59 years with a good performans status (ECOG =50% of leukemic blasts in bone marrow (BM) or peripheral blood, and with no contra-indications to apheresis. Vaccines were produced by Nantes UTCG according to good manufacturing practices for cell and gene based therapies in order to comply to the French AFSSAPS (currently ANSM) agency guidelines. Patients had to be pre-included (refractory or not) at diagnosis or at time of first relapse in order to collect sufficient leukemic cells (〉2.4 108) prior to chemotherapy after 1 or 2 days of collection. After blasts collection, the choice of chemotherapy regimen was at the discretion of the investigator. Few courses of chemotherapy were allowed before vaccine production but not after. Patients were definitively included only in case of CR to allow collecting autologous non-leukemic peripheral monocytes by apheresis to generate the DC vaccine. Patients were programmed to receive up to 5 doses of vaccine (days +1 +7 +14 +21 and +35 +2) which consisted of 10 millions pulsed DC, including 9 millions administered subcutaneously (1 mL) and 1 million administered intradermally (0.1mL). Minimal residual disease (MRD) was studied after vaccines using flow cytometry. Results: Between November 2009 and March 2015, 23 patients were pre-included but 2 patients were excluded for analyses because blast collection was finally not performed. Thus, overall, 21 elderly AML patients (male n=14; median age: 74 years (range: 65-84), secondary AML n=8) were considered either at time of diagnosis or at time of first relapse. The median % of BM blasts was 63% (range: 20-92), including 3 and 4 cases with less than 40% and between 40-49%, respectively (protocol deviation). Although it was not the case for one patient with 〉50% of BM blasts, all patients between 40-49% of BM blasts reached the threshold of 2.4x108blasts required for the study. Two patients out of 3 with less than 40% BM blasts had insufficient blast collection to pursue the protocol. After blast collection, the majority of patients (n=19) received non-intensive chemotherapy. 5/21 (24%) cases achieved CR, a rate that was expected for this very old population. All of CR patients could proceed to apheresis after 2 (n=4) or 4 (n=1) courses of non-intensive consolidation. Production of the 5 vaccines was possible for all of them and first infusion was made at a median of 25 days (range: 20-28) from the apheresis. However, a median of 27 vaccines (range: 8-85) could have been theoretically produced in CR patients, suggesting the possibility to realize a longer maintenance therapy to prevent relapse in the future. All patients received as expected the 5 vaccines and no adverse events were documented. Durations of response from CR were: +8.5, +8, +4.5, +4, +12 months and from first vaccine: +5.5, +4.5, +1.8, +1.8, and +9 months. Two patients had relapsed before day+55. At this time, the 3 other patients were documented with negative MRD. In July 2016, 2 patients are still alive, 1 at +30 months from CR in relapse and 1 at +13 months in CR. The 3 other cases died of relapse at +15.5, +8 and +5.5 months from CR. The median OS from pre-inclusion was significantly higher for vaccinated CR patients (13 months (9-41) vs 4.75 months (1-24), p=0.009). Conclusion: Our strategy seems promising for elderly AML patients achieving CR in terms of relapse prevention. Vaccine production is reproducible and compliant for clinical use. Larger Phase 2 studies are required to confirm our results in younger and older AML population. The trial is registered at Clinicaltrials.gov NCT01146262. This study was supported by a grant from the French National Cancer Institute. Disclosures Moreau: Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Janssen: Honoraria, Speakers Bureau; Novartis: Honoraria; Takeda: Honoraria; Amgen: Honoraria.

Description: Introduction Various Peripheral T-cell lymphoma (PTCL) entities are recognized in the World Health Organization (WHO) classification based on clinical, histopathological, phenotypic and molecular criteria. Their diagnosis is however often challenging for pathologists, and up to 30% of cases, currently not classifiable, are recognized as not otherwise specified (NOS). Recent gene expression profiling (GEP) studies have significantly improved the molecular and ontogenic characterization of these tumors, but such high-throughput technologies are hardly feasible in the routine clinical setting. There is therefore an important need for alternative diagnostic strategies to allow for the development of specific therapies. Here, we sought to create a parsimonious and robust GEP assay to differentiate the different PTCL entities and to better characterize the heterogeneity of PTCL-NOS. Methods A Reverse Transcriptase-Multiplex Ligation dependant Probe Amplification (RT-MLPA) assay addressing 20 markers was applied to a cohort of 227 PTCLs biopsies enriched in PTCL-NOS (n=126). This assay determines the expression of seventeen genes routinely tested as immunohistochemical (IHC) markers or selected from high throughput GEP studies, together with the EBV infection status (EBER1 expression) and the presence of RHOAG17V and IDH2R172K/T mutations. Results Unsupervised hierarchical clustering analysis of 101 control cases representing the main PTCL entities other than PTCL-NOS by RT-MLPA accurately classified 28/29 Angioimmunoblastic T-cell lymphomas (AITL), 21/21 Anaplastic large T-cell lymphomas (ALCL) ALK+, 16/16 NK/T-cell lymphomas (NKTCL), 6/6 Hepatosplenic T-cell lymphomas (HSTL) and 12/12 Adult T-cell Leukemia/Lymphomas (ATLL)(Figure). AITL were classified according to the expression of Tfh markers (CXCL13, CXCR5, ICOS, BCL6) and RHOA mutations (n=18); NKTCLs according to EBER1, GZMB and Th1 markers (TBX21, IFNγ); HSTLs to CD56, GATA3, TBX21 and BCL6; ALCL ALK+ according to CD30, ALK and cytotoxic markers (PRF, GZMB); ATLLs to ICOS and Th2 markers (GATA3, CCR4). Interestingly, ALCL ALK- cases (n=17) were divided into 2 subgroups: one, associated with high expression of CD30 and cytotoxic markers (PRF, GZMB), clustered with ALCL ALK+ cases (n=11), the other showed absence of PRF and GZMB, but expression of CD30 and Th2 markers (n=6). Applied to 126 nodal PTCL-NOS, the RT-MLPA classifier identified 33 AITL-Like cases expressing Tfh markers and often presenting with RHOA mutations (15 cases). It also identified 5 NKTCL-like cases (EBV-cytoxic) and 1 ALCL-like case (cytotoxic-CD30).The CD30-Th2 signature was found in 15 cases, reinforcing the hypothesis that it may delineate a novel PTCL entity, at the frontier between ALCL ALK- and other PTCLs. In agreement with previous GEP studies, 23 cases expressed Th2 markers but no CD30 (often in association with a significant Tfh signature, probably reflecting a contribution from the microenvironment). Twenty-five other cases showed a hybrid cytotoxic-Th1 signature. The remaining 14 cases did not reveal any recognizable gene expression profile. Finally, we observed a strong correlation between RT-MLPA and IHC for most markers evaluated by both methods (p

Description: Abstract 1607 Background: Lymphoid malignancies derived from T and NK cells (PTCLs) constitute a heterogeneous group of uncommon disease entities, with marked geographic variation in their distribution. The most recent data from the International T-cell Lymphoma Project based on a retrospective analysis of PTCLs diagnosed between 1990 and 2002 (Blood. 2011;117(25): 6756–67) indicate that PTCL, not otherwise specified (PTCL,NOS) represents the most common PTCL worldwide (25,9%), followed by angioimmunoblastic T-cell lymphoma (AITL) (18,5%). Over the last few years, a better characterization of the cellular origin and pathophysiology of PTCLs has led to the development of new diagnostic markers. Aim of the study: To characterize the epidemiology of mature T/NK-cell malignancies in Western Europe (France and bordering countries) and to examine whether the availability of new tools/antibodies and changing concepts over the past years might have translated into an apparent change in the relative distribution of PTCL entities. Materials and methods: The histopathological diagnosis of PTCL entities according to the 2008 WHO classification were collected through two independent sets of PTCLs in France and bordering countries. Results: Over the past two years (2010–2011), 933 newly diagnosed non-cutaneous PTCLs were reviewed in reference centers through the prospective network “Lymphopath” aiming to review any newly diagnosed lymphoma in France. According to the 2008 WHO classification, the 933 PTCLs comprised: 314 AITL (33,6%), 239 PTCL,NOS (25,6%), 78 ALK-positive anaplastic large cell lymphoma (ALCL) (8,3%), 72 ALK-negative ALCL (7,7%), 59 extranodal NK/T-cell lymphomas (ENKTL 6%), 33 enteropathy-associated T-cell lymphoma (4%), 32 HTLV1+ adult T leukemia/lymphoma (3%), 7 hepatosplenic T-cell lymphoma (1%) and 99 cases of other entities or unclassifiable (11%). A high prevalence of AITL was also found in an independent set of PTCLs retrospectively collected in the framework of a multicentric T-cell lymphoma research consortium “Tenomic” where non-cutaneous PTCL with frozen material available (n=623) from 1999 to 2009 in France and Belgium were retrieved and collegially reviewed for consensus diagnosis. In this collection, AITL (n=288, 46%) also outnumbered PTCL, NOS (n=215, 35%). Of the 196 AITL cases extensively investigated for CD10, TFH markers (PD1, CXCL13), CD21/CD23 follicular dendritic cells (FDC) and EBV, the initial diagnosis was recorded in 178 cases as: AITL in 155 cases (87%), PTCL, NOS in 21 cases (12%), and intermediate between PTCL,NOS and AITL (PTCL,NOS/AITL) in 2 cases, indicating the impact of additional stainings for the diagnosis of AITL. The 107 PTCL,NOS cases also extensively immunostained included 9 follicular variant of PTCL,NOS, 8 PTCL,NOS/AITL cases, 5 cases intermediate between PTCL,NOS and ALK-negative ALCL (of which 2 had been diagnosed as such and two as PTCL,NOS), and 85 remaining cases truly unspecified. Of these, 60 had been initially diagnosed as PTCL,NOS; 2 as PTCL,NOS/AITL; 4 as ALK-negative ALCLs and 1 as ENKTL. Conclusion: This study based on two independent large cohorts of non-cutaneous PTCLs highlights AITL as the most prevalent entity in Western Europe. It shows that extensive studies including investigation for CD10, TFH markers, FDC and EBV can at least partly contribute to the reclassification of some PTCL, NOS into the AITL spectrum category. Disclosures: No relevant conflicts of interest to declare.

Description: Gene expression profiling has provided new insights into the understanding of mature B cell neoplasms by relating each one to its normal counterpart, so that they can be to some extent classified according to the corresponding normal B-cell stage. Thus, diffuse large B cell (DLBCL) and follicular lymphoma (FL) have been related to a germinal center precursor whereas mantle cell lymphoma (MCL) or marginal zone lymphoma (MZL) are more likely to derive from naïve and memory B cell, respectively. However, little is still known about the physiopathology of B-cell lymphomas and particularly the deregulated pathways involved in their oncogenesis. To further investigate that point, we performed laser capture microdissection (LCM) of the three anatomic lymphoid compartments (i.e germinal center, mantle zone and marginal zone) taken from nine normal spleens and lymph nodes and magnetic cell separation of the four normal B cell subpopulations (i.e naïve B cells, centroblasts, centrocytes and memory B cells) purified from twelve normal tonsils for gene expression profiling by cDNA microarray. These molecular profiles have been compared to those of the four most frequent mature B cell neoplasms in adult (i.e DLBCL, FL, MZL and MCL), each one isolated from five previously untreated patients. Unsupervised analysis by hierarchical clustering of the normal anatomic and cellular populations could discriminate the germinal from the extra-germinal populations by genes involved in cell proliferation (e.g. E2F5, CCNB2, BUB1B and AURKB), DNA repair (e.g. PCNA and EXO1), cytokine secretion (e.g. IL8, IL10RB, IL4R and TGFBI) and apoptosis (e.g. CASP8, CASP10, BCL2 and FAS). Supervised analysis of the comparison between each B-cell lymphoma and its anatomic and cellular physiologic equivalent identified molecular deregulations concerning several genes’families characterizing the different histologic subtypes. Genes associated with cellular adhesion and ubiquitin cycle were significantly up-regulated in MCL (FCGBP, ITGAE, USP7, VCAM1) and MZL (CTGF, CDH1, ITGAE) whereas germinal center derived lymphomas (i.e. DLBCL and FL) mainly showed up-regulation of genes involved in cell proliferation (TNFRSF17, SEPT8) and immune response (FCER1G, XBP1, IL1RN). Few deregulated genes were common to the four subtypes, principally associated with cell proliferation (CYR61, GPNMB), cytosqueleton organization (EPB41L3) and carbohydrates metabolism (GNPDA1), suggesting potential similar oncogenic pathways. Those preliminary results are compatible with both subtype-specific and overall mechanisms of lympomagenesis and should be verified in a wider range of samples to confirm the oncogenic events involved in this heterogeneous disease.

Description: Background: Angioimmunoblastic T cell lymphoma (AITL) is one of the most frequent peripheral T cell lymphoma, and has a poor prognosis. Neoplastic T cells originate from T follicular helper cells, and are admixed among a prominent microenvironment, making their identification sometimes difficult. IDH2 mutations are present in 20-30% AITL patients, where they often co-exist with TET2, DNMT3A and RHOA mutations. They affect almost exclusively the codon R172 of IDH2, providing to the IDH2 enzyme a neo-activity that converts α ketoglutarate (αKG) to D 2-hydroxyglutarate (2HG). D-2HG, the dextrogyre form of 2HG, is an oncometabolite present at very low level under physiological condition, which inhibits many αKG dependent dioxygenases, including TET proteins and is involved in oncogenesis of various cancers such as gliomas or acute myeloid leukemias (AML). Preliminary data, based on small series, showed that increased level of 2HG was detectable in tumor and in serum of IDH2 mutated AITL. However, 2HG level, as well as D/L ratio, has not been evaluated as a surrogate marker of IDH2 mutation in AITL, at diagnosis or during the follow-up. Patients and Methods: Serum from 69 AITL patients, collected in REVAIL trial (NCT00169156) (N=48), RAIL trial (NCT01553786) (N=9) or TENOMIC collection (N=12) were included in this study. IDH2 mutations were assessed in formalin fixed paraffin embedded tumor tissue by deep next generation sequencing of exon 4, using PGM technology (N=63) or allele specific PCR (N=6). For the purpose of the study the cohort was enriched in mutated patients. Serum was collected at diagnosis and at the end of the frontline treatment in 6 patients, 5 of them being IDH2 mutated. D and L 2HG was measured in serum using a liquid tandem mass spectrometry method as previously described (Poinsignon et al. J Chromatogr B 2016) to determine total (D+L) 2HG and D/L 2HG ratio. Results: Twenty-four patients (35%) were IDH2 mutated. Median IDH2 variant allele frequency (VAF) was 7% (IQR, 4%-12.5%). Median total 2HG was 3.63 µM (IQR, 1.6-6.1) in mutated patients versus 1.17 µM (IQR, 0.85-1.68) in wild type patients (p

Description: Abstract 4103 Background. Acute GVHD after allogeneic stem cell transplantation (allo-SCT) is an exaggerated immune response against alloantigens involving dysregulation of inflammatory cytokine cascades. Previous studies established an important role of Th1 cells in acute GVHD pathophysiology. However, the identification of proinflammatory Th17 cells which contribute to autoimmune diseases pathophysiology, raised the issue of the role of Th17 cells in human acute GVHD. Indeed, the contribution of Th17 cells in acute GVHD was assessed in GVHD mouse models with conflicting results. In addition, the role of the PDC subset (the professional type I IFN-secreting cells), which play an important role in triggering Th17-related cytokines and autoimmune diseases, is not yet established in the acute GVHD setting. This report investigated the role of Th17 cells and their interaction with PDC in cutaneous biopsies taken from patients with or without acute GVHD. Patients and Methods. Studies described in this report were performed in a single centre series of 38 patients who underwent allo-SCT for different hematological malignancies (n=37) and severe aplastic anemia (n=1). The median age of patients was 52 years (range, 17–70). The stem cell source was PBSCs in 27 cases (71%), CB in 6 cases and BM in 5 cases. 11 patients received transplant from a matched-related donor, and 27 patients from an unrelated donor. A reduced-intensity conditioning regimen was used in the majority of cases (n=29; 76%) Immunohistochemistry was performed on deparaffinized tissues sections using an indirect immunoperoxydase method. A quantitative evaluation of antigens expression was performed by counting the number of positive cells in the whole biopsy at 200 magnifications for each sample. Results. In this cohort, based on standard pathology criteria, 29 patients had a histologically proven skin acute GVHD. In all cases, biopsies were taken before initiation of systemic corticosteroid therapy. The remaining 9 patients did not have histological signs of acute GVHD (and did not develop clinical signs of acute GVHD) and thus, were used as controls. In order to identify the Th17 cell population, biopsies were tested for expression of the CD161 and CCR6 markers, and ROR-gamma-t, the key transcription factor that orchestrates the differentiation of Th17 cells. Significantly higher numbers of ROR-gamma-t+, CD161+ and CCR6+ cells were counted in the skin of patients with acute GVHD compared with intestinal mucosa of patients without acute GVHD, mainly found in the lamina propria but also in the epithelium of altered glands (p=0.001, p

Description: Background. Peripheral T-cell lymphomas (PTCLs) are rare and heterogeneous diseases with dismal outcome when treated with chemotherapy alone. Because allogeneic stem cell transplantation (allo-SCT) can cure relapse-refractory patients, we hypothesised that upfront allo-SCT may be less toxic and provide a better outcome. Therefore, all patients that presented with advanced PTCL in our institution at diagnosis were scheduled to undergo upfront allo-SCT after induction chemotherapy. Patients and methods. The aim of the present work was to assess the feasibility and toxicity of upfront allo-SCT. From 2004 to 2012, 49 newly diagnosed PTCL patients were scheduled to receive upfront allo-SCT. An HLA-matched donor was found for 42 patients: related to the patient in 15 cases, unrelated in 20 cases, and suitable cord blood units were used in seven cases. Results. After induction chemotherapy, 17 patients reached complete remission and 29 (60%) proceeded to upfront allo-SCT. For all patients, the one and two-year overall survival (OS) rates were 59% (CI95%; 47-75) and 55 % (CI95%; 43-71), respectively. The most frequent reason we did not proceed to allo-SCT was disease progression or insufficient response after induction. For transplanted patients, the one- and two-year OS were 76% (CI95%; 62-93) and 72.5% (CI95%; 58-91), respectively. Toxicity related mortality one year after allo-SCT was only 8.2% (CI95%; 0-18.5). The two-year progression-free survival (PFS) rate of patients who did not proceed to allo-SCT (n= 20) was below 30%. The disease status at the time of transplantation was a strong predictive marker for both PFS and OS in transplant patients. Conclusions. Upfront allo-SCT in PTCLs is feasible with low TRM, and it provides long-term disease control. However one third of patients remain chemo-refractory, and thus, new therapeutic approaches are warranted. The role of upfront allo-SCT compared to other therapeutic approaches in PTCLs requires investigation in randomized studies. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2968 Background. Acute GVHD after allogeneic stem cell transplantation (allo-SCT) is an exaggerated immune response against alloantigens involving dysregulation of inflammatory cytokine cascades. Previous studies established an important role of Th1 cells in acute GVHD pathophysiology. However, the identification of proinflammatory Th17 cells which contribute to autoimmune diseases pathophysiology, raised the issue of the role of Th17 cells in human acute GVHD. Indeed, the contribution of Th17 cells in acute GVHD was assessed in GVHD mouse models with conflicting results. In addition, the role of the PDC subset (the professional type I IFN-secreting cells), which play an important role in triggering Th17-related cytokines and autoimmune diseases, is not yet established in the acute GVHD setting. This report investigated the role of Th17 cells and their interaction with PDC in gastrointestinal (GI) biopsies taken from patients with or without acute GVHD. Patients and Methods. Studies described in this report were performed in a single centre series of 21 patients who underwent allo-SCT for different hematological malignancies (n=19) and severe aplastic anemia (n=2). The median age of patients was 53 years (range, 16–69). The stem cell source was PBSCs in 19 cases (85%), CB in 2 cases and BM in one case. Ten patients received transplant from a matched-related donor, and 11 patients from a matched-unrelated donor. A reduced-intensity conditioning regimen was used in the majority of cases (n=19; 90%) Immunohistochemistry was performed on deparaffinized tissues sections using an indirect immunoperoxydase method. A quantitative evaluation of antigens expression was performed by counting the number of positive cells in the whole biopsy at 200 magnifications for each sample. Results. In this cohort, based on standard pathology criteria, 16 patients had a histologically proven gastrointestinal acute GVHD. In all cases, biopsies were taken before initiation of systemic corticosteroid therapy. The remaining 5 patients did not have histological signs of acute GVHD (and did not develop clinical signs of acute GVHD) and thus, were used as controls. In order to identify the Th17 cell population, biopsies were tested for expression of the CD161 and CCR6 markers, and ROR-gamma-t, the key transcription factor that orchestrates the differentiation of Th17 cells. Significantly higher numbers of ROR-gamma-t+ and CD161+ cells were counted in the intestinal mucosa of patients with acute GVHD compared with intestinal mucosa of patients without acute GVHD, mainly found in the lamina propria but also in the epithelium of altered glands (p=0.016 and p=0.009 for ROR-gamma-t and CD161 expression respectively). Given the role of PDCs in triggering Th17-related cytokines, we sought next to determine the proportion of PDCs in intestinal biopsies from these same patients. This analysis showed a significant increase of CD123+ PDCs in the intestinal mucosa of patients with acute GVHD compared with mucosa of patients without acute GVHD (p=0.017). Moreover, we observed a significant correlation between the number of CD123+ PDCs and ROR-gamma-t or CD161 expressing cells in the intestinal mucosa of acute GVHD patients, highlighting the link between PDC and Th17 cells. Conclusion. The current study shed some light on the role of Th17 cells in the context of gastro-intestinal acute GVHD. Using well-established specific markers, we show that Th17 cells infiltrate intestinal biopsies from patients with acute GVHD. In addition, Th17 infiltration was paralleled by the infiltration of PDCs, suggesting a potential new pathophysiological link between PDCs and Th17 response in the context of gastro-intestinal acute GVHD. This is consistent with studies showing that PDCs can drive the differentiation of Th17 cells. Functional analyses are currently ongoing. Although the exact mechanism that links type I IFN production to PDC-mediated Th17 responses is still unclear in acute GVHD, these data raise the prospect of future innovative approaches to optimize immunosuppression regimens for the treatment or prophylaxis of acute GVHD by targeting PDCs and the Th17 response. Disclosures: No relevant conflicts of interest to declare.