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Purpose:We analyzed the utility of cell-free DNA (cfDNA) in a prospective population-based cohort to determine the mutational profile, assess tumor burden, and estimate its impact in response rate and outcome in patients with diffuse large B-cell lymphoma (DLBCL).Experimental Design:A total of 100 patients were diagnosed with DLBCL during the study period. Mutational status of 112 genes was studied in cfDNA by targeted next-generation sequencing. Paired formalin-fixed, paraffin-embedded samples and volumetric PET/CT were assessed when available.Results:Appropriate cfDNA to perform the analyses was obtained in 79 of 100 cases. At least one mutation could be detected in 69 of 79 cases (87%). The sensitivity of cfDNA to detect the mutations was 68% (95% confidence interval, 56.2–78.7). The mutational landscape found in cfDNA samples was highly consistent with that shown in the tissue and allowed genetic classification in 43% of the cases. A higher amount of circulating tumor DNA (ctDNA) significantly correlated with clinical parameters related to tumor burden (elevated lactate dehydrogenase and β2-microglobulin serum levels, advanced stage, and high-risk International Prognostic Index) and total metabolic tumor volume assessed by PET/CT. In patients treated with curative intent, high ctDNA levels (>2.5 log hGE/mL) were associated with lower complete response (65% vs. 96%; P < 0.004), shorter progression-free survival (65% vs. 85%; P = 0.038), and overall survival (73% vs. 100%; P = 0.007) at 2 years, although it did not maintain prognostic value in multivariate analyses.Conclusions:In a population-based prospective DLBCL series, cfDNA resulted as an alternative source to estimate tumor burden and to determine the tumor mutational profile and genetic classification, which have prognostic implications and may contribute to a future tailored treatment.

1 - Serum and tumor cytokines according to MYD88 mutational status 2- Discrepancies in cell of origin distribution between gene expression and immunohistochemistry

Purpose: Mutations in MYD88 are found in different lymphoproliferative disorders associated with particular biologic characteristics and clinical impact. The aim of this study was to analyze the incidence of MYD88 mutations and its clinical impact in diffuse large B-cell lymphoma (DLBCL).Experimental Design: The incidence, clinicobiological features, and outcome of 213 patients (115 M/98 F; median age, 65 years) with DLBCL treated with immunochemotherapy in a single institution according to MYD88 mutational status as assessed by an allele-specific PCR assay were analyzed. The cell of origin (COO) was determined in 129 cases by gene expression.Results: MYD88 mutations were found in 47 cases (22%), including L265P in 39 and S219C and M232F in 4 cases, respectively. Patients with MYD88 L265P were older, presenting frequent extranodal involvement, and mostly corresponded to activated B-cell like (ABC) subtype, whereas no preference in COO was observed in patients with other MYD88 mutations. Five-year overall survival (OS) for MYD88 wild-type, MYD88 L265P, and other variants was 62%, 52%, and 75%, respectively (P = 0.05). International Prognostic Index (IPI) (HR, 2.71; P < 0.001) and MYD88 L265P (HR, 1.786; P = 0.023) were independent variables predicting OS in the multivariate analysis. However, MYD88 L265P lost its independent value when COO was included in the model.Conclusions: Our findings indicate that MYD88 L265P mutations, but no other variants, identify a subgroup of DLBCL mainly of ABC origin, with extranodal involvement and poor outcome. Clin Cancer Res; 22(11); 2755–64. ©2016 AACR.

Supplementary tables S1 to S6

Suppplementary Methods and Figures

This is a randomized phase-2 trial aimed to compare consolidation vs. maintenance in untreated patients with follicular lymphoma (FL) responding to induction. 146 patients were enrolled from 25 Spanish institutions (ZAR2007; ClinicalTrials.gov #NCT00662948). Patients in PR or CR/CR[u] after R-CHOP were randomized 1:1 to 90Y-ibritumomab-tiuxetan 0.4 mCi/kg (arm A) vs. rituximab 375 mg/m2 every 8 weeks for 2 years (arm B). After a median follow-up of 10.55 years, 53 patients eventually progressed with a 10-year PFS of 50% vs. 56% for patients in arm A and B, respectively (HR = 1.42; p > 0.1). No significant differences were seen in OS (10-year OS 78% vs. 84.5%; HR = 1.39, p > .1). Patients receiving 90Y-ibritumomab-tiuxetan showed higher incidence of second neoplasms than those in arm B (10-year cumulative incidence 18.5 vs. 2%, respectively; p = .038). In conclusion, in FL patients responding to R-CHOP, no significant differences were found between consolidation and maintenance, although with higher late toxicity for consolidation.

Relapsed or refractory diffuse large B-cell lymphoma (DLBCL) cases have a poor outcome. Here we analysed clinico-biological features in 373 DLBCL patients homogeneously treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP), in order to identify variables associated with early failure to treatment (EF), defined as primary refractoriness or relapse within 12 months from diagnosis. In addition to clinical features, mutational status of 106 genes was studied by targeted next-generation sequencing in 111 cases, copy number alterations in 87, and gene expression profile (GEP) in 39. Ninety-seven cases (26%) were identified as EF and showed significantly shorter overall survival (OS). Patients with B symptoms, advanced stage, high levels of serum lactate dehydrogenase (LDH) or beta 2-microglobulin, low lymphocyte/monocyte ratio and higher Revised International Prognostic Index (R-IPI) scores, as well as those with BCL2 rearrangements more frequently showed EF, with R-IPI being the most important in logistic regression. Mutations in NOTCH2, gains in 5p15 center dot 33 (TERT), 12q13 (CDK2), 12q14 center dot 1 (CDK4) and 12q15 (MDM2) showed predictive importance for EF independently from R-IPI. GEP studies showed that EF cases were significantly enriched in sets related to cell cycle regulation and inflammatory response, while cases in response showed over-representation of gene sets related to extra-cellular matrix and tumour microenvironment.

This is a randomized phase-2 trial aimed to compare consolidation vs. maintenance in untreated patients with follicular lymphoma (FL) responding to induction. 146 patients were enrolled from 25 Spanish institutions (ZAR2007; ClinicalTrials.gov #NCT00662948). Patients in PR or CR/CR[u] after R-CHOP were randomized 1:1 to

Pediatric large B-cell lymphomas (LBCLs) share morphological and phenotypic features with adult types but have better prognosis. The higher frequency of some subtypes such as LBCL with IRF4 rearrangement (LBCL-IRF4) in children suggests that some age-related biological differences may exist. To characterize the genetic and molecular heterogeneity of these tumors, we studied 31 diffuse LBCLs (DLBCLs), not otherwise specified (NOS); 20 LBCL-IRF4 cases; and 12 cases of high-grade B-cell lymphoma (HGBCL), NOS in patients ≤25 years using an integrated approach, including targeted gene sequencing, copy-number arrays, and gene expression profiling. Each subgroup displayed different molecular profiles. LBCL-IRF4 had frequent mutations in IRF4 and NF-κB pathway genes (CARD11, CD79B, and MYD88), losses of 17p13 and gains of chromosome 7, 11q12.3-q25, whereas DLBCL, NOS was predominantly of germinal center B-cell (GCB) subtype and carried gene mutations similar to the adult counterpart (eg, SOCS1 and KMT2D), gains of 2p16/REL, and losses of 19p13/CD70. A subset of HGBCL, NOS displayed recurrent alterations of Burkitt lymphoma–related genes such as MYC, ID3, and DDX3X and homozygous deletions of 9p21/CDKN2A, whereas other cases were genetically closer to GCB DLBCL. Factors related to unfavorable outcome were age >18 years; activated B-cell (ABC) DLBCL profile, HGBCL, NOS, high genetic complexity, 1q21-q44 gains, 2p16/REL gains/amplifications, 19p13/CD70 homozygous deletions, and TP53 and MYC mutations. In conclusion, these findings further unravel the molecular heterogeneity of pediatric and young adult LBCL, improve the classification of this group of tumors, and provide new parameters for risk stratification.

The T Cell Project was the largest prospective trial to explore the incidence, treatment patterns, and outcomes for T cell lymphomas. The rare subtypes of T cell lymphomas, including hepatosplenic T cell lymphoma (HSTCL), enteropathy associated T cell lymphoma (EATL), and peripheral gamma delta T cell lymphomas (PGDTCLs) are poorly represented in most studies and there is little data regarding treatment patterns. We report results from 115 patients with hepatosplenic (n = 31), enteropathy associated (n = 65), and PGDTCLs (n = 19). While anthracycline regimens were most commonly used as first line therapy, response rates ranged from 20%−40% and were suboptimal for all groups. Autologous stem cell transplantation was performed as a consolidation in first remission in a small number of patients (33% of HSTCL, 7% of EATL, and 12% of PGDTCL), and four patients with HSTCL underwent allogeneic stem cell transplantation in first remission. The progression free survival at 3 years ranged from 28%−40% for these rare subtypes, and the overall survival at 3 years was most favorable for PGDTCL (70%). These data highlight the need for novel treatment approaches for rare subtypes of T cell lymphomas and for their inclusion in clinical trials.

Angioimmunoblastic T-cell lymphoma (AITL) is a unique subtype of peripheral T-cell lymphoma (PTCL) with distinct clinicopathologic features and poor prognosis. We performed a subset analysis of 282 patients with AITL enrolled between 2006 and 2018 in the international prospective T-cell Project (NCT01142674). The primary and secondary end points were 5-year overall survival (OS) and progression-free survival (PFS), respectively. We analyzed the prognostic impact of clinical covariates and progression of disease within 24 months (POD24) and developed a novel prognostic score. The median age was 64 years, and 90% of patients had advanced-stage disease. Eighty-one percent received anthracycline-based regimens, and 13% underwent consolidative autologous stem cell transplant (ASCT) in first complete remission (CR1). Five-year OS and PFS estimates were 44% and 32%, respectively, with improved outcomes for patients who underwent ASCT in CR1. In multivariate analysis, age ≥60 years, Eastern Cooperative Oncology Group performance status >2, elevated C-reactive protein, and elevated β2 microglobulin were associated with inferior outcomes. A novel prognostic score (AITL score) combining these factors defined low-, intermediate-, and high-risk subgroups with 5-year OS estimates of 63%, 54%, and 21%, respectively, with greater discriminant power than established prognostic indices. Finally, POD24 was a powerful prognostic factor with 5-year OS of 63% for patients without POD24 compared with only 6% for patients with POD24 (P < .0001). These data will require validation in a prospective cohort of homogeneously treated patients. Optimal treatment of AITL continues to be an unmet need, and novel therapeutic approaches are required.

GELTAMO and GENOSEN group., [Introduction] To assess the management of immunocompetent patients with primary central nervous system lymphomas (PCNSL) in Spain., [Methods] Retrospective analysis of 327 immunocompetent patients with histologically confirmed PCNSL diagnosed between 2005 and 2014 in 27 Spanish hospitals., [Results] Median age was 64 years (range: 19–84; 33% ≥ 70 years), 54% were men, and 59% had a performance status (PS) ≥ 2 at diagnosis. Median delay to diagnosis was 47 days (IQR 24–81). Diagnostic delay > 47 days was associated with PS ≥ 2 (OR 1.99; 95% CI 1.13–3.50; p = 0.016) and treatment with corticosteroids (OR 2.47; 95% CI 1.14–5.40; p = 0.023), and it did not improve over the years. Patients treated with corticosteroids (62%) had a higher risk of additional biopsies (11.7% vs 4.0%, p = 0.04) but corticosteroids withdrawal before surgery did not reduce this risk and increased the diagnostic delay (64 vs 40 days, p = 0.04). Median overall survival (OS) was 8.9 months [95% CI 5.9–11.7] for the whole series, including 52 (16%) patients that were not treated, and 14.1 months (95%CI 7.7–20.5) for the 240 (73.4%) patients that received high-dose methotrexate (HD-MTX)-based chemotherapy. Median OS was shorter in patients ≥ 70 years (4.1 vs. 13.4 months; p, [Conclusions] Corticosteroids withdrawal before surgery does not decrease the risk of a negative biopsy but delays diagnosis. In this community-based study, only 73.4% of patients could receive HD-MTX-based chemotherapy and OS remains poor, particularly in elderly patients ≥ 70 years., This work was partially supported by Grant PI1501303 from ISCIII and Fondo Europeo de Desarrollo Regional (FEDER).

The clinical behavior of FL patients is heterogeneous. The levels of sIL-2R have been correlated with tumor burden and outcome in FL. However, the impact of IL-6 and TNF-α in this disease is unclear. We studied 253 patients diagnosed with grade 1-3a FL between 2002 and 2018, with available information on serum levels of sIL-2R, IL-6, and TNF-α at diagnosis. Patients with cytokine levels above the cutoff had features of a higher tumor burden and higher-risk disease. Levels of any of the studied cytokines above the cutoff and a higher number of cytokines above the cutoff impacted on a shorter PFS and OS. TNF-α levels were an independent predictor of a poorer PFS. No differences were observed in the risk of histological transformation or second malignancies. The determination of cytokine levels in FL patients is feasible in clinical practice, and elevated levels are associated with a higher tumor burden and poorer survival.

Follicular lymphoma (FL) is an indolent disease characterized by long survival but frequent relapses. Before the introduction of rituximab, the clinical course of these patients showed a shorter response duration (RD) after each relapse. In this study, we analysed if this pattern of shortened responses remains in patients treated in the rituximab era. We selected 348 patients newly diagnosed with FL in two institutions between 2001 and 2014 that received chemoimmunotherapy. After a median follow-up of 6 center dot 3 years, 10-year progression-free and overall survivals were 53% and 72%, respectively. All patients received first-line, 111 second-line and 41 third-line treatments, with a 5-year RD of 62%, 39% and 24%, respectively (P < 0 center dot 0001). Variables predicting longer RD after first-line treatment were normal beta 2microglobulin, complete remission achievement and maintenance with rituximab. Patients with longer RD after first-line showed significantly longer RD after second-line therapy. Autologous stem-cell transplantation after second-line therapy did not significantly impact RD. Median survival after first, second and third therapies was not reached, 7 center dot 6 and 4 center dot 8 years, respectively, whereas relative survival with respect to a sex- and age-matched Spanish population, the decrease in the life expectancy at 10 years was 17%, 45% and 79%, respectively. Thus, RD still shortens after each relapse in patients with FL treated in first line with rituximab combinations.

High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements, mostly known as double-hit lymphoma (DHL), is a rare entity characterized by morphologic and molecular features between Burkitt lymphoma and the clinically manageable diffuse large B-cell lymphoma (DLBCL). DHL patients usually undergo a rapidly progressing clinical course associated with resistance to standard chemo-immunotherapy. As a consequence, the prognosis of this entity is particularly poor with a median overall survival inferior to 1 year. ABT-199 (venetoclax) is a potent and selective small-molecule antagonist of BCL-2 recently approved for the treatment of a specific subtype of lymphoid neoplasm. In this study, we demonstrate that single-agent ABT-199 efficiently displaces BAX from BCL-2 complexes but fails to maintain a significant antitumor activity over time in most MYC+/BCL2+DHL cell lines and primary cultures, as well as in a xenograft mouse model of the disease. We further identify the accumulation of the BCL2-like protein BFL-1 to be a major mechanism involved in acquired resistance to ABT-199. Noteworthy, this phenomenon can be counteracted by the BET bromodomain inhibitor CPI203, since gene expression profiling identifies BCL2A1, the BFL-1 coding gene, as one of the top apoptosis-related gene modulated by this compound. Upon CPI203 treatment, simultaneous downregulation of MYC and BFL-1 further overcomes resistance to ABT-199 both in vitro and in vivo, engaging synergistic caspase-mediated apoptosis in DHL cultures and tumor xenografts. Together, these findings highlight the relevance of BFL-1 in DH lymphoma-associated drug resistance and support the combined use of a BCL-2 antagonist and a BET inhibitor as a promising therapeutic strategy for patients with aggressive DHL.

Context Scarce data on clinical features and treatment outcomes of elderly patients is presented in clinical practice. The outcomes of elderly patients are worse, especially with aggressive lymphoma types. The conventional CHOP-based chemotherapy is a standard frontline treatment for peripheral T-cell lymphomas (PTCLs). Objective In this report, we aimed to compare outcomes in elderly patients with PTCLs in the global International T-Cell Project (TCP) after treatment with combination chemotherapy, reduced doses, or palliative single-agent approaches. The primary and secondary endpoints were 5-year OS and PFS. Patients or Other Participants We performed a sub-analysis of 246 patients >70 years with PTCLs out of 1,553 cases enrolled between 2006 and 2018 in the TCP, involving 74 institutions in 13 countries in Europe, North/South America, and Asia. All patients received frontline therapy for anaplastic large cell lymphoma (ALCL) ALK–, ALCL ALK +, angioimmunoblastic T-cell lymphoma (AITL), and PTCL not otherwise specified (NOS). Baseline patient characteristics were summarized with descriptive statistics. Survival analyses were performed using Kaplan–Meier method. The TCP is registered on ClinicalTrials.gov (NCT01142674). Results Of 246 patients ≥70 years, 217 (88%) patients received frontline therapy with curative intent, and 29 (12%) patients with palliative intent. The median age at diagnosis was 76 years (range 70–89) in the curative intent arm and 77 years (range 70–85) in the palliative arm. Stage III-IV had 77.4% of patients, and 54% were males. High IPI and PIT scores were found in 62% and 65% of patients, respectively. Median OS of curative vs palliative intent was 59 vs 4.7 months (p=0.001). The 3- and 5-year OS for patients with curative treatment arm were 71% and 50%, vs 28% and 22% with palliative treatment, respectively. Median PFS of curative vs palliative intent patients was 37 vs 3.7 months (p=0.004). The 3- and 5-year PFS rates for patients with curative and palliative approaches were 54% and 37% vs 21% and 16%, respectively. Conclusions In this prospective registry cohort of patients 70 years and older, we observed improved survival for patients treated with curative combination therapy compared to palliative single agents. Based on our findings, the inclusion of otherwise fit older patients in ongoing clinical trials of frontline therapies incorporating targeted agents is warranted.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoma with heterogeneous outcomes. To improve accuracy of the international prognostic index score, new biological variables are being investigated. The aim of this study was to determine the prognostic significance of serum levels of different cytokines, namely soluble interleukin-2 receptor (sIL2-R), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF). We analyzed 197 de novo DLBCL patients (91 M/106 F; median age 66 years) treated with immunochemotherapy in a single institution. Serum cytokine determination was performed with ELISA, using the upper normal values as cut-offs. sIL-2R, IL-6 and TNF were elevated in 133, 130 and 144 cases, respectively. Elevation of each of these cytokines correlated with worse performance status, presence of B symptoms, advanced stage, elevated LDH and β2-microglobulin (P

MYC translocation is a defining feature of Burkitt lymphoma (BL), and the new category of high-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 translocations, and occurs in 6% to 15% of diffuse large B-cell lymphomas (DLBCLs). The low incidence of MYC translocations in DLBCL makes the genetic study of all these lymphomas cumbersome. Strategies based on an initial immunophenotypic screening to select cases with a high probability of carrying the translocation may be useful. LMO2 is a germinal center marker expressed in most lymphomas originated in these cells. Mining gene expression profiling studies, we observed LMO2 downregulation in BL and large B-cell lymphoma (LBCL) with MYC translocations, and postulated that LMO2 protein expression could assist to identify such cases. We analyzed LMO2 protein expression in 46 BLs and 284 LBCL. LMO2 was expressed in 1/46 (2%) BL cases, 146/268 (54.5%) DLBCL cases, and 2/16 (12.5%) high-grade B-cell lymphoma cases with MYC and BCL2 and/or BCL6 translocations. All BLs carried MYC translocation (P

Objectives Diffuse large B-cell lymphoma (DLBCL) is an aggressive heterogeneous lymphoma with standard treatment. However, 30%-40% of patients still fail, so we should know which patients are candidates for alternative therapies. IPI is the main prognostic score but, in the rituximab era, it cannot identify a very high-risk (HR) subset. The MD Anderson Cancer Center reported a score in the prerituximab era exclusively considering tumor-related variables: Tumor Score (TS). We aim to validate TS in the rituximab era and to analyze its current potential role. Methods From GELTAMO DLBCL registry, we selected those patients homogeneously treated with R-CHOP (n = 1327). Results Five-years PFS and OS were 62% and 74%. All variables retained an independent prognostic role in the revised TS (R-TS), identifying four different risk groups, with 5-years PFS of 86%, 71%, 50%, and very HR (28%). With a further categorization of three variables of the original TS (Ann Arbor Stage, LDH and B2M), we generated a new index that allowed an improvement in HR assessment. Conclusions (a) All variables of the original TS retain an independent prognostic role, and R-TS remains predictive in the rituximab era; (b) R-TS and additional categorization of LDH, B2M, and AA stage (enhanced TS) increased the ability to identify HR subsets.

Summary Background Extranodal natural killer (NK) T-cell lymphoma (ENKTL) is a unique clinicopathological entity, typically associated with poor survival outcomes. Most published data have come from east Asian study groups, with little information available from international cohorts. The effects of treatment advances on routine clinical practice across continental territories has not been clear. We aimed to improve understanding of the clinical characteristics and outcomes of patients with ENKTL. Methods We did a substudy of patients with ENKTL from the T-cell Project, a global prospective cohort study. The T-cell Project registered consecutively diagnosed adults (>18 years) with newly diagnosed, untreated mature T-cell or NK lymphomas (WHO 2001 or 2008 classifications) from 74 centres in 13 countries (in Asia, Europe, North America, and South America). In total, 1695 patients with mature T-cell or NK lymphomas were enrolled between Oct 12, 2006 and Feb 28, 2018 in the T-cell Project. The first patient with ENKTL was enrolled on Feb 15, 2007, and the last on May 26, 2017. Data on baseline characteristics, first-line treatment, treatment response, and survival outcomes were recorded in a central database (locked March 30, 2019). The primary outcome was 5-year overall survival. The T-cell Project is registered on ClinicalTrials.gov , NCT01142674 . Findings 166 patients were diagnosed with ENKTL, comprising 11% of 1553 eligible registered cases and distributed across 40 participating centres in four continents. At a median follow-up of 44 months (IQR 20–61), overall survival at 5 years was 54% (95% CI 44–63) in patients with nasal disease (n=98) and 34% (27–46) in patients with extranasal disease (n=68). Interpretation To our knowledge, this study presents the largest international cohort of patients with ENKTL. We describe a clinically significant improvement in the survival of patients with ENKTL treated in routine clinical practice over the past decade, likely to be attributable to the increasing use of treatment protocols specific for ENKTL. Funding The Fondazione Cassa di Risparmio di Modena, the Associazione Angela Serra per la Ricerca sul Cancro, the Fondazione Italiana Linfomi, Allos Therapeutics, Spectrum Pharmaceuticals, Associazione Italiana per la Ricerca sul Cancro, and the National Cancer Institute at the National Institutes of Health.

Diffuse large B cell lymphoma (DLBCL) patients carrying hepatitis C virus (HCV) have higher risk of treatment toxicity and complications. The aim of this study was to assess the impact of HCV in a series of DLBCL patients treated with immunochemotherapy. 321 patients (161 M/160F; median age, 66 years) diagnosed with de novo DLBCL in a single center between 2002 and 2013 were included. Immunodeficiency-related lymphomas were excluded. HCV+ cases were defined by the presence of IgG anti-HCV. Main clinico-biological characteristics and outcome were analyzed according to the viral status. Two hundred ninety patients were HCV− and 31 HCV+. HCV+ patients were older (median age 71 vs. 64 years, P = 0.03), had more often B symptoms (P = 0.013), spleen (P = 0.003), and liver (P = 0.011) involvement, higher rate of early death (

Peripheral T-cell lymphomas (PTCLs) comprise a heterogeneous group of neoplasms that are derived from post-thymic lymphoid cells at different stages of differentiation with different morphological patterns, phenotypes and clinical presentations. PTCLs are highly diverse, reflecting the diverse cells from which they can originate and are currently sub-classified using World Health Organization (WHO) 2008 criteria. In 2006 the International T-Cell Lymphoma Project launched the T-Cell Project, building on the retrospective study previously carried on by the network, with the aim to prospectively collect accurate data to improve knowledge on this group of lymphomas. Based on previously published reports from International Study Groups it emerged that rendering a correct classification of PTCLs is quite difficult because the relatively low prevalence of these diseases results in a lack of confidence by most pathologists. This is the reason why the T-Cell Project requested the availability of diagnostic material from the initial biopsy of each patient registered in the study in order to have the initial diagnosis centrally reviewed by expert hematopathologists. In the present report the results of the review process performed on 573 cases are presented. Overall, an incorrect diagnosis was centrally recorded in 13.1% cases, including 8.5% cases centrally reclassified with a subtype eligible for the project and 4.6% cases misclassified and found to be disorders other than T-cell lymphomas; 2.1% cases were centrally classified as T-Cell disorders not included in the study population. Thus, the T-Cell Project confirmed the difficulties in providing an accurate classification when a diagnosis of PTCLs is suspected, singled out the major pitfalls that can bias a correct histologic categorization and confirmed that a centralized expert review with the application of adequate diagnostic algorithms is mandatory when dealing with these tumours. Copyright © 2016 John Wiley & Sons, Ltd.

Purpose: Mutations in MYD88 are found in different lymphoproliferative disorders associated with particular biologic characteristics and clinical impact. The aim of this study was to analyze the incidence of MYD88 mutations and its clinical impact in diffuse large B-cell lymphoma (DLBCL). Experimental Design: The incidence, clinicobiological features, and outcome of 213 patients (115 M/98 F; median age, 65 years) with DLBCL treated with immunochemotherapy in a single institution according to MYD88 mutational status as assessed by an allele-specific PCR assay were analyzed. The cell of origin (COO) was determined in 129 cases by gene expression. Results: MYD88 mutations were found in 47 cases (22%), including L265P in 39 and S219C and M232F in 4 cases, respectively. Patients with MYD88 L265P were older, presenting frequent extranodal involvement, and mostly corresponded to activated B-cell like (ABC) subtype, whereas no preference in COO was observed in patients with other MYD88 mutations. Five-year overall survival (OS) for MYD88 wild-type, MYD88 L265P, and other variants was 62%, 52%, and 75%, respectively (P = 0.05). International Prognostic Index (IPI) (HR, 2.71; P < 0.001) and MYD88 L265P (HR, 1.786; P = 0.023) were independent variables predicting OS in the multivariate analysis. However, MYD88 L265P lost its independent value when COO was included in the model. Conclusions: Our findings indicate that MYD88 L265P mutations, but no other variants, identify a subgroup of DLBCL mainly of ABC origin, with extranodal involvement and poor outcome. Clin Cancer Res; 22(11); 2755–64. ©2016 AACR.

Diffuse large B-cell lymphoma (DLBCL) represents a heterogeneous group of lymphomas that can be divided in two gene expression profiles by means of microarray gene expression profiling (GEP), germi...

Background The co-existence at diagnosis of follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) components (FL/DLBCL) has been considered a transformed lymphoma and accordingly treated although clinicobiological information on these patients is scarce. The aim of this study was to analyze the initial features and outcome of FL/DLBCL patients in the rituximab era. Patients and methods All patients consecutively diagnosed at a single institution with FL/DLBCL (n = 40), as well as those with pure FL (n = 328) or de novo DLBCL (n = 510) as controls. Results The proportion of the DLBCL component was highly variable (median 50%). In 29 FL/DLBCL cases analyzed, the cell of origin was GCB in 86%, ABC in 10% and unclassifiable in 4%. NOTCH1-2 was mutated in 10% of these cases. The proportion of DLBCL component did not impact on overall survival (OS). Regarding initial characteristics, patients with FL/DLBCL were closer to FL in terms of primary nodal origin, good performance status and advanced stage, whereas the other features were intermediate between FL and DLBCL. FL/DLBCL patients were treated as DLBCL with no further intensification. Complete response and primary refractory rates were 65% and 20%, respectively, with these figures being similar to DLBCL and worse than FL. Progression-free survival and OS were intermediate between FL and DLBCL (5-year OS: 85%, 73% and 63% for FL, FL/DLBCL and DLBCL, respectively). FL/DLBCL histology did not reach independent prognostic value for OS in the multivariate analyses. Conclusions The outcome of FL/DLBCL patients is not worse than that of de novo DLBCL. These cases should be treated with immunochemotherapy as DLBCL, but intensification with ASCT may not be necessary. The biological insights of FL/DLBCL warrants further genetic and molecular studies.

Introduction: Previous studies have highlighted the potential of cell-free DNA (cfDNA) to assess the mutational profile and copy number alterations (CNA) in diffuse large B-cell lymphoma (DLBCL), usually performed in tissue biopsies, both at diagnosis and relapse. In addition, the quantitative levels of cfDNA might be a surrogate of the lymphoma tumor burden and could therefore predict response to therapy, progression-free survival (PFS) and overall survival (OS). The aim of this study was to analyze the mutational profile and CNA at diagnosis using cfDNA, to compare these results with those obtained from formalin-fixed paraffin-embedded (FFPE) biopsies, and to estimate the correlation of pre-treatment levels of cfDNA with FDG-PET/CT Total Metabolic Tumor Volume (TMTV) and their impact in the outcome of DLBCL patients. Methods: We included 79 patients (41M/38F , median age 63 years) diagnosed with DLBCL according to the WHO criteria in a single institution between 2016 and 2018. All patients received chemoimmunotherapy. After frontline treatment, 56 patients achieved a complete (CR) response, 4 partial response, 16 were refractory, including 7 early deaths, and in 3 cases the response was not yet evaluable. Samples were obtained at diagnosis before starting treatment. cfDNA extraction was performed from 2-4 mL of plasma collected in PAXgene Blood ccfDNA tubes (Qiagen) and size distribution of DNA fragments was analyzed using the Agilent 2100 Bioanalyzer. Tumor genomic DNA (gDNA) was isolated from FFPE diagnostic tissue biopsies. A panel of 115 genes was enriched using a hybridization capture-based protocol from 10-30 ng of cfDNA and 150 ng of gDNA (ThruPlex Tag-seq kit and SureSelectXT enrichment reagents, Agilent Technologies) and sequenced in a MiSeq instrument (Illumina). CNA were examined using CNVkit software toolkit. cfDNA levels were reported as haploid genome equivalents per mL of plasma and expressed as a base 10 logarithm (log hGE/mL). Quantitative analysis of TMTV was performed using the semiautomatic MIM software, with a fixed SUV>2.5 thresholding method for segmentation. Results: A median of 15.6 ng/mL (range: 4-754 ng/mL) of cfDNA was obtained. At least one mutation could be detected in 69/79 cases (87.3%). The median number of mutations per sample was 6 (range: 0-41 mutations). The genes most frequently mutated at diagnosis in plasma samples were KMT2D, TP53, TNFRSF14, MYD88, BCL2, SOCS1, CREBBP, MYC and EP300. In 45 cases, paired FFPE samples were available. Sensitivity of cfDNA to detect mutations in baseline FFPE samples was 69% (95%CI: 64.1-73.9). In 28 of the 45 cases, >70% of the mutations were observed both in the cfDNA and FFPE samples. In the remaining 17 cases, the number of mutations identified in cfDNA was lower than the one observed in the FFPE sample (Figure 1a). Of note, most of the cases in which mutations were not detected in the cfDNA corresponded to localized stages (11/17, 65%) and/or primary extranodal DLBCL (12/17, 71%). The CNA profile in cfDNA was similar to that reported in tissue, with recurrent deletions of TNSFRS14 (11%), TNFAIP3 (14%), CDKN2A (7%), or TP53 (7%), as well as gains of REL (7%) or BCL2 (5%), among others (Figure 1b). Median pre-treatment TMTV (N=48) was 292 cm3 (0-4,171 cm3). Higher TMTV predicted for a poorer PFS (HR 4.85; p=0.005). cfDNA concentration significantly correlated with TMTV (R=0.546, p3 log hGE/mL) had a significantly lower CR rate, PFS, and OS than those with low levels (CR rate, 47% vs. 92%, p Conclusions: cfDNA represents a valuable tool to easily assess the genomic landscape and tumor burden in DLBCL, as well as to predict response to therapy and outcome in these patients. Figure 1 Disclosures Gine: Janssen: Other: Travel expenses, Research Funding; Roche: Other: Travel expenses, Research Funding; Gilead: Other: Travel expenses, Research Funding. Lopez-Guillermo:Gilead: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Janssen: Research Funding.

Introduction: Peripheral T-cell lymphomas (PTCL) are a heterogeneous group of mature T-cell neoplasms with aggressive behavior and dismal outcomes. Anthracycline-based chemotherapy is commonly used upfront; hematopoietic stem-cell transplantation (HSCT) is employed as consolidation by some groups. Refractory/relapsed cases (R/R) have a median survival of less than 6 months. Our objective was to analyze the outcomes of all consecutive PTCLs diagnosed and treated in a single center during 16 years according to histological subtype, disease phase (first line and R/R) and treatment strategies. Patients and methods: All adult PTCL patients referred to our center between 2003 and 2019 were included. All cases were locally diagnosed based on the current WHO classification by an expert hemato-pathologist. Initial clinical features, treatment and outcomes were analyzed, as well as salvage strategies. Results: A total of 188 patients were included (118 male, 70 female; median age 62 years). Median OS was 22.7 months, with a median follow up of 54 months. Histological subtypes, initial features, response to treatment and survival are detailed in table 1. Briefly, most cases were diagnosed at advanced stages, with extranodal involvement in 69% and elevated beta-2 microglobulin (B2m) in 77%; half had B symptoms. OS according to different subtypes is shown in figure 1. Of note, anaplastic large cell lymphoma (ALCL)-ALK+ patients had a 5-year OS of 88%. Interestingly, fifteen patients had circulating lymphoma cells (12 PTCL not otherwise specified [NOS], 2 hepatosplenic lymphoma and 1 angioimmunoblastic lymphoma [AITL]), with no impact on outcome. PTCL-NOS was the most prevalent subtype (40%) followed by AITL; unexpectedly, the proportion of PTCL-NOS cases increased while AITL cases decreased after 2008. Seven cases belonging to the recently recognized nodal PTCL with T follicular helper (TFH) phenotype were observed, with baseline characteristics and outcome similar to other PTCLs. IPI score index stratified patients into 4 groups with 24-month OS of 71%, 55%, 42% and 16% for low, int-low, int-high and high risk patients, respectively (P=.049). Only B2m and IPI score maintained independent significance for OS (HR= 3.2 and 1.8, respectively, P Conclusion: Initial features, treatments and outcomes for PTCL have not significantly changed in a 16-year period. Only a minority of cases underwent HSCT or received new agents. Ideal salvage regimens are not defined and, in our experience, single agent Gemcitabine or BV performed better than more aggressive combinations. Although promising new drugs have been recently approved for PTCL, their impact on outcome is still not clear. Recent progresses in molecular characterization of the disease may translate into better outcomes through prospective collaborative efforts in the near future. Disclosures Silva: Gilead Sciences: Consultancy, Other: Travel support, Research Funding; Janssen Cilag: Consultancy, Other: Travel support; Abbvie: Consultancy, Other: Travel support; Celgene: Consultancy; Roche: Consultancy, Other: Travel support.

Peripheral T-cell lymphomas (PTCLs) comprise a heterogeneous group of neoplasms that are derived from post-thymic lymphoid cells at different stages of differentiation with different morphological patterns, phenotypes and clinical presentations. PTCLs are highly diverse, reflecting the diverse cells from which they can originate and are currently sub-classified using World Health Organization (WHO) 2008 criteria. In 2006 the International T-Cell Lymphoma Project launched the T-Cell Project, building on the retrospective study previously carried on by the network, with the aim to prospectively collect accurate data to improve knowledge on this group of lymphomas. Based on previously published reports from International Study Groups it emerged that rendering a correct classification of PTCLs is quite difficult because the relatively low prevalence of these diseases results in a lack of confidence by most pathologists. This is the reason why the T-Cell Project requested the availability of diagnostic material from the initial biopsy of each patient registered in the study in order to have the initial diagnosis centrally reviewed by expert hematopathologists. In the present report the results of the review process performed on 573 cases are presented. Overall, an incorrect diagnosis was centrally recorded in 13.1% cases, including 8.5% cases centrally reclassified with a subtype eligible for the project and 4.6% cases misclassified and found to be disorders other than T-cell lymphomas; 2.1% cases were centrally classified as T-Cell disorders not included in the study population. Thus, the T-Cell Project confirmed the difficulties in providing an accurate classification when a diagnosis of PTCLs is suspected, singled out the major pitfalls that can bias a correct histologic categorization and confirmed that a centralized expert review with the application of adequate diagnostic algorithms is mandatory when dealing with these tumours. Copyright © 2016 John WileySons, Ltd.

We have analyzed in lymph nodes at diagnosis of 75 patients with follicular lymphoma (FL) the relationship between different T-cell subpopulations, assessed by immunohistochemistry (IHC) and flow cytometry (FC), with the outcome. CD4+ cells were the most abundant T-cells in tumor tissue sections, whilst CD57+ cells were the less frequent. In addition to nonambulatory performance status, advanced stage and FLIPI, low CD4+CD57+/CD4+ ratio (p = .041), and low CD4+/CD8+ ratio (p = .008) predicted poor overall survival (OS). Multivariate analysis showed that CD4+CD57+/CD4+ ratio was the most important variable for OS. In conclusion, T-cell subpopulations, including CD4+CD57+/CD4+ ratio analyzed by FC, could identify FL patients with favorable outcome.

The study included 1848 diffuse large B-cell lymphoma (DLBCL)patients treated with chemotherapy/rituximab. The aims were to validate the National Comprehensive Cancer Network International Prognostic Index (NCCN-IPI) and explore the effect of adding high Beta-2 microglobulin (β2M), primary extranodal presentation and intense treatment to the NCCN-IPI variables in order to develop an improved index. Comparing survival curves, NCCN-IPI discriminated better than IPI, separating four risk groups with 5-year overall survival rates of 93%, 83%, 67% and 49%, but failing to identify a true high-risk population. For the second aim the series was split into training and validation cohorts: in the former the multivariate model identified age, lactate dehydrogenase, Eastern Cooperative Oncology Group performance status, Stage III-IV, and β2M as independently significant, whereas the NCCN-IPI-selected extranodal sites, primary extranodal presentation and intense treatments were not. These results were confirmed in the validation cohort. The Grupo Español de Linfomas/Trasplante de Médula ósea (GELTAMO)-IPI developed here, with 7 points, significantly separated four risk groups (0, 1-3, 4 or ≥5 points) with 11%, 58%, 17% and 14% of patients, and 5-year overall survival rates of 93%, 79%, 66% and 39%, respectively. In the comparison GELTAMO IPI discriminated better than the NCCN-IPI. In conclusion, GELTAMO-IPI is more accurate than the NCCN-IPI and has statistical and practical advantages in that the better discrimination identifies an authentic high-risk group and is not influenced by primary extranodal presentation or treatments of different intensity.

MYC rearrangement can be detected in a subgroup of diffuse large B-cell lymphoma characterized by unfavorable prognosis. In contrast to Burkitt lymphoma, the correlation between MYC rearrangement and MYC protein expression in diffuse large B-cell lymphoma is less clear, as approximately one-third of rearranged cases show negative or low expression by immunohistochemistry. To better understand whether specific characteristics of the MYC rearrangement may influence its protein expression, we investigated 43 de novo diffuse large B-cell lymphoma positive for 8q24 rearrangement by FISH, using 14 Burkitt lymphoma for comparison. Different cell populations (clones), breakpoints (classical vs non-classical FISH patterns), partner genes (IGH vs non-IGH) and immunostaining were detected and analyzed using computerized image systems. In a subgroup of diffuse large B-cell lymphoma, we observed different clones within the same tumor distinguishing the founder clone with MYC rearrangement alone from other subclones, carrying MYC rearrangement coupled with loss/extra copies of derivatives/normal alleles. This picture, which we defined MYC genetic heteroclonality, was found in 42% of cases and correlated to negative MYC expression (P=0.026). Non-classical FISH breakpoints were detected in 16% of diffuse large B-cell lymphoma without affecting expression (P=0.040). Non-IGH gene was the preferential partner of rearrangement in those diffuse large B-cell lymphoma showing MYC heteroclonality (P=0.016) and/or non-classical FISH breakpoints (P=0.058). MYC heteroclonality was not observed in Burkitt lymphoma and all cases had positive MYC expression. Non-classical FISH MYC breakpoint and non-IGH partner were found in 29 and 20% of Burkitt lymphoma, respectively. In conclusion, MYC genetic heteroclonality is a frequent event in diffuse large B-cell lymphoma and may have a relevant role in modulating MYC expression.

We evaluated the efficacy and toxicity of retreatment with purine analogs (PA) in 62 patients with relapsed chronic lymphocytic leukemia. Median OS and PFS after retreatment were 60 and 26 months, respectively. By multivariate analysis, minimal residual disease status, ZAP-70 expression and age had independent predictive power in terms of OS. Toxicity was mainly neutropenia (21%) and infections (39%). Second malignancies were observed in 10 (16%) patients. Our results outline that retreatment with PA is remarkably effective in patients with relapsed CLL, but with a significant toxicity.

BACKGROUND Pediatric aggressive large B-cell lymphomas (LBCL) share morphological and phenotypic features with adult types but seem to have better prognosis. Additionally, a specific subtype carrying IRF4 translocations (LBCL-IRF4) has been recently identified in this age group. In adults, the cell-of-origin (COO) distinction of diffuse large B-cell lymphoma (DLBCL) based on gene expression signatures (germinal center B-cell like, GCB and activated B-cell like, ABC) and more recently clusters of genetic alterations have identified molecularly distinct DLBCL subsets that may benefit from novel therapeutic targets. The integration of pediatric population in these clinically relevant molecular subgroups and its clinical importance is not well known. The aim of this study was to characterize the molecular heterogeneity of LBCL in pediatric and young adult patients and evaluate their potential clinical impact. DESIGN Sixty-one LBCL diagnosed in patients ≤25 years-old (median age 14 years old, male/female 38/23) were included in the study. Molecular analyses included fluorescence in situ hybridization for MYC, BCL2, IRF4 and BCL6, copy number (CN) analysis (Oncoscan, Affymetrix), COO Lymph2Cx assay (NanoString) and targeted next generation sequencing of 96 B-cell lymphoma driver genes (SureSelect XT, Agilent Technologies). CNA and mutational profiles were compared to those previously published in adult DLBCL. Correlation of molecular features and event free survival (EFS) was performed using Kaplan-Meier curves. RESULTS Histologically, 33 were DLBCL, 20 LBCL-IRF4 and 8 high grade B-cell lymphomas, not otherwise specified (HGBCL). Nodal disease was present in 57%, mainly in the cervical region. COO distribution was: 68% GCB, 18% ABC and 13% unclassified. Most of LBCL-IRF4 cases were GCB-COO (73%). The IRF4 translocation was demonstrated in 16 out of 19 cases diagnosed as LBCL-IRF4 and an IGH rearrangement was seen in the other 3. Five cases carried MYC-breaks (3 DLBCL and 2 HGBCL) and two cases carried BCL6-breaks (1DLBCL and 1HGBCL). BCL2 rearrangements were absent. CN analysis detected alterations in 46/51 cases with recurrent gains (>15%) of 1q, 2p16, 11q, trisomies 7 and 12, and recurrent losses (>10%) of 1p36, 6q21-q22, 15q24, 17p13 and 19p13. Recurrent homozygous deletions were observed at 19p13/CD70 (6 cases), 9p13/CDKN2A (3 cases) and 13q14/RB1 (2 cases). Alteration patterns suggestive of chromothripsis were found in 10% (5/51) of the cases. No ABC-DLBCL related alterations such as 3p21-p14, 6q21-q25, 9p21.3 and 17p13 losses were seen. Targeted sequencing detected a total of 434 variants in 44 of 47 cases (mean 9.2 mutations/case). A pipeline for selection of driver mutations revealed a total of 270 mutations (62%) with potential functional effect. Recurrent mutations found in >15% of the cases affected IRF4, SOCS1, PIM1, CARD11, ACTB and CCND3 genes. In comparison to adult DLBCL, pediatric and young adult cases had significantly lower incidence of MYD88 (5 cases), CREBBP,TP53 (3 cases each) and TNFRSF14 (2 cases) mutations which are strongly associated with the definition of established mutational clusters in adult DLBCL. In our cohort, the morphological subtypes displayed different molecular profiles. IRF4 variants (some cases with >7 variants) and mutations in NF-kB pathway (CARD11, CD79B and MYD88-non L265P) were found specifically in the LBCL-IRF4 subgroup, whereas mutations in GCB related genes such as SOCS1 and EZH2 were particularly seen in cases with DLBCL diagnosis. All 49 patients with available follow-up received chemotherapy as first line treatment (41% containing Rituximab). Variables significantly associated with poor EFS in univariate analysis were age >18 years, ABC-COO, Stage IV, high genetic complexity (chromothripsis and/or >10 CN alterations), 2p16/REL gain/amplification, 9p21.3/CDKN2A homozygous deletions, MYC rearrangements and mutations in MYC, TP53 and DDX3X genes. CONCLUSION Despite pediatric/young-adult LBCL having overlapping features with adult disease our findings suggest that LBCL in young age have specific molecular mechanisms and highlight potential key drivers of these lymphomas in this age group. Disclosures No relevant conflicts of interest to declare.

Purpose The objective of this study was to investigate clinicopathologic features and prognostic factors of patients diagnosed with PTCL in 13 sites across Spain. Patients and Methods A multicenter, retrospective study was carried out between September 2015-November 2017.Medical charts of patients diagnosed with PTCLs between January 2008 and December 2013 that have signed the approved informed consent form were reviewed. PTCLs were then classified according to the 2016 revision of the WHO classification of lymphoid neoplasms. Clinical characteristics,history, standard immunohistochemistry (IHC) data, International Prognostic Index (IPI) and Prognostic Index for T-cell lymphoma (TCL) (PIT) were also assessed. Medians (range), mean (standard deviation) and frequency as the number of patients (n) and percentages (%) with confidence intervals at 95% (CI95%) were calculated. Overall Survival (OS) and Progression Free Survival (PFS) were analyzed using the Kaplan Meier method. Results 175 (88.4%) patients were successfully analyzed, the male/female ratio was 1.7:1.0, and the median age was 67.2 years (range: 24.8 years -95.8 years). ECOG performance status >1 was reported for 31.9% patients. Ann Arbor stages were III and IV 27.4% and 45.7%, respectively, and LDH levels were elevated to 92 patients (52.6%). Those with B symptoms accounted for 39.4%, while soft tissue was the most frequent location (23,7%) among the 76 patient with extranodal disease; bone marrow infiltration was confirmed in 18.3% patients. Relevant clinical antecedents related to immunological aspects were also frequently reported, including previous neoplasia (18.9%), autoimmune disease (16%), immunosuppressive treatments (7.3%) and previous viral diseases (HIV, HBV or HCV, 5.7%, 4.6% and 7.4%, respectively). Most patients presented with angioimmunoblastic TCL (31.4%); similar proportions of patients were observed among nodal PTCL with TFH phenotype (13.1%, PTCL not otherwise specified (12.0%) and extranodal NK/TCL nasal type (11.4%). CD30 expression and staining pattern (ranged 1-4) allowed the stratification of patients according CD30 intensity (n= 121; weak: 35, moderate: 57, and intense: 29); Patients were also classified based on CD30 expression considering the median value of quantitative CD30 in our sample (15%) the cut-off point: n=132; Negative First-line treatment with a CHOP/CHOP-like regimen was the most common finding (69.7%). Best response was observed after a median of 4 months since the start of first-line treatment (range 0.0 months - 65.2 months). Overall response rate after first-line treatment was 66.9%, with 61/151 patients reaching complete response (CR). Median PFS (n=157) and OS (n=175) of this series were 7.87 months (CI95%: 4.98 months-10.75months) and 15.77 months (CI95%: 10.23 months -21.30 months), respectively. Overall, IPI and PIT scores influenced the PFS and OS (p Reaching a CR was associated with a better PFS (CR: 62.6m; CI95%: 20.2 months -105.1 months) than the rest of patients (3.97m: CI95%: 3.08 months -4.85m; p Conclusion This is the largest series of T cell Lymphoma reported in Spain and has allowed the description of distribution of PTCL subtypes, analyzed through central hematopathologists reanalysis and reclassification of samples from 175 PTCL patients, according to the WHO 2016 classification of lymphoid neoplasms. Our data confirm the poor prognosis of these patients, as well as the impact of prognostic indexes and the response to first line treatment on their outcome. Disclosures Rodriguez-Pinilla: Takeda: Honoraria. Piris:Takeda: Honoraria; Janssen: Honoraria; Gilead: Honoraria; Kura: Honoraria. Ruiz-Zorrilla:Takeda: Employment. Montoto:Takeda: Employment.

On behalf of the Grupo Español de Linfomas y Trasplante Autólogo de Médula Ósea (GELTAMO)., Diffuse large B-cell lymphoma (DLBCL) patients failing rituximab-containing therapy have a poor outcome with the current salvage regimens. We conducted a phase 1b trial to determine the maximum tolerated dose (MTD) of lenalidomide in combination with R-ESHAP (rituximab, etoposide, cisplatin, cytarabine, methylprednisolone) (LR-ESHAP) in patients with relapsed or refractory DLBCL. Efficacy data were collected as a secondary objective. Subjects received 3 cycles of lenalidomide at escalating doses (5, 10 or 15 mg) given on days 1-14 of every 21-day cycle, in combination with R-ESHAP. Responding patients received BEAM (carmustine, etoposide, cytarabine, melphalan) followed by autologous stem-cell transplantation. Lenalidomide 10 mg/d was identified as the MTD because, in the 15 mg cohort, one patient experienced dose-limiting toxicity (grade 3 angioedema) and two patients had mobilization failure. A total of 19 patients (3, 12 and 4 in the 5, 10 and 15 mg cohorts, respectively) were evaluable. All toxicities occurring during LR-ESHAP cycles resolved appropriately and no grade 4-5 non-haematological toxicities were observed. The complete remission and overall response rates were 47·4% and 78·9%, respectively. With a median follow-up of 24·6 (17·4-38·2) months, the 2-year progression-free survival and overall survival were 44% and 63%, respectively. In conclusion, the LR-ESHAP regimen is feasible and yields encouraging outcomes., This study was supported (in part) by research funding from Celgene Corporation.

Introduction Diffuse large B cell lymphoma (DLBCL) is a heterogeneous group of aggressive B cell lymphomas, considering their biologic, pathological and clinical backgrounds. However, treatment of DLBCL is relatively homogeneous and standard, mainly based in the R-CHOP regimen. Several prognostic scores have been proposed for categorizing the risk and finding those with worse results with standard treatment, suitable to be treated with new schemes or drugs. The most important and widely used is the International Prognostic Index (IPI) proposed in 1993 and lately validated in the rituximab era (R-IPI). However, being a good prognostic score lacks the ability to identify a very high risk prognostic subset in the rituximab era. Trying to improve this situation several attempts have been made including NCCN-IPI or GELTAMO-IPI. In 1992, the MD Anderson Cancer Centre (MDACC) reported a prognostic score considering exclusively variables related to the tumor: the Tumor Score (TS). Two of them already present in IPI: high LDH and Ann Arbor stage III-IV but three different: high beta-2-microglobulin (B2M), bulky mass and presence of B symptoms. This index has not been studied in the rituximab era. Our aim is to validate the TS in the rituximab era and analyze its current potential role. METHODS From the nation-wide database of DLBCL of the Grupo Español de Linfoma y Trasplante de Médula Ósea (GELTAMO), we included for the validation those patients homogenously treated with R-CHOP and with all five TS variables available (n=1294). Patients had to be ≥ 18 years-old and a minimum of 1 year of follow-up; all histological subtypes of DLBCL and primary extranodal cases were acceptable, with the only exclusion of primary testicular or CNS sites. Failure-free survival (FFS) (including disease progression, no response to treatment or death events) and overall survival (OS) were analyzed with the Kaplan-Meier method and compared with the log-rank test. Cox Regression models were used for univariate and multivariate analysis. Comparisons between scores were performed with concordance probability estimates (CPEs). RESULTS Median follow-up was 60 months (12-176). 5y-FFS and OS of the series were 62% and 74%, respectively. All the variables of the original TS retained an independent prognostic role in our series. The TS in the rituximab era (R-TS) remains predictive and clearly identifies four different risk groups (Figure 1A), finding a particularly high risk subset with a worse outcome (5y-FFS of 29%). Comparison between TS and the other indexes (IPI, NCCN-IPI or GELTAMO-IPI) showed similar CPEs for FFS in our series: 0.64 vs 0.64, 0.64 and 0.65, respectively (Figure 1B). However, TS has a better discrimination of the higher risk subgroup than IPI (5y-FFS of 47%) and NCCN-IPI (5y-FFS of 39%), and the same that GELTAMO-IPI but with a better consistency between points of the score. As R-IPI and R-TS have complementary information, another option was combining R-IPI and R-TS: patients within high risk by R-IPI (5y-FFS of 47%) when tested with R-TS can be further subdivided in three risk categories (intermediate low, intermediate high and high with 5y-FFS of 75%, 61% and 37%, respectively). To further improve the ability of finding a very high risk subset with the variables included in TS, we tested categorizing B2M normalized (normal, >1 and >3), LDH normalized (normal, >1 and >3) and AA stage (I, II and III-IV). With these changes the new enhanced TS could identify a higher risk group with a 5y-FFS of 20% and a median FFS of 7 months (Figure 1C and 1D). CONCLUSIONS 1) All variables included in the original MD. Anderson TS retain an independent prognostic role in the rituximab era, including B symptoms, B2M and bulky mass; 2) TS remains predictive of FFS and OS in the rituximab era with a similar CPE in discrimination when compared to previously reported prognostic scores; 3) TS and GELTAMO-IPI showed a better identification of patients with high risk prognosis compared to IPI or NCCN-IPI; 4) R-IPI and R-TS may be combined in order to easily improve risk classification of DLBCL patients; 5) Further categorization of LDH, B2M and AA stage increased the ability of TS to identify high risk subsets of DLBCL; 6) TS could be a backbone for introducing other new molecular or biologic tumor related prognostic factors. Figure 1. FFS using R-TS (1A), R-IPI (1B) and enhanced TS (1C). OS using enhanced TS (1D). Figure 1 Figure 1. Disclosures Gutierrez: SERVIER: Speakers Bureau; GILEAD: Honoraria; TAKEDA: Speakers Bureau; PFIZER: Consultancy; JANSSEN: Consultancy, Research Funding, Speakers Bureau; ROCHE: Research Funding, Speakers Bureau. Diaz-Lopez: TFS: Employment. Lopez-Guillermo: Roche: Consultancy, Other: Research grant; Celgene: Consultancy; Gilead: Consultancy; Janssen: Consultancy; Novartis: Consultancy. Martín: Roche: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Servier: Consultancy, Honoraria; Janssen: Honoraria; Gilead: Consultancy. Salar: Roche: Speakers Bureau; Janssen: Speakers Bureau; Servier: Speakers Bureau.

Double-hit lymphoma (DHL) is a rare subtype of B-cell lymphoma characterized by MYC and either BCL2 or BCL6 chromosomal rearrangements. DHL cases may have different morphology and are included in the updated 2016 WHO classification as a new category of high grade B-cell lymphoma with rearrangements. DHL patients usually undergo a rapidly progressing clinical course and are poorly responsive to standard chemo-immunotherapy. As a consequence, the prognosis of this entity is particularly poor with a median overall survival of approximately one year. ABT-199 (venetoclax) is a potent and selective small-molecule antagonist of BCL-2 recently approved for the treatment of some lymphoid malignancies. In this study, we demonstrate that single-agent ABT-199 efficiently displaces pro-apoptotic BAX from BCL-2 complexes but fails to maintain a significant antitumor activity over time in most MYC+/BCL2+ DHL cell lines and primary cultures, as well as in a xenograft mouse model of DHL. We further identify the accumulation of the BCL2-like protein, BFL-1, to be a major mechanism involved in acquired resistance to ABT-199. We show this phenomenon to be manageable by the BET bromodomain inhibitor CPI203, since gene expression profiling identifies BCL2A1 as one of the top apoptosis-related gene modulated by the compound. Upon CPI203 treatment, simultaneous downregulation of MYC and BFL-1 at both mRNA and protein levels further overcomes resistance to ABT-199 both in vitro and in vivo, allowing DHL cultures and tumor xenografts to undergo synergistic caspase-mediated apoptosis. Together, these findings highlight the relevance of BFL-1 in DHL-associated drug resistance and support the combined use of BCL-2 antagonist and BET inhibitor as a promising therapeutic strategy in patients with aggressive DHL. Citation Format: Anna Esteve-Arenys, Juan Garcia-Valero, David Gonzalez, Aránzazu Chamorro-Jorganes, Vanina Rodriguez, Ivan Dlouhy, Itziar Salaverria, Elias Campo, Dolors Colomer, Antonio Martinez, Grzegorz Rymkiewicz, Patricia Pérez-Galán, Armando Lopez-Guillermo, Gaël Roué. Pharmacological downregulation of BFL-1 by the BET bromodomain inhibitor CPI203 overcomes ABT-199 resistance in MYC+/BCL2+ double hit lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2161. doi:10.1158/1538-7445.AM2017-2161