Peripheral T-cell Lymphomas Entities Research Articles

How molecular advances may improve the diagnosis and management of PTCL patients.

Peripheral T-cell lymphomas (PTCL) comprised more than 30 rare heterogeneous entities, representing 10 to 15% of adult non-Hodgkin lymphomas. Although their diagnosis is still mainly based on clinical, pathological, and phenotypic features, molecular studies have allowed for a better understanding of the oncogenic mechanisms involved and the refinement of many PTCL entities in the recently updated classifications. The prognosis remains poor for most entities (5-year overall survival < 30%), with current conventional therapies based on anthracyclin-based polychemotherapy regimen, despite many years of clinical trials. The recent use of new targeted therapies appears to be promising for relapsed/refractory patients, such as demethylating agents in T-follicular helper (TFH) PTCL. However further studies are needed to evaluate the proper combination of these drugs in the setting of front-line therapy. In this review, we will summarize the oncogenic events for the main PTCL entities and report the molecular targets that have led to the development of new therapies. We will also discuss the development of innovative high throughput technologies that aid the routine workflow for the histopathological diagnosis and management of PTCL patients.

Distinct and overlapping features of nodal peripheral T-cell lymphomas exhibiting a follicular helper T-cell phenotype: a multicenter study emphasizing the clinicopathological significance of follicular helper T-cell marker expression

Nodal peripheral T-cell lymphoma (PTCL) is a heterogeneous category including angioimmunoblastic T-cell lymphoma (AITL), PTCL of follicular helper T-cell (Tfh) phenotype (PTCL-Tfh), and PTCL, not otherwise specified (PTCL-NOS). We explored Tfh marker profiles in nodal PTCL. Nodal PTCLs (n=129) were reclassified into AITL (58%; 75/129), PTCL-Tfh (26%; 34/129), and PTCL-NOS (16%; 20/129). Histologically, clear cell clusters, high endothelial venules, follicular dendritic cell proliferation, EBV+cells, and Hodgkin-Reed-Sternberg (HRS)-like cells were more common in AITL than PTCL-Tfh (HRS-like cells, P=.005; otherwise, P<.001) and PTCL-NOS (HRS-like cells, P=.028; otherwise, P<.001). PTCL-NOS had a higher Ki-67 index than AITL (P=.001) and PTCL-Tfh (P=.002). Clinically, AITL had frequent B symptoms (versus PTCL-Tfh, P=.010), while PTCL-NOS exhibited low stage (versus AITL+PTCL-Tfh, P=.036). Positive Tfh markers were greater in AITL (3.5±1.1) than PTCL-Tfh (2.9±0.9; P=.006) and PTCL-NOS (0.5±0.5; P<.001). Tfh markers showed close correlations among them and AITL-defining histology. By clustering analysis, AITL and PTCL-NOS were relatively exclusively clustered, while PTCL-Tfh overlapped with them. Survival was not different among the PTCL entities. By Cox regression, sex and ECOG performance status (PS) independently predicted shorter progression-free survival in the whole cohort (male, P=.001, HR=2.5; PS≥2, P=.010, HR=1.9) and in 'Tfh-lymphomas' (ie, AITL+PTCL-Tfh) (male, P=.001, HR=2.6; PS≥2, P=.016, HR=2.1), while only PS predicted shorter overall survival (OS) in the whole cohort (P=.012, HR=2.7) and in 'Tfh-lymphomas' (P=.001; HR=3.2). ICOS predicted favorable OS in 'Tfh-lymphomas' (log-rank; P=.016). Despite the overlapping features, nodal PTCL entities could be characterized by Tfh markers revealing clinicopathologic implications.

Gene Expression Signatures for the Accurate Diagnosis of Peripheral T-Cell Lymphoma Entities in the Routine Clinical Practice.

Peripheral T-cell lymphoma (PTCL) includes heterogeneous clinicopathologic entities with numerous diagnostic and treatment challenges. We previously defined robust transcriptomic signatures that distinguish common PTCL entities and identified two novel biologic and prognostic PTCL-not otherwise specified subtypes (PTCL-TBX21 and PTCL-GATA3). We aimed to consolidate a gene expression-based subclassification using formalin-fixed, paraffin-embedded (FFPE) tissues to improve the accuracy and precision in PTCL diagnosis. We assembled a well-characterized PTCL training cohort (n = 105) with gene expression profiling data to derive a diagnostic signature using fresh-frozen tissue on the HG-U133plus2.0 platform (Affymetrix, Inc, Santa Clara, CA) subsequently validated using matched FFPE tissues in a digital gene expression profiling platform (nCounter, NanoString Technologies, Inc, Seattle, WA). Statistical filtering approaches were applied to refine the transcriptomic signatures and then validated in another PTCL cohort (n = 140) with rigorous pathology review and ancillary assays. In the training cohort, the refined transcriptomic classifier in FFPE tissues showed high sensitivity (> 80%), specificity (> 95%), and accuracy (> 94%) for PTCL subclassification compared with the fresh-frozen-derived diagnostic model and showed high reproducibility between three independent laboratories. In the validation cohort, the transcriptional classifier matched the pathology diagnosis rendered by three expert hematopathologists in 85% (n = 119) of the cases, showed borderline association with the molecular signatures in 6% (n = 8), and disagreed in 8% (n = 11). The classifier improved the pathology diagnosis in two cases, validated by clinical findings. Of the 11 cases with disagreements, four had a molecular classification that may provide an improvement over pathology diagnosis on the basis of overall transcriptomic and morphological features. The molecular subclassification provided a comprehensive molecular characterization of PTCL subtypes, including viral etiologic factors and translocation partners. We developed a novel transcriptomic approach for PTCL subclassification that facilitates translation into clinical practice with higher precision and uniformity than conventional pathology diagnosis.

Evaluation of the NCCN-IPI As a Prognostic Tool in PTCLs

Background: The majority of systemic peripheral T-cell lymphomas (PTCLs) have an aggressive behavior and compared to diffuse large B-cell lymphoma, survival is inferior using standard anthracycline-based treatment. Patient and disease factors impact survival, and the International Prognostic Index (IPI) is the most widely used clinical tool for prognostication for aggressive lymphomas. The IPI has shown prognostic utility in some PTCL entities such as systemic anaplastic large cell lymphoma (ALCL) and PTCL not otherwise specified (PTCL NOS), but less usefulness for others such as angioimmunoblastic T-cell lymphoma (AITL) as well as the rarer predominantly extranodal PTCL subtypes. T-cell specific prognostic indices such as the Prognostic Index for PTCL-U (PIT) have been explored but the IPI remains the most commonly used prognostic tool. The NCCN-IPI prognostic index has recently been developed for DLBCL. The NCCN-IPI builds on much the same parameters as the IPI, but with age stratified into four groups, lactate dehydrogenase (LDH) ratio using the upper limit of normal (ULN) separated into three categories (<1 ULN, 1-3 ULN, >3 ULN) and it also incorporates specific extranodal sites (CNS, gastrointestinal, lung or bone marrow involvement) but not absolute number of sites, with a maximum score of 8 points. In this study we evaluated the NCCN-IPI in comparison to the IPI and PIT in the three main nodal PTCL subgroups: ALK-neg ALCL, AITL, and PTCL NOS.Methods: A cohort of newly diagnosed adult PTCL patients treated with anthracyline-based or other curative intent chemotherapy regimens was assembled from the Swedish Lymphoma Registry (SWE), University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER), and British Columbia Cancer Agency (BCCA). Patients with complete prognostic data and ALK-neg ALCL, AITL or PTCL NOS subtypes were included. Event-free survival (EFS) was defined as time from pathologic diagnosis to progression/relapse, retreatment, or death due to any cause. Kaplan-Meier curves and Cox proportional hazards models were used to examine prognostic utility of indices; concordance was measured via Harrell's c-index.Results: In total, 603 patients were included. The majority of AITL presented with advanced stage disease (92%) and elevated LDH (75%) while ALK-neg ALCL patients less often exhibited adverse factors (advanced disease [67%] and elevated LDH [50%]) and PTCL NOS patients showed characteristics in between. At a median follow-up of 81 months (range 1-185), 449 patients (75%) had an event and 400 patients (66%) had died. Kaplan-Meier curves for EFS by NCCN-IPI are shown in figure 1. In patients with AITL (N=145), the NCCN-IPI had limited prognostic utility (EFS c-index=0.580) which was worse than the IPI (c-index=0.597) and similar to the PIT (c=0.585). The number of AITL patients with low-risk disease by NCCN-IPI was very small (N=2 [1%]). The NCCN-IPI had excellent prognostic utility in N=152 patients with ALK-neg ALCL (c-index=0.683), which was superior to the IPI and PIT (both c-index=0.665). However, a limited number of patients were classified as low risk (15%) or high risk disease (8%). The NCCN-IPI had modest prognostic utility in 306 patients with PTCL, NOS (c-index=0.629), which was inferior to the IPI (c-index=0.645) and similar to the PIT (c-index=0.631). Similar to ALK-neg ALCL, a limited number of patients were classified as low risk (12%) or high risk (14%) disease.Conclusions: The NCCN-IPI performed similarly to the IPI with the exception of ALK-neg ALCL where it appeared to be better at defining low and very high risk patients. In AITL, the NCCN-IPI had very limited prognostic ability and performed even worse than the IPI (which also had limited prognostic ability). Better risk stratification is needed in this disease. For PTCL-NOS, the NCCN-IPI performed similarly to the IPI and PIT. While regrouping of scores could potentially improve this situation, the integration of other clinically defined prognostic factors and molecular characteristics is needed for more precise prognostic models in PTCLs. [Display omitted] DisclosuresEllin:CTI: Consultancy; ROCHE: Consultancy, Research Funding. Connors:NanoString Technologies, Amgen, Bayer, BMS, Cephalon, Roche, Genentech, Janssen, Lilly, Merck, Seattle Genetics, Takeda,: Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Research Funding; Seattle Genetics: Research Funding; NanoString Technologies: Research Funding; Lilly: Research Funding; Merck: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Takeda: Research Funding; F Hoffmann-La Roche: Research Funding; Bayer Healthcare: Research Funding. Smedby:Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding. Ansell:Bristol-Myers Squibb: Research Funding; Celldex: Research Funding; Merck: Research Funding; Seattle Genetics: Research Funding; Affimed: Research Funding. Slack:Seattle Genetics: Consultancy. Cerhan:Janssen: Other: Scientific Advisory Board (REMICADELYM4001); Janssen: Other: Multiple Myeloma Registry Steering . Savage:Celgene: Consultancy; Roche: Research Funding; Seattle Genetics: Consultancy, Honoraria; Bristol-Myers Squibb: Honoraria; Merck: Honoraria.

Genome-Wide microRNA Expression Profiling in Molecular Subgroups of Peripheral T-Cell Lymphoma Identified Role of Mir-126 in T-Cell Lymphomagenesis

Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of non-Hodgkin lymphomas and approximately 30% of PTCLs are designated as not-otherwise specified (PTCL-NOS). Gene expression profiling (GEP) identified molecular classifiers for PTCL entities and identified 2 novel biological subgroups within PTCL-NOS (PTCL-GATA3 and PTCL-TBX21), associated with T-cell differentiation subsets. To further investigate molecular oncogenesis, we performed microRNA expression profiling (miR-EP) in several molecular subtypes of PTCL including angioimmunoblastic T-cell lymphoma (AITL), PTCL-GATA3 and PTCL-TBX21 using formalin fixed paraffin embedded tissues. We also performed miR-EP of normal T-cell subsets polarized to represent different differentiation stages (TFH, TH1 and TH2). We performed miR-EP on 102 PTCL cases using either quantitative real time PCR (ABI, Biosystem) or ultra-sensitive direct miRNA counting (nCounter, NanoString). Corresponding GEP (mRNA) were available for 67 PTCL cases. Normal T-cells were polarized in-vitro with different cytokine milieu and examined by flow cytometry. We observed distinct miRNA profiles, with miRNA being uniquely expressed in TFH polarized cells (miR-26a-5p, miR-17-5p, miR-30d-5p, miR-22-3p, miR-222-3p, miR-142-3p, let-7i-5p and miR-29b-3p). In contrast, the TH1 lineage was enriched for expression of miR-155-5p, miR-146a-5p, miR-1246, miR-93-5p, miR-16-5p, miR-21-5p, miR-363-3p, miR-1260a, miR-186-5p, miR-148a-3p and miR-579-3p, whereas TH2 polarized cells expressed miR-181a-5p, let-7a-5p, miR-191-5p, miR-15b-5p, let-7d-5p, let-7b-5p, miR-140-5p, miR-98-5p, miR-423-5p and miR-630. Several of these miRNA expressed in the T-cells subsets showed corresponding expression in their respective PTCL entity such as miR-142-3p, let7i-5p, miR-21-5p and miR-29b-3p with AITL, miR-146-5p, miR-155-5p and miR-16-5p in PTCL-TBX21 and miR-181a-5p, miR-630 and let7a-5p in PTCL-GATA3. We also performed the MiRNA Enrichment Analysis and Annotation (miEAA) for miRNA signatures and observed an enrichment of miRNA regulating epigenetic modifications in TFH cells (p=0.028), whereas TH1 showed an enrichment of miRNA regulating IFN-g signaling (p=0.0024), and miRNA signatures in TH2 showed negative regulation of TGF-b signaling (p=0.023). Supervised analysis (p=0.05) of the miRNA profiles identified significant association of miR-126, miR-145, and let-7c-5p with AITL, when compared to other PTCLs. Similarly, miR-92a, miR-25, miR-636, miR-210, miR-222 and miR-491-5p significantly associated with PTCL-GATA3 and miRNA 126-3p, 145-5p, miR-26a-5p and miR-34a-5p associated with PTCL-TBX21. The miEAA for tumor miRNA signatures revealed enrichment of miRNAs regulating histone methylation (h3 k4 methylation) and chemokine receptor signaling in AITL, whereas miRNA regulating T-cell receptor were enriched in PTCL-TBX21 and TP53 signaling pathway in PTCL-GATA3. We validated the expression of miR-126 in AITL by qRT-PCR and also observed its increased expression in IL21 stimulated CD4+ T-cells. Ectopic expression of miR-126 resulted in a ~3 fold increased expression in T-cell lines and led to reduced proliferation and increased apoptosis with expression of T-cell exhaustion makers PD1 and TIM3. Computational algorithmic programs identified relevant biological targets of miR-126, including p85/PIK3R2, S1PR2 and DNMT3A that were further validated in-vitro. We observed an inverse correlation of miR-126 expression with S1PR2 expression (r=-0.64). S1PR2 is a crucial G protein-coupled receptor regulating B and T-cell migration in the germinal center (GC) reaction. Migration assays demonstrated significant decreases in T to B-cell migration, when B-cells (Raji) were co-cultured with Jurkat cells with ectopic expression of miR-126. With the GC reaction holding an important role in AITL, we investigated the biological significance of miRNA-126 in the context of the AITL microenvironment. High expression of miRNA-126 significantly associated with inferior survival in AITL (p=0.008) and significant differences in tumor microenvironment signatures. We identified distinct miRNA signatures for AITL and molecular subgroups of PTCL-NOS. Furthermore, elevated expression of miR-126 may contribute to the dysregulation and the homing of TFH cells in GC reaction through S1PR2 and warrants further mechanistic investigation. Disclosures No relevant conflicts of interest to declare.

Integration of transcriptional and mutational data simplifies the stratification of peripheral T-cell lymphoma.

The histological diagnosis of peripheral T-cell lymphoma (PTCL) can represent a challenge, particularly in the case of closely related entities such as angioimmunoblastic T-lymphoma (AITL), PTCL-not otherwise specified (PTCL-NOS), and ALK-negative anaplastic large-cell lymphoma (ALCL). Although gene expression profiling and next generations sequencing have been proven to define specific features recurrently associated with distinct entities, genomic-based stratifications have not yet led to definitive diagnostic criteria and/or entered into the routine clinical practice. Herein, to improve the current molecular classification between AITL and PTCL-NOS, we analyzed the transcriptional profiles from 503 PTCLs stratified according to their molecular configuration and integrated them with genomic data of recurrently mutated genes (RHOA G17V , TET2, IDH2 R172 , and DNMT3A) in 53 cases (39 AITLs and 14 PTCL-NOSs) included in the series. Our analysis unraveled that the mutational status of RHOA G17V , TET2, and DNMT3A poorly correlated, individually, with peculiar transcriptional fingerprints. Conversely, in IDH2 R172 samples a strong transcriptional signature was identified that could act as a surrogate for mutational status. The integrated analysis of clinical, mutational, and molecular data led to a simplified 19-gene signature that retains high accuracy in differentiating the main nodal PTCL entities. The expression levels of those genes were confirmed in an independent cohort profiled by RNA-sequencing.

Molecular and Genomic Landscape of Peripheral T-Cell Lymphoma.

Peripheral T-cell lymphoma (PTCL) is an uncommon group of lymphoma covering a diverse spectrum of entities. Little was known regarding the molecular and genomic landscapes of these diseases until recently but the knowledge is still quite spotty with many rarer types of PTCL remain largely unexplored. In this chapter, the recent findings from gene expression profiling (GEP) studies, including profiling data on microRNA, where available, will be presented with emphasis on the implication on molecular diagnosis, prognostication, and the identification of new entities (PTCL-GATA3 and PTCL-TBX21) in the PTCL-NOS group. Recent studies using next-generation sequencing have unraveled the mutational landscape in a number of PTCL entities leading to a marked improvement in the understanding of their pathogenesis and biology. While many mutations are shared among PTCL entities, the frequency varies and certain mutations are quite unique to a specific entity. For example, TET2 is often mutated but this is particularly frequent (70-80%) in angioimmunoblastic T-cell lymphoma (AITL) and IDH2 R172 mutations appear to be unique for AITL. In general, chromatin modifiers and molecular components in the CD28/T-cell receptor signaling pathways are frequently mutated. The major findings will be summarized in this chapter correlating with GEP data and clinical features where appropriate. The mutational landscape of cutaneous T-cell lymphoma, specifically on mycosis fungoides and Sezary syndrome, will also be discussed.

New insights in the pathogenesis of T-cell lymphomas.

Peripheral T-cell lymphomas (PTCLs) represent diverse and aggressive malignancies, with few recent therapeutic improvements. Recent high-throughput genomic studies have revealed the complex mutational landscape of these rare diseases. These novel findings provide the grounds to a more comprehensive classification of these diseases, reflected in the 2017 WHO classification. Our review is focused on selected PTCL entities. Angioimmunoblastic T-cell lymphoma and other lymphomas derived from T follicular helper cells feature a rather homogeneous mutational landscape. These neoplasms recapitulate a multistep oncogenic process associating epigenetic deregulation, and second hit mutations affecting the T-cell receptor signaling pathway. This model inferred from comprehensive analyses of patients samples, was confirmed in mouse models. Among ALK-negative anaplastic large-cell lymphomas, translocation-associated subsets are found in both systemic and cutaneous types, and the newly described breast implant-associated type is usually indolent. Extranodal lymphomas of the innate immune system also harbor a combination of mutations affecting different classes of epigenetic modifiers, and mutation-induced activation of the Janus Kinase/signal transduction and activator of transcription pathway. Understanding of PTCL pathogenesis has substantially improved, and oncogenic events have been identified. The current challenge is to mount efficient therapeutic strategies targeting these aberrations to improve patients' outcome.

Molecular Insights Into Pathogenesis of Peripheral T Cell Lymphoma: a Review

Peripheral T cell lymphoma (PTCL) is a heterogeneous group of lymphoproliferative neoplasms, with at least 29 distinct entities described in current WHO classification. Using present diagnostic approaches, more than a third of PTCL cases cannot be classified, hence designated as PTCL-not otherwise specified (PTCL-NOS). Herein, we summarize the current genomic findings and their role in the molecular pathogenesis in different PTCL entities. Gene expression profiling (GEP) studies have identified distinct molecular signatures for accurate diagnosis and elucidated oncogenic pathways enriched in major PTCL entities. Furthermore, genomic characterization has identified recurrent somatic mutations and potential therapeutic targets. Further efforts are underway to develop genetically faithful murine models. GEP studies have identified molecular subgroups of PTCL, characterized by distinct genetic and epigenetic alterations. Understanding the molecular mechanisms of T cell lymphomagenesis using in vivo model will help to reveal novel therapeutic targets.

How I manage peripheral T-cell lymphoma, not otherwise specified and angioimmunoblastic T-cell lymphoma: current practice and a glimpse into the future.

Peripheral T-cell lymphoma (PTCL), not otherwise specified (NOS) and angioimmunoblastic T-cell lymphoma (AITL) are the most frequent of more than 20 mature PTCL entities featuring a broad spectrum of morphological, immunophenotypic, molecular and clinical characteristics. Unfortunately, recent progress in understanding the (epi)genetic background of PTCL has not been met with similar advances in treatment. Thus, CHO(E)P [cyclophosphamide, doxorubicin, vincristine, and prednisone (plus etoposide)] remains standard first-line therapy. Patients without comorbidities achieving complete or partial remission proceed to autologous stem cell transplantation. With this approach about 50% of patients survive long-term. Patients relapsing after or progressing during first-line therapy have a dismal prognosis. They receive salvage gemcitabine-therapy followed by allogeneic transplantation whenever possible. After allografting, approximately half of the patients survive long-term; any other treatment is palliative. New drugs investigated in phase II studies achieved response rates between 10% and 30%; long-term remissions are the exception to the rule. While most new drugs are not licensed and not readily available, a plethora of other innovative drugs targeting (epi-)genetic abnormalities are in early development. These, together with combinations of new and old drugs, will hopefully increase response to first-line therapy, bridge more patients to transplantation, and finally improve prognosis for all patients with PTCL.

Gene Expression Profiling Using a Reverse Transcriptase-Multiplex Ligation Dependant Probe Amplification Assay Allows for an Accurate Classification of Peripheral T-Cell Lymphoma and Highlights Novel Subgroups within the PTCL-NOS Category

IntroductionVarious 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.MethodsA 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.ResultsUnsupervised 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<10-3 for CD8, CD30, GZMB, PRF, ALK, CD56, CXCL13, ICOS and GATA3), indicating that our assay is reliable and may constitute a valuable alternative to IHC.ConclusionThis study demonstrates the applicability of a parsimonious RT-MLPA classifier for the classification of PTCLs. Its simplicity of use and its applicability on FFPE samples makes it an attractive alternative to high throughput GEP approaches and IHC. Its implementation in clinical trials, in combination with conventional pathological evaluation, may thus participate to improve the classification of PTCLs and therefore the management of PTCL patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Functional Characterization of BCL2-Family Members Identifies BH3 Mimetics As Potential Therapeutics in T-Cell Lymphomas

Peripheral T-cell lymphomas (PTCL) are a heterogeneous group of lymphoid malignancies with generally poor outcomes when treated with current regimens. The pro-survival BCL-2 family members BCL-2, BCL-xL, and MCL-1 contribute to tumor maintenance, progression, and chemoresistance across a range of cancers but their contributions in distinct subtypes of PTCL are poorly understood. Immunohistochemical analyses of PTCL specimens have revealed a distinct expression pattern of BCL-2 family members, most notably the high level expression of BCL-2 in up to 50% of certain PTCL entities (Rassidakis et al., J Pathol 2003). In fact, high BCL-2 expression has been associated with unfavorable prognosis (Ling et al., Biomed Environ Sci 2011).We amassed a collection of 21 T-cell lymphoma cell lines (representing Alk+ ALCL, Alk- ALCL, PTCL-NOS, cutaneous T-cell lymphoma (CTCL) and rare subtypes) and 7 patient-derived xenograft (PDX) models of T-cell lymphoma. The latter include models of Alk+ ALCL, Alk- ALCL, ATLL, NK-T cell lymphoma and AITL (available at http://www.PRoXe.org) (Townsend et al. Cancer Cell 2016). To assess the expression level and protein abundance of BCL2 family members, we performed RNA-Seq and immunoblotting. To functionally characterize dependence on BCL-2 family members, we utilized BH3 profiling, a technique that allows for assessment for how "primed" or close to the cell death threshold cells are by evaluating the degree of mitochondrial outer membrane permeabilization (MOMP), induced by a panel of BH3 domain peptides (Ryan and Letai, Cell Death and Differentiation 2013). Binding specificity of BH3 domain peptides allows for determination of which pro-survival Bcl-2 family members cells are dependent on for survival and thus makes it a powerful tool to predict response to BH3 mimetics. Finally, we assessed in vitro the cytotoxicity induced by the BH3 mimetics venetocxlax (ABT-199, a BCL-2 specific agent) and navitoclax (ABT-263, which targets both BCL-2 and BCL-xL) in PTCL cell lines.Gene expression and protein levels of the anti-apoptotic BCL-2 family members showed a distinct pattern in the cell lines that closely recapitulated immunohistochemical analysis of clinical samples (Rassidakis et al., J Pathol 2003). Specifically, both MCL-1 and BCL-xL were ubiquitously expressed, with higher levels of MCL-1 in ALCL cell lines and the PTCL-NOS cell line SMZ-1, while BCL-xL was highly expressed predominately in CTCL cell lines. While cell lines and PDX models from Alk+ ALCL and CTCL universally did not express BCL-2, approximately two-thirds of cell lines and PDX models representing Alk- ALCL, PTCL-NOS, AITL, NK/T-cell lymphoma, ATLL and rare subtypes of T-cell lymphomas did express BCL-2. Despite this expression, only 3 of 8 BCL2-expressing cell lines were sensitive to ABT-199 (IC50<1 µM), indicating that BCL2 expression is an inadequate biomarker for ABT-199 sensitivity. In contrast, BH3 profiling of these models identified either exclusive BCL-2 dependence, which correlated with sensitivity to ABT-199 in vitro, or exclusive MCL-1 dependence, which correlated with resistance to ABT-199. Alk+ and Alk- ALCL cell lines and PDX models were predominately MCL-1 dependent, but some also showed co-dependence on BCL-xL that correlated with sensitivity to ABT-263 in vitro. Among CTCL cell lines, we identified a dominant BCL-xL dependence that correlated with low nanomolar IC50 to ABT-263. In line with this, primary samples of CTCL (n=3) also showed BCL-xL dependence, offering a novel therapeutic strategy for this disease. Table 1 shows a representative illustration of these data in a selection of cell lines.In summary, we have defined distinct classes of BCL-2 family member dependence that are revealed by BH3 profiling and predict sensitivity or resistance to available clinical agents. [Display omitted] DisclosuresLetai:AbbVie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding. Weinstock:Novartis: Consultancy, Research Funding.

Molecular pathogenesis of peripheral T cell lymphoma (2): extranodal NK/T cell lymphoma, nasal type, adult T cell leukemia/lymphoma and enteropathy associated T cell lymphoma

Peripheral T-cell lymphomas (PTCL) belong to the group of non-Hodgkin lymphoma and particularly that of mature T /NK cells lymphoproliferative neoplasms. The 2008 WHO classification describes different PTCL entities with varying prevalence. With the exception of histologic subtype "ALK positive anaplastic large cell lymphoma", PTCL are characterized by a poor prognosis. The mechanisms underlying the pathogenesis of these lymphomas are not yet fully understood, but development of genomic high-throughput analysis techniques now allows to extensively identify the molecular abnormalities present in tumor cells. This review aims to summarize the current knowledge and recent advances about the molecular events occurring at the origin or during the natural history of main entities of PTCL. The first part published in the October issue was focused on the three more frequent entities, i.e. angioimmunoblastic T-cell lymphoma, peripheral T-cell lymphoma, not otherwise specified, and anaplastic large cell lymphoma. The second part presented herein will describe other subtypes less frequent and of poor prognosis : extranodal NK/T-cell lymphoma, nasal type, adult T-cell leukemia/lymphoma, and enteropathy-associated T-cell lymphoma.

Molecular pathogenesis of peripheral T-cell lymphoma (1): angioimmunoblastic T-cell lymphoma, peripheral T-cell lymphoma, not otherwise specified and anaplastic large cell lymphoma

Peripheral T-cell lymphomas (PTCL) belong to the group of non-Hodgkin lymphoma and particularly that of mature T/NK cells lymphoproliferative neoplasms. The 2008 WHO classification describes different PTCL entities with varying prevalence. With the exception of the histological subtype "ALK positive anaplastic large cell lymphoma", PTCL are characterized by a poor prognosis. The mechanisms underlying the pathogenesis of these lymphomas are not yet fully understood, but development of genomic high-throughput analysis techniques now allows to extensively identify the molecular abnormalities present in tumor cells. This review aims to summarize the current knowledge and recent advances about the molecular events occurring at the origin or during the natural history of main entities of PTCL. It will be published in two parts: the first is focused on the three more frequent entities, angioimmunoblastic T-cell lymphoma, peripheral T-cell lymphoma, not otherwise specified, and anaplastic large cell lymphoma. The second (which will appear in the november issue) will describe other subtypes less frequent and of poor prognosis: extranodal NK/T-cell lymphoma, nasal type, adult T-cell leukemia/lymphoma, and enteropathy-associated T-cell lymphoma. T or NK cell lymphoproliferative disorders with leukemic presentation, primary cutaneous T-cell lymphoma and very rare subtypes of PTCL whose prevalence is less than 5% (hepatosplenic T-cell lymphoma and subcutaneous panniculitis-like T cell lymphoma) will not be discussed herein.

Abstract IA02: Principles of lymphoma classification.

Abstract IA02: Principles of lymphoma classification.

Immunohistochemistry as a valuable tool to assess CD30 expression in peripheral T-cell lymphomas: high correlation with mRNA levels

AbstractThe extended use of brentuximab-vedotin was reported for CD30+ nonanaplastic peripheral T-cell lymphomas (PTCLs) with promising efficacy. CD30 status assessment is thus a critical factor for therapeutic decision, but the reliability of immunohistochemistry (IHC) in evaluating its expression remains to be defined. This prompted us to investigate the correlation between semiquantitative CD30 protein assessment by IHC and messenger RNA (mRNA) assessment by microarrays in a cohort of 376 noncutaneous PTCLs representative of the main entities. By IHC, CD30 expression was heterogeneous across and within entities and significantly associated with large tumor cell size. In addition to 100% anaplastic large-cell lymphomas, 57% of other PTCL entities were CD30-positive at a 5% threshold. CD30 protein expression was highly correlated to mRNA levels. mRNA levels were bimodal, separating high from low CD30-expressing PTCL cases. We conclude that IHC is a valuable tool in clinical practice to assess CD30 expression in PTCLs.

Evaluation of clinical trial eligibility and prognostic indices in a population-based cohort of systemic peripheral T-cell lymphomas from the Danish Lymphoma Registry.

Clinical trials (CTs) are needed to improve the outcome for peripheral T-cell lymphomas (PTCL), and accrual into CTs is one of the main recommendations in international treatment guidelines. The use of risk-adapted strategies has been suggested as a way to optimize treatment outcome in PTCL. The aim of the present study was to evaluate CT eligibility and selected prognostic indices in a population-based PTCL cohort of 481 PTCL patients identified from the Danish Lymphoma Registry in the period 2000-2010. According to five predefined parameters (age, performance status, P-creatinine, P-ALAT and measurable tumour lesion), patients were subdivided into four groups: 'younger fit', 'elderly fit', 'frail' and 'not CT eligible'. International prognostic index (IPI), prognostic index for T-cell lymphoma (PIT) and anaplastic lymphoma kinase (ALK) protein expression were tested at subtype-specific level. Overall, 41% of the patients were considered eligible for interventional CTs implicating curatively intended multiagent chemotherapy, including, if considered appropriate, consolidating stem cell transplantation (SCT), as part of the upfront management strategy. Moreover, 28% was elderly fit and eligible for interventional CT, including those with SCT as part of the trial design. Approximately 7% were defined as 'too frail' for aggressive treatment schedules, whereas 24% were deemed not to be eligible for any CT. Both overall and progression-free survivals were effectively predicted by IPI and PIT (p < 0.001). ALK-positive anaplastic large cell lymphoma patients were significantly younger (median age 40 vs. 62, p < 0.001) and had a better outcome than their ALK-negative counterparts (p < 0.001). However, ALK expression lost its prognostic significance when adjusting for age. In a population-based cohort of adult Caucasian PTCL patients, approximately half were eligible for multiagent chemotherapy with or without consolidating SCT. Both IPI and PIT are useful prognostic indices in all 'primary nodal' PTCL entities. The prognostic value of ALK protein expression in anaplastic large cell lymphoma is significantly downsized when adjusting for age.

Pathology of Peripheral T-Cell Lymphomas: Where Do We Stand?

Peripheral T-cell lymphomas (PTCLs) are heterogeneous and uncommon malignancies characterized by a usually aggressive clinical course. The current World Health Organization (WHO) classification delineates many entities grouped according to the clinical presentation as predominantly leukemic, cutaneous, extranodal, or nodal diseases. Yet, few genetic lesions serve as entity-defining markers. Using high-throughput methods, new recurrent genetic and molecular alterations are being discovered that are expected to refine the current classification and serve as diagnostic genetic markers and targets for novel therapies. There is increasing evidence that certain cellular subsets, in particular follicular helper T cells and gamma delta T cells, represent important defining markers and/or determinants of the biology of certain entities; nevertheless, the cellular derivation of many PTCL entities remains poorly characterized and there is evidence of plasticity in terms of cellular derivation (alpha-beta, gamma-delta, natural killer [NK]) especially in several extranodal entities with a cytotoxic profile. While most clonal NK/T-cell proliferations are in general highly malignant, some more indolent forms of NK or T-cell lympho-proliferations are being identified.

Small Nucleolar RNA Expression Profiling Identifies Potential Prognostic Markers in Peripheral T-Cell Lymphoma.

AbstractAbstract 2678 Introduction:Peripheral T-cell Lymphomas (PTCL) are types of rare and heterogeneous Non-Hodgkin's Lymphoma (NHL) that, in general, are associated with a poor clinical outcome. Discovery of new prognostic tools is thus a current and major challenge. In a cohort of 122 cases of PTCL collected from a multicentric T-cell lymphoma consortium (TENOMIC), we analyzed the expression of 80 non-coding small nucleolar RNAs (snoRNA) using high-throughput quantitative PCR (Fluidigm). SnoRNAs belong to the non-coding RNA family, and participate to diverse biological processes, most importantly ribosomal RNA maturation. Patients and methods:PTCL samples (n=122, 32 ALCL (22 ALK+, 10 ALK-) and 90 non-ALCL cases) were collected from TENOMIC. For each case, a consensus diagnosis was made by a panel of expert hemopathologists (some of the patients participated in a GELA-sponsored clinical trial, the others benefited from a national review protocol for T-cell lymphomas). Total RNA from sorted healthy donor-derived T-lymphocytes (n=35) and tumor samples were extracted (Trizol), integrity was verified using Agilent NanoChip, and reverse transcribed and assessed for snoRNA expression levels using The Fluidigm high-throughput quantitative PCR method. Medical cases were used to calculate progression-free and overall survival (PFS and OS) in 78 non-ALCL patients (26 PTCL-NOS, 46 AITL, 6 other rare entities) who received CHOP or CHOP-like regimen first-line. Results:First, we used unsupervised hierarchical clustering to show that, irrespectively from cell of the origin of the T cells, PTCL cells had a significant down-regulation of snoRNA expression in 63% of the snoRNA tested. In particular, there was a clear delineation between AITL, a tumor of T-follicular Helper (TFH) origin, and normal TFH cells from healthy donors. Among PTCL entities, ALCL had a specific snoRNA profile, but unsupervised clustering was not able to distinguish ALK- from ALK+ patients. However, a supervised comparison identified snoRNA U3 as a discriminant marker that sorted ALK+ from ALK- ALCL samples. Unsupervised or supervised snoRNA clustering failed to distinguish AITL from the other subgroups of PTCL.Second, we assessed prognostic impact of snoRNA expression in 78 non-ALCL patients. Characteristics of the cohorts were as follows: median age 65y/74y, Ann-Arbor stage III-IV in 94%/88%, elevated LDH in 76.7%/73.1%, IPI score 3–5 in 70%/61%, respectively. Although the snoRNA expression profiles of AITL and other PTCL subtypes appeared very similar, unsupervised clustering revealed the over-expression of 8 snoRNA in a subgroup of 31 patients with 45% OS at 5y (vs 47 patients with 18% OS at 5y). In AITL and PTCL-NOS cases, median OS was 26mo and 13mo, respectively. These thresholds were used in a supervised analysis to try to identify a specific prognostic snoRNA signature. A three snoRNA set was found significantly over-expressed in patients with the best prognosis, both in AITL patients (HBII-239, U59B, U90 impacted both on OS and PFS), and in PTCL-NOS patients (HBII-239, HBII-438A, U80 impacted on OS). Very interestingly, these signatures were not correlated neither with IPI or IPI-T scores, nor with overall response rates after CHOP. Lastly, we investigated the prognostic impact of HBII-239 snoRNA as a single marker. HBII-239 over-expression was statistically associated with prolonged PFS and OS in the entire cohort of 78 patients.Third, we investigated the impact of HBII-239 over-expression on cellular pathways in the FEPD cell line. This snoRNA is a precursor for microRNA-768, which is processed from the 3p strand of HBII-239. The FEPD cell line had a weaker expression of miR-768-3p as compared to patients' samples. Transfection of a miR-768-3p microRNAmimic induced a significant reduction of proliferation rate (as assessed by an MTS assay), due to a block at the G1/S checkpoint. In patients with low HBII-239 level, it is therefore expected that proliferation rate of lymphomatous cells would be increased, translating into decreased OS. Conclusions:Our study showed that snoRNAs are differentially regulated in normal compared to malignant T-cell populations. Besides a global down-regulation of these molecules, specific signatures may have a prognostic significance in PTCL. The different snoRNAs that can be regarded as potential biomarkers in these tumors may play a direct role in different cellular pathways. Disclosures:No relevant conflicts of interest to declare.

Gene Expression Signatures That Delineate Biologic and Prognostic Subgroups in Peripheral T-Cell Lymphoma

AbstractAbstract 679 Background:Peripheral T-cell lymphoma (PTCL) represents approximately 10–12% of all non-Hodgkin lymphoma (NHL) in the Western world, with a higher incidence in Asian populations. The World Health Organization classification recognizes a number of distinctive subtypes of PTCL including angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), adult T-cell leukemia/lymphoma (ATLL), extranodal NK/T-cell lymphoma of nasal type (ENKTCL), and many other rare entities that present mainly as extranodal PTCL. However, with current immunophenotypic and molecular markers, about 30–50% of PTCL cases are not classifiable and are categorized as PTCL-not otherwise specified (PTCL-NOS). With the exception of ALK(+)ALCL, the PTCLs generally have a poor outcome and, thus a better understanding of the biology of these diseases is greatly needed to improve the long-term survival of these patients. Methods:In the current study, we performed gene expression profiling analysis on a large and well- characterized series of PTCL and ENKTCL cases (n=372) from the Lymphoma Leukemia Molecular Profiling Project (LLMPP), the International Peripheral T-cell Lymphoma Project (IPTCL) and other major institutions to define robust molecular classifiers, oncogenic pathways and prognosticators for the more common PTCL entities, as well as unique molecular and prognostic subgroups within PTCL-NOS. Molecular signatures for diagnosis and prognosis were generated in training data sets and validated in separate cohorts. Results:Robust molecular classifiers for AITL, two types of systemic ALCL (ALK(+) and ALK(-)), ATLL and ENKTCL were identified (Figure 1). These classifiers reflect the pathobiology of the tumor cells, as well as their microenvironment, and represent a refinement of what we reported previously (Iqbal et.al Blood, 2010; Iqbal et.al Leukemia. 2011). Importantly, ALK(-)ALCL can be differentiated from ALK(+)ALCL and PTCL-NOS with a unique gene expression signature. Approximately 14% of PTCL-NOS were re-classified as ALK(-)ALCL and showed expression of CD30 protein, TIA-1 or granzyme B by immunohistochemistry. ENKTCL can be separated molecularly into NK-cell lymphoma and gd-PTCL, the latter of which was also identified in 9% of PTCL-NOS. The remaining PTCL-NOS cases could be separated into two major subgroups related to T-cell differentiation and characterized by either high expression of GATA3 (30%) or TBX21(T-BET) (45%) and many of the corresponding target genes (Figure 2). Cases with high expression of GATA3 had poor overall survival and showed enriched Wnt and mTOR pathways, but no prominent microenvironment signature. The high TBX21 subgroup had a remarkably good outcome for patients with a high plasma cell-like gene expression signature, but poor overall survival when expressing a high cytotoxic signature (Figure 3). The molecular prognosticator for AITL largely reflected the role of the tumor microenvironment, with the presence of a high B-cell signature correlating with favorable outcome, whereas high dendritic cell/monocyte signatures were associated with inferior survival. Conclusion:We have organized the most comprehensive molecular profiling study of PTCL, and have not only refined the molecular diagnostic and prognostic signatures for the common subtypes of PTCL, but also segregated PTCL-NOS in meaningful biological and prognostic subtypes. Molecular diagnostic and prognostic signatures of PTCL frequently include components of the tumor-host interactions, highlighting the importance of the microenvironment in PTCL biology. This study provides an important framework for additional analysis to identify novel therapeutic targets to improve the outcome of patients with PTCL. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.