ALBERT

Description: The risk of developing post transplant lymphoproliferative disease (PTLD) following solid organ transplantation (SOT) in children is in large part dependent on the specific organ transplanted and the degree of immune suppression post SOT (Webber et al Lancet 2006; Dharnidharka et al Transplantation 2001; Newell et al Transplantation 1996). However, the importance of expression of CD20 and/or Epstein-Barr virus (EBV) as prognostic factors for development and survival of pediatric PTLD after SOT in young patients are poorly understood. We previously demonstrated the safety and efficacy of cyclophosphamide, prednisone and rituximab (CPR) in CD20+ PTLD (Orjuela/Cairo, CCR 2005). We now report on our experience in children, adolescents and young adults with PTLD following SOT treated at a single institution from 1990–2008 and on the prognostic significance of expression of CD20 and EBV in this population. 45 SOT pts (28 heart, 11 liver, 6 kidney) were diagnosed with PTLD (53.3 % female) at a mean onset of 45±43 months (mo) post primary SOT (4–153). Three patients had multiple SOT. Age at diagnosis of PTLD ranged from14 to 287 mo with mean 118 (SD=77) mo. EBV and CD20 status were evaluated in all evaluable tumor sites. CD20 status was categorized as positive when all tumor sites expressed CD20 (by IHC) and negative when only some or no tumor sites expressed CD20. EBV status was categorized as positive when any tumor sites were EBV positive (by ISH), and negative when no tumor sites were EBV positive. Of 40 evaluable tumors (11 monomorphic, 19 polymorphic, 5 early lesions, 2 T-cell lymphomas, and 3 rarer types (2 HD, 1 multiple myeloma like), 32 (80%) had detectable EBV, while 28 (70%) were classified as CD20 positive. EBV expression was unrelated to age at SOT. Those patients whose PTLD expressed CD20 and/or EBV had shorter time interval between last SOT and the onset of PTLD (CD20 positive vs negative (mean±SD): 28±31 mo vs 64±44 mo, p

Description: Background Post-transplant lymphoproliferative disorders (PTLDs) encompass heterogeneous entities with different histomorphology and behavior. T/NK-cell PTLDs (T/NK-PTLDs) are rare, accounting for 2-15% of all PTLD, which are often EBV-negative and associated with a poor prognosis. The pathogenesis of T/NK-PTLDs and the underlying molecular alterations are presently unknown. In order to better understand their pathogenesis, we performed high resolution DNA profiling for copy number alterations and targeted mutational analyses of 18T/NK-PTLDs. Methods Cases of T/NK-PTLD were selected from the archives of two institutions. Morphological features were assessed and immunohistochemistry (IHC) was performed to classify lymphomas according to the WHO 2008 criteria. DNA was extracted from formalin-fixed paraffin-embedded tissue. Copy number analysis (n=16; Affymetrix Oncoscan FFPE Assay) and targeted next-generation sequencing for 467 cancer-associated genes, after capture of all exons or select whole genes (n=17; Illumina HiSeq 2500), were performed. Variants with allele frequency 1% in 1000 genomes project, read depth

Description: Introduction : Post-Transplant Lymphoproliferative Disorders (PTLD) are a group of heterogeneous disorders that arise as a consequence of iatrogenic immunosuppression for solid organ or allogeneic bone marrow/stem cell transplantation. Though they all arise in a common clinical context, different types of PTLD differ with respect to their underlying biology, clinical presentation and treatment. The aims of this study were to: (1) Define the cell of origin (COO) of monomorphic Diffuse Large B-cell Lymphoma (DLBCL) PTLD and evaluate their impact on clinical presentation and survival and (2) assess the impact of different Rituximab containing treatment regimens on survival outcomes in monomorphic DLBCL PTLD patients. Methods : We conducted a retrospective review of our institutional databases to identify all the cases of monomorphic PTLD (DLBCL) diagnosed and treated at our medical center from 2000-2013. COO classification into germinal center B-cell like (GCB) and non-germinal center B-cell like (non-GCB) type was performed by immunohistochemistry using the Hans algorithm. Results : Cell of origin: 40 cases of monomorphic PTLD (DLBCL) were diagnosed during the study interval. Tissue material for COO subtyping was available for 25 patients. By immunohistochemistry 16/25 (64%) were non-GCB and 9/25 (36%) were GCB subtype, median age of presentation being 46 years (range 3-75) and 48 years (range 3-64), respectively. A trend towards EBV positivity (by in situ hybridization for EBV encoded RNA) was noted in the non-GCB group (75% vs. 33%) [p=0.09]. Non-GCB DLBCL PTLD presented earlier post-transplant at a median 1.5 years (range 0.2-15) vs. 3.9 years (range 0.7-17) for GCB cases. When comparing immunosuppressive therapy at the time of PTLD presentation, an association between Tacrolimus therapy and non-GCB phenotype was identified [p=0.03]. Non-GCB DLBCL PTLD demonstrated a trend towards higher rates of extra nodal involvement (88% vs. 44%) [p=0.06] and advanced stage disease (Stage III/IV 75% vs. 33%) [p=0.09]. No significant differences in organ transplanted, LDH, ECOG performance and IPI were observed. While acknowledging the heterogeneity of therapies administered, no significant differences in Progression Free Survival (PFS) (median PFS non-GCB = 17 months vs. GCB = 15 months [p=0.36]) and Overall Survival (OS) (median OS non-GCB = 33 months vs. GCB = 27 months [p=0.22]) were identified. Impact of Treatment: 35 adults (age≥18) were treated at our center. The four most common first line therapies administered were R-CHOP (14), R-EPOCH (7), Palliative Care (5) and Rituximab monotherapy (4). Five patients were given 4 other different therapies. In patients given Rituximab monotherapy, two patients presenting with stage I disease responded while two with stage IV disease progressed. When focusing on patients who commenced R-CHOP or R-EPOCH as their initial therapy, no significant differences in age, stage, LDH, extra nodal disease, ECOG performance status, IPI and immunosuppression therapy was identified between the groups. The complete response (CR) rate for R-CHOP was 50% vs. 71% for R-EPOCH. Primary refractory disease was present in 29% of patients receiving R-CHOP vs. 14% with R-EPOCH. Death during first line therapy occurred equally in both groups (14%). All four primary refractory disease patients in the R-CHOP arm died, while the one patient who was primary refractory to R-EPOCH is alive 3 years post autologous stem cell transplant. R-EPOCH demonstrated prolonged PFS (median PFS R-CHOP = 15 months vs. R-EPOCH not reached [p=0.049]) and prolonged OS (Figure 1) [p=0.036]. Conclusions : (1) In monomorphic PTLD (DLBCL), the non-GCB subtype predominates and is associated with the use of Tacrolimus. It commonly presents with advanced stage disease and extra nodal involvement however, no difference in PFS and OS was noted when compared to GCB DLBCL PTLD. (2) R-EPOCH demonstrated prolonged PFS and OS when compared to R-CHOP. The survival differences reflect the higher rates of primary refractory disease in the R-CHOP group and the inability to salvage patients once they become relapse/refractory. Given the retrospective nature of our analysis, further studies in a larger cohort of patients are ongoing to validate these results. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Introduction B-cell post-transplant lymphoproliferative disorders (B-PTLD) represent a spectrum of lymphoid neoplasms arising as a consequence of impaired T-cell surveillance secondary to immunosuppressive medications. Epstein-Barr Virus (EBV) has been implicated in the development of B-PTLD. B-PTLDs diagnosed within one year post-transplant are more likely to be associated with EBV whereas B-PTLD diagnosed after a year are more likely to be EBV-negative and are associated with worse prognosis. Studies have shown that EBV up-regulates CD30 expression, a member of the TNF-receptor superfamily (Haque et al 2011). Novel agents are now available that target cells expressing CD30. Hence, knowledge of the expression profiles and the clinical characteristics of CD30+ neoplasms is warranted. In this study, we evaluated CD30 expression in 59 cases of B-PTLD and correlated CD30 and EBV status with clinical characteristics. Methods Consecutive cases of B-PTLD diagnosed between 1997 and 2013 were retrieved from our archives. Immunohistochemistry (ISH) for CD30 was performed on formalin-fixed, paraffin embedded biopsies using the BerH2 monoclonal antibody (BioSB, Santa Barbara, CA) according to standard methods. Positive expression was pre-defined as CD30 expression by ≥20% of the lesional cells (Figure 1). Log-rank and Fischer's Exact 2-sided Test were performed in STATA 11, as appropriate. Results The 59 B-PTLDs occurred in 54 patients. Three patients had two different sites biopsied during the diagnostic workup. Two other patients had recurrent B-PTLD. The median age at diagnosis was 22.8 years old (range 1.2 to 75.9 years); 65% were male. The median time from transplant to diagnosis was 2.6 years (range 6 weeks to 15.9 years, mean 4.2 years). Eighteen cases (35%) were diagnosed within the first year of transplantation. The organs transplanted are detailed in Table 1 with the percentage of CD30+ cases noted. Prior to transplantation, only 25/44 (57%) patients were EBV IgG seropositive, likely reflecting the number of children who were younger than age ten when transplanted (21/52; 40%). (Orjuela et al 2011). Serum EBV Polymerase Chain Reaction (PCR) was positive in 18/27 (67%) cases analyzed. Pathological data are summarized in Table 2. In total, 39 of 59 cases (66%) showed 20% or more CD30 expression. Positive EBV ISH was significantly associated with CD30 expression (r=0.38, p〈 0.01). Of 59 tumors informative for CD30 and EBV (by ISH), 32/41 of EBV-positive tumors were CD30 positive (78%) while 7 of 18 tumors that were EBV negative were CD30 positive (39%) (r=0.38 for the association of EBV and CD30 status, p=0.01). Detectable serum viremia was associated with CD30 status (r=0.49, p=0.02). There was no association found between CD20 status and CD30 status. There was no significant association between CD30 status and ki67 index or c-myc expression either (data not shown). Conclusion We observed a high frequency of CD30 expression in patients with B-PTLD. Expression of CD30 correlates with ISH evidence of EBV infection in B-PTLD. In addition, positive serum EBV PCR was associated with CD30 expression. These two findings further support the link between EBV infection and CD30 expression. CD30 positivity was still found in 39% of EBER – negative cases, suggesting that CD30 ISH should be considered on all cases of B-PTLD. A monoclonal antibody conjugated to the cytotoxic agent MMAE,Brentuximab Vedotin (SGN-35), has shown activity in clinical trials against CD30 positive lymphomas (Jacobson et al 2012). Considering the relatively common prevalence of CD30+ expression in PTLD, brentuximab vedotin could be considered for study in combination with traditional front-line therapies or as an alternative therapy for relapsed/refractory disease. A prospective clinical trial (NCT 01805037) at Northwestern University is ongoing. Disclosures: Schecter: Seattle Genetics: Research Funding, Speakers Bureau. O'Connor:Seattle Genetics: Research Funding.

Description: To ascertain the genetic basis of pediatric Burkitt lymphoma (pBL), we performed clinical-grade next-generation sequencing of 182 cancer-related genes on 29 formalin-fixed, paraffin embedded primary pBL samples. Ninety percent of cases had at least one mutation or genetic alteration, most commonly involving MYC and TP53. EBV(−) cases were more likely than EBV(+) cases to have multiple mutations (P 〈 .0001). Alterations in tumor-related genes not previously described in BL were identified. Truncating mutations in ARID1A, a member of the SWI/SNF nucleosome remodeling complex, were seen in 17% of cases. MCL1 pathway alterations were found in 22% of cases and confirmed in an expanded panel. Other clinically relevant genomic alterations were found in 20% of cases. Our data suggest the roles of MCL1 and ARID1A in BL pathogenesis and demonstrate that comprehensive genomic profiling may identify additional treatment options in refractory disease.

Description: Background Infectious etiologies have been established for a variety of lymphomas whereby viral, bacterial or ricketssial organisms can induce or promote lymphomagenesis either indirectly through antigen stimulation or by inflicting immune dysregulation or by directly infecting lymphocytes mimicking or co-opting signaling pathways leading to cellular transformation. EBV infection represents the most well studied pathogen that can directly infect lymphocytes, and induce neoplasia via cellular immortalization. An infectious etiology for a variety of EBV negative (EBV-) lymphoproliferative neoplasms/disorders, occurring in either immunocompromised or immunocompetent individuals has long been considered, but never been established. To investigate the possibility of novel lymphotropic pathogens as causative agents of lymphomagenesis, we used next-generation, high throughput sequencing (HTS) to analyze subsets of suspect T- and B-cell lymphomas for the presence of non-human genetic material. Methods Forty-nine lymphomas, representing 5 different entities were evaluated: 9 EBV- negative post-transplant lymphoproliferative disorders, monomorphic type (EBV- PTLD), 10 EBV-negative classical Hodgkin lymphomas (cHL), 10 peripheral T-cell lymphomas (PTCL), 10 marginal zone lymphomas (MZL) and 10 chronic lymphocytic leukemia/small lymphocytic lymphomas (CLL/SLL). Frozen sections of non-Hodgkin lymphoma tumor blocks were evaluated and samples with tumor representation 〉70% were selected for analysis. EBV status was determined by using in situ hybridization (ISH) for EBV encoded small RNAs (EBER). For next-generation sequencing, RNA (0.5 µg) was extracted from frozen tumors, DNase I digested (DNA-free; Ambion, Austin, TX) and reverse transcribed using Superscript II kit (Invitrogen) with random octamer primers (MWG, Huntsville, AL). The cDNA was RNase H treated prior to random amplification by PCR. Products of 70 bp were purified (MinElute, Qiagen) and ligated to linkers for sequencing using a GS FLX sequencer (454 Life Sciences, Branford, CT). Primer sequences were removed, followed by multiple filtering steps and sequences obtained were compared with those of known infectious agents using software available at the BLAST website (www.ncbi.nlm.nih.gov/BLAST). Results Sequencing was successful in 46 cases. Microbial sequences were detected in 5 specimens (9%). In the remaining 41 cases, including all EBV- PTLDs and cHLs no non-human genetic material was identified. Human herpes virus 4 (EBV) was detected in one PTCL harboring an EBV+ B-cell lymphoma which embodied 20% of the total tumor mass in the specimen (as evaluated by ISH). EBV sequences were not detected in 4 other PTCL exhibiting EBV+ B-cells (range 1-10% involvement by ISH). These 4 cases represented angioimmunoblastic T-cell lymphomas. Human immunodeficiency virus -1 sequences (HIV) were detected in a lung MZL occurring in a known HIV+ patient. Sequences corresponding to propionebacterium sp., tetracyclin resistant streptococcus sp. and acinetobacter sp., were identified in one case each: MZL, EBV-PTLD and CLL; and were considered contaminants, likely acquired during biopsy procurement. Conclusion No novel lymphotropic microbial pathogens were identified in non-EBV associated T- and B-cell lymphoproliferations. Our findings argue against a clonal infectious etiology, which has previously been hypothesized for subsets of the lymphomas analyzed. Inability in detecting EBV sequences in samples containing low levels of EBV infected cells, suggests that this methodology might not be suitable for investigating lymphoproliferations with low tumor burden (e.g. cHL) or those arising as a consequence of chronic antigen stimulation due to a low-frequency intratumoral microbial pathogens (e.g. MZL). Further studies in a larger cohort of lymphoproliferative neoplasms will be helpful to further validate our results. Disclosures: Schecter: Seattle Genetics: Honoraria, Research Funding.

Description: Key Points We investigated all 3 subtypes of BL by WGS and transcriptome sequencing. Experimental validation through CRISPR screening and mouse models provides a better functional understanding of BL genetic drivers.

Description: Abstract 899 While the majority of children with Burkitt lymphoma (BL) are cured with conventional chemotherapy, outcome for patients with relapsed disease is poor (overall survival 1 alteration compared with 2/13 EBV(+) cases with 〉1 alteration (p

Description: Abstract 4151 Post-transplant lymphoproliferative disorder (PTLD) is a broad spectrum of lymphoproliferative disorders that can occur after solid organ transplant or hematopoietic stem cell transplant. The incidence ranges from 1% to 20% depending on the type of organ graft, the intensity of the immunosuppression and the Epstein-Barr virus (EBV) serostatus. It represents a serious and potentially life threatening complication, with reported mortality rate up to 40–50%. According to the World Health Organization classification system, PTLD are classified as early lesions (EL), polymorphic (p-PTLD), monomorphic (m-PTLD), and Hodgkin-like (HL). Here we analyzed the prognosis and clinical characteristics of 120 patients diagnosed and treated over a 19-year period (from 1990 to 2009). To the best of our knowledge, this is the largest series of PTLD to be reported by a single Institution. The cases were classified as follows: 70 (58.3%) m-PTLD, 34 (28.3%) p-PTLD, 14 (11.7%) EL, and 2 (1.7%) HL. In the m-PTLD group, 59 were of B-cell origin (52 DLBCL, 4 BL, 2 plasmacytoma-like, 1 multiple myeloma and 1 pleural effusion) and 10 were of T/NK cell lineage (4 peripheral T-cell lymphomas, 2 CTCL, 2 HSTCL, 1 T-ALL and 1 NK-cell lymphoma). The age of the patients ranged from 1 to 76 years, with 39 pediatric patients (

Description: EBV-associated B-cell Post-Transpant Lymphoproliferative Disorders (PTLDs) represent a diverse group of lesions morphologically, in clinical presentation and behaviour, ranging from early reversible lesions to monomorphic aggressive lymphomas. Polymorphic cases, which represent the focus of our analysis, contain a mixture of cells in various EBV latency stages, defined by EBNA1, EBNA2 and LMP1 immunostaining. LMP1 is a key viral protein for cellular transformation and, analogously to CD40, engages TNF Receptor Associated Proteins and activates NF-kB and NF-kB-responsive genes. We analyzed the protein signature of LMP1 in PTLDs and non-PTLD tonsils by double staining for LMP1, CD30, CD20, Pax5 and signaling molecules. A remarkably conserved set of proteins, associated with LMP1/CD40 signaling and NF-kB activation is expressed both in the EBV-infected lymphoid population in polymorphic PTLDs and in a normal B-cell subset(s) in reactive tonsils. These proteins include highly expressed CD30, JunB, nuclear cRel, TRAF-1, Bcl-XL, MUM1, CCL22 and downregulated BCL6 and CD10. We observed that EBV infection, possibly through LMP1 and LMP2A signaling, results in varioius degrees of differentiation within the neoplastic clone. EBER+ terminally differentiated mucosa-associated IRTA-1+ marginal zone B-cells and CD138+ plasma cells were identified in most cases, including control post-transplant tonsils with no overt disease. We document for the first time in situ, in-vivo evidence of EBV latently infected post-Germinal Center B cells of marginal and plasma cell types in PTLDs. Polymorphic PTLD cases represent EBV-induced expansion of B cells, mimicking CD40L-like activated Peri/Interfollicular CD30+ normal B-cells.