ALBERT

Description: Abstract 731 Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired disorder of hemopoietic stem cells (HSCs). Affected individuals experience intravascular hemolysis and have a predisposition to thromboembolism, renal impairment and pulmonary hypertension. These symptoms vary in severity, but in general, the higher the proportion of PNH blood cells produced, the more severe the symptoms. The molecular pathogenesis in PNH is known to relate to a single gene mutation in the X-linked phosphatidylinositol glycan class A gene (PIG-A) which causes a complete or partial deficiency of glycophosphatidylinositol (GPI) anchored proteins leading to the symptoms of the disease. The recent development of eculizumab therapy in PNH has had a dramatic impact in reducing both morbidity and mortality in the disease but PNH remains incurable. A sub-optimal response to eculizumab, certainly when assessed by transfusion requirements, is often due to the underlying bone marrow failure that is considered to be universally present in PNH. The factors that lead from the development of a mutant clone to clonal expansion and symptomatic disease are poorly understood but are the key to improving responses and potentially to move towards a cure. Bone marrow failure appears to provide the environment necessary for expansion of PNH clones and small PNH clones are often detected in aplastic anemia and less commonly in myelodysplasia. There is no evidence that PNH HSCs have an intrinsic proliferative advantage compared to normal HSCs and an immune-mediated extrinsic suppression of normal hematopoiesis with a selective advantage for the PNH cells over residual normal stem cells is likely to explain the preferential development of PNH clones concurrent with bone marrow failure. To gain a better understanding of clonal expansion in PNH we have developed an in vitro PNH bone marrow culture model using a stromal cell line which allows PNH stem cells to be maintained in long term cultures and their capacity to form progenitor cells in myeloid colony forming assays to be assessed. We have evaluated bone marrow from 11 patients with PNH (median age 47 years, median granulocyte clone size 95.3%) and 10 normal controls (median age 42 years) within these long term bone marrow culture experiments. Unmanipulated bone marrow mononuclear cells (MNCs), CD34 selected cells and MNCs with their T-cell component depleted were used in this model. This in vitro model provides the environment for PNH stem cells to be maintained for up to eight weeks and, unlike previous studies, produce progenitor cells. When the patient's MNC's are used to seed the culture system there is poor growth of the culture which is only maintained for a median of 2 weeks. However if CD34 selected cells are used then the cultures are maintained for up to 8 weeks (similar to the normal controls) suggesting that there is a component within the MNC's that is responsible for suppressing the marrow culture which is removed by CD34 select. We next selectively removed the T-cells from the PNH MNC's and demonstrated that the marrow cultures now survived to the extent of the controls (see Figure). This demonstrates that the immune suppression in PNH resides in the T-cells. The progenitor cells produced in both the CD34 selected or the T-cell depleted MNCs over the course of the long term culture experiments show an increase in the proportion of normal progenitors the longer the cultures are maintained. This supports the hypothesis that PNH stem cells have no intrinsic proliferative advantage over normal HSCs and that T-cells are the critical cell suppressing the normal hematopoiesis in PNH. We are now examining the specific cell type that causes the myelosuppression in PNH and which will facilitate a targeted approach to the treatment of bone marrow failure in PNH and related disorders. In conclusion we have developed an in vitro PNH bone marrow culture model that allows the immune insult in PNH to be evaluated. Furthermore, this work confirms the important role that T-cells play in the etiology of PNH and provides a model with which to define the exact mechanism of suppression of hematopoiesis in PNH (and aplastic anemia). This information is essential to develop targeted therapies for the marrow failure seen in PNH and aplastic anemia. Disclosures: Kelly: Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Richards:Alexion Pharmaceuticals: Honoraria, Speakers Bureau. Arnold:Alexion Pharmaceuticals: Honoraria. Hill:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hillmen:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: Based on the profile of genetic alterations occurring in tumor samples from selected diffuse large B-cell lymphoma (DLBCL) patients, 2 recent whole-exome sequencing studies proposed partially overlapping classification systems. Using clustering techniques applied to targeted sequencing data derived from a large unselected population-based patient cohort with full clinical follow-up (n = 928), we investigated whether molecular subtypes can be robustly identified using methods potentially applicable in routine clinical practice. DNA extracted from DLBCL tumors diagnosed in patients residing in a catchment population of ∼4 million (14 centers) were sequenced with a targeted 293-gene hematological-malignancy panel. Bernoulli mixture-model clustering was applied and the resulting subtypes analyzed in relation to their clinical characteristics and outcomes. Five molecular subtypes were resolved, termed MYD88, BCL2, SOCS1/SGK1, TET2/SGK1, and NOTCH2, along with an unclassified group. The subtypes characterized by genetic alterations of BCL2, NOTCH2, and MYD88 recapitulated recent studies showing good, intermediate, and poor prognosis, respectively. The SOCS1/SGK1 subtype showed biological overlap with primary mediastinal B-cell lymphoma and conferred excellent prognosis. Although not identified as a distinct cluster, NOTCH1 mutation was associated with poor prognosis. The impact of TP53 mutation varied with genomic subtypes, conferring no effect in the NOTCH2 subtype and poor prognosis in the MYD88 subtype. Our findings confirm the existence of molecular subtypes of DLBCL, providing evidence that genomic tests have prognostic significance in non-selected DLBCL patients. The identification of both good and poor risk subtypes in patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) clearly show the clinical value of the approach, confirming the need for a consensus classification.

Description: Abstract 2187 Plasma cells, the terminal effectors of the B-cell lineage include both short- and long-lived cells. The latter persist for extended periods in the absence of cell division, supported in niche environments. No model system has successfully recapitulated the function of the niche to allow the in vitro generation of long-lived plasma cells. This limits investigation into the factors controlling and targeting plasma cell populations. Here we describe the generation of mature human plasma cells with extended lifespan from peripheral blood B-cells. Cell division accompanies phenotypic maturation between plasmablasts and plasma cells. These cells then persist in the absence of cell division, remaining functional and viable in extended culture, currently limited solely by elective termination. Extended survival is accompanied by maturation to a phenotype consistent with human bone marrow plasma cells. By establishing a set of conditions sufficient to allow the development and persistence of mature human plasma cells in vitro, we recapitulate the essential function of the plasma cell niche. We definitively link phenotypic maturation to lifespan and provide the first platform with which to explore and manipulate the full trajectory of human plasma cell differentiation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2484 Diffuse large B cell lymphoma (DLBCL) is a heterogenous disease, which has been subclassified into germinal centre (GCB) and activated B-cell (ABC) type using gene expression profiling. This has been shown to separate DLBCL into distinct prognostic sub-groups in patients treated with either CHOP or CHOP-R therapy. A number of published immunohistochemistry algorithms have attempted to replicate this subclassification using a limited number of markers, however, despite being widely adopted, the reproducibility of the algorithms has proven difficult, possibly due to the subjective nature of interpreting immunohistochemistry results. The aim of this study was therefore to evaluate the most widely accepted immunohistochemistry algorithms, and validate the results using RQ-PCR on RNA extracted from paraffin sections in a large series of well characterised formalin fixed paraffin embedded (FFPE) biopsies of R-CHOP treated DLBCLs. RNA was extracted using the Ambion Recoverall extraction kit. Applied Biosytems Taqman probes were used to evaluate gene expression of CD10, BCL6, GCET1, FOXP1 and IRF4. RQ-PCR was run on an Applied Biosystems 7500Fast cycler, and results were calculated using the deltadeltaCt method, using PGK1 as the reference housekeeper gene and either RAJI cell line or commercial RNA as the standard. Using the Hans criteria, 130/277 (47%) presentation DLBCL biopsies were classified as GCB and 147/277 (53%) were ABC. Further classification of a subset of cases using the Choi algorithm showed concordant results in 48/61 (78.7%) cases, with 1 (1.6%) case classified as GCB using Hans and ABC using Choi, and 12 (19.7%) cases classified as ABC using Hans and GCB using Choi. RQ-PCR data showed excellent correlation with immunohistochemistry for all genes incorporated into the algorithms (CD10, p

Description: Germinal Center (GCB) and activated (ABC) B-cell subtypes of DLBCL can be identified by Gene expression profiling (GEP). These subgroups are biologically distinct, harboring mutations in different pathways. Patients classified as ABC more often have mutations of the NF-kB pathway and an inferior response to standard R-CHOP therapy. The REMoDL-B trial utilised GEP to stratify patients for the addition of bortezomib to R-CHOP, based on the hypothesis that this agent may selectively improve the outcome of the ABC subtype. GEP was performed on RNA extracted from diagnostic formalin-fixed paraffin-embedded (FFPE) biopsies using Illumina WG-DASLTM during the 1st cycle of R-CHOP. Patients were classified as GCB, ABC or Unclassified before cycle 2 using the cross-platform DAC classifier (Care et al, PLOS ONE 2013) and randomised to continue R-CHOP+/-bortezomib. Work is underway to compare data generated on Affymetrix arrays and targeted RNA-seq (Illumina TRex), as well as validation by targeted mutational analysis of 18 genes associated with DLBCL (TNFAIP3, CARD11, CD79A, CD79B, MYD88, TRAF3, TNFRSF11A, PRDM1, TP53, FAS, B2M, CD58, EZH2, MLL2, MEF2B, EP300, CREBBP, KDM2B) using Fluidigm multiplex PCR and Illumina MiSeq on DNA also from the FFPE blocks. The trial closed to recruitment in May 2015 and 1147 samples have been analysed. One hundred and fifty three (13%) biopsies were unsuitable for GEP (for insufficient tumor tissue, inappropriate block sent). The remaining samples were classified as ABC (n=261, 23%), GCB (n=471, 44%) and Unclassified (n=214, 19%), with only 11 samples (1%) failing to yield a GEP result. GEP was successful in a range of sample types, including needle and endoscopic biopsies, bone marrow trephines and formal biopsies, with results obtained from as little as 40ng of total RNA, all from FFPE samples. Mutational data were available in 199 samples, with 73% of these having a mutation detectable in 1 or more genes (range 0-5) at a AAF (alternative allele frequency) cutoff at 10%. MYD88 was most commonly mutated (in 30% of ABC and 7% of GCB). EZH2 mutations were restricted to the GCB category (26%) and MYD88, CD79a/b and PRDM1 were more commonly associated with the ABC group. MYD88/PRDM1 were the most frequently associated events, with MYD88/CD79a/b and MYD88/NF-kB being mutually exclusive. Where MYD88 was seen in GCB cases, coexisting mutations imply an origin from transformed follicular lymphoma. B2M mutations were commonly identified across all subtypes (n=26), but specifically enriched in Type III (unclassified) cases (25%), which supports the hypothesis that mutational immune escape may be a feature of DLBCL, in common with other tumor types. Cross platform validation is highly concordant using Affymetrix arrays from a pilot series (27/27 gave the same classifier output, with correlating confidences also seen between platforms). RNA-seq analysis is ongoing, however initial analysis shows 86% concordance with the DASL output. Comprehensive cross platform comparison data will be available for presentation at the meeting. This study demonstrates the feasibility of GEP classification of DLBCL at diagnosis in a large international trial. The molecular classification can also be replicated using different technologies. Mutational analysis confirmed the association between DLBCL subtype and specific mutational hotspots. Disclosures Sharon: Johnson & Johnson: Other: Funded the laboratory work for the REMoDL-B trial (ISRCTN 51837425). Davies:Takeda: Honoraria; Seattle Genetics: Research Funding. Jack:Jannsen: Research Funding. Johnson:Johnson & Johnson: Other: Funded the laboratory work for the REMoDL-B trial (ISRCTN 51837425).

Description: Introduction: Plasmablastic lymphoma (PBL) is a rare, aggressive lymphoma of differentiated B lymphocytes. PBL sits at the boundary between Diffuse Large B Cell Lymphoma (DLBCL) and plasmablastic myeloma, and is more frequent amongst HIV positive patients. With an increasing divergence between therapy for myeloma and lymphoma and a deficit of prospective studies, the nature of PBL and outcome on current therapy merits further assessment. Methods: We conducted a retrospective analysis of consecutive patients diagnosed between 1st January 2004 and 1st July 2015 with PBL at the HMDS, a regional NHS diagnostic centre serving a population in excess of 3 million and representative of the wider UK population. Using the Haematological Malignancy Research Network (HMRN), a population based epidemiological study overlapping with the population served by HMDS, patient demographics and clinical outcomes were investigated. Results: 53 patients (18 female, 35 male) were diagnosed with PBL. The median age was 67 years (19-95 years). 56.8% were 65 years or older. Six patients were HIV positive and three patients were immunosuppressed following solid organ transplantation. 37/53 (69.8%) patients had extranodal PBL at presentation, including 16/53 (30.2%) with gastrointestinal tract disease. Patient demographics are summarised below. Table 1. Plasmablastic lymphoma Number Male (%) Median age in years (range) Extranodal disease (%) Total 53 35(66.7) 67 (19-95) 37 (69.8) HIV positive 6 4 (66.7) 47 (39-56) 4 (66.7) HIV negative 44 38 (86) 70 (19-95) 33 (75) Post solid organ transplant 3 3 (100) 60 (55-68) 1 (33.3) B cell markers (CD19, CD20, PAX-5) were negative or weakly expressed in all cases. One third demonstrated weak CD45 positivity. The neoplastic cells retained CD79a and expressed plasma cell marker CD138 but were negative for CD56. IRF4/MUM1 and BLIMP1 were expressed in all tumours examined. A high KI67 index was common, with 60.4% of cases having a KI67 index of 80% or greater. Bone marrow involvement was found in eight patients, raising the question of re-assignment to plasmablastic myeloma. Three additional patients had small monoclonal B cell populations detected by flow cytometry. Where suitable material was available, FISH for MYC was performed. MYC/IgH translocation was detected in 23%. The MYC/IgH translocation occurred as a sole abnormality in one third of positive cases and as part of a complex karyotype in15.4%. Extra copies of MYC were found in 15.4% and MYC amplification occurred in a 7.7%. LMP-1 was performed in 17/53 cases, of which three (17.6%) were positive. EBER-ISH was positive in 60% cases tested. HHV8 was detected in a single HIV positive patient. Eight patients were managed with palliative intent, with a median survival of 6 days (95% CI 1-30). Of the patients receiving at least one cycle of chemotherapy, the median was 474 days (95% CI 174-1580). Cyclophosphamide, doxorubicin, vincristine and prednisolone (CHOP) was used most frequently. The median survival in the overall group is 241 days (95% CI 141-613). The five year overall survival of the overall population was 21.4%. Conclusion: Our unselected cohort represents a large population based series of a rare and challenging disease and describes the experience in a predominantly elderly, HIV negative UK population. The male predominance, propensity for extranodal sites and association with EBV infection mirrors published reports in HIV positive patients. Survival in this population remains dismal. Further investigation is warranted to refine diagnostic criteria and explore molecular pathogenesis of PBL. Innovative therapeutic modalities guided by insights into molecular pathogenesis and exploring options across both lymphoma and myeloma therapeutic strategies are required to improve patient outcome. Given the rarity of PBL, prospective randomised studies are unlikely to be achievable. Our cohort serves as a historical control for future phase II clinical trials. Figure 1. Kaplan Meier survival curve for plasmablastic lymphoma patients Figure 1. Kaplan Meier survival curve for plasmablastic lymphoma patients Disclosures O'Connor: Celgene: Research Funding.

Description: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder of hemopoietic stem cells (HSC) that is characterized by intravascular hemolysis and venous thrombosis. The PNH HSC’s (and their progeny) have a somatic mutation of the X-linked phosphatidylinositol glycan class A gene (PIG-A) which causes a complete or partial deficiency of glycophosphatidylinositol (GPI) anchored proteins, which in turn produces the symptoms of the disease. PIG-A mutations account for all of classical acquired PNH cases thus far reported as a single acquired PIG-A mutation on the only active X-chromosome results in GPI-deficiency. A novel mutation in the promoter of the phosphatidylinositol glycan class M gene (PIG-M) has recently been reported in two unrelated families exhibiting autosomal recessive inheritance of congenital GPI-deficiency. This point mutation reduces PIG-M transcription and causes partial deficiency of GPI-anchored proteins. It is possible that this point mutation in PIG-M occurs at low frequency in the population but predisposes to mutation of the other PIG-M allele and could result in acquired PNH. To determine the prevalence of the PIG-M in classical acquired PNH the PIG-M promoter region spanning the proposed mutation was sequenced to look for heterozygotes with the C → G substitution at position −270. This mutation was not identified in 36 patients with PNH. There is convincing evidence that GPI-deficient HSC’s have no intrinsic proliferative advantage over normal HSC’s suggesting that other factors underlie the clonal expansion of PNH cells. The most likely explanation appears to be due to factors which are extrinsic to the PNH clone such as immune attack on normal HSC. Recently secondary molecular events within the PNH clone have been suggested to provide the growth advantage allowing expansion of the PNH clone. One such proposed event is a translocation affecting the HMGA2 gene, which has been described in 2 cases of PNH. HMGA2 is a member of the high motility group of proteins and acts as an architectural transcription factor. HMGA2 proteins are associated with gene activation and are mainly expressed during embryonic development. Rearrangements of HMGA2 commonly occur in benign mesenchymal tumours, but have also been identified as infrequent events in myeloid malignancies. To gain insight into the potential role of HMGA2 deregulation in the pathogenesis of PNH we evaluated the expression of HMGA2 mRNA by means of quantitative RT-PCR in peripheral blood samples of 42 PNH patients (median age: 42, range: 18–81) and ten normal controls. All PNH samples had large granulocyte clones (median size 97.81%) but showed no increase in HMGA2 mRNA. In nine of the 42 PNH patients blood samples underwent additional enrichment for CD15 positive cells to maximize the proportion of PNH granulocytes present. This again showed no aberrant expression of HMGA2 mRNA. This is the largest reported group with PNH evaluated for HMGA2 expression. Despite two case reports suggesting that the deregulation of HMGA2 may be a pathophysiological factor in occasional cases of PNH our data indicates that this mechanism accounts for the growth advantage of PNH cells in, at most, only a small minority of patients. Similarly, the PIG-M promoter mutation observed in congenital GPI-deficiency was not found in any of our patients with classical acquired PNH indicating that it has no or little role in the pathophysiology of PNH.

Description: Abstract 2485 Diffuse large B cell lymphoma (DLBCL) is a heterogenous disease, which has been subclassified into germinal centre (GCB) and activated B-cell (ABC) type using gene expression profiling. This has been shown to separate DLBCL into distinct prognostic sub-groups in patients treated with either CHOP or CHOP-R therapy. Previous studies have required the use of fresh or frozen samples for the extraction of RNA of sufficient quality to permit whole genome expression analysis. The Illumina ‘DASL' platform allows for highly reproducible gene expression data to be generated from FFPE material, which opens up large series' of retrospective data for detailed expression studies. The aim of this study was therefore to determine whether the Illumina DASL platform could yield reproducible results on formalin fixed paraffin embedded (FFPE) biopsies from a large series of archival CHOP-R treated DLBCL samples. RNA was extracted from paraffin sections using the Ambion Recoverall extraction kit, with 179/206 (87%) of cases yielding 〉200ng of RNA sufficient for DASL analysis. The DASL assay was performed according to Illumina protocols. Using stringent exclusion criteria, 157/179 (88%) cases yielding results that were considered to be of sufficiently high quality to be included in the analysis. To fully assess the reproducibility of the assay, 35 cases were analysed on 2–8 occasions across multiple experimental days. Using Pearson's correlation, with full-linkage clustering, four discrete clusters were identified (n=28, 40, 46 and 43). Of important note, 95% of the samples were seen to cluster more tightly with their repeats than with any other sample, with all duplicated samples being called in the same cluster with 100% accuracy, suggesting that the technique is highly reproducible. Univariate Kaplan-Meier survival analysis showed that the clusters identified patients with very different outcomes. Two of the clusters showed identical survival curves and therefore these clusters were merged to give 3 clusters with 2-year overall survivals (OS) of 51% (n=71), 65% (n=46) and 77% (n=40), log rank p=0.03, with a 3.7 year follow-up. This data supports the use of gene expression profiling to classify DLBCL patients into clinically relevant prognostic groups. The Illumina DASL assay allows for highly reproducible gene expression data to be produced in valuable, archival data series, and also in the context of clinical trials, where the majority of the tissue available for study is FFPE. The patients identified in this study as having a sub-optimal response to CHOP-R should be considered for alternative therapies, which should be validated in the context of a clinical trial. Disclosures: No relevant conflicts of interest to declare.