Maguire, Alanna

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

Introduction: HIV infected individuals are 17x more likely to receive a diagnosis of Diffuse Large B cell Lymphoma (DLBCL) compared to their uninfected counterparts. Moreover, DLBCL is more aggressive in HIV infected individuals. However, the molecular pathology driving the aggressive nature of HIV related DLBCL is poorly understood. Previously, we demonstrated that HIV-associated [HIV(+)] germinal center B-cell (GCB) DLBCL tumors are more proliferative, with enhanced genomic stability and increased expression of DNA repair genes compared to their HIV-not associated [HIV(-)] counterparts (Maguire et al, Int J Cancer,2019). Given the immunocompromised nature of these patients, herein we assessed whether specific immunological signaling pathways are also altered in HIV(+) GCB DLBCL tumors, that may work in conjunction with, or independently of, increased DNA repair, to drive the enhanced aggressive nature of HIV related DLBCL. Methods: We used our previously studied cohort of patient samples, which included a total of 39 molecularly defined GCB DLBCL cases. Of these, 18 were HIV(+) cases from the AIDS Cancer Specimen Resource Network (https://acsr.ucsf.edu/) and 21 were HIV(-) institutional cases. Samples underwent hematopathologist review for diagnosis confirmation and tumor content assessment. Samples with <60% tumor content were macrodissected, with a total of 4x 5µm FFPE sections per sample used for RNA/DNA extraction. Digital gene expression profiling was used to assess differential expression of 579 immune related genes using the NanoString PanCancer Immune Profiling panel. The data was normalized using nSolver, and differential expression analysis was performed using the R statistical software package NanoStringDiff. The resulting expression data were then analyzed using Gene Set Enrichment Analysis (GSEA), and GSEA Molecular Signatures Database (MSigDB) overlap analyses. Results: A total of 110 genes were differentially expressed between the HIV(+) and HIV(-) cohorts at p<0.05. As expected due to immunosuppression in HIV(+) individuals, 100 genes (91%) were significantly reduced and 10 genes (9%) were significantly increased in the HIV(+) cohort compared to the HIV(-) cohort. GSEA and MSigDB analyses revealed that the genes with reduced expression in the HIV(+) cohort were associated with losses in both adaptive and innate immune signaling including cytokine interactions, the IL1 pathway and B-cell receptor signaling amongst others. MSigDB overlap analyses of the 10 genes upregulated in the HIV(+) cohort identified 3 Gene Ontology molecular functions, all of which were receptor signaling related; including Receptor Binding (CD8A, CD160, CCL8, C1QBP, MIF), Receptor Activity (CD8A, CD160, CCRL1, TFRC, KLRC2), and Cargo Receptor Activity (CCRL1, TFRC). Gene overlaps between the subsets include genes associated with NK and cytolytic T cells (CD8A, CD160), as well as the transferrin receptor TRFC/CD71, which with a fold increase of 3.9, was the most significantly increased gene in the HIV(+) cohort (p=0.002, FDR=0.0025). Conclusions: As expected, the results demonstrate a loss in both adaptive and innate immune signaling in the HIV(+) cohort compared to the HIV negative cohort as well as alterations in receptor signaling. In line with our previous observation that HIV(+) GCB DLBCL tumors have higher expression of proliferation genes and the Ki67 antigen than their HIV(-) counterparts, the results of this study also reveal overexpression of TRFC/CD71 mRNA in HIV(+) GCB DLBCL tumors compared to the HIV(-) cases. TFRC/CD71 is well known as a mediator of iron uptake in erythroid cells. Iron uptake through TFRC/CD71 is also necessary for, and positively regulates T and B cell proliferation; and is commonly expressed on aggressive B cell lymphomas. We suggest that TFRC/CD71 may play a role in the enhanced proliferative capabilities of HIV(+) GCB DLBCL. These effects may be mediated through the HIV protein Nef, which has known effects on TFRC/CD71 protein recycling in lymphoid populations (Madrid et al, J Biol Chem 2005), and has been shown to be transferred from HIV infected macrophages to uninfected B-cells via contact-dependent cellular conduits (Xu et al, Nat Immunol 2009). Nef transfer between cells has also been described to occur via microvesicle transfer and trogocytosis (reviewed in Ellwanger et al, Infect Genet Evol 2017). Further analysis of these pathways is ongoing. Disclosures Rimsza: NanoSting: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution].

Abstract Introduction: HIV infected individuals are 17x more likely to receive a diagnoses of Diffuse Large B cell Lymphoma (DLBCL) compared to their uninfected counterparts. Moreover, DLBCL is more aggressive in HIV infected individuals, with up to 70% of patients being primary refractory to chemotherapeutic regimens. However, the molecular pathology driving the aggressive nature of HIV related DLBCL is poorly understood. Here, we have assessed the genomic and transcriptional differences between HIV(+) and HIV(-) DLBCL in order to identify the mechanisms driving the enhanced aggressive and refractory nature of HIV related DLBCL. Methods: A total of 66 cases, including 27 HIV(+) from the AIDS Cancer Specimen Resource Network (https://acsr.ucsf.edu/) and 39 HIV(-) institutional cases were included in this study. Fresh H&Es were reviewed by a hematopathologist to validate diagnosis and determine tumor content. Samples with <60% tumor content were macro-dissected. A total of 4x 5µm FFPE sections per sample were used for DNA and RNA extraction. DNA damage at the gross aberration level was assessed using array comparative genomic hybridization (aCGH). NanoString digital gene expression profiling (GEP) was used to perform the Lymph2Cx DLBCL Cell of Origin molecular subtyping assay (Germinal Center B-cell/ GCB, Activated B-Cell/ ABC, Unclassifiable/UNC) and to assess the differential expression of genes with known roles in cancer using the PanCancer Pathways panel. Results: Both the HIV(+) and HIV(-) cohorts were found to be GCB enriched with GCB-ABC-UNC distributions of 70%(19/27)-11%(3/27)-19%(5/27) and 54%(21/39)-33%(13/39)-13%(5/39) respectively. For both statistical and biological reasons, analysis was restricted to the GCB-DLBCL subtype only. Array CGH revealed the HIV(+) tumors have less DNA aberrations than their HIV(-) counterparts, indicative of enhanced genomic stability. GEP GSEA analysis revealed significant differences in the HIV(+) cohort compared to the HIV(-) cohort; including increases in DNA replication (MCM2, MCM4, MCM7) and Cell Cycle progression (CDC25A, CCNA2, CCNB1, E2F1) related genes as well as decreases in cell cycle negative regulators (CDKN1A, CDKN1B, CDKN2B, RB1), pro-apoptotic BCL2 genes (BAX, BIM, BMF, PUMA, BNIP3) and pro-survival BCL2 genes (BCL2, BCLW). Analyses also revealed significant increases in DNA repair related genes, particularly those with roles in the Fanconi Anemia-Homologous Recombination-Translesion Synthesis axis (FANCA, FANCD1/BRCA2, FANCE, FANCG, FANCR/RAD51, FANCS/BRCA1, FANCT, FANCV/MAD2L2). Conclusions: Our results show, for the first time, that GCB DLBCL arising in HIV infected individuals possesses a distinct molecular pathology to that which arises in uninfected individuals. These results indicate that the aggressive nature of HIV(+) GCB DLBCL is mediated by increased proliferation in conjunction with reduced cell cycle inhibitory capabilities and potentiated by heightened DNA repair that promotes genomic stability. Moreover, it is probable that the bolstered DNA repair capabilities confer an innate ability to repair DNA damage resulting from the administration of genotoxic chemotherapeutic agents and may be the mechanism underlying the high primary refractory rate to chemotherapeutics in HIV related DLBCL. Disclosures Rimsza: NanoString: Other: Inventor on the patent for the Lymph2Cx assay.

Abstract Introduction: Primary central nervous system lymphoma (PCNSL) is a rare intracranial lymphoma that accounts for less than 1% of all non-Hodgkins lymphomas and 3% of all brain tumors. Histopathologically, approximately 90% of PCNSL cases are categorized as a diffuse large B-Cell lymphoma (DLBCL). DLBCL malignancies are subdivided by Cell of Origin (COO), with the vast majority of PCNSL categorized as non-germinal center B cell (non-GCB) by immunohistochemistry. Gene expression profiling (GEP), however, has shown that immunohistochemically defined non-GCB resolves into two distinct subtypes, namely activated B-cell (ABC) and unclassified (UNC) subtypes. Using the Lymph2Cx molecular COO subtyping assay, we have found that 91% of PCNSL are ABC (unpublished data). Unlike systemic-DLBCL, PCNSL is largely confined to and rarely metastasizes outside of the immune privileged central nervous system. Despite this, PCNSL is one of the most aggressive forms of DLBCL. Given the immune privileged milieu in which PCNSL arises, we hypothesized that this milieu elicits a transcriptional profile that contributes to the enhanced aggressive nature of PCNSL compared to systemic-DLBCL. To investigate this hypothesis, this study assessed the gene expression differences between ABC-PCNSL and ABC-systemic-DLBCL, in order to identify novel players in the pathogenesis of ABC-PCNSL. Methods: A total of 35 HIV negative samples, with proven ABC-subtype COO as per the GEP Lymph2Cx assay, were employed; including 10 ABC systemic-DLBCL and 25 primary ABC PCNSL cases with no concurrent or prior history of systemic DLBCL. Samples were reviewed by a hematopathologist to confirm diagnoses and determine tumor content. Samples with <60% tumor content were macro-dissected before nucleic acid extraction, which was performed using the Qiagen AllPrep DNA/RNA FFPE Kit. Extracted DNA and RNA were quantified using the Qubit HS-kit and NanoDrop respectively. Digital gene expression technology was used to perform the PanCancer Pathways panel (NanoString, Seattle, WA). Differential gene expression analysis was performed using the NanoString specific statistical method NanoStringDiff. Identified gene sets were analyzed using the online Gene Set Enrichment Analysis (GSEA) Molecular Signatures Database (MSigDB). Results: Of the 739 cancer related genes targeted by the PanCancer panel, 256 were found to be significantly differentially expressed in the ABC-PCNSL cohort compared to the ABC-DLBCL cohort (p<0.05). Fifty six genes were upregulated and 200 were downregulated. With a 4.9 fold change, the most significantly overexpressed gene was FGF1 (p=4.7E-11). FGF1 encodes a primary ligand for the fibroblast growth factor receptors (FGFR) -1, -2, -3 and -4; of which, FGFR2 (p=1.0E-7) and FGFR3 (p=0.003) were also significantly overexpressed. Moreover, MSigDB identified the FGF signaling pathway as enriched in the upregulated gene set (5 genes, p=7.4E-9, FDR=6.6E-7). FGFRs are a family of receptors that activate known mitogenic signaling pathways including MAPK signaling, which MSigDB identified as the most enriched pathway in the upregulated gene set (14 genes, p=1.65E-19, FDR=2.2E-16). MSigDB analysis of the 200 down regulated genes revealed that 5 of the top 20 enriched signaling pathways were immune related and included Signaling by interleukins (26 genes, p=2.9E-38, FDR=3.2E-36), Immune cytokine signaling (31 genes, p=1.1 E-34, FDR=1.1E-32), chemokine signaling (28 genes, p=1.5E-34, FDR=1.4E-32), T-cell receptor signaling (24 genes, p=2.4E-34, FDR=1.9E-32) and Toll-like receptor signaling (23 genes, p=4.1E-33, FDR=3.0E-31). Conclusions: We show, for the first time, that ABC-PCNSL and ABC-systemic-DLBCL possess significantly different transcriptional profiles despite identical, molecularly determined, COO status. A principle difference between these DLBCL malignancies is their anatomical location related immune privilege status which is reflected as reduced immune related signaling in the CNS-DLBCL cohort and may have important mitogenic signaling implications. Indeed, the results suggest that the enhanced aggressive nature of PCNSL compared to systemic-DLBCL is mediated, at least in part, by enhanced FGF signaling; a pathway with known roles in cell survival and proliferation. Disclosures Rimsza: NanoString: Other: Inventor on the patent for the Lymph2Cx assay.

Abstract Objectives: Primary central nervous system lymphoma (PCNSL) is a rare aggressive B-cell lymphoma, particularly in HIV-negative individuals, that represents a clinical challenge due to its location and lack of comprehensive molecular and biologic description. Histopathologic features are that of diffuse large B-cell lymphoma with expression of pan-B-cell markers as well as cell of origin (COO) germinal center B cell (GCB) and post-germinal center B cell (non-GCB) markers. Previous studies using immunohistochemistry (IHC) suggest that the majority of PCNSL cases are non-GCB. However, gene expression profiling has revealed non-GCB to be comprised of two distinct subtypes, namely activated B-cell (ABC) and unclassified (UNC) subtypes that are indistinguishable by IHC. To date COO testing using the highly accurate Lymph2Cx gene expression profiling assay has not been reported in PCNSL. Methods: IRB approval was obtained and HIV negative patients diagnosed with PCNSL, who had given informed consent and for whom archived tumor tissue was available for testing were identified. COO testing was performed using the Lymph2Cx NanoString assay on RNA extracted from formalin-fixed, paraffin embed tissues using our established laboratory protocols. Clinical data including patient demographics, lines of treatment and survival outcomes were collected and correlated with each other and Lymph2Cx COO results. Results: Thirty-two HIV-negative patients diagnosed between January 2005 and June 2015 were included. Median age was 61 years (30-82) and 53% were male. Radiographic information was available for 18/32 patients. Eleven (61%) had a single brain lesion at diagnosis, while 7 (39%) had >1 brain lesion. Lines of systemic therapy were 1 (91%) and 2 (9%). All patients received methotrexate-based induction therapy (44% received methotrexate, rituximab and temozolomide, 16% received single-agent methotrexate, 31% received methotrexate and rituximab, and 9% received the modified Bonn regimen; methotrexate and cytarabine-based induction). A total of 10 patients (31%) received high-dose chemotherapy and autologous stem cell transplant (ASCT) for consolidation. Of the 10 patients that underwent consolidation therapy, 9 underwent ASCT after first line induction, and 1 underwent ASCT after second line therapy. None of the patients received whole-brain radiation therapy. At a median follow-up of 29 months (range of 2-107) median event-free survival (EFS) was 16.3 months (95% CI, 8.8-45.7), and median overall survival (OS) was 41.2 months (95% CI, 28.5-NE). COO testing using the Lymph2Cx assay revealed that 91% (29/32) were ABC, 9% (3/32) were GCB, and none were UNC. Histopathology reports described COO using the Hans algorithm in 11 of the 32 cases. Of the 3 determined to be GCB on Lymph2Cx, 1 was denoted GCB by the Hans algorithm and 2 were not stained to determine COO. Of the 29 determined to be ABC by Lymph2Cx, 9 were denoted non-GCB and 1 was denoted GCB by the Hans algorithm, and 19 were not stained to determine COO. Conclusions: This series of HIV-negative patients with PCNSL showed median survival consistent with previous studies. In this first series using the Lymph2Cx assay, we confirmed that over 90% of PCNSLs are of ABC subtype, which concurs with previous reports that PCNSL tumors are predominately non-GCB by the Hans algorithm. These findings provide biological rationale forthat pharmacologic interventions targeting B-cell receptor signaling to be explored in clinical trials in the majority of patients with PCNSL. Figure. Figure. Disclosures Rimsza: NanoString: Other: Inventor on the patent for the Lymph2Cx assay.

Diffuse large B‐cell lymphoma (DLBCL) is up to 17‐fold more likely to occur, follows a more aggressive clinical course and frequently presents at advanced stages in HIV infected (+) individuals compared to HIV negative (−) individuals. However, the molecular pathology underpinning the clinical features of DLBCL in HIV(+) patients relative to the general population is poorly understood. We performed a retrospective study examining the transcriptional, genomic and protein expression differences between HIV(+) and HIV(−) germinal center B‐cell (GCB) DLBCL cases using digital gene expression analysis, array comparative genomic hybridization (CGH) and immunohistochemistry (IHC). Genes associated with cell cycle progression ( CCNA2 , CCNB1 , CDC25A , E2F1 ), DNA replication ( MCM2 , MCM4 , MCM7 ) and DNA damage repair, including eight Fanconi anemia genes ( FANCA , FANCD1/BRCA2 , FANCE , FANCG , FANCR/RAD51 , FANCS/BRCA1 , FANCT/UBE2T , FANCV/MAD2L2 ), were significantly increased in HIV(+) GCB‐DLBCL tumors compared to HIV(−) tumors. In contrast, genes associated with cell cycle inhibition ( CDKN1A , CDKN1B ) as well as apoptosis regulating BCL2 family members ( BCL2 , BAX , BIM , BMF , PUMA ) were significantly decreased in the HIV(+) cohort. BCL2 IHC confirmed this expression. Array CGH data revealed that HIV(+) GCB‐DLBCL tumors have fewer copy number variations than their HIV(−) counterparts, indicating enhanced genomic stability. Together, the results show that HIV(+) GCB‐DLBCL is a distinct molecular malignancy from HIV(−) GCB‐DLBCL; with an increased proliferative capacity, confirmed by Ki67 IHC staining, and enhanced genomic stability, the latter of which is likely related to the enhanced expression of DNA repair genes.

Tissue microarrays (TMAs) are important tools to conserve precious tissue resources from increasingly smaller biopsies and to control experimental costs and variation across sample sets. The quality assurance assessment of TMA materials created at centralized biobanks has not been standardized. Herein, we outline 2 processes for the construction of TMAs (“recipient block” and “tape” methods) and the associated preconstruction quality control measures (pathology review, protein and RNA assessment, map creation, and storage conditions) developed by the AIDS Cancer Specimen Resource (ACSR) Network’s Science and Technology Core. These steps provide a suggested framework for quality assessment that allows end-users, receiving materials from tissue banks, confidence in their experimental results.