Ramsower, Colleen

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 mediastinal large B-cell lymphoma (PMLBCL) is recognized as a distinct lymphoid neoplasm. However, differentiation from diffuse large B-cell lymphoma (DLBCL) can be difficult due to the need for close clinicopathologic correlation and variability in morphology and immunophenotype. In addition, a recent study identified lymphomas that share molecular and morphological features with PMBCL, yet do not involve the mediastinum. Correct classification of PMBCL may impact therapeutic decision making, increasing the demand for more accurate diagnostic methods. Recent research identified a new gene expression signature that robustly differentiated PMLBCL from DLBCL in both training and independent validation cohorts using RNA isolated from routinely available formalin-fixed, paraffin-embedded (FFPE) tissues. Methods This assay, known as the “Lymph3Cx,” utilizes the nCounter platform (NanoString Technologies, Seattle, WA) and consists of probes for 58 target genes (including both discriminatory and housekeeping genes) that can distinguish PMLBCL cases from DLBCL as well as the “cell-of-origin” subtypes of DLBCL. In the current study reported here, the Molecular Diagnostics–Arizona Laboratory (MDAZL) performed a full validation of the Lymph3Cx assay for use in our clinical diagnostic laboratory. Results Assay performance included accuracy (92.9%), precision (100%), analytical sensitivity (100 ng RNA limit of detection), analytical specificity (measures are in place to prevent interfering substances), reproducibility between technologists and technical replicates (100%), specimen stability (18 months for unstained tissue sections), and RNA Isolation Kit Utility (two kits with equal performance) in order to become the first CLIA-certified, CAP-accredited clinical diagnostics laboratory to offer a molecular assay for distinction of PMLBCL from DLBCL. Conclusion The Lymph3Cx assay will be prospectively used for evaluation of Mayo Clinic patients and for use as an integrated biomarker in clinical trials.

Abstract Introduction Lymph2Cx lymphoma cell-of-origin assay (LM2CX) was developed by the Lymphoma/Leukemia Molecular Profiling Project (LLMPP) to better categorize diffuse large B-cell lymphoma (DLBCL). NanoString has licensed the assay and is currently pursuing FDA approval. In the meantime, the test is offered exclusively as a lab-developed test (LDT) by the Mayo Clinic Molecular Diagnostics–Arizona Lab (MDAZL) to Mayo Clinic Enterprise patients. In order to comply with these restrictions, the normal workflow for Mayo Clinic Rochester has been modified as cases are sent to Arizona. Rochester consultants order LM2CX using their local laboratory information system (LIS). Slides are prepared by Rochester histology and then shipped to Arizona, where they are entered into the Arizona LIS and processed. In July 2018, we discovered numerous cases that were ordered but not shipped. Because this step took place at the LIS transition between Arizona and Rochester, it was not detected immediately. Methods Allied Health Staff (AHS) colleagues in Rochester and Arizona had the unique opportunity to collaborate. After Arizona AHS identified the problem, they reached out to the Rochester pathology reporting specialists (PRSs) and began a joint improvement project. Together, we were able to measure the impact of the problem, with Arizona auditing digitally, while Rochester audited physical cases. We found 29% of the cases were handled improperly over a 6-month time period. In order to eliminate the gap, Rochester implemented several improvements, including training, tagging all LM2CX cases, and huddle discussions, while Arizona AHS monitored the process digitally. Results Since implementation of improvements, we have had zero defects. Modifications to AHS/consultant training in Rochester will ensure continued success. Conclusions This intervention illustrates the importance of strong collaborations in order to quickly respond to testing issues and provide the greatest value to patients.

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.

Abstract Primary mediastinal large B-cell lymphoma (PMBCL) is recognized as a distinct entity in the World Health Organization classification. Currently, the diagnosis relies on consensus of histopathology, clinical variables, and presentation, giving rise to diagnostic inaccuracy in routine practice. Previous studies have demonstrated that PMBCL can be distinguished from subtypes of diffuse large B-cell lymphoma (DLBCL) based on gene expression signatures. However, requirement of fresh-frozen biopsy material has precluded the transfer of gene expression–based assays to the clinic. Here, we developed a robust and accurate molecular classification assay (Lymph3Cx) for the distinction of PMBCL from DLBCL subtypes based on gene expression measurements in formalin-fixed, paraffin-embedded tissue. A probabilistic model accounting for classification error, comprising 58 gene features, was trained on 68 cases of PMBCL and DLBCL. Performance of the model was subsequently evaluated in an independent validation cohort of 158 cases and showed high agreement of the Lymph3Cx molecular classification with the clinicopathological diagnosis of an expert panel (frank misclassification rate, 3.8%). Furthermore, we demonstrate reproducibility of the assay with 100% concordance of subtype assignments at 2 independent laboratories. Future studies will determine Lymph3Cx’s utility for routine diagnostic purposes and therapeutic decision making.