Metagenomic Analysis to Identify Novel Infectious Agents in Systemic Anaplastic Large Cell Lymphoma

Anaplastic large cell lymphoma (ALCL) is a rare T-cell non-Hodgkin lymphoma (NHL). Risk of systemic ALCL is markedly increased among immunosuppressed people, such as those with human immunodeficiency virus (HIV) infection or solid organ transplant recipients. This increased risk in immunosuppressed populations suggests a viral etiology, given similar elevations for diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, and Hodgkin lymphoma (caused by Epstein-Barr virus [EBV]) and other virus-related cancers in immunosuppressed individuals. EBV might be a plausible candidate, but most reported ALCL tumors are EBV-negative. Therefore, we conducted a metagenomic analysis of microbial associations with ALCL to determine whether a known or novel pathogen is associated with the tumor. We obtained archived formal fixed paraffin embedded (FFPE) ALCL tumor tissue from the Iowa cancer registry and an additional case from an immunosuppressed individual infected with human immunodeficiency virus. Controls included 3 diffuse large B-cell lymphomas (DLBCLs) in transplant recipients (shown to be related to Epstein-Barr virus [EBV] by in situ hybridization) and 3 breast cancers (no known viral etiology). RNA was extracted from FFPE specimens, and total RNA libraries were prepared using the KAPA RNA HyperPrep Kit (Roche). Five samples (IA01, IA53, IA57, IA07, and IA17) were sequenced on a NextSeq 500 (Illumina) and the remainder on a NovSeq 6000 (Illumina). Using novel computational approaches, we eliminated human reads and aligned residual non-human reads to known microbial references using GATK-PathSeq or conducted de novo assembly of unassigned reads using virID pipeline. Although EBV was detected in the 3 DLBCL controls, we did not detect EBV or any novel pathogen in ALCL tumors or in the breast cancers. Our study had a modest sample size and only one case arising in HIV-infected individual. Therefore, further studies are required to characterize the metagenome of systemic ALCLs, especially cases arising in immunosuppressed people.]]>
Cases: We identified systemic anaplastic large cell lymphoma (ALCL) cases from the cancer registry using International Classification of Diseases for Oncology (3rd edition, ICD-O-3) morphology code 9714, and excluded cases with ICD-O-3 topography codes C44.x (skin) or C50.x (breast), or B-cell, pre-B-cell, or B-cell precursor cell types. We included an additional systemic ALCL cases from an HIV-infected person. Controls: For positive controls, we selected diffuse large B-cell lymphoma (DLBCL) cases arising in solid organ transplant recipients because most DLBCL cases in immunosuppressed individuals are associated with Epstein-Barr virus (EBV) infection. We leveraged a previous linkage between the US Scientific Registry of Transplant Recipients (SRTR) and cancer registries to identify DLBCL cases in transplant recipients. We identified 3 EBV+ DLBCL cases linked to the transplant registry with adequate tissue available for the analysis. We selected 3 breast cancer cases as negative controls, because breast cancer does not have a known viral etiology. In addition, one case was reclassified as DLBCL and included as an EBV- control.]]>
December, 2017: Study approved by the National Cancer Institute January, 2018-January, 2019: Retrieving tumor specimens January, 2019-April, 2019: Histopathology confirmation of ALCL and DLBCL cases April, 2019-January, 2020: RNA extraction, sequencing, and analyses of data January 2020-May 2020: Completion of manuscript ]]>