Flow Cytometric Characterization of 129 Cases of Peripheral T Cell Lymphoma Not Otherwise Specified (PTCL NOS) and Angioimmunoblastic T Cell Lymphoma (AITL)
Authors: Hapgood Greg , Mottok Anja , Slack Graham W , Gascoyne Randy D. , Steidl Christian , Savage Kerry J. , Weng Andrew P. 
Published: October 2019

Abstract
Background: Peripheral T cell lymphoma not otherwise specified (PTCL NOS) and angioimmunoblastic T cell lymphoma (AITL) together comprise approximately half of all peripheral T cell lymphomas. Malignant T cells in AITL and in a subset of PTCL-NOS exhibit a phenotype mimicking that of normal T-follicular helper (TFH) cells. Immunohistochemical (IHC) studies performed on paraffin-embedded tissues are a mainstay of diagnostic histopathology, but can be difficult to interpret when the malignant T cells show limited cytological atypia and when there are abundant infiltrating reactive T cells. Flow cytometry represents an alternate means to define the cellular immunophenotype, but requires access to single cell suspensions of viable tumor cells. Flow cytometry has certain additional benefits over IHC including highly quantitative measurement of multiple antigens simultaneously and statistical power afforded by analyzing tens of thousands of individual cells. We report here immunophenotypic characterization of a large cohort of cases of PTCL NOS and AITL using a 12-color flow cytometry assay and correlation of immunophenotypic features with clinical outcomes.
Methods: Cases of PTCL-NOS and AITL spanning a 24 year period (1990-2014) for which viably frozen cell suspensions from diagnostic lymph node biopsies were available were identified within the British Columbia Cancer Agency (BCCA) lymphoma database. Cryopreserved cell suspensions were thawed and stained with a 12-color panel including 11fluorochrome-conjugated antibodies against lineage (CD45, CD19, CD3, CD4, CD8), pan-T cell (CD2, CD5, CD7), and TFH cell (CD10, CD279, CXCR5) markers, plus DAPI for gating of live cells. Flow cytometric data was acquired on a Becton Dickinson FACSAria3 instrument as part of a sorting experiment to isolate tumor cell subpopulations. Data was analyzed by conventional gating and bivariate plot display using FlowJo software and correlated with clinical outcome data.
Results: 74 cases of PTCL-NOS and 55 cases of AITL were analyzed. The median age at diagnosis was 57 years (y) for PTCL NOS (male:female 1.6) and 75 y for AITL (male:female 1.0). The median follow up for living patients was 5.15 y. The median specimen viability was 36.5% (range 0.8-89.3%) and median specimen tumour content was 64.3% of viable events (range 0.98-91.8%). Aberrant T cell immunophenotypes were identified in 50 of 74 cases (68%) of PTCL NOS and 36 of 55 cases (65%) of AITL. Five specimens had more than one identifiable immunophenotypically aberrant T cell population.
For the 50 PTCL NOS cases with an aberrant immunophenotype, 31 (62%) demonstrated loss of CD3 and 42 (84%) demonstrated loss of CD7. About half of cases were CD4+CD8- (27, or 54%) including 11 (22%) that exhibited a TFH-like phenotype (positive for at least 2 of the 3 assayed TFH markers), while the remaining were CD4-CD8- (23, or 46%). TFH-like cells were also identified in 11 of 24 (46%) cases lacking an aberrant T cell immunophenotype.
For the 36 AITL cases with an aberrant immunophenotype, 21 (58%) demonstrated loss of CD3 and 29 (80%) demonstrated loss of CD7. The majority of cases were CD4+ (30, or 83%) including 21 (58%) that exhibited a TFH-like phenotype, while the remaining were either CD8+ (4, or 11%) or CD4-CD8- (2, or 6%). TFH-like cells were also identified in 7 of 19 (37%) cases lacking an aberrant T cell immunophenotype.
Similar to other patient cohorts, the 5 y PFS and 5 y OS was 21% and 40%, respectively, for PTCL NOS and 17% and 28%, respectively, for AITL. The presence of an aberrant phenotype, CD3 status, and CD4/CD8 status were not associated with prognosis in either PTCL subtype. A preliminary analysis suggests loss of CD7 expression in PTCL NOS is associated with an inferior outcome. Analysis of archival material and exploration in a validation cohort is ongoing.
Discussion: An aberrant population of varying abundance was detected in >65% of specimens for PTCL NOS and AITL. The aberrant immunophenotype in PTCL NOS was evenly split between CD4+CD8- and CD4-CD8- cases. Interestingly, nearly half of CD4+ cases showed evidence of TFH-like differentiation, possibly corresponding to the TFH-like variant of PTCL NOS. The aberrant immunophenotype in AITL was typically CD4+ and often with co-expression of TFH-associated markers. Loss of CD7 and CD3 were the most common abnormalities. Loss of CD7 may demonstrate a poor-risk group of patients with inferior outcomes in PTCL NOS.

Disclosures
Savage: Seattle Genetics: Honoraria, Speakers Bureau; BMS: Honoraria; Infinity: Honoraria; Roche: Other: Institutional research funding.