Critical Role of Flow Cytometric Immunophenotyping in the Diagnosis, Subtyping, and Staging of T-Cell/NK-Cell Non-Hodgkin's Lymphoma in Real-World Practice: A Study of 232 Cases From a Tertiary Cancer Center in India

Abstract

Background: T-cell/NK-cell non-Hodgkin's lymphoma (T/NK-NHL) is an uncommon heterogeneous group of diseases. The current classification of T/NK-NHL is mainly based on histopathology and immunohistochemistry. In practice, however, the lack of unique histopathological patterns, overlapping cytomorphology, immunophenotypic complexity, inadequate panels, and diverse clinical presentations pose a great challenge. Flow cytometric immunophenotyping (FCI) is a gold standard for the diagnosis, subtyping, and monitoring of many hematological neoplasms. However, studies emphasizing the role of FCI in the diagnosis and staging of T/NK-NHL in real-world practice are scarce.

Methods: We included T-cell non-Hodgkin's lymphoma (T-NHL) patients evaluated for the diagnosis and/or staging of T/NK-NHL using FCI between 2014 and 2020. We studied the utility of FCI in the diagnosis and subtyping of T/NK-NHL and correlated the FCI findings with the results of histopathology/immunohistochemistry. For correlation purposes, patients were categorized under definitive diagnosis and subtyping, inadequate subtyping, inadequate diagnosis, and misdiagnosis based on the findings of each technique.

Results: A total of 232 patients were diagnosed with T/NK-NHL. FCI findings provided definitive diagnoses in 198 patients and subtyping in 187/198 (95.45%) patients. The correlation between FCI and histopathological/immunohistochemistry results (n = 150) demonstrated an agreement on the diagnosis and subtyping in 69/150 (46%) patients. Of the remaining cases, the diagnosis and subtyping were inadequate in 64/150 (42.7%), and 14/150 (9.33%) were misdiagnosed on histopathology/immunohistochemistry results. FCI provided definitive diagnosis and subtyping in 51/64 (79.7%) patients. Among these, 13 patients diagnosed with peripheral T-cell lymphoma not-otherwise-specified were reclassified (angioimmunoblastic T-cell lymphoma (AITL)-11 and prolymphocytic leukemia-2) on FCI. It corrected the diagnosis in 14 patients that were misdiagnosed (6 B-cell NHL (B-NHL), 3 Hodgkin's lymphoma, 1 acute leukemia, and 1 subcutaneous panniculitis-like T-cell lymphoma) and misclassified (3 T-NHL) on histopathological results. AITL was the commonest T-NHL misclassified on histopathological results. FCI also confirmed the definite involvement in 7/83 (8.4%) and 27/83 (32.5%) bone marrow (BM) samples reported as suspicious and uninvolved, respectively, on histopathological evaluation.

Conclusion: AITL was the most frequently diagnosed T/NK-NHL in this study. FCI provided a distinct advantage in detecting BM involvement by T/NK-NHL, especially in patients with low-level involvement. Overall, our study concluded that FCI plays a critical role in the diagnosis, subtyping, and staging of T/NK-NHL in real-world practice.

Keywords: T cell; flow cytometry; immunophenotyping; non-Hodgkin’s lymphoma; real-world practice.

Figure 1

(A) Flowchart representing the distribution of T-cell/NK-cell NHL patients studied. BM, bone marrow; BF, body fluids; FCI, flow cytometric immunophenotyping; FNA, fine-needle aspiration; Histo, histopathology; IHC, immunohistochemistry; PB, peripheral blood; NHL, non-Hodgkin’s lymphoma. (B) Distribution of subtypes of T-cell/NK-cell NHL patients. AITL, angioimmunoblastic T-cell lymphoma; ALCL, anaplastic large T-cell lymphoma; ATLL, adult T-cell leukemia/lymphoma; CTCL, cutaneous T-cell lymphoma; FCM, flow cytometry; FHTCL, follicular helper type T-cell lymphoma; HSTCL, hepatosplenic T-cell NHL; I, intestinal; IN, inadequate; LEP, lupus erythematosus panniculitis; LGLL, large granular lymphocytic leukemia; M, male; MF, mycosis fungoides; NK/NKTCL, NK-/T-cell lymphoma; PTCL-NOS, peripheral T-cell lymphoma not otherwise specified; PLT, platelets; PLL, prolymphocytic leukemia; SPTCL, subcutaneous panniculitis-like T-cell lymphoma.

Figure 2

Flowchart demonstrates an immunophenotypic approach for assessing common T-cell markers using a primary antibody panel followed by the selection of an additional antibody panel based on the CD4 and/or CD8 distribution. The latter half of the flowchart demonstrates the utility of selective markers for the subtyping of T-cell NHL. AITL, angioimmunoblastic T-cell lymphoma; ALCL, anaplastic large T-cell lymphoma; ATLL, adult T-cell leukemia/lymphoma; CTCL, cutaneous T-cell lymphoma; EATCL, enteropathy-associated T-cell lymphoma; FCM, flow cytometry; FHTCL, follicular helper type T-cell lymphoma; Gz, granzyme; HSTCL, hepatosplenic T-cell NHL; I, intestinal; IN, inadequate; KIR, killer immunoglobulin-like receptors; LEP, lupus erythematosus panniculitis; LGLL, large granular lymphocytic leukemia; M, male; MF, mycosis fungoides; NK/NKTCL, NK-/T-cell lymphoma; PFR, perforin; PTCL-NOS, peripheral T-cell lymphoma not otherwise specified; PLT, platelets; PLL, prolymphocytic leukemia; SPTCL, subcutaneous panniculitis-like T-cell lymphoma; (+) positive expression; (++) strong positive expression; (+/−) positive or negative or heterogeneous expression; (−) negative expression.

Figure 3

Flow cytometric immunophenotyping (FCI) findings (A, B) and lymph node histopathology and immunohistochemistry results (C) from a representative case of angioimmunoblastic T-cell-lymphoma (AITL) that was misdiagnosed as B-cell non-Hodgkin’s lymphoma (B-NHL). (A) The dot-plots of FCI from the lymph node fine-needle aspiration sample. (B) The dot-plots of FCI from the bone marrow aspiration sample from the same patient. In these dot-plots, the abnormal T cells (dark blue dots) show bright CD45, moderate CD5, bright CD2, moderate CD4, partial CD10, bright CD279 (PD1), and moderate CD185 (CXCR5) expressions but aberrant loss of surface CD3 and CD7 expressions. The orange dots represent normal T cells, and light blue dots show polyclonal B cells. (C) Microscopic pictures of histological (H&E staining) and immunohistochemistry results from the lymph node biopsy demonstrating B cell [highlighted by CD20 and PAX5 immunohistochemistry (IHC)] and plasma cell hyperplasia (highlighted by CD138 IHC). T cells were highlighted by CD5, CD7, CD3, CD4, and CD8 IHC.

Figure 4

Flow cytometric immunophenotyping (FCI) findings (A, B) and lymph node histopathology and immunohistochemistry results (C) from a representative case of angioimmunoblastic T-cell-lymphoma (AITL) that was misdiagnosed as classical Hodgkin’s lymphoma (cHL). (A) the dot-plots of FCI from the lymph node fine-needle aspiration sample. (B) The dot-plots of FCI from the bone marrow aspiration sample from the same patient. In these dot-plots, the abnormal T cells (dark blue dots) show bright CD45, bright CD2, bright CD5, moderate-to-dim CD4, moderate-to-dim CD10, moderate CD279 (PD1), moderate CD185 (CXCR5), and moderate HLA-DR expressions but abnormal loss of surface CD3 expression. The orange dots represent normal T cells. (C) Microscopic pictures of histological (H&E staining) and immunohistochemistry results from the lymph node biopsy showing follicles highlighted by CD20 and expanded paracortex in the CD3 immunohistochemistry (IHC). Scattered Reed–Sternberg (RS)-like cells are indicated with an arrow.

Figure 5

Flow cytometric immunophenotyping (FCI) findings (A) and histopathology and immunohistochemistry results (B) from a representative case of primary cutaneous γδT-cell lymphoma (γδCTCL) that was misclassified as peripheral T-cell lymphoma not otherwise classified (PTCL-NOS). (A) The dot-plots of FCI of fine-needle aspiration sample from the subcutaneous nodule. In these dot-plots, the abnormal γδT cells (dark blue dots) shows bright CD45, bright CD3 bright CD5, moderate CD4, and bright γδT-cell receptor (TCRγδ) expressions but abnormal loss of CD7 expression. On additional immunophenotyping, these cells also show TCRVδ1 restriction but negative TCRVδ2 and TCRVγ9 expressions. The orange dots represent normal T cells. (B) Microscopic pictures of histological (H&E staining) and immunohistochemistry results from the surgical biopsy from the same nodule show significantly increased proportion of CD4-positive T cells with decreased expressions of CD3 and CD7.