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An 18-year-old African American male with chronic rhinosinusitis presented with a 3 cm nasal mass. Imaging studies and complete blood count (CBC) were unavailable for review. Physical examination revealed no lymphadenopathy or organomegaly. He underwent surgical resection of the nasal mass. Frozen section evaluation (performed at an outside laboratory) demonstrated a “probable small cell neoplasm.” Fresh tissue in RPMI was submitted for flow cytometric analysis.
FLOW CYTOMETRIC STUDIES
Flow cytometric immunophenotyping (FCI); (Becton-Dickinson Biosciences, San Jose, CA; FACS Canto II) was performed using the antibody/fluorochrome combinations shown in Table 1. List mode files and example analysis are available in the Supporting Information.
All antibodies were purchased from BD Biosciences (San Jose, CA) except CD16, kappa, lambda by Dako (Carpinteria, CA), CD38, CD5, CD10, CD2, CD7, CD13, MPO, CD79a by Beckman Coulter (Miami, FL) and 7AAD by Invitrogen (Carlsbad, CA).
FCS 3.0 files were generated using BD FACSDiva Software Version 6.0 for data acquisition, and FCS Express 4.01.0026 (De Novo Software, Los Angeles California) was used for data analysis. FCS files and example analysis are available as Supporting Information.
FCI revealed a predominant NK-type cell population (72% of nucleated cells, 91% of lymphocytes) expressing bright CD45, CD2, CD7, and CD56 (partial) and negative for expression of CD3, CD4, CD5, CD8, CD16, CD57, TdT, and the T-cell receptor (TCR) (Fig. 1, red population). Down-regulation of CD56 and absence of normal CD16 expression were significant antigenic aberrancies.
Hematoxylin and eosin staining (Fig. 2) revealed soft tissue with diffuse lymphoid infiltrates. The lymphoid cells were predominantly small in size with mature-appearing chromatin. Mucus-glands appeared to be spared of the lymphoid infiltrates. A small focus of coagulative necrosis was noted. Mitotic figures were not increased. There was no definitive evidence of an angiocentric/angiodestructive growth pattern. Immunohistochemical staining and in situ hybridization (ISH) studies (Table 2) showed that the tumor cells stained positively for CD3 (ε chain), CD7, CD56 (weak), granzyme B, and Ki-67 (20%) (Figs. 3a–3c). They stained negatively for CD5, CD20, CD30, AE1/AE3, and LMP-1. ISH for Epstein-Barr encoded RNA (EBER) showed diffuse positivity (Fig.3d). PCR clonality studies were negative for clonal TCR gene rearrangement.
Table 2. Results of Immunohistochemical/ISH Studies
Correlation of all clinical data, morphologic, immunophenotypic, ISH, and molecular studies suggests the best classification of this neoplasm to be extranodal NK/T-cell lymphoma (ENKTL), nasal-type, intranasal variant (Stage Ia). ENKTL, nasal type, is a rare lymphoid neoplasm, representing approximately 1.5% of all lymphomas in the United States a ([1-3]). It is more prevalent in Asian populations and native Americans of Central and South America ([1-4]). ENKTL is typically seen in adults but can occur in children as well ([1-3]). Approximately 70% of ENKTL are reported to be of NK cell origin ().
Recognizing abnormal NK cell expansions by FCI is challenging, as NK cells lack a uniquely identifiable immunophenotype. Derivation of NK cells from a bipotential T-/NK-cell progenitor leads to variable expression of T-lineage-associated antigens (). Normal NK cells typically coexpress CD2, CD7, and CD8 ([1-5]). They are negative for surface CD3 expression but express the cytoplasmic CD3ε chain ([1-5]). Anti-CD3 antibodies used in FCI recognize the fully assembled CD3 complex. NK cells are therefore negative for CD3 expression by FCI. Conversely, the antibodies used in immunohistochemical analysis of paraffin sections react positively with the cytoplasmic CD3ε subunit of NK cells ([3, 5, 6]). NK cells also express the “NK lineage-associated markers,” CD16, CD56, and CD57 ([1-5]).
The typical immunophenotype of ENKTL is described as CD2+, CD56+, surface CD3−, cytoplasmic CD3ε+, granzyme B+, TIA-1+, and perforin + ([2, 4-6]). Occasional cases also show CD7 positivity, as is seen in this specimen ([2, 4, 6]). The CD56 positivity in this case is notably weak and partial. According to the 2008 WHO Classification, CD3ε+/CD56− lymphomas that express cytotoxic molecules and demonstrate EBV positivity should be classified as ENKTL ().
Stage Ia ENKTL is characterized by mucosal sparing (). This intramucosal variant may lack the prototypical angiocentricity and angiodestruction previously considered to be absolute characteristics (). Although classically an aggressive lymphoma, intramucosal ENKTL appears to be less so ([1, 7, 8]). Some reports also suggest that ENKTL with a significant small cell component may portend a more favorable clinical prognosis ([2, 5]). Standard treatment regimens utilize combined radiotherapy and chemotherapy ([1-3, 5, 8]). Timing of the radiation appears to be important for intramucosal ENKTL, with earlier initiation yielding better results (). The complete remission rate for Stage I is 73% compared to the abysmal rate of 15% for stage IV (). However, early diagnosis is vital to achieving this favorable clinical outcome.
Diagnosis of NK/T-cell lymphomas by FCI differs significantly from that of B-cell lymphomas. Unlike B-cells, clonality in NK and T-cells is more challenging. While T-cell Vβ NK-cell KIR analyses are available by FCI, these methodologies are complex and not readily available in all clinical laboratories. Rather, T/NK-cell clonality is typically inferred in FCI by aberrant antigen expression. Additionally, FCI is limited in its ability to differentiate NK cells from abnormal T-cells lacking the functional CD3 complex, as described above.
Despite its limited diagnostic utility in T/NK-cell neoplasms, FCI is a valuable methodology for rapidly identifying abnormal T/NK-cell populations. T-cell populations restricted to either CD4 or CD8 expression are potentially abnormal and warrant further characterization. Similarly, dual absence or expression of CD4/CD8 should prompt further evaluation. Detection of aberrant antigen expression by FCI allows for the diagnosis of such neoplasms as CD10+ follicular T-helper lymphoma, CD103+ primary intestinal intraepithelial natural killer-like T-cell lymphoma and CD30+ anaplastic T-cell lymphoma ([9-11]). FCI is also the ideal modality for identifying residual and recurrent disease in T/NK-cell malignancies.
Certain aspects of ENKTL, such as EBV involvement, necrosis, and angiodestruction, are best highlighted by morphological/IHC evaluation. However, as is highlighted by this case, the application of FCI to the analysis these tumors allows for rapid diagnosis and prompt therapeutic intervention. It has become an invaluable tool in the pathologist's repertoire for the evaluation of abnormal T/NK-cell populations.
CASE 3 DIAGNOSIS: Extranodal NK/T-cell Lymphoma, Nasal Type, Stage Ia
The author is indebted to Teri Oldaker, MLS (ASCP), QCYM and Carol Moore, H (ASCP) for technical assistance.