Marginal zone lymphoma-derived interfollicular diffuse large B-cell lymphoma harboring 20q12 chromosomal deletion and missense mutation of BIRC3 gene: a case report

The H&E-stained sections demonstrated a dense nodular lymphoid infiltrate in the superficial and deeper dermis without involvement of the epidermis (Fig. 1a). The infiltrate was composed of predominantly small lymphocytes with scattered larger transformed cells with immunoblast morphology (Fig. 1b). Immunohistochemical stains demonstrated nodules of CD20 positive B cells around and within follicles, with scattered larger interfollicular B cells also present (Fig. 1c). CD3 stain highlighted abundant interfollicular T lymphocytes (not shown). Essentially all neoplastic B cells as well as many of the interfollicular T cells were positive for BCL2 (Fig. 1d). BCL6 was negative in the tumor cells. The findings were consistent with cutaneous involvement by marginal zone lymphoma.

The H&E stained sections showed a lymph node with an overall well-preserved nodal architecture including reactive lymphoid follicles with well-demarcated mantle zones (Fig. 2a). The interfollicular zones were, however, expanded by numerous large atypical mononuclear cells admixed with abundant eosinophils, small lymphocytes, plasma cells and histiocytes (Fig. 2a and b). The larger cells had oval to slightly irregular nuclear contours, vesicular chromatin, prominent nucleoli, and scant to moderate amount of clear to amphophilic cytoplasm (Fig. 2c and d). Immunohistochemical stains showed that the large cells were positive for B-cell markers CD20 (Fig. 2e), PAX5 (Fig. 2f), and CD79a, as well as BCL6 (subset), CD30 (Fig. 2g), MUM1(Fig. 2h), CD23, c-MYC (40–50%), p53 (dim in 40% of the large cells), BOB.1 (variable) and Oct-2 (variable). The Ki-67 proliferation index in the interfollicular areas was high, exceeding 70%. Key negative stains included BCL2, CD10, and CD15. CD138 highlighted plasma cells with a kappa to lambda ratio of 3:1. An in-situ hybridization study for Epstein-Barr virus (EBER) was negative. CD20, PAX5 and CD79a stains also marked the abundant reactive follicles with CD10+, BCL6+ and BCL2- germinal centers and BCL2+ mantle zones. The proliferation index within the germinal centers was high as expected (>90%) and highlighted the polarized light and dark zones. The infiltrating small T lymphocytes were positive for CD2, CD3, CD5 and CD7 (major subset). The CD4 to CD8 ratio was increased (>5:1). CD57 and PD-1 were positive in a subset of germinal center T cells whereas TIA1, perforin, and granzyme B were positive in scattered small interfollicular T cells with a pattern similar to CD8 staining. CD56 and ALK-1 stains were negative. CD68, CD163, and CD1a stained scattered histiocytes and accessory cells respectively.

Flow cytometry studies were performed on representative tissue from the right inguinal lymph node. As shown in Fig. 3a–c, there was a large population of CD19+ B lymphocytes (51% of all lymphoid cells) with a strong lambda bias bordering on overt restriction (kappa: lambda ratio of 0.33 and 0.27 for the CD19+ and CD20+ cells, respectively). Minor subsets of all B lymphocytes were CD5+ (Fig. 3a) or CD10+ (Fig. 3d); these cells expressed predominantly kappa and, hence, were not part of the apparent lambda clone.

Molecular studies for IGH gene rearrangement performed on the skin biopsy showed a 281 base pair (bp) peak in framework 2, as well as 94 bp and 115 bp peaks in framework 3 (Fig. 4). Examination of tissue obtained from the right inguinal lymph node demonstrated the same 281 bp peak in framework 2 and the same 115 bp peak in framework 3 whereas the 94 bp peak seen in the skin specimen was not seen in the lymph node (Fig. 4b). The overall results indicate that the lymphoproliferative disorders involving lymph node and skin are clonally related. The significance of the 94 bp peak is unclear, but it may represent a separate (sub-)clonal expansion. No clonal T-cell receptor (TCR) rearrangement was detected in the lymph node specimen.

FISH studies for rearrangements of MYC, BCL2, and BCL6 performed on the lymph node were also negative. Previous report described del(20q) in MZL with aggressive features [12]. Since this patient’s DLBCL was thought be derived from a MZL, additional FISH analysis for a 20q12 deletion was performed on both skin and lymph node material to determine whether this abnormality may be present. The analysis revealed that 38/150 (25.3%) of cells in the involved lymph node were harboring del(20q12) (Fig. 5), whereas the skin lesion was negative for this deletion (data not shown).

Targeted DNA sequencing studies using our hematologic malignancy panel on the lymph node tissue did not reveal any known disease-associated mutations, however it did identify two variants of uncertain significance (Table 1): a missense variant in BIRC3 (Baculoviral IAP repeat-containing protein 3) at amino acid 572 converting the wild type residue, cysteine, to tyrosine in BIRC3 (p.C572Y → c.1715G > A) in 491 reads out of a total 9155 sequence reads for an allele frequency of 5.36% and a missense variant in EZH2 gene (Enhancer of Zeste Homolog 2) at amino acid 322 converting the wild type residue, Asparagine, to Serine (p.N322S → c.965A > G) in 818 reads out of a total 1561 sequence reads for an allele frequency of 52.40% . Subsequent sequencing studies of the skin revealed the same missense variant in EZH2 as in the lymph node in 1097 reads out of a total 2165 sequence reads for an allele frequency of 50.67% but no other alterations, including the BIRC3 mutation, were identified.