Distinct pattern of lymphoid neoplasms characterizations according to the WHO classification (2016) and prevalence of associated Epstein–Barr virus infection in Nigeria population

Formalin-fixed paraffin-embedded (FFPE) biopsies, including their demographic data, were collected from tertiary facilities of three different geopolitical areas in Nigeria: University College Hospital, Ibadan, Oyo State, Enugu State University of Science and Technology Teaching Hospital (ESUTH), and Meena Histopathology and Cytology Laboratory, Jos, Plateau State. Ethical committee approvals were obtained from the University of Nigeria Teaching Hospital, Enugu (UNTH-NHREC/05/01/2008B), Ministry of Health, Ibadan, Oyo State (AD13/479/1138), and Meena Histopathology and Cytology Laboratory, Jos, Plateau State (MEENALAB/AEC/177). A total of 152 FFPE morphologically diagnosed as lymphoid neoplasms in the already listed institutions between 2008 and 2018 were obtained. The revised classification was made according to the latest WHO classification of Tumors of Lymphoid Tissues. The statistical analysis was done with IBM, SPSS version 25.0 software package (IBM® Software). The level of statistical significance was set at p < 0.05. Inferential statistics were t-test in case of 2 groups and ANOVA in case of 3 or more groups with continuous normally distributed variables. Significant ANOVA results were followed by Tukey post hoc test for paired comparison. Association between variables was by Pearson product-moment correlation (r) and Chi-square (X²) test for independence.

4 μm sections were cut and used for H&E staining and immunohistochemistry (IHC) from each block. All slides were reviewed by two expert hematopathologists (LL, SL). Cases with insufficient or not optimal material were excluded from the study. The immunohistochemical analysis was performed with a panel of 25 antibodies commonly used in NHL and HL cases, using the Ventana staining system (Ventana, Tucson, AZ, USA).

According to the manufacturer’s instruction, EBV was detected by in-situ-hybridization (ISH) using the INFORM EBER probe (Ventana Medical Systems, Inc., Tucson, AZ, USA). The slides were stained using an automated stainer (Benchmark Ultra, Ventana Medical Systems, Inc., Tucson, AZ, USA). The visualization was by ISH iView Blue Detection kit (Ventana Medical Systems, Inc., Tucson, AZ, USA) with alkaline phosphatase 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP NBT/) substrate. Nuclear Fast red (Ventana Medical Systems, Inc., Tucson, AZ, USA) was used as a contrast. Appropriate positive and negative controls were set up parallel to the section analyzed.

A total of 152 cases were analyzed. From the study cohort’s total number, 50 cases were excluded due to insufficient tissue materials or inadequate antigen reactivity. Of the remaining 102 samples, after using IHC, in only 66 cases, a lymphoma diagnosis was established. The remaining 36 cases were confirmed with the following diagnosis: 22 reactive lymphadenopathies, 2 Castleman disease (CD), 2 Rosai-Dorfman disease (RDD), and 10 cases as non-lymphoid malignancies. Among the 66 lymphoma cases, 44 (66.7%) and 22 (33.3%) were male and female, respectively, with a male to female ratio of 2:1 (Table 1). All the lymphoma subtypes showed a male predominance with higher ratios in HL (3.3:1) and BL (4:1). The lymphoma cases showed an age range between 2 and 85 years with a mean age of 44.4 ± 20.1 years that was not significantly different (p = 0.295) among the two genders (male: 42.6 years; female: 48.1 years). As shown in Fig. 1, most lymphoid neoplasm (34.8%) occurred within the age group of 41–60 years. The mean age of chronic lymphocytic leukemia (CLL) patients (55.7 years) was significantly different from the mean ages of patients with BL (27.6 years; p = 0.009) and HL (33.8 years; p = 0.001) as opposed to the mean age of patients with DLBCL, NOS (p = 0.304).

Also, in Table 1, nodal involvement (83.3%) compared to extranodal sites (16.7%) was more frequently observed in the different tumor types. Instead, the extranodal localizations according to the different lymphoid neoplasms were: 4 cases of CLL (omental mass, ovarian mass, breast, and colon), 2 for DLBCL, NOS (jaw and non-nodal tissue mass), 1 for BL (ovary), 2 for splenic marginal zone lymphoma (SMZL) (spleen), 1 for extranodal marginal zone lymphoma (ENMZL) (conjunctiva), and 1 for lymphoplasmacytic lymphoma (LPL) (scalp mass).

The number of cases that were concordant and discordant from the previous diagnosis is shown in Table 2. The list of the antibody that was employed to reclassify are shown in Table 3. A total of 65 (98.5%) cases originated by B lymphocytes while only one by T lymphocytes. The majority of the reclassified lymphomas were CLL (n = 23; 34.8%), and a greater proportion (65.2%) of these cases came from the Hausa ethnic group (Table 4) involving mostly nodal sites (34.5%). Only one case of the 23 CLL cases presented a concordant diagnosis while the other 22 (95.6%) presented a discordant diagnosis (Supplementary Table 1). 17 cases of HL/HD (25.8%) were identified with 5 (29.4%) cases that presented a concordant diagnosis and the remaining 12 (70.6%) cases of HL/HD were actually discordant from the original diagnosis (Supplementary Table 2). The highest incidence of HL/HD occurred within the age group of 21–40 years (Fig. 2), with a major frequency in the Yoruba ethnic group (52.9%) as shown in Table 4. The distribution of the HL subtypes was: 4 (23.5%) nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL), 1 (5.9%) lymphocyte rich classical Hodgkin lymphoma (LRCHL), 10 (58.8%) of mixed cellularity classical Hodgkin lymphoma (MCCHL) and 2 (11.8%) nodular sclerosis classical Hodgkin lymphoma (NSCHL). EBV infection was noticed in 6 cases of MCCHL. Following IHC and expert revision, 10 cases (71%) out of 14 of the DLBCL group presented a discordant diagnosis (Supplementary Table 3). Instead, all the BL cases showed a concordant diagnosis in 2 cases (40%) and the rest were discordant (n = 3; 60%), as shown in Supplementary Table 4. For the Igbo ethnic group, there was an identical distribution in the number of CLL (33.3%), DLBCL, NOS (33.3%), and HL/HD (33.3%) as presented in Table 4.

The histologic diagnosis of the remaining 6 entities (Supplementary Table 5) were discordant with the final diagnosis according to the latest WHO classification: 2 SMZL (3%), 1 ENMZL (1.5%), 1 (1.5%) follicular lymphoma (FL), 1 LPL (1.5%), 1 (1.5%) B-lymphoblastic leukemia/lymphoma (B-LBL) and 1 (1.5%) Angioimmunoblastic T-cell lymphoma (AITL). The representative slides of the H&E and IHC are shown in Figs. 3, 4 and 5.

The non-lymphoid malignancies and other rare conditions that were more accurately diagnosed using IHC are shown in Supplementary Table 6. Apart from the identified rare conditions such as Castleman Disease (n = 2; 14.3%), Rosai Dorfman Disease (n = 2; 14.3%), the rest of the cases were metastases of carcinomas (n = 6; 43%) and sarcomas (n = 4; 28.6%).

The COO determined by the Hans algorithm demonstrated that 10 (71.4%) cases were of the Germinal Center type (GCB), 3 (21.4%) were Non-Germinal Centre (non-GCB) type, and 1 (7.1%) case could not be determined (Supplementary Table 3). Also, in both GCB and non-GCB subtypes, two of the DLBCL, NOS (14.3%), presented co-expression of c-MYC protein and BCL2 (“double expressors”).