The clinical characteristics of PTFL, PNMZL, and MTT patients were summarized in Table 1, and no significant difference was found among the 3 groups. Totally, there were 52 male and 7 female patients, with a male to female ratio of 7.43:1. The average and median age at diagnosis was 18 and 17 years, respectively (range, 10–35 years), with 70% of the patients ranging from 10 to 20 years. Most patients (96.6%) had head and neck lymph node involvement and were diagnosed with stage I disease. Three of 59 patients (5%) presented with B symptoms. Among the 50 patients with available telephone follow-up, 44 (88%) underwent a watch-and-wait approach after surgical resection of the lesions. Complementary chemotherapy (with regimens of R-CHOP in 4 patients and CHOP in one patient) and radiotherapy (in one patient) were given in the remaining 6 patients. All of the 50 patients were alive at the last follow-up, with a follow-up time ranging from 12 to 90 months (median 40 months); the other 9 patients were lost to follow-up. Only one PTFL patient experienced a relapse 6 months after diagnosis.

Microscopically, the 39 PTFL cases exhibited enlarged follicles, with thin or absent mantle zones and serpiginous margins. Notably, focal or occasional follicles with a PTGC-like pattern, that is, with hyperplastic mantle zone and disrupted and regressed germinal centers, were always present. The tumor cells featured centroblast/centrocyte-like appearance, and more frequently, exhibited blastoid appearance without prominent nucleoli. Mitotic figures were readily seen. CD10, BCL6, and CD43 expression was observed in 97%, 97%, and 23% of the cases, respectively. BCL2 and MUM1 were negative in most cases but weak positivity was observed in 21% and 8% of cases, respectively (Table 2). The Ki-67 proliferation index ranged from 60 to 90% in atypical germinal centers (Fig. 1). Thirty-six percent of cases demonstrated light chain restriction by immunohistochemical or flow cytometric detection of Kappa/Lambda expression. The 5 PNMZL cases morphologically featured atypical small centrocyte-like lymphocyte proliferation that surrounding follicles, extending into the interfollicular areas, and sometimes, colonizing into follicles together with the mantle cells, thus producing a PTGC-like pattern. Phenotypically, the tumor cells were negative for CD10 and BCL6, whereas the residual germinal centers expressed these markers. Eighty percent of PNMZL cases expressed BCL2, and 60% expressed CD43. MUM1 was negative in all cases. The Ki-67 proliferation index of neoplastic cells ranged from 20 to 60%, which was much lower than that of the germinal centers (Fig. 2). Light chain restriction was demonstrated in 40% of cases by immunohistochemical or flow cytometric analysis. The 15 MTT cases were composed of both enlarged CD10-positive, BCL2-negative germinal centers with a serpiginous growth pattern and marked MZL components with a prominent PTGC-like pattern, indicating the presence of a composite form of PTFL and PNMZL (Figs. 3 and 4).

Histologic findings of one representative PTFL case. The H&E-stained slide shows marked expansile follicles with thin or absent mantle zones (a, e). The follicular growth pattern is further highlighted by the CD20 staining (b). Tumor cells are intermediate in size, which demonstrate a blastoid appearance (f) and CD10 + (c), BCL2 − (d), and BCL6 + (g) phenotype. The Ki-67 proliferation index is approximately 90% in neoplastic germinal centers (h)

Histologic findings of one representative PNMZL case. The H&E-stained slide shows interfollicular expansion and PTGC-like changes of the follicles (a, e). Tumor cells are atypical small centrocyte-like in appearance (f), which are BCL2 + (b) and CD10 − (c). IgD staining highlights the presence of PTGC-like follicles (g). The Ki-67 index of tumor cells is approximately 30–40% (d, h)

Histologic findings of one representative MTT case. The H&E-stained slide shows approximately one-half each of the area of the whole lymph node demonstrating PNMZL-like and PTFL-like features (a, b, c). Staining with CD20 (d, e), CD10 (f, g), BCL2 (h, i), and Ki-67 (j, k) antibodies highlights distinctive growth patterns of the PNMZL-like and PTFL-like components

Histologic findings of another MTT case. The H&E-stained slide shows composite PTFL-like (on the right) and PNMZL-like (on the left) components (a, e), which is further highlighted by the BCL2 (b), CD10 (c), and Ki-67 (d) stainings. Tumor cells within the neoplastic germinal centers are large to intermediate in size (f), with a BCL6 + phenotype (g) and relatively high Ki-67 proliferation index (90%), in contrast to the index of 30–40% in the interfollicular areas (d, h)

Thirty-one PTFL cases underwent the detection for IG gene rearrangements, and all showed positive results. The positive rates of IGH, IGK, and IGL were 90.3%, 74.2%, and 9.7%, respectively. All of the 5 PNMZL cases showed IG gene rearrangements, with a positive rate of 60% for IGH, 60% for IGK, and 20% for IGL. Eleven cases with MTT underwent the test, and 10 (91%) showed IG rearrangements, with positive rates of IGH, IGK, and IGL being 90.9%, 72.7%, and 36.4%, respectively. No significant differences were found among the 3 groups (Table 3).

The DNA-based targeted NGS results were obtained in 7 cases with available high-quality DNA materials, including 3 PTFL (PTFL1-3), 2 PNMZL (PNMZL1-2), and 2 MTT (MTT 1–2) (Fig. 5). There were totally 61 mutations that have been identified, with a mean of 8.7 mutations per case. The most frequently mutated gene was TNFRSF14, with 6 mutations in 3 PTFL and 2 MTT patients (2 mutations in PTFL1, 71%), followed by MAP2K1, with 4 mutations in 2 PTFL, 1 PNMZL, and 1 MTT patients (57%), and IRF8, MAPK1, and MUC16, each with 3 patients (43%, 43%, 43%) containing mutations. Other mutated genes included MUC4, TTN (two patients each, 29%), ABCA13, ADGRV1, APP, ARID1B, BLM, BPTF, BRAF, CD28, CREBBP, DOT1L, DUSP2, EBF1, EPPK1, FAT1, FOXO1, GNAS, ITPKB, KIT, LYST, MSH6, MYOM2, NCOR2, NOTCH2, PAX5, PTPN13, PTPRC, PTPRS, RECQL4, ROBO1, and RP1L1 (one patient each, 14%).

Results for targeted next-generation sequencing of 571 lymphoma-related genes

As for the mutational signatures, the TNFRSF14 mutations included 2 nonsynonymous SNVs and 2 stop-gain mutations, p.E46Q and p.Y47* in PTFL1, p.C57* in PTFL2, and p.L23P in PTFL3. The MAP2K1 p.F53U and p.K57E nonsynonymous SNVs were found in PTFL1 and PTFL2, respectively. MAPK1 and TTN mutations were respectively detected in 3/3 and 2/3 PTFL patients but not in PNMZL or MTT patients. No common mutations were found in 2 PNMZL cases. PNMZL1 displayed IRF8 p.K66R hotspot mutation. PNMZL2 featured a MAP2K1 p.S200F nonsynonymous SNV, a CREBBP non-frameshift deletion mutation, multiple missense mutations in the MUC4 gene (p.T2991I and p.P3794L), and a stop-gain mutation in MUC16. The 2 MTT cases showed IRF8 p.K66R hotspot mutations, TNFRSF14 mutations (p.E46K in MTT1 and p.L23P in MTT2), and MUC16 mutations (p.F12236U in MTT1 and p.P1456R in MTT2). In addition, MTT2 had a MAP2K1 p.C121S mutation.

To compare the genetic mutational profiles of PTFL, PNMZL, and MTT, previously published mutational data of 96 PTFLs, 25 PNMZLs, and 46 MTTs were collected (Table 4) and analyzed (Table 5) [12,13,14,15,16,17,18,19,20]. TNFRSF14 (41.9%) and MAP2K1 (39.6%) were the two most frequently mutated genes in PTFLs. In PNMZLs, the mutation frequencies of MAP2K1 (32%) and IRF8 (28%) were slightly higher than that of TNFRSF14 (20%). And the MTTs had the highest frequency of MAP2K1 (54.3%), TNFRSF14 (43.6%), and IRF8 (32.4%) mutations among the 3 groups. However, no statistically significant differences regarding the mutational frequency of MAP2K1, TNFRSF14, and IRF8 were found among the 3 groups except for the mutational frequency of TNFRSF14 in PTFL vs. PNMZL (P2 = 0.046), which might be due to the relatively small sample size of PNMZL.