Data from four patients of PTFL were obtained from the Clinical Pathology Center of the Affiliated Cancer Hospital of Zhengzhou University and the Department of Pathology of Shanghai Cancer Center of Fudan University between June 2019 and August 2021. Two pathologists blinded to the molecular results reviewed the cases, and a consensus diagnosis was achieved based on the revised WHO classification of lymphoma (2021 version). One of the specimens was obtained by needle biopsy, and the other two were obtained through excisional biopsy. Relevant clinical and survival data were obtained using the electronic medical record system and through a telephone follow-up. This study was approved by the Institutional Review Boards of the two hospitals and was conducted according to the Declaration of Helsinki.

Tissues were fixed in a 3.7% neutral formaldehyde solution, dehydrated, embedded in paraffin, and sectioned at 4 μm thickness. H&E staining was used to evaluate lymph node biopsies. IHC staining was performed for CD20, CD3, CD5, CD10, BCL2, BCL6, MUM1, CD21, CyclinD1, and Ki-67 as part of the diagnostic workup. Immunostaining was performed on Ventana BenchMark ULTRA following the protocol of the manufacturer. CD20, CD21, CD3, CD5, and CD10 were located in the cell membrane; BCL2 was located in the cell membrane/cytoplasm; and MUM1, BCL6, CyclinD1, and Ki-67 were located in the nucleus.

The expression of EBER in PTFL paraffin sections was also detected using Ventana BenchMark ULTRA. Epstein–Barr virus (EBV) in situ hybridization was performed using the EBER probe, which was purchased from Roche Diagnostics (Shanghai) Co., Ltd. (Shanghai, China). The experimental procedures were performed according to the instructions of the manufacturer. The nucleus was stained dark blue (positive signal), while the cytoplasm was stained light red. EBV-positive nasopharyngeal carcinoma was selected as positive control. In addition, the BCL2 and IRF4 gene rearrangements were detected using fluorescence in situ hybridization (FISH) assays using commercial reagent kits (BCL2 and IRF4 Dual Color Break Apart Rearrangement probe, Anbiping Co., Ltd.). A double-color breakage probe contains two probes labeled with green and red fluorescence, respectively. In cells without gene breakage, a yellow fusion signal appears (red and green signals overlap to form a yellow signal), or red and green signals adhere to each other (the interval is less than two signal diameters). In broken genes, a fused yellow signal, a separate green signal, and a separate red signal appear in the nucleus.

Genomic DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue sections using the Maxwell 16 FFPE Tissue LEV DNA Purification Kit and the Maxwell 16 Instrument, following the instructions of the manufacturer. Polymerase chain reaction amplification was performed to detect clonal immunoglobulin (Ig) heavy and/or Ig kappa light chain gene rearrangements, following the BIOMED-2 protocol. The criteria for defining a positive band are as follows: Products generated from diagnostic samples that fall within the valid size range and are at least three times the amplitude of the third largest peak in the polyclonal background are consistent with a positive peak. One or two prominent positive bands within the valid size range are reported as “positive for the detection of clonal immunoglobulin heavy chain or kappa light chain gene rearrangement(s) consistent with the presence of a clonal cell population. In the context of overall diagnostic criteria, clonal cell populations can indicate the presence of hematologic malignancy.” In contrast, an absence of positive bands within the valid size range is reported as “negative for the detection of clonal immunoglobulin heavy chain or kappa light chain gene rearrangement(s).”

Targeted NGS was performed on the FFPE tissue samples of the patients using a customized panel of 93 lymphoma-related genes, related to auxiliary diagnosis, prognostic assessment, and targeted drug use of lymphoma. Genomic DNA was extracted from FFPE tissues using the Gene+OncoLym FFPE Kit. Single-nucleotide variants (SNVs) and small insertions and deletions (indels) were detected using an in-house developed pipeline, SNVer and LoFreq. Translocations were detected using Delly and Mantana. Mutations were annotated using SnpEff.

Case 1 involves a 6-year-old male patient who presented to our hospital for the evaluation of a non-tender mass over the right neck area. A positron emission tomography–computed tomography (CT) scan showed multiple soft tissue nodule shadows in the right cervical regions Ⅱ and Ⅲ with increased metabolism, indicating the presence of malignant lesions of multiple lymph nodes. A biopsy of the right neck was performed for pathological diagnosis. No operation or chemotherapy was performed after the diagnosis using a needle biopsy. The patient underwent follow-up for 28 months without disease progression (Table 1).

Case 2 involves an 18-year-old male patient who presented to our hospital for the evaluation of a slowly growing and non-tender mass over the left parotid gland area. No systemic symptoms, such as fever, night sweats, and weight loss, were present. A preoperative ultrasound showed a solid mass in the left parotid gland but no abnormal lymphadenopathy in the bilateral neck. There was a solid space-occupying lesion but no abnormal enlargement of lymph nodes. Mass removal with superficial parotidectomy was performed with routine precautions to preserve the facial nerve and the parotid duct. No further treatment following the simple surgical resection was performed. The patient underwent follow-up for 16 months and survived without a tumor (Table 1).

Case 3 involves a 13-year-old male patient who presented with a mass behind the left ear area that had been present for 3 months. No systemic symptoms, such as fever, night sweats, and weight loss, were present. After surgical resection, the patient underwent follow-up for 12 months and survived without a tumor (Table 1).

Case 4 involves a 15-year-old male patient who presented with a non-tender mass on the right neck area that had been present for 2 months. A preoperative CT scan showed that the lymph nodes in the right submaxillary region were swollen, and there were numerous small lymph nodes in the two necks, some of which were slightly larger. After surgical resection, the patient underwent follow-up for 16 months and survived without a tumor (Table 1).

Taking Case 2 as an example, H&E staining showed extensive proliferation of follicular cells. Under a low-power microscope, it was observed that the structure of the lymph nodes was destroyed, and crowded, swollen, and irregular follicle-like structures with different sizes were seen, which were creeping forms (Figure 1A). The “starry sky” pattern was easily observed, normally a feature of reactive follicles caused by tangible body macrophages. The polar distribution of the light and dark areas of the follicles disappeared, and the mantle area became thinner or disappeared in some areas (Figure 1B). At high magnification, neoplastic follicles were found to be composed of uniform, medium-to-large blastoid cells (Figure 1C). In addition, there is no histologic heterogeneity throughout the lymph node. Follicle centers were strongly positive for BCL6 and CD20 (Figure 1D) and negative for BCL2 (Figure 1G), CD3 (Figure 1E), CD5, and CyclinD1. Neoplastic follicles of two cases were positive for CD10 (Figure 1F), and one case was positive for MUM1 (Figure 1H). CD21 showed dilated growth of a follicular dendritic cell network in the neoplastic follicles. In addition, Ki-67 was highly expressed in all four cases (Figure 1I).

EBER hybridization, BCL2 gene breaks, and IRF4 gene breaks were all negative in the four cases (Figures 2A–C), while the IG gene rearrangements were all positive (Figure 2D). There were polyclonal backgrounds, but no oligoclonal backgrounds, in the four cases. Products generated from the four diagnostic samples that fall within the valid size range and are at least three times the amplitude of the third largest peak in the polyclonal background are consistent with a positive peak; therefore, the detection of clonal immunoglobulin gene rearrangements was positive.

Targeted NGS was performed on all four cases. A total of 15 SNVs or indels were detected in 12 genes, including PCLO, CD79B, MYD88, GNA13, KMT2D, TCF3, MAP2K1, IRF8, FOXO1, POLE, INPP5D, and FAT4. Two of the four cases had IRF8 gene mutation (Table 2), and one case had a dual mutation of the MAP2K1 gene.