This study included three patients diagnosed with PTCL, each harboring germline mutations in FHL-related genes. Baseline clinical characteristics, treatment modalities, and response assessments of the 3 cases were reviewed.

Genomic DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Libraries were prepared using the KAPA HTP Library Preparation Kit (Kapa Biosystems, Boston, USA). For targeted enrichment, a customized panel of biotinylated oligoprobes (Roche NimbleGen) was designed to capture the coding regions of 700 genes associated with hematological hereditary disorders and immunodeficiency disorders. The captured DNA library was amplified and sequenced on Illumina Novoseq 6000 sequencer for paired reads at 150 bp.

Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood samples using the Ficoll-Hypaque density centrifugation method. CD8+ T-cells were isolated from PBMCs via magnetic-activated cell sorting, using CD8 microbeads as per the manufacturer’s instructions (Miltenyi Biotech, Germany).

Total RNA was extracted from the suspension of isolated CD8+ T-cells by Trizol reagent (Invitrogen). The cDNA libraries were constructed using the VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina (vazyme, Inc.). Clean reads were aligned to the human genome (GRCh38, Ensembl104) using the Hisat2 (7). EBSeq algorithm was applied to filter the differentially expressed genes (DEGs) under the following criteria: i) fold change (FC) > 1.5 or < 0.67; ii) false discovery rate (FDR) < 0.05 (8). Pathway annotation of DEGs was performed using the KEGG database. Fisher’s exact test was used to identify significant pathways with a threshold of significance defined by P < 0.05.

CD107a degranulation assay and NK-cell activity were analyzed utilizing the flow cytometry method. For the CD107a degranulation assay, PBMCs isolated from peripheral blood samples of the patients and cultivated K562 cells were co-cultured at an effector-to-target ratio of 10:1. A basal degranulation group (PBMCs + culture medium) and a positive control group (PBMC + PMA and ionomycin containing medium) were also prepared. Cells were labeled with anti-human anti-CD107a-PE antibody. Anti-CD56-APC and anti-CD3-PerCP antibody were used to differentiate T- and NK-cells. After co-culture and staining, the samples were analyzed by flow cytometry. Surface expression of CD107a was assessed in the T- and NK-cells and compared before and after stimulation. Results are reported as ΔCD107a (percentage CD107a+ cells of stimulated – percentage CD107a+ cells of unstimulated sample) and measured by mean fluorescence intensity (MFI).

For the NK-cell activity test, the K562 target cell line with an enhanced stable expression fluorescent was established using the lentivirus-mediated transfection method. PBMCs of the patients and transfected K562 cells were co-cultured. The annexin V-PE/7AAD double staining marker was used for apoptosis labeling. After co-culture and staining, the samples were analyzed by flow cytometry. The percentage of apoptotic target cells reflected the cytotoxic activity of NK cells.

Case 1: A 45-year-old male presented with a 12-year history of cytopenia and splenomegaly and 1 month of limb weakness. In 2010, leukopenia, thrombocytopenia, and splenomegaly were first recognized in a routine blood test without any symptoms. Atypical lymphocytes were noticed on bone marrow smear whereas a trephine biopsy showed no abnormalities. A splenectomy was performed in 2019 due to worsening fatigue and cytopenia. Pathological findings showed only splenic congestion without evidence of lymphoma. The patient’s symptoms were relieved and the blood cell count returned to normal after splenectomy. However, in 2021, the patient developed progressive limb numbness and weakness. He had no fever and tests for ferritin, triglyceride, and fibrinogen were within normal range. A repeated bone marrow assessment indicated an elevated proportion of lymphocytes (88%) with abnormal morphology ( Figure 1A ). Clonal rearrangement of the TCR Vβ gene was confirmed by PCR method. The diagnosis of T-cell lymphoma was confirmed by bone marrow biopsy. The immunophenotype of the T-lymphocytes was positive for CD2, CD3, CD5, CD8, and TCRαβ with weak expression of CD57 by flow cytometry. Positron emission tomography (PET)/computed tomography (CT) showed increased ¹⁸F-fluorodeoxyglucose (FDG) uptake in the lymph nodes, left cervical plexus, and bone marrow ( Figure 1E ). The patient was treated with six cycles of bendamustine and achieved complete remission. As of the latest follow-up in October 2023, the patient remains in remission.

Case 2: A 34-year-old male presented with a 2-year history of cytopenia and splenomegaly and a 3-month history of cervical lymphadenopathy. Leukopenia and splenomegaly were first noticed in 2021 without symptoms. He then developed cervical lymphadenopathy and multiple subcutaneous nodules with mild anemia and thrombocytopenia in 2022. The patient did not show fever during the clinical course and results for HLH-related tests (complete blood count, ferritin, triglyceride, and fibrinogen) were unremarkable. Bone marrow smear revealed an elevated proportion of lymphocytes with abnormal morphology. The abnormal T-lymphocytes were positive for CD3, CD7, CD8, and TCRαβ, with weak expression of CD5 and CD57 by flow cytometry. The patient was diagnosed with T-cell lymphoma following bone marrow biopsy. Further biopsies of the cervical lymph node and subcutaneous nodule were consistent with T-cell proliferative disease/lymphoma. PET/CT showed multiple FDG-avid lymph nodes in the neck, supraclavicular, right parotid gland, and subcutaneous nodules on the shoulders and back. The patient received 6 cycles of brentuximab vedotin combined with bendamustine and achieved partial remission. However, the disease progressed 5 months later with liver involvement. The patient was treated with thalidomide combined with cyclophosphamide and dexamethasone and was preparing for allogeneic hematopoietic stem cell transplantation (allo-HSCT).

Case 3: A 20-year-old male presented with a 1-year history of swelling of the tongue and a 3-month history of right maxillofacial mass. Since May 2022, the patient has experienced progressive swelling of the tongue with surface ulceration and movement restriction. Corticosteroid treatment was initially effective, but symptoms worsened after discontinuation. The patient subsequently developed swelling in the right maxillofacial region and a soft tissue mass was palpable. He did not experience fever and hepatosplenomegaly during the course and results for HLH-related tests were unremarkable. A biopsy of the lower lip confirmed the diagnosis of PTCL. Immunohistochemical staining of the tumor cells was positive for CD3, CD5, CD7, CD8, granzyme B, and TIA ( Figures 1B–D ). PET/CT revealed significant ¹⁸F-FDG uptake in the nasopharyngeal, cervical lymph nodes, and muscles of the neck, right shoulder, and right erector spinae ( Figure 1F ). The patient received two cycles of cyclophosphamide, doxorubicin, vincristine, and prednisone therapy but showed no improvement. The patient was now treated with etoposide combined with dexamethasone and was preparing for allo-HSCT.

NGS performed on the blood sample of case 1 revealed homozygous mutation of UNC13D c.2588G>A:p.Gly863Asp. NGS was also performed on blood samples of both parents of case 1 using the same panel. Both parents were determined as heterozygous carriers of UNC13D c.2588G>A mutation which confirmed the germline origin of this mutation. NGS performed on the bone marrow sample of case 2 identified compound heterozygous mutations of UNC13D c.2588G>A and UNC13D c.1259_1260 del:p.S420Cfs*40 with a variant allele frequency of around 50%. Genotyping of the buccal mucosa further confirmed the germline origin of these 2 mutations. NGS performed on the blood sample of case 3 identified two heterozygous mutations of UNC13D c.1352C>T: p.Thr451IlE and STX11 c.401C>G: p.Ala134Gly. Germline origin of these two mutations was confirmed using the same method as case 1 Schematic locations of the mutations in UNC13D and STX11 gene products of the 3 cases are depicted in Figures 2A, B .

The impact of germline mutation in the 3 cases was evaluated according to the American College of Medical Genetics and Genomics (ACMG) guideline (9). As shown in Table 1 , variant UNC13D c.2588G>A, UNC13D c.1352C>T, and STX11 c.401C>G were all classified as uncertain significance according to the ACMG guideline. According to the gnomAD database, the frequencies of variant UNC13D c.2588 G>A and STX11 c.401C>G were much higher in the East Asian population than in the European population (0.54% and 0.06% versus 0% and 0%). In silico analysis suggests that the amino acid position of UNC13D p.G863D and UNC13D p.T451I were predicted to be damaging by the SIFT (Sorting Intolerant From Tolerant) sorting algorithm. Variant UNC13D c.1259_1260 del is novel which resulted in a frameshift mutation starting at codon 420 and leading to a premature stop codon at the region that interacted with Rab27a. It is classified as likely pathogenic for FHL type 3 according to the ACMG guideline.

We further compared the gene expression profile of the peripheral blood cytotoxic T-cells between PTCL group (case 2 and case 3), and 3 healthy controls. A total of 369 DEGs were identified between the PTCL group and healthy controls, including 188 up-regulated and 181 down-regulated genes ( Figures 2C, D ). For pathway analyses, the up-regulated DEGs were mainly enriched in autoimmune disease-related pathways and the down-regulated DEGs were mainly enriched in NK-cell mediated cytotoxicity and T-cell receptor signaling pathway. The top 15 significant KEGG pathways are shown in Figures 2E, F . For cytotoxic function analysis, CD107a degranulation assay and NK-cell activity were performed utilizing flow cytometry analysis ( Figure 3 ). Following stimulation with K562 cells, the mean fluorescence intensity (MFI) of CD107a of cytotoxic T-cells of case 2 increased only 2.2 fold, indicating impaired cytotoxic activity (the normal range reference was defined as  > 2.8 fold). The CD107a degranulation assay result of case 3 was within the normal range. NK-cell activity of case 3 was decease, while that of case 2 was within the normal range.