Abstract
Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare primary cutaneous non-Hodgkin lymphoma involving CD8+ T cells, the genetic underpinnings of which remain incompletely understood. Here we report two unrelated patients with B cell Expansion with NF-κB and T cell Anergy (BENTA) disease and a novel presentation of SPTCL. Patient 1 presented early in life with recurrent infections and B cell lymphocytosis, linked to a novel gain-of-function (GOF) CARD11 mutation (p.Lys238del). He developed SPTCL-like lesions and membranoproliferative glomerulonephritis by age 2, treated successfully with cyclosporine. Patient 2 presented at 13 months with splenomegaly, lymphadenopathy, and SPTCL with evidence of hemophagocytic lymphohistiocytosis. Genetic analysis revealed two in cis germline GOF CARD11 variants (p.Glu121Asp/p.Gly126Ser). Autologous bone marrow transplant resulted in SPTCL remission despite persistent B cell lymphocytosis. These cases illuminate an unusual pathological manifestation for BENTA disease, suggesting that CARD11 GOF mutations can manifest in cutaneous CD4+ and CD8+ T cell malignancies.
Keywords: CARD11, BENTA, SPTCL, lobular panniculitis, B cell lymphocytosis
1. Introduction
Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare and distinct primary cutaneous lymphoma. Accounting for >1% of non-Hodgkin lymphomas, SPTCL is characterized by infiltration of the subcutaneous tissue by cytotoxic TCRαβ T cells, mimicking panniculitis (1). This lymphoma presents in both children and adults, and tends to affect females more than males. Up to 20% of cases are associated with autoimmune disorders. Immunophenotypically, the neoplastic T cells are CD3+CD8+ with strong expression of cytotoxic proteins (granzyme B, TIA-1, perforin). Although patients with SPTCL have an excellent prognosis (5-year overall survival (OS) >80%), ~20% of patients develop hemophagocytic lymphohistiocytosis (HLH), which drastically impairs patient survival (5-year OS of 46%) (2).
Genetic studies of SPTCL have identified germline biallelic loss-of-function (LOF) HAVCR2 (encoding the immunomodulatory receptor TIM-3) in ~60–85% of cases (3–8). However, the genetic underpinnings of SPTCL still remain incompletely understood. Recent reports have further characterized SPTCL through the study of genes often associated with other cutaneous T cell lymphomas (CTCLs) (5, 9). CARD11, encoding for a lymphocyte-restricted scaffold protein required for antigen receptor signaling, is one such candidate with frequent somatic mutations found in CD4+ CTCLs, including Sézary syndrome (10) and adult T cell leukemia/lymphoma (ATL) (11). Heterozygous, germline gain-of-function (GOF) mutations in CARD11 cause B cell Expansion with NF-κB and T cell Anergy (BENTA) disease, a congenital lymphoproliferative disorder associated with mild immunodeficiency (12, 13). Here we report two unrelated BENTA patients with a novel presentation of SPTCL-like disease. These cases illuminate an unusual pathological manifestation for BENTA disease, and further suggest that CARD11 GOF mutations can contribute to the development of CTCLs involving both CD4+ and CD8+ lineages.
2. Materials and Methods
2.1. Patients
Patients provided written informed consent to participate in this research in accordance with the Declaration of Helsinki. Patient 1 was consented to NIH protocol 06-I-0015.
2.2. Whole Genome/Exome Sequencing
Genomic DNA were extracted from patient’s whole blood samples and used for whole genome or exome sequencing. Whole genome sequencing libraries are generated from fragmented DNA using the Illumina TruSeq DNA PCR-Free HT Library Preparation Kit and sequencing on an Illumina NovaSeq 6000 using a S4 Reagent Kit (300 cycles).
All sequencing data were processed with Burrows–Wheeler Aligner (BWA) and the Genome Analysis Toolkit (GATK) best-practice pipeline to reference genome hg19 for alignment and variants call. An in-house custom analysis pipeline modified from GEMINI (GEnome MINIng) were used for variant annotation, filtered and prioritized for disease causal variants.
2.3. Histopathology
Formalin fixed paraffin embedded (FFPE) H&E-stained slides were reviewed. The following immunohistochemical stains were performed using an automated immunostainer BenchMark Ultra (Roche) according to the manufacturer instructions. Antibodies were used against the following markers: CD20 (predilute Clone L26, Roche), CD3 (predilute Clone 2GV6, Roche), CD4 (predilute Clone SP35, Roche), CD8 (predilute Clone SP57, Roche), CD138 (predilute CloneB-838, Roche), CD123 (dilution 1:100 Clone 7G3, BD Pharmingen), TIA-1(dilution 1:100 Clone TIA1, Abcam), Beta-F1 (dilution 1:80 Clone 8A3, Thermo Scientific) and TCR-delta (dilution 1:150 Clone H-41, Santa Cruz Biotechnology). The UltraView Universal DAB Detection kit was used for signal detection. Images were taken with an Olympus Bx41 microscope, objective U-PLanFI 4x/0.13, 10x/0.30, 20x/0.50 ∞/0.17, 40x/0.75∞/0.17 with an adaptor U-TV0.5xC using an Olympus DP27 camera with an Olympus U-VTO.63xC adaptor, using “CellSens, XV Image Processing” imported into Adobe Photoshop CC 2019.
2.4. Plasmid DNA cloning
The wild-type (WT) human pUNO-CARD11 plasmid tagged with a 3X FLAG tag (WT CARD11-FLAG) (Invivogen) was previously described (14). Patient-derived mutations were introduced into the WT CARD11-FLAG construct by site-directed mutagenesis using the following oligonucleotides: K238del forward, 5’-CTAAAGCACCGGAATAAGATGGAG-3’; K238del reverse, 5’-CTCCATCTTATTCCGGTGCTTTAG-3’; E121D/G126S forward, 5’-TCCACCATTGTGGTGGACGAAGGCCACGAGAGCCTCACGCACTTCCTG-3’; E121D/G126S reverse, 5’-CAGGAAGTGCGTGAGGCTCTCGTGGCCTTCGTCCACCACAATGGTGGA-3’. Linear amplification was performed using Phusion High-Fidelity Polymerase (Thermo) followed by digestion with DpnI (Thermo) to destroy methylated DNA. To facilitate simultaneous detection by immunoblot of co-expressed WT CARD11 and patient-derived mutant constructs, the WT CARD11-FLAG plasmid was modified to include a V5 epitope tag (WT CARD11-V5). The V5 tag was generated using annealed, overlapping oligonucleotides encoding the V5 epitope tag and overlapping DNA homologous to the WT CARD11-FLAG construct (V5 tag forward, 5’-GGACGAGGACCAGCTGGGATCCCGGGCTGGAAAACCAATACCAAATCCATTATTGGGATTAGATTCTACA-3’; V5 tag reverse, 5’-CAATGTATCTTATCATGTCTGGCCAGCTAGCGGATCACTATGTAGAATCTAATCCCAATAATGGATTTGGTATTGGTTTTCC-3’). The WT CARD11-FLAG plasmid was digested with BamHI and NheI to remove the 3X FLAG tag. Subsequently, the pUNO-CARD11 backbone and the V5 epitope tag were assembled using the NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs). Mutations and V5 tag insertion were confirmed by Sanger sequencing. All plasmids were transformed into NEB 5-alpha competent E. coli (New England BioLabs), selected with blasticidin (InvivoGen) and purified using the GenElute HP Plasmid Maxiprep Kit (Sigma).
2.5. CARD11 transfections
CARD11-deficient Jurkat T cells (JPM50.6) were originally provided by Dr. Xin Lin. JPM50.6 cells were maintained in RPMI 1640 (Gibco) supplemented with 2 mM glutamine, 100 U/mL each of penicillin and streptomycin (Life Technologies), and 10% fetal bovine serum (FBS; Sigma). Transfections of CARD11 plasmid DNA were performed as previously described (14). Briefly, JPM50.6 cells (5×106/cuvette) were transfected with 5 μg plasmid DNA in 400 μL RPMI 1640 supplemented with 2 mM glutamine and 10% FBS (no antibiotics) using an ECM 630 Electroporator (BTX Harvard Apparatus) using 260 V (LV) and 950 μF. After resting the cells overnight, a portion of the cells were collected to assess CARD11 protein expression by immunoblot, and the remainder of cells were stimulated for analysis by flow cytometry. For the analysis of NF-κB-driven GFP expression by flow cytometry, cells were left unstimulated or stimulated with 1 μg/mL of anti-CD3 and anti-CD28 (BD Biosciences) for 24 hours. NF-κB-driven GFP expression was measured using an Accuri C6 flow cytometer (BD Biosciences).
2.6. Immunoblotting
Cell lysates were prepared in ice-cold 1% NP-40 lysis buffer (50 mM Tris [pH7.4], 150 mM NaCl, 0.5 mM EDTA, 1% NP-40, 0.5% deoxycholate, protease and phosphatase inhibitors) and separated on 4–20% Tris-Glycine SDS gels (Bio-Rad) followed by transfer to nitrocellulose membranes (TransBlot Turbo, Bio-Rad). After blocking, membranes were immunoblotted using the following antibodies: anti-FLAG (M2, Sigma), anti-β-actin (AC-15, Sigma), anti-V5 (D3H8Q, Cell Signaling). Antibodies were detected using IRDye-conjugated secondary antibodies and immunoblots were imaged on an Odyssey CLx Imaging System (LI-COR Biosciences).
2.7. Statistical analysis
For JPM50.6 transfections, one-way ANOVAs were utilized to test whether the GFP mean fluorescence intensity (MFI) for each putative GOF CARD11 variant was significantly greater than WT. The results were adjusted for multiple comparisons using Dunnett’s multiple comparison test. P values are included in each figure legend.
3. Results and Discussion
3.1. Patient 1 Clinical Description
Patient 1 presented at three months for a tonsillectomy due to severe obstructive apnea. He had recurrent infections during the first two years of life: two pneumococcal pneumonias, influenza A, varicella, and candidiasis. Clinical laboratory values were initially remarkable for significantly elevated CD19+ B lymphocytes (61%), 95% of which were transitional (CD19+ CD5+ CD10+) (Table 1). He had normal immunoglobulin levels, with defective anti-pneumococcal but normal anti-tetanus toxoid responses. At two years old he presented with glomerulopathy and diffuse skin lesions (Fig 1A, D), featuring dense, proliferative CD3+ TIA1+ T cell infiltrates surrounding subcutaneous adipose tissue (Fig 1D), prompting an initial diagnosis of SPTCL. He was subsequently treated with four cycles of CHOP chemotherapy, with remission of SPTCL and glomerulopathy. Whole exome/genome sequencing (WES/WGS) for the proband discovered a novel heterozygous variant of unknown significance (VUS) in CARD11 (NM_032415:c.713–715del; NP_115791:p.Lys238del; Fig 2A), which was not detected in either parent by Sanger sequencing. This de novo variant demonstrated GOF activity by inducing constitutive NF-κB activation in previously described T cell transfection assays (Fig 2B) (Snow et al, 2012), confirming a diagnosis of BENTA disease. Six months after CHOP treatment, he experienced a recurrence of membranous glomerulopathy, followed by skin lesions that reappeared one month later. Histopathological review of skin biopsy identified reactive panniculitic lesions without evidence of TCR clonality, inconsistent with recurrent SPTCL. Glomerulopathy and skin lesions rapidly resolved after starting low doses of cyclosporine. These results imply a novel disease diagnosis recently coined “atypical lymphocytic lobular panniculitis (ALLP),” whose features overlap with both SPTCL and lupus panniculitis (LP) (15, 16). The patient remains in remission on cyclosporine and TMP-SMX prophylaxis with no infectious complications.
Table 1.
Pertinent laboratory results and lymphocyte counts for Patients 1 and 2.
| Patient 1 (age: 1 year). | ||
|---|---|---|
| Immunologic Parameter | % | Abs count |
| Lymphocytes | 75% of PBMC | 4.23 (6–16) |
| CD3+ T cells | 32% of lymphocytes (51–77%) |
3.08 (2.10–6.20)* |
| CD3+ Naïve T cells | 32% of CD3+ | ND |
| CD3+ Memory T cells | 67% of CD3+
(4–24%) |
ND |
| CD3+ Central Memory T cells | 67% of CD3+ | ND |
| CD3+ Effector Memory T cells | 0.2% of CD3+ | ND |
| CD4+ T cells | 48.7% of lymphocytes (35–56%) | 2.04 (1.30–3.40)* |
| CD8+ T cells | 40.6% of lymphocytes (12–23%) | 0.85 (0.62–2.00)* |
| CD19+ B cells | 61% of lymphocytes (11–41%) |
9.25 (0.72–2.60)* |
| CD19+CD5+CD10+
Transitional B cells |
95% of CD19+
(11.4–38.4%) |
ND |
| NK cells | 5.6% of lymphocytes (3–14%) |
0.28 (0.18–0.92)* |
| IgG (mg/dL) | N/A | 622 |
| IgA (mg/dL) | N/A | 55 |
| IgM (mg/dL) | N/A | 68 |
| IgE (mg/dL) | N/A | 14 |
| Patient 2 | ||
| Laboratory Parameter (pre-HSCT) | Age 13 mos | Age 25 mos |
| Absolute lymphocyte count (x103 cells/μL) | 1.8 (2.7–12) | 0.4 (3.5–9) |
| Platelet count (x103 cells/μL) | 164 (150–400) | 16 (150–400) |
| Hemoglobin (g/dL) | 9.6 | 8.3 |
| Ferritin (μg/L) | 2463 (6–110) | 2267 (6–110) |
| Triglycerides (mmol/L) | 3.71 (0.4–1.3) | 4.36 (0.4–1.3) |
| LDH (U/dL) | 1960 (125–320) | 1018 (125–320) |
| ALT (U/L) | 40 (1–35) | 63 (1–35) |
| AST (U/L) | 111 (10–55) | 122 (10–55) |
| Fibrinogen (g/L) | 2.8 (1.6–4.1) | 1.2 (1.6–4.1) |
| D-dimer (mg/L FEU) | ND | >10 (<0.46) |
| Absolute lymphocyte count (x103 cells/μL) | 1.8 (2.7–12) | 0.4 (6–16) |
| Immunologic Parameter (post-HSCT) | Age: 3 years | Age: 11 years |
| T cells | ||
| CD3+ T cells (x103 cells/μL) | 2.81 (1.40–3.70) | 2.93 (1.20–2.60) |
| CD4+ T cells (x103 cells/μL) | 1.89 (0.70–2.20) | 2.17 (0.65–1.50) |
| CD8+ T cells (x103 cells/μL) | 0.67 (0.49–1.30) | 0.52 (0.37–1.10) |
| CD3+CD4+CD45RA+ (x103 cells/μL) | 1.26 (0.43–1.50) | ND |
| CD3+CD8+CD45RA+ (x103 cells/μL) | 0.43 (0.38–1.10) | ND |
| T cell subsets | ||
| CD3+CD4+CD45RA+CD27+
Naïve T cells (x103 cells/μL) |
ND | 1.172 (0.2–1.7)† |
| CD3+CD4+CD45RA+CD27−
Terminally differentiated T cells (x103 cells/μL) |
ND | 0.004 (0.0–0.051)† |
| CD3+CD4+CD45RA−CD27+
Central Memory T cells (x103 cells/μL) |
ND | 0.91 (0.12–0.74)† |
| CD3+CD4+CD45RA−CD27−
Effector Memory T cells (x103 cells/μL) |
ND | 0.084 (0.005–0.21)† |
| CD3+CD8+CD45RA+CD27+
Naïve T cells (x103 cells/μL) |
ND | 0.415 (0.078–0.64)† |
| CD3+CD8+CD45RA+CD27−
Terminally differentiated T cells (x103 cells/μL) |
ND | 0.024 (0.035–0.42)† |
| CD3+CD8+CD45RA−CD27+
Central Memory T cells (x103 cells/μL) |
ND | 0.069 (0.002–0.086)† |
| CD3+CD8+CD45RA−CD27−
Effector Memory T cells (x103 cells/μL) |
ND | 0.013 (0.016–0.81)† |
| B cells | ||
| CD19+ B cells (x103 cells/μL) | 7.44 (0.39–1.40) | 1.77 (0.27–0.86) |
| CD19+IgD+CD27−
Naïve B cells (x103 cells/μL) |
6.99 (0.28–1.33) | 1.624 (0.12–0.43) |
| CD19+IgD−CD27+
Memory B cells (x103 cells/μL) |
0.05 (0.05–0.39) | 0.011 (0.05–0.20) |
| CD19+IgD+CD27+
Transitional B cells** (x103 cells/μL) |
0.2 (0.02–0.18) | 0.036 (0.02–0.07) |
| B cell subsets | ||
| CD19+ B cells (x103 cells/μL) | ND | 2.437 (0.27–0.86) |
| CD19+CD20+ B cells (x103 cells/μL) | ND | 2.432 (0.12-0.74)† |
| CD19+CD27+
Memory B cells (x103 cells/μL) |
ND | 0.046 (0.05–0.20) |
| CD19+CD27−IgD+
Naïve B cells (x103 cells/μL) |
ND | 2.162 (0.12–0.43) |
| CD19+CD27+IgM+IgD+CD38dimCD24+
Memory, Non-switched B cells (x103 cells/μL) |
ND | 0.036 (0.02–0.07)† |
| CD19+CD27+IgM−IgD−CD38dimCD24− Memory, Class-switched B cells (x103 cells/μL) | ND | 0.01 (0.03–0.11) |
| CD19+CD27+IgM+IgD−
Memory, IgM+ B cells (x103 cells/μL) |
ND | 0.001 (0.0019–0.013)† |
| CD19+IgM+CD38++
Transitional B cells (x103 cells/μL) |
ND | 0.098 (0.01–0.06) |
| CD19+CD21lowCD38low Activated B cells (x103 cells/μL) |
ND | 0.049 (0.01–0.03) |
| CD19+CD27−IgM+IgD+CD21lowCD24++
Immature B cells (x103 cells/μL) |
ND | 0.13 (0.01–0.05) |
| CD19+CD27++IgM−IgD−CD38++
Plasmablasts (x103 cells/μL) |
ND | 0.022 (0.00–0.02) |
| NK cells (x103 cells/μL) | 0.53 (0.13–0.72) | 0.72 (0.10–0.48) |
| Immunoglobulins | ||
| IgG (mg/dL) | 742 (540–1600) | 994 (640–1700) |
| IgA (mg/dL) | 32 (35–240) | 196 (50–380) |
| IgM (mg/dL) | 129 (20–210) | 117 (30–240) |
| IgE (mg/dL) | 3.2 (mean 9) | 8.7 (mean 26) |
Cells (x103/μL); ND = Not Determined. N/A = not applicable.
Reference range from clinical laboratory at Alberta Children’s Hospital, University of Calgary
Non-switched memory/marginal zone-like.
Figure 1.
Clinical presentation of two unrelated children with SPTCL and B cell lymphocytosis, harboring novel heterozygous CARD11 GOF variants. (A) Subcutaneous nodules (black arrows) on back of Patient 1. (B) PET imaging of Patient 2 using fluoro-deoxyglucose (FDG, 555MBq) with CT demonstrating extensive areas of subcutaneous thickening and induration with abnormal moderate hypermetabolic activity before transplant (left), with complete resolution achieved post-HSCT (right). (C) Enhanced CT of Patient 2 demonstrating prominent soft tissue density and stranding within the subcutaneous fat pre-HSCT (top), which resolved post-HSCT (bottom). (D) Patient 1: Immunohistochemical staining of skin biopsy for CD3+ TIA1+ atypical neoplastic T cells (brown, 40x magnification), plus immunofluorescence of kidney biopsy showing IgG deposition in the glomerulus. (E) Patient 2: immunohistochemical staining of skin biopsy showing infiltration of neoplastic CD3+CD8+ granzyme B+ cytotoxic T cells (brown, 20x). (F) Absolute cell counts for peripheral blood lymphocyte subsets since initial diagnosis; gray zone delineates normal ranges for age-matched individuals (19). (G) Schematic of CARD11 protein: variants highlighted for each patient. (H-I) Top; Representative histograms depicting NF-κB-driven GFP MFI in JPM50.6 cells transfected with empty vector (EV), wild-type (WT) or mutant CARD11-FLAG expression constructs in the absence (H) or presence of WT CARD11-V5 (I), −/+ anti-CD3/CD28 stimulation. Numbers represent %GFP+ cells. Middle; Bar graphs of GFP MFI −/+ SEM (n=3); asterisks denote statistical significance vs. WT (H) or WT+WT (I) (p < 0.01). Bottom; Representative western blots showing comparable expression of CARD11-FLAG protein for each plasmid without (H) or with (I) WT CARD11-V5; β-actin serves as a loading control.
3.2. Patient 2 Clinical Description
Patient 2 was healthy with normal growth and development until she presented at 13-months-old with a three-month history of rash on her abdomen and back, initially appearing as bruises with lumpy skin underneath. The rash spread to the upper chest, upper arms, diaper area with excoriation of her labial folds, and legs. Mild splenomegaly, noted before skin presentation, worsened during this time. Additional symptoms included intermittent night sweats, fatigue, irritability, and fevers. Physical examination showed thick subcutaneous skin that was firm on palpation despite normal outward appearance. She also had hepatosplenomegaly and mild hemihyperplasia of the left arm. PET imaging revealed significant thickening of subcutaneous tissue, with avid metabolic activity noted in her abdomen and upper thighs (Fig 1B). Staging CT showed prominent soft tissue density, hepatosplenomegaly and widespread lymphadenopathy, with most lymph nodes (LNs) ~1 cm in size (Fig 1C). Bone marrow aspirate/biopsy and cerebral spinal fluid were normal. Laboratory tests showed mild anemia, lymphopenia and absent monocytes, as well as elevated ferritin, triglycerides, lactate dehydrogenase and liver enzymes (Table 2). Coagulation, fibrinogen and renal function were normal. Histopathological examination of skin biopsy revealed evidence of SPTCL (including highly proliferative, polyclonal CD3+CD8+TIA1+ CTL infiltrates) (Fig 1E) with some features of hemophagocytosis.
Prednisone therapy resulted in marked improvement of rash, resolution of fevers, increased energy and playfulness, and improvement in laboratory markers despite persistent splenomegaly. Because PET scan showed ongoing extensive involvement of subcutaneous tissues and likely involvement of the spleen, CHOP chemotherapy was started, which she tolerated well with only one admission for Staphylococcus epidermidis bacteremia. Despite significant clinical response within four cycles, a subsequent PET scan and skin biopsy revealed ongoing residual disease in her head, neck and thighs. Methotrexate was substituted for doxorubicin after six cycles due to concerns of risk for long-term cardiac toxicity, and G-CSF was added due to prolonged periods of neutropenia. After two more cycles, PET scan showed increased activity in the head and neck, with one area corresponding to a new nodule on her right cheek; biopsy confirmed persistent SPTCL. Salvage therapy with DHAP (dexamethasone, high dose cytarabine, and cisplatin) was initiated before proceeding to autologous hematopoietic stem cell transplant (HSCT) at 26 months of age. Prior to conditioning, the patient developed fevers and laboratory features of HLH (Table 2), including new significant hemophagocytosis in bone marrow aspirate. Although symptoms resolved spontaneously, dexamethasone and cyclosporine were added to the transplant regimen. She received etoposide (60mg/kg) and 1200 cGy total body irradiation as conditioning. Transplant infusion was well-tolerated, but post-transplant course was complicated by mucositis, diarrhea, and episodes of Enterococcus faecium, coagulase negative Staphylococcus bacteremia, and Clostridium difficile infection. With no further evidence of HLH, dexamethasone and cyclosporine were weaned over four and six months post-transplant, respectively. Post-HSCT PET and CT imaging showed metabolic remission and resolution of soft tissue density (Fig 1B–C); bone marrow aspirate was free of hemophagocytosis.
Four months post-HSCT, she required admission to intensive care for non-invasive ventilation with pneumonia and pleural effusions. PET scan at 8 months post-HSCT demonstrated increased uptake in the tonsils/adenoids, with multiple enlarged cervical and mesenteric LNs. Biopsy of a cervical node showed reactive lymphoid hyperplasia with a follicular pattern of progressive transformation of germinal centers (PTGC). PET scan at 14 months post-therapy showed increased uptake in cervical, supraclavicular, subclavian, axillary and inguinal LNs (1–1.5 cm). Similar to Patient 1, laboratory tests demonstrated elevated circulating B cells with low memory B cells (Table 1, Fig 1F). Whole exome sequencing revealed two in cis, de novo missense CARD11 VUSs (NM_032415:c.363G>C, NP_115791:p.Glu121Asp; NM_032415:c.376G>A, NP_115791:p. Glu126Ser) (Fig 2A). Both variants were predicted to be damaging based on CADD score (27.2 and 24.3, respectively). Indeed, these in cis CARD11 variants exhibited GOF activity in our T cell transfection studies, comparable to a confirmed BENTA patient mutation Glu134Gly (13), when expressed alone (Fig 2B) or with WT CARD11 to mimic a heterozygous state (Fig 2C). The patient developed several long-term complications from therapy, including bilateral cataracts, several absent adult teeth, high frequency hearing loss, short stature, ADHD, insulin-resistant diabetes mellitus complicated by severe hepatic steatosis/fibrosis, and bilateral slipped capital femoral epiphysis requiring surgery. She has not had recurrent sinopulmonary infections and has never been exposed to EBV; several warts responded to standard therapy. Mild lymphadenopathy, splenomegaly and B cell lymphocytosis (Table 2) persist with intercurrent illnesses.
4. Discussion
To our knowledge, these are the first cases of SPTCL reported in BENTA disease patients, or in any primary immune disorder other than TIM-3 deficiency. These findings are significant, as CARD11 mutations have not been previously linked to CD8+ T cell dyscrasias like SPTCL. These patients presented with markedly different disease courses, prompting divergent clinical management to attain stable disease. Indeed, disease features in Patient 1 may suggest ALLP, a recently described distinct disease entity with overlapping features of both SPTCL and LP. We do not yet appreciate if differences in clinical presentation could be attributed to localization of GOF variants within the CARD11 protein, or specific downstream effects on signaling pathways beyond NF-κB. It also remains unclear if impactful CARD11 GOF variants are true oncogenic drivers that preferentially predispose patients to B versus T cell malignancies. Indeed, B cell lymphomas do not occur in many BENTA patients, and somatic CARD11 variants often segregate with other specific gene mutations in ATL (11, 17). Nevertheless, our study unequivocally confirms the link between purported germline CARD11 GOF variants and selective B cell lymphocytosis; Patient 2 displayed prominent BENTA disease post-HSCT with resolution of SPTCL, suggesting additional somatic variants may have contributed to the latter Although the variant allele frequencies for CARD11 GOF mutations in both P1 (0.382) and P2 (0.417) are consistent with normal germline variant frequencies derived from whole blood WES, it remains possible (albeit unlikely) that a somatic variant arising early in lymphocyte differentiation could provide a survival/growth advantage to B and T cell descendants, presenting clinically as BENTA and/or SPTCL (18). Additional genetic modifiers (germline or somatic) and environmental factors undoubtedly influence disease penetrance and expressivity for many monogenic immune disorders. Further analysis of WGS/WES data from both patients revealed no potentially damaging private or low frequency variants (<1% MAF) in any overlapping genes/pathways (data not shown).
4.1. Conclusions
Although more mechanistic studies are required to answer outstanding questions, our novel findings highlight SPTCL and/or ALLP as a significant potential manifestation of BENTA disease that clinicians must be aware of. Our experience suggests such patients may require more aggressive immunosuppressive treatments beyond those with stable/waning polyclonal B cell lymphocytosis, particularly when HLH is observed.
Highlights:
We describe 2 patients with BENTA disease and a novel presentation of SPTCL/ALLP.
Though unusual, SPTCL noted in BENTA may require aggressive treatment if severe.
CARD11 GOF variants can occur in both CD4+ and CD8+ T cell dyscrasias.
5. Acknowledgments
We thank the patients and their families for participating in this research and Helen Matthews for regulatory assistance. This work was supported by grants from the Jeffrey Modell Foundation Specific Defect Research Program (A.L.S.). Y.Z. and H.C.S. are supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. The opinions and assertions expressed herein are those of the authors and are not to be construed as reflecting the views of Uniformed Services University of the Health Sciences or the United States Department of Defense.
6. Funding Sources
This work was supported by the National Institutes of Health (intramural award to H.C.S.) and the Jeffrey Modell Foundation (Specific Defect Research Program award to A.L.S.).
Abbreviations:
- ALLP
Atypical lymphocytic lobular panniculitis
- ATL
adult T cell leukemia/lymphoma
- BENTA
B cell Expansion with NF-kB and T cell Anergy
- CARD11
caspase activation and recruitment domain 11
- GOF
gain-of-function
- G-CSF
granulocyte colony stimulating factor
- HLH
hemophagocytic lymphohistiocytosis
- HSCT
hematopoietic stem cell transplant
- OS
overall survival
- SPTCL
Subcutaneous panniculitis-like T-cell lymphoma
- TCR
T cell receptor
- TIM-3
T cell immunoglobulin mucin 3
Footnotes
Declarations of interest: None.
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