1 Introduction
Epstein–Barr virus (EBV)-associated lymphoproliferative disease (EBV-LPD) refers to a group of lymphoid diseases, ranging from inert polyclonal proliferation to invasive lymphoma. This condition commonly occurs in immunocompromised patients, particularly after solid-organ transplantation (SOT) or hematopoietic stem-cell transplantation (HSCT) [
1] but is rarely reported after autologous HSCT (ASCT) [
2,
3] and never after B cell-directed chimeric antigen receptor-modified T (CAR-T) cell therapy.
CAR-T cell therapy has unprecedented efficacy in refractory or relapsed B-cell malignancies, with high treatment responses and a manageable safety profile. With an extended follow-up, previously unknown long-term adverse events can be recognized and managed. Here, we report for the first time two patients who developed EBV-LPDs after CAR-T cell therapy with ASCT.
2 Cases report
2.1 Case 1
In July 2016, a 33-year-old female patient was diagnosed as double-hit EBV-positive diffuse large B-cell lymphoma (DLBCL), nongerminal center B-cell-like subtype, manifesting as multiple nodal and extranodal involvements, with Ann Arbor stage of IVB and NCCN-IPI score of 3.
After failure of four lines treatment for lymphoma, the patient enrolled in a clinical trial of ASCT in tandem with CAR-T cell therapy targeting CD19 and CD22 (ChiCTR-OPN-16009847) [
4] at our center in June 2017. In this trial, after myeloablative chemotherapy, autologous stem cells were infused on day 0, followed by the infusion of CD19 and CD22-directed CAR-T cells (CAR19/22 T cell) cocktail on day 5. Fig.1 depicts the lentiviral copies of CAR19 and CAR22 transgenes in peripheral blood. The patient had partial remission after three months and gradually achieved complete remission (CR) at month 17; the CR was maintained thereafter, as assessed by positron emission tomography/computed tomography (PET/CT) or contrast-enhanced CT. Tab.1 describes the detailed clinical features, pathologic changes, and treatments.
After 44 months, PET/CT scanning revealed multiple diseases with upregulated metabolism, involving nasopharynx, bilateral cervical lymph nodes, muscle, and spleen. After the biopsy of involved cervical lymph node and nasopharynx, the patient was diagnosed as pleomorphic B cell EBV-LPD, grade 2, with monoclonal plasmacytic hyperplasia. Germline missense mutations in genes LYST (NM_001301365.1:c452A>G[p.H151R]) and BCL10 (NM_003921.4:c485C>T[p.T162M]) were detected by next-generation sequencing and predicted to be potentially pathogenic. The pathologic changes are described in Fig.2 and Tab.1. When EBV-LPD was diagnosed, CAR19 and CAR22 transgenes were not detected by droplet digital polymerase chain reaction (PCR) of the peripheral blood, and the levels of peripheral B cells and serum IgG were restored to normal. EBV-DNA measured 8.05 × 106 and 1.41 × 106 copies/mL in the plasma and peripheral blood mononuclear cells (PBMCs), respectively, according to the quantitative real-time PCR (EBV-qPCR). This finding was further confirmed in peripheral B cells enriched by magnetic activated cell sorting. The patient then received pembrolizumab treatment (200 mg, once a month, four cycles). Four months later, contrast-enhanced CT showed that the swollen cervical lymph nodes significantly shrunk and partially disappeared. Copies of EBV-DNA in plasma and PBMC gradually declined and turned negative (Fig.3). After 17 months, the patient achieved and maintained CR according to PET/CT assessment.
2.2 Case 2
Presented an enlarged right axillary lymph node, a 71-year-old male patient was diagnosed as EBV-positive Burkitt’s lymphoma with IVB Ann Arbor stage due to multiple nodal, bone marrow, and extranodal involvements in May 2019.
After failure to two lines of treatment, the patient was enrolled in the clinical trial of ASCT in tandem with CAR19/22 T cell cocktail infusion (ChiCTR-OPN-16009847) [
4] in November 2019. Fig.1 depicts the lentiviral copies of CAR19 and CAR22 transgenes in peripheral blood. Tab.1 describes the detailed clinical features, pathologic changes, and treatments. The patient showed CR at month 6, and such condition was maintained thereafter, as assessed by PET/CT or contrast-enhanced CT.
After 33 months, enlarged lymph nodes in the left neck with upregulated metabolism were revealed by PET/CT scanning. After biopsy, the patient was diagnosed as CD20-negative pleomorphic B cell EBV-LPD, grade 2, with infiltration of reactive T cells (Tab.1). EBV-DNA in the plasma and PBMCs were confirmed by EBV-qPCR. Although CAR transgenes were not detected at this point, B cell aplasia in peripheral blood was sustained. The patient received two cycles of sintilimab (200 mg, once a month) and one cycle of tafasitamab (200 mg). The levels of EBV-DNA in the plasma and PBMCs gradually decreased to values lower than the detection threshold (Fig.3). Four months later, CR was achieved, as demonstrated by PET-CT, and sustained thereafter.
3 Discussion
EBV represents a widely disseminated herpesvirus (human herpes virus 4) that persists asymptomatically as a latent infection for life in most adults; the presence of this virus is associated with the development of nasopharyngeal carcinoma and lymphoproliferative disorders, especially in individuals with congenital or acquired immunodeficiency. We reported the prevalence of genetic aberrations in epigenetic modifiers and retinoic acid-inducible gene I-like receptor pathway in adult patients with T/natural killer (NK)-cell EBV-LPDs and their association with unfavorable prognosis [
5], which suggest the genetic pathogenesis of EBV-LPDs. Germline mutations in genes
LYST and
BCL10 were detected in case 1. LYST is involved in the regulation of lysosome transport, and its mutation may lead to hemophagocytic lymphohistiocytosis (HLH) and immunodeficiency syndrome. BCL10 is involved in the regulation of NF-κB signal pathway, and its mutation can cause immunodeficiency and repeated bacterial or viral infection [
6]. These aberrations may contribute to the underlying susceptibility and pathogenesis of the patient’s successive EBV-positive DLBCL and pleomorphic EBV-LPD.
EBV-LPD commonly affects immunocompromised patients, particularly after SOT or HSCT. Most EBV-associated post-transplant LPDs (PTLDs) are derived from B cells, and 5% are derived from T or NK cells. Although T/NK-cell-derived PTLD is rare, it is generally associated with poor prognosis. Germline mutations of genes involved in familial HLH and primary immunodeficiencies have been identified in various EBV-LPDs [
7,
8], which suggests that persistent EBV infection may be the result of viral evasion strategies to host immune responses. CAR-T cells targeting CD19 or CD22 eliminate B cells
in vivo, which leads to severe immunodeficiency and impaired generation of EBV-specific antibody or memory B cells [
9], and may drive and accelerate the development of EBV-LPD post-CAR-T cell therapy, particularly when CAR-T cell therapy is combined with ASCT.
Current treatment strategies for PTLD include the reduction of immunosuppressants, rituximab, chemotherapy, and EBV-specific cytotoxic T cell or CAR-T cell therapy. Immune checkpoint blocking with programmed death receptor 1/programmed death ligand 1 (PD-1/PD-L1) inhibitors has been adopted in the treatment of EBV-LPDs, EBV-related HLH, and extranodal NK/T cell lymphoma [
10]. PD-1/PD-L1 inhibitors block the activity of PD-1 and PD-L1, expand PD-1-positive T cells, and restore the expressions of degranulation and costimulatory genes in CD8
+ T cells, which normalize the cytotoxic activation program that correlates with EBV clearance [
11]. Thus, in addition to B-cell depleting agents, immune checkpoint inhibitors represent a rational treatment choice for EBV-LPDs post-autologous CAR-T cell therapy. Consistent with the definite pathological diagnosis, the indolent clinical course and robust treatment response induced by B-cell depleting agents or immune checkpoint inhibitors further support the diagnosis of pleomorphic B-cell EBV-LPD post-CAR-T cell therapy in the studied patients.
In addition, plasma EBV-DNA, which is released from EBV-infected cells to the plasma, is a valuable biomarker for disease monitoring, efficacy assessment, and prognostication in patients with EBV-positive lymphomas [
12]. An increasing trend of EBV-DNA copies has been observed in the plasma and PBMCs before the diagnosis of EBV-LPDs in the studied patients, which suggests that monitoring of EBV-DNA copies in peripheral blood post-CAR-T cell treatment can alert clinicians regarding the occurrence of EBV-LPDs after CAR-T cell therapy, especially in patients with previous history of EBV-positive lymphoma.
In summary, this work is the first report on EBV-LPD after B-cell-directed CAR-T cell therapy in tandem with ASCT, which may represent a long-term AE after CAR-T cell therapy. More evidence is needed to gain insights into the pathogenesis of EBV-LPD. Immune checkpoint agents are effective in this setting, although large-scale studies and extended follow-up are warranted.
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