Background

Immunotherapy has become one of the most promising treatments for refractory/relapsed B cell lymphoma [1, 2]. Among immunotherapies, chimeric antigen receptor T (CAR T) cell immunotherapy has recently been found to be a highly effective treatment for common pre-B cell acute lymphoblastic leukemia and for relapsed or refractory diffuse large B-cell lymphoma (DLBCL), resulting in approximately a 40% durable response [3–6]. Our preliminary unpublished results showed that sequential infusion of CAR 19/22 T-cells is safe and well tolerated in patients with refractory/relapsed B-cell malignancies. The safety of CAR T cell therapy and the risk of the reactivation of hepatitis B virus (HBV) in DLBCL patients who are HBV inactive carriers (HBsAg-positive with undetectable HBV-DNA) has not yet been assessed.

The reactivation of HBV is a well-known complication in patients undergoing chemotherapy or immunosuppressive therapy for hematologic malignancies, particularly in the event of stem cell transplantation or when using monoclonal antibodies against the CD20 protein, which is found on the surface of immune system B cells, such as rituximab [7–10]. The reactivation of HBV is defined as a more than 10-fold increase in HBV-DNA, the detection of HBV-DNA in a patient who previously had undetectable HBV-DNA, or when reverse seroconversion occurs with liver damage, which is seldom life-threatening [11]. Guidelines suggest that antiviral prophylaxis should be initiated at least 1 week before or when starting chemotherapy. Antiviral prophylaxis should be continued for the duration of chemotherapy and should be administered for at least 12 to 24 months after the discontinuation of the immunosuppressive regimen [12]. No guidelines are available that provide a clear consensus regarding the management of patients with resolved HBV infections undergoing CAR T cell therapy. The safety of CAR T cell therapy in patients with B-cell lymphoma and HBV infection remains completely unexplored. Here, we report a case of early HBV reactivation in a patient diagnosed with diffuse large B-cell lymphoma who was treated with the sequential infusion of anti-CD 19 and anti-CD 22 CAR T cells.

Case report

A 64-year-old woman was diagnosed with diffuse large B-cell lymphoma at the IIIB stage (Ann Arbor staging system) 5 years ago and received a standard dose of R-CHOP (rituximab, cyclophosphamide, vincristine, adriamycin and prednisone) for 8 cycles and achieved complete remission. She had an enlargement of the cervical lymph nodes and suspected remission four and half years after initial diagnosis. The patient underwent rebiopsy of the cervical lymph nodes. The pathology revealed a relapse of the primary disease. Next-generation sequencing (NGS) of the resected lymph nodes revealed a CARD11 K215 T mutation without any other mutations. After relapse, she received a standard dose of R-ICE (rituximab, ifosfamide, carboplatin, and etoposide) for 2 cycles and intermittently took lenalidomide, but the disease still progressed. She had a history of HBV infection, and blood tests were positive for HBsAg, anti-HBc and anti-HBe, with undetectable serum HBV-DNA levels. Anti-hepatitis C virus (HCV) antibody results were negative. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were consistently normal, and no hematochemical and liver ultrasound findings were indicative of chronic active hepatitis. The patient received antiviral prophylaxis with entecavir (0.5 mg per day) during chemotherapy and had discontinued antiviral prophylaxis 1 year ago.

In the presence of relapsed disease, we tried to use CAR T therapy with anti-CD19 and anti-CD22 CAR constructs to generate CAR T 19 and CAR T 22 cells, respectively. Although the HBV-DNA level remained undetectable, we reintroduced entecavir (0.5 mg per day) 2 months before CAR T cell therapy. Autologous peripheral blood mononuclear cells (PBMCs) were cultured with an anti-CD3 monoclonal antibody to induce T cell proliferation. The anti-CD22 CAR T and anti-CD19 CAR T cells were cultured for 14 days before infusion. Subsequently, she was conditioned with a standard lymphodepleting chemotherapy regimen consisting of fludarabine (25 mg/m²) and cyclophosphamide (20 mg/kg) on day − 4 ~ − 2. The sequential infusion of CAR T cells was performed as follows: 4 × 10⁶ cells/kg of CAR T 22, divided into two infusions on day 0 to day + 1 (7/3/2018 and 7/4/2018), followed by 4 × 10⁶ cells/kg CAR T 19, divided into two infusions on day + 2 and day + 3 (7/5/2018 and 7/6/2018). The autologous CAR T cells proliferated in vitro, and the tumor-cytotoxic effect of CAR T 19 and CAR T 22 was up to 53.36 and 57.71%, respectively, with an effector/target ratio of 25:1 (Fig. 1a). After CAR T infusions, she had 1~2 degree of cytokine release syndrome with elevated IL-6 and ferritin. The levels of IL-6 and ferritin gradually returned to the baseline levels 2 weeks after CAR T therapy (Fig. 1b–c). On day + 10 after CAR T cell infusion, the WBC increased to 1.26 × 10⁹/L with 0.33 × 10⁹/L neutrophil granulocytes, 0.51 × 10⁹/L lymphocytes and 0.27 × 10⁹/L monocytes. The dynamic changes in white blood cells and lymphocytes after CAR T cell therapy are depicted in Fig. 1d. Lentivirus copies detected by PCR and CD19⁺CD22⁺ CAR T cells detected by flow cytometry from the CAR T cell infusion significantly increased with a decline in B lymphocytes, indicating that CAR T cells reached their peak levels after 3 weeks (Fig. 1e–f). In addition, the ratio of CD4⁺/CD8⁺ T cells in the peripheral blood was significantly below normal two and 3 weeks after CAR T cell infusion (Fig. 1g).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 1

Sequential infusion of anti-CD 19 and anti-CD22 CAR T cell therapy. a In vitro tumor-cytotoxicity effect of CART 19 and CART 22 cells at effector/target ratios of 25:1, 5:1 and 1:1. b Levels of IL-6 after CAR T cell therapy. c Levels of ferritin after CAR T cell therapy. d Dynamic white blood cell numbers and lymphocyte numbers before and after CAR T cell therapy. e Copies of lentivirus-containing CARs in the peripheral blood after CAR T cell therapy. f CAR T cell and B cell numbers after CAR T cell therapy. g The ratio of CD4+/CD8+ T cells in the peripheral blood after CAR T cell therapy

The patient was instructed to remain on antivirals. The patient, however, became noncompliant and discontinued entecavir 1 month after CAR T immunotherapy. She was admitted with a 7-day history of vomiting, jaundice, itching, and dark urine two and half months after CAR T cell therapy. The primary disease was stable two and half months after treatment. No superficial lymph nodes could be palpated. Cervical lymph node and abdominal ultrasonography revealed that no superficial and deep lymph nodes could be detected. The evaluation of CAR T therapy by flow cytometry revealed 1.4% CD19⁺CD22⁺ CAR T cells. After excluding other possible causes of acute liver damage, HBV reactivation was suspected. Blood tests were positive for HBsAg, anti-HBc HBeAg and anti-HBe. The level of HBV-DNA was 2.57 × 10⁸ IU/mL. Anti-HAV IgM, anti-HCV and anti-HIV showed negative results. The blood tests performed after admission showed elevated ALT and AST. BUN and creatinine were normal at that time. No ascites was found by ultrasonography. Following the reintroduction of entecavir (1 mg once daily), a decline in HBV-DNA copies was observed, but the ALT, AST and bilirubin levels continued to increase (Fig. 2a–b), and there was no improvement in the clinical condition of the patient. The patient was then transferred to the department of infectious disease. Plasma exchange therapy with an artificial liver support system was used periodically every five to 6 days. The patient soon entered a stage of hepatic coma, and the phenomenon of enzyme bilirubin separation was observed, which is associated with poor outcomes. Unfortunately, her symptoms worsened. She passed away because of deteriorated hepatic function 40 days after admission. The brief chronology of the key clinical events in this case is depicted in Table 1.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 2

Longitudinal evaluation of hepatitis B virus (HBV)–DNA, liver enzymes, and bilirubin. a The dynamic changes in ALT, AST and total bilirubin before and after CAR T cell therapy. b HBV-DNA copies before and after CAR T cell therapy

Funding

This work was supported by the Key Program of the National Natural Science Foundation of China (81830008, 81630006 and 81873427).