Authors: Yuehui LIN, Shuangyou LIU, Chunrong TONG
We have successfully treated a lady with advanced diffuse large B-cell lymphoma (DLBCL) by sequentially murinized CD19-CART, humanized CD20-CART and humanized CD22-CART. The patients lymphoma cells had complex chromosomal abnormalities and TP53 gene variation of function loss, and She had higher hepatitis B virus(HBV) DNA load in peripheral blood(PB). CART therapy made her survive for nearly 2 years. From the patients treatment, we have learned the following experience and lessons:
1. CART therapy is a revolutionary therapy for refractory/relapsed DLBCL (rr-DLBCL), the patient can achieve complete remission(CR) even though there exist complex abnormal chromosomes and gene variations indicating a very poor prognosis, and the high load of HBV-DNA in PB.
2. Our team has almost 4 years experience to treat the side effects of CART cells, it is relatively safer during CART therapy. We recommended that the patients who failed the first-line chemotherapy (or plus CD20 antibody/and other antibody therapy) receive CART therapy earlier to avoid more serious chromosomal abnormalities, genetic variations or immune system damage caused by excessive chemotherapy, which make the patient more difficult be cured.
3. Lymphoma cells are heterogeneous, which means they have different antigens, gene variations, chromosome abnormalities etc. in one patient, the patient is therefore hard to be cured just by single-target CART therapy. It is rational that multi-target CART therapy may significantly prolong the duration of CR, even cure the patient.
4. Under the therapy of strong anti-virus drug and immune globin, CART therapy is safe for patients with high HBV-DNA loads in PB.
5. The side effects of CART therapy are related with the tumor burden.
6. Whether triple-targeted CART therapy can cure patients with complex chromosomal abnormalities and poor gene variations such as TP53 and whether target drugs are needed for maintenance remain to be further researched.
DLBCL accounts for about 30-40% of non-Hodgkin lymphoma (NHL) in adults, its standard treatment is anti-CD20 monoclonal antibody (mab) combined with chemotherapy (also known as immunochemotherapy) with a cure rate of 50-70%. However, 30-50% patients are primary resistant or relapse after the first line therapy (called as rrDLBCL). The 2-year overall survival (OS) is only<20% even after the second or more chemotherapy combined with radiotherapy, targeted therapy or hematopoietic stem cell transplantation (HCT) etc. In addition, long-term repeated high dose chemotherapy, radiotherapy or HCT will seriously affect the patients quality of life and dramatically increase the cost of medicine.
The efficacy of CD19 specific chimeric antigen receptor cell (CD19-CART) therapy for rrDLBCL has been confirmed, and it has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of rrDLBCL. However, several clinical reports have showed that the CR rate is only about 50% for rrDLBCL by CD19-CART therapy, and most patients relapsed. Our clinical data have confirmed that CD22-CART, 20-CART were also effective in the treatment of rrB-cell tumors, thus, we designed sequential multiple target CART therapy for rrB-cell tumors to overcome the shortcomings of single-targeted CART therapy, thereby improve the CR rate, prolong the duration of CR, and then cure the patient.
We hereby reported the process of sequential multi-target CART therapy for an elderly female rrDLBCL patient with complex chromosomal abnormalities, TP53 gene variation, high HBV-DNA load in PB, splenomegaly and bone marrow involvement.
I. The diagnosis and treatment before our hospital
In August 2016, a 54-year-old female patient was admitted to a famous hospital for her enlarged lymph nodes in right retro auricular. Complete blood count showed hemoglobin (HGB) 61g/L and platelet (PLT) 76×109 / L. The bone marrow(BM) cells were tested by morphological and flow cytometry(FCM) and found 42.6% abnormal B lymphocytes with the antigens expression of CD45dim, CD19, CD20, CD22, HLA-DR and cCD79a. Chromosome karyotype of BM cells by G-banding was 81 (4n), XXXX, -1, -1, del (1) (p36.1) × 2, del (20) (q11.2), de (?)t(1:?(p13:?) ×2. Ultrasound showed splenomegaly and enlarged lymph nodes in the retroperitoneum, both sides of the neck, bilateral armpits and groins. PET-CT showed increased FDG metabolism in multiple lymph nodes, bone marrow cavity and splenomegaly. Pathological examination of resected right inguinal lymph nodes diagnosed as DLBCL, NGC origin, CD20(+), CD79a(+) CD3(-), CK(-), CD10(-),CD21(FDC+), CyclinD1(-), c-myc(-), Bcl-6(-), MUM1(+), PAX-5(+) , bcl-2 (+), CD23 (FDC+), CD5 (weakly +), CD38 (-), KI-67 (50% +). Fluorescence in situ hybridization(FISH) showed EBER (-).
Several siblings of the patient's mother carried the hepatitis B virus. The patient's mother died from "lung cancer".
The patient received R-CHOP (rituximab, cyclophosphamide, liposomal doxorubicin, vincristine and prednisone or dexamethasone) therapy, After 4 cycles of R-CHOP therapy，PET-CT and BM results showed partial remission (PR). Then she received 2 cycles of R-DA-EPOCH (rituximab with etoposide, cyclophosphamide, liposomal doxorubicin, vincristine and dexamethasone). In May 2017, her lymph node enlarged again in the neck, which means DLBCL progression. Then, ibrutinib and lenalidomide were given for 4 months. In the first 3 months, the lymph nodes shrank, but in the fourth month, they no longer shrank. Thus, on October 16, 2017, the patient came to our hospital.
II. Diagnosis and treatment after the first admission to our hospital
1. Inspection at admission
1) Physical examination: anemic appearance with an ECOG score 2, multiple enlarged lymph nodes on both sides of the neck with the largest about 3.5CM in diameter, the spleen was the 13CM from the second line and 7CM from the third line under rib.
2) PB tests: WBC 1.42×109/L, HGB 65g/L, and PLT 59×109/L. HBV-DNA 7.917×105 IU/ml.
3) BM tests: Morphological examination showed 14% medium to large abnormal lymphocytes, FCM showed 22.35% abnormal medium to large mature monoclonal B cells with strong expression of CD19, CD22，weak expression of CD20. 121 kinds of common hematological malignancies gene screening showed TP53 and NOTCH2 variations. G-Banding chromosome analysis showed complex chromosomal abnormalities: 72-76<3n>, XXX, -1, del(1)(p36)×2, +3, del(3)(p21)×2, -5, +6, +9, +10, +12, Add(16)(q12), +18, +3mar, inc[cp5]/46, and XX. FISH demonstrated amplification of the genes BCL2, BCL6 and MYC, which was possibly associated with the change in the number of chromosomes.
BM cell morphology showed 14% medium to large abnormal lymphocytes
BM cell immunophenotype by FCM: 22.35% medium to large abnormal mature B cells with strong expression of CD19, CD22, dim expression of CD20.
BM cell genes showed variations as follows:
Bone-marrow chromosomes: 72-76<3n>, XXX, -1, del(1)(p36)×2, +3, del(3)(p21)×2, -5, +6, +9, +10, +12, Add(16)(q12), +18, +3mar, inc[cp5]/46, and XX.
BM cells FISH: Amplification of the genes BCL2, BCL6 and MYC, which was possibly associated with the change in the number of chromosomes.
3) Imaging examination:
l Color Doppler ultrasound: enlarged multiple lymph nodes on both sides of the neck, bilateral supraclavicular regions, bilateral armpits and groins, diffusely enlarged spleen with an abnormal shape with approximately 11.1cm in thickness, slightly enlarged liver.
l ECG: Sinus rhythm and ST-T wave changes.
l Chest CT: 1. Infection in both lungs, pleural thickening & adhesion on both sides; 2. multiple enlarged mediastinal lymph nodes; 3. splenomegaly.
l Head and Brain MRI: 1. Ischemic lesions in frontal and parietal lobes on both sides; 2. significant thickening of external and inferior orbital walls on both sides (pls combine it with the clinical diagnosis for further confirmation); 3. mastoiditis developing from otitis media.
Because the patient previously failed multiple lines of chemotherapy and target therapy, with complex chromosomal abnormalities (including -5 abnormalities mostly caused by chemotherapy) and TP53 variation of function loss, poor general condition, lower WBC, HGB and PLT, ineffective transfusion, it was unlikely to achieve CR by chemotherapy alone, CART therapy may be her only choice now. But the high tumor burden and HBV-DNA may result in life-threatening side effects such as severe cytokine release syndrome (CRS), uncontrolled HBV infection or other kinds of infection, bleeding etc. The patient and her family were repeatedly informed of such risks, and they still asked for CART therapy.
Based on immunophenotyping of tumor cells in bone marrow and previous experience, we first selected murinized CD19-CART therapy for this patient, because murinized CD19-CART has been the most effective CART up to now, the patient's tumor cells strongly express CD19.
First we treated the patients HBV infection with entecavir, lamivudine and gamma globulin for intermittent infusion, and lung infection with antibiotics. Then we harvested her PB lymphocytes(PBL) by blood separator (COBE Spectrum) for murinized CD19-CART preparation. According to our previous experience, the lower the tumor burden, the milder the CRS, we treated the patient with VLD chemotherapy (vincristine for 1d, dexamethasone 30mg/d x 4d, and L-asparaginase 10000 units/d ⅹ2d) to reduce tumor burden. Then she received the pre-conditioning chemotherapy with cyclophosphamide (CTX) 0.2ⅹ3d (lower than the standard dose because of the patient’s poor heart function), fludarabine (Flu) 45 mg/d ⅹ3d. To prevent serious CRS, only 0.5ⅹ106/kg of CD19-CART cells was infused initially, 7 days later, no severe CRS occurred, another 0.5ⅹ106/kg of CD19-CART cells was infused again.
On the 6th day after the first infusion of CART, multiple cytokines increased in PB, including interleukin-6 [IL-6] 62.66 pg/mL ↑, interleukin-10 [IL-10] 449.36 pg/mL ↑, interferon-γ [IFN-γ] 49.29 pg/mL ↑, soluble CD25 18424 pg/mL ↑, tumor necrosis factor-α [TNF-α] 53.52 pg/mL ↑. Total bilirubin [TBIL] 28.78 µmol/L ↑, albumin [ALB] 30.55 g/L ↓, direct bilirubin [DBIL] 9.90 µmol/L ↑, total protein [TP] 51.46 g/L ↓, indirect bilirubin [IBIL] 18.88 µmol/L ↑, prealbumin [PA] 46.40 mg / L ↓, albumin to globulin [A/G] ratio 1.46 ↓, calcium [CA] 1.87 mmol / L ↓, and complement C4 [C4] 0.05 g / L ↓. On the 7th day, the patient felt chest distress. On the 9th day, she developed low fever with the maximum T: 37.6°C. On the 10th day, her maximum T 38.1°C, which was relieved by oral Motrin. On the 12th day, her maximum T 39.3°C, which was relieved by oral Motrin. On the 13th day, the tests in PB showed IL-6 83.04 pg /mL ↑, IL-10 448.84 pg/mL ↑, IFN-γ 136.26 pg/mL↑, soluble CD25 25620 pg/mL ↑, and TNF-α 31.11 pg/mL ↑. 2mg dexamethasone was infused and her body temperature became normal.
After the first infusion of CART, the lymph nodes and spleen gradually shrank. On the 15th day, BM morphological and FCM tests showed no lymphoma cells and the chromosomes became normal. On the 24th day, PLT increased to 43×109/L, HGB to 92 g/L, and WBC 1.04×109/L and transfusion was no longer needed. During the CART therapy, the HBV-DNA in PB was between 104 and 105 IU/ml, transaminase and bilirubin remained normal. On the 28th day, the patient was discharged.
Figure1: WBC in PB
Figure 2: HGB change
Figure3: PLT change
The patient was followed up after discharge. At 3 months after the first CART infusion, her WBC、HGB、PLT in PB returned to normal. Ultrasound tests in a local hospital showed that the spleen and the lymph nodes throughout the body were normal. The patient had good condition and traveled outside several times. she refused further therapy and examinations.
III. The second hospitalization after relapse
In October 2018, nearly 1 year after the initial infusion of CART, the patient found lymph nodes enlarged again in the neck and felt distention in abdomen. On October 29, 2018, she was admitted to our hospital again.
1. Inspection at admission
1) Physical examination: good general condition, ECOG score 1. multiple lymph nodes enlarged on both sides of her neck (the largest one 2.5cm×2.0cm). The spleen was felt under the ribs.
2) PB tests: WBC, HGB, PLT were little lower than normal.
3) BM tests: morphological examination and FCM showed 7.79% abnormal B lymphocytes which expressed CD19, CD22 and CD20. Chromosome karyotype by G-banding were 75, XXX, +del(1)(q25), +del(3)(q21),-4,-5,+6,+10,+12,+16,-17,+20, +2mar,inc [cp3]/46. 339 kinds of common genes screening for hematological malignancies found the following genes variation: TP53 p.R248Q, PRL10 p.K39fs, TNFAIP 3 p.Q700X, and DNMT3A p.G543V. The HBV-DNA in PB was 9.5×105 IU/ml.
3) Imaging tests: Ultrasound showed multiple lymph nodes enlarged on both sides of the neck, right supraclavicular, the largest one 2.6×0.9×2.0 cm, a diffusely enlarged spleen. Chest CT showed lung infection.
Because the patient achieved CR for approximately 1 year after CD19-CART therapy with minor side effects, her family required CART therapy. According to our previous observation, if the patient had received CD19-CART therapy, even though the tumor cells express CD19, the efficiency of the second CD19-CART therapy would not be good. Our previous experience has demonstrated that the efficiency of humanized CD20-CART therapy is better than humanized CD22-CART if the tumor cells express the same strength of antigen. The patient already discontinued anti-CD20 mab for more than 1 year, and her lymphoma cells strongly expressed CD20, so we selected humanized CD20-CART as the second CART therapy.
First the antibiotics and two anti-HBV drugs were used to treat lung infection and control HBV infection. The patient's tumor burden was not as high as the first admission, thus, the patient received pre-conditioning chemotherapy Flud 45mg d1-3 and CTX 0.2g d1-3, dexamethasone 15mg q12h d1-3. On November 7, 2018, total 1.1×10^6/kg of humanized CD20-CART cells was infused. On 7th day, the patient developed mild chills and high fever with the maximum T 39.7°C, which were relieved for the moment after Motrin. Due to repeated hyperpyrexia, 2 mg dexamethasone was given on day 8, then, the temperature turned to normal.
At approximately 20 days after infusion of CD20-CART cells, the enlarged lymph nodes and the spleen cant be touched. BM morphology and FCM showed no lymphoma cells, chromosome was normal. The patient was then discharged from hospital.
IV: CART therapy for the third time
Considering that the patient relapsed after the first CART therapy, she was adviced to receive humanized CD22-CART to prevent relapse again. 4 months after CD20-CART therapy, she came to receive the third CART therapy.
1. Inspection at admission
1) Physical examination: normal.
2) PB tests: WBC, HGB and PLT were normal, HBV-DNA in PB was 103 IU/ml.
3) BM tests: morphology and FCM showed no lymphoma cells.
4) Imaging tests: Ultrasound showed multiple lymph nodes in the neck, the largest one is 0.89×0.40×0.58cm, and the spleen normal.
The two kinds of anti-HBV drugs continued to be used, after FC chemotherapy as before, On March 13, 2019, 1.7×105/kg of humanized CD22-CART cells was infused. During treatment, the patient had no side effects.
The patient was followed up by telephone until now. She feels very well, the WBC, HGB and PLT have been normal. But she refused PET-CT scan.