재발성혹은불응성신경모세포종환자에서의활성화 T- 림프구세포치료 2 예

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1 Clinical Pediatric Hematology-Oncology Volume 25 ㆍ Number 2 ㆍ October 2018 CASE REPORT 재발성혹은불응성신경모세포종환자에서의활성화 T- 림프구세포치료 2 예 최정윤ㆍ안홍율ㆍ홍경택ㆍ홍채리ㆍ강형진ㆍ신희영 서울대학교의과대학소아과학교실, 서울대학교암연구소 Two Cases of Adjuvant Immunotherapy with Cytokine-Induced Killer Cells for Relapsed or Refractory Neuroblastoma Jung Yoon Choi, M.D., Hong Yul An, M.D., Kyung Taek Hong, M.D., Chery Hong, M.D., Hyoung Jin Kang, M.D. and Hee Young Shin, M.D. Department of Pediatrics, Seoul National University College of Medicine, Seoul National University Cancer Research Institute, Seoul, Korea The treatment outcomes of relapsed or refractory neuroblastoma have been unsatisfactory till date. We reported two cases of adoptive immunotherapy using cytokine-induced killer (CIK) cells against relapsed or refractory neuroblastoma. CIK cell production was attempted in three patients, out of which two patients exhibited adequate levels of CIK cell production. Two patients completed full term of CIK cell infusions (weekly for 6 weeks and then biweekly for 8 wk) without serious adverse events. The progression-free survivals for the two patients were 1.9 and 4.1 months. Their overall survivals were 16.7 and 28.7 months. Although the efficacy was unclear, CIK cell infusion combined with other treatment strategies may have prolonged overall survival in refractory neuroblastoma patients. Further studies are needed to determine the exact role of CIK cell-based immunotherapy in relapsed or refractory neuroblastoma patients. Key Words: Neuroblastoma, Refractory, Cytokine-induced killer cells pissn / eissn Clin Pediatr Hematol Oncol 2018;25: Received on September 21, 2018 Revised on September 30, 2018 Accepted on October 15, 2018 Corresponding Author: Hee Young Shin Department of Pediatrics, Seoul National University College of Medicine, Seoul National University Children s Hospital, Seoul National University Cancer Research Institute, 101, Daehak-ro, Jongno-gu, Seoul 03080, Korea Tel: Fax: hyshin@snu.ac.kr ORCID ID: orcid.org/ Introduction Neuroblastoma is a heterogeneous childhood malignancy. Low-risk patients can be cured only by surgery, but high-risk patients, which account for about half of the cases, have only 20% long-term survival rates with surgery and chemotherapy [1]. In addition to conventional chemotherapy, treatment strategies, such as high-dose chemotherapy (HDC) and autologous stem cell transplantation (ASCT), local radiotherapy (RT), 13-cis-retinoic acid (CRA), interleukin-2 (IL-2), and 131 I-Metaiodobenzylguanidine ( 131 I-MIBG), have 202

2 CIK Cell Immunotherapy for Neuroblastoma improved the survival rates of high-risk neuroblastoma by 50-70% [2-4]. However, some patients with relapsed or refractory neuroblastoma are still difficult to cure despite multimodal treatments, and develop treatment toxicities due to cumulative doses of several chemotherapeutic agents. Allogeneic hematopoietic stem cell transplantation has also been proposed as a therapeutic alternative, but it is not yet widely used [5]. Therefore, it is necessary to use a novel treatment strategy with potential antitumor effects and fewer side effects. Cytokine-induced killer (CIK) cell-based immunotherapy has been reported to exhibit antitumor effects in some malignant tumors such as hepatocellular carcinoma (HCC) and pancreatic cancer in adults [6,7]. Based on these effects, an autologous CIK cell-based immunotherapeutic agent (Immuncell-LC; Green Cross Cell Crop, Seoul, Korea) was developed using standard protocol. CIK cells were approved as an adjuvant therapy in patients who had undergone curative treatment for HCC with surgery, radiofrequency ablation, or percutaneous ethanol injection [6]. CIK cells are produced by extracorporeal cultures of patients peripheral blood mononuclear cells through co-stimulation with interleukin-2 (IL-2) and anti-cd3 antibody [6]. We reported the immunotherapy mediated by CIK cells in relapsed or refractory neuroblastoma. We investigated the efficacy and safety of immunotherapy using CIK cells in these patients. Informed consent was obtained from patients and their parents for the treatments and procedures. This study was approved by the institutional review boards (IRB number: H ). Case Report We employed CIK cell therapy for three patients with relapsed or refractory neuroblastoma from January 2013 to Table 1. Individual characteristics of patients Patient No. Sex Age at diagnosis Primary site Involved site Stage N-myc amplification Previous treatments Adequate production of CIK cells 1 M 12.2 yr Adrenal gland 2 F 3.2 yr Adrenal gland Retroperiteneum, multiple bone metastasis Multiple bone metastasis, BM IV (+) CDDP, etoposide, ADR, CPM 13 partial mass excision RT (retroperitenoum, residual lesion) 1 st relapse para-aortic LN dissection CPM, topotecan, etoposide 23 mass excision ifosfamide, carboplatin, etoposide 5 etoposide, vincristine 6 mass excision 2 nd relapse IV ( ) CDDP, etoposide, ADR, VCR 5 partial mass excision CDDP, etoposide, ADR, CPM 4 RT (retroperiteneum, 15Gy) ITT 1 st relapse, off 1 yr 2 mo CDDP, etoposide, ADR, CPM 4 progression CPM, topotecan, etoposide 26, 131 I-MIBG 400mCi persistence Yes No 3 M 2.9 yr Adrenal gland Retroperitoneum, multiple bone metastasis, BM IV ( ) CDDP, etoposide, ADR, CPM 6 partial mass excision RT (retroperiteneum) CPM, etoposide 2 apbsct #1 (TopoThioCarbo) apbsct #2 (MEC) 1 st relapse mass excision CDDP, etoposide, ADR, CPM 10 CPM, topotecan, etoposide 21 2 nd relapse left iliac LN excision ifosfamide, carboplatin, etoposide 21 rinotecan, temozolomide 8 persistence Yes ADR, doxorubicin; apbsct, autologous peripheral blood stem cell transplantation; BM, bone marrow; CDDP, cisplatin; CPM, cyclophosphamide; MIBG, metaiodobenzylguanidine; MEC, melphalan, etoposide, and carboplatin; LN, lymph node; RT, radiation; TTC, topotecan, thiotepa, and carboplatin; VCR, vincristine. Clin Pediatr Hematol Oncol 203

3 Jung Yoon Choi, et al December Table 1 summarizes the clinical characteristics of the patients and their treatment history before CIK cell immunotherapy. Sixty milliliters of blood was collected from patients for the production of CIK cells at least three weeks prior to the first CIK cell infusion. CIK cells were manufactured by Green Cross Corp in Korea, and activated T-lymphocytes were mixed with normal saline and 1% human serum albumin to make a total volume of 200 ml. CIK cells were successfully produced in two of the three patients. When CIK cell production was successful, cells were administered at a time, and 10 times in total. The number of injected CIK cells, viability, and the number of CD3+, CD8+, CD56+, CD14+, and CD20+ cells are shown in Table 2. Based on RECIST criteria, patients were evaluated as exhibiting either complete remission, partial remission, stable disease (SD), or progressive disease (PD) [8]. We presented the response, progression-free survival (PFS) overall survival (OS), and side effects of two patients who completed CIK cell therapy. Patient 1 was diagnosed with stage IV neuroblastoma at the age of 12.2 years and N-myc gene amplification was confirmed by fluorescence in situ hybridization. He was treated with chemotherapy consisting of cisplatin, etoposide, doxorubicin, and cyclophosphamide, and radiotherapy for residual tumor bed. At two years and two months off treatment, one of the soft tissue masses around the aorta increased from 5.3 mm to 7.6 mm and the other one increased from 8.7 mm to 15.6 mm, and surgical resection was performed. Pathological report revealed relapse of neuroblastoma. After 23 cycles of chemotherapy consisting of cyclophosphamide, topotecan, and etoposide, resection of the residual tumors was performed. Pathological reports showed partially differentiated ganglioneuroblastoma cells. Five cycles of chemotherapy consisting of ifosfamide, carboplatin, and etoposide were performed. Chemotherapy regimen was changed to etoposide and vincristine due to ifosfamide-induced nephrotoxicity. Six additional cycles of chemotherapy consisting of etoposide and vincristine were performed. However, the size of the lymph nodes near the aorta increased and surgical resection was done. Two months after the surgery, magnetic resonance imaging (MRI) revealed increased sizes of the para-aortic, common iliac, and left aor- Table 2. Composition of cytokine-induced killer cell injected into the 2 patients Patient No. Number Total cell count, 10 9 cells Cell viability (%) CD3+ cell, 10 9 (%) CD8+ cell, CD56+ cell, CD14+ cell, CD20+ cell, (99.7) 5.40 (94.8) 2.16 (37.9) 0 (0.0) 0.00 (0.0) (99.9) 4.94 (95.0) 1.13 (21.8) 0 (0.0) 0.01 (0.1) (99.7) 8.21 (95.5) 2.89 (33.6) 0 (0.0) 0.00 (0.0) (99.8) 5.97 (93.3) 1.87 (29.2) 0 (0.0) 0.00 (0.0) (99.8) 6.68 (95.4) 2.39 (34.1) 0 (0.0) 0.00 (0.0) (99.8) 5.19 (91.0) 1.81 (31.8) 0 (0.0) 0.01 (0.1) (99.6) 7.20 (91.2) 2.53 (32.0) 0 (0.0) 0.00 (0.0) (99.7) 5.04 (91.6) 1.74 (31.7) 0 (0.0) 0.00 (0.0) (99.7) 5.43 (92.1) 1.75 (29.6) 0 (0.0) 0.00 (0.0) (99.7) 4.42 (90.3) 1.41 (28.7) 0 (0.0) 0.00 (0.0) (99.8) (93.6) 6.83 (42.7) (0.1) 0.02 (0.1) (99.8) 4.06 (92.2) 1.44 (32.7) 0.0 (0.0) 0.00 (0.1) (99.8) 5.69 (93.2) 2.20 (36.1) 0.0 (0.0) 0.00 (0.0) (99.8) 7.52 (94.0) 1.75 (21.9) 0.0 (0.0) 0.02 (0.2) (99.9) 6.40 (92.7) 1.78 (25.8) 0.0 (0.0) 0.00 (0.0) (99.9) 6.18 (92.2) 2.19 (32.7) (0.1) 0.01 (0.2) (99.8) 5.15 (90.3) 1.65 (28.9) 0.0 (0.0) 0.0 (0.0) (99.9) 5.79 (90.5) 1.94 (30.3) 0.01 (0.2) 0.03 (0.5) (99.9) 7.68 (90.4) 2.76 (32.5) 0.0 (0.0) 0.01 (0.1) (99.9) 7.43 (94.1) 2.52 (31.9) 0.01 (0.1) 0.02 (0.3) 204 Vol. 25, No. 2, October 2018

4 CIK Cell Immunotherapy for Neuroblastoma Table 3. Treatment outcomes after CIK cell infusion Patient No. Age at enrollment Treatments PFS (mo) OS (mo) Outcome yr CIK infusion #1-5 progression CIK infusion #6-10, irinotecan, temozolomide stable disease 131 I-MIBG 200mCi progression ifosfamide, carboplatin, etoposide 5 arsenic trioxide radiotherapy (C, T spine) progression yr CIK infusion #1-10 progression mass excision CDDP, etoposide, CPM 22 partial response irinotecan, temozolomide 3 stable disease DOD AWD AWD, alive with disease; CDDP, cisplatin; CIK cell, cytokine-induced killer cell; CPM, cyclophosphamide; DOD, died of disease; MIBG, metaiodobenzylguanidine; OS, overall survival; PFS, progression-free survival. tocaval lymph nodes. The patient underwent CIK cell immunotherapy. Patient received CIK cells 6 times every week, followed by 4 times every 2 weeks. One month after CIK cell therapy, tumor progressed on MRI, and combination therapy with CIK cells and chemotherapy consisting of irinotecan and temozolomide were started. After 5 cycles of CIK cells, the patient complained of moderate pain which ameliorated within a week. The relationship with pain and CIK cell immunotherapy is yet not clear. After completion of 10 cycles of CIK cell therapy, only irinotecan and temozolomide chemotherapy were continued along with 131 I-MIBG 200 mci treatment. The patient showed PD at 7.1 months after CIK cell therapy. He underwent chemotherapy consisting of ifosfamide, carboplatin, etoposide, and arsenic trioxide, but died due to disease progression. The PFS was 1.9 months and OS was 16.7 months after CIK cell immunotherapy. Patient 3 was diagnosed with stage IV neuroblastoma at the age of 2.9 years and underwent chemotherapy consisting of cisplatin, etoposide, doxorubicin, and cyclophosphamide, along with surgical resection, and radiotherapy. Thereafter, tandem HDC and ASCT, and 13-CRA treatment were performed. At 1 year and 4 months off treatment, relapse developed in the left sacrum, sacral nerve, and left common iliac lymph node. Ten cycles of chemotherapy consisting of cisplatin, etoposide, doxorubicin, and cyclophosphamide were performed. For the remaining tumors, patient was then treated with 21 cycles of chemotherapy consisting of cyclophosphamide, topotecan, and etoposide. After 2 years and 3 months, he received surgical resection due to increased sizes of left iliac lymph node, and second relapse was confirmed by pathological analyses. The patient received 21 cycles of chemotherapy consisting of ifosfamide, carboplatin, and etoposide and 131 I-MIBG 200 mci treatment. Due to ifosfamide-induced nephrotoxicity, chemotherapy regimen was changed to irinotecan and temozolomide. After 8 cycles of irinotecan and temozolomide treatment, he possessed residual tumors. Therefore, we decided to administer CIK cells. The patient completed a total of 10 CIK cell infusions in a similar fashion as the previous patient, and did not show moderate and severe side effects. MRI after completion of the CIK cell therapy showed PD in dthe left sacrum, and he underwent surgical resection. The patient continues to receive chemotherapy consisting of cisplatin, etoposide, and cyclophosphamide. The PFS was 4.1 months and OS was 28.7 months after CIK cell therapy. Table 3 summarizes the treatment outcomes. Discussion In this study, we tried to produce autologous CIK cells in three patients. Two of them succeeded in producing enough CIK cells and completed the planned CIK cell therapy. Long-term survival was not expected for any of the three patients using conventional treatments. One of the two patients who had completed CIK cell therapy survived 16.7 months after CIK cell therapy, and the other has survived and receive chemotherapy till date. In high-risk neuroblastoma, 13-CRA and IL-2 therapy for minimal residual tumors after HDC and ASCT has been performed as standard therapy. However, there is a need for Clin Pediatr Hematol Oncol 205

5 Jung Yoon Choi, et al better treatment owing to unsatisfactory treatment results [2,9]. Yu AL et al. reported that anti-disialoganglioside(gd)2 antibody with granulocyte-macrophage colony-stimulating factor and IL-2 significantly improved the survival rate of neuroblastoma [10]. Anti-GD2 antibodies are known to kill tumor cells using antibody-dependent cell mediated cytotoxicity. An anti-gd2 antibody, dinutuximab, was approved by the FDA in However, it is still not easily available in Korea due to the cost. In addition, the efficacy in patients with refractory disease other than minimal residual tumor has not been proven. Therefore, there is a need for novel therapeutic strategy for relapsed and refractory neuroblastoma. Previous studies have shown that immunotherapy using CIK cells improves relapse-free survival and OS in HCC patients [6]. The development and progression of HCC is known to be associated with chronic inflammation [11]. After tumor cells develop in the presence of chronic inflammation, the interaction between existing tumor cells and inflammatory immune cells may produce more favorable environments for tumor cell survival. Enhancing the activity of direct cytotoxic effector cells with major histocompatibility complex unrestricted in HCC could act against tumors, and CIK cell therapy was one such strategy [6]. CD3- and CD56-positive CIK cells have a higher proportion of CD8+ cells and are known to exert potent antitumor effects with granzyme and perforin-mediated tumor cell lysis [12,13]. They were expected to have an antitumor effect in neuroblastoma by the same mechanism, but the two neuroblastoma patients who received CIK cell immunotherapy did not show any significant extension of PFS. This is the first study to employ CIK cell immunotherapy in childhood cancer, although there is a limitation in small-scale studies. This study showed that CIK cell production was possible in patients who had been heavily pretreated. CIK cell therapy is a safe method for neuroblastoma patients. Although the antitumor response is not satisfactory, it has shown to have potential as a safe alternative treatment to increase survival. Further studies are required to overcome the limitations of CIK cell therapy by investigating the micro-environment of neuroblastoma and its interaction with immune cells. Acknowledgments This study was supported by the Foundation for the Seoul National University Children s Hospital and Green Cross Cell Crop (Seoul, Korea). References 1. Matthay KK, Villablanca JG, Seeger RC, et al. Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid. Children's Cancer Group. N Engl J Med 1999;341: George RE, Li S, Medeiros-Nancarrow C, et al. High-risk neuroblastoma treated with tandem autologous peripheral-blood stem cell-supported transplantation: long-term survival update. J Clin Oncol 2006;24: Sung KW, Son MH, Lee SH, et al. Tandem high-dose chemotherapy and autologous stem cell transplantation in patients with high-risk neuroblastoma: results of SMC NB-2004 study. Bone Marrow Transplant 2013;48: Lee JW, Lee S, Cho HW, et al. Incorporation of high-dose 131I-metaiodobenzylguanidine treatment into tandem high-dose chemotherapy and autologous stem cell transplantation for high-risk neuroblastoma: results of the SMC NB-2009 study. J Hematol Oncol 2017;10: Sung KW. Allogeneic stem cell transplantation for neuroblastoma. Korean J Hematol 2012;47: Lee JH, Lee JH, Lim YS, et al. Adjuvant immunotherapy with autologous cytokine-induced killer cells for hepatocellular carcinoma. Gastroenterology 2015;148: e6. 7. Chung MJ, Park JY, Bang S, Park SW, Song SY. Phase II clinical trial of ex vivo-expanded cytokine-induced killer cells therapy in advanced pancreatic cancer. Cancer Immunol Immunother 2014;63: Eisenhauer EA, Therasse P, Bogaerts J, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 2009;45: Rossi AR, Pericle F, Rashleigh S, Janiec J, Djeu JY. Lysis of neuroblastoma cell lines by human natural killer cells activated by interleukin-2 and interleukin-12. Blood 1994;83: Yu AL, Gilman AL, Ozkaynak MF, et al. Anti-GD2 antibody with GM-CSF, interleukin-2, and isotretinoin for neuroblastoma. N Engl J Med 2010;363: Korangy F, Höchst B, Manns MP, Greten TF. Immune responses in hepatocellular carcinoma. Dig Dis 2010;28: Jamieson AM, Diefenbach A, McMahon CW, Xiong N, Carlyle 206 Vol. 25, No. 2, October 2018

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