Clinical and Experimental Immunology

OR I G INA L A RTI CLE

doi:10.1111/cei.12755

Autologous cytokine-induced killer (CIK) cell immunotherapy combined with cyclophosphamide in five patients with POEMS syndrome

L. Ma,*1 Y. Wang,†1 J. Bo,‡1 W. Han,† Y. Wang,* L. Zhang,* X. Wu,* S. Yu* and R. Liu* *Department of Neurology, Chinese PLA General Hospital, †Department of Immunology, Institute of Basic Medicine, School of Life Sciences, and ‡Department of Haematology, Chinese PLA General Hospital, Beijing, China

Accepted for publication 7 December 2015 Correspondence: R. Liu, Department of Neurology, Chinese PLA General Hospital, Beijing 100853, China. E-mail: [email protected] Co-correspondence: S. Yu, Department of Neurology, Chinese PLA General Hospital, Beijing 100853, China.

Summary The primary objective of this study was to evaluate the safety and clinical efficacy of autologous cytokine-induced killer (CIK) cells combined with cyclophosphamide in the treatment of polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy and skin changes (POEMS) syndrome patients. We evaluated five POEMS syndrome patients treated with autologous CIK cell immunotherapy combined with cyclophosphamide from 1 May 2012 to 30 November 2014. The Overall Neuropathy Limitation Scale (ONLS), computed tomography of the chest and abdomen, ultrasound of the abdomen, serum vascular endothelial growth factor (VEGF) level and lymphocyte count findings in the five patients were recorded. The median age of the patients was 40 years (range: 25–62), and all the patients were male. CIK cells were generated routinely from peripheral blood mononuclear cells (PBMCs) of all five patients, and the numbers of CIK cells increased by approximately 105-fold after 14 days of culture. All five patients (100%) responded to their neuropathy treatment, the ONLS scores were reduced by at least 1 and a paired-sample t-test revealed a significant difference (t 5 5
715, P 5 0
003 < 0
01). The extravascular volume overload responses indicated partial remission (PR 5 60%) or stable disease (SD 5 40%), and no cases of progressive disease (PD) or complete remission (CR) were observed. During clinical treatment, the serum VEGF of patient 5 decreased after one cycle of transfusion within 1 month. The lymphocyte counts of all the patients increased significantly after CIK transfusion, and a paired-sample t-test revealed a significant difference (t 5 5
101, P 5 0
004 < 0
01). Autologous CIK cell infusion combined with cyclophosphamide was found to be highly safe and elicited no adverse reactions. CIK cells can improve both the symptoms and quality of life, decrease serum VEGF levels and increase lymphocyte counts in patients with POEMS syndrome.

E-mail: [email protected] 1

These authors contributed equally to this

work.

Keywords: clinical efficacy and safety, cyclophosphamide, cytokine-induced killer (CIK) cell, lymphocyte, POEMS syndrome, vascular endothelial growth factor (VEGF)

Introduction Polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy and skin changes (POEMS) syndrome is a paraneoplastic syndrome that results from an underlying plasma cell neoplasm. The major criteria for the diagnosis of this syndrome are polyradiculoneuropathy, clonal plasma cell disorder (PCD), sclerotic bone lesions, elevated vascular endothelial growth factor (VEGF) levels and the

presence of Castleman disease. Minor features include organomegaly, endocrinopathy, characteristic skin changes, papilloedema, extravascular volume overload and thrombocytosis [1]. POEMS syndrome can cause polyneuropathy, but can also involve many of the organs of the body and cause organomegaly (usually in the liver and spleen) and extravascular volume overload (usually pleural effusion and ascites) [2]. Its cause is not well known. The

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quality of life of people with POEMS syndrome deteriorates due to progressive neuropathy and the accumulation of fluid in the limbs or organs [3]. No randomized controlled trials of treatments for POEMS syndrome have been conducted, so treatment recommendations are based on case series and anecdotal evidence [4]. There is a variety of therapeutic options for POEMS syndrome, including alkylator-based chemotherapy, corticosteroids, autologous stem cell transplantation and immunomodulatory drugs, such as lenalidomide and bortezomib, but there is no standard treatment. Local irradiation and low-dose alkylators have constituted the core of the treatment of POEMS syndrome with steroids. However, local irradiation is not indicated for patients with widespread osteosclerotic lesions or patients without bone lesions [4]. Alkylators and corticosteroid regimens are less toxic, but these treatments have low response rates and short response durations [4]. Recently, high-dose melphalan with autologous peripheral stem cell transplantation has resulted in impressive progress in the treatment of POEMS syndrome. The neuropathy response rate is almost 100%, and the response rates of other specific features of POEMS syndrome range from 70 to 90% [5,6]; however, this treatment is expensive. Cytokine-induced killer (CIK) cells are a heterogeneous subset of ex-vivo-expanded T lymphocytes that exhibit phenotypical and functional properties of both natural killer (NK) and T cells, predominantly CD31CD81 T cells [7], and exhibit major histocompatibility complex (MHC)unrestricted anti-tumour activity [8]. Additionally, CIK cells exhibit a high proliferation rate [9], minimal toxicity [10], do not cause any vascular adverse events (AEs) [11,12] and are readily available [13] and well tolerated [14] in both autologous and allogeneic contexts [15]. CIK cells are becoming an important and effective method of cancer therapy after primary surgery, radiotherapy and chemotherapy [16]. Numerous clinical trials of the use of CIK cells for the treatment of solid tumours [17] and haematological malignancies [18–20] have been conducted. In the last few years, the application of CIK cells has developed from experimental observations to clinical studies. CIK cells have been demonstrated to constitute an effective immunotherapy against most malignancies [21], including multiple myelomas, in clinical testing [8]. Cyclophosphamide is one of the most frequently used alkylating antineoplastic drugs. The greatest POEMS syndrome-related experience has been with alkylator-based therapy [1]. Therefore, cyclophosphamide can not only enhance the effects of CIK cell transfusion but can also can treat POEMS syndrome by itself. CIK cells are expanded easily in vitro due to their high proliferation rate, and they exhibit strong anti-tumour cytotoxic activity [22]. CIK cells are also sensitive to some multi-drug-resistant tumour cells [23]. However, to date, there are no reports of the therapeutic effects of CIK treatment for POEMS syndrome patients. The therapeutic 84

methods applied to POEMS syndrome are borrowed from other plasma cell disorders, especially multiple myeloma and light chain amyloidosis [3]. One case report evaluated the safety and efficacy of cellular immunotherapy with CIK cells for multiple myelomas with lung cancer [25]. The multiple myelomas and lung cancer remained stable, and the concomitant paraneoplastic dermatoses were improved markedly after treatment [25,26]. Based on the efficacy of CIK cell immunotherapy for multiple myelomas, we conducted a single-centre prospective study to evaluate the therapeutic efficacy and safety of autologous CIK cell immunotherapy combined with cyclophosphamide for patients with POEMS syndrome. The safety assessment was based primarily on the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) version 3. The response assessments were divided into the following three parts: (1) polyneuropathy (ONLS), organomegaly (computed tomography or ultrasound of the abdomen) and extravascular volume overload (computed tomography or ultrasound of the abdomen and chest); (2) serum VEGF levels; and (3) lymphocyte counts.

Patients and methods This study was approved by the Ethics Committee of the Chinese General Hospital of the People’s Liberation Army. All patients signed consent forms prior to participating in the study in accordance with the Declaration of Helsinki. No commercial sponsors were involved in this study.

Patient profiles All the patients were enrolled from 1 May 2012 to 30 November 2014 at the Chinese PLA General Hospital according to the diagnostic criteria. The patients’ liver and renal functions were required to be normal. All patients signed informed consent forms prior to receiving CIK cell therapy. All the patients were required to stop corticosteroid therapy at least 2 weeks before peripheral blood was collected.

Methods Therapeutic method. Venous blood was obtained from each patient for each CIK cell treatment. The CIK cells were generated by culturing steady-state apheresis products for 13–15 days. The CIK cells were required to meet the following criteria prior to transfusion: the proportion of CD31 cells was > 90%, and the cells were viable. After culturing, the CIK cells were transfused for 2 consecutive days for each course of treatment. If any toxicity occurred during the cell infusion, the patient was required to discontinue subsequent CIK cell treatments immediately, and 20 mg diphenhydramine and 25 mg promethazine hydrochloride were administered intramuscularly. The autologous CIK cells (3– 3 109) were reinfused into the patients. The regimen was repeated every 4 weeks.

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Fig. 1. Flow-chart of one treatment cycle for the polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy and skin changes (POEMS) patients. Peripheral blood mononuclear cells (PBMCs) were collected from 50 ml of fresh venous blood. The next day, the patients were treated with cyclophosphamide (600 mg, intravenously). PBMC expansion required 14 days. The autologous cytokine-induced killer (CIK) cells were then transfused twice. Cyclophosphamide was infused every 2 weeks.

For the chemotherapy regimen, cyclophosphamide [600 mg via intravenous (i.v.) drip] was administered every 2 weeks. The cyclophosphamide i.v. drips began on the second day after the collection of the venous blood and again after 14 days. The second cyclophosphamide i.v. drip and the CIK transfusion were performed on the same day. The treatment flow-chart is illustrated in Fig. 1. Generation and phenotypical analysis of the CIK cells. After signing the informed consent form, 50 ml of blood was collected from each patient into evacuated tubes containing heparin. Human peripheral blood mononuclear cells (PBMCs) were isolated from the fresh blood via FicollHypaque density gradient centrifugation. The PBMCs were washed three times, adjusted to a final concentration of 2 3 106 cells/ml with CIK medium (Takara, Tokyo, Japan) supplemented with 0
6% autogeneic serum and then cultured in 175-cm2 culture flasks coated with 15 ml of phosphate-buffered saline (PBS) that contained 5 mg/ml anti-human CD3 monoclonal antibody (Takara) at 48C overnight. On day 0 of culture, we added 1000 U/ml recombinant human interferon (IFN)-g (PeproTech, Rocky Hill, NJ, USA) and 1000 U/ml recombinant human interleukin (IL)22 (rhIL-2; PeproTech) to the culture medium. The cells were cultured in a humidified 5% CO2 incubator at 378C. The cells were transferred from the coated flasks to fresh flasks after 4 days. Every 3 days, fresh CIK medium and 1000 U/ml rhIL-2 were added. The composition and purity were assessed with fluorescence activated cell sorting (FACS) on days 13–15, and the cells were then harvested. The total cells were harvested and washed twice with PBS and then resuspended in 100 ll of PBS, labelled with 15 ml of antibodies against CD4/8/3 [fluorescein isothio-

cyanate (FITC)-conjugated anti-CD4, phycoerythrin (PE)-conjugated anti-CD8 and peridinin chlorophyll (PerCP)-conjugated anti-CD3; Becton Dickinson, Franklin Lakes, NJ, USA] and 5 ml of anti-CD5 antibody [allophycocyanin (APC)-conjugated anti-CD56; BD Biosciences] in the dark for 30 min at 48C. The phenotypes of the PBMCs and CIK cells from the patients were examined with FACS [24]. The phenotypes of the PBMCs were analysed as a control.

Response assessments Safety assessment. Toxicity and adverse events during chemotherapy were documented and classified according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) version 3
0. Therapeutic effect assessments. For polyneuropathy, the POEMS syndrome patients underwent neurological examinations administered by two specialists with senior professional qualifications. We used the ONLS to assess neurological disability; this test was derived from the Overall Disability Sum Score and was designed to assess the physical limitations of patients with immune-mediated peripheral neuropathies. The ONLS consists of scores for the arms and legs; the arm scores range from 0 (normal) to 5 (disability), and the leg scores ranges from 0 (normal) to 7 (restricted to a wheelchair or bed for most of the day and unable to make any purposeful movements of the legs). The total ONLS score is the sum of the arm and leg scores. The total scores range from 0 (no disability) to 12 (maximal disability) [25]. Nerve function was assessed with the ONLS before treatment and during the final visit. The ONLS is a concise and straightforward scale that enables easy follow-up in out-patient settings. The ONLS is

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L. Ma et al. Table 1. Generation of autologous cytokine-induced killer (CIK) cells. PBMCs (%) Patient cycle 1–1 2–1 2–2 2–3 2–4 3–1 3–2 3–3 3–4 4–1 5–1 5–2 AVE (%) STD (%) AVE STD

Total cells (3107)

CD31

7
63 6
86 5
86 7
82 6
67 6
59 5
64 5
74 7
83 5
22 6
14 7
04 – – 6
59 0
96

44
43 60
82 65
67 24
97 36
94 42
49 80
13 65
27 54
80 83
88 32
35 79
53 55
94 18
87 3
68 0
88

CIK cells (%)

CD81

CD31 CD41

CD31 CD81

CD31 CD561

CD81 CD561

Total cells (3107)

CD31

CD81

CD31 CD41

CD31 CD81

CD31 CD561

CD81 CD561

12
68 9
11 21
83 5
31 12
62 8
16 35
38 23
09 16
72 36
93 18
01 18
35 18
18 11
04 1
20 0
55

14
62 34
40 17
68 14
37 12
75 25
04 11
62 29
80 21
77 10
19 18
15 20
29 19
22 8
24 1
27 0
57

23
43 17
72 42
10 8
82 19
08 14
46 67
03 45
96 31
05 71
24 33
05 35
13 34
09 21
88 2
25 1
10

7
40 5
48 12
28 2
01 22
30 1
78 17
60 4
78 1
97 3
23 6
97 8
79 7
88 7
19 0
52 0
45

3
22 2
24 8
59 1
26 9
78 1
14 17
85 3
27 2
65 1
38 4
21 6
07 5
14 5
40 0
34 0
31

355 412 346 489 427 396 314 349 465 377 367 419 – – 393 55
79

99
70 98
26 96
52 98
50 99
15 99
70 98
30 97
41 99
72 96
78 98
11 98
69 98
40 1
20 386
73 57
05

85
65 56
49 65
99 66
54 69
14 85
65 82
41 55
09 88
03 71
34 70
03 75
23 72
63 12
18 285
45 63
23

12
95 39
81 28
49 31
10 27
05 12
95 15
45 39
60 08
47 15
03 22
04 24
14 23
09 11
57 90
74 48
54

85
6 56
89 66
27 66
96 69
23 85
60 82
28 54
85 87
60 71
35 70
22 75
10 72
66 12
03 285
57 62
83

22
22 28
92 6
91 11
82 07
24 22
22 10
13 8
23 6
59 14
16 12
04 15
64 13
84 7
90 54
41 31
88

18
46 14
73 6
09 8
82 4
38 18
46 9
43 4
72 5
1 8
62 9
03 10
33 9
88 5
45 38
83 20
97

PBMCs 5 peripheral blood mononuclear cells; 2–1 5 the first treatment cycle on patient 2; CIK 5 autologous cytokine-induced killer. Peripheral blood mononuclear cells (PBMCs) were isolated from 50 ml fresh blood from four polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy and skin changes (POEMS) patients, and CIK cells were harvested after 13–15 days of culture. AVE 5 Average; STD 5 standard deviation.

recommended as an outcome measurement for clinical trials dealing with various peripheral neuropathies [25]. Computed tomography of the chest and abdomen and ultrasound of the abdomen were performed to assess organomegaly (splenomegaly and hepatomegaly) and extravascular volume overload (ascites and pleural effusion) before and after the treatment. The organomegaly and extravascular volume overload responses were defined as CRs or PRs. The curative nature of the clinical responses were categorized as follows: complete response (CR), partial remission (PR), progressive disease (PD) and stable disease (SD). A complete response (CR) was defined as by the complete disappearance of clinical symptoms, a partial response (PR) was defined as greater than 50% remittance in clinical symptoms, progressive disease (PD) was defined as an increase of greater than 50% in these symptoms and stable disease (SD) included all statuses other than CR, PR and PD. The serum VEGF levels were measured with an enzymelinked immunosorbent assay (ELISA) before treatment, 14 days after treatment and 1 month after treatment. Serum VEGF levels below 600 pg/ml are in the normal range [26]. Routine blood tests for white blood cells (WBCs) and lymphocyte counts were performed before treatment and after CIK transfusion.

sons. Paired-sample t-tests were used for the statistical analyses of the ONLS scores and the lymphocyte counts before and after treatment. P-values below 0
05 based on one-sided tests indicated statistical significance. All calculations were performed using SPSS version 17
0 software.

Results Clinical-grade expansion of the CIK cells First, we established a stable in-vitro system for the expansion of the CIK cells from the POEMS patients. After 14 days of culture, the total numbers of cells from all five patients increased by 105-fold. The phenotypes of the PBMCs and CIK cells were examined with FACS. The CIK cell counts increased significantly as follows: CD31 (3
68 6 0
88 3 107 versus 386
73 6 57
05 3 107), CD81 (1
20 6 0
55 3 107 versus 285
45 6 63
23 3 107), CD31CD41 (1
27 6 0
57 3 107 versus 90
74 6 48
54 3 107), CD31CD81 (2
25 6 1
10 3 107 versus 285
57 6 62
83 3 107), CD31CD561 (0
52 6 0
45 3 107 versus 54
41 6 31
88 3 107) and CD81CD561 (0
34 6 0
31 3 107 versus 38
83 6 20
97 3 107). These data are illustrated in Table 1 and Fig. 2.

Response assessment Statistical analysis The data are reported as the means with the standard deviations. Student’s t-tests were applied for the comparisons. The Mann–Whitney U-test was used for median compari86

Safety. During the CIK cell therapy, no severe adverse reactions were observed. No patient discontinued therapy due to drug-related adverse events. No treatment-related deaths were observed in this study.

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Fig. 2. Phenotypical analyses of the cells from the polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy and skin changes (POEMS) syndrome patients as assessed with fluorescence activated cell sorter (FACS) analysis. All cell samples taken for phenotypical analysis were stained with peridinin chlorophyll (PerCP)-conjugated antibodies against CD3, fluorescein isothiocyanate (FITC)-conjugated antibodies against CD4, phycoerythrin (PE)-conjugated antibodies against CD8 and allophycocyanin (APC)-conjugated antibodies against CD56. (a) A typical phenotypical analysis of the peripheral blood mononuclear cells (PBMCs) and autologous cytokine-induced killer (CIK) cells from patient 2. (b) Comparison of the phenotypical analyses of the PBMCs and CIK cells. The phenotypical comparisons were performed for all five patients and a total of 12 cycles, and the results are expressed as the means 6 standard deviation.

Therapeutic effects. Among our five patients, the neurological response rate was perfect (100%), and the ONLS scores all decreased by at least 1 point. The ONLS results revealed a three-point difference in the arm grades and a four-point difference in the leg grades following treatment in all five patients. The ONLS scores were divided into two groups according to the treatment time. Paired t-tests revealed that the differences in the arm grades (t 5 2
449, P 5 0
035 < 0
05), leg grades (t 5 4
000, P 5 0
008 < 0
05) and total

grades (t 5 5
715, P 5 0
003 < 0
05) across all five patients were significant. Because POEMS syndrome is a rare disease and the cost of treatment with CIK cells is high, the number of cases that we were able to collect was limited. The data were too limited for statistical analyses; thus, we were unable to identify a criterion for the number of treatment cycles. A greater sample size would have been required to determine whether a cycledependent anti-POEMS effect occurred in this clinical trial.

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L. Ma et al. Table 2. Clinical response of five patients. ONLS (before treatment/ last visit) Patient 1 2 3 4 5

Age, gender 62 54 40 25 49

M M M M M

Cycles of treatment

Paired t-test

Arm grade

Leg grade

Total grade

P (< 0
05)

1/1 2/1 5/4 4/3 4/4 0
035

4/3 6/5 7/6 7/7 7/6 0
008

5/4 8/6 12/10 11/10 11/10 0
003

1 4 4 1 2

Lymphocyte count (3109/l) Before treatment 1
28 1
72 1
80 0
86 1
59 0
004

Last visit

Response rate

2
09 2
63 3
66 1
58 3
21 PR%

Organomegaly

Extravascular volume overload

SD SD SD SD SD 0%

PR PR PR SD SD 60%

M 5 male; ONLS 5 Overall Neuropathy Limitation Scale; PR 5 partial remission; SD 5 stable disease.

For organomegaly and extravascular volume overload, partial remission PR and SD statuses were present in 60 and 40%, respectively, of the cases of extravascular volume overload (ascites and pleural effusion). SD statuses occurred in 100% of the organomegaly cases (splenomegaly and hepatomegaly). In this prospective study, we enrolled only patients with records of serum VEGF levels before and after treatment. The serum VEGF levels of patient 5 were 800 pg/ml before the CIK treatment, 756 pg/ml after 14 days of treatment and 688 pg/ml after 1 month of treatment. Unfortunately, the other patients declined to undergo these tests. The lymphocyte counts across all five patients increased significantly following treatment (paired t-test, t 5 5
101, P 5 0
004). The clinical responses of the five patients are illustrated in Table 2.

Discussion The pathogenesis of POEMS syndrome is not understood fully. CIK cells are expanded easily in vitro and exhibit high proliferation rates and strong anti-tumour cytotoxic activity [22]. CIK cells are also sensitive to some multi-drugresistant tumour cells [23]. In this study, we provided the first clinical evidence related to the use of autologous CIK cell immunotherapy combined with cyclophosphamide for the treatment of POEMS patients. This study revealed 100% safety and indicated that this treatment is very well tolerated by patients. Autologous CIK cells combined with cyclophosphamide for the treatment of POEMS syndrome patients exhibited a high level of safety and elicited no adverse reactions. Our data indicate that autologous CIK cells combined with cyclophosphamide is a very effective therapy for POEMS syndrome patients; we observed a perfect neurological response rate (100%) and were able to achieve a high extravascular volume overload response rate of 60% PR in the patients. Thus, autologous CIK cells combined with cyclophosphamide can improve the symptoms 88

of patients with POEMS syndrome, improve their quality of life, decrease their serum VEGF levels and increase their lymphocyte counts. Overall, we conclude that CIK cells combined with cyclophosphamide represents a safe and effective method for the treatment of POEMS syndrome. Some evidence suggests that the high serum VEGF levels contribute to some of the specific features of POEMS syndrome, including extravascular volume overload, organomegaly and haemangioma. The VEGF level has become one of the major revised POEMS criteria because a high VEGF level is considered to be characteristic of the disease, and this serum marker is correlated highly with POEMS disease activity. CIK cells combined with cyclophosphamide for the treatment of POEMS syndrome can reduce serum VEGF levels and simultaneously improve significantly the extravascular volume overload symptoms of patients. We infer that this treatment improves extravascular volume overload by reducing the level of VEGF. The characteristics of polyneuropathy include distal symmetrical sensory motor damage, which contrasts with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). CIDP affects mainly nerve roots, which have the weakest blood–nerve barriers, or the distal nerve stem is involved significantly due to segmental demyelination. Biopsies of the sural nerves of patients with POEMS syndrome have revealed that axonal degeneration and demyelination or axonal degeneration and small vessel lesions without inflammation are present. The treatment detailed here can improve polyneuropathy symptoms significantly and reduce serum VEGF levels. Therefore, we speculate that the nerve involvement of POEMS syndrome may be related to peripheral nerve vascular lesions mediated by VEGF. Autologous CIK cells combined with cyclophosphamide reduced the lymphocyte count effectively, as evidenced by the perfect response rate (100%). CIK cells are different from ordinary T cells and belong predominantly to the type II class of NK T cells. Large numbers of CIK cells were acquired via in-vitro amplification and subsequently

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reinfused into the patients to supplement the quantity of CD31CD81 cells in the PB of patients with POEMS syndrome. Cyclophosphamide is one of the most frequently used alkylating antineoplastic drugs, but it is associated with the side effect of reducing the numbers of white blood cells and lymphocytes. Therefore, we infer that lymphocyte recovery represented a response to the CIK cell therapy. In conclusion, CIK cell infusion might be a useful treatment strategy for POEMS syndrome. Autologous CIK cells combined with cyclophosphamide for the treatment of POEMS syndrome patients exhibited a high level of safety and elicited perfect clinical efficacy responses. Therefore, as an adoptive cell immunotherapy, CIK cells are expected to become a promising method for the treatment of POEMS syndrome. Studies with greater patient numbers and longer follow-up times are needed to assess the relationships between clinical responses, neurological responses, VEGF levels and relapse-free and/or overall survival rates.

Disclosure The authors declare that they have no disclosures.

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