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Oncology 1991;48:387-391

Hyperimmunoglobulinemia D following Cancer Chemotherapy Eiichi Azuma, Shin-ichi Masíala, Motoi Hañada, Yan-Wen Zhou, Shao-Li Zhang, Takeo Shihata, Yoshihiro Komada, Minoru Sakurai Department of Pediatrics. Mic University School of Medicine. Mie-Ken. Japan

Key Words. Hyperimmunoglobulinemia D • Cancer • Chemotherapy

Introduction Although the humoral response following cancer chemotherapy has been well documented, clinical re­ ports have been few with the majority focusing on major serum immunoglobulin (IgG, IgA, and IgM) alterations during treatment. Little is known about IgD and IgE during and after chemotherapy. Hyper­ immunoglobulinemia D (hyper-IgD) has been report­ ed in patients with IgD myeloma [1]. recurrent bac­ terial infections [2], periodic fever [3], and celiac disease [4]. It is intriguing that hyper-IgD has also been observed in cigarette smokers [5] and immuno­ deficiency such as hyper-IgE syndrome [6], since an increased risk of developing cancer has been reported in patients with immune deficiency. Here, we report the immunoglobulin (Ig) levels in 107 children with tumors during and after chemotherapy.

Patients and .Methods Patients. At the Mic University Hospital, immunological studies of 107 children with acute leukemias (47 cases), neuroblastomas (13). non-Hodgkin's lymphomas (10). brain tumors (5). retino­

blastomas (5). histiocytosis syndrome (4), Wilms' tumor (2), hepatoblastomas (2). and miscellaneous tumors (19) were con­ ducted during a 1-ycar period. December 1988 through November 1989. They were subclassified into three groups. Group A: no che­ motherapy group. Twelve patients with tumors, including thymic cyst, ganglioneuroblastoma. benign ovarian tumor, teratoma, nonI lodgkin's lymphoma, or low-grade astrocytoma, were treated with either operation or irradiation, or both. Group B; on-chemotherapy group. Thirteen patients with malignant diseases, including com­ mon acute lymphoblastic leukemia, neuroblastoma, and nonHodgkin's lymphoma, received chemotherapy with or without irra­ diation. Group C: off-chemotherapy group. F.ighty-two children with malignant tumors described above have been followed up regularly after cessation of chemotherapy. Informed consent was obtained according to institutional guidelines. Cytochemical anti Immunological Studies. The diagnosis of leukemias was based on the French-American-British criteria [7], The diagnosis of solid tumors was based on the pathological spe­ cimen using hematoxylin-eosin and other different staining meth­ ods. IgG, IgA. IgM. and IgD were measured by scrum protein electrophoresis and immunoclcctrophoresis. IgD was measured at Biomedical Laboratories, Tokyo, and normal control values from 157 healthy subjects showed that mean IgD level was 2.3 mg/dl (SD = 2.7). In the current study. hyper-IgD was defined as serum IgD levels over the average plus 3 SD (i.e. over 10.4 mg/dl). Ig studies were performed on fresh scrum samples (i.e.. within 48 h of venepuncture), or in sera stored at 20 C after addition of epsilonaminocaproic acid (final concentration 0.5%) to prevent spon­ taneous degradation of IgD [3. 8). IgF. was measured by radioim­ munosorbent test. Immunophcnotypmg of peripheral blood lym-

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Abstract. Five classes of serum immunoglobulin levels were investigated in 107 children with malignant or benign tumors. Hyperimmunoglobulinemia D (hyper-IgD) was observed in 31 of 82 children who were in complete remission off chemotherapy with a median follow-up of 4.5 years after cessation of chemotherapy. On the other hand, hyper-IgD was not found among 12 children with malignant or benign tumors treated without chemotherapy and a low incidence of hyper-IgD was observed during chemotherapy (1 of 13 cases). The result indicates that hyper-IgD is not uncommon in children off chemotherapy, suggesting that dysrégula­ tion of IgD synthesis persists long after cessation of antineoplastic drugs.

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Azuma/Masuda/Hanada/Zhou/Zhang/Shibata/Komada/Sakurai

Table 1. Abnormal serum imm unoglobulin levels in patients ofT chemotherapy Abnormality

Patients n

IgG. mg/dl (600 1.900)

IgA. mg/dl (40 430)

IgM. mg/dl (50-350)

IgD. mg/dl (0.1-10.4)

IgE. IU/ml (1-450)

Hyper-IgG Hyper-IgA Hyper-IgM Hyper-IgD Hyper-IgE

6 1 3 31 17

2.180 1,310 1.533 1.410 1.340

211 480 147 211 219

196 (62) 39 430 (71) 151 (60) 185(123)

10.5 0.1 3.5 23.5 12.9

175 599 643 270 1077

(251) (198) (375) (347)

(114) (77) (78) (96)

(9.1) (4.2) (20.2) (22.6)

(178) (513) (543) (635)

phocytes using a panel of monoclonal antibodies was also inves­ tigated. All patients appeared clinically well at the time of blood collection. Tw’o-tailed Student t test was used for statistical analy­ sis [9], Treatment. The treatment of ALL patients consisted of standard chemotherapy (vincristine, prednisone, methotrexate. 6-mcrcaptopurinc) plus subcutaneous injections of allogeneic or autologous leukemic 80 Gy-irradiatcd lymphoblasts with bacillus CalmetteGuerin (BCG; Japan BCG Manufacturing Company. Japan) or the streptococcal product OK432 (Chugai Pharmaceutical Co. Ltd.. Japan) as a biological response modifier [10], The treatment of AML consisted of anthracycline antibiotic, cytosine arabinoside. and ctoposide. Non-Hodgkin’s lymphomas were treated with cyclo­ phosphamide. anthracycline, vincristine, prednisone, and high-dose methotrexate. The duration of chemotherapy for leukemia and lymphoma was 3 years. Actinomycin D and anthracycline were used in Wilms’ tumor [11], Cisplatin. anthracycline, cyclophosphamide, ctoposide. and vincristine were administered in neuroblastomas [12]. small round cell tumor, or hepatoblastoma. Nitrosourea, cis­ platin, cyclophosphamide, and anthracycline were used in brain tumors after operation, irradiation, or both [13]. Prednisone, vin­ blastine. anthracycline, cyclophosphamide, and ctoposide were ad­ ministered in histiocytosis syndrome [14], The doses of antineoplastic drugs in each cancer were determined according to the published protocols. The duration of chemotherapy for solid tumors was usually 2 years (1-3). depending on the extent and the histology of the tumor. The treatment results are similar to the published reports [unpubl. data].

Results Abnormalities in IgG. IgA. IgM, and IgE. We con­ firm previous reports that the major Ig (IgG. IgA and IgM) levels are normal in most patients who continue complete remission [15, 16]. However, a small portion of the 82 patients who are in complete remission off chemotherapy showed abnormal major Ig levels (table 1). Hyper-IgG (> 1.900 mg/dl) was found in 6 of 82 children. In 3 of these 6 patients. hyper-IgG was as­

sociated with hyper-IgD (tables 1, 2). One patient showed hyper-IgA with hypo-IgM and hyper-IgE. Three patients showed hyper-IgM with hyper-IgE. Hyper-IgE was found in 17 patients in whom 2 pa­ tients showed atopic dermatitis; the rest had no known allergic disorders or parasitic infections. Abnormality in IgD Levels. Hyper-IgD was found in 31 patients (31/82 = 37.8%) who continue in com­ plete remission off chemotherapy, as shown in tables 1 and 2. There were 22 males and 9 females; mean age was 11 years (SD = 4.2), ranging from 5 to 19 years. The median follow-up duration after cessation of che­ motherapy was 4.5 years, with ranges from 1.5 to 11.1 years. Among children with hyper-IgD, accompany­ ing hyper-IgG (3/31=9.7%) and hyper-IgE (5/28 cases tested = 17.9%) were found, as shown in table 2. The result of surface marker analysis of peripheral blood lymphocytes in children with hyper-IgD indica­ tes that reversed CD4/CD8 ratio (< 1.0) was observed in 8 out of 22 tested [data not shown]. There were no monoclonal gammopathy or frequent infections in these 31 patients. Figure 1 depicts IgD levels in can­ cers treated with or without chemotherapy. The mean IgD levels in 12 children with malignant or benign tumors who did not receive chemotherapy (mean = 3.4 mg/dl, SD = 3.2) and in 13 patients receiving chemo­ therapy (mean = 7.0 mg/dl, SD = 810) are shown in lanes 2 and 3, respectively. In 82 patients who are in complete remission off chemotherapy, IgD levels were higher (lane 4; mean= 11.6 mg/dl. SD = 16.1). Thirtyone our of 82 patients showed hyper-IgD, ranging from 10.6 to 117.0 mg/dl (mean = 23.5 mg/dl, SD = 20.2). Lanes 3 and 4 were statistically significant as compared with the controls (p = 0.03 and p < 0.001, respectively); no statistical significance was found bet­ ween other combinations. The frequency of hyper-

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Values are mean with SD in parentheses.

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Hyper-IgD in Childhood Cancer

Table 2. Hyper-IgD in patients off chemotherapy Patient No.

Sex/Age

Disease

IgD mg/dl

IgG mg/dl

IgA mg/dl

IgM mg/dl

IgE IU/ml

Time after Tx. years

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

F/9 M/7 M/16 M/6 M/13 M/5 M /l 1 M/13 M/8 F/13 M/5 F/8 M/16 F/l 1 F/18 M/l 9 M/8 F/7 M/14 F /16 M/16 F/l 1 M/8 M/14 M/14 M/12 F/12 M/9 M/14 M/14 M/8

pontine glioma common ALL common ALL common ALL pinealoma ganglioncuroblastoma retinoblastoma common ALL hepatoblastoma common ALL ganglioneuroblastoma common ALL common ALL small round cell tumor T cell ALL common ALL common ALL eosinophilic granuloma retinoblastoma common ALL neuroblastoma common ALL common ALL suprasellar germinoma common ALL common ALL common ALL neuroblastoma common ALL non-Hodgkin"s lymphoma common ALL

117.0 69.4 42.5 29.6 27.4 26.6 26.2 23.5 22.4 19.8 19.7 18.7 18.7 18.2 17.7 16.6 16.3 16.3 16.2 15.1 14.9 14.4 13.6 13.5 13.5 12.7 12.6 11.8 11.6 10.9 10.6

1.300 1.190 1.110 1.380 1.980 963 1.530 955 NT 1.120 1.900 1.150 1.560 1.300 1.850 1.200 1.090 1.450 1.610 1.800 1.990 1.700 1.430 1.250 1.600 2.610 1.020 1.130 1.300 1.060 1.180

127 104 170 148 354 167 215 188 NT 164 226 211 301 280 260 241 135 191 382 419 201 218 130 153 140 251 213 156 236 155 102

172 90 149 160 206 80 92 86 NT 196 311 228 71 200 109 159 129 109 183 145 157 119 199 105 297 92 179 133 113 166 182

2150 9 58 1 411 39 41 2 NT 383 596 13 15 74 103 34 10 222 48 110 13 2.100 10 53 139 22 545 503 NT NT 104

3 1 9 1 4 4 8 4 3 5 2 3 9 3 5 6 1 2 ii 8 9 5 4 3 6 5 6 2 5 3 3

IgD increased following chemotherapy and reached a plateau 1-3 years after cessation of chemotherapy. After cessation of chemotherapy, hyper-IgD was found in 3 of 11 patients in the first year, 9 of 23 patients between 1 and 3 years, 7 of 19 patients bet­ ween 3 and 5 years, and 12 of 29 patients after 5 years.

Control

No On Off chemotherapy chemotherapy chemotherapy

Fig. 1. IgD levels in patients treated with or without chemo­ therapy. Lane I represents normal values from healthy 157 subjects. The mean IgD levels in various conditions are shown in lane 2(12 children with malignant or benign tumors who did not receive chemotherapy), lane 3(13 cancer patients receiving chemotherapy), and lane 4 (82 patients who arc in complete remission off chemo­ therapy).

It is well documented that Ig levels, as measured during the course of acute leukemia, have shown an immediate drop in IgG, IgA, and IgM during the induction phase with gradual return to normal or slightly subnormal levels thereafter [15, 16]. In the current study we confirmed the previous reports and extended them by pointing out that hyper-IgD was

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Discussion

not uncommon among patients who were in complete remission off chemotherapy. The role of IgD in the immune response has re­ mained elusive, although IgD was discovered as early as 1965 [1], Cord levels of IgD are very low and rise slowly in the first year, achieving levels of 10-25% of adult levels by 12 months. Adult levels are achieved by about age 15 [17]. IgD has been shown to have some antibody activity to benxylpenicilloyl acid, diphtheria toxoid, bovine gamma globulin, and cell nuclei [18]. An important clue to the biologic role of IgD made up a disproportionately high percentage (up to 10%) of Ig bound to the membrane of B cells from newborns [19] , Subsequent studies have identified IgD as being present on the majority of normal B lymphocytes. This led to the proposal that IgD serves as an antigen receptor on the lymphocytes surface, particularly during immune development [17]. In addition to the predominance of IgD on the B cell surface, Coico et al. [20] reported that IgD receptors were found on CD4 + T cells and could be induced by IgD itself or interleukin-2 in a dose-dependent manner, resulting in enhanced antibody response upon antigen stimula­ tion. However, hyper-IgD in the study was not neces­ sarily associated with other hypergammaglobulinemia (tables 1, 2). Thus, the relationship between CD4" T cells and IgD still remains to be determined. Table 1 shows that major Igs (IgG, IgA, IgM) are within normal limits in most long-term survivors, whereas hyper-IgD was found in 37.8% of them. These find­ ings may suggest that the defect occurs early in B cell differentiation from IgM-bearing precursors [21], As shown in table 2, hyper-IgD was associated with hyper-IgE in 5 of 29 cases tested (17.1%). Abnor­ malities of IgD and IgE levels, both high and low values, are not uncommon in incomplete antibody deficiency syndromes such as hyper-IgE syndrome [6], IgE levels often parallel IgA levels, and IgD levels often parallel IgM levels and isolated deficiencies of IgD and IgE are rare and of not much clinical signifi­ cance [17]. Although a close correlation between the duration of immunosuppression and abnormal serum Ig levels has been suggested, its relationship to immunosup­ pressive agents is often difficult to assess. The agents used to achieve and maintain complete remission (prednisone, cyclophosphamide, etc.) are directed at modifying T cell function and lymphokine production that in turn modify B cell function [22]. Pollock et al. [22] reported in the long-term follow-up study (up to

Azuma Vlasuda Hanada/Zhou/Zhang Shibata Komada/Sakurai

214 months) of renal transplant recipients that abnor­ mal serum Ig levels (IgG. IgA. IgM) are not uncom­ mon in recipients who were treated with immunosup­ pressive agents and time from transplantation was significantly longer in those with abnormal serum Igs, suggesting that total immunosuppressive dose rather than maintenance dose may be an important factor in its pathogenesis. Thus, antineoplastic drugs used in this study may be blamed for hyper-IgD, although it has been reported that chemotherapy is immunosup­ pressive, but such suppression is almost always acute and not prolonged [23]. In the present study, BRMs were administered in ALL patients. However, the role of BRM in hyper-IgD may be minimal since other patients treated with antineoplastic drugs alone also showed hyper-IgD. Reversed CD4/CD8 ratio often indicates abnormal T cell regulation as in acquired immunodeficiency syndrome [24], which may lead to abnormal Ig production. This may be the case in 8 of 22 tested who showed reversed CD4/CD8 ratio. Further study will be needed to elucidate the outcome of hyper-IgD in these patients. It is intriguing that hyper-IgD in cigarette smokers returned to the normal level after quitting smoking [5], Although hyper-IgD the study did not appear to be associated with par­ ticular diagnoses, it is tempting to speculate that hyper-IgD might be associated with (secondary) can­ cer in some patients with hyper-IgD in light of the fact that hyper-IgD has been observed in some type of immunodeficiency and cigarette smokers. In conclusion, this paper indicates that dysrégula­ tion of immunoglobulins, especially IgD, is not un­ common in patients who had received antineoplastic drugs. It is of note that dysrégulation of IgD persists long after cessation of chemotherapy.

Acknowledgment The authors thank Toshie Ueno. RN at outpatient clinic of Department of Pediatrics for sample collection and Ms. Yoshiko Kanda for technical assistance.

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Eiichi Azuma. MD Department of Pediatrics Mie University School of Medicine 174-2 Edobashi Isu Mie-Ken 514 (Japan)

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