In VitroChromosomal Radiosensitivity in Common Variable Immune Deficiency

CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY

Vol. 86, No. 2, February, pp. 180–182, 1998 Article No. II974478

In Vitro Chromosomal Radiosensitivity in Common Variable Immune Deficiency Sukru Palanduz,* Ayse Palanduz,† Isik Yalcin,† Ayper Somer,† Ulker Ones,† Duran Ustek,* Sukru Ozturk,* Nuran Salman,† Nermin Guler,† and Hatice Bilge‡ *Istanbul Faculty of Medicine, Department of Internal Medicine, Division of Medical Genetics, †Istanbul Faculty of Medicine, Department of Pediatrics, and ‡Institute of Oncology, Istanbul University, 34390, Capa, Istanbul, Turkey

Common variable immune deficiency (CVID) is characterized by low immunoglobulin levels and recurrent infections in patients with a period of normal immune function several years after birth. It is associated with diarrhea, malabsorption, bronchiectasis, and lymphoreticular malignancies. Radiation-induced chromosome instability may contribute to the high degree of susceptibility to neoplasia. Peripheral blood lymphocyte cultures were obtained from six patients with CVID and the healthy control group matched by age and sex. The groups did not differ in the frequency of spontaneous chromosome aberrations. After exposure to X-ray radiation, mitotic indices were found to be significantly low and incidence of chromosomal alterations were high in the CVID group. We conclude that chromosomes of cells from patients with CVID are significantly more radiosensitive than those of controls. Thus these patients must be protected from unnecessary X-ray examinations and in case of radiosensitive tumour, the dose of irradiation should be carefully monitored. q 1998 Academic Press Key Words: radiosensitivity; common variable immune deficiency.

INTRODUCTION

Common variable immune deficiency (CVID) is a delayed-onset immune deficiency characterized by recurrent infections, bronchiectiasis, diarrhea, and malabsorption (1, 2). It is associated with autoimmune disorders (3). In a prospective study Kinlen et al. reported that they had observed 5-fold increase of cancer and 30-fold increase of lymphoma among CVID patients (4). Hermans et al. recorded seven neoplasms (15%), only one of which was a lymphoma in a study of 46 patients with CVID (1). In contrast Cunningham-Rundles et al. found that 11 individuals of a total group of 98 (11.2%) had developed cancer and 8 of them (8.2%) involved lymphoid tissue (5). Ataxia telangiectasia (AT) is an other primary immune deficiency with increased risk of tumors. Cells

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MATERIALS AND METHODS

Patients and controls. Six CVID patients and six healthy controls, matched for age and sex, were involved in this study. All of the patients were regularly seen at the Istanbul University, Faculty of Medicine, Department of Pediatric Allergy, Clinical Immunology and Infectious Diseases. They were aged 9 to 19 years and four of six were boys. Clinical features of these patients are summarized in Table 1. Family consents were taken for all of the patients and controls. Cultures. Heparinized blood samples were obtained and cultured in Ham’s F10 medium supplemented by 20% fetal bovine serum, 1% penicillin–streptomycin, 1.5% PHA-M, 1% L-glutamine by a modification of Moorhead’s method (7). Two cultures were prepared per each child: one for irradiation and the other for unirradiated samples. Irradiation and harvesting of cells. After an incubation period of 72 h, lymphocytes were exposed to a dose of 250 cGy of X rays (Siemens 200 kV, 10 mA, 1 mm Cu, 34.2 cGy/min). Thirty minutes after irradiation colcemide was added at a concentration of 0.1 mg/ml for 60 min. They were then centrifuged for 9 min at 150g. An additional 6 min was allowed before adding 75 mM KCl for 20 min. An additional 15 min was allowed for the second centrifugation. The total time between irradiation and fixation was 140 min. Metaphase chromosomes were prepared according to the standard methods and stained in Giemsa (5%) for 3 min (8). Cytogenetic studies. Fifty metaphase cells were examined per person in each irradiated and unirradiated

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from AT patients exhibit an increased number of chromosome aberrations after radiation (6). This is probably the common reason for the higher incidence of cancer in both AT and CVID. In this study we exposed the lymphocytes of CVID patients to X-ray irradiation to detect the chromosomal aberrations as an indicator of radiosensitivity.

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CHROMOSOMAL RADIOSENSITIVITY

TABLE 1

TABLE 3

Clinical Features of Patients

Frequency of Aberrant Cells after Irradiation (Mean { Standard Deviation)

Case

Age (years)

Sexa

Clinical features

1

19

F

2 3 4 5

19 10 9 14

F M M M

6

17

M

Recurrent pulmonary infections, recurrent bacterial meningitis Bronchiectasis, diarrhea, malabsorption Bronchiectasis, chronic sinusitis Recurrent pulmonary infections Bronchiectasis, recurrent pulmonary infections, short stature Bronchiectasis

a

CVID patients (n Å 6)

Controls (n Å 6)

300

300

182

89

61 { 26 26–82

30 { 7 20–42

Number of cells analyzed Number of cells with aberration Frequency of cells with aberrations (%) Range (%)

Significance

P ú 0.05

F, female; M, male.

cultures. The recorded aberrations were chromatid breaks, chromatid gaps, chromosome breaks, chromosome gaps, and acentric fragments. Statistical analysis. We used the SPSS statistical program. RESULTS

All of the patients and controls had normal karyotypes (46,XY or 46,XX). No spontaneous aberration was recorded either in the patient or control groups. The mean mitotic index (MI) of patients and controls is seen in Table 2. The mean MI of CVID patients was significantly lower than that of controls in both irradiated and unirradiated samples. After irradiation MI decreased significantly more than that of controls. The mean frequency of the cells with aberration was higher in the CVID patients than in the control group but it was not found statistically significant (Table 3). The number of aberrations (chromosome breaks, chromosome gaps, chromatid gaps, and acentric fragments) per cell and total number of aberrations in the CVID patients were significantly more than that of controls (Table 4).

with AT, Bloom’s Syndrome, and Fanconi’s anemia after irradiation. This is termed as chromosomal radiosensitivity (9). Individuals with genetic diseases predisposing to cancer or hereditary neoplasm such as xeroderma pigmentosum, familial polyposis, Gardner’s syndrome, hereditary malignant melanoma, dysplastic nevus syndrome, and cancer family members exhibit the same response (10). The higher incidence of chromatid breaks and gaps in metaphase cells after X-ray irradiation has been shown to be a result of deficient DNA repair. Gantt et al. reported the biochemical evidence for that DNA repair deficiency (11). Sanford et al. stated that deficient DNA repair could provide genetic instability resulting in point mutations or deletions at cancer predisposing sites (10). AT is a complex syndrome with a very high cancer risk. In an article by Morrell et al. cancer incidence was reported to be 61 times higher for white probands and 184 times higher for black probands. The cancer excess was most pronounced for lymphoma, with 252and 750-fold excesses observed for whites and blacks, respectively (12). Of the AT patients that are homozygous for the A-T gene located at 11q23, 30–40% develop cancer. Of these cancers 80% are lymphoid. Genetic predisposition to cancer for heterozygotes of A-T gene is also remarkable.

DISCUSSION

Frequency of chromosomal aberrations was found to be increased in the cultured lymphocytes of individuals

TABLE 4 Comparison of Number of Aberrations per Cell (Mean { Standard Deviation) in the Irradiated Samples of CVID Patients and Controls

TABLE 2

CVID patients

Comparison of MI (Mean { Standard Deviation) in CVID Patients and Controls before and after Irradiation Unirradiated sample CVID patients (n Å 6) Controls (n Å 6) Significance

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2.68 { 1.92 5.67 { 0.65 P õ 0.05

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Irradiated sample 0.71 { 0.74 3.06 { 0.67 P õ 0.005

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Chromosome breaks Chromosome gaps Chromatid breaks Chromatid gaps Acentric fragments Total number of aberrations

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3.4 1.63 10.25 4.77 1.4

{ { { { {

2.14 0.88 3.44 1.88 0.69

21.45 { 4.92

Controls 0.84 0.1 6.87 0.85 0.35

{ { { { {

0.52 0.08 2.45 0.34 0.26

9.01 { 2.76

Significance P P P P P

õ õ ú õ õ

0.05 0.05 0.05 0.05 0.05

P õ 0.05

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Breast cancer is seen 6.8 times more in female carriers (13) and the overall risk of cancer is three times that of the norm (14). In AT nonrandom chromosome breaks and rearrangements in lymphocytes involving the sites of immunoglobulin and T cell receptor genes are increased. The propensity to progress to a malignant transformation is found to be great when the rearrangements involve certain sites, especially a locus within 14q32 (13). We observed a significant decrease in mitotic indices and increase in chromosome aberrations after irradiation of cells of CVID patients. Since no spontaneous aberration was recorded in those patients, this response is interpreted as radiosensitivity. Vorechovsky et al. demonstrated bleomycin-induced chromosome instability in patients with CVID. They claimed increased chromatid instability after DNA damage to be responsible for cancer susceptibility (15). The genomic instability is the main, but not the only, cause of malignant transformation. Viruses including Ebstein–Barr virus and hepatitis B virus may contribute to the oncogenic transformation. It is not clear yet whether either virus-infected cells show chromosomal aberrations or viral insertion and integration is facilitated by genomic instability (16). Chromosomal damage in the G2 lymphocytes of 24 CVID patients and 21 controls after X-irradiation in vitro was analyzed by Vorechovsky et al. There was a significant difference in mean aberration yields between patients and controls. They observed that the patient with the highest chromosomal radiosensitivity subsequently developed lymphoma (17). We conclude that cells from CVID patients exhibit increased chromosomal radiosensitivity. We should concern to avoid unnecessary irradiation for diagnosis and treatment unless sufficient clinical data is obtained about the contrary. REFERENCES 1. Hermans, P. E., Diaz-Buxo, J. A., and Stobo, J. D., Idiopathic late-onset immunoglobulin deficiency: Clinical observations in 50 patients. Am. J. Med. 11, 221–237, 1976. 2. Hausser, C., Virelizier, J. L., Buriot, D., and Griscelli, C., Common variable hypogammaglobulinemia in children: Clinical and immunologic observations in 30 patients. Am. J. Dis. Child. 137, 833–837, 1983.

3. Conley, M. E., Park, C. L., and Douglas, S. D., Childhood common variable immune deficiency with autoimmune disease. J. Pediatr. 108(6), 915–922, 1986. 4. Kinlen, L. J., Webster, A. D. B., Bird, A. G., Haile, R., Peto, J., Soothill, J. F., and Thompson, R. A., Prospective study of cancer in patients with hypogammaglobulinemia. Lancet 1, 263–266, 1985. 5. Cunningham-Rundles, C., Siegal, F. P., Cunningham-Rundles, S., and Lieberman, P., Incidence of cancer in 98 patients with common varied immunodeficiency. J. Clin. Immunol. 7(4), 294– 299, 1987. 6. Mozdarani, H., and Bryant, P. E., Cytogenetic response of normal human and ataxia telangiectasia G2 cells exposed to X-rays and ara-C. Mutat. Res. 226, 223–228, 1989. 7. Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battips, D. M., and Hungerford, D. A., Chromosome preparations of leukocyte cultures from human peripheral blood. Exp. Cell. Res. 20, 613– 616, 1960. 8. Gustashow, K. M., Chromosome stains. In ‘‘The ACT Cytogenetics Laboratory Manual’’ (M. J. Barch, Ed.), p. 205, Raven Press, New York, 1991. 9. Higurashi, M., and Conen, P. E., In vitro chromosomal radiosensitivity in chromosomal breakage syndromes. Cancer 32(2), 380– 383, 1973. 10. Sanford, K. K., Parshad, R., Gantt, R., et al., Factors affecting and significance of G2 chromatin radiosensitivity in predisposition to cancer. Int. J. Radiat. Biol. 55(6), 963–981, 1989. 11. Gantt, R., Parshad, R., Price, F. M., and Sanford, K. K., Biochemical evidence for deficient DNA repair leading to enhanced G2 chromatid radiosensitivity and susceptibility to cancer. Radiat. Res. 108, 117–126, 1986. 12. Morrell, D., Cromartie, E., and Swift, M., Mortality and cancer incidence in 263 patients with ataxia-telangiectasia. J. Natl. Cancer Inst. 77(1), 89–92, 1986. 13. Peterson, R. D., Funkhouser, J. D., Tuck-Muller, C. M., and Gatti, R. A., Cancer susceptibility in ataxia-telangiectasia. Leukemia 6(Suppl. 1), 8–13, 1992. 14. Bay, J. O., Udar, N., Bignon, Y. J., and Gatti, R. A., Ataxia telangiectasia and genetic predisposition to cancer. Bull. Cancer Paris 83(3), 171–175, 1996. 15. Vorechovsky, I., Munzarowa, M., and Lokaj, J., Increased bleomycin-induced chromosome damage in lymphocytes of patients with common variable immunodeficiency indicates an involvement of chromosomal instability in their cancer predisposition. Cancer Immunol. Immunother. 29, 303–306, 1989. 16. Vorechovsky, I., Litzman, J., Jindrich, L., Hauner, P., and Poch, T., Common variable immunodeficiency and malignancy: a report of two cases and possible explanation for the association. Cancer Immunol. Immunother. 31, 250–254, 1990. 17. Vorechovsky, I., Scott, D., Haeney, M. R., and Webster, D. A. B., Chromosomal radiosensitivity in common variable immune deficiency. Mutat. Res. 290, 255–264, 1993.

Received January 22, 1997; accepted with revision September 19, 1997

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