Int. J. Cancer (Pred. Oncol.): 69, 265-267 (1996) Q 1996 Wiley-Liss, Inc. ( Publication of the International Union Against Cancer Publication de I'Union InternationaleContre le Cancer MUTAGEN SENSITIVITY AS A PREDISPOSING FACTOR IN FAMILIAL ORAL CANCER Ravindran ANKATHIL1,4, Narayanan V. BHATTATHIRI?, James V. FRANC IS^, K. RATHEESAN~, B. JYOTHISH',R. CHANDINI~, Dinesh D. ROY', Abraham K. ELIZABETH3 and M. Krishnan NAIR? Depts. of 'Cancer Research, ?Radiotherapy and 3Cytopathology, Regional Cancer Centre, Trivandrum, India. Pedigree analysis of the oral cancer (OC) patients registered at our Centre had disclosed familial aggregation of oral cancer which hitherto has not been largely reported. There is a paucity of information on the genetic determinism for familial oral cancer predisposition. Therefore, we investigated constitutional chromosome abnormalities and bleomycin-induced chromosome sensitivity of 7 familial and 10 sporadic oral cancer patients and 14 unaffected family members (first-degree relatives) to determine whether these factors could give any clues regarding cancer-predisposingfactors. Neither the oral cancer patients nor the unaffected family members showed any constitutional chromosomal abnormalities. However, with regard to bleomycin sensitivity, there was significant difference between the oral-cancer patients and unaffected relatives. The mean b/c value was I .68 f 0.48 for familial OC patients, I. I 2 f 0.36 for sporadic OC patients and 0.52 f 0. I 8 for the unaffected family members (p < 0.00 I). A noteworthy observation was that one unaffected family member also showed bleomycin hypersensitivity and expressed a mean b/c value of I .32, at the initiation of the study. That patient later developed oral carcinoma. This clearly demonstrates that mutagen hypersensitivity among unaffected relatives in OC families may be related to cancer predisposition. The mutagen sensitivity study is being continued in a larger series of subjects, for the development of a cytogenetic marker for predictionof cancer susceptibility. 8 1996 Wiley-Liss,Inc. Carcinoma of the human oral cavity comprises a highly prevalent cancer in India. The majority of Indian oral cancers have been associated with habits such as chewing and/or smoking of tobacco and consumption of alcohol. At the Regional Cancer Centre, Thiruvananthapuram (S. India), oral cancer constitutes about 25% of the total 6,000 new cancer cases registered annually (Nair, 1994). A preliminary analysis of the pedigrees of oral cancer patients registered at our Centre had revealed familial aggregation of oral cancer (Ankathil et al., 1996). This shows that inherited factors are also important in oral cancer. But the genetic determinism for oral-cancer predisposition among the family members remains unknown. In the absence of complete information on the genes involved, it is of interest to search for criteria to detect individuals at risk for inherited forms of oral cancer. One criterion may be genomic instability. Genomic instability can be detected at the cytogenetic level by either a high rate of spontaneous alterations or an increased sensitivity to mutagenic or clastogenic agents (Delhanty et al., 1983; Hsu et al., 1989; Pathak et al., 1991). This study was developed on familial and non-familial (sporadic) oral cancer patients before treatment and unaffected first-degree relatives from oral-cancer families. The aim was to quantify the constitutional genetic instability of these subjects through the bleomycin-induced chromosome sensitivity assay and to determine whether this may give any clues to cancer predisposition in oral-cancer families. Pedigree analysis All the oral-cancer patients, on their first attendance at the Clinics of Regional Cancer Centre, Trivandrum, were interviewed via a prestructured questionnaire aimed at the oncological history of first-degree relatives, with particular attention being paid to oral cancer. O n the basis of family history analysis, we identified 5 site-specific oral cancer families. Three families showed aggregation of buccal mucosal cancer, 1 family showed aggregation of carcinoma of the lip and 1 showed aggregation of carcinoma of the tongue. Detailed pedigrees of these families were prepared. The pedigree of one of the oral cancer families is shown in Figure 1. MATERIAL AND METHODS In the present study, we employed short-term microcultures of peripheral blood lymphocytes from 10 sporadic and 7 familial oral-cancer patients and 14 unaffected first-degree relatives of familial oral-cancer patients. Lymphocytes from each sample were cultured under 2 different sets of conditions. The culture medium used was RPMI 1640 (DIFCO; Detroit, MI) supplemented with 20% FBS and 0.5 ml phytohaemagglutinin (DIFCO). In culture A, cells were incubated in culture medium alone to study the constitutional chromosome markers and the spontaneous chromosome aberrations. In culture B, lymphocytes were cultured in culture medium and the mutagen bleomycin was added to induce chromosomal breakage, according to the method of Hsu et al. (1991). The total incubation time for all these cultures was 72 hr at 37°C. Bleomycin treatment (0.03 units/ml, Nippon Kayaku, Tokyo, Japan) was given during the last 5 hr in culture, ensuring that damage induced in the late S and G2-phases of the cell cycle could be evaluated at metaphase. During the last 2 hr, the cells were treated with colcemid (0.04 Fgiml) to accumulate mitoses before being harvested for conventional air-dried preparations. All cultures were harvested by standard cytogenetic procedures (Moorhead et al., 1960). Chromosome preparations were stained with conventional Giemsa and banded with trypsin-Giemsa (Seabright, 1971). From each sample, 20-30 metaphases were counted and 5-10 metaphases were karyotyped to detect constitutional chromosome abnormalities. Karyotypes were prepared according to the International System for Human Cytogenetic Nomenclature (ISCN, 1991). For mutagen sensitivity, chromosome breaks were scored on 100 metaphases per sample by one of us (R.A.) who was blinded as to the case control and exposure status of the individual. Only frank chromatid breaks or exchanges were recorded. Each chromatid break was recorded as one break and each chromatid exchange was recorded as 2 breaks. The mean number of breaks per cell (b/c), based on evaluation of 100 metaphases, was taken as a measure of mutagen sensitivity. Any individual expression > 0.8 b / c was considered sensitive to bleomycin-induced chromosome damage and any individual expression above 1.0 b/c was considered hypersensitivc (Spitz et al., 1989). RESULTS Constitutional chromosome abnonnalities G-banded karyotype analysis of the lymphocytes from 10 sporadic and 7 familial oral-cancer patients and 14 unaffected JTo whom correspondence and reprint requests should be sent, at the Division of Cancer Research, Regional Cancer Centre, Trivandrum, India--695 011. Fax: 91 0471 447454. Received: December 6 , 1995 and in revised form March 28, 1996. 266 ANKATHIL E T A L . family members did not show any constitutional chromosome abnormalities of either numerical or structural types. All these subjects showed the normal karyotype pattern, with 46 chromosomes. Spontaneous and bleomycin-induced chromosomal breakage The frequencies of spontaneous chromosomal breaks were generally low in all the subjects, ranging from 0 to 5 breaks per 100 metaphases. However, there were great variations in bleomycin-induced chromosome breakage frequencies among the patients and their unaffected relatives. The most predominant type of aberration was chromatid breaks. All the 17 oral cancer patients were hypersensitive to bleomycin and expressed between 88 and 192 breaks per 100 metaphases. The mean b / c value was 1.68 -t 0.48 for the 7 familial oral-cancer patients and 1.12 f 0.36 for the 10 sporadic oral-cancer patients. All the unaffected family members (except one) expressed a lower number of breaks with a mean b / c value of 0.52 ? 0.18. The mean b / c values of the familial oral-cancer patients were significantly higher than the values of unaffected close relatives ( p < 0.001), as shown in Table I. A significant observation was made in one of the families studied (Fig. 1). Bleomycin-induced chromosome sensitivity of one familial oral-cancer patient (patient 4) and her 3 first-degree relatives (numbers 12, 13 and 14) had been studied 18 months previously. In this family, the proband, a female aged 59, who did not chew, smoke or consume alcohol, had carcinoma of the buccal mucosa and expressed a b / c value of 1.45. One of her daughters, aged 35 (number 13 in pedigree), who also had no habits of chewing or smoking tobacco, showed chromosome hypersensitivity to bleomycin with a b / c value of 1.32. This value was as high as that observed for oral-cancer patients and so she was regularly followed up. Subsequently, 18 months later, she presented with symptoms of buccal rnucosal cancer, at a younger age. DISCUSSION It appears that genetic instability may participate in cancer predisposition in addition to other factors and may account for the heterogeneity of the pathologic status for a given genetic constitution. Available reports indicate that individuals with certain chromosomal abnormalities are more susceptible to the development of specific types of cancer (Cohen et al., 1979; Johnson et al., 1982; Orkin et al., 1984; Bolger et al., 1985). In the current study, we investigated the constitutional chromosomal abnormalities, if any, associated with lymphocytes of familial and/or sporadic oral-cancer patients. However, our results did not confirm the existence of a direct relationship between a given acquired chromosome alteration and a disease state. Hsu et al. (1989) developed the mutagen-sensitivity assay as an indirect indicator of DNA repair competence and cancer predisposition. It has been suggested that cancer-prone individuals have a deficiency in some step of D N A repair, which TABLE I - BLEOMYCIN-INDUCED CHROMOSOMAL BREAKSICELL VALUES OF THE 3 DIFFERENT SUBJECT GROUPS $$ f~~~~ h i c range Mean blc Subjects Number Unaffected relatives Sporadic oral cancer Familial oral cancer 14 717 30-55 0.27-0.7 10 416 53-72 0.88-1.44 1.12 7 3/4 48-59 1.21-1.92 1.68 f 0.48* 0.52 2 ? SD 0.18 * 0.36 *p < 0,001 significantly different (Student’s t-test) from unaffected relatives. 32 30 28 26 37 3 5 3 3 28 26 33 31 28 31 28 25 0 FIGURE 1 - Pegidree of an oral cancer family. Squares, males; circles, females; numbers above symbols, identifying numbers of family members; numbers below symbols, age at tumour diagnosis or at last observation; U, age unknown; symbols with a diagonal, deceased; open symbols, no neoplasm detected; solid symbols,oral cancer. may represent a type of genetic predisposition to cancer (Hsu et al., 1991). In order to determine whether mutagen sensitivity o r resistance correlates with cancer predisposition in the oral-cancer families described here, the ability of relatives’ cells to repair chromosomal breakage induced by the mutagen bleomycin was assayed. All the 7 familial oral-cancer patients expressed high mutagen sensitivity with a mean b / c value of 1.68, which was significantly higher than the values of unaffected family members ( p < 0.001). All the 10 sporadic oral-cancer patients also showed mutagen sensitivity, with a mean b / c value of 1.12. The observation that these high frequencies of breakage were of the chromatid type indicates that the breakages are the result of the patients’ hypersensitivity to the mutagen bleomycin. Our study subjects included smokers and non-smokers. But tobacco chewing and smoking, age, gender and stage of disease did not have any impact on the bleomycin-sensitivity profile of our study subjects. Liang et al. (1989) reported that the levels of bleomycin-induced breaks are generally unchanged before, during and after chemotherapy, thereby indicating that therapy has no effect on the mutagen sensitivity of cancer patients. Even though a few reports are available regarding increased bleomycin sensitivity of head-and-neck cancer patients (Spitz et al., 1989; Schantz et al., 1990; Cloos et al., 1994) no previous reports exist on the bleomycin sensitivity of familial oral-cancer patients. Mutagen-induced chromosome sensitivity is considered as a constitutional factor which may be used to identify persons with a genetic predisposition to cancer (Hsu, 1987; Cloos et al., 1994). This has been clearly demonstrated in the oral-cancer family shown in Figure 1. In this family, a woman aged 35, who had no habit of chewing or smoking tobacco, showed chromosome hypersensitivity to bleomycin. She expressed a b / c value of 1.32 which was as high as that of oral-cancer patients. Her mother and maternal grandfather had carcinoma of the buccal mucosa. O n follow-up, this woman later developed buccal mucosal cancer. This was a noteworthy observation which indicates that an elevated bleomycin clastogenicity score may identify individuals who have a constitutional hypersensitivity towards certain genotoxicants and may show an increased cancer susceptibility. All other unaffected first-degree relatives in other oral-cancer families studied were hyposensitive to bleomycin, indicating genetic stability and/or efficient D N A repair capabilities. Bondy et al. (1993) evaluated the selfreported cancer histories of 669 first-degree relatives of 108 patients with cancers of the upper aerodigestive tract and MUTAGEN SENSITIVITY IN FAMILIAL ORAL CANCER observed familial aggregation of cancer in mutagen-sensitive patients. There are reports stating that cells of patients with chromosome breakage syndromes such as Ataxia telangiectasia or Fanconi’s anemia are sensitive t o ionizing radiation and a variety of alkylating agents, and these patients are more prone to develop malignancies (Heddle et al., 1978; Paterson and Smith. 1979; Harnden, 1985). All these observations reiterate that genetic susceptibility t o malignancies may manifest itself a s chromosomal instability. It may be because of the genetic instability that somatic-cell chromosomes are liable t o spontaneous or induced breaks. Through the bleomycin-sensitivity study, the ability of a n individual’s cells to repair bleomycin-induced chromosome breakage can b e assayed. T h e mutagen hypersensitivity of the 7 patients in these 5 oral-cancer families suggests a common genetic determinant that may b e an important predisposing factor for cancer development. It is likely that the affected 267 individuals have deficiencies in their general cancer-protective mechanisms such as those of DNA repair and recombination which are essential for maintaining the integrity of their DNA. Bleomycin-hypersensitive individuals may be deficient in the repair genes that affect the efficiency of repair of the DNA lesions induced by bleomycin. Hence it is reasonable to assume that, in oral-cancer families, unaffected members who are bleomycin-hypersensitive may be at an increased risk of cancer susceptibility. We are continuing serial studies of mutagen sensitivity in oral-cancer families with the aim of developing a cytogenetic marker for prediction of cancer susceptibility. ACKNOWLEDGEMENT The authors thank Ms. P.T. Latha and Ms. Anitha Nayar, social investigators of our centre for their support and Ms. L. Remadevi, for secretarial assistance. REFERENCES ANKATHIL, R., MATHEW,A., JOSEPH,F. and KRISHNAN NAIR,M., Is oral cancer susceptibility inherited? Report of 5 oral cancer families. Oral Oncol., 32B, 63-67 (1996). BOLGER,G.B., STAMBERG, J., KIRSCH,I.R., HOLLIS,G.F. and SCHWARTZ-THOMAS, G.H., Chromosome translocation t(14;22) and oncogene variant in a pedigree with familial meningioma. New Engl. J. Med., 312,564-567 (1985). BONDY,M.L., SPITZ.M.R., HALABI, S., FUEGER,J.J., SCHANTZ, S.P., SAMPLE,D. and Hus, T.C., Association between family history of cancer and mutagen sensitivity in upper aerodigestive tract cancer patients. Cancer Epidemiol. Biomarkers Prev., 2,103-106 (1993). CLOOS,J., BRAAKHUIS, B.J., STEEN,I., COPPER,M.P., DE VRIES,N., NAUTA,J.J. and SNOW,G.B., Increased mutagen sensitivity in head and neck squamous cell carcinoma patients, particularly those with multiple primary tumours. Int. J. Cancer, 56,816-819 (1994). COHEN,A.J., LI, F.P., BERG,S., MARCHETTO, D.J., TSAI,S., JACOBS, S.C. and BROWN, R.S., Hereditary renal cell carcinoma associated with a chromosomal translocation. New Engl. J. Med., 301,592-595 (1979). DELHANTY, J.D.A., DAVIS,M.B. and WOOD,J., Chromosome instability in lymphocytes, fibroblasts and colon epithelial-like cells from patients with familial polyposis coli. Cancer Genet. Cyfogenet.,8,27-50 (1983). HARNDEN, D.G., Inherited factors in leukaemia and lymphoma. Leuk. Rex, 9,705-707 (1985). HEDDLE, J.A., LUE,C.B., SAUNDERS, E.F. and BENZ,R.D., Sensitivity to five mutagens in Fanconi’s anemia as measured by the micronucleus method. Cancer Res., 38,2983-2988 (1978). Hsu, T.C., Genetic predisposition to cancer with special reference to mutagen sensitivity.In vitro Cell Devel. Biol., 23,591-603 (1987). Hsu, T.C., JOHNSTON, D.A., CHERRY, L.M., RAMKISSOON, D., SCHANTZ, S.P., JESSUP,J.M., WINN,R.J., SHIRLEY, L. and FURLONG, C., Sensitivity to genotoxic effects of bleomycin in humans. Possible relationship to environmental carcinogenesis. Int. J. Cancer, 43,403409 (1989). Hsu, T.C., SPITZ,M.R. and SCHANTZ,S.P., Mutagen sensitivity-a biological marker for cancer susceptibility. Cancer Epidemiol. Biomark. Prev., 1 , 8 3 4 9 (1991). ISCN, Guidelines for cancer cytogenetics: Supplement to An International System for Human Cytogenetic Nomenclature. F. Mitelman (ed.), S. Karger, Basel (1991). JOHNSON, M.P., RAMSAY, N.. CERVANKA, J. and WANG,N., Retinoblastoma and its association with a deletion in chromosome 13. A survey using high-resolution chromosome techniques. Cancer Genet. cy60genet., 6,29-37 (1982). LIANG,J.C., PINKEL, D.P., BAILEY,N.M. and TRUJILLO, J.M., Mutagen sensitivity and cancer susceptibility. Report of a cancer prone family. Cancer, 64,1474-1479 (1989). MOORHEAD, P.S., NOWELL,P.C., MELLMANN, W.J., BATTIPPS,D.W. and HUNGERFORD, D.A., Chromosome preparations of leukocytes cultured from human peripheral blood. Exp. Cell Res., 20, 613-616 (1960). NAIR,M.K., Regional Cancer Centre-Trivandrum, India. Annual Report (1994). ORKIN,S.H., GOLDMAN, D.S. and SALLAN,S.E., Development of homozygosity for chromosome tip markers in Wilms’ tumour. Nature (Lottd.), 309,172-174 (1984). PATERSON, M.C. and SMITH,P.J., Ataxia telangiectasia-an inherited human disorder involving hypersensitivity in ionizing radiation and related DNA-damaging chemicals. Ann. Rev. Genet., 13, 291-318 (1979). PATHAK,S., HOPWOOD,V.L., HUGHES.J.I. and JACKSON,G.L., Identification of colon cancer-predisposed individuals. A cytogenetic analysis.Amer. J. Gastroenterol., 86, 676-684 (1991). SCHANTZ, S.P., SPITZ,M.R. and HSU,T.C., Mutagen sensitivity in head and neck cancer patients: a biologic marker for risk of multiple primary malignancies. J. naf. Cancer Znst., 82, 1773-1775 (1990). SEABRIGHT, M., A rapid banding technique for human chromosomes. Lancet., ii, 971-972 (1971). SPITZ,M.R., FUEGER, J.J., BEDDINGFIELD, N.A., ANNEGERS, J.F., Hsu, T.C. and NEWALL,G.R., Chromosome sensitivity to bleomycininduced mutagenesis-an independent risk factor for upper aerodigestive tract cancers. Cancer Res., 4 9 , 4 6 2 H 6 2 8 (1989).