- Email: [email protected]
HLA-A, B, C, DR and DQ expression and hepatocellular carcinoma: study of 205 Italian subjects
ELSEVIER
Cancer Letters 98 (1995) 121-125
CANCER LETTERS
HLA-A, B, C, DR and DQ expression and hepatocellular carcinoma: study of 205 Italian subjects Giorgio Ricci a,*, Clara Colombo a, Barbara Ghiazza b, Camillo Porta c, Mauro Moroni c, Maria Teresa Illeni d a2nd Geriatric Division, Azienda Ospedale San Gerardo, 20052, Monza, Milan, Italy bGeriatric Valuational Unit, Azienda USSL 8, 22055, Merate, Lecco, Italy CDepartment of Internal Medicine and Medical Therapy, University of Pavia, IRCCS-Policlinico San Matteo, 27100, Pavia, Italy dDivision of lmmunohematology, Istituto Nazionale dei Tumori di Milano, 20122, Milano, Italy
Received 23 July 1995; revised version received 15 September 1995; accepted 28 September 1995
Abstract
We have evaluated the frequency of HLA class I and II antigens in 205 Italian patients with hepatocellular carcinoma (HCC) and 749 blood donors (controls). Moreover we have looked for correlations between HLA antigen frequencies and HBV and/or HCV infections in HCC patients. We found great differences in HLA antigen frequencies considering only two groups: HCC patients and controls. The polymorphism is smaller when we consider the different groups of HCC patients in regard to the previous viral infections (HBV and/or HCV). The most interesting finding is the higher frequency of Cw7, B8 and DR3 in almost all groups of HCC patients. It is well known, that the HLA A1, Cw7, B8, DR3 antigen haplotype is associated with a rapid decline of CD4 cells, and HLA B8, DR3 positive subjects may display some changes in immune parameters and are prone to develop several immunological diseases. Thus HCC might be the result of a lower sensitivity (genetically given) to mitogenic stimuli of HBV and HCV. Keywords: Hepatocellular carcinoma; Hepatitis B virus; Hepatitis C virus; HLA
1. I n t r o d u c t i o n
Even if environmental and viral factors seem to be the most important aetiological factors of hepatocellular carcinoma (HCC) [1-4], it is likely that genetic factors may also be involved. It is known, for example, that in Singapore, the age standardized incidence of HCC is higher in the Chinese than in Malay or in * Corresponding author. Via Saffi 7, 20058-Villasanta (MI), Italy. Tel.: +39 39 303621; fax: +39 39 2050491.
Indian population [5]. The prevalence of hepatitis B virus surface antigen (HBsAg) in the different ethnic groups, follows the same trend [6]. Moreover, it is known that hepatitis C virus (HCV) has a very high prevalence in patients afflicted with liver cirrhosis and by HCC, and is often associated with markers of hepatitis B virus infection (HBV). In the same ethnic group there are patients afflicted by HCC with high or low levels of a-fetoprotein, with different prevalence of serum level of HBsAg and with different immune response to HBV infection [7,8]. Moreover,
0304-3835/95/$09.50 9 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3835(95)04005-0
122
G. Ricci et al. / Cancer Letters 98 (1995) 121-125
some authors have shown an association between the persistence of infection and HLA [9,10]. and between HLA and liver cirrhosis [11-17], even if the results are indefinite. These data suggest that the different prevalence of HCC in the different ethnic groups could be determined by a different reaction of the host to HBV and/or HCV infection. The aim of our work has been to evaluate the phenotypic frequency of 1st and 2nd class HLA antigens in a group of Italian patients with HCC. Moreover, we have looked for correlations between allelic frequencies and HBV and or HCV infections in HCC subjects.
The correlation between HLA in the group of HCC patients (n = 205) and in controls (n = 749), showed higher frequency of HLA-A3, A19, Cw4, Cw7, B35, DR3 and DQ1, and a lower frequency of HLA-A9(23;24) in patients with HCC. The statistical significance (P < 0.0001), is maintained after multiplying the P value by the number of comparisons (<0.006) (Table 1).
2. Patients and methods
3.1. H C C and H B V infection (Table 2)
Two hundred and five patients with HCC and 749 healthy controls were studied. All were Italian (148 males, 57 females), with age ranging from 46 to 81 years (mean _+SD 63.2 _+5.75). The diagnosis of HCC was based on: (a) pathological findings typical of HCC; (b) space taking lesion in the liver detected by ultrasonography or computed tomography with an a-fetoprotein level greater than 100 IU/ml; (c) increased a-fetoprotein level, greater than 100 IU/ml, and malignant cells found by aspiration. Among the 205 patients with HCC, 148 were tested for HBV markers of infection (HBsAg, antiHBsAg, anti HBcAg, anti-HBeAg, HBeAg). Only 10 males were drinkers (alcohol intake higher than 80 g/day); 183 had liver cirrhosis. All the 749 controis were negative for previous HBV and or HCV infections. All the 205 patients were typed for HLA-A, B, C; 181 patients were typed for HLA-DR, and 139 for HLA-DQ antigens. The typing was performed by microdroplet cytotoxicity test, according to the recommendation of the National Institute of Health [ 18]. The T-lymphocytes for HLA class I typing and Blymphocytes for class II typing were separated by the use of immunomagnetic beads (Dynal A.S., N-0212, Oslo, Norway) from 10ml of heparinized whole blood. The differences in HLA frequencies between the patients and controls were evaluated by the chi squared test according to Woolf [19]. Haldane's
(a) Patients with no previous HBV infection and HCC, showed a higher frequency of HLA-Cw4 and Cw7 (P < 0.0001; P • n < 0.006) when compared to controls. (b) Patients HBsAg positive showed a higher frequency of HLA-Cw7, B8, DR3 and DQ2 than controis. (c) Patients HBsAg negative but with at least one marker of HBV infection (antiHBcAg and/or antiHBsAg and/or antiHBeAg and/or HBeAg) showed higher frequency of HLA-Cw4, Cw7, B8, DR3 and DQ 1 than controls. (d) Patients positive for at least one marker of
method was also employed when one of the compared numbers was equal to zero, and P values were corrected for the number of comparisons (P x n). 3. Results
Table 1 HLA antigens frequenciesin HCC patients (HCC) and in controls (C) HLA
HCC (N 205) %
C X2 (N 749) %
P
Px
A3 A9(23,24) A19 Cw4 Cw7 B35 DR3 DQ1
28.78 19.02 38.53 34.14 28.78 33.65 29.83 52.17
16.55 33.64 21.89 22.02 10.14 20.29 16.15 34.16
0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001
0.006 0.006 0.006 0.006 0.006 0.006 0.006 0.006
15.52 16.23 23.48 12.73 46.00 16.15 17.86 36.44
ap • n is P correctedfor the numberof comparisons.
na
G. Ricci et al. I Cancer Letters 98 (1995) 121-125
123
Table 2
Table 3
HLA antigens frequencies and H B V in HCC patients
HLA antigens frequencies and anti-HCV in H C C patients
HLA
A3 Cw4 Cw7
HCC patients
(C)
(l)
(2)
(3)
(4)
%
%
%
%
%
38.2 ~
16.55 22.03
33.92 ^
37.50* 30.43*
50.00 +
38.92 ^
41.170
10.14
B8
30.48 +
26.78 ^
28.43 ~
10.14
DR3
48.78 +
36.17 ^
42.08 ~
16.15
DQ1 DQ2
69.04 ^ 46.51 +
34.16 24.67
X2
27.25 ~ 13.49 *a 28.31 ^a 17.77 +a 63.14 +a 28.31 ^a 72.37 ~ 17.77 +a 14.39 ^a 27.75 ~ 28.22 +a 12.36 ^a 34.47 ~ 19.00 ^a 19.00 +a
(1) HBV negative, n = 46; (2) H B s A g positive, n = 46, (3) antiH B s / a n t i H B c A g positive, n = 56; (4) (2)+(3), n = 102. (C) Controis, n = 749. a p < 0 . 0001; P • n < 0. 006.
HBV infection showed higher frequency of HLA-A3, Cw7, B8 and DR3 than controls.
3.2. HCC and HCV infection (Table 3) (a) Patients without previous HCV infection and HCC, showed higher frequency of HLA-DR3 than patients with HCC and previous HCV infection and controls. (b) HCC patients without HCV infection showed higher frequency of HLA-A1, Cw7 and DR3 than controls. (c) Patients with HCC and markers of HCV infection, showed a higher frequency of HLA-Cw7, B35 and DQ1 than controls.
3.3. HCC and HBV and HCV infections combined (Table 4) (a) Patients with HCC without any marker of HBV and HCV infection show higher frequency of HLACw7 than controls (P < 0.0001; P • n < 0.006).
HLA
HCC patients
Controls
antiHCV(n = 66) %
antiHCV+ (n = 74) %
%
A1 Cw7
42.42 ^ 47.93 ^
33.74 ~
22.03 10.14
B8 B18 B35 DR3
31.82 ^ 24.246 40.54 ~ 16.60 ~
10.14 10.01 20.29 16.15
62.31 ~
34.16
49.19 ^
DQ1
aP < 0.0001; P • n < 0.006. ^ a n t i H C V - versus controls; ~ H C V - versus anti HCV+.
X2
(n = 749)
13.96 ^a 61.71 ^a 39.95 ~ 14.88 ^a 12.38 ^a 16.03 ~ 40.56 ^a 16.16 ~ 34,16 ~
versus controls; ^~
(b) HCC patients with positivity for antiHCV antibodies (but without markers of HBV infection) show higher frequency of HLA-Cw4 and DQ1 than controis (P < 0.0001;
P •
n < 0.006).
(c) HCC patients with at least one marker of HBV infection and antiHCV antibodies negative, have higher frequency of HLA-Cw7, B8, DR3 (P < 0.0001; P • n < 0.006) than controls. (d) HCC patients with positivity for both HBV and HCV markers of infection show a higher freTable 4 HLA, HBV markers and antiHCV in HCC patients HLA
HCC patients (104) B-/C(14) %
Cw4 Cw7
B8 DR3 DQ1
B-/C+ (21) %
Controls (749)
B+/C(33) %
B+/C+ (36) %
%
46.93 +
38.77*
22.03 10.14
65.21 ~ 42.10 ^
36.73 + 54.56 + 88.88 ~
10.14 16.15 34.26
X2
23.28 ~ 19.43 ^a 57.29 +a 35.94 *a 31.29 +a 43.33 +a 22.92 ~
B+, HBV markers positive (at least one marker); B - , HBV markers negative; C+, antiHCV positive; C - , antiHCV negative. a p < 0. 0001; P • n < 0. 006.
124
G. Ricci et al. / Cancer Letters 98 (1995) 121-125
quency of HLA-Cw7 in comparison with controls (P < 0.0001; P x n < 0.006).
4. Discussion There is a great difference in H L A antigen distribution between controls and HCC patients (Table 1). These differences are smaller when we consider the individual subpopulations of patients with HCC. Evaluating patients on the grounds of viral aetiology, we have observed a lower frequency of HLA-B8 and DR3 in patients with HCC and negative for HBV markers of infection, when compared to HBV positive patients (HBsAg and/or antiHBsAg and/or antiHBcAg) (Table 2). The comparisons of allelic frequencies in subgroups of patients, evaluated on the basis of antiHCV antibody positivity is more complex, because differences between the two groups of patients (HCV+ and H C V - ) and between the two groups of patients and controls, do not always agree (Table 3). The same problem appears if we consider patients on the grounds of positivity and negativity for HBV and H C V markers of infection (Table 4). The most interesting point, in our opinion, is the high frequency of Cw7 (P < 0.0001; P • n < 0.006), B8 ( P < 0 . 0 0 1 ; P • DR3 ( P < 0 . 0 0 0 1 ; P • n < 0.006) in patients with HCC compared to controls. This difference is present in almost all groups of HCC patients subdivided on the basis of the positivity to HBV and HCV markers of infection (Tables 1-4). These data are of some importance, because it is well known that the haplotype HLA-A1, Cw7, B8, DR3 is associated with a quick decline of CD4 cells [20]. Moreover subjects which are HLA-B8 and/or DR3 positive, show differences in immunological parameters when compared to subjects who are HLA-B8 and/or DR3 negative. It was shown that subjects HLA-B8 a n d / o r DR3 positive have a lower catabolic activity of macrophages, lower levels of natural antibodies and immunoglobulins (A and D) [21-24], an altered response to tetanus toxoids [25], a low cytokine production (in vitro) [26], an absolute reduction of lymphocytes [27-29], and other changes in immunological response that would give higher sensitiveness to mito-
gens and, on the other side, a higher predisposition to immunological diseases, because they are genetic low responders [30-37]. Taken together our data suggest that HCC could be the result of a lower excitability to mitogenic stimuli of HBV and/or HCV. The lower excitability might be determined, however it will be necessary to conduct further studies to confirm these data.
References [1] Shanmugaratnam,K. (1976) Epidemiologyof liver cancer in Singapore. In: Proc. 2nd Asian Cancer Conf., Singapore, pp. 23-28. Editors: K. Shanmugaratnam, R. Nambiar, K.K. Tan and L.K. Cha. Stanford College Press. [2] Porta, C., Moroni, M., Nastasi, G., Casagrande, I. and Ricci, G. (1993) Antibodies to hepatitis C virus and hepatocellular carcinoma in a Northern Italian population. Uppsala J. Med. Sci., 97, 261-266. [3] Ricci,G., Ghiazza, B. and Tieghi, B. (1991) Risk factors for hepatocellular carcinoma. Med. Biol. Environ., 19, 333336. [4l Beasley, R.P., Hwang, L.Y., Lin, C.C. and Chien, C.S. (1981) Hepatocellular carcinoma and hepatitis B virus. Lancet, ii, 1129-1133. [5] Shanmugaratnam, K. (1973) Cancer in Singapore; ethnic and dialect group variations in cancer incidence. Singapore Med. J., 14, 69-81. [6l Yap, E.H., Ong, Y.W., Simons, M.J., Okoci, Y., Mayumi, M. and Nishioka, K. (1972) Australian antigen in Singapore. II -Differential frequency in Chinese, Malays and Indians. Vox Sanguinis (Basel), 22, 371-375. [7] Simons, M.J., Yu, M., Shanmugaratnam, K. (1975) Immunodeficiency to hepatitis B virus infection and genetic susceptibility to development of hepatocellular carcinoma. Proc. Conf. on Carcinofetal Proteins: Biology and Chemistry. Ann. N. Y. Acad. Sci., 259, 181-195. [8l Simons, M.J., Chan, S.H., Wee, C.B. and Mathews, J. (1978) HLA and hepatocellular carcinoma. In: Proc. Hepatitis Symp., Tokyo, 1976, pp. 257-268. University of Tokyo Press. [9] Boettcher, B. (1975) Some possible causes of associations between HLA antigens and disease. Immunogenetics, 2, 485. [10] Hillis, W.B., Hillis, A. and Bias, W.B. (1977) Associations of hepatitis B surface antigenemia with HLA locus B specificities. N. Engl. J. Med,, 296, 1310. [11] Eddleston, A. and Williams, R. (1978) HLA and liver disease. Br. Med. Bull., 34, 295. [12] Madsen, M., Johnsen, H.E. and Kissmeyer-Nielsen, F. (1979) Separation of human T and B lymphocytes using AET-treated sheep red blood cells. Transpl. Proc., 11, 1381. [13] Sampliner, R.E., Bias, W.B. and Camey, E. (1981) HLA antigens and HBV infection: evaluation in the chronic carrier state and in a large family. Tissue Antigens, 18, 248.
G. Ricci et al. / Cancer Letters 98 (1995) 121-125
[14] Saunders, J.B., Wodak, A.D. and Haines, A. (1982) Accelerated development of alcoholic cirrhosis in patients with HLA B8. Lancet, i, 1381. [15] Sengar, D.P.S., Rashia, A., Jindal, S.L. and Christie, C.T. (1979) HLA antigens in HBsAg infection. Vox Sanguinis, 36, 353. [16] Watanabe, H., Okumura, M., Hirayama, K. and Sasukai, T. (1990) HLA-Bw54, DR4, DRw53, DQw4 haplotype controls non responsiveness to hepatitis B surface antigen via CD8positive suppressor T-cells. Tissue Antigens, 36, 69. [17] Ricci, G., Ghiazza, B., Bombelli, G., Russo, E. and Illeni, M.T. (1993) HLA-A, B, C and DR in hepatitis B virus (HBV)-related liver cirrhosis: a study of 851 elderly subjects. Uppsala J. Med. Sci., 98, 179-183. [18] Mittal, K. (1968) Standardization of the HLA typing method and reagents. Vox Sanguinis, 34, 58--63. [19] Woolf, B. (1955) On estimating the relation between blood group and disease. Ann. Hum. Genet., 19, 251-253. [20] Caruso, C., Candore, G. and Modica, M.A (1990) HLA-DR3 and immunoresponsiveness. Lancet, 336, 506-507. [21] Caruso, C., Palmieri, P., Ferraro, A. and Modica, M.A. (1985) Effect of HLA-DR phenotype on allohemoglutinins versus A and B erythrocyte antigens. IRCS Med. Sci., 13, 354. [22] Modica, M.A., Freddi, S. and Caruso, C. (1989) Blood IgA, IgM and IgE levels are influenced by sex and HLA phenotype. Exp. Clin. Immunogenet., 6, 251. [23] Frazer, P.A. and Schur, P.H. (1981) Hypoimmunoglobulinemia D: frequency family studies and association with HLA. Clin. lmmunol. Immunopathol., 19, 67. [24] Cryan, E.M., Stevens, F.M., Skehill, R., Biurke, M., Grimes, H. and McCarthy, C.F. (1985) Immunoglobulins in healthy controls: HLA-BS and sex differences. Tissue Antigens, 26, 254. [25] Feehally, J., Brenchley, P.E.C., Coupes, B.M., Mallick, N.P., Morris, D.M. and Short, C.D. (1986) Impaired response to tetanus toxoid in human membranous nephropathy: association with HLA-DR3. Clin. Exp. Immunol., 63, 367. [26] Caruso, C., Modica, M.A. and Salerno, A. (1986) Reduced rate of interleukin 2 (IL2) production in vitro in the B8, DR3
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36] [37]
125
haplotype. In: Proc. 18th Congress of Italian Society of Pathology, pp. 270. Editors: L. Frail, M. Piccoli and A. Santoni. Rome, Italy. Caruso, C., Lio, D., Palmed, P. and Cillari, E. (1984) HLADR phenotypes and blood levels of T-cells subsets. Tissue Antigens, 24, 320. Caruso, C., Cillari, E., Palmeri, P., Lio, D. and Salerno, A. (1985) OKT4+ and OKT8+ cell subset values and HLA-DR phenotypes. J. Clin. Lab. Immunol., 16, 161. Caruso, C., Modica, M.A., Lio, D. and Cillari, E. (1987) HLA-DR linked genes are involved in the control of Tlymphocytes blood levels. Clin. Immunol. Immunopathol., 44, 160. McCoombs, C.C. and Michalski, J.P. (1982) Lymphocyte abnormality associated with HLA-B2 in healthy young adults. J. Exp. Med., 156, 936. McCoombs, C.C., Michalski, J.P., DeShazo, R., Bozelka, S. and Lane, J.T.L. (1986) Immune abnormalities associated with HLA-B8: lymphocyte subsets and functional correlates. Clin. Immunol. Immunopathol., 39, 112. Ambinder, H.J., Chiorazi, N., Gibofsky, A., Fofino, M. and Kunkel, H.G. (1982) Special characteristics of cellular function in normal individuals of the HLA-DR3 type. Clin. Immunol. Immunopathol., 23,269. Lawley, T.J, Hail, R.P., Fauci, A.S., Katz, S.I., Hamburger, M.I. and Frank, M.M. (1981) Defective Fc-receptor functions associated with the HLA-B8, DR3 haplotype. N. Engl. J. Med., 304, 185. Amer, A., Singh, G., Darke, C. and Dolhy, A.E. (1986) Impaired lymphocyte responsiveness to phytohaemagglutinin associated with the possession of HLA-B8, DR3. Tissue Antigens, 28, 193. Kallemberg, C.G.M. and Klaasen, R.J.L. (1985) HLA-B8, DR3 phenotype and the primary immune response. Clin. Immunol. Immunopathol., 34, 135. Crumpton, M.J. (1987) HLA in medicine. Br. Med. Bull., 43, 1-245. Modica, M.A., Camrnarata, G. and Caruso, C. (1990) HLA88, DR3 phenotype and lymphocyte responses to phytohaemagglutinin. J. Immunogenet., 17, 101-107.
Cancer Letters 98 (1995) 121-125
CANCER LETTERS
HLA-A, B, C, DR and DQ expression and hepatocellular carcinoma: study of 205 Italian subjects Giorgio Ricci a,*, Clara Colombo a, Barbara Ghiazza b, Camillo Porta c, Mauro Moroni c, Maria Teresa Illeni d a2nd Geriatric Division, Azienda Ospedale San Gerardo, 20052, Monza, Milan, Italy bGeriatric Valuational Unit, Azienda USSL 8, 22055, Merate, Lecco, Italy CDepartment of Internal Medicine and Medical Therapy, University of Pavia, IRCCS-Policlinico San Matteo, 27100, Pavia, Italy dDivision of lmmunohematology, Istituto Nazionale dei Tumori di Milano, 20122, Milano, Italy
Received 23 July 1995; revised version received 15 September 1995; accepted 28 September 1995
Abstract
We have evaluated the frequency of HLA class I and II antigens in 205 Italian patients with hepatocellular carcinoma (HCC) and 749 blood donors (controls). Moreover we have looked for correlations between HLA antigen frequencies and HBV and/or HCV infections in HCC patients. We found great differences in HLA antigen frequencies considering only two groups: HCC patients and controls. The polymorphism is smaller when we consider the different groups of HCC patients in regard to the previous viral infections (HBV and/or HCV). The most interesting finding is the higher frequency of Cw7, B8 and DR3 in almost all groups of HCC patients. It is well known, that the HLA A1, Cw7, B8, DR3 antigen haplotype is associated with a rapid decline of CD4 cells, and HLA B8, DR3 positive subjects may display some changes in immune parameters and are prone to develop several immunological diseases. Thus HCC might be the result of a lower sensitivity (genetically given) to mitogenic stimuli of HBV and HCV. Keywords: Hepatocellular carcinoma; Hepatitis B virus; Hepatitis C virus; HLA
1. I n t r o d u c t i o n
Even if environmental and viral factors seem to be the most important aetiological factors of hepatocellular carcinoma (HCC) [1-4], it is likely that genetic factors may also be involved. It is known, for example, that in Singapore, the age standardized incidence of HCC is higher in the Chinese than in Malay or in * Corresponding author. Via Saffi 7, 20058-Villasanta (MI), Italy. Tel.: +39 39 303621; fax: +39 39 2050491.
Indian population [5]. The prevalence of hepatitis B virus surface antigen (HBsAg) in the different ethnic groups, follows the same trend [6]. Moreover, it is known that hepatitis C virus (HCV) has a very high prevalence in patients afflicted with liver cirrhosis and by HCC, and is often associated with markers of hepatitis B virus infection (HBV). In the same ethnic group there are patients afflicted by HCC with high or low levels of a-fetoprotein, with different prevalence of serum level of HBsAg and with different immune response to HBV infection [7,8]. Moreover,
0304-3835/95/$09.50 9 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3835(95)04005-0
122
G. Ricci et al. / Cancer Letters 98 (1995) 121-125
some authors have shown an association between the persistence of infection and HLA [9,10]. and between HLA and liver cirrhosis [11-17], even if the results are indefinite. These data suggest that the different prevalence of HCC in the different ethnic groups could be determined by a different reaction of the host to HBV and/or HCV infection. The aim of our work has been to evaluate the phenotypic frequency of 1st and 2nd class HLA antigens in a group of Italian patients with HCC. Moreover, we have looked for correlations between allelic frequencies and HBV and or HCV infections in HCC subjects.
The correlation between HLA in the group of HCC patients (n = 205) and in controls (n = 749), showed higher frequency of HLA-A3, A19, Cw4, Cw7, B35, DR3 and DQ1, and a lower frequency of HLA-A9(23;24) in patients with HCC. The statistical significance (P < 0.0001), is maintained after multiplying the P value by the number of comparisons (<0.006) (Table 1).
2. Patients and methods
3.1. H C C and H B V infection (Table 2)
Two hundred and five patients with HCC and 749 healthy controls were studied. All were Italian (148 males, 57 females), with age ranging from 46 to 81 years (mean _+SD 63.2 _+5.75). The diagnosis of HCC was based on: (a) pathological findings typical of HCC; (b) space taking lesion in the liver detected by ultrasonography or computed tomography with an a-fetoprotein level greater than 100 IU/ml; (c) increased a-fetoprotein level, greater than 100 IU/ml, and malignant cells found by aspiration. Among the 205 patients with HCC, 148 were tested for HBV markers of infection (HBsAg, antiHBsAg, anti HBcAg, anti-HBeAg, HBeAg). Only 10 males were drinkers (alcohol intake higher than 80 g/day); 183 had liver cirrhosis. All the 749 controis were negative for previous HBV and or HCV infections. All the 205 patients were typed for HLA-A, B, C; 181 patients were typed for HLA-DR, and 139 for HLA-DQ antigens. The typing was performed by microdroplet cytotoxicity test, according to the recommendation of the National Institute of Health [ 18]. The T-lymphocytes for HLA class I typing and Blymphocytes for class II typing were separated by the use of immunomagnetic beads (Dynal A.S., N-0212, Oslo, Norway) from 10ml of heparinized whole blood. The differences in HLA frequencies between the patients and controls were evaluated by the chi squared test according to Woolf [19]. Haldane's
(a) Patients with no previous HBV infection and HCC, showed a higher frequency of HLA-Cw4 and Cw7 (P < 0.0001; P • n < 0.006) when compared to controls. (b) Patients HBsAg positive showed a higher frequency of HLA-Cw7, B8, DR3 and DQ2 than controis. (c) Patients HBsAg negative but with at least one marker of HBV infection (antiHBcAg and/or antiHBsAg and/or antiHBeAg and/or HBeAg) showed higher frequency of HLA-Cw4, Cw7, B8, DR3 and DQ 1 than controls. (d) Patients positive for at least one marker of
method was also employed when one of the compared numbers was equal to zero, and P values were corrected for the number of comparisons (P x n). 3. Results
Table 1 HLA antigens frequenciesin HCC patients (HCC) and in controls (C) HLA
HCC (N 205) %
C X2 (N 749) %
P
Px
A3 A9(23,24) A19 Cw4 Cw7 B35 DR3 DQ1
28.78 19.02 38.53 34.14 28.78 33.65 29.83 52.17
16.55 33.64 21.89 22.02 10.14 20.29 16.15 34.16
0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001
0.006 0.006 0.006 0.006 0.006 0.006 0.006 0.006
15.52 16.23 23.48 12.73 46.00 16.15 17.86 36.44
ap • n is P correctedfor the numberof comparisons.
na
G. Ricci et al. I Cancer Letters 98 (1995) 121-125
123
Table 2
Table 3
HLA antigens frequencies and H B V in HCC patients
HLA antigens frequencies and anti-HCV in H C C patients
HLA
A3 Cw4 Cw7
HCC patients
(C)
(l)
(2)
(3)
(4)
%
%
%
%
%
38.2 ~
16.55 22.03
33.92 ^
37.50* 30.43*
50.00 +
38.92 ^
41.170
10.14
B8
30.48 +
26.78 ^
28.43 ~
10.14
DR3
48.78 +
36.17 ^
42.08 ~
16.15
DQ1 DQ2
69.04 ^ 46.51 +
34.16 24.67
X2
27.25 ~ 13.49 *a 28.31 ^a 17.77 +a 63.14 +a 28.31 ^a 72.37 ~ 17.77 +a 14.39 ^a 27.75 ~ 28.22 +a 12.36 ^a 34.47 ~ 19.00 ^a 19.00 +a
(1) HBV negative, n = 46; (2) H B s A g positive, n = 46, (3) antiH B s / a n t i H B c A g positive, n = 56; (4) (2)+(3), n = 102. (C) Controis, n = 749. a p < 0 . 0001; P • n < 0. 006.
HBV infection showed higher frequency of HLA-A3, Cw7, B8 and DR3 than controls.
3.2. HCC and HCV infection (Table 3) (a) Patients without previous HCV infection and HCC, showed higher frequency of HLA-DR3 than patients with HCC and previous HCV infection and controls. (b) HCC patients without HCV infection showed higher frequency of HLA-A1, Cw7 and DR3 than controls. (c) Patients with HCC and markers of HCV infection, showed a higher frequency of HLA-Cw7, B35 and DQ1 than controls.
3.3. HCC and HBV and HCV infections combined (Table 4) (a) Patients with HCC without any marker of HBV and HCV infection show higher frequency of HLACw7 than controls (P < 0.0001; P • n < 0.006).
HLA
HCC patients
Controls
antiHCV(n = 66) %
antiHCV+ (n = 74) %
%
A1 Cw7
42.42 ^ 47.93 ^
33.74 ~
22.03 10.14
B8 B18 B35 DR3
31.82 ^ 24.246 40.54 ~ 16.60 ~
10.14 10.01 20.29 16.15
62.31 ~
34.16
49.19 ^
DQ1
aP < 0.0001; P • n < 0.006. ^ a n t i H C V - versus controls; ~ H C V - versus anti HCV+.
X2
(n = 749)
13.96 ^a 61.71 ^a 39.95 ~ 14.88 ^a 12.38 ^a 16.03 ~ 40.56 ^a 16.16 ~ 34,16 ~
versus controls; ^~
(b) HCC patients with positivity for antiHCV antibodies (but without markers of HBV infection) show higher frequency of HLA-Cw4 and DQ1 than controis (P < 0.0001;
P •
n < 0.006).
(c) HCC patients with at least one marker of HBV infection and antiHCV antibodies negative, have higher frequency of HLA-Cw7, B8, DR3 (P < 0.0001; P • n < 0.006) than controls. (d) HCC patients with positivity for both HBV and HCV markers of infection show a higher freTable 4 HLA, HBV markers and antiHCV in HCC patients HLA
HCC patients (104) B-/C(14) %
Cw4 Cw7
B8 DR3 DQ1
B-/C+ (21) %
Controls (749)
B+/C(33) %
B+/C+ (36) %
%
46.93 +
38.77*
22.03 10.14
65.21 ~ 42.10 ^
36.73 + 54.56 + 88.88 ~
10.14 16.15 34.26
X2
23.28 ~ 19.43 ^a 57.29 +a 35.94 *a 31.29 +a 43.33 +a 22.92 ~
B+, HBV markers positive (at least one marker); B - , HBV markers negative; C+, antiHCV positive; C - , antiHCV negative. a p < 0. 0001; P • n < 0. 006.
124
G. Ricci et al. / Cancer Letters 98 (1995) 121-125
quency of HLA-Cw7 in comparison with controls (P < 0.0001; P x n < 0.006).
4. Discussion There is a great difference in H L A antigen distribution between controls and HCC patients (Table 1). These differences are smaller when we consider the individual subpopulations of patients with HCC. Evaluating patients on the grounds of viral aetiology, we have observed a lower frequency of HLA-B8 and DR3 in patients with HCC and negative for HBV markers of infection, when compared to HBV positive patients (HBsAg and/or antiHBsAg and/or antiHBcAg) (Table 2). The comparisons of allelic frequencies in subgroups of patients, evaluated on the basis of antiHCV antibody positivity is more complex, because differences between the two groups of patients (HCV+ and H C V - ) and between the two groups of patients and controls, do not always agree (Table 3). The same problem appears if we consider patients on the grounds of positivity and negativity for HBV and H C V markers of infection (Table 4). The most interesting point, in our opinion, is the high frequency of Cw7 (P < 0.0001; P • n < 0.006), B8 ( P < 0 . 0 0 1 ; P • DR3 ( P < 0 . 0 0 0 1 ; P • n < 0.006) in patients with HCC compared to controls. This difference is present in almost all groups of HCC patients subdivided on the basis of the positivity to HBV and HCV markers of infection (Tables 1-4). These data are of some importance, because it is well known that the haplotype HLA-A1, Cw7, B8, DR3 is associated with a quick decline of CD4 cells [20]. Moreover subjects which are HLA-B8 and/or DR3 positive, show differences in immunological parameters when compared to subjects who are HLA-B8 and/or DR3 negative. It was shown that subjects HLA-B8 a n d / o r DR3 positive have a lower catabolic activity of macrophages, lower levels of natural antibodies and immunoglobulins (A and D) [21-24], an altered response to tetanus toxoids [25], a low cytokine production (in vitro) [26], an absolute reduction of lymphocytes [27-29], and other changes in immunological response that would give higher sensitiveness to mito-
gens and, on the other side, a higher predisposition to immunological diseases, because they are genetic low responders [30-37]. Taken together our data suggest that HCC could be the result of a lower excitability to mitogenic stimuli of HBV and/or HCV. The lower excitability might be determined, however it will be necessary to conduct further studies to confirm these data.
References [1] Shanmugaratnam,K. (1976) Epidemiologyof liver cancer in Singapore. In: Proc. 2nd Asian Cancer Conf., Singapore, pp. 23-28. Editors: K. Shanmugaratnam, R. Nambiar, K.K. Tan and L.K. Cha. Stanford College Press. [2] Porta, C., Moroni, M., Nastasi, G., Casagrande, I. and Ricci, G. (1993) Antibodies to hepatitis C virus and hepatocellular carcinoma in a Northern Italian population. Uppsala J. Med. Sci., 97, 261-266. [3] Ricci,G., Ghiazza, B. and Tieghi, B. (1991) Risk factors for hepatocellular carcinoma. Med. Biol. Environ., 19, 333336. [4l Beasley, R.P., Hwang, L.Y., Lin, C.C. and Chien, C.S. (1981) Hepatocellular carcinoma and hepatitis B virus. Lancet, ii, 1129-1133. [5] Shanmugaratnam, K. (1973) Cancer in Singapore; ethnic and dialect group variations in cancer incidence. Singapore Med. J., 14, 69-81. [6l Yap, E.H., Ong, Y.W., Simons, M.J., Okoci, Y., Mayumi, M. and Nishioka, K. (1972) Australian antigen in Singapore. II -Differential frequency in Chinese, Malays and Indians. Vox Sanguinis (Basel), 22, 371-375. [7] Simons, M.J., Yu, M., Shanmugaratnam, K. (1975) Immunodeficiency to hepatitis B virus infection and genetic susceptibility to development of hepatocellular carcinoma. Proc. Conf. on Carcinofetal Proteins: Biology and Chemistry. Ann. N. Y. Acad. Sci., 259, 181-195. [8l Simons, M.J., Chan, S.H., Wee, C.B. and Mathews, J. (1978) HLA and hepatocellular carcinoma. In: Proc. Hepatitis Symp., Tokyo, 1976, pp. 257-268. University of Tokyo Press. [9] Boettcher, B. (1975) Some possible causes of associations between HLA antigens and disease. Immunogenetics, 2, 485. [10] Hillis, W.B., Hillis, A. and Bias, W.B. (1977) Associations of hepatitis B surface antigenemia with HLA locus B specificities. N. Engl. J. Med,, 296, 1310. [11] Eddleston, A. and Williams, R. (1978) HLA and liver disease. Br. Med. Bull., 34, 295. [12] Madsen, M., Johnsen, H.E. and Kissmeyer-Nielsen, F. (1979) Separation of human T and B lymphocytes using AET-treated sheep red blood cells. Transpl. Proc., 11, 1381. [13] Sampliner, R.E., Bias, W.B. and Camey, E. (1981) HLA antigens and HBV infection: evaluation in the chronic carrier state and in a large family. Tissue Antigens, 18, 248.
G. Ricci et al. / Cancer Letters 98 (1995) 121-125
[14] Saunders, J.B., Wodak, A.D. and Haines, A. (1982) Accelerated development of alcoholic cirrhosis in patients with HLA B8. Lancet, i, 1381. [15] Sengar, D.P.S., Rashia, A., Jindal, S.L. and Christie, C.T. (1979) HLA antigens in HBsAg infection. Vox Sanguinis, 36, 353. [16] Watanabe, H., Okumura, M., Hirayama, K. and Sasukai, T. (1990) HLA-Bw54, DR4, DRw53, DQw4 haplotype controls non responsiveness to hepatitis B surface antigen via CD8positive suppressor T-cells. Tissue Antigens, 36, 69. [17] Ricci, G., Ghiazza, B., Bombelli, G., Russo, E. and Illeni, M.T. (1993) HLA-A, B, C and DR in hepatitis B virus (HBV)-related liver cirrhosis: a study of 851 elderly subjects. Uppsala J. Med. Sci., 98, 179-183. [18] Mittal, K. (1968) Standardization of the HLA typing method and reagents. Vox Sanguinis, 34, 58--63. [19] Woolf, B. (1955) On estimating the relation between blood group and disease. Ann. Hum. Genet., 19, 251-253. [20] Caruso, C., Candore, G. and Modica, M.A (1990) HLA-DR3 and immunoresponsiveness. Lancet, 336, 506-507. [21] Caruso, C., Palmieri, P., Ferraro, A. and Modica, M.A. (1985) Effect of HLA-DR phenotype on allohemoglutinins versus A and B erythrocyte antigens. IRCS Med. Sci., 13, 354. [22] Modica, M.A., Freddi, S. and Caruso, C. (1989) Blood IgA, IgM and IgE levels are influenced by sex and HLA phenotype. Exp. Clin. Immunogenet., 6, 251. [23] Frazer, P.A. and Schur, P.H. (1981) Hypoimmunoglobulinemia D: frequency family studies and association with HLA. Clin. lmmunol. Immunopathol., 19, 67. [24] Cryan, E.M., Stevens, F.M., Skehill, R., Biurke, M., Grimes, H. and McCarthy, C.F. (1985) Immunoglobulins in healthy controls: HLA-BS and sex differences. Tissue Antigens, 26, 254. [25] Feehally, J., Brenchley, P.E.C., Coupes, B.M., Mallick, N.P., Morris, D.M. and Short, C.D. (1986) Impaired response to tetanus toxoid in human membranous nephropathy: association with HLA-DR3. Clin. Exp. Immunol., 63, 367. [26] Caruso, C., Modica, M.A. and Salerno, A. (1986) Reduced rate of interleukin 2 (IL2) production in vitro in the B8, DR3
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36] [37]
125
haplotype. In: Proc. 18th Congress of Italian Society of Pathology, pp. 270. Editors: L. Frail, M. Piccoli and A. Santoni. Rome, Italy. Caruso, C., Lio, D., Palmed, P. and Cillari, E. (1984) HLADR phenotypes and blood levels of T-cells subsets. Tissue Antigens, 24, 320. Caruso, C., Cillari, E., Palmeri, P., Lio, D. and Salerno, A. (1985) OKT4+ and OKT8+ cell subset values and HLA-DR phenotypes. J. Clin. Lab. Immunol., 16, 161. Caruso, C., Modica, M.A., Lio, D. and Cillari, E. (1987) HLA-DR linked genes are involved in the control of Tlymphocytes blood levels. Clin. Immunol. Immunopathol., 44, 160. McCoombs, C.C. and Michalski, J.P. (1982) Lymphocyte abnormality associated with HLA-B2 in healthy young adults. J. Exp. Med., 156, 936. McCoombs, C.C., Michalski, J.P., DeShazo, R., Bozelka, S. and Lane, J.T.L. (1986) Immune abnormalities associated with HLA-B8: lymphocyte subsets and functional correlates. Clin. Immunol. Immunopathol., 39, 112. Ambinder, H.J., Chiorazi, N., Gibofsky, A., Fofino, M. and Kunkel, H.G. (1982) Special characteristics of cellular function in normal individuals of the HLA-DR3 type. Clin. Immunol. Immunopathol., 23,269. Lawley, T.J, Hail, R.P., Fauci, A.S., Katz, S.I., Hamburger, M.I. and Frank, M.M. (1981) Defective Fc-receptor functions associated with the HLA-B8, DR3 haplotype. N. Engl. J. Med., 304, 185. Amer, A., Singh, G., Darke, C. and Dolhy, A.E. (1986) Impaired lymphocyte responsiveness to phytohaemagglutinin associated with the possession of HLA-B8, DR3. Tissue Antigens, 28, 193. Kallemberg, C.G.M. and Klaasen, R.J.L. (1985) HLA-B8, DR3 phenotype and the primary immune response. Clin. Immunol. Immunopathol., 34, 135. Crumpton, M.J. (1987) HLA in medicine. Br. Med. Bull., 43, 1-245. Modica, M.A., Camrnarata, G. and Caruso, C. (1990) HLA88, DR3 phenotype and lymphocyte responses to phytohaemagglutinin. J. Immunogenet., 17, 101-107.