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High incidence of oral precancerous lesions in a hyperendemic area of hepatitis C virus infection
Y
HeP Res Hepatology
Research
8 (1997) 173- 177
High incidence of oral precancerous lesions in a hyperendemic area of hepatitis C virus infection Yumiko
Nagao a,*, Michio Sata b, Kunitaka Fukuizumi Kyuichi Tanikawa b, Tadamitsu Kameyama a
’ Drpurtmmt of Oral Surgery. Kurume Unicersity School of Medicine, 67 Asahi-muchi, h Secotrd Department
Received
13 June
Fukuoka 830, Jopan Medicine, Kurume Uniwrsity Fukuoka 830, Japan
oj Internal
1997; received
in revised
School of Medicine,
form 28 July 1997; accepted
4 August
b, Kurume, Kurume.
1997
Abstract We have investigated the incidence of oral precancerous lesions among the inhabitants in C virus (HCV) hyperendemic area. Two oral surgeons examined the oral lesions of 685 (295 men, 390 women; mean age of 56.1 years) adult inhabitants, hyperendemic area of HCV infection. All sera were examined for antibodies to HCV (anti-HCV) and serum HCV RNA. Anti-HCV or HCV RNA were detected in sera from 84 (12.3%) or 61 (8.9%) of all. Oral lichen planus (OLP), leukoedema, or leukoplakia were observed in 10 (1.5%), 82 (12%), or 47 (6.9%) subjects, respectively. The incidences of OLP, leukoedema, and leukoplakia in the subjects with HCV infection were significantly higher than those without HCV. These results indicated that HCV may play an important role in oral cancer and related precancerous lesions. Moreover, our study also emphasized the need for periodic examination of the oral cavity in patients with HCV. 0 1997 Elsevier Science Ireland Ltd. a hepatitis
Kewords:
Oral lichen planus;
Leukoplakia;
Oral cancer:
Hepatitis
area
* Corresponding 1386-6346/97.‘$17.00
author.
Tel.:
+ 81 942 353311; fax:
0 1997 Elsevier
PII S 1386-6X46(97)00067-3
Science
Ireland
+ 81 942 317704.
Ltd. All righls
reserved.
C virus; Hyperendemic
174
Y. Nagao et al. /Hepatology
Research 8 (1997) 173-l 77
1. Introduction Hepatitis C virus (HCV) leads to serious consequences such as liver cirrhosis and hepatocellular carcinoma. Besides, liver disease HCV infection is associated with various extrahepatic manifestations and immunological disorders which include membranoproliferative glomerulonephritis, cryoglobulinemia, autoimmune thyroiditis, Sjiigren syndrome, malignant lymphoma, and lichen planus [l-3]. Many have reported a relationship between pathogenesis of lichen planus and HCV infection [4-61. We have previously described evidence of high prevalence HCV infection in patients with oral cancer or oral lichen planus (OLP) [7,8]. However, the prevalence of these diseases in patients with HCV infection is unknown. Therefore, to clarify the rate of diseases in patients with HCV infection, we analyzed the relationship between disease and HCV infection, this involved the mass screening of inhabitants of a specific geographic area.
2. Subjects and methods A total of 685 adult inhabitants in H town [9,10], hyperendemic area of HCV infection participated in this study, 295 men and 390 women (mean age f S.D.: 56.1 + 16.1). Oral disease in all adults was observed by a ratio of two oral surgeons to one inhabitant. The diagnosis was made on the basis of clinical features. Sera from all inhabitants was provided for the following liver function tests; serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), y-glutamyl transpeptidase (y-GTP), total protein, albumin and y-globulin. Sera in all adults was tested for antibodies to HCV (anti-HCV) by second-generation passive hemagglutination assay (PHA kit, DAINABOT, Tokyo, Japan) according to the manufacturer’s instructions [11,12]. Ultrasonographic examination, computed tomography and liver biopsy was performed on some inhabitants. HCV RNA in sera was determined by HCV-specific oligonucleotides and reverse transcription-nested polymerase chain reaction (RT-nested PCR). RNA was extracted from sera in all cases using ISOGEN-LS (Wako, Tokyo, Japan) according to the manufacturer’s instructions. For extraction of RNA, 0.75 ml of ISOGEN-LS and 0.2 ml chloroform was added to 0.25 ml of serum. The mixture was centrifuged at 1.2 x lo4 t-pm for 15 min and the aqueous phase containing 0.5 ml of isopropanol was centrifuged at 1.2 x lo4 rpm for 10 min. Nucleic acids in the solution were precipitated with 75% ethanol, vacuum-dried, then dissolved in distilled water. cDNA was subjected to PCR amplification using a set of 5’-non coding region primers [ 131, 5’-GGCGACACTCCACCAT GAATCACT-3’ (senseprimer), and 5’CACGAATTCAGTCTTCTT TGTCGCGCGCACACCCAA-3’ (antisenseprimer) for the first round PCR, and 5’-ATAGGATCCACTCCCCTGTGAGGAACTA C TGTC-3’ sense, 5’-ATGAATTCATGGTGCACGGTCTGAGACCTCCCG-3’ (antisenseprimer) for the second round PCR. The conditions for the first round PCR amplification were 20 cycles (94”C/l min, 45”C/l min, 72”C/l min) followed by 20 cycles (94”C/l min, 6O”C/l min, 72”C/l min). Those for the second round PCR
Y. Nagao et al. /Hepatology
175
Research 8 (1997) 173-177
amplification were 35 cycles (94”C/l min, 6O”C/l min, 72”C/l min). The PCR products were analyzed by agarose gel electorophoresis and ethidium bromide staining.
3. Statistical
analysis
The x2 test was used for statistical analysis. Any P value less than 0.05 was considered statistically significant.
4. Results
Anti-HCV antibodies or HCV RNA were detected in sera from 84 (12.3’Yo)or 61 (8.9%) of 685 patients (Table 1). The other 591 patients (86.3%) were negative for both anti-HCV and HCV RNA. The prevalence of OLP in all subjects was 1.5% (10/685), while that of OLP in anti-HCV positive or anti-HCV and HCV RNA negative subjects was 4.8% (4/84, P < 0.01 vs. anti-HCV and HCV RNA negative OLP subjects) or 1.0% (6/591), respectively. Although none had oral cancer, the incidences of leukoedema and leukoplakia in the subjects with HCV infection were significantly higher than those without HCV as shown in Table 1. The male-to-female ratio in anti-HCV or HCV RNA positive inhabitants with OLP was 3:l. While the ratio in leukoedema was 2:l or 2.5:1, respectively. However, there was no significant gender-related difference in the prevalence of oral lesions in anti-HCV or HCV RNA positive subjects. Furthermore, we investigated the relationship between the progression of liver disease and appearance of oral lesions (Table 2). However, there was no significant correlation in these two factors. Table Subject Clinical
I characteristics features
Total
OLP
Leukoedema
Leukoplakia
Subjects (‘XI) Age (year) (mean k S.D.) Sex (male/female)
685 56.1 k 16.1 2951390
IO (1.5) 60.8 k Il.6 812
82 (12.0) 62.0 k IS.7 58:‘24
47 (6.9) 58.1 + 16.5 311’16
Anti-HCV (+ )(0/o) Male/female
84 (12.3) 42!42
4184 (4.8)” 3/l
18,‘84 (21.4) l2:6
l8/84 (21 .4)d IO/S
61 (8.9) 33/28
4/6l 3/l
14;61 (23.0)’ to:4
l5/‘61 (24.6) 8,‘7
591 (86.3)
6/59l
60:59 (10.2)“.’
26:59 I (4.4)“.’
247/344
5/l
42:‘18
l8,‘8
HCV RNA Male/female
( +)(‘%I)
Anti-HCV (-) and HCV RNA (-) (%) Male/female
(6.6)’
(I .O)a.b
“ P
SD.,
standard
deviation;
(+),
positive:
176
Y. Nagao et al. /Hepatology
Research 8 (1997) 173-l 77
Table 2 Liver disease of anti-HCV positive patients with OLP, leukoedema and leukoplakia Diagnosis of liver disease HCV carrier HCV carrier complicated with fatty liver Chronic hepatitis C Chronic hepatitis C complicated with alcoholic liver disease HCV related-liver cirrhosis HCV related-liver cirrhosis complicated with alcoholic liver disease Total
OLP
Leukoedema
Leukoplakia
3
5 1 10 1 1
3 1 13
1
1 4
18
18
-
5. Discussion
Previously we have reported a high incidence of HCV among patients with oral cancer and OLP [7,8]. Furthermore, the results of our previous studies provided evidence of the high prevalence of HCV infection in patients with head and neck SCC at national level in Japan [14]. Almost all cases of OLP were benign, but malignant transformation was evident in a very small number of people. The suggested association between OLP and the development of malignancies has been reported [15- 171, although there has been considerable controversy as to whether OLP has a premalignant potential. There is no doubt that a small percentage of leukoplakia are premalignant and some may be invasive carcinoma at presentation [18,19]. Leukoedema is an abnormality of the buccal mucosa which clinically resembles early leukoplakia, but appears to differ from it in certain respects. The incidence of oral cancer and its precancerous lesions such as OLP and leukoplakia among the general population with HCV is still unclear. We originally investigated the incidence of precancerous lesions among the inhabitants in a HCV hyperendemic area. There were high incidences of OLP and leukoplakia subjects with HCV infection in these inhabitants. In addition, the prevalence of leukoedema in subjects with HCV infection was significantly higher than those without HCV. Our results showed that OLP, leukoplakia, and leukoplakia was observed not only in patients with severe liver dysfunction but in patients with mild liver dysfunction. These findings suggest that HCV infection by itself may play an important role in the development of oral cancer and related precancerous lesions, although the mechanisms are unclear. Moreover, our study emphasized the importance of periodic examination of oral cavity among patients with HCV and contributed to the investigation of oral cancer and HCV.
Y. Nagao et al. 1 Hepatology Research 8 (1997) 173-l 77
111
References [I] Johnson RJ, Gretch DR, Yamabe H. Membranoproliferative glomerulonephritis associated with hepatitis C virus infection. New Engl J Med 1993;328:465-70. [2] Gumber SC, Chopra S. Hepatitis C: A multifaced disease. Ann Intern Med 1995;123:615520. [3] Ferric C: Civita LL, Monti M. Can type C hepatitis infection be complicated by malignant lymphoma?. Lancet 1995;346:1426-7. [4] Jubert C. Pawlotsky JM, Pouget F. Lichen planus and hepatitis C virus-related chronic active hepatitis. Arch Dermatol 1994;130:73-6. [5] Gandolfo S, Carbone M, Carrozzo M, Gallo V. Oral lichen planus and hepatitis C virus (HCV) infection: is there a relationship? A report of 10 cases. J Oral Pathol Med 1994;23:119-22. [6] Carrozzo M, Gandolfo S, Carbone M. Hepatitis C virus infection in Italian patients with oral lichen planus: a prospective case-control study. J Oral Pathol Med 1996;25:527733. [7] Nagao Y, Sata M, Tanikawa K, Itoh K, Kameyama T. High prevalence of hepatitis C virus antibody and RNA in patients with oral cancer. J Oral Pathol Med 1995;24:354-60. [S] Nagao Y. Sata M, Tanikawa K, Itoh K, Kameyama T. Lichen planus and hepatitis C virus in the Northern Kyushu region of Japan. Eur J Clin Invest 1995;25:910-4. [9] Yamakawa Y, Sata M, Suzuk H, Noguchi S, Tanikawa K. Higher elimination rate of hepatitus C virus among women. J Viral Hepatitis 1996;3:317-21. [IO] Noguchi S, Sata M, Suzuki H, Mizokami M, Tanikawa K. Routes of transmission of hepatitis C virus infection in an endemic rural area of Japan. Molecular epidemilogical study of hepatitis C virus infection. Stand J Infect Dis 1997;29:23-E. [I I] Iino S, Koike K, Yasuda K. Detection of HCV antibody with 2nd generation EIA (HCV EIA II) (Japanese). Prog Med 1991;11:1911-21. [I 21 Watanabe J, Matsumoto C, Fujimura K. Predictive value of screening tests for persistent hepatitis C virus infection evidenced by viraemia. VOX Sang 1993;65:199-203. [13] Okamoto H, Okada S, Sugiyama Y. Detection of hepatitis C virus RNA by a two stage polymerase chain reaction with two pairs of primers deduced from the 5’noncoding regions. Jpn J Exp Med 1990;60:215522. 1141 Nagao Y. Sata M, Itoh K. High prevalence of hepitits C virus antibody and RNA in patients with head and neck squamous cell carcinoma. Heptology Res 1997:7:206- 12. [I51 Krutchkoff DJ, Cutler L, Laskowski S. Oral lichen planus: the evidence regarding potential malignant transformation. J Oral Path01 1978;7:1-7. [16] Silverman S, Gorsky M, Lozada-Nur F. A prospective follow-up study of 570 patients with oral lichen planus: persistence, remission, and malignant association. Oral Surg Oral Med Oral Pathol 1985;60:30 --4. [17] Barnard NA, Scully C, Eveson JW, Cunningham S, Porter SR. Oral cancer development in patients with oral lichen planus. J Oral Pathol Med 1993;22:421-4. [18] WHO Collaborating Reference Centre for Oral Precancerous Lesions: Definition of leukoplakia and related lesions: and aid to studies on oral precancer. Oral Surg 1978;46:5177538. [19] Silverman S, Gorsky M, Lozada F. Oral leukoplakia and malignant transformation: A follow-up study of 257 patients. Cancer 1984;53:56338.
HeP Res Hepatology
Research
8 (1997) 173- 177
High incidence of oral precancerous lesions in a hyperendemic area of hepatitis C virus infection Yumiko
Nagao a,*, Michio Sata b, Kunitaka Fukuizumi Kyuichi Tanikawa b, Tadamitsu Kameyama a
’ Drpurtmmt of Oral Surgery. Kurume Unicersity School of Medicine, 67 Asahi-muchi, h Secotrd Department
Received
13 June
Fukuoka 830, Jopan Medicine, Kurume Uniwrsity Fukuoka 830, Japan
oj Internal
1997; received
in revised
School of Medicine,
form 28 July 1997; accepted
4 August
b, Kurume, Kurume.
1997
Abstract We have investigated the incidence of oral precancerous lesions among the inhabitants in C virus (HCV) hyperendemic area. Two oral surgeons examined the oral lesions of 685 (295 men, 390 women; mean age of 56.1 years) adult inhabitants, hyperendemic area of HCV infection. All sera were examined for antibodies to HCV (anti-HCV) and serum HCV RNA. Anti-HCV or HCV RNA were detected in sera from 84 (12.3%) or 61 (8.9%) of all. Oral lichen planus (OLP), leukoedema, or leukoplakia were observed in 10 (1.5%), 82 (12%), or 47 (6.9%) subjects, respectively. The incidences of OLP, leukoedema, and leukoplakia in the subjects with HCV infection were significantly higher than those without HCV. These results indicated that HCV may play an important role in oral cancer and related precancerous lesions. Moreover, our study also emphasized the need for periodic examination of the oral cavity in patients with HCV. 0 1997 Elsevier Science Ireland Ltd. a hepatitis
Kewords:
Oral lichen planus;
Leukoplakia;
Oral cancer:
Hepatitis
area
* Corresponding 1386-6346/97.‘$17.00
author.
Tel.:
+ 81 942 353311; fax:
0 1997 Elsevier
PII S 1386-6X46(97)00067-3
Science
Ireland
+ 81 942 317704.
Ltd. All righls
reserved.
C virus; Hyperendemic
174
Y. Nagao et al. /Hepatology
Research 8 (1997) 173-l 77
1. Introduction Hepatitis C virus (HCV) leads to serious consequences such as liver cirrhosis and hepatocellular carcinoma. Besides, liver disease HCV infection is associated with various extrahepatic manifestations and immunological disorders which include membranoproliferative glomerulonephritis, cryoglobulinemia, autoimmune thyroiditis, Sjiigren syndrome, malignant lymphoma, and lichen planus [l-3]. Many have reported a relationship between pathogenesis of lichen planus and HCV infection [4-61. We have previously described evidence of high prevalence HCV infection in patients with oral cancer or oral lichen planus (OLP) [7,8]. However, the prevalence of these diseases in patients with HCV infection is unknown. Therefore, to clarify the rate of diseases in patients with HCV infection, we analyzed the relationship between disease and HCV infection, this involved the mass screening of inhabitants of a specific geographic area.
2. Subjects and methods A total of 685 adult inhabitants in H town [9,10], hyperendemic area of HCV infection participated in this study, 295 men and 390 women (mean age f S.D.: 56.1 + 16.1). Oral disease in all adults was observed by a ratio of two oral surgeons to one inhabitant. The diagnosis was made on the basis of clinical features. Sera from all inhabitants was provided for the following liver function tests; serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), y-glutamyl transpeptidase (y-GTP), total protein, albumin and y-globulin. Sera in all adults was tested for antibodies to HCV (anti-HCV) by second-generation passive hemagglutination assay (PHA kit, DAINABOT, Tokyo, Japan) according to the manufacturer’s instructions [11,12]. Ultrasonographic examination, computed tomography and liver biopsy was performed on some inhabitants. HCV RNA in sera was determined by HCV-specific oligonucleotides and reverse transcription-nested polymerase chain reaction (RT-nested PCR). RNA was extracted from sera in all cases using ISOGEN-LS (Wako, Tokyo, Japan) according to the manufacturer’s instructions. For extraction of RNA, 0.75 ml of ISOGEN-LS and 0.2 ml chloroform was added to 0.25 ml of serum. The mixture was centrifuged at 1.2 x lo4 t-pm for 15 min and the aqueous phase containing 0.5 ml of isopropanol was centrifuged at 1.2 x lo4 rpm for 10 min. Nucleic acids in the solution were precipitated with 75% ethanol, vacuum-dried, then dissolved in distilled water. cDNA was subjected to PCR amplification using a set of 5’-non coding region primers [ 131, 5’-GGCGACACTCCACCAT GAATCACT-3’ (senseprimer), and 5’CACGAATTCAGTCTTCTT TGTCGCGCGCACACCCAA-3’ (antisenseprimer) for the first round PCR, and 5’-ATAGGATCCACTCCCCTGTGAGGAACTA C TGTC-3’ sense, 5’-ATGAATTCATGGTGCACGGTCTGAGACCTCCCG-3’ (antisenseprimer) for the second round PCR. The conditions for the first round PCR amplification were 20 cycles (94”C/l min, 45”C/l min, 72”C/l min) followed by 20 cycles (94”C/l min, 6O”C/l min, 72”C/l min). Those for the second round PCR
Y. Nagao et al. /Hepatology
175
Research 8 (1997) 173-177
amplification were 35 cycles (94”C/l min, 6O”C/l min, 72”C/l min). The PCR products were analyzed by agarose gel electorophoresis and ethidium bromide staining.
3. Statistical
analysis
The x2 test was used for statistical analysis. Any P value less than 0.05 was considered statistically significant.
4. Results
Anti-HCV antibodies or HCV RNA were detected in sera from 84 (12.3’Yo)or 61 (8.9%) of 685 patients (Table 1). The other 591 patients (86.3%) were negative for both anti-HCV and HCV RNA. The prevalence of OLP in all subjects was 1.5% (10/685), while that of OLP in anti-HCV positive or anti-HCV and HCV RNA negative subjects was 4.8% (4/84, P < 0.01 vs. anti-HCV and HCV RNA negative OLP subjects) or 1.0% (6/591), respectively. Although none had oral cancer, the incidences of leukoedema and leukoplakia in the subjects with HCV infection were significantly higher than those without HCV as shown in Table 1. The male-to-female ratio in anti-HCV or HCV RNA positive inhabitants with OLP was 3:l. While the ratio in leukoedema was 2:l or 2.5:1, respectively. However, there was no significant gender-related difference in the prevalence of oral lesions in anti-HCV or HCV RNA positive subjects. Furthermore, we investigated the relationship between the progression of liver disease and appearance of oral lesions (Table 2). However, there was no significant correlation in these two factors. Table Subject Clinical
I characteristics features
Total
OLP
Leukoedema
Leukoplakia
Subjects (‘XI) Age (year) (mean k S.D.) Sex (male/female)
685 56.1 k 16.1 2951390
IO (1.5) 60.8 k Il.6 812
82 (12.0) 62.0 k IS.7 58:‘24
47 (6.9) 58.1 + 16.5 311’16
Anti-HCV (+ )(0/o) Male/female
84 (12.3) 42!42
4184 (4.8)” 3/l
18,‘84 (21.4) l2:6
l8/84 (21 .4)d IO/S
61 (8.9) 33/28
4/6l 3/l
14;61 (23.0)’ to:4
l5/‘61 (24.6) 8,‘7
591 (86.3)
6/59l
60:59 (10.2)“.’
26:59 I (4.4)“.’
247/344
5/l
42:‘18
l8,‘8
HCV RNA Male/female
( +)(‘%I)
Anti-HCV (-) and HCV RNA (-) (%) Male/female
(6.6)’
(I .O)a.b
“ P
SD.,
standard
deviation;
(+),
positive:
176
Y. Nagao et al. /Hepatology
Research 8 (1997) 173-l 77
Table 2 Liver disease of anti-HCV positive patients with OLP, leukoedema and leukoplakia Diagnosis of liver disease HCV carrier HCV carrier complicated with fatty liver Chronic hepatitis C Chronic hepatitis C complicated with alcoholic liver disease HCV related-liver cirrhosis HCV related-liver cirrhosis complicated with alcoholic liver disease Total
OLP
Leukoedema
Leukoplakia
3
5 1 10 1 1
3 1 13
1
1 4
18
18
-
5. Discussion
Previously we have reported a high incidence of HCV among patients with oral cancer and OLP [7,8]. Furthermore, the results of our previous studies provided evidence of the high prevalence of HCV infection in patients with head and neck SCC at national level in Japan [14]. Almost all cases of OLP were benign, but malignant transformation was evident in a very small number of people. The suggested association between OLP and the development of malignancies has been reported [15- 171, although there has been considerable controversy as to whether OLP has a premalignant potential. There is no doubt that a small percentage of leukoplakia are premalignant and some may be invasive carcinoma at presentation [18,19]. Leukoedema is an abnormality of the buccal mucosa which clinically resembles early leukoplakia, but appears to differ from it in certain respects. The incidence of oral cancer and its precancerous lesions such as OLP and leukoplakia among the general population with HCV is still unclear. We originally investigated the incidence of precancerous lesions among the inhabitants in a HCV hyperendemic area. There were high incidences of OLP and leukoplakia subjects with HCV infection in these inhabitants. In addition, the prevalence of leukoedema in subjects with HCV infection was significantly higher than those without HCV. Our results showed that OLP, leukoplakia, and leukoplakia was observed not only in patients with severe liver dysfunction but in patients with mild liver dysfunction. These findings suggest that HCV infection by itself may play an important role in the development of oral cancer and related precancerous lesions, although the mechanisms are unclear. Moreover, our study emphasized the importance of periodic examination of oral cavity among patients with HCV and contributed to the investigation of oral cancer and HCV.
Y. Nagao et al. 1 Hepatology Research 8 (1997) 173-l 77
111
References [I] Johnson RJ, Gretch DR, Yamabe H. Membranoproliferative glomerulonephritis associated with hepatitis C virus infection. New Engl J Med 1993;328:465-70. [2] Gumber SC, Chopra S. Hepatitis C: A multifaced disease. Ann Intern Med 1995;123:615520. [3] Ferric C: Civita LL, Monti M. Can type C hepatitis infection be complicated by malignant lymphoma?. Lancet 1995;346:1426-7. [4] Jubert C. Pawlotsky JM, Pouget F. Lichen planus and hepatitis C virus-related chronic active hepatitis. Arch Dermatol 1994;130:73-6. [5] Gandolfo S, Carbone M, Carrozzo M, Gallo V. Oral lichen planus and hepatitis C virus (HCV) infection: is there a relationship? A report of 10 cases. J Oral Pathol Med 1994;23:119-22. [6] Carrozzo M, Gandolfo S, Carbone M. Hepatitis C virus infection in Italian patients with oral lichen planus: a prospective case-control study. J Oral Pathol Med 1996;25:527733. [7] Nagao Y, Sata M, Tanikawa K, Itoh K, Kameyama T. High prevalence of hepatitis C virus antibody and RNA in patients with oral cancer. J Oral Pathol Med 1995;24:354-60. [S] Nagao Y. Sata M, Tanikawa K, Itoh K, Kameyama T. Lichen planus and hepatitis C virus in the Northern Kyushu region of Japan. Eur J Clin Invest 1995;25:910-4. [9] Yamakawa Y, Sata M, Suzuk H, Noguchi S, Tanikawa K. Higher elimination rate of hepatitus C virus among women. J Viral Hepatitis 1996;3:317-21. [IO] Noguchi S, Sata M, Suzuki H, Mizokami M, Tanikawa K. Routes of transmission of hepatitis C virus infection in an endemic rural area of Japan. Molecular epidemilogical study of hepatitis C virus infection. Stand J Infect Dis 1997;29:23-E. [I I] Iino S, Koike K, Yasuda K. Detection of HCV antibody with 2nd generation EIA (HCV EIA II) (Japanese). Prog Med 1991;11:1911-21. [I 21 Watanabe J, Matsumoto C, Fujimura K. Predictive value of screening tests for persistent hepatitis C virus infection evidenced by viraemia. VOX Sang 1993;65:199-203. [13] Okamoto H, Okada S, Sugiyama Y. Detection of hepatitis C virus RNA by a two stage polymerase chain reaction with two pairs of primers deduced from the 5’noncoding regions. Jpn J Exp Med 1990;60:215522. 1141 Nagao Y. Sata M, Itoh K. High prevalence of hepitits C virus antibody and RNA in patients with head and neck squamous cell carcinoma. Heptology Res 1997:7:206- 12. [I51 Krutchkoff DJ, Cutler L, Laskowski S. Oral lichen planus: the evidence regarding potential malignant transformation. J Oral Path01 1978;7:1-7. [16] Silverman S, Gorsky M, Lozada-Nur F. A prospective follow-up study of 570 patients with oral lichen planus: persistence, remission, and malignant association. Oral Surg Oral Med Oral Pathol 1985;60:30 --4. [17] Barnard NA, Scully C, Eveson JW, Cunningham S, Porter SR. Oral cancer development in patients with oral lichen planus. J Oral Pathol Med 1993;22:421-4. [18] WHO Collaborating Reference Centre for Oral Precancerous Lesions: Definition of leukoplakia and related lesions: and aid to studies on oral precancer. Oral Surg 1978;46:5177538. [19] Silverman S, Gorsky M, Lozada F. Oral leukoplakia and malignant transformation: A follow-up study of 257 patients. Cancer 1984;53:56338.