Prognostic determinants in extrahepatic bile duct cancer

BACKGROUND/AIMS: The understanding of histopathological prognostic factors is critical to improving surgical outcome. This study investigated the microscopic features of cancer of the extrahepatic bile duct in order to clarify the prognostic determinants affecting surgical outcome. METHODOLOGY: In 90 cancers of the extrahepatic bile duct, the correlation between several microscopic parameters and survival was investigated. Lymphatic, venous, and perineural invasion, and the surgical margin (tumor-free or tumor-positive) were examined with serial step-wise sectioned specimens. RESULTS: Seven pT1-tumors showed no venous or perineural invasion and no lymph node involvement and were associated with prolonged survival (5 year survival, 86%) compared with pT2,3 tumors (23%). In pT2,3 tumors, lymphatic, venous, and perineural invasion was found in 80%, 47%, and 88%, respectively, with no significant differences in occurrence of these parameters according to the origin of the primary tumor. As for survival with pT2,3 tumors, lymph node involvement (58%) and status of the surgical margin were significant parameters (p=.0330 and p=.0309, respectively). In addition, these latter parameters differed significantly according to the origin of the primary tumor. CONCLUSION: In cancer of the extrahepatic bile duct, lymph node involvement and status of the surgical margin were the most important microscopic parameters affecting prognosis.     

Prognostic significance of Ki-67 and p53 antigen expression in carcinomas of bile duct and gallbladder

Ki-67 and p53 protein expression was evaluated immunohistochemically in 32 patients with intrahepatic, extrahepatic bile duct and gallbladder carcinomas, who underwent surgery at First Department of Surgery, The University of Tokushima School of Medicine. p53 expression was found more in the well differentiated group than poorly differentiated group (p = 0.007). MIB1 labelling index (MIB1 LI) was higher in EHC than in GBC (p = 0.0061). MIB1 LI (T), (MIB1 LI in tumor) was higher in cases with lymph node metastasis than in those without lymph node metastasis (p = 0.0189). Moreover, MIB1 LI (L) (MIB1 LI in metastasized lymph node) was higher in poorly differentiated than in well differentiated carcinoma (p = 0.0404). Prognostically, patients with high MIB1 LI (T) (> 56.93) had a worse prognosis after surgery than those with low MIB1 LI (T) (p < 0.05). There was no association between p53 positive tumors and MIB1 expression. These results suggest that cancer cell proliferative activity was markedly increased in cases with EHC compared to those with GBC and the poorly differentiated and lymph node metastasis group. MIB1 LI in tumor was found to be a good prognostic indicator whereas there was no association of p53 positive tumor with MIB1 expression and prognosis of the patients.     

Extrahepatic peribiliary glands express alpha-amylase isozymes, trypsin and pancreatic lipase: an immunohistochemical analysis

We examined expression of alpha-amylase isozymes (pancreatic and salivary), trypsin and pancreatic lipase on the epithelium of extrahepatic peribiliary glands immunohistochemically using 53 autopsied normal extrahepatic bile ducts. Three parts of the extrahepatic bile duct (common bile duct, intrapancreatic bile duct and bile duct at the ampulla of Vater) were examined in each case. Histologically, the extrahepatic bile duct harbored branched tubular glands (extrahepatic peribiliary glands). Extrahepatic peribiliary glands were few in the common bile duct and intrapancreatic bile duct and numerous in the bile duct at the ampulla of Vater. Immunohistochemically, pancreatic alpha-amylase was expressed in the epithelium of extrahepatic peribiliary glands in 42 cases (79%). Salivary alpha-amylase was expressed in the epithelium of the glands in 38 cases (72%). Trypsin was expressed in the epithelium of the glands in 32 cases (60%). Pancreatic lipase was expressed in the epithelium of the glands in 45 cases (85%). The immunoreactivity of these enzymes was granular and located in the supranuclear cytoplasm (corresponding to the Golgi apparatus) of the epithelium of the glands. We confirmed the specificity of the immunoreactivity of these enzymes with various methods. These results suggest that extrahepatic peribiliary glands produce alpha-amylase isozymes, trypsin and pancreatic lipase and secrete these enzymes into lumens of the extrahepatic bile duct. The secreted enzymes may play an important role in the physiology of the extrahepatic bile duct and bile.     

Comparison of p53 expression in proximal and distal gastric cancer: histopathologic correlation and prognostic significance

BACKGROUND: The overexpression of p53 has been found to be correlated with prognosis of some carcinomas, including gastric cancer, but no studies have reported on its relationship to the location of gastric cancer. In the present study, we compared the p53 expression of proximal and distal gastric cancer concerning histopathology and prognosis. METHODS: A total of 170 tumors in the patients with proximal (80 cases) and distal (90 cases) gastric cancer were studied by immunohistochemical methods. RESULTS: p53 immunopositivity was detected in 28.8% of all tumors. The p53-positive expression in proximal gastric cancer was higher than in distal gastric cancer (38.8% vs. 20.0%, p < 0.05). A 5-year survival analysis showed that there is no significant difference between tumors that are p53 positive and p53 negative. No correlation was found between p53 expression and histopathology of gastric cancer. CONCLUSION: p53 nuclear staining is not useful as a prognostic indicator or as a parameter in gastric cancer.     

The developing human biliary system at the porta hepatis level between 29 days and 8 weeks of gestation: a way to understanding biliary atresia. Part 1

The developing biliary system in normal human embryos from 29 days to 8 weeks post-fertilization was studied. The primitive extrahepatic bile duct that originates from the embryonic hepatic foregut diverticulum is in contact with the hepatic anlage from the start of organogenesis and remains so throughout the gestational ages examined. The primitive extrahepatic bile duct maintains continuity with the ductal plate from which intrahepatic bile ducts are eventually formed. Contrary to long-held concepts of biliary development, no 'solid stage' of entodermal occlusion of the common bile duct lumen was found at any stage of gestation in the material investigated. Therefore, biliary atresia is not caused by incomplete vacuolization of the 'solid stage'.     

Expression of a novel antiapoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas

A novel inhibitor of apoptosis designated survivin has recently been found in many common human cancers but not in normal tissues. A potential distribution of survivin in gastric cancer and its implication for apoptosis inhibition have been investigated. Recombinant survivin expressed in Escherichia coli as a glutathione S-transferase fusion protein was used to raise a novel panel of mouse monoclonal antibodies. In an immunohistochemical analysis of 174 cases of gastric carcinomas (stages I-III), anti-survivin monoclonal antibody 8E2 (IgG1) reacted with 34.5% of cases (60 of 174 cases) with a variable number of tumor cells stained (20-100%). In contrast, no expression of survivin in neighboring normal tissues was observed. When stratified for p53 and bcl-2 expression and apoptotic index, the expression of survivin significantly segregated with p53- and bcl-2-positive cases [56.1 versus 15.2% (P = 0.001) and 69.2 versus 31.6% (P = 0.006), respectively] and with a decreased apoptotic index as compared with that of survivin-negative tumors (0.97 +/- 0.64 versus 0.62 +/- 0.39%, P < 0.001). These data identify a role for survivin in promoting aberrantly increased cell viability in gastric cancer and suggest a potential correlation between accumulated p53 and survivin expression in neoplasia.     

Metallic stents in malignant biliary obstruction: prospective long-term clinical results

OBJECTIVE: The purpose of this prospective study was to evaluate the long-term clinical efficacy of metallic stents when used as the initial palliative treatment of patients with inoperable malignant biliary obstruction. SUBJECTS AND METHODS: From August 1991 through May 1995, 100 consecutive patients with malignant biliary obstruction were treated with percutaneous placement of metallic stents. The causes of obstruction were bile duct carcinoma (n = 50), pancreatic carcinoma (n = 17), gallbladder carcinoma (n = 6), hepatocellular carcinoma (n = 2), and metastatic lymphadenopathy in the hepatoduodenal ligament (n = 25). We used 123 stents: 64 Gianturco Z stents, 39 Hanaro spiral stents, 16 Wallstents, two tantalum Strecker stents, one Endocoil stent, and one Memotherm nitinol stent. Every 3 months we followed up all patients except those who died. The average length of follow-up was 220 days (range, 4-1125 days). Patient survival and stent patency rates were estimated by life-table analysis. RESULTS: The median length of survival for the entire patient group was 246 days: 25-week and 50-week survival rates were 62% and 25%, respectively. We found no statistically significant difference in the median length of survival between patients with hilar obstruction (256 days) and patients with common bile duct (CBD) obstruction (227 days). Patients with bile duct carcinoma had longer median survival (269 days) than did patients with other conditions (197 days). The overall median length of patency for all stents was 360 days; the 25-week and 50-week patency rates were 81% and 53%, respectively. The stent patency rate at the median length of survival was 71%. The median length of stent patency in patients with hilar obstruction (617 days) was nearly double that of patients with CBD obstruction (324 days). However, the median length of stent patency in patients with bile duct carcinoma showed no statistically significant difference from the median length in patients with other disease. Four patients (4%) died within 1 month after stent placement. Twenty-one patients (21%) developed recurrent jaundice or cholangitis. In order of frequency, the causes of recurrent jaundice were tumor overgrowth, incrustation of bile sludge, duodenal obstruction due to tumor invasion, stent impaction into the bile duct wall, stent malposition, and tumor ingrowth. CONCLUSION: Metallic stents showed a favorable patency rate with regard to patient survival. In patients with hilar obstruction, the clinical efficacy of metallic stents was superior to that in patients with CBD obstruction. We believe that placement of metallic stents is the procedure of choice for palliation of malignant biliary obstruction.     

Value of intraoperative cholangiography in laparoscopic cholecystectomy

By introduction of laparoscopic cholecystectomy, an increase of accidental common bile duct injuries up to 1.2% has been reported. In the present study of 325 cholecystectomies we evaluated whether mandatory intraoperative cholangiography (IOC) can reduce the rate of accidental bile duct injuries or, at least, identify them early in order to make an adequate repair possible. In addition 163 patients underwent preoperative intravenous cholangiography (IVC). Both imaging techniques were compared with regard to their sensitivity in the detection of anatomic variations and stones of the extrahepatic bile duct system. Our results demonstrated a great advantage of the IOC. The IOC was feasible in 98.1% of the cases and presented a complete depiction of the extrahepatic bile duct system in 99.3%. IVCs showed the biliary system in 91.4% of the cases but without visualization of the cystic duct in 51.5% and the hepatic confluence in 16%. Anatomic variations of the bile duct system which consecutively influenced the operative management were found in additional 27.6% exclusively by IOC. 71.4% of bile duct stones were not detected by IVC. The intraoperative time consumption of IOC was unimportant. The x-ray-load was clearly lower by a factor of 3.5. There was no complication after IOC. In comparison, 6.1% of patients demonstrated an anaphylactic reaction by IVC. One common duct injury (0.3%) was detected intraoperatively by IOC and at the same operation treated without postoperative complications. In conclusion, we recommend an IOC in addition to a thorough preoperative ultrasound-examination. By this technique intraoperatively identified stones of the common bile duct can be sufficiently treated by postoperative endoscopic extraction and anatomic variations of the bile duct system will be visualized and therefore accidental injuries will be avoided.     

Evaluation of a new rapid detection system for mycobacteria using an oxygen sensitive fluorescent sensor

Gallbladder carcinoma is one of the most frequent neoplasms diagnosed in Chile. Although the premalignant lesions have been extensively studied and are well characterized, there is only limited information about the genetic abnormalities that might be important in the pathogenesis of gallbladder carcinoma or that might have prognostic implications. The present study evaluates the immunohistochemical expression of p53 protein in premalignant lesions and invasive carcinoma of the gallbladder, and correlates the p53 expression with histological type, grade of differentiation, and level of invasion of the tumor. The authors studied the immunohistochemical p53 protein overexpression in 52 gallbladder carcinomas, 47 carcinomas in situ (CISs), 34 dysplasias, and 10 specimens with chronic cholecystitis containing normal and metaplastic epithelium. A semiquantitative scoring system was used to assess the p53 reactivity. p53 overexpression was found in 34 of 52 (65.4%) carcinomas, 21 of 47 (44.7%) CISs, and 11 of 34 (32.4%) dysplasias. There were no significant differences in p53 expression in premalignant lesions associated with invasive carcinoma and those that were not. Normal and metaplastic epithelium did not overexpress p53 protein. In adenocarcinomas, no correlation was found between p53 protein overexpression and histological subtype, grade of differentiation, or level of invasion. The high incidence of p53 overexpression in gallbladder carcinoma and its presence in dysplasia, even in specimens without invasive carcinomas, suggests that this abnormality is an important and early event in the pathogenesis of the tumor. The progressively increasing incidence of p53 overexpression observed from premalignant lesions to invasive tumor provides additional support to the view that this is the usual route for the development of infiltrating gallbladder carcinoma.     

Intraductal ultrasonography for the diagnosis of proximal invasion in extrahepatic bile duct cancer

Intraductal ultrasonography (IDUS) was performed on 22 patients with extrahepatic bile duct cancer, using the percutaneous transhepatic approach. Intraductal ultrasonography images of the proximal invasion of the bile duct cancer were defined. In addition, three patients were examined through the peroral approach, to try to diagnose whether or not the cancer invaded to the bifurcation of the hepatic duct. Intraductal ultrasonography images obtained through the percutaneous approach could be classified into three patterns, types 1, 2 and 3, according to the features of the interior surface of the bile duct and the thickness of the bile duct wall. Type 1 images, which did not show protrusions into the bile duct lumen and had a bile duct wall of even thickness, were not likely to show bile duct cancer. Type 2 images showed protrusions of the tumour into the bile duct lumen and the surfaces of the protrusions were irregular. Type 3 images showed single or multiple low echoic papillary masses in the bile duct. Using the peroral technique, we considered all three cases to be type 1 and could diagnose that cancer had not invaded to the bifurcation of the hepatic ducts. From the results of this study, we suggest that proximal invasion of extrahepatic bile duct cancer can be diagnosed using IDUS.     

Pancreatic adenocarcinomas frequently show p53 gene mutations

Thirty-four pancreatic adenocarcinomas were studied for the presence of p53 gene mutations by the single-strand conformation polymorphism method and by direct sequencing of PCR-amplified fragments. p53 protein expression was immunohistochemically evaluated using monoclonal PAb1801 and polyclonal CM1 antibodies. Mutations were detected in 14 cases. The transitions were six G to A and two A to G; the transversions were one C to G and two A to C; the remaining three were frameshift mutations. Immunostaining results were identical with both antibodies. Nuclear immunohistochemical p53-positive cells were found in nine p53 mutated cases and in 12 cases in which no mutation was detected. In most of these latter cases only a minority of cancer cells showed immunohistochemical positivity. Twenty-nine cases, including all p53 mutated cancers, were known to contain codon 12 Ki-ras gene mutations. Also in the light of the demonstrated cooperation of ras and p53 gene alterations in the transformation of cultured cells, our data suggest that p53 mutation is one of the genetic defects that may have a role in the pathogenesis of a proportion of pancreatic cancers.     

Assessment of tumor extent in extrahepatic bile duct cancers--utility of intraductal ultrasonography

Intraductal ultrasonography (IDUS) were performed in patients with extrahepatic bile duct cancer and compared to other diagnostic modalities and to resected specimens. Endoscopic ultrasonography (EUS) is a non-invasive diagnostic method useful for screening patients with bile duct cancers and determining whether they are resectable or not. While, EUS was not useful for the differential diagnosis of advanced and early tumors, and less useful in case of bile duct tumors located at the hilus hepatitis. IDUS proved useful without blind spot even in case of bile duct cancers at the hilus hepatis. IDUS was especially useful for the differential diagnosis of advanced and early tumors. IDUS is the very accurate diagnostic modality which make up for EUS and essential to determine the appropriate operation plan.     

An uncertain role for p53 gene alterations in human prostate cancers

Inactivation of the p53 gene has been implicated in prostate cancer progression. To determine the role of p53 inactivation in the progression of clinical prostatic carcinomas, we assessed 67 tumors derived from patients with clinically localized disease for chromosome 17p and p53 gene allelic loss, p53 gene mutations using single-strand conformational polymorphism and direct sequencing, and p53 protein expression using immunohistochemical staining. Of 55 informative tumors, 10 demonstrated loss of 17p or the p53 gene; however, only a single tumor had a mutation in its remaining p53 allele. Significant p53 overexpression was observed in 2 of 38 tumors, and 9 others had faint staining of a few nuclei ( < 1%). p53 overexpression occurred in no informative tumor with allelic loss or mutation. In a 1-7-year follow-up, positive immunohistochemical staining did not confer an increased risk of recurrence (risk of recurrence, 0.86, P = 0.78), whereas allelic loss of chromosome 17p appeared to be highly correlated with recurrence (risk of recurrence, 3.7, P = 0.003). In an unrelated group of 42 patients with metastatic prostate cancer, p53 overexpression was found in 26 tumors (62%), and 15(36%) had high grade staining. Neither the presence nor the degree of expression correlated with time to progression or time to death. This series suggests that p53 gene inactivation is rare in primary prostatic tumors, not essential to the development of prostate cancer metastases, and of limited use as a prognostic marker in patients with primary or metastatic disease. Another gene or genes on chromosome 17p may be involved in prostate cancer progression.     

Group B Streptococcal septicaemia/meningitis in neonates in a Singapore teaching hospital

Several studies have indicated that frequent allelic losses in some specific chromosomal regions occur during colorectal cancer (CRC) progression. To clarify the correlation between such allelic losses and metastatic potential, the allelotype of lymph node-positive early CRCs, which are small but extremely malignant cancers consisting of metastatically competent cells, were investigated. Nineteen paraffin-embedded specimens of early CRC (pT1 tumors according to TNM classification) with positive lymph nodes were collected. The tumor tissues were examined for loss of heterozygosity (LOH), using microsatellite markers on chromosomes 1p34-36, 8p21-22, 14q32, 18q21 and 22q12-13. The relationship between p53 protein expression and the metastatic status was also investigated by immunohistochemical staining. A group of 20 early CRCs with negative lymph nodes having a similar distribution of macroscopic appearance were used as controls. Among the 19 node-positive tumors, LOH at 8p21-22 and 18q21 was detected in 11 cases (57.9%) and 17 cases (89.4%), respectively. Allelic losses within these 2 regions in node-positive tumors were significantly more frequent than that in node-negative ones (p < 0.01). No significant correlation was found between LOH at 1p34-36, 14q32 or 22q12-13 and lymph node metastasis. p53 protein expression was not significantly associated with lymph node metastasis. Our results suggest that putative tumor suppressor genes, which may be involved in the metastatic process of CRC, are located on chromosomes 8p21-22 and 18q21. Allelic losses in these regions are possible risk factors for lymph node metastasis of early CRC.     

p53 in node-negative breast carcinoma: an immunohistochemical study of epidemiologic risk factors, histologic features, and prognosis

PURPOSE: The present study explores p53 in relation to the following four aspects of node-negative breast carcinoma: epidemiologic risk factors, tumor histopathology, prognosis, and HER2/neu (HER) expression. MATERIALS AND METHODS: Immunohistochemical (IH) staining for p53 was performed on formaldehyde-fixed, paraffin-embedded primary invasive carcinomas from 440 node-negative patients with a median follow-up duration of 119 months. RESULTS: The IH expression, or lack thereof, of p53 separately or in combination with HER did not prove to be prognostically significant and there was no consistent association of p53 with epidemiologic risk factors. p53 was expressed in 68% of medullary carcinomas (MEDs), which is a significantly higher frequency (P < .001) than in lobular (9%) and duct (23%) carcinomas. p53 was not found in some types of low-grade carcinomas (tubular and papillary), and was observed in a minority of mucinous carcinomas. p53 was present significantly more often in carcinomas with high-grade or poorly differentiated nuclear grade than in low- or intermediate-grade tumors. There was an inverse statistically significant relationship between estrogen receptor (ER) positivity and p53 expression. Tumors with the p53(+)/HER(-) immunophenotype tended to be MEDs or duct carcinomas with a marked lymphoplasmacytic reaction. Infiltrating lobular carcinomas (IFLCs) were largely p53(-)/HER(-). p53(+)/HER(+) carcinomas had the best prognosis. The poorest outcome was associated with the p53(-)/HER(+) immunophenotype. This trend was statistically significant for recurrence-free and overall survival in patients with T1NOMO infiltrating duct carcinoma (IFDC). CONCLUSION: The IH demonstration of p53 was not a reliable prognostic indicator in the node-negative breast carcinoma patients studied and it was not associated with major epidemiologic risk factors. The combined immunophenotypic expression of p53 and HER was significantly associated with some histologic types of breast carcinoma and with prognosis in T1NOMO breast carcinoma.     

Intraductal ultrasound of the bile ducts

Intraluminal ultrasound with 12.5 and 20 MHz transducer allows precise analysis of the extrahepatic bile ducts and the adjacent structures. Inflammatory and malignant thickening of the bile duct wall can be assessed without difficulty. It is not possible, however, to differentiate between benign and malignant bile duct thickening, and for this purpose biopsy is required in addition. In tumor staging intraluminal ultrasound is superior to conventional ultrasound, because tumor infiltration into the adjacent tissues can be assessed and lymph nodes along the hepaticoduodenal ligament can be detected. Owing to the ultrasound frequencies available, local staging is limited to a circumference of 1.5/2 cm. Intraluminal ultrasound provides additional information that is useful in the diagnosis and therapy of bile duct stones and in different types of palliative tumor therapy. The procedure is simple and not too time consuming, and it involves no risks to the patient.     

P53 overexpression in head and neck carcinoma and radiotherapy results

PURPOSE: P53 gene mutations are the common genetic changes encountered in human cancers, and there is extensive evidence that the P53 status may determine tumor response to therapy. This study was carried out to investigate whether there is any correlation between accumulation (overexpression) of P53 protein and poor prognosis in patients with head and neck carcinomas treated with radical radiotherapy. METHODS AND MATERIALS: Seventy-nine patients with head and neck carcinomas who were diagnosed and treated in 1989-90 with curative radiotherapy were studied retrospectively. Paraffin sections from archival material were studied using immunohistochemical staining (IHC) with mouse monoclonal antibodies (D0-7) to human P53 protein. Univariate and multivariate analysis of loco-regional tumor control and patient survival were performed on possible prognostic factors. RESULTS: Forty-two (53%) patients showed positive IHC staining in their tumors. Fifty-three percent of the laryngeal, 64% of the oropharyngeal, and 43% of the oral cavity carcinomas showed P53 overexpression. All tumor specimens with vascular, lymphatic, and/or sarcolemmal invasion showed P53 overexpression. The proportion of tumor-stained nuclei was higher in the poorly differentiated than in the well and moderately differentiated tumors (p < 0.05), but there was no correlation with the patient overall or disease-free 5-year actuarial survival. There was no difference in the 5-year actuarial survival and disease-free survival between patients with P53 immunostaining in their tumors and those with no immunostaining (59% vs. 65% and 57% vs. 51%, respectively). The TNM tumor stage was the most significant prognostic factor with 5-year actuarial survival of 87% for early and 14% for late stages (p < 0.0001). There was a significant correlation between immunostaining and history of smoking (p = 0.02). CONCLUSION: The data demonstrate that the P53 accumulation as detected by immunohistochemical staining in a group of head and neck carcinomas was not predictive of patient's poor survival or disease-free survival. Multivariate statistical analysis showed that the TNM tumor stage was the only significant prognostic factor. There was a significant association between P53 accumulation and smoking.     

Needs, demands and manpower balance. Dentists/populations. FDI Commission, 1996

BACKGROUND:The cause of extrahepatic portal vein obstruction in childhood is unknown. We investigated the anatomical features of extra hepatic portal vein obstruction to clarify its cause. STUDY DESIGN: We studied portal venous anatomy in 10 patients with extrahepatic portal vein obstruction without hepatic disturbances ranging in age from 1 to 7 years (mean age, 4.2 years) using ultrasonography, portal venography, cholangio-computed tomography, and magnetic resonance imaging. RESULTS: The extrahepatic portal vein was not obliterated, but it crossed over the common bile duct from the left to the right side at the cranial level of the pancreas and ran in a cranial direction along the right side of the common bile duct or coiled itself around the bile duct. Thus, the extrahepatic portal vein formed a tortuous eta-shape. CONCLUSIONS: The portal vein was not obstructed in patients with extrahepatic portal vein obstruction but formed a characteristic eta-shape by coiling itself around the common bile duct, suggesting that extrahepatic portal vein obstruction has an embryological cause.     

Abnormalities and the implication of retinoblastoma locus and its protein product in head and neck cancers

Abnormalities in the Retinoblastoma tumor suppressor gene (Rb) have been observed in a large number of human cancers. Loss of heterozygosity (LOH) is a common mode of allelic inactivation of Rb and other tumor suppressor genes. We investigated DNA from 45 primary human head and neck cancers to determine LOH at the Rb locus using a polymerase chain reaction-based restriction fragment length polymorphism assay. Of informative cases, we found LOH in 4 of 28 (14%) head and neck cancers. Of immunohistochemical staining of Rb protein, we found that in one of ten LOH negative cases the nuclei of fibroblasts were stained with anti-Rb antibody but there was no nuclear staining tumor cells. These results suggest that inactivation of Rb protein is involved in the carcinogenesis of head and neck cancer at all levels of the process of protein expression: DNA, mRNA and protein.