Bile duct adenoma and von Meyenburg complex-like duct arising in hepatitis and cirrhosis: pathogenesis and histological characteristics.

Morphologic features and neoplastic potentials of bile duct adenoma (BDA) and von Meyenburg complex (VMC)-like duct arising in chronic liver disease were unknown. Thirty-five BDAs and 12 VMC-like duct lesions were observed in 39 cases with chronic liver disease. BDAs were divided into the EMA-cytoplasmic type (n = 14) and EMA-luminal type (n = 21). EMA-cytoplasmic BDA composed of a proliferation of cuboidal to low-columnar cells forming an open lumen with NCAM(+)/MUC6(-), resembling an interlobular bile duct. EMA-luminal BDA showed uniform cuboidal cells with narrow lumen, and NCAM(++)/MUC6(++), resembling a ductular reaction. VMC-like duct showed positive MUC1 expression and negative MUC6. The expression of S100P, glucose transporter-1 (GLUT-1) and insulin-like growth factor II mRNA-binding protein 3 (IMP-3) were not detected in three lesions. p16 expression was higher than those of the ductular reaction, and the Ki67 and p53 indexes were very low (<1.0%). Large-sized EMA-luminal BDA shows sclerotic stroma. We classified small nodular lesions of ductal or ductular cells in chronic hepatitis and cirrhosis into the following groups: BDA, interlobular bile duct type; BDA, ductular/peribiliary gland type; and VMC-like duct. They may be reactive proliferation rather than neoplastic lesions.

Hepatocellular and cholangiolar carcinoma-derived cell lines reveal distinct sets of chromosomal imbalances.

OBJECTIVES: Hepatocellular carcinoma (HCC) and cholangiolar carcinoma (CC) cell lines are used to analyze the basic mechanisms of carcinogenesis and target therapies. However, it is not yet clear which chromosomal aberrations are to be typically expected in such cell lines. It is also not clear whether there are prerequisites for in vitro growth on the genomic and/or expression level. We therefore analyzed HCC and CC cell lines for typical genetic settings. METHODS: The HCC cell lines HLE, HLF, Huh7, HepG2 and Hep3b and the CC cell lines EGI1, MzCha1 and TFK-1 were analyzed using high-density arrays for comparative genomic hybridization (aCGH; 244,000 oligonucleotides). Additional fluorescence in situ hybridization analyses were done to confirm the aCGH results and to add information regarding the aneuploidy of cell lines. RESULTS: The gain of 1q, in particular q21-22, was detected in all HCC cell lines also as a partial loss of 13q. In contrast, a loss of 8p in combination with a relative gain of 8q was seen in all CC but no HCC cell lines. Interestingly, a gain of 17q was seen in all cell lines. These aberrations are also well documented for surgical tumor specimens. Besides these imbalances, the cell lines revealed imbalances for 11p, 12p, 14q, 16p, 16q, 21q and 22q, respectively, only rarely seen in surgical tumor specimens. These aberrations could be of importance for the in vitro cultivation of tumor cells. Structural aberrations were accompanied by aneuploidy in 3 of 5 HCC cell lines and 2 of 3 CC cell lines. Ploidy status was not correlated to any of the imbalances mentioned above. CONCLUSIONS: HCC and CC cell lines revealed characteristic chromosomal imbalances similar to those seen in surgical tumor specimens including chromosomes 1, 8, 13 and 17, respectively. These aberrations are characteristic of the histogenetic origin of the tumor cells. However, the chromosomal imbalances that occurred probably led to the ability of tumor cells to grow in vitro.

Intraductal papillary neoplasm arising from peribiliary glands connecting with the inferior branch of the bile duct of the anterior segment of the liver.

Intraductal papillary neoplasms of the bile duct are generally thought to arise from neoplastic papillary proliferation of epithelial cells lining the bile duct. We herein report a case with findings that strongly suggested that the biliary cystic tumor might have derived from a peribiliary gland. A 69-year-old female was found to have a cystic lesion with intracystic protrusions at the anterior segment of the right hepatic lobe and underwent hepatic anterior segment resection. Fluoroscopy of the resected specimen injected with contrast medium into the cyst revealed a connection between the cystic lesion and the bile ducts. The cyst was multilocular in appearance. On microscopic examination, the cyst was located within the portal tract of the inferior branch of the anterior segment and connected with the inferior branch of the bile duct. The wall of the hepatic cyst lacked an ovarian-like stroma. The tumor was composed of papillary and glandular components, and the tumor cells were similar to gastric foveolar and pyloric gland epithelia and regarded as adenoma. These tumor cells were positive for MUC 5AC, MUC6, and HIK1083. The tumor was finally diagnosed as an intraductal papillary neoplasm of the bile duct (adenoma, gastric type) arising from a peribiliary gland.

An immunohistochemical profile of the so-called bile duct adenoma: clues to pathogenesis.

The so-called bile duct adenoma and peribiliary glands are characterized by the expression of two foregut antigens (designated D10 and 1F6) and secretion of acid mucin. On account of this similarity in phenotype and their frequent close association with a large caliber bile duct, it was earlier suggested that bile duct adenoma represent a peribiliary gland hamartoma. Here, we compare the expression of 13 tissue antigens in bile duct adenomas, other benign bile duct lesions, and various foregut-derived tissues, to further investigate the bile duct adenoma phenotype and pathogenesis. Antibodies to 4 intestinal mucins, 3 cytokeratins, and CDX2, were not informative. Five foregut antigens (D10, 1F6, MUC6, MUC5AC, and TFF2) and secretion of acid mucin were of use in distinguishing bile duct adenoma from other hepatic lesions. 1F6 and MUC6 were normally present in bile ductules and canals of Hering, whereas the epithelium lining the larger bile ducts stained focally for D10, MUC5AC, MUC6, or TFF2 in, respectively, 21%, 36%, 43%, and 100% of the livers examined. Thirty-six bile duct adenomas examined were distinguished by expression of MUC6 (94% of bile duct adenoma), MUC5AC (90%), TFF2 (80%), D10 (67%), and 1F6 (61%), and varying degrees of acid mucin secretion (100%). Of 30 bile duct adenoma tested for all 5 antigens, 40% expressed all 5, 27% expressed 4, 17% expressed 3, 13% expressed 2, and 1 expressed only MUC6. Peribiliary glands invariably expressed D10, 1F6, MUC6, and TFF2, and showed acid mucin secretion, with MUC5AC present in the inflamed peribiliary glands of 3/4 livers with recurrent pyogenic cholangitis, but none of the glands of the other 23 normal or diseased livers tested. The acini of pyloric gland metaplasia in gallbladder and terminal ileum also stained for D10, 1F6, MUC6, and TFF2, with MUC5AC focally present in the gastric foveolar metaplasia overlying the pyloric gland metaplasia but not in the metaplastic glands. MUC6 was expressed in 92% of ductular reactions, 1F6 in 42%, and D10 in 25%. Focal expression of MUC6, or TFF2 was observed in 1 or 2 examples of 14 von Meyenburg complexes and 6 polycystic livers, with staining for acid mucin generally obvious only in the glycocalyx of the epithelium of these two types of lesions. The distinguishing feature of so-called bile duct adenoma is their display of the same phenotype as pyloric gland metaplasia. It is concluded that they develop as a localized biliary healing response equivalent to the function of a peribiliary gland or pyloric gland metaplasia in the foregut.

The MicroRNA let-7a modulates interleukin-6-dependent STAT-3 survival signaling in malignant human cholangiocytes.

The inflammation-associated cytokine interleukin-6 (IL-6) can contribute to tumor growth and resistance to therapy by the activation of survival mechanisms. In several human cancers, IL-6-activated survival signaling involves the signal transducers and activators of transcription (Stat) factors or protein kinase cascades. microRNAs (miRNAs) are endogenous regulators of gene expression that are altered in expression in many cancers. However, the effect of inflammatory cytokines on miRNA expression and the role of miRNA in modulating IL-6-mediated cell survival are unknown. We investigated the involvement of miRNA in malignant cholangiocytes stably transfected to overexpress IL-6, which enhances tumor growth in vivo by inhibition of apoptosis. We provide evidence that (i) miRNA expression both in vitro and in vivo is altered by overexpression of IL-6; (ii) selective miRNAs including let-7a are up-regulated and contribute to the survival effects of enforced IL-6 activity; and (iii) let-7a contributes to the constitutively increased phosphorylation of Stat-3 by a mechanism involving the neurofibromatosis 2 (NF2) gene. These findings reveal a novel mechanism by which IL-6 mediates tumor cell survival that may be therapeutically targeted and emphasize the presence of complex interrelationships between deregulated expression of miRNA and transcription factors in human cancers.

CD56 expression aids in the differential diagnosis of cholangiocarcinomas and benign cholangiocellular lesions.

CD56 (neuronal cell adhesion molecule, N-CAM) has been reported in neuroendocrine tumours and as a marker of reactive biliary epithelial cells. However, up to date, it is not used to distinguish malignant from non-malignant biliary lesions. In this study, we systematically examined CD56 expression on 98 tumours arising from the biliary tree as well as intrahepatic conditions with reactive neoductules. When neuroendocrine carcinomas are excluded, only 4 of 32 (12.5%) cholangiocarcinomas expressed CD56, 2 of which showed clear cell morphology. Reactive bile ductules adjacent to cirrhotic nodules as well as in focal nodular hyperplasia were CD56 positive. Twelve of 17 (70.5%) bile duct adenomas were CD56 positive, whereas von Meyenburg complexes expressed CD56 only very focally in less than 5% of lesional cells. Bile duct cysts were negative for CD56 with the exception of focally interspersed neuroendocrine cells, similar to that seen in segmental bile ducts. Thus, if van Meyenburg complexes are excluded, CD56 can be used to differentiate intrahepatic non-neoplastic from neoplastic proliferations, which is a helpful diagnostic tool in small liver biopsies.

Frequent p16ink4a inactivation is an early and frequent event of intraductal papillary neoplasm of the liver arising in hepatolithiasis.

Intraductal papillary neoplasm of the liver (IPNL) is a precursor lesion of intrahepatic cholangiocarcinoma (ICC) arising in hepatolithiasis. In this study, 98 foci of IPNL identified in 39 surgically resected hepatolithiatic livers were investigated for expression of p16INK4a, cyclin D1, p21WAF1/CIP1, p53, mouse double-minute 2 (MDM2), and pRb. In addition, methylation-specific polymerase chain reaction (MSP) for p16 INK4a promoter region was performed in these foci. Nonneoplastic bile ducts from 11 hepatolithiatic livers, 5 histologically normal livers, and 9 cases of nonpapillary conventional ICC were used as controls. Decreased expression of p16INK4A was seen in IPNL group 1 with mild dysplasia and continued along the progression of IPNL to ICC. The expression of cyclin D1, p21WAF1/CIP1,and pRb gradually increased along the progression of IPNL to ICC and became significantly high in IPNL of group 3 (carcinoma in situ). The expression of p53 and MDM2 was increased in IPNL group 3 and group 4 with evident invasive carcinoma. MSP revealed that 54.6% of 44 IPNL foci harbored p16INK4a promoter hypermethylation, and such foci were significantly correlated with decreased expression of p16INK4a protein. Ki-67 labeling index exhibited a stepwise increase from IPNL group 1 to group 4. We conclude that p16INK4a inactivation, due mainly to its promoter hypermethylation, is a frequent and early event of IPNL and may be responsible for genetic and epigenetic alterations of other cell cycle regulators in IPNL.

Immunohistochemical analysis of biliary tract lesions.

The distinction among inflammatory, benign, and malignant lesions of the biliary tract can at times be difficult. Several methods have been used, including immunohistochemistry (IHC), with variable success. We evaluated a panel of IHC stains to determine their utility in discriminating between bile duct lesions. Formalin-fixed, paraffin-embedded 4-microm sections from 12 inflammatory lesions, 10 bile duct adenomas, and 13 bile duct carcinomas were immunostained using a modified avidin-biotin-complex technique after epitope enhancement using antibodies for p53, Ki-67, and bcl-2. For p53 and bcl-2, greater than 1% of cells staining positive was interpreted as positive. The proliferation index was calculated by determining the number of Ki-67-positive cells in a 1000 cell count. In the inflammatory group, 0 of 12 reacted with anti-p53, 2 of 12 were positive with anti-bcl-2, and the proliferation index with was 22.9% +/- 3.9%. Two of 10 bile duct adenomas showed reactivity with anti-bcl-2, and none were decorated with anti-p53 or Ki-67. In the carcinoma group, 6 of 13 were positive with anti-p53, 9 of 12 were positive with anti-bcl-2, and the proliferation index was 35.3% +/- 5.5%. The proliferation rates differed significantly between groups (P < 0.05). The presence of bcl-2 and p53 immunoreactivity coupled with a high proliferative rate in a biliary tract lesion suggests a malignant process. A panel using these antibodies may be useful in difficult cases.

Endoscopic papillary balloon dilation causes transient pancreatobiliary and duodenobiliary reflux.

BACKGROUND: Endoscopic papillary balloon dilation reduces sphincter function at least transiently or partially, which may allow pancreatobiliary and duodenobiliary reflux to occur. This study prospectively evaluated pancreatobiliary and duodenobiliary reflux after endoscopic papillary balloon dilation. METHODS: In 12 patients with choledocholithiasis, ductal bile was sampled for amylase concentration and bacterial culture during ERCP, before and at 7 days to 5 years after endoscopic papillary balloon dilation. To provide comparative and control data, ductal bile was sampled in 12 patients with gallbladder cholesterol polyps and 6 with anomalous pancreaticobiliary junction who did not undergo endoscopic papillary balloon dilation. RESULTS: Amylase concentrations in ductal bile from patients with choledocholithiasis before endoscopic papillary balloon dilation were marginally significantly higher (before Bonferroni correction) compared with concentrations in bile from patients with gallbladder polyps. The concentration of amylase in bile was significantly increased at 7 days after endoscopic papillary balloon dilation compared with that before endoscopic papillary balloon dilation; the level was comparable with that of patients with an anomalous pancreaticobiliary junction. Subsequently, the amylase concentration gradually decreased and was approximately equal to the pre-endoscopic papillary balloon dilation level at 1 year. Bacteriocholia was frequent (67%-92%) for up to 3 months after endoscopic papillary balloon dilation but was rare thereafter. CONCLUSIONS: Endoscopic papillary balloon dilation causes transient pancreatobiliary and duodenobiliary reflux. However, reflux is no longer present at 1 year after endoscopic papillary balloon dilation.

Atypical bile duct adenoma, clear cell type: a previously undescribed tumor of the liver.

A variable proportion of bile duct adenomas of the liver are still confused with metastatic well-differentiated adenocarcinoma by surgeons and pathologists. We present here three examples of previously undescribed primary hepatic bile duct tumors that were composed almost entirely of clear cells that closely mimicked metastatic renal cell carcinoma. They were interpreted as atypical bile duct adenomas and occurred in two males and one female whose ages ranged from 25 to 64 years. All three tumors were incidental findings and measured from 0.8 to 1.1 cm. The clear neoplastic cells showed mild nuclear atypia and no mitotic activity. They were arranged in tubules and nests that focally infiltrated the hepatic parenchyma. For comparison, a case of clear cell cholangiocarcinoma and 13 conventional bile duct adenomas were examined. The clear cell cholangiocarcinoma was larger (6.0 cm) and had the tubular pattern of conventional cholangiocarcinoma and an abundant desmoplastic stroma. The clear cells of this tumor exhibited greater nuclear atypia and increased mitotic activity. All three atypical bile duct adenomas expressed cytokeratin (CK) 7, p53 protein, epithelial membrane antigen (EMA), and carcinoembryonic antigen (CEA); they were negative for CK20, vimentin, Hep Par 1, chromogranin, and prostatic specific antigen (PSA) and exhibited less than 10% of Ki-67-positive nuclei. One atypical bile duct adenoma displayed luminal immunoreactivity for villin. With the exception of Ki-67 reactivity, the 13 conventional bile duct adenomas and the clear cell cholangiocarcinoma had essentially a similar immunohistochemical profile as that of the atypical clear cell bile duct adenomas. The absence of an extrahepatic primary tumor, the histologic features, the immunohistochemical profile, and the fact that all patients are symptom-free 2 months to 18 years after wedge liver biopsy support the interpretation of atypical clear cell bile duct adenoma. The differential diagnosis with clear cell hepatocellular carcinoma and metastatic clear cell carcinomas is discussed.

Papillary adenoma of the distal common bile duct.

A 73-year-old man with a papillary adenoma located in the distal common bile duct is reported. He underwent pylorus-preserving pancreatoduodenectomy. The lesion in the common bile duct featured papillary proliferation of the epithelium and fibrous elements with diffuse infiltration by inflammatory cells. Positive staining for MIB-1 (Ki-67) and p53 was identified in the nuclei of the proliferative epithelium. These findings suggested the malignant potential of this lesion. Further progress in imaging diagnostic techniques should increase the frequency with which such lesions are discovered. Even now, if mural irregularities and defects are found in the extrahepatic biliary system, especially the distal common bile duct, the possibility of such borderline biliary adenoma should be taken into consideration when making a diagnosis.

NEU overexpression in the furan rat model of cholangiocarcinogenesis compared with biliary ductal cell hyperplasia.

Immunohistochemical studies have suggested that the tyrosine kinase growth factor receptor p185neu is overexpressed in a high percentage of human cholangiocarcinomas. To establish the specificity and temporal relationship between the expression of this receptor in cholangiocarcinogenesis, we investigated c-neu expression in precancerous cholangiofibrotic tissue and subsequently derived primary and transplantable cholangiocarcinomas originated in the livers of furan-treated rats. Proliferated bile ductules formed in rat models of bile ductular hyperplasia and the cell types of normal adult rat liver were also analyzed for c-neu expression. c-neu expression was not detected in normal adult rat liver by either Western blotting, immunohistochemistry, or in situ hybridization. In comparison, all of the cholangiocarcinomas analyzed, which were characterized by intestinal-type mucin-producing neoplastic glands, exhibited a prominent band with a molecular weight 185 kd, corresponding to p185neu. Only the neoplastic glandular epithelia of the cholangiocarcinomas showed a strong immunoreactivity for p185neu, which was predominantly localized to their cell surface but also observed cytoplasmically. In situ hybridization further revealed the cytoplasm of the tumor glandular epithelial cells to be strongly positive for c-neu mRNA transcripts. Of particular interest was our finding that c-neu is expressed early in furan cholangiocarcinogenesis, being more pronounced in the metaplastic intestinal glands of cholangiofibrotic tissue than in hyperplastic biliary epithelial cells in either the same tissue or in hyperplastic bile ductule tissue. Our results demonstrate that c-neu overexpression is a prominent feature of intestinal-type cholangiocarcinomas as well as of metaplastic intestinal glands that precede their development and is detected at lower levels in hyperplastic biliary epithelia. The overexpression of c-neu in the metaplastic and malignant neoplastic glands also correlated with their increased proliferating cell nuclear antigen (PCNA) labeling indices relative to those of hyperplastic biliary ducts and ductules and also appeared to correlate with their intestinal glandular pattern of differentiation.

p53 protein expression and prognosis in gallbladder carcinoma and premalignant lesions.

BACKGROUND/AIMS: p53 protein expression in gallbladder carcinoma has recently been detected by immunohistochemical techniques, but the relationship between p53 expression and prognosis or clinico-pathological factors is still obscure. MATERIALS AND METHODS: We investigated 48 gallbladder carcinoma, 7 adenoma and 11 dysplasia cases for p53 expression by immunohistochemical techniques. RESULTS: p53 expression was positive in 39.6% of gallbladder cancer cases, but in no adenoma or dysplasia cases. No significant correlation was found between p53 overexpression and prognosis or recurrence in 20 patients with carcinoma involvement up to the subserosal layer. p53 overexpression was correlated with DNA aneuploidy pattern and the absence of stones, but was not correlated with clinical staging or lymph node metastasis. CONCLUSION: These results suggest that p53 gene mutation is related to the transition from premalignancy to malignancy in gallbladder carcinogenesis, as well as DNA ploidy alterations and carcinogenesis unassociated with gallstones, but has no bearing on the prognosis.

[The role of alpha-tocopherol and retinol in correcting disorders of lipid peroxidation in patients with malignant liver neoplasms]. / Rol' al'fa-tokoferola i retinola v korrektsii narushenii perekisnogo okisleniia lipidov bol'nykh so zlokachestvennymi opukholiami pecheni.

Patients with liver tumors are known to reveal antioxidant system disorders which lead to accumulation of products of lipids peroxidation and lower resistance. Levels of malonic dialdehyde as well as the antioxidant system (superoxide dismutase, catalase, alpha-tocopherol and retinol) in liver and tumor have been followed in 28 patients in whom liver was removed to treat malignant tumors. Liver and tumor tissue were shown to contain more dialdehyde and less superoxide dismutase and catalase than in the livers of accident victims. Treatment with alpha-tocoferol (600 mg), retinol (100,000 MU) and ascorbic acid (1.5 g) for 7 days before surgery was found to significantly reduce dialdehyde level in the liver. Also, the catalase level increased. Treatment with alpha-tocoferol and retinol resulted in their selective accumulation in the liver. No changes in lipid peroxidation or accumulation of alpha-tocoferol in tumor were recorded. Purulent and septic complications were 1.6 times less frequent after preoperative antioxidant treatment than in controls. It is recommended that said antioxidant treatment should be used to correct lipid peroxidation and to improve the effectiveness of therapy of liver cancer.

Integrins as differential cell lineage markers of primary liver tumors.

This study analyzed new cell lineage markers for the differential diagnosis between hepatocellular carcinoma (HCC) and cholangiocarcinoma (ChC), as well as the potential pathways of cell-cell and cell-extracellular matrix interactions of neoplastic liver cells during tumor spread and invasion, by comparing the expression of (VLA) integrins, vitronectin receptor, and neural cell adhesion molecule in normal, inflamed, and neoplastic human liver biopsies. All cases of liver cell adenoma and well-differentiated HCC expressed the same set of integrins as observed in normal liver tissue, i.e., VLA-alpha 1 and VLA-beta 1. Poorly differentiated HCC also expressed VLA-alpha 1 and VLA-beta 1, but in addition de-novo expressed VLA-alpha 2, VLA-alpha 3, VLA-alpha 6 and vitronectin receptor. All cases of well-differentiated ChC expressed an identical integrin immunoprofile as observed in normal bile duct epithelium, i.e., VLA-alpha 2, VLA-alpha 3, VLA-alpha 6, VLA-beta 4 and vitronectin receptor, whereas poorly differentiated ChC showed a markedly decreased expression of these integrin subunits. VLA-alpha 1 was constantly absent from all cases of ChC, whereas VLA-beta 4 was never expressed by HCC. Neural cell adhesion molecule, exclusively expressed by proliferating reactive bile ductules in cholestatic and regenerating liver, was constantly absent from both malignant neoplasms. In conclusion, the integrin make up of various liver tumors closely follows that of their normal counterparts. Differences in integrin receptor expression vary according to the cellular origin of the tumors and are associated with a poor differentiation. Our findings suggest that immunohistochemical staining for VLA-alpha 1 and VLA-beta 4 integrin subunits, which highlight the cellular phenotype of the two neoplasms, might be a helpful tool in the differential diagnosis between HCC and ChC.

Rapidly growing mucinous cholangiocarcinoma.

A 65-year-old man was hospitalized due to an abdominal tumor. Several imaging studies showed multilocular tumors in the right hepatic lobe and in the pancreatic head, 4 and 7 cm in size, respectively. The hepatic tumor rapidly grew to 9 cm in 4 weeks, associated with a rapid increase in the serum carcinoembryonic antigen level from 125 ng/ml to 1,000 ng/ml. The pathologic diagnosis of the resected liver tumor was mucinous cholangiocarcinoma. This hepatic tumor produced a large amount of mucin, but did not secrete mucin into the bile ducts. Therefore, there was no obstructive jaundice. The incidentally accompanied pancreatic tumor was a typical serous cystadenoma. The present case suggested that mucinous cholangiocarcinoma could present rapid growth of the tumor. The clinicopathological features of mucinous cholangiocarcinoma are demonstrated for the first time, and the differences between mucinous cholangiocarcinoma and mucin-producing papillary adenocarcinoma of duct-spreading type are discussed.

Micropharmacology of monoclonal antibodies in solid tumors: direct experimental evidence for a binding site barrier.

Monoclonal antibodies (MAbs) often distribute nonuniformly in tumors. In part, that observation reflects intrinsic heterogeneity within the tumor; in part, it reflects poor penetration through tumor substance. Several years ago, we proposed the "binding site barrier" hypothesis (J.N. Weinstein, R.R. Eger, D.G. Covell, C.D.V. Black, J. Mulshine, J.A. Carrasquillo, S.M. Larson, and A.M. Keenan, Ann. NY Acad. Sci., 507: 199-210, 1987; K. Fujimori, D.C. Covell, J.E. Fletcher, and J.N. Weinstein, Cancer Res., 49: 5656-5663, 1989), the idea that antibodies (and other ligands) could be prevented from penetrating tumors by the very fact of their successful binding to target antigen. Calculations suggested that this might be a significant factor in the therapy of even microscopic nodules. The higher the affinity and the higher the antigen density, the greater the barrier. Here, we provide direct experimental evidence of such a barrier to the percolation of D3 MAb through intradermally implanted line 10 carcinoma of guinea pigs. After affinity purification using glutaraldehyde-fixed line 10 cells, the D3 had an average immunoreactivity of 88%, a binding constant of 1.6 +/- 0.3 (SEM) x 10(10) M-1, and saturation binding of 355,000 +/- 15,000 molecules/cell. Using a combination of double-label autoradiography and double-chromagen immunohistochemistry, we determined simultaneously the distribution of (a) i.v. injected D3 MAb; (b) coinjected isotype-matched control IgG (BL3); (c) D3 antigen; (d) blood vessels. The previously developed mathematical models aided in the design of these experiments. Double immunochemical staining of the tumors showed antigen-rich patches 100-800 microns across, surrounded by blood vessels. At a low MAb dose (30 micrograms), binding to antigen severely hindered penetration into antigenic patches as small as 200 microns, even at 72 h. Explanation of this finding by a physical barrier was ruled out by the observation that BL3 distributed uniformly in the same patches. At a higher dose (1000 micrograms), the binding site barrier could be partially overcome. The same general principles of micropharmacology may apply to biological ligands other than antibodies, including those secreted by genetically modified cells.

A cholangiocellular carcinoma nude mouse strain showing histologic alteration from adenocarcinoma to squamous cell carcinoma.

BACKGROUND: Adenosquamous carcinoma and squamous cell carcinoma (SCC) occur rarely in the liver compared with adenocarcinoma, and the histogenesis and biologic behaviors of these tumors remain unknown. The authors addressed these issues in the current article. METHODS: A specimen aseptically obtained from the surgically resected cholangiocellular carcinoma (CCC) was cut into pieces and inoculated into the back of a nude mouse, bilaterally. The developed tumors were resected and serially transplanted into nude mice. The morphologic features and growth kinetics of the nude mouse tumors at different passages were compared. RESULTS: The authors established a new human CCC nude mouse strain, designated nuKMC-2, from a 64-year-old woman. The original tumor of the patient showed the features of moderately differentiated tubular adenocarcinoma with small sheet-like arrangement of polygonal cells. The initial tumor developed in a nude mouse showed morphologic features similar to the original tumor. With the serial transplantation to nude mice, the components of tubular adenocarcinoma diminished, and all of the nuKMC-2 was replaced by SCC. Doubling times of nuKMC-2 at the 5th and 11th passages were 9.9 and 7.4 days, respectively, which suggested that the tumor with squamous components were more aggressive biologically than the adenocarcinoma. CONCLUSIONS: The results suggested that adenosquamous carcinoma might be a transitional form from adenocarcinoma to SCC and that some of the primary hepatic SCC might originate from adenocarcinomas.

Immunohistochemical study on epidermal growth factor (EGF) receptor during carcinogenesis in the rat liver.

The expression and localization of epidermal growth factor (EGF) receptor were investigated immunohistochemically using anti-EGF receptor antibody in the normal rat liver and 3'-methyl-4-dimethylaminoazobenzen (3'-Me-DAB) induced tumors in rats. In 8 weeks after 3'-Me-DAB treatment, multiple nodules of cholangiocarcinoma were found in the rat liver, and atypical nodules of hepatocytes were also found 14 weeks later. Immunoreactive products against EGF receptor were only slightly positive in the normal liver, the nodule of cholangiocarcinoma, and atypical nodule of hepatocytes. It was noted that EGF receptor immunoreactivity was more intense in non-cancerous tissue adjacent to tumorous nodules than in the cancerous tissue. The present finding suggests that the expression of EGF receptor may be associated with regenerating as well as carcinogenetic processes in the rat liver.