What's New Archive

September 2009

Shell Ridge Summit walk for Bile Duct Cancer Research

On April 11, 2009, the family of Levan Gray organized a walk to support research on bile duct cancer. The walk was successful beyond all expectations, and raised over twelve thousand dollars for biliary cancer research! A second annual walk is now planned for Saturday, April 2, 2010. To learn more about this wonderful undertaking, please follow this link to the website http://bileductcancerwalk.org.

May 2008

Genetic Analysis of Gallbladder Cancer Reveals Novel Biomarkers

The Hopkins’ biliary cancer group hopes to utilize global gene expression profiling to identify new prognostic markers and therapeutic targets within gallbladder and bile duct cancers. Our goal is to use genetics to identify factors which make only the cancer cells grow, and which can therefore be specifically blocked via nontoxic but highly effective therapies.

In the May 1st , 2008 issue of Clinical Cancer Research, Hector Alvarez from our group has published the first study of gallbladder cancer using serial analysis of gene expression (SAGE). This method, first developed at Johns Hopkins, allows for an unbiased and quantitative assessment of mRNA expression from different tissue samples. This methodology facilitates the discovery of cancer biomarkers, imaging targets, and therapeutic avenues. For example, in 2001, our group used SAGE to identify mesothelin and prostate stem cell antigen as biomarkers and therapeutic targets for pancreatic cancer. Parenthetically, both of these proteins are also expressed in biliary cancer, allowing patients with biliary tract cancer to benefit from these discoveries.

Alvarez et al. performed serial analysis gene expression (SAGE) on three stage-matched gallbladder cancers (representing Hispanic/Latino, Native American, and Caucasian ethnicities, respectively) and one histologically normal gallbladder. In this study, Alvarez et al. determined that connective tissue growth factor (CTGF) was overexpressed in gallbladder cancer compared to normal gallbladder, and was associated with a relatively favorable prognosis. While the latter is an important piece of information, the true importance of this study is that the data generated from this exhaustive genetic analysis is publicly available online (http://cgap.nci.nih.gov) , and should allow for further analysis of genetic pathways which are altered in gallbladder cancer. This should allow for our group and others to identify pathways which can be targeted for therapy. For example, preliminary analysis of the gallbladder cancer SAGE libraries demonstrated upregulation of several genes in the mTOR pathway, suggesting that this may be a pathway which can be targeted. We look forward to using this incredible online dataset to further study this biliary tract cancer, and find novel, nontoxic avenues of treatment.

PDF here

Reference:

Clinical Cancer Research 2008;14: 2631-8.

June 2007

Hopkins Fellow Wins International Award for Biliary Cancer Research

Congratulations to Donna E. Hansel, MD Ph.D. Dr. Hansel received the 2007 F. Stephen Vogel Award for outstanding paper by a resident or fellow published in one of the United States and Canadian Academy of Pathology Journals. This award was given for her paper titled "Tumor length variation in biliary tract metaplasia, dysplasia and carcinoma" (Modern Pathology 2006;19:772-779).

Telomeres are functional caps at the ends of each of the linear human chromosome. Telomeres consist repetitive DNA sequences and proteins that protect the chromosome ends from undergoing breakage or recombination. Hence, functional telomeres help maintain the stability and integrity of the human genome. A variety of human cancers demonstrate short telomeres, which correlates with their genetic instability. Recently, at Johns Hopkins, a novel technique to identify telomere lengths in situ (TEL-FISH) (American Journal of Pathology 2002;160: 1259-1268) was developed.

In this paper, Dr. Hansel and colleagues at Hopkins demonstrated telomere shortening in biliary carcinomas, as well as pre-invasive lesions such as dysplasia and metaplasia, a lesion traditionally thought to be non-neoplastic. This paper offers insight into the pathogenesis of biliary tract cancer. This study represents the first in situ assessment in tumor length in biliary cancer. Prior assessments of human cancers have used whole tissue bulk techniques, which include contaminating normal tissue, and hence their results are reliable. This study clearly demonstrates that telomere shortening is a key process in biliary tract neoplasia and occurs at the pre-invasive, asymptomatic state. This study has potential for early detection of biliary carcinomas, since cells with shortened telomeres should be detectable in screening techniques. It also suggests that any telomerase inhibitors that become available for clinical use may be effective in biliary tract neoplasia, since tumors with shortened telomeres should be most sensitive to the effects of inhibition of telomerase. For these reasons, this study, which was supported by donations to our program, was recognized with this prestigious international award.

Click here to view the pdf copy of "Tumor length variation in biliary tract metaplasia, dysplasia and carcinoma".

March 2005

Potential New Target for Therapy in Biliary Cancer Discovered at Johns Hopkins

October 2004

New Diagnostic Marker of Biliary Tract Carcinoma Discovered at Hopkins

It is clear that further studies involving larger patient populations will be needed to thoroughly evaluate the diagnostic accuracy of Mac-2BP, particularly in patients at high risk of developing biliary carcinoma, such as those with primary sclerosing cholangitis. However, this study demonstrates that screening for this tumor may be possible using bile, the bodily fluid in direct contact with tumor cells. Examination of the proteins within bile in addition to the routine brush cytology obtained from patients who undergo ERCP (endoscopic retrograde cholangiopancreatography) for suspected biliary tract tumors may improve diagnostic accuracy, and allow biliary malignancies to be detected at an early and thus potentially curable stage. These studies also show the power of new technologies (in this case proteomics) in identifying new diagnostic and potential therapeutic targets in biliary tract carcinoma.

April 2004

Progression of Gene Hypermethylation in Gallstone Disease Leading to Gallbladder Cancer

The alteration of function of several types of genes can promote the development of cancer. Genes that promote growth are usually termed “oncogenes”; these are often overexpressed or hyperactive in the cancer. Inactivation of genes that promote DNA repair creates genetic instability, which selects for tumor growth in other cancers. Other genes that normally block cell growth are inactivated in cancers; these are called tumor suppressor genes. Tumor suppressor genes can be inactivated by several mechanisms, including direct damage to the DNA (mutation), or by overt loss of the coding DNA (gene deletion). A more novel mechanism of inactivation of tumor suppressor genes is that of DNA methylation. Here, the DNA is reversibly altered such that the expression of the gene is lost, even though the coding DNA remains intact. Hence, methylation is the functional equivalent of mutation or deletion, with the exception that it is potentially reversible. Methylation of and inactivation of tumor suppressor genes is a known cancer-promoting mechanism in a variety of different cancers, including pancreas, breast, lung, and gastric carcinomas. Very little is known about methylation and its role in the development of gallbladder cancer.

To address this, our group at Hopkins, led by Dr. House, studied 54 gallbladder tumors from both the United States and Chile, where gallbladder carcinoma is endemic. Six candidate tumor suppressor genes (p16, APC, methylguanine methyltransferase, hMLH1, retinoic acid receptor beta-2 and p73) were studied. We found that while only 1 of 15 normal gallbladder controls showed methylation of these genes, 28% of gallbladders with chronic cholecystitis and 72% of gallbladder neoplasms demonstrated aberrant methylation. The most commonly methylated genes in gallbladder cancers were p16 (56%), p73 (28%), APC (27%), and hMLH1 (14%). Interestingly, significant differences in methylation were discovered between gallbladder cancers from the United States and those from Chile. APC gene methylation was more common in cancers from the United States, whereas p73 gene methylation was more common in Chilean cancers.

This study shows that the acquisition of hypermethylation at multiple tumor-suppressor gene-promoter sites may contribute to tumor formation and progression from chronic cholecystitis to gallbladder cancer. These findings open up the potential for screening for aberrant methylated genes in bile specimens, with the goal of detecting gallbladder cancers when they are small and are curable. Also, since methylation can be reversed with certain drugs, one could envision treating these tumors with these drugs to try to re-express the tumor suppressor genes, and thereby block the growth of the tumor. Since gallbladder cancers from Chile and the United States demonstrated genetic differences; it is possible that the identical cancers arising in two different parts of the world have a different and unique biology.

October 2003

New Pathway for Cancer Discovered in Hedgehog

Researchers at Johns Hopkins have made a remarkable discovery regarding the activation of the "sonic hedgehog gene" in biliary and pancreatic cancers.

"Sonic hedgehog" is part of a cell pathway that plays an important role in embryogenesis. Cell pathways are groups of proteins within cells that interact/communicate with each other. Embryogenesis is the formation of the embryo. Dr. Beachy discovered several of the key components of this pathway, studying, of all things, fruit fly genetics. It was shown that alterations in the sonic hedgehog pathway caused fruit fly larva to look like, you guessed it, the computer game character "sonic hedgehog". Dr. Beachy's team then went on to show that this same pathway is important in human embryogenesis and that the sonic hedgehog pathway plays a key role in maintaining stem cells in our bodies. Dr. Beachy was elected to the National Academy of Science for this work. Now, Drs. Beachy, Maitra and Berman team together to show that the sonic hedgehog pathway is activated in human cancers, including cancers of the pancreas and biliary tree. Furthermore, they show that when they block the pathway using a drug called cyclopamine, that they completely block tumor growth.

This paper is very exciting to me for several reasons. First, it helps us understand the fundamental biology of pancreatic and biliary cancer. Their data show that a pathway that regulates stem cells is altered in these cancers (for scientists this is a very exciting idea). Second, the discovery of this pathway's role in human pancreatic and biliary cancer opens an entire new area for treating these cancers-- targeting pancreatic and biliary cancers using drugs, such as cyclopamine, that specifically inhibit the sonic hedgehog pathway. The team now plans to test a large panel of blockers ("inhibitors") of the sonic hedgehog pathway to determine which inhibitor provides the maximum anti-tumor effect and the minimum side effects. Third, the study is an example of what, in my opinion, makes Hopkins such a special place- the willingness of creative scientists from very diverse fields to work together to tackle big problems. Fourth, the creation of the cell lines and cancer xenografts that were central to this research was made possible by private donations. This is a wonderful example of how private philanthropy can support cutting edge research and have a significant impact in the war on cancer.

July 2003

Opportunity for Collaboration from New Biliary Cancer Laboratory

February 2003

Analysis of novel tumor markers in pancreatic and biliary carcinomas using tissue microarrays.

Using global gene expression analyses (such SAGE and oligonucleotide or cDNA microarray analysis), multiple novel tumor markers have been identified in infiltrating ductal adenocarcinoma of the pancreas. These markers have potential in early diagnosis, screening and treatment of this disease. Infiltrating adenocarcinoma of the biliary tract is pathologically similar to pancreatic adenocarcinoma; however, the expression of these markers in biliary carcinoma has not been studied. To assess each of the newly identified markers, we performed immunohistochemistry according to standard techniques on our recently constructed biliary tissue microarrays (TMA). The TMA contain four 1.4 mm tissue cores of each of 10 intrahepatic cholangiocarcinomas, 15 distal bile duct carcinomas, and 15 gallbladder carcinomas, along with normal gallbladder. The new markers studied were: 14-3-3 , fascin, mesothelin, prostate stem cell antigen (PSCA), cdc2/p34, topoisomerase II (topo II), mucin 4, (muc4), and heat shock protein 47(hsp47). The results were quite promising. Seven of the eight markers were overexpressed by biliary carcinomas, including cdc2/p34 (84% of cases), topo II (76%), 14-3-3 (72%), mesothelin (42%) muc4 (42%), fascin (84%), and PSCA (11%). However, in contrast to the findings in normal pancreas, focal strong positivity for muc4 and 14-3-3 was also identified in normal biliary epithelium. The eighth marker, hsp47, was overexpressed in the reaction to the tumor (peritumoral stroma) in 58% of cases.

To determine the timing of overexpression of the most promising markers in the progression of these cancers from early precursors to fully invasive lesions, we studied a large series of whole tissue sections of invasive gallbladder carcinoma. On these whole tissue sections, mesothelin was always absent from normal epithelium, was expressed in 44% percent of carcinoma in situ (8 of 18 cases), and was expressed in all of the invasive carcinomas (22 of 22). Fascin was essentially absent in normal epithelium, but was expressed in pyloric metaplasia (2 of 2 cases), a subset of carcinoma in situ (8 of 19 cases), and in most (18 of 27) invasive carcinomas. Interestingly, in several cases, expression of mesothelin and fascin was upregulated during the progression from carcinoma in situ to invasive carcinoma, implicating a role in cancer progression.

In conclusion, this study demonstrates that several novel tumor markers of pancreatic adenocarcinoma demonstrate similar but not identical expression patterns in biliary carcinoma. These markers are potentially useful in diagnosis and treatment of biliary carcinomas. For example, the consistent expression of mesothelin in biliary carcinomas suggest that experiments seeking to develop a mesothelin-targeted vaccine for pancreatic adenocarcinoma may also be useful for patients with biliary tract carcinomas. Also, any of these markers which are secreted into the blood are potential targets for a screening test.

Identification of novel targets of biliary tract cancers using global gene expression technology.

Previously, identification of novel tumor specific markers was extremely difficult. Many markers were discovered more by accident than by true science, leading to frustrating delays in their identification. The availability of global RNA-based gene expression platforms has rapidly changed all of this. With this technology, one compares the RNA expression of the DNA genes of a tumor versus that of its corresponding normal cell. Genes that are overexpressed at the RNA level are likely overexpressed at the protein level, and hence potentially identify new targets for treatment of the cancer. While this method has been successful for several different cancers, is has yet to be used in biliary tract carcinomas. To address this issue, we used the Affymetrix GeneChip U133A arrays containing over 22,000 unique transcripts to compare gene expression profiles of 20 biliary cancers (11 primary tumors and 9 cell lines) versus normal gall bladder and bile duct epithelial scrapings obtained from specimens with a normal biliary tree. The immense data obtained from the microarrays was normalized using computer software and genes expressed three-fold or higher in cancers versus the normal tissues were identified.

The results were that 512 RNA transcripts were overexpressed at three-fold or higher in the resected primary tumors and cell lines compared to normal tissue. These overexpressed genes included proliferation antigens (cyclins A2, D2 and E2, topoisomerase II), transcription factors (homeobox B7, islet-1, dickkopf 1), cell adhesion molecules (integrin alpha6), growth factors and receptors (hepatocyte growth factor and its receptor, met) and potential therapeutic factors (ribosomal protein S6 kinase, a key downstream element of the mammalian target of rapamycin [mTOR]). Overexpression of selected genes was confirmed by immunohistochemistry in four cases, or by in situ hybridization in three cases, using the previously described biliary cancer tissue microarrays, and by reverse transcriptase polymerase chain reaction (RT-PCR) in biliary cancer cell lines.

This study represents the first systemic gene profiling of biliary cancers. Overexpression of a small subset of selected genes has been validated using conventional methods such as immunohistochemistry, in situ hybridization and RT-PCR. Many of the upregulated genes have not previously been identified in biliary cancer, and may represent potential diagnostic and therapeutic targets for this lethal cancer type. This wealth of data will provide the launching point for a variety of studies identifying and exploring the validity of each of these markers, and determining which have potential diagnostic and therapeutic applications in biliary cancer.

Telomere length variation in biliary tract metaplasia, dysplasia and carcinoma.

Telomeres are functional caps at the ends of each of the linear human chromosome. Telomeres consist repetitive DNA sequences and proteins that protect the chromosome ends from undergoing breakage or recombination. Hence, functional telomeres help maintain the stability and integrity of the human genome. A variety of human cancers demonstrate short telomeres, which correlates with their genetic instability. Recently, at Johns Hopkins, a novel technique to identify telomere lengths in situ (TEL-FISH) (American Journal of Pathology 2002;160: 1259-1268) was developed. We have now used this technique to study a variety of inflammatory, metaplastic and neoplastic processes of the biliary tract.

We found that the biliary epithelium from 5 of 5 normal gallbladders and 15 of 15 cases of cholecystitis demonstrated normal telomere lengths, identical to that of the surrounding normal connective tissue (stroma). In contrast, the vast majority of the 65 invasive adenocarcinomas of the biliary tract demonstrated marked telomere shortening, although intratumoral variability did exist. Of note, 9 of 10 examples of dysplastic epithelium and 50 percent of metaplastic biliary epithelium also demonstrated telomere shortening, supporting prior data that metaplastic lesions are genetically neoplastic in the biliary tree.

This study represents the first in situ assessment in tumor length in biliary cancer. Prior assessments of human cancers have used whole tissue bulk techniques, which include contaminating normal tissue, and hence their results are reliable. This study clearly demonstrates that telomere shortening is a key process in biliary tract neoplasia and occurs at the pre-invasive, asymptomatic state. This study has potential for early detection of biliary carcinomas, since cells with shortened telomeres should be detectable in screening techniques. It also suggests that any telomerase inhibitors that become available for clinical use may be effective in biliary tract neoplasia, since tumors with shortened telomeres should be most sensitive to the effects of inhibition of telomerase.

September 2002

A New Lab for Biliary Tract Cancers at Johns Hopkins

Overview  | Research  | People | Support our Research

Biliary tract cancers encompass cancers of the gallbladder and bile ducts. The diagnosis of biliary tract cancers is often difficult since tumors may reach a large size before symptoms are present, and serum markers are currently not available for screening. In addition, there is no "gene test" to determine who is at risk for these cancers. Therefore, in order to fight these cancers, we need to understand the biology of these tumors better and use this knowledge to develop new methods for screening, early detection, and treatment for patients suffering from this disease.

The goal of our lab is to identify genes and proteins that are abnormally expressed in biliary tract cancers. Once we have identified these "candidate genes" that are responsible for transforming normal bile duct cells into cancer cells, we can use this information to determine how we can prevent biliary cancers, and identify new strategies for diagnosis and treatment.

For example, certain subgroups of individuals are at high risk for developing biliary cancers; in this country the single most important risk factor is the presence of an uncommon disease called primary sclerosing cholangitis. Patients with this condition have a one in three chance of developing bile duct cancers within 10 years. We plan to use information gained from our molecular studies in biliary cancers to develop novel screening and early detection techniques for this at-risk group of patients.

Research

We are using state-of-the-art "gene chip" technology to study changes in gene expression between normal samples and biliary cancer specimens. Using specialized chips, we can detect genes that are up- or down-regulated in cancer specimens compared to normal. Secondly, we are using biliary cancer cells in tissue culture to manipulate genes in order to identify the functional changes important in biliary tumor cell growth and invasion. Finally, we are growing human biliary tumors in mice (a technique called "xenografting") in order to develop a model where we can test novel treatment strategies for biliary cancer using an animal model.

Our ultimate goal is to utilize the basic knowledge we gain through our molecular studies to develop new methods of screening and treatment for patients with biliary tract cancers. In addition, the findings from biliary tract cancer studies may help people with other cancers, since often the pathways of tumor development are often similar between different cancer types.

People

Anirban Maitra, MD is the Principal Investigator of the new biliary tract cancer lab and an Instructor of Pathology and Genetic Medicine at The Johns Hopkins University School of Medicine. Dr. Maitra graduated from the All India Institute of Medical Sciences in New Delhi, India. He completed his residency and fellowship training in Anatomic Pathology at the University of Texas Southwestern Medical Center in Dallas, followed by a fellowship in Gastrointestinal and Liver Pathology at The Johns Hopkins University School of Medicine. Dr. Maitra has authored numerous papers in the field of pancreatic and biliary cancer research, and is an Associate Editor for the peer-reviewed journal Current Molecular Medicine.

Donna Hansel, MD/PhD is a fellow in the division of Gastrointestinal Pathology at The Johns Hopkins University School of Medicine. Dr. Hansel received her MD/PhD from The Johns Hopkins University School of Medicine and has trained in anatomic pathology at the Josephine Nefkins Instituut, Erasmus Universiteit, Rotterdam, The Netherlands. Dr. Hansel is interested a career in basic research in gastrointestinal malignancies.

Ayman Rahman, BA is the research assistant for the new biliary tract cancer lab and is responsible for harvesting tissue for creating biliary tumor xenografts. He received his degree in biology from the University of Virginia. Ayman is an expert in immunohistochemistry and in situ hybridization techniques.

Pedram Argani, MD is an Assistant Professor of Pathology at The Johns Hopkins University School of Medicine and an attending pathologist at The Johns Hopkins Hospital. Dr. Argani is a graduate of the University of Pennsylvania School of Medicine and received his pathology training at the Hospital of the University of Pennsylvania. He also completed fellowships in Oncologic Pathology and Molecular Pathology at Memorial Sloan-Kettering Cancer Center in New York. Dr. Argani is Director of the Immunopathology Laboratory of The Johns Hopkins Hospital. He has a special diagnostic interest in biliary cancer pathology. He has created the first biliary cancer tissue microarrays, which facilitates the high-throughput molecular analysis of abnormal proteins expressed by biliary cancers. He has also published on biliary tract cancers in the internationally-recognized journal Cancer.
Connie Knapik provides assistance with manuscript preparation and lab organization.

Support

Since the molecular biology of biliary cancers has been studied for only a short period of time, there is little organized funding available to support innovative research in this area. In order to move forward in our understanding of these cancers, we need support from many arenas, including both federal funding and private donations. You can make a difference with your contribution to this cause! If you would like to contribute to our understanding of biliary tract cancers, please address your donations to:

Biliary Cancer Research
c/o Dr. Pedram Argani
Johns Hopkins Hospital
Weinberg Building/Room 2242
401 North Broadway
Baltimore, MD 21231-2410

February 2002

CREATION OF GALLBLADDER AND BILE DUCT CANCER TISSUE MICROARRAYS

Through the use of high throughput genetic technologies, such as cDNA microarrays and serial analysis of gene expression (SAGE), numerous new markers of cancer have been identified. This has been particularly true for cancer of the pancreas (see the What's New section on the pancreas cancer Website). With the discovery of so many novel markers of disease and targets for potential therapy, there needs to be a way to quickly screen a large number of cancers for their expression of these new markers. Tissue microarray technology allows for such high-volume screening. In tissue microarrays, numerous cores (spots) of a large number of different tumors are placed into a single paraffin tissue block. This block can yield up to 300 or so slides, each containing samples of multiple (up to 300) tumors, which can be analyzed using a variety of technologies including immunohistochemistry for protein expression, in situ hybridization for RNA expression, and florescence in situ hybridization for DNA copy number. The pathology department of The Johns Hopkins Hospital has created a tissue microarray facility, which allows for construction of microarrays from tumors of any organ system. Such arrays have been constructed for prostatic and pancreatic adenocarcinomas, and preliminary results with these arrays have shown that tissue arrays can greatly speed the analysis of new cancer markers.

This past month, we have just completed the construction of, to our knowledge, the first gallbladder and bile duct carcinoma tissue microarray (See Figure below). This microarray contains spots of 40 different gallbladder and bile duct carcinomas, along with five normal gallbladder specimens and nine normal unrelated tissues as controls. We have made duplicate blocks such that each tumor is represented in four spots on the two blocks. These blocks will allow us to rapidly screen gallbladder and bile duct carcinomas for novel tumor markers that we identify through analysis of gene expression using gene chips. Preliminarily, we can report that these arrays have proven quite useful for evaluating markers identified in pancreatic cancers in gallbladder and bile duct carcinomas, and hold promise for identifying new ways to make the diagnosis earlier and potentially identify targets for rational therapy.

Our research team is greatly indebted to the friends and family of Margaret Lee for their generous donation, which funded the creation of these tissue microarrays. Because biliary tract cancers are not common, it is difficult to obtain government funding for this sort of study. Because of the Lee's generous gift, we were able to rapidly create these tissue microarrays to spur a new wave of biliary tract cancer research at Johns Hopkins. The patients and families who will benefit from the discoveries that result from these arrays, as well as the investigators themselves, are greatly indebted to the Lee family for their generosity.

January 2002

IMMUNOHISTOCHEMICAL ANALYSIS OF CYCLOOXYGENASE ENZYMES IN THE SEQUENTIAL PATHOGENESIS OF GALLBLADDER CARCINOMA.

A Maitra, II Wistuba, JF Miquel, RH Hruban, P Argani, R Ashfaq
Modern Pathology 2002; 15: 137A (abstract 571)

It is known that gallbladder carcinoma is associated with gallstones (cholelithiasis), and chronic inflammation. Recently, it has been discovered that the proinflammatory enzymes cyclooxygenase (COX1 and COX2) are overexpressed in a variety of human cancers, and may contribute to their formation. Analysis of COX expression in gallbladder carcinoma, which is associated with inflammation, has not yet been studied.

In this study, Dr. Maitra and colleagues at Johns Hopkins, University of Texas Southwestern Medical Center, and Catholic University in Chile studied a series of gallbladder carcinomas for expression of COX1 and COX2. The study showed that COX1 and COX2 were overexpressed in gallbladder carcinoma as compared to normal epithelium. In fact, the level of expression increased progressively from normal epithelium to low-grade dysplasia, to carcinoma in situ to invasive gallbladder carcinoma.

These studies demonstrate that COX overexpression is associated with tumor progression in gallbladder carcinoma. Since COX inhibitors are well characterized and well tolerated medications, these findings raise the possibility that COX2 and COX1 may be targets for prevention of gallbladder carcinoma.

MICROSATELLITE INSTABILITY IN INTRADUCTAL PAPILLARY MUCINOUS NEOPLASMS OF THE BILIARY TREE

Abraham SC, Lee JH, Boitnott JK, Argani P, Furth EE, Wu TT. Microsatellite instability in intraductal papillary neoplasms of the bilairy tract. Mod Pathol 2002;15: 1309-1317.

  

Intraductal papillary neoplasms of the biliary tree are unusual and rare bile duct tumors. They are analogous to the intraductal papillary mucinous neoplasms of the pancreas. They may be purely intraductal and non-metastasizing lesions, but may in time develop into invasive adenocarcinomas with the capacity to metastasize. Even if they are noninvasive, biliary intraductal papillary mucinous neoplasms may diffusely involve the liver, leading to liver failure.

Some cancers, particularly a subset of adenocarcinomas of the colon, have been shown to demonstrate genetic microsatellite instability. This finding essentially means that the cancer is characterized by frequent mistakes in replication of its DNA, which leads to mutations that promote the tumor's growth. Microsatellite instability is not well described in the cancers of the biliary tree. However, in this study, Dr. Wu and colleagues at MD Anderson Cancer Center in Houston, The University of Pennsylvania School of Medicine in Philadelphia, and Johns Hopkins University School of Medicine in Baltimore show that microsatellite instability was identified and present in almost 50% of biliary intraductal papillary mucinous neoplasms. Over 10% of these cases showed high levels of microsatellite instability. The authors also showed genetic differences between the intraductal and infiltrating bile duct carcinomas in these patients, indicating that these tumors are genetically heterogeneous.

This study is one of the first to identify genetic abnormalities in this rare form of biliary cancer. Once again, it is hoped that this further understanding will help us develop targeted therapy for these lethal neoplasms.

IMMUNOHISTOCHEMICAL AND GENETIC ANALYSIS OF NON-SMALL CELL AND SMALL CELL GALLBLADDER CARCINOMA AND THEIR PRECURSOR LESIONS

Parwani AV, Geradts J, Caspers E, Offerhaus GJ, Yeo CJ, Cameron JL, Klimstra DS, Maitra A, Hruban RH, Argani P. Modern Pathology 2003 April;16(4): 299-308

Gallbladder carcinoma is a highly lethal malignancy, yet little is known about its pathogenesis. It is hoped that a further understanding of the genetic abnormalities associated with gallbladder carcinoma will allow specific targeted therapies to be employed to treat this deadly disease.

Towards this end, Dr. Parwani and colleagues at The Johns Hopkins Hospital have studied a series of 35 gallbladder carcinomas for genetic abnormalities involving the p53, p16, Rb, and DPC4 tumor suppressor genes, as well as activation of the K-ras oncogene. These results are to be presented at the International Academy of Pathology meeting in Chicago in late February, 2002. Briefly, this study showed that p53 and p16 were very frequently (over 65% of the time) inactivated in gallbladder adenocarcinomas, while inactivation of Rb was infrequent. DPC4 was inactivated in 18% of gallbladder carcinomas, while 30% of cases harbored K-ras activating mutations. In contrast, small cell carcinomas of the gallbladder consistently showed p53 overexpression, while p16 and DPC4 were intact. Interestingly, Rb was inactivated in the majority of small cell carcinomas. K-ras mutations were not identified in small cell carcinomas.

These findings demonstrate that there are significant genetic differences between adenocarcinoma and small cell carcinoma of the gallbladder. This study is the first to demonstrate Rb inactivation in small cell carcinoma and DPC4 inactivation in adenocarcinoma. What is more interesting is that all the genetic changes identified appear to be present at the early clinical stage of in situ (noninvasive) carcinoma in the gallbladder. In fact, one case of carcinoma in situ of the gallbladder harbored alterations in p53, p16, and K-ras, indicating that carcinoma in situ of the gallbladder, while clinically early, is a genetic advanced lesion. These studies further our understanding of gallbladder carcinoma and highlight the fact that markers of tumor progression remain to be discovered in this neoplasm.

July 2001

MOLECULAR AND IMMUNOHISTOCHEMICAL ANALYSIS OF INTRADUCTAL PAPILLARY NEOPLASMS OF THE BILIARY TRACT

Abraham SC, Lee JH,Hruban RH, Argani P, Furth EE, Wu TT. The Johns Hopkins University School of Medicine and the University of Pennsylvania Medical Center.
Human Pathology. 2003 Sep;34(9):902-10.

  

Intraductal papillary neoplasms of the biliary tract are rare lesions that may extensively colonize the biliary tree. They are histologically and radiologically similar to the intraductal papillary mucinous neoplasms of the pancreas (IPMNs). Like IPMNs, these biliary intraductal lesions present a risk of progression to invasive carcinoma, in this case invasive cholangiocarcinoma. While IPMNs typically obstruct the pancreatic ducts and lead to chronic pancreatitis and pancreatic failure, intraductal papillary neoplasms of the biliary tract obstruct the bile ducts and therefore lead to hepatic damage. While IPMNs of the pancreas have been analyzed molecularly, little is known about the molecular pathogenesis of intraductal papillary neoplasms of the biliary tract. A group at Johns Hopkins, led by Dr. Susan Abraham, has recently studied fourteen cases of intraductal papillary neoplasms of the biliary tree, including five with associated invasive cholangiocarcinoma. They microdissected tissue from the intraductal and invasive carcinoma and compared it to normal tissue. Specific genes analyzed included the K-ras gene and the b-catenin gene, while allelic loss was analyzed on chromosome 18q (the site of DPC4), 17p (the site of p53), and 5q (the site of the APC gene).

Immunohistochemistry for p53, b-catenin, and Dpc 4 was also performed. The Hopkins group found activating mutations in K-ras in four of fourteen (29%) of intraductal papillary neoplasms of the biliary tree. Of these four cases, two occurred in patients with both intraductal papillary neoplasms and invasive cholangiocarcinoma, and the identical K-ras gene mutation was present in both the intraductal and invasive tumors. Three cases showed allelic loss on chromosome 18q, but no case demonstrated loss of Dpc 4 expression. b-catenin mutations were not detected by sequencing, and nuclear accumulation of b-catenin protein was similarly not identified in any case. Nuclear p53 accumulation never exceeded 20%, and allelic loss on chromosome 17p and 5q were not identified.

These results show that intraductal papillary neoplasms of the biliary tract are associated with K-ras gene mutations, which appear to arise early in their pathogenesis. The frequency detected in the biliary tree is lower than that seen in pancreatic IPMNs, but similar to that reported in hepatic cholangiocarcinomas. These results further our understanding of this unusual form of biliary tract carcinoma.

May 2001

CONSISTENT OVEREXPRESSION OF FATTY ACID SYNTHASE (FAS) IN BILIARY TRACT CARCINOMAS: A NOVEL TARGET FOR ANTI-BILIARY TRACT CANCER DRUG DEVELOPMENT?

P Argani, FP Kuhajda, RE Wilentz, GH Su, TA Sohn, CJ Yeo, JL Cameron, RH Hruban The Johns Hopkins University, Baltimore, MD.
Modern Pathology 2001;14: 192A.

Fatty Acid Synthase (FAS) is the primary enzyme involved in the anabolic conversion of dietary carbohydrates to long chain fatty acids. Specifically, FAS catalyzes the NADPH-dependent reductive synthesis of palmitate from acetyl CoA and malonyl CoA.

FAS is normally expressed in the liver, lactating breast, and adipose tissue (where it functions in anabolic energy storage pathways), but minimally expressed in other adult tissues which preferentially utilize circulating lipids.

FAS has been demonstrated to be overexpressed in several human cancers (breast, endometrial, prostate, colon). In some cancers, high levels of FAS expression have been associated with poor prognosis, suggesting that FAS expression may promote tumor growth and virulence. Recently-synthesized inhibitors of FAS have demonstrated antitumor activity in xenografts without concurrent toxicity to normal proliferative tissues (bone marrow, gastrointestinal mucosa, lymph nodes), and hence hold promise as therapy for tumors that overexpress FAS (Cancer Res 2000; 60: 213-218) (PNAS 2000; 97: 3450-3454).

FAS expression had not been studied in biliary tract carcinomas, which are highly aggressive and often unresponsive to conventional therapy.

We recently examined 107 biliary tract carcinomas for FAS overexpression using an immunohistochemical assay on formalin-fixed, paraffin-embedded tissue. FAS was overexpressed in 93% of carcinomas of the biliary tract. Therefore, FAS inhibitors hold promise as therapy for biliary tract carcinomas.

Figure 1: FAS immunolabeling of a bile duct carcinoma. The carcinoma surrounds a benign remnant of bile duct. While the bile duct carcinoma is strongly positive for FAS(brown staining), the normal bile duct does not label.

March, 2001

DIFFERING RATES OF LOSS OF DPC4 EXPRESSION AND OF p53 OVEREXPRESSION AMONG CARCINOMAS OF THE PROXIMAL AND DISTAL BILE DUCTS: EVIDENCE FOR A BIOLOGIC DISTINCTION

Pedram Argani MD1, Aasma Shaukat MD MPH2, Manju Kaushal M.Sc.1, Robb E. Wilentz MD1, Gloria H. Su PhD¹, Taylor A. Sohn MD3, Charles J. Yeo MD3,4, John L. Cameron MD3, Scott E. Kern MD1,4, and Ralph H. Hruban MD1,4 From the Departments of Pathology1, Surgery3, and Oncology4, and The Johns Hopkins School of Public Health2, The Johns Hopkins Medical Institutions, Baltimore, Maryland. USA
Cancer 2001;91: 1332-41

  

We had previously referred to a study performed at Hopkins (Genes, Chromosomes, and Cancer 1999;26; 185-191) in which our group analyzed the overall genetic composition of distal bile duct cancers using conventional cytogenetics and comparative genomic hybridization. The analysis of distal bile duct cancers revealed frequent loss of genetic material on the long arms of chromosomes 18, 6, and 12 (18q, 6q, and 12q), and the short arms of chromosomes 10, 8, and 17 (10p, 8p, and 17p). Of great interest was the fact that many of these changes are similar to those previously identified in pancreatic cancers, suggesting that these two tumors share a number of genetic changes. Some of the sites identified suggested specific genes that might be involved in bile duct cancers. For example, the p53 gene is located on chromosome 17p and is known to be mutated in the majority of pancreatic cancers.

We have recently completed a follow-up study that appeared in the April 1, 2001 issue of Cancer in which we have identified several specific genes involved in bile duct carcinoma. Using immunohistochemical labeling, we were able to show that the DPC4, a tumor suppressor gene discovered at Hopkins that is known to play a major role in pancreas cancer, is also involved in bile duct carcinomas. Loss of Dpc4 protein was identified in a significant number of bile duct carcinomas. However, not all bile duct carcinomas are equal: we were able to demonstrate that distal common bile duct carcinomas (those located near the pancreas) were far more likely to demonstrate loss of DPC4 than proximal bile duct cancers (Klatskin tumors and cholangiocarcinomas of the liver). In fact, the frequency of DPC4 loss that we demonstrated in distal bile duct carcinomas (55%) is identical to that which was demonstrated in pancreatic cancer. Similarly, we were able to show that the p53 gene product was abnormally expressed far more frequently in distal bile duct cancers than proximal ones. These results show that distal common bile duct cancers have some of the same genetic alterations as pancreatic cancers, while other bile duct cancers are biologically distinct. It is hoped that these results will allow us to develop more rational therapies for these tumors.

Figure 1: Moderately differentiated distal common bile duct adenocarcinoma demonstrating loss of Dpc4 expression and p53 overexpression (160 times magnification). A. Hematoxylin and eosin shows the adenocarcinoma infiltrating between a fibrotic reactive stroma. B. Dpc4 antibody labels the fibrotic stroma surrounding the carcinoma (brown staining is positive), but the carcinoma does not express Dpc4. C. p53 labeling yields the inverse pattern, as the infiltrating carcinoma overexpresses p53 (brown staining in the nucleus), while the stroma does not express p53.

Fig 1a

Fig 1b

Fig 1c

Fig 2

Figure 2: Summary of the results of the study, showing the pattern of Dpc4 and p53 expression in intrahepatic (top), perihilar (middle) and distal (bottom) bile duct carcinomas.