Gallbladder and Bile Duct Cancer

2003 News Archive

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.

Full Text

~October 2003



Opportunity for Collaboration from New Biliary Cancer Laboratory

Johns Hopkins Gallbladder and Bile Duct Cancer Research Laboratory
Principal Investigators: Anirban Maitra, MD and Pedram Argani, MD

The biliary cancer cell lines used in this study were obtained from the following sources:

please contact the cell line repository or Investigator listed directly if you are interested in obtaining the cell lines.

HuCCT1 - Health Science Research Resources Bank, Osaka, Japan

HuH28 - Health Science Research Resources Bank, Osaka, Japan

EGI-1 - German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany - https://www.dsmz.de/

TFK-1 - German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany - https://www.dsmz.de/

GBD1 - Prof. Kohje Miyazaki, Saga University, Saga, Japan -

GBH3 - Prof. Kohje Miyazaki, Saga University, Saga, Japan -

SNU245 - Korean Cell Line Bank, Seoul, Korea

SNU1079 - Korean Cell Line Bank, Seoul, Korea

SNU308 - Korean Cell Line Bank, Seoul, Korea

For Scientists Interested in Biliary Tract Cancer Research:
The Affymetrix U133A hybridization data for the nine cell lines listed above and five normal biliary epithelial samples (NBE) is freely available for academic researchers only in Excel format. The data has been normalized using dCHIP.

~July 2003

Johns Hopkins Biliary Cancer Tissue Resources

We have established and harvested multiple low-passage Human Gallbladder and Bile Duct Cancer nude mice xenografts from resected primary and metastatic biliary cancers. In addition, we have established tissue microarrays from archival human biliary tract cancers (gallbladders, intrahepatic and distal bile duct carcinomas).

Nucleic acid and/or tissues from these xenografts, and tissue microarrays are available for academic researchers on a collaborative basis.

Please contact .

~July 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.

PubMed Abstract | Full Text
Swierczynski SL, Maitra A, Abraham SC ,Iacobuzio-Donahue CA , Ashfaq R, Cameron JL, Schulick RD, Yeo CJ, Rahman A, Hinkle DA, Hruban RH, Argani P. Hum Pathol. 2004 Mar;35(3):357-66.

~February 2003



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.

PubMed Abstract | Full Text
Hansel DE, Rahman A, Hidalgo M, Thuluvath PJ, Lillemoe KD, Shulick R, Ku J-L, Park J-G, Miyazaki K, Ahsfaq R, Wistuba II, Geradts J, Argani P, Maitra A. Identification of Novel Cellular Targets in Biliary Tract Cancers using Global Gene Expression Technology. Am J Pathol 2003;163: 217-229.

~February 2003



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.

~February 2003