Familial Pancreatic Cancer and the Genetics of Pancreatic Cancer

Familial Pancreatic Cancer and the Genetics of Pancreatic Cancer


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FAMILIAL PANCREATIC CANCER AND THE GENETICS OF PANCREATIC CANCER Jeanne A. Lumadue, MD, PhD, Constance A. Griffin, MD, Medhat O~man, MS, MD, and Ralph H. Hruban, MD

With the advent of recombinant DNA technology, the past two decades have witnessed an exponential growth in our understanding of the structure and function of the human genome. Nowhere have these advances been more dramatic than in the identification of genetic alterations that occur in association with human neoplasms. These genetic alterations not only shed light on the events that cause cancer to develop, but also suggest new methods to diagnose, detect, and treat cancer. Clearly, the need to improve the diagnosis, detection, and treatment of pancreatic cancer is great. Adenocarcinoma of the pancreas is the fifth leading cause of cancer deaths in the United States,11 and in the majority of cases, the carcinoma has spread beyond the gland by the time of initial presentation, rendering surgical and medical interventions relatively ineffective. 60 It is in these types of cancers, in which the mortality is so great and the clinical detection so difficult, that the recent advances in the understanding of cancer genetics will have the greatest impact. Genetic alterations can be detected at different levels. With classic cytogenetic techniques, individual chromosomes can be examined. With molecular techniques alterations can be detected at the level of the DNA sequence, such that alterations in single nucleotides can be identified. This article focuses on the cytogenetic changes associated with sporadic and familial adenocarcinoma of the pancreas and some of the data on the inheritance of pancreatic cancer; the following article by Hahn and Kern focuses on the molecular changes.

From the Departments of Pathology (JAL, CAG, RHH), Oncology (CAG, RHH), and Medicine (CAG), and The Johns Hopkins Oncology Center (CAG, MO, RHH), The Johns Hopkins Medical Institutions, Baltimore, Maryland






One way to detect chromosome abnormalities in cancer is to measure the DNA content in each tumor cell. This can be done by either flow cytometry or absorption photocytometry, two techniques that measure the nucleic acid content of the individual cells. Aneuploidy, a deviation in chromosome number from the normal 46, has been associated with a poorer prognosis in a number of neoplasms, including colorectal carcinoma, melanoma, and node-negative breast carcinoma. 3, 14,27,33,36,55 Similarly, the DNA content of cancer cells within pancreatic adenocarcinomas has been shown to be an important predictor of survival in patients who undergo pancreatoduodenectomy.2, 7, 51 Allison et aF examined the DNA content of 47 pancreatic adenocarcinomas by absorption photocytometry and found that the 19 patients with diploid carcinomas had a mean survival of 25 months, whereas the mean survival of the remaining 28 patients with aneuploid tumors was significantly lower (mean survival of 10.5 months, P = 0.003). Other groups have reported similar findings. Porschen· et a151 have reported that the survival rate of patients with diploid pancreatic cancers was significantly higher than in those with aneuploid neoplasms, and in a study by Bottger et aV tetraploid tumors (e.g., tumors with twice the normal amount of DNA) were found to have a better outcome than nontetraploid tumors. Although these findings need to be verified in larger studies, the association of aneuploidy with poorer patient outcome may one day aid in the management of patients with pancreatic cancer. For example, ploidy studies performed on tumor aspirates obtained by fineneedle aspiration may identify those individuals with a poorer prognosis, and ploidy could be used as one of the criteria in deciding who is a candidate for surgical resection. Ploidy analyses are, however, only a gross measurement of the overall loss or gain of large numbers of chromosomes. The identification of specific chromosomes lost or gained by a neoplasm can be made directly by karyotypic analysis of metaphase spreads of cells obtained from each tumor. Several groups have examined G-banded metaphase chromosome spreads prepared from fresh tumors to identify specific chromosomal alterations· that occur in pancreatic adenocarcinomas. Analysis of a total of 34 pancreatic adenocarcinomas, reported by Johansson et aV' 32 has shown 12 (35%) to have no detectable cytogenetic abnormalities, 8 (24%) to have relatively "simple" abnormalities (including 4 with a loss of the Y chromosome only), and the remaining 14 (41%) to have markedly abnormal karyotypes with triploid or neartriploid modal chromosome complements. The most frequent numerical abnormalities that they identified were loss of 18, loss of 12, loss of the Y chromosome, and extra copies of chromosomes 7, 11, and 20. Structural abnormalities were most frequent in chromosome arms 1, 8, 17, and 6. In an even larger series of 62 pancreatic cancers from our institution,21, 23 normal karyotypes were seen in only nine (15%) tumors.



Twenty-seven (44%) of the adenocarcinomas contained both normal and abnormal clones. The remaining 26 (42%) were thought to have markedly abnormal karyotypes. These included gains of chromosome 20 in 7 tumors and of chromosome 7 in 5 tumors. Losses of chromosomes were much more frequent, with losses of chromosome 18 found in 22 tumors, chromosome 13 in 15 tumors, chromosome 12 in 13 tumors, and chromosome 6 in 12 tumors. Numerous intrachromosomal breakpoints were identified, and these occurred (in order of decreasing incidence) on the long arm of chromosome 6, the short arms of chromosomes 1 and 3, the short arms of chromosomes 11 and 17, the short arm of chromosome 8, and the long arm of chromosome 19.23 Taken together with the studies of Johansson et al., these cytogenetic data suggest that gains in chromosomes 7 and 20 and loss of chromosome arms 1p and 6q are important in the pathogenesis of pancreatic cancer. The alterations defined cytogenetically can be more precisely defined using a panel of molecular probes specific for each chromosome arm. This technique identifies the specific chromosomal areas that have lost genetic material (so-called allelic loss studies). These studies are valuable for two primary reasons. First, just as ploidy provides prognostic information, so can allelic loss studies. For example, the number pf chromosomes lost in the tumor (fractional allelic loss, FAL) provides an estimate of global genetic damage within the tumor, and this loss has been associated with patient outcome in breast and colon cancer. 16,35 Seymour et a156 studied seven pancreatic tumors and found that the three patients with a low FAL were alive after 17 months, whereas the four patients with a high FAL died from presumably more biologically aggressive disease. Second, these allelic loss studies can be useful in the identification of tumor suppressor genes. Tumor suppressor genes are genes that normally function as genetic barriers to uncontrolled cellular proliferation and are particularly vulnerable sites for allelic 10ss.26 If one copy of the tumor suppressor gene is lost (by genetic events such as mutations, deletions, chromosomal rearrangements, or mitotic recombinations), only one copy remains to carry out the tumor-suppressing activity of the gene within the cell. Should this second copy become mutated or deleted, the cell can undergo uncontrolled proliferation. These allelic loss studies have been used to examine pancreatic adenocarcinomas. Seymour et a156 examined seven tumors and found the most frequently lost chromosome arms to be the short arm of 17 (called 17p) and the long arm of chromosome 18(called 18q). These data have been expanded by the same group to include 17 tumors. In this expanded series of pancreatic cancer, high frequencies of allelic loss were found on chromosome arms 1p (50%), 6p (50%), 6q (50%), 8p (56%), 9p (76%), lOp (58%), 10q (50%), 12p (50%), 12q (67%), 17p (95%), 18q (88%), 21q (61 %), and 22q (61%).24 These allelic loss data directly correlate with known genomic locations of tumor suppressor genes. For example, chromosome 17p, which was lost in 95% of the pancreatic cancers, is the normal location of the p53 tumor suppressor gene, a gene that has been implicated in the

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development of about half of all human malignancies. 26 In pancreatic cancers, not only is one copy of the p53 gene frequently lost, but the second copy is frequently mutated. 5 , 12, 17, 18, 52-54 Similarly, the long arm of chromosome 18, which was lost in 88% of the cases studied, is the normal site of the DCC (deleted in colon cancer) tumor suppressor gene. DCC, as its name implies, is important in the development of colon cancer. 20 As was true for p53, the second copy of the DCC gene is frequently mutated in pancreatic cancers. 30,57 Furthermore, Hohne et apo have shown an absence of or reduction in DCC expression in 8 of 11 pancreatic carcinoma cell lines and in 4 of 8 primary tumors. Thus, these allelotype studies also point to DCC as a candidate gene for the development of pancreatic adenocarcinomas. The third most frequently lost chromosome arm in the allelic loss studies was 9p. The loss of chromosome 9p has long been implicated in the development of human tumors,8,34 and in the allelic loss study cited above, it was shown to be deleted in 76% of the primary pancreatic tumors examined. The short arm (p arm) of chromosome 9 has recently been shown to contain at least one tumor suppressor gene, the MTS1 (multiple tumor suppressor; p16) and is speculated to contain additional suppressor 10ci.8 Caldas et a19 examined pancreatic carcinomas and found deletions of 9p21-22 in 85% of cases. Moreover, analysis of the MTS1 gene sequence in these tumors revealed homozygous deletions in the gene in 41% of cancers and sequence changes in the gene in 38%. These mutations were coexistent with frequent loss of heterozygosity and mutational inactivation of the p53 gene. Taken together, these data suggest that involvement of the DCC, MTS1, and p53 tumor suppressor genes may be important in the development of pancreatic cancer. The DNA studies mentioned above require tissue disruption for analysis. However, neoplasms are often composed of different subpopulations, and the examination of the genetic changes in each of these subpopulations can be important in our understanding of oncogenesis. This is especially true in the study of in situ carcinomas, or in early lesions in which the tumor cells represent a relatively small population of the entire specimen. Fluorescent in situ hybridization (FISH) is a technique that has recently been developed in which genetic analysis can be performed on intact tissue sections. With FISH, DNA probes that are specific for particular regions of the chromosome are labeled with a fluorescent marker and hybridized to intact tissue sections of the tumor. These sections can be examined using a fluorescence microscope, providing a visual correlation between the tumor cells, their morphologic characteristics, and the presence or absence of DNA sequences from their chromosomes. Using these techniques, Chou et al13 have examined six pancreatic cystic neoplasms (three adenomas and three carcinomas); four of the six tumors (two adenomas and two carcinomas) were trisomic for chromosome 16, and two of the six had alterations in the copy number of the X chromosome. Similar studies applied to in situ pancreatic cancers may provide insight into the earliest genetic changes in the development of cancer of the pancreas.22



From the previous studies, it is apparent that our understanding of the genetics of pancreatic cancer is increasing. Initial cytogenetic studies have identified chromosome abnormalities in pancreatic cancers. These studies have been expanded by allelic loss studies, which have more precisely defined the segments of DNA that are abnormaL Current studies are now focusing on determining which genes are actually responsible for the transformation from the normal to the malignant phenotype. FAMILIAL PANCREATIC CARCINOMA

Family studies can add a great deal to our understanding of the genetic alterations in cancer. In these "cancer families," the genetic alteration(s) leading to tumor development are inherited in the germ line. Thus, these patients have a uniform inherited abnormality that leads to the development of their carcinomas. Patients with cancer in their families presumably inherited a germ-line genetic mutation in a cancer-causing gene, whereas patients in families without cancers presumably did not. When multiple family members are afflicted with the same tumor type, these tumors become extremely useful for genetic analyses, in that the mutations responsible for the tumor are present in the cancers of each afflicted individuaL By utilizing this type of material, the genetic alterations not related to tumor development (that is, the genetic noise) can be sorted out from the seminal alterations that are responsible for cancer development. Two classic examples of the usefulness of familial forms of cancer are familial adenomatous polyposis and the Li-Fraumeni syndrome. 6,37, 38, 49 Familial adenomatous polyposis is characterized by the autosomal dominant transmission of the propensity to develop multiple colonic adenomas (polyps), which frequently progress to adenocarcinoma of the colon. Genetic studies first localized the gene responsible for this syndrome to chromosome 5, and later the gene itself, called "APC," was identified and cloned. 28,29 Family members affected by familial adenomatous polyposis inherit a mutated APC gene, whereas nonaffected family members have a normal APC gene. Li-Fraumeni syndrome is a familial cancer syndrome in which afflicted individuals develop sarcomas and carcinomas at an early age. 39 Like familial adenomatous polyposis, the Li-Fraumeni syndrome has been shown to be caused by inherited mutations, this time within the p53 tumor suppressor gene. 6,49 Families afflicted with either of these syndromes can now be identified using simple genetic tests, and members who carry the mutations that cause these syndromes can be carefully screened for the development of early cancers. In the case of familial adenomatous polyposis, prophylactic colectomy may be warranted in patients who carry the mutated gene. Anecdotal data and case reports have long suggested that familial clustering may occur in cancer of the pancreas (the family of a former head of state is a notable example), and it is hoped that these families

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will provide clues to the development of sporadic cases. Patients with pancreatic cancer are statistically significantly more likely to have a close relative with pancreatic cancer: Studies have shown that as many as 7.8% of persons with pancreatic cancer have a family history of pancreatic cancer, compared with only 0.6% of controlsY· 42 These reports clearly indicate that if an individual has a family member afflicted with pancreatic adenocarcinoma, he or she has a small but increased risk for developing a cancer. Although the cause of these familial pancreatic cancers is not yet known, several inherited disorders have been shown to predispose one to the development of pancreatic cancer (Table 1). These include hereditary pancreatitis, ataxia-telangiectasia, a form of hereditary nonpolyposis colorectal carcinoma (HNPCC: Lynch syndrome II or the cancer family syndrome), and a subset of the familial atypical mole-multiple melanoma (FAMMM) syndrome. 41 Hereditary pancreatitis is a syndrome in which the afflicted individuals suffer from relapsing pancreatitis, often at an early age of onset. IS Frequently, there is an association with splenic and portal vein thrombosis, and patients often manifest the sequelae of chronic pancreatitis, such




Hereditary pancreatitis




HNPCC (hereditary non polyposis colon cancers; cancer family syndrome; Lynch syndrome II)


FAMMM (familial atypical mole-malignant melanoma syndrome)


Relapsing pancreatitis, frequently at a young age of onset Frequently associated thrombosis of the portal/splenic veins Associated pancreatic insufficiency, diabetes, and pseudocysts Progressive cerebellar ataxia Multiple telangiectasias (primarily conjunctival) Tendency to sinopulmonary infections Oculomotor apraxia Frequently associated thymic hypoplasia and immune deficiencies Tendency to chromosomal breakage Tendency to form malignancies Adenocarcinoma of the colon associated with adenocarcinoma of the endometrium, ovary, or pancreas Increased frequency of other malignancies Replication error phenotype Predisposition to form malignant melanomas Frequent congenital nevi Frequent dysplastic nevi Increase in other malignancies such as colon and pancreatic cancer

'AD = autosomal dominant; AR = autosomal recessive.



as pancreatic insufficiency, diabetes, and pseudocysts. The biochemical or anatomic nature of the anomaly is unknown. so The mechanism by which hereditary pancreatitis predisposes affected individuals to pancreatic cancer is not clear, but because these persons are not predisposed to all cancers, it may be that the increased risk of pancreatic cancer observed in these patients is ca~sed by the chronic pancreatitis itself.19,4O Ataxia-telangiectasia is inherited as an autosomal recessive trait, and it is characterized by progressive cerebellar ataxia, telangiectasias, increased susceptibility to sinopulmonary infections, and oculomotor apraxia. 50 Persons with ataxia-telangiectasia are believed to harbor a mutation in a DNA repair or· processing pathway and, thus, are at increased risk for developing a number of types of cancer, including leukemias, breast cancer, gastric cancers, and paI)creatic cancers. 41,50 These neoplasms presumably develop secondary to the inefficient repair and accumulation of altered DNA sequences within the genome. The Lynch cancer family syndrome II (hereditary nonpolyposis, HNPCC) is another syndrome that predisposes individuals to pancreatic Cancers. Pedigree studies have clearly documented the transmission of an autosomal dominant factor that predIsposes the affected individuals to colonic cancer in association with other forms of cancer (primarily of the breast, endometrium, and ovary, but also of the pancreas).44--48 Recently, individuals afflicted with HNPCC have been shown to have widespread genetic alterations in their DNA. These alterations, called dinucleotide repeats, replication errors, or microsatellite instability, I, 59 are believed to be caused by mutations in the human mismatch repair system that stabilizes nucleotide repeatsP Pancreatic cancers have been examined for replication errors such as those found in HNPCC and thus far no consensus exists: Although one report describes frequent dinucleotide repeats in pancreatic cancer,2s a group here at Johns Hopkins found only one tumor with replication errors in 35 pancreatic cancers (5 Kern, personal communication). Thus, the role of this type of "mutator phenotype" remains controversial. . Finally, a subset of individuals with the FAMMM syndrome and pancreatic carcinomas has been reported. 43 The FAMMM syndrome is an autosomal dominantly inherited syndrome in which patients have numerous normal nevi, multiple atypic::al nevi, and cutaneous malignant melanomas. 43 Once again, the underlying genetic basis for the abnormality is unknown, but it is presumably secondary to a uniform underlying genetic tendency to form neoplasms. Of interest, the MT51 gene is frequently mutated in melanoma cell lines and in pancreatic cancers.9,34 Also, because genes such as pq3, DCC, MT51, and the K-ras oncogene appear to be altered in the sporadic cases of pancreatic cancer, these are excellent candidates for mutated genes in familial cases of pancreatic cancer. We are in the process of examining these genes in familial cases of pancreatic cancer. For example, recently a prophylactic pancreatectomy was performed at our institution on a person with a strong family history of pancreatic carcinoma. This resection specimen provided us with an opportunity to study the molecular alterations in




familial forms of pancreatic cancer. Although no invasive carcinoma was identified in the patient's pancreas, there was multifocal mucinous metaplasia within the pancreatic ducts. Molecular analysis of these areas of mucinous metaplasia revealed two distinct activating point mutations in codon 12 of the K-ras oncogene, a gene associated with the development of pancreatic carcinoma. 1S In contrast, no K-ras mutations were found in histologically normal ducts. Mutations were not present in the p53 tumor suppressor gene in any of the ducts. Thus, we can conclude that this individual did not have a germ-line mutation of K-ras or p53 (but perhaps had mutations in other loci), but that she had already begun to accumulate genetic defects that can lead to the development of pancreatic cancer. Alternatively, familial cases of pancreatic cancer occur independently of a known syndrome. Lynch's group is currently exarnirring 30 extended families with multiple cases of pancreatic carcinoma, and they have identified an autosomal dominant mode of transmission in some of these families. They estimate that up to 3% to 5% of pancreatic cancers have a hereditary origin but admit that the transmission is complex and that common exposures and environmental factors cannot be rigorously excluded. 41,42 For example, it is possible that several members of a family all developed cancer because they all smoked. Moreover, to date there are insufficient data on the genetic penetration (percent of persons who carry the gene and are affected) or expressivity (how severely the trait affects each carrier of the trait) in these families. Additional population genetic data are needed to sort out these issues. Because of the relative rareness but extreme value of families afflicted with pancreatic cancer, a national registry for familial pancreatic cancer has been formed here at Johns Hopkins. The registry is currently collecting families in which more than one family member is afflicted with pancreatic cancer. Persons wishing to enroll patients or themselyes may contact the registry at: National Familial Pancreatic Tumor Registry The Johns Hopkins Hospital Department of Pathology 600 N. Wolfe Street Baltimore, MD 21287 Phone: 410-955-9132 Fax: 410-955-0125 This registry will be used to determine whether or not there is a familial form of pancreatic cancer. Once families are registered, they can be very useful in helping to identify the genes responsible for the development of cancer of the pancreas. If a gene is identified, it can in tum be used to identify family members at risk for the development of pancreatic cancer, and a gene may also be found that could form the basis for the development of a screening program for pancreatic cancer in this population. This is possible because molecular techniques such



as the polymerase chain reaction may be used to detect genetic alterations in extremely small samples of body tissues and fluids.lO Microliter volumes of pancreatic secretions obtained at ERCP, needle biopsies, and even blood or stool samples may be used to detect mutations within the DNA of cells. lO, 58, 61 Those individuals most at risk for developing the cancers are, of course, the most likely population for the screening. SUMMARY

In our current understanding of pancreatic carcinoma, these neoplasms can arise either sporadically or in familial clusters. Extensive chromosome abnormalities are frequent, as is loss of heterozygosity at loci known to contain the tumor suppressor genes DCC, p53, and MTSl. Although the genetic examination of all pancreatic cancers is important, the examination of familial cases is especially useful in that these allow the identification of uniform genetic alterations that are inherited through the germ line. Much additional work needs to be done before the genetic basis of pancreatic cancer is completely understood. Although our knowledge is limited, it is clear that genetic analyses can be used to establish the prognosis for a patient with pancreatic cancer and, it is hoped, will someday be used in the management, treatment, and detection of pancreatic cancer. References 1. Aaltonen LA, Peltomaki P, Mecklin J-p, et al: Replication errors in benign and malignant tumors from hereditary nonpolyposis colorectal cancer patients. Cancer Res 54:1645-1648, 1994 2. Allison De, Bose KK, Hruban RH, et al: Pancreatic cancer cell DNA content correlates with long-term survival after pancreatoduodenectomy. Ann Surg 214:648-{i56, 1991 3. Armitage Ne, Robins RA, Evans OF: The influence of tumour cell DNA abnormalities on survival in colorectal cancer. Br J Surg 72:828-830, 1985 4. Bardi G, Johansson B, Pandis N, et al: Karyotypic abnormalities in tumours of the pancreas. Br J Cancer 67:1106-1112, 1993 5. Barton CM, Staddon SL, Hughes CM, et al: Abnormalities of the p53 tumour suppressor gene in human pancreatic cancer. Br J Cancer 64:1076-1082, 1991 6. Birch J, Hartley A, Tricker K, et al: Prevalence and diversity of constitutional mutations in the p53 gene among 21 Li-Fraumeni families. Cancer Res 54:1298-1304, 1994 7. Bottger Te, Starkel S, Wellek S, et al: Factors influencing survival after resection of pancreatic cancer: A DNA analysis and histomorphologic study. Cancer 73:63-73,1994 8. Cairns P, Li M, Merlo A, et al: Rates of p16 (MTS1) mutations in primary tumors with 9p loss. Science 265:415-417, 1994 9. Caldas C, Hahn SA, da Costa LT, et al: Frequent somatic mutations and homozygous deletions of the MTS1 gene in pancreatic adenocarcinoma. Nature Genetics, 1994, in press 10. Caldas e, Hahn SA, Hruban RH, et al: Detection of K-ras mutations in the stool of patients with pancreatic adenocarcinoma and pancreatic ductal hyperplasia. Cancer Res 54:3568-3573, 1994 11. Cancer Facts and Figures-1994. Atlanta, American Cancer Society, 1994, pp 1-28



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Address reprint requests to Ralph H. Hruban, MD Meyer 7-181 Department of Pathology The Johns Hopkins Hospital 600 North Wolfe Street Baltimore, MD 21287