Multi-step pancreatic carcinogenesis and its clinical implications

European Journal of Surgical Oncology 1999; 25: 562–565

REVIEW

Multi-step pancreatic carcinogenesis and its clinical implications G. H. Sakorafas and A. G. Tsiotou Department of Surgery, 251 Hellenic Air Force (HAF) General Hospital, Athens, Greece

The poor prognosis of pancreatic cancer relates mainly to its delayed diagnosis. It has been repeatedly shown that earlier diagnosis of pancreatic cancer is associated with a better outcome. Molecular diagnostic methods (mainly detection of K-ras mutations in pure pancreatic or duodenal juice, on specimens obtained by percutaneous fine-needle aspirations or in stool specimens) can achieve earlier diagnosis in selected subgroups of patients, such as patients with chronic pancreatitis (especially hereditary), adults with recent onset of non-insulin-dependent diabetes mellitus and patients with some inherited disorders that predispose to the development of pancreatic cancer. There is increasing evidence that pancreatic carcinogenesis is a multi-step phenomenon. Screening procedures for precursor lesions in these selected subgroups of patients may reduce the incidence and mortality from pancreatic cancer. Key words: pancreatic cancer; multi-step carcinogenesis; pancreas; K-ras mutations; pancreatic intraepithelial neoplasia.  1999 Harcourt Publishers Ltd

Introduction

Molecular biology of pancreatic cancer

Pancreatic adenocarcinoma is typically a very aggressive carcinoma and has the worst prognosis of more than 60 cancers. Median survival is 4.1 months and 5-year survival is only 3%.1 This dismal prognosis is a result not only of biological aggressiveness but also of diagnosis late in the chronological progression of the tumour. More than 85% of patients have disease extending beyond the pancreas at the time of diagnosis, rendering surgical and medical interventions relatively ineffective. However, if pancreatic cancer can be resected when it is small (<2 cm), the prognosis is much better, with a 5-year survival of approximately 40%.2 A better understanding of the pathogenesis of pancreatic cancer and more effective screening techniques are required to increase the proportion of patients presenting with early resectable disease and to improve current survival rates. It is well known that identifiable genetic alterations occur in association with human neoplasms.3 These genetic alterations both shed light on the events that cause cancer to develop and suggest novel strategies to diagnose and treat this disease at earlier stages, with better results.4

The study of the genetics of pancreatic cancer is hampered by several characteristics.

Correspondence to: George H. Sakorafas, MD, Consultant, Department of Surgery, 251 Hellenic Air Forces (HAF) General Hospital, Panormou 5, 115 22 Athens, Greece. 0748–7983/99/060562+04 $12.00/0

(1) Precursor lesions to pancreatic cancer are relatively rare. There are no available tumour markers or diagnostic methods of sufficient sensitivity and specificity to aid in its earlier diagnosis. Therefore, the early genetic changes associated with the development of the tumour are difficult to access. The molecular genetic analyses of this tumour have mainly been done on advanced lesions.5,6 (2) The molecular study of tumour specimens requires tissue samples that are enriched for cancer cells. In primary pancreatic carcinoma there is often a strong desmoplastic reaction by the host tissues.7 (3) There is generally a low number of fresh primary tumours accessible for research, because of the low rate of surgical resection in most centers. Many abnormalities in structure and function of several oncogenes (most notably K-ras; c-erbB-3, c-erbB-2), tumour suppressor genes (mainly p53, p16; DCC, DPC4, etc.), growth factors and their receptors (EGF, TGF-a, TGF-b, aFGF and bFGF, EGFR, TGF-bR, FGFR, etc.) have been described in pancreatic cancer (Table 1).1,4–10 The main oncogene involved in pancreatic carcinogenesis seems to be the K-ras oncogene, which is mutated at a frequency higher than in other tumours. Several recent studies have reported that up to 95% of pancreatic cancer (PC) tissues contain  1999 Harcourt Publishers Ltd

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Multi-step pancreatic carcinogenesis Table 1. Molecular alterations in pancreatic cancer Oncogenes (amplification, overexpression or mutation)

Tumour-suppressor genes (mutations or deletions)

Growth factors and their receptors

K-ras c-erbB-2 c-erbB-3 c-myc (?) c-fos (?) Others (?)

p53 p16 p15 DCC DPC4 Rb (?) APC (?) Others (?)

EGFR EGF TGF-a TGF-b FGF (a & b) FGFR TGF-bR Others

mutated K-ras gene.11 Tumour-suppressor gene p53 is also frequently mutated in pancreatic cancer (40–70%). This frequency is similar to that reported in other common human malignancies, such as colorectal, breast, lung and hepatocellular cancer.12 Other tumour-suppressor genes that have been suggested to play a role in pancreatic carcinogenesis are: p16 (a member of the INK family), which is mutated in 38% of pancreatic cancers,13 DCC (Deleted in Colon Cancer),14 DPC4 (Deleted in Pancreatic Cancer, locus 4)15 and BRCA2.16 Moreover, many growth factors and growth factor receptors are involved in the molecular carcinogenesis of pancreatic cancer [EGF, EGFR, TGF-a, TGF-b (and isoforms 1, 2, 3), TGF-bR, TGF (a and b), HGF, PDGF, pS2, etc.].17–20 These observations about the role of growth factors and their receptors illustrate the importance of autocrine and paracrine (i.e. tumourstroma) influences in the development of PC.

Multi-stage process in pancreatic carcinogenesis The multi-step concept of carcinogenesis may explain many forms of neoplasia.21 For example, in the uterine cervix squamous intraepithelial lesions (or cervical intraepithelial neoplasia) precede the development of infiltrating squamous carcinoma and screening procedures for these precursor lesions have reduced substantially the incidence of and mortality from cervical cancer in the last three decades. Analogous precursor lesions to infiltrating carcinoma also have been described in the breast, colon, lung and prostate, where these lesions are markers for an increased risk of later development of infiltrating cancer. The best studied epithelial gastrointestinal tumour is colon cancer, in which genetic alterations occur in a preferred order, genetically defining the progression of a colonic adenoma toward carcinoma.22–24 The total accumulation of genetic changes rather than their order appears to be important.22–24 The colorectal model might also prove relevant to pancreatic neoplasms.14 Most (80–90%) pancreatic cancers arise from duct epithelium, although about 10% of all pancreatic cells are duct cells. Main pancreatic ductal cells are reported to have a higher turnover rate than other cell types in the pancreas25,26 and give rise to the most common type of human pancreatic carcinoma (ductal adenocarcinoma). Normal pancreatic ducts and ductules are lined by a single layer of cyboidal epithelial cells. The

ducts adjacent to infiltrating pancreatic cancers frequently show a spectrum of morphological changes, ranging from ‘flat hyperplasia’ to ‘atypical papillary hyperplasia or papillary dysplasia’.27–30 Some lesions exhibit classic signs of atypia, such as nuclear pleomorphism, an increased nucleus-to-cytoplasm ratio and a loss of cellular polarity. Ductal papillary hyperplasia is three times more frequent in pancreatic cancer specimens than in non-pancreatic cancer controls.29 Pancreatic ductal hyperplasia can be found in the normal pancreas27 and in patients with chronic pancreatitis. Necropsy studies have found an increasing incidence of ductal hyperplasia with advancing age.27 The frequent finding of ductal hyperplasia in pancreatic cancer may represent a progression from hyperplasia to carcinoma, analogous to the adenoma–carcinoma progression in the colon. Pancreatic intraepithelial neoplasia (PIN) has been increasingly noted.31,32 The dysplastic epithelium may be flat, micropapillary or grossly papillary. This may be analogous to adenomatous polyps of the colorectum, suggesting a progression from pancreatic intraepithelial neoplasia (PIN) to infiltrating carcinoma of the pancreas.22,23,31–34 K-ras mutation is considered as an early event in pancreatic carcinogenesis and has been identified in PIN.35–38 The K-ras mutations are as frequent in PIN as in ductal adenocarcinoma.38 A step-wise increase in the frequency of K-ras mutations correlates with the stage of neoplastic evolution to cancer.38 In vitro, normal human epithelial cells may be partially transformed by activated ras oncogenes alone, but for full malignant transformation an additional genetic event associated with immortalization is necessary.39 This additional genetic event may involve the loss or inactivation of a tumour-suppressor gene. An overexpression of mutant p53 has been described in PIN.40 In patients with Li-Fraumeni syndrome, characterized by a hereditary predisposition to various tumours, including pancreatic cancer, germ-line mutations of the p53 gene have been described.41,42 It may be that in these patients p53 mutations are a late event and that the order of events is not important, the net result being dependent on the accumulation of genetic abnormalities.43,44

Future perspectives The recognition of patients at high risk for the development of pancreatic cancer will allow the rational application of the newer molecular diagnostic modalities in carefully selected and well-defined groups of patients. Such groups are patients with chronic pancreatitis (especially hereditary pancreatitis), adults with recent onset of non-insulindependent diabetes and patients with some inherited disorders which predispose to the development of pancreatic cancer (Table 2).4,45–48 Detection of mutant K-ras in pancreatic disease might provide a diagnostic tool. Molecular techniques, such as the polymerase chain reaction (PCR), may be used to detect these genetic alterations even in very small samples of tissue and fluid. K-ras mutations can be detected in microlitre volumes of pancreatic secretions (pure pancreatic juice

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Table 2. Inherited disorders predisposing to the development of pancreatic cancer Hereditary pancreatitis (autosomal dominant) Multiple endocrine adenomatosis (autosomal dominant) Glycagonoma syndrome (possible autosomal dominant) Pancreatic cancer as part of the tumour spectrum in hereditary non-polyposis colorectal cancer, Lynch II variant (autosomal dominant Gardner’s syndrome (autosomal dominant) Hippel–Lindau’s disease (autosomal dominant) Neurofibromatosis (Recklinghausen’s disease) (autosomal dominant) Ataxia-telangiectasia (autosomal recessive) Familial atypical multiple mole melanoma (FAMMM) syndrome

obtained during ERCP or in duodenal juice), on specimens, obtained by percutaneous fine-needle aspirations or in stool specimens.8–10,17,49–57 Moreover, a method for the measurement of serum p53 protein concentration (ELISA assay) has been described which does not require tissue specimen and is easy to perform.58 There is evidence that molecular techniques (especially K-ras assay) can achieve earlier diagnosis of pancreatic cancer. Currently, these molecular diagnostic assays are not recommended for mass screening of asymptomatic individuals, but mainly as a complementary method to the established radiological and pathological techniques in selected subgroups of patients. Screening procedures for precursor lesions in these patients with the aid of molecular techniques may hopefully reduce the incidence and mortality from pancreatic cancer, as has occurred with cervical, breast, colon, lung and prostate cancer in recent decades.

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Accepted for publication 19 October 1998