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The Familial Atypical Multiple Mole Melanoma (FAMMM) syndrome is caused by a germline mutation of p16. More than 90% of the sporadic pancreatic carcinomas contain genetic alterations that inactivate p16. Patients with the FAMMM syndrome have an increased risk of developing pancreatic cancer. Ductal adenocarcinoma is the most common cancer of the pancreas and the one encountered in patients with FAMMM syndrome. Undifferentiated carcinoma with osteoclastic giant cells, also referred to as UCOCGC of the pancreas, is a rare variant of pancreatic cancer. An UCOCGC of the pancreas associated with FAMMM syndrome is described in this report. Molecular analysis confirmed a germline p16- Leiden deletion in the UCOCGC, accompanied by somatic loss of heterozygosity (LOH) of the second p16 allele, and absence of p16 protein expression in the neoplastic cells. It is the first case reported and it provides additional evidence that UCOCGC can be considered as a variant of conventional ductal adenocarcinoma of the pancreas.
The p16 gene on chromosome 9p is a tumor suppressor that inhibits the cell cycle by binding Cdk4 and Cdk6 and interfering with their ability to phosporylate RB1.6,7 More than 90% of the sporadic pancreatic adenocarcinomas show complete loss of function of p16 due to a combination of deletion, inactivating mutation and/or hypermethylation of the promoter region.9,13 The Familial Atypical Multiple Mole Melanoma (FAMMM) syndrome can be caused by germline mutations of p16.3 Families with the FAMMM syndrome are not only at risk for melanoma but also for the development of pancreatic cancer.4 FAMMM patients have a 20- to 34-fold higher risk of pancreatic cancer compared to the general population, although germline p16 mutations per se contribute to significantly less than 5% of all familial pancreatic cancer cases. To the best of our knowledge, there are no significant differences in the age of onset, histology, and prognosis of FAMMM-associated ductal adenocarcinomas compared to those occurring in the non-syndromic setting, although the paucity of cases renders a definitive conclusion as unwarranted.
A variety of different types of pancreatic cancer exists, but the most common type is pancreatic ductal adenocarcinoma and this is also the entity that typically is encountered in patients with the FAMMM syndrome.2 An unusual variant of pancreatic cancer is the anaplastic carcinoma that is accompanied by non-neoplastic osteoclastic like multinucleated giant cells, originally described by Rosai.8 The giant cells in this neoplasm resemble cells observed in giant cell tumors of bone. A growing body of evidence suggests that UCOCGC of the pancreas is best regarded as a variant of ductal adenocarcinoma with an associated non-neoplastic giant cell reaction.1
In this report a case of UCOCGC is described associated with the FAMMM syndrome due to a p16-Leiden deletion.2 It is the first reported case of UCOCGC associated with FAMMM. The case provides indirect support for the notion that UCOCGC can be considered as a variant of ductal adenocarcinoma.
A 39 year-old male was referred to us from another hospital. The patient had presented with abdominal discomfort, pain, jaundice and weight loss and had a history of a previous hospitalization for pancreatitis due to alcohol abuse. Computed tomography (CT) and magnetic resonance imaging (MRI) showed heterogeneity and calcification in the pancreas consistent with chronic pancreatitis and no evidence for malignancy although a tumor in the head of the pancreas could not be ruled out with certainty. Endoscopic retrograde cholangiopancreaticography (ERCP) revealed an ampullary lesion (Figure 1A), and the biopsy was initially interpreted as reactive, since epithelial markers were negative on immunohistochemistry.
The patient’s family history was remarkable for the occurrence of multiple melanomas and occasionally pancreatic cancer due to the FAMMM syndrome caused by a p16-Leiden deletion.
Deeper sections of the ampullary biopsy (Figure 1B) showed osteoclast like giant cells which expressed CD68 on immunohistochemistry (Figure 1D), whereas immunolabeling for CAM 5.2, pancytokeratin, and CK19 was negative. Immunolabeling for S-100 protein, Melan-A, and HMB-45, performed to rule out melanoma, were also negative. A diagnosis of UCOCGC was considered and the decision was made to do a mutational analysis of KRAS2 codon 12, since a previous study by our group had demonstrated that the neoplastic cells of UCOCGC often harbour KRAS2 gene mutations and that this mutant DNA can be phagocytosed by, and detected in, the non-neoplastic giant cells.11 A KRAS2 gene mutation was found (Figure 1C) and, in combination with the family history, it was interpreted as sufficient evidence to justify surgery. A pylorus preserving partial pancreaticoduodenectomy was performed and the surgical specimen grossly revealed a soft hemorrhagic neoplasm in the pancreatic head. The tumor measured 3cm in diameter with involvement of the ampulla (Figure 2A), adjacent to the distal common bile duct. On cut section the tumor was partially cystic and had necrotic areas (Figure 2B). The resection margins were free. Microscopically, the neoplasm had the typical features of an UCOCGC with anaplastic pleomorphic mononucleated cells and scattered osteoclast like multinucleated giant cells without atypia (Figure 2C). Focal ostechondroid differentiation was present. PanIN lesions of various degrees were seen, ranging from PanIN 1A-3. Mutation analysis of p16 performed on the neoplastic cells confirmed the germ line p16-Leiden deletion and demonstrated loss (LOH) of the remaining wild-type allele (Figure 2D). Immunolabeling for the p16 protein revealed that the neoplastic anaplastic cells were negative, while the non-neoplastic osteoclast-like giant cells were positive (Figure 2C, inset).
The patient had an uneventful recovery and was discharged from the hospital after 10 days.
Genomic DNA was isolated from formalin fixed, paraffin embedded tissue of the initial biopsy, the resected tumor and normal tissue respectively.
The DNA extracted from the biopsy was subjected to PCR amplification of the KRAS2 gene and subsequent sequence analysis for codon 12 mutations was performed as described elsewhere.5
The DNA extracted from the surgically resected tumor and normal tissue was PCR amplified for mutational analysis for the p16-Leiden deletion, as previously described.2 This deletion extends 19 bp and removes nucleotides at positions 225-243 of exon 2.2 Loss of heterozygosity (LOH) was defined as a decrease of at least 40% in the intensity of one allele in the tumor sample compared to the matched wild type allele in the normal tissue. The quotient of the peak height ratios of normal and tumor tissue was considered as indicative for LOH if <0.6 and >1.6 corresponding with 40% reduction of one allele.10 It was estimated that from the micro-dissected tumor tissue for LOH analysis 50-70% of the cells were tumor cells.
The FAMMM syndrome is characterized by the familial segregation of multiple melanomas and atypical precursor nevi and can be caused by germline mutations of the p16 tumor suppressor gene.2-4 Carriers of this germline mutation are not only at risk for melanomas and its precursors, but they also have a significantly increased risk of pancreatic carcinoma.3,4 The 20- to 34-fold higher risk of pancreatic cancer in FAMMM patients translates into a 10- 20% risk of developing pancreatic cancer by the age of 70 years. This is not surprising, since the large majority of sporadic (non-syndromic) pancreatic adenocarcinomas harbor acquired p16 gene mutations, leading to loss of function of this tumor suppressor.6,7,9,13 FAMMM family members with a specific 19 bp deletion of p16, the so called p16-Leiden deletion, have an estimated risk of almost 20% of developing pancreatic cancer by the age of 75 years.2 Pancreatic cancers that are encountered in association with the FAMMM syndrome are typically conventional ductal adenocarcinomas, although unusual cases of adenosquamous and neuroendocrine carcinoma have been reported.2,12 The UCOCGC of the pancreas is considered a rare variant of ductal adenocarcinomas, characterized by the presence of reactive multinucleated osteoclast like giant cells. Accumulating evidence suggests that UCOCGCs are undifferentiated ductal carcinomas arising from precursor ductal lesions.1 In addition to morphologic observations, this hypothesis is supported by immunolabeling for the p53 protein which reveals abnormal accumulation of the protein only in the undifferentiated cells. The osteoclast-like giant cells do not accumulate the p53 protein, suggesting that they are TP53 gene wild-type. Interestingly, we have previously shown that the non-neoplastic multinucleated osteoclast like giant cells can phagocytose degraded neoplastic cells, and, as a result, some of these non-neoplastic cells contain phagocytosed mutant KRAS2 genes. KRAS2 gene analysis was used in the current case to arrive at the proper diagnosis.11 It is at the same time yet another example of the great value of molecular DNA analysis as an adjunct to conventional microscopy.
To our knowledge this is the first case of UCOCGC of the pancreas in a patient from a family with FAMMM syndrome. DNA analysis of the neoplasm and normal tissue in the resection specimen confirmed the expected 19 bp deletion typical for the p16-Leiden deletion, accompanied by LOH of the remaining wild-type allele. Accordingly, p16 immunohistochemistry was negative in the neoplastic cells consistent with loss of function of the protein product. By contrast, the osteoclast-like giant cells in this case showed intact labelling for the p16 protein, further supporting the hypothesis that these are non-neoplastic reactive cells. The loss of the wild-type p16 allele in this case supports the contention that the UCOCGC of the pancreas in this patient was causally related to the underlying FAMMM syndrome and not a coincidence. Indirectly, the case therefore also shows that it appears indeed legitimate to consider UCOCGC of the pancreas as a variant of ductal adenocarcinoma.
Supported by the NIH SPORE (Specialized Programs of Research Excellence) in Gastrointestinal Cancer Grant CA62924 and The Dutch Cancer Foundation KWF (Koningin Wilhelmina Fonds).