American Journal of Surgical Pathology:
Carcinoid Tumors of the Extrahepatic Bile Ducts: A Study of Seven Cases
Maitra, Anirban M.D.; Krueger, Jo Ellen M.D.; Tascilar, Metin M.D.; Offerhaus, G. Johan A. M.D., Ph.D.; Angeles[ndash]Angeles, Arturo M.D.; Klimstra, David S. M.D.; Hruban, Ralph H. M.D.; Albores[ndash]Saavedra, Jorge M.D.
From the Department of Pathology (A.M., J.E.K., J.A.-S.), University of Texas Southwestern Medical Center, Dallas, Texas, U.S.A.; the Academic Medical Center (M.T., G.J.A.O.), Amsterdam, The Netherlands; the Instituto Nacional de la Nutricion Salvador[ndash]Zubiran (A.A.-A.), Tlalpan, Mexico; Memorial Sloan[ndash]Kettering Cancer Center (D.S.K.), New York, NY, U.S.A.; and the Johns Hopkins Medical Institution (R.H.H.), Baltimore, Maryland, U.S.A.
Address correspondence and reprint requests to Jorge Albores[ndash]Saavedra, MD, Department of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, U.S.A.; e-mail[colon] albores-saavedra.jorge[commat]pathology.swmed.edu
The authors report seven patients with carcinoid tumors of the extrahepatic bile ducts (EHBDs). All patients were women, with an average age at diagnosis of 49.8 years (range, 37[ndash]67 yrs). The most common presenting symptom was painless jaundice with or without pruritus. Although one patient had peptic ulcer disease before the onset of obstructive jaundice, none had systemic endocrine manifestations. These neoplasms were most often located in the common bile duct. Grossly, the carcinoid tumors were usually nodular and poorly demarcated, and ranged from 1.1 to 2.7 cm in size. Only one of the neoplasms was polypoid. Microscopically, the tumors had a trabecular or nesting pattern with occasional tubule formation, and were composed of relatively small cells with granular chromatin. All of the neoplasms expressed chromogranin and two expressed synaptophysin. Three expressed serotonin and two of the three were also immunoreactive for pancreatic polypeptide or somatostatin. Two tumors were focally positive for gastrin and one of these two tumors was also positive for serotonin and pancreatic polypeptide. All seven carcinoid tumors showed no immunoreactivity for p53, and assays for p53 loss of heterozygosity analysis were negative in two, suggesting that p53 mutations do not play a role in the pathogenesis of EHBD carcinoids. A mutation in codon 12 of K-ras was found in one carcinoid tumor whereas two of two showed immunoreactivity for Dpc4 protein. In view of the small number of carcinoids studied, the importance of these findings in the pathogenesis of these tumors is unclear. Ultrastructural examination of three of the tumors revealed numerous membrane-bound, round neurosecretory granules. Clinically, these lesions had an indolent course. Even in the presence of lymph node metastases (noted in two patients), all of the patients remained disease free 2 to 11 years (average follow up, 6.6 yrs) after segmental resection or pancreaticoduodenectomy (Whipple's procedure). Because carcinoid tumors of the EHBD are of low malignant potential, they should be separated from the more common adenocarcinomas in this location.
Carcinoid tumors of the extrahepatic bile ducts (EHBDs) are exceedingly rare, and account for 0.1[percnt] to 0.2[percnt] of all carcinoids of the gastrointestinal tract. 2 Given the rarity of these endocrine neoplasms, most of the cases published to date have been in the form of isolated case reports. 3[ndash]6,8[ndash]13,15[ndash]21,23[ndash]25,28[ndash]33,36,37,40,43[ndash]46,48[ndash]50,54,57 Therefore, information about the natural history, pathologic, immunohistochemical, and ultrastructural features of these neoplasms is limited. We report the clinical and pathologic features of seven patients with carcinoid tumors of the EHBDs, most with long-term follow up. Additionally, we describe the immunohistochemical characteristics, ultrastructural features, and molecular analysis of selected cases.
MATERIALS AND METHODS
Case Selection and Immunohistochemistry
The seven carcinoid tumors of the EHBDs were retrieved from the consultation files of one of the authors (J.A.-S.) and the surgical pathology archives of Memorial Sloan[ndash]Kettering Cancer Center, New York. Ampullary and duodenal carcinoids were excluded from the study. Two of the patients (patient nos. 1 and 3) have been reported previously, 4,31 but are included with extended follow up. Hematoxylin and eosin-stained sections were available for review in all cases, as were immunohistochemical stains using a panel of antibodies (Table 1). Clinical data and follow-up information were obtained from medical records and physicians responsible for patient care.
When possible, the original paraffin blocks were recut, and immunohistochemical stains using commercially available antibodies (Table 1) were performed on routinely processed, formalin-fixed, paraffin-embedded surgical material. An automated immunostainer (Ventana Biotek System, Tucson, AZ, USA) and the avidin[ndash]biotin[ndash]peroxidase complex technique were used. All cases examined were subjected to heat-induced epitope retrieval. Because of the limited availability of slides and blocks, not all antibodies were used in each case. Positive and negative controls were included with each assay.
For Dpc4 immunohistochemistry, a monoclonal antibody (clone B-8, Santa Cruz Biotechnology, CA, USA) was used at a concentration of 1[colon]100 in phosphate-buffered saline (PBS). For p16 immunohistochemistry, a monoclonal antibody (PharMingen International, San Diego, CA, USA) was used at a concentration of 1[colon]50 in PBS. For Dpc4 and p16, adjacent normal tissue in both cases served as the internal positive control.
Deoxyribonucleic Acid (DNA) Preparation for Genetic Analysis
From the formalin-fixed, paraffin-embedded tumor, 5-[mgr]m slides were cut and microdissected. At least 75[percnt] of the microdissected tissue consisted of neoplastic cells. The tissue was then incubated in proteinase K solution (100 [mgr]g/mL proteinase K, 50 mM Tris [lsqb]pH, 8.5[rsqb], 0.2[percnt] Tween-20, 1 mM ethylenediaminetetraacetic acid) overnight at 56[deg]C, after which the proteinase K solution was inactivated by heating the solution to 96[deg]C for 10 minutes.
Loss of Heterozygosity (LOH) and Microsatellite Instability (MI) Analyses
LOH and MI were analyzed using several highly polymorphic microsatellite markers (Table 2). Primer sequences are described in the Genome Database (http[colon]//http://www.gdb.org).
Forty cycles of polymerase chain reaction (PCR) were performed, consisting of 1 minute at 94[deg]C, 2 minutes at 55[deg]C, and 1 minute at 72[deg]C. The reaction volume was 20 [mgr]L, in which 40 ng of each primer (one of which was tagged with a fluorescent marker) was used, along with 0.2 mM dNTPs, 1.5 mM MgCl2, and 1.0 U Platinum Taq (BRL, Rockville, MD, USA) in the buffer supplied by the manufacturer. PCR was performed in a PTC100 cycler (MJ Research, Waltham, MA, USA), and the PCR products were then electrophoresed and analyzed using an automated ABI 377 sequencer and Genescan 2.1 software (Perkin[ndash]Elmer, Norwalk, CT, USA). Ratios of allelic peak heights, defined as the allelic imbalance factor (AIF), were calculated in the control and tumor samples. LOH was defined by an AIF for a specific marker higher than 1.7 or lower than 0.6. All AIFs meeting these criteria were repeated in independent experiments for confirmation.
K-ras Codon 12 Analysis
The protocol for K-ras codon 12 analysis has been described. 14 DNA is subjected to PCR amplification using primers centered around codon 12. One of the primers introduces a restriction site in the PCR products derived from wild-type codon 12 alleles but not in those derived from mutant codon 12 alleles. Digestion of the PCR products is followed by a second round of PCR amplification, which then yields a PCR product enriched for K-ras codon 12 mutations. The resulting DNA products are denatured, dot-blotted onto nylon membranes, and subjected to allele-specific oligonucleotide (ASO) hybridization with radioactive-labeled probes, specific for each possible K-ras codon 12 mutation, followed by autoradiography. Cell suspensions with mutant-to-wild-type ratios of 1[colon]100 and 1[colon]1000 were used as positive controls in every PCR. The suspensions were made of the human colon cancer cell line SW 480 (containing a homozygous GGT-to-GTT mutation at codon 12 of the K-ras) and the human colon cancer cell line HT 29 (with wild-type K-ras). We have previously validated the aforementioned mutant-enriched PCR with ASO hybridization through comparison with sequence analysis. 26
Fresh tissue from three carcinoid tumors was fixed in 3[percnt] glutaraldehyde. Thin sections were stained with lead citrate and uranyl acetate, and were examined with a JEOL 100 SX electron microscope.
The clinicopathologic features of the seven cases are summarized in Table 3. All patients reported in this study were women. The average age at diagnosis was 49.8 years (range, 37[ndash]67 yrs). The presenting complaint was, most commonly, painless jaundice with or without pruritus. One patient was diagnosed with peptic ulcer disease before the onset of obstructive jaundice, and was subsequently found to have a gastrin-secreting carcinoid of the common bile duct (CBD). One patient had a carcinoid tumor found incidentally during follow up for previous colon cancer. All of the patients are alive without evidence of recurrent or metastatic disease, with an average follow up period of 6.6 years (range, 2[ndash]11 yrs).
Four of the seven carcinoid tumors (57[percnt]) arose in the CBD, whereas the remaining three were located in the common hepatic duct (CHD). The neoplasms had an average size of 2.0 cm (range, 1.1[ndash]2.7 cm), and most often formed ill-defined submucosal nodules associated with thickening of the bile duct wall. Only one tumor (patient no. 2) appeared as a well-defined polypoid mass that projected into the bile duct lumen.
Microscopic examination of the neoplasms revealed a submucosal proliferation of cells characterized by granular or clear cytoplasm, round to ovoid nuclei with small inconspicuous nucleoli, granular chromatin, minimal pleomorphism, and rare mitotic figures (one to two mitotic figures per 10 high-power fields). The cells grew in nests, clusters, or trabeculae, occasionally forming tubular structures (Figs. 1[ndash]5). The cell nests infiltrated the intramural glands and in two carcinoid tumors some of these glands were replaced by neoplastic endocrine cells (Fig. 6). The intervening stroma ranged from highly vascular loose connective tissue to dense, acellular hyalinized collagen. One case demonstrated stromal amyloid deposits, whereas another case had focal calcifications. Microscopic foci of hemorrhage and necrosis were identified in the central portion of only one tumor (patient no. 2). All seven tumors infiltrated the wall of the CBD or CHD, and two extended into the periductal adipose tissue. Perineural invasion (n [equals] 4) and lymphovascular invasion (n [equals] 2) were identified (Fig. 7). Metastatic carcinoid tumor was found in the portal lymph nodes of two cases. No involvement of hepatic or pancreatic parenchyma was noted in any of the cases examined.
Immunohistochemical results are summarized in Tables 4 and 5. Chromogranin reactivity was diffusely and strongly positive in all seven cases, whereas synaptophysin expression was noted in two cases (Fig. 8). Serotonin was identified in three cases (43[percnt]), gastrin in two cases (14[percnt]), and insulin and glucagon were uniformly negative in all seven tumors (Figs. 9 and 10). Two cases labeled for pancreatic polypeptide and two stained for somatostatin. None of the carcinoid tumors expressed p53 protein at immunohistochemically detectable levels. Additional immunohistochemical stains performed, as listed in Table 5, included cytokeratin (positive in four of four cases), epithelial membrane antigen (positive in one of one case), and MIB-1, which showed rare, scattered reactivity (patient no. 6). Loss of p16 expression was seen in patient no. 6, whereas p16 expression was retained in patient no. 7. Dpc4 immunoreactivity was present in both cases examined (patient nos. 6 and 7).
Ultrastructural examination of three of the neoplasms revealed numerous cytoplasmic, membrane-bound, dense-core neurosecretory granules measuring approximately 200 to 600 [mgr]m in diameter.
LOH, MI, and K-ras Mutation Analysis
In the two tumors analyzed (patient nos. 6 and 7), no LOH was detected at D18S64, D9S171, PYGM, or TP53. Both tumors were noninformative at the p53-Alu marker, and no MI was detected in either case by BAT26 assay. The tumor from patient no. 6 demonstrated wild-type K-ras, whereas patient no. 7 had a codon 12 mutation (GGT[rarr]GAT transition) in exon 1 of the K-ras oncogene.
Approximately 98[percnt] of all malignant neoplasms arising in the EHBDs are carcinomas, predominantly well-to moderately differentiated adenocarcinomas. 1,2 Carcinoids of the EHBDs are exceedingly rare, representing only 0.2[percnt] to 0.3[percnt] of tumors at this site. 2 Because so few cases of EHBD carcinoids have been reported, little is known about the clinical features and natural history of these neoplasms. Nevertheless, certain distinguishing clinicopathologic characteristics of EHBD carcinoids have been identified.
Both adenocarcinomas and carcinoids may have similar presenting clinical features, including painless jaundice with or without pruritus. However, whereas adenocarcinomas of the EHBDs have a slight male predilection and typically present in the seventh or eighth decades of life, 1 carcinoid tumors in this location are nearly twice as common in women, and present at a younger age. In addition, although adenocarcinomas occur with increased frequency in patients with inflammatory bowel disease and primary sclerosing cholangitis, carcinoids have rarely been reported to occur in association with multiple endocrine neoplasia type 1 (MEN-1) and the von Hippel[ndash]Lindau syndromes. 16 Unlike carcinoid tumors in some other locations, carcinoid tumors of the EHBDs do not give rise to systemic manifestations. Lastly, the prognosis for these tumors is markedly different[colon] Although adenocarcinomas are aggressive tumors and are almost always fatal, carcinoid tumors are far more indolent. Indeed, patients with carcinoid tumors of the EHBDs have long-term life expectancy, even in the face of hepatic metastases. 15
Our series of seven patients supports the clinicopathologic features described earlier[colon] All of the patients were women, and the average age at presentation (49.8 yrs) was nearly a decade younger than that typical for adenocarcinomas of the EHBDs. The most common presenting complaint was painless jaundice with or without pruritus. Although one of the seven patients had persistent peptic ulcer disease in association with her gastrin-secreting carcinoid, there was no evidence of Zollinger[ndash]Ellison syndrome. None of the patients had inflammatory bowel disease, primary sclerosing cholangitis, the MEN-1 syndrome, or von Hippel[ndash]Lindau syndrome.
The carcinoid tumors in our patients were most often located in the CBD (57[percnt]), with the remaining tumors arising in the CHD. None of the neoplasms in this series occurred in the cystic duct, although this site accounts for seven cases reported in the literature. 13,17,21,24,33,43,46 At the time of surgery, the neoplasms were locally invasive but did not involve the liver or the pancreas. Although two patients showed lymph node metastases, none had distant metastatic disease. Tumor size did not correlate with the development of lymph node metastasis. A 5-mm carcinoid tumor of the cystic duct that metastasized has been reported. 24 All seven patients are alive and disease free after curative surgical resections, with an average follow-up period of 6.6 years (range, 2[ndash]11 yrs).
Histologic features of EHBD carcinoids are similar to those of intestinal carcinoids. They tend to grow in cords, nests, or trabeculae, and usually, as seen in our patients, invade the ductal wall (100[percnt]). Moreover, perineural (57[percnt]) and lymphovascular (29[percnt]) invasion is common.
In view that most carcinoid tumors of the EHBDs often secrete more than one hormone 23 or are immunoreactive only for chromogranin, functional classification of carcinoid tumors by immunohistochemistry is of little value.
The differential diagnosis for EHBD carcinoids principally includes poorly differentiated adenocarcinomas with numerous endocrine cells and small cell carcinomas. Because as many as one third of adenocarcinomas in this location demonstrate increased numbers of endocrine cells, immunohistochemical reactivity for chromogranin and synaptophysin in an EHBD lesion should not cause an erroneous diagnosis of carcinoid[colon] Such mistakes have been made in the past with adenocarcinomas of the gallbladder, 55 which show extensive gland formation and greater cytologic atypia. In addition, diffuse and strong positivity for chromogranin and synaptophysin in all of the tumor cells favors a diagnosis of carcinoid. Conversely, focal, scattered positivity is seen with adenocarcinoma. By the same token, small cell carcinomas may occasionally demonstrate a trabecular growth pattern with rosette formation, leading to diagnostic confusion with carcinoid tumors. However, a diffuse chromatin pattern, a high mitotic rate, extensive tumor necrosis, and only focal positivity for chromogranin and synaptophysin favor a diagnosis of small cell carcinoma.
As manifested by their clinicopathologic characteristics, carcinoid tumors of the EHBDs appear to have fundamentally different biologic features from adenocarcinomas in this location. Not only do the EHBD carcinoids demonstrate limited propensity for growth and metastatic spread, but patient survival is also far greater for these neoplasms than for biliary adenocarcinomas. We would suspect, therefore, that carcinoids would have different molecular changes than adenocarcinomas. Contrary to our expectations, however, one of the two carcinoid tumors examined molecularly contained an activating G-A mutation in codon 12 of the K-ras oncogene, which is the most common mutation seen in biliary adenocarcinomas (50[percnt][ndash]75[percnt] of tumors, depending on the series). 27,39,52 In contrast to our findings in this series, K-ras mutations have not been detected in typical carcinoids arising in the duodenum, appendix, lung, and cervix. 34,35,51,53,58 It is worth noting that, as is noted in adenocarcinomas of the EHBDs, 39 the presence of the K-ras mutation in carcinoids does not appear to influence the clinical course, because the patient in whom the mutation was detected remains alive and disease free 24 months after surgery. Although we were limited by the paucity of archival material available for mutational analysis, these findings suggest that critical genetic alterations contributing to the aggressive clinical course in adenocarcinomas (compared with carcinoid tumors) do not lie in the K-ras oncogene. Additional studies are warranted, however, before definitive conclusions may be drawn.
In contrast to biliary adenocarcinomas, which demonstrate p53 mutations in as many as 45[percnt] of cases, 38,47 assays for p53 mutations by LOH analysis and immunohistochemistry in two and seven carcinoid tumors respectively were negative. These findings support similar studies of typical carcinoids arising in other sites, in which only higher-grade neuroendocrine tumors have demonstrated p53 mutations. 34,53 Despite the small number of cases analyzed, these findings suggest that p53 mutations do not play a role in the pathogenesis of biliary carcinoids. Whether the absence of p53 abnormalities carries prognostic significance is a matter of speculation.
Loss of expression of wild-type p16 was present in one of the carcinoid tumors. p16 functions at the G1 cell cycle checkpoint, and the P16 gene has been found to be inactivated by mutations, methylation, or deletion in a large number of epithelial malignancies, 41 including bile duct adenocarcinomas. 56 Loss of p16 expression in an EHBD carcinoid suggests that alterations in this gene may be involved in the genesis of some carcinoid tumors at this site. Interestingly, no concurrent LOH at the P16 locus (D9S171) could be demonstrated in this neoplasm, raising the possibility that epigenetic mechanisms, such as promoter hypermethylation, may be causing the decreased expression of this gene. 7
Dpc4 is a newly described protein that is important in the signaling pathway for transforming growth factor beta. 22 Mutations of this DPC4 gene have been identified in a majority of pancreatic adenocarcinomas. 42 We found persistent immunoreactivity for Dpc4 in both of the carcinoid tumors examined, and LOH at chromosome 18q21.1 was not seen. The importance of this finding is unclear in the absence of a larger series of patients, but it suggests that DPC4 alterations may not be critical in the pathogenesis of biliary tract carcinoids.
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Carcinoid tumors; Extrahepatic bile ducts; Immunohistochemistry; Electron microscopy; p53; k-ras genes
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