Genetic, Immunohistochemical, and Clinical Features of Medullary Carcinoma of the Pancreas
2000; Elsevier BV; Volume: 156; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)65035-3
ISSN1525-2191
AutoresRobb E. Wilentz, Michael Goggins, Mark Redston, Victoria Marcus, Volkan Adsay, Taylor A. Sohn, Shrihari S. Kadkol, Charles J. Yeo, Michael A. Choti, Marianna Zahurak, Karen A. Johnson, Metin Tascilar, G.J.A. Offerhaus, Ralph H. Hruban, Scott E. Kern,
Tópico(s)Cholangiocarcinoma and Gallbladder Cancer Studies
ResumoMedullary carcinomas of the pancreas are a recently described, histologically distinct subset of poorly differentiated adenocarcinomas that may have a unique pathogenesis and clinical course. To further evaluate these neoplasms, we studied genetic, pathological, and clinical features of 13 newly identified medullary carcinomas of the pancreas. Nine (69%. of these had wild-type K-ras genes, and one had microsatellite instability (MSI). This MSI medullary carcinoma, along with three previously reported MSI medullary carcinomas, were examined immunohistochemically for Mlh1 and Msh2 expression, and all four expressed Msh2 but did not express Mlh1. In contrast, all of the medullary carcinomas without MSI expressed both Msh2 and Mlh1. Remarkably, the MSI medullary carcinoma of the pancreas in the present series arose in a patient with a synchronous but histologically distinct cecal carcinoma that also had MSI and did not express Mlh1. The synchronous occurrence of two MSI carcinomas suggests an inherited basis for the development of these carcinomas. Indeed, the medullary phenotype, irrespective of MSI, was highly associated with a family history of cancer in first-degree relatives (P < 0.001). Finally, one medullary carcinoma with lymphoepithelioma-like features contained Epstein-Barr virus-encoded RNA-1 by in situ hybridization. Therefore, because of medullary carcinoma's special genetic, immunohistochemical, and clinical features, recognition of the medullary variant of pancreatic adenocarcinoma is important. Only by classifying medullary carcinoma as special subset of adenocarcinoma can we hope to further elucidate its unique pathogenesis. Medullary carcinomas of the pancreas are a recently described, histologically distinct subset of poorly differentiated adenocarcinomas that may have a unique pathogenesis and clinical course. To further evaluate these neoplasms, we studied genetic, pathological, and clinical features of 13 newly identified medullary carcinomas of the pancreas. Nine (69%. of these had wild-type K-ras genes, and one had microsatellite instability (MSI). This MSI medullary carcinoma, along with three previously reported MSI medullary carcinomas, were examined immunohistochemically for Mlh1 and Msh2 expression, and all four expressed Msh2 but did not express Mlh1. In contrast, all of the medullary carcinomas without MSI expressed both Msh2 and Mlh1. Remarkably, the MSI medullary carcinoma of the pancreas in the present series arose in a patient with a synchronous but histologically distinct cecal carcinoma that also had MSI and did not express Mlh1. The synchronous occurrence of two MSI carcinomas suggests an inherited basis for the development of these carcinomas. Indeed, the medullary phenotype, irrespective of MSI, was highly associated with a family history of cancer in first-degree relatives (P < 0.001). Finally, one medullary carcinoma with lymphoepithelioma-like features contained Epstein-Barr virus-encoded RNA-1 by in situ hybridization. Therefore, because of medullary carcinoma's special genetic, immunohistochemical, and clinical features, recognition of the medullary variant of pancreatic adenocarcinoma is important. Only by classifying medullary carcinoma as special subset of adenocarcinoma can we hope to further elucidate its unique pathogenesis. Most, but not all, poorly differentiated carcinomas arising in the pancreas are conventional ductal adenocarcinomas.1Hruban RH Wilentz RE The pancreas.in: Weidner N Cote RJ Suster S Weiss LM Modern Surgical Pathology. Saunders, Philadelphia2000Google Scholar, 2Solcia E Capella C Klöppel G Tumors of the pancreas.Atlas of Tumor Pathology, Third Series, Fasicle 20. Armed Forces Institute of Pathology, Washington, DC1997Google Scholar, 3Cubilla A Fitzgerald PJ Morphological patterns of primary nonendocrine human pancreas carcinoma.Cancer Res. 1975; 35: 2234-2248PubMed Google Scholar Some poorly differentiated pancreatic carcinomas, however, are medullary carcinomas.4Goggins M Offerhaus GJ Hilgers W Griffin CA Shekher M Tang D Sohn TA Yeo CJ Kern SE Hruban RH Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar Although historically grouped with poorly differentiated ductal adenocarcinomas, medullary carcinomas are histologically distinct. As first described in 1998, they have poorly defined cellular boundaries (syncytial growth pattern), expanding tumor borders, and extensive necrosis, features similar to their histological counterparts arising in the colorectum (Figure 1).4Goggins M Offerhaus GJ Hilgers W Griffin CA Shekher M Tang D Sohn TA Yeo CJ Kern SE Hruban RH Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar, 5Lynch HT Smyrk TC Lynch JF Overview of natural history, pathology, molecular genetics, and management of HNPCC (Lynch syndrome).Int J Cancer. 1996; 69: 38-43Crossref PubMed Scopus (187) Google Scholar, 6Lothe RA Peltomaki P Meling GI Aaltonen LA Nystrom-Lahti M Pylkkanen L Heimdai K Andersen TI Moller P Rognum TO Fossa SD Haldorsen TI Langmark F Brogger A de la Chapelle A Borresen A-L Genomic instability in colorectal cancer: relationship of clinicopathological variables and family history.Cancer Res. 1993; 53: 5849-5852PubMed Google Scholar, 7Jass JR Smyrk TC Stewart SM Lane MR Lanspa SJ Lynch HT Pathology of hereditary non-polyposis colon cancer.Anticancer Res. 1994; 14: 1631-1634PubMed Google Scholar, 8Graham DM Appelman HD Crohn's-like lymphoid reaction and colorectal carcinoma: a potential histologic prognosticator.Mod Pathol. 1990; 3: 332-335PubMed Google Scholar Medullary carcinomas may also be genetically distinct from conventional ductal adenocarcinomas of the pancreas. Indeed, of five medullary carcinomas first described by Goggins et al,4Goggins M Offerhaus GJ Hilgers W Griffin CA Shekher M Tang D Sohn TA Yeo CJ Kern SE Hruban RH Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar three (60%. had both microsatellite instability (MSI) and a wild-type K-ras gene. (One of the six medullary carcinomas reported by Goggins et al4Goggins M Offerhaus GJ Hilgers W Griffin CA Shekher M Tang D Sohn TA Yeo CJ Kern SE Hruban RH Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar was later determined to be a metastatic renal cell carcinoma.) These data are atypical for nonmedullary ductal adenocarcinomas of the pancreas, which nearly universally harbor K-ras gene mutations and seldom if ever have MSI.4Goggins M Offerhaus GJ Hilgers W Griffin CA Shekher M Tang D Sohn TA Yeo CJ Kern SE Hruban RH Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar, 9Hruban RH van Mansfeld ADM Offerhaus GJ van Weering DHJ Allison DC Goodman SN Kensler TW Bose KK Cameron JL Bos JL K-ras oncogene activation in adenocarcinoma of the human pancreas: a study of 82 carcinomas using a combination of mutant-enriched polymerase chain reaction analysis and allele-specific oligonucleotide hybridization.Am J Pathol. 1993; 143: 545-554PubMed Google Scholar, 10Almoguera C Shibata D Forrester K Martin J Arnheim N Perucho M Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes.Cell. 1998; 53: 549-554Abstract Full Text PDF Scopus (1918) Google Scholar, 11Smit VT Boot AJM Smits AMM Fleuren GJ Cornelisse CJ Bos JL K-ras codon 12 mutations occur very frequently in pancreatic adenocarcinomas.Nucleic Acids Res. 1988; 16: 7773-7782Crossref PubMed Scopus (544) Google Scholar, 12Barbacid M Ras genes.Annu Rev Biochem. 1987; 56: 779-827Crossref PubMed Scopus (3787) Google Scholar, 13Grünewald K Lyons J Frohlich A Feichtinger H Weger RA Schwab G Janssen JW Bartram CR High frequency of Ki-ras codon 12 mutations in pancreatic adenocarcinomas.Int J Cancer. 1989; 43: 1037-1041Crossref PubMed Scopus (309) Google Scholar Although these preliminary findings favor a distinct pathogenetic course for medullary pancreatic carcinomas, the small number of cases studied so far has required a cautious view. To further elucidate the clinical and genetic features associated with the medullary morphology, we studied a larger series of medullary pancreatic carcinomas. Specifically, our goals were to determine 1) the frequency of wild-type K-ras genes and MSI among medullary carcinomas; 2) whether immunohistochemistry for the DNA-repair gene products Mlh1 and Msh2 could identify the medullary carcinomas with MSI; and 3) whether the medullary carcinomas were associated with any other immunohistochemical, pathological, or clinical variables, including latent Epstein-Barr (EBV) infection or family history of cancer. Answering these three questions would help us to gauge the utility of classifying medullary carcinomas as a special subset of ductal adenocarcinomas of the pancreas. The surgical pathology files of The Johns Hopkins Hospital and the Academic Medical Center (University of Amsterdam) were screened for pancreatic carcinomas with the medullary histological pattern.4Goggins M Offerhaus GJ Hilgers W Griffin CA Shekher M Tang D Sohn TA Yeo CJ Kern SE Hruban RH Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar A histological review of 450 randomly chosen pancreatic cancers revealed 18 (4.0%) possible medullary carcinomas, each originally diagnosed as a poorly differentiated ductal adenocarcinoma. Five of these cases were those originally identified by Goggins et al.4Goggins M Offerhaus GJ Hilgers W Griffin CA Shekher M Tang D Sohn TA Yeo CJ Kern SE Hruban RH Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar One of the medullary carcinomas also contained adenosquamous carcinoma comprising approximately half of the neoplasm. All 17 of the remaining medullary carcinomas were pure medullary carcinomas. The 18 medullary carcinomas were then pooled by a reference pathologist (REW) with 17 other (nonmedullary) pancreatic carcinomas. The 17 nonmedullary carcinomas included 10 poorly differentiated ductal adenocarcinomas, four moderately differentiated ductal adenocarcinomas, two mucinous adenocarcinomas, and one adenosquamous carcinoma. One slide from each case was coded, and the set of slides was reviewed by three other pathologists (GJAO, RHH, and NVA), one of whom (NVA) was from an institution not associated with our original study on medullary carcinomas.4Goggins M Offerhaus GJ Hilgers W Griffin CA Shekher M Tang D Sohn TA Yeo CJ Kern SE Hruban RH Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar Each pathologist graded the cases according to criteria originally proposed by Goggins et al4Goggins M Offerhaus GJ Hilgers W Griffin CA Shekher M Tang D Sohn TA Yeo CJ Kern SE Hruban RH Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar (gland formation, invasion pattern, syncytial growth pattern, necrosis). Each neoplasm then received an overall grade from 1 (most similar) to 5 (least similar), based on its similarity to the index medullary carcinoma case identified in the original study by Goggins et al.4Goggins M Offerhaus GJ Hilgers W Griffin CA Shekher M Tang D Sohn TA Yeo CJ Kern SE Hruban RH Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar A neoplasm was considered a medullary carcinoma only when it met the following two criteria: first, an average grade less than 3; and second, at least two of the three pathologists providing a grade of 2 or lower. The three pathologists were said to disagree when any of the pathologists gave a score that was more than one point away from either of the other two pathologists. When available, sections containing nonneoplastic pancreatic tissue surrounding each medullary carcinoma were histologically analyzed by the reference pathologist (REW) for associated pancreatic intraepithelial neoplasias (PanINs), the putative precursors of pancreatic ductal adenocarcinoma.14Hruban RH Wilentz RE Goggins M Offerhaus GJA Yeo CJ Kern SE Pathology of incipient pancreatic cancer.Ann Oncol. 1999; 10: 9-11Abstract Full Text PDF PubMed Scopus (73) Google Scholar, 15Wilentz RE Geradts J Maynard R Offerhaus GJA Kang M Goggins M Yeo CJ Kern SE Hruban RH Inactivation of the p16 (INK4A) tumor-suppressor gene in pancreatic duct lesions: loss of intranuclear expression.Cancer Res. 1998; 58: 4740-4744PubMed Google Scholar, 16Wilentz RE, Iacobuzio-Donahue CA, Argani P, McCarthy DM, Parsons JL, Yeo CJ, Kern SE, Hruban RH: Loss of expression of Dpc4 in pancreatic intraepithelial neoplasia (PanIN): evidence that DPC4 inactivation occurs late in neoplastic progression. Cancer Res (in press) 2000Google Scholar PanINs were identified and graded according to accepted criteria established at a National Cancer Institute-sponsored Pancreas Cancer Think Tank, held September, 1999, in Park City, Utah. These criteria are available on the Worldwide Web (http://pathology.jhu.edu/pancreas/panin). Because they share some features with EBV-containing lymphoepitheliomas of the nasopharynx, all 18 medullary carcinomas were assayed by in situ hybridization for Epstein-Barr virus-encoded RNA-1. In addition, five of the 17 nonmedullary carcinomas were also studied for latent EBV infection. These five nonmedullary carcinomas included two poorly differentiated conventional adenocarcinomas, one moderately differentiated conventional adenocarcinoma, and two mucinous adenocarcinomas. Formalin-fixed, paraffin-embedded tissue sections were deparaffinized and hydrated in graded ethanol. Sections were washed in water, digested with 10 μg/ml of proteinase K in 50 mmol/L Tris-HCl (pH 7.5) for 30 minutes at 37°C, and washed again in water. Sections were hybridized at 37°C for 16 to 18 hours with 20 μl of fluorescein isothiocyanate-labeled Epstein-Barr virus-encoded RNA riboprobe in hybridization solution (Novocastra, Newcastle on Tyne, UK). A parallel section was hybridized with a negative control probe. The sections were washed in Tris-buffered saline (pH 8.0) with 0.05% Tween 20 (TBS-T. Sigma Chemical Co., St. Louis, MO) and then immersed in 0.2× sodium chloride-sodium citrate/0.1% sodium dodecyl sulfate solution at 52°C for 10 minutes. Sections were incubated in 10% normal rabbit serum for 30 minutes and then in a 1:200 dilution of anti-fluorescein isothiocyanate-alkaline phosphatase conjugate (In Situ Hybridization Detection Kit, Novocastra) in Tris-buffered saline (pH 8.0) with 0.05. Tween 20 for 30 minutes. After three washes, sections were placed in 1× alkaline-phosphatase buffer (100 mmol/L Tris-HCl, pH 9.5, 100 mmol/L NaCl, 50 mmol/L MgCl2) for 5 minutes. Chromogenic detection was then performed with nitroblue tetrazolium and X-phosphate. Adequate color development occurred within 2 hours of incubation in the chromogen solution. The slides were rinsed in water, counterstained with hematoxylin, and mounted with aqueous mounting medium (GlycerGel; DAKO, Carpinteria, CA). An intense bluish-purple color indicated a positive hybridization signal. The five medullary carcinomas originally identified by Goggins et al4Goggins M Offerhaus GJ Hilgers W Griffin CA Shekher M Tang D Sohn TA Yeo CJ Kern SE Hruban RH Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar had already been analyzed genetically. Therefore, 13 genetically unanalyzed medullary carcinomas remained. We also genetically analyzed five of the 17 nonmedullary pancreatic carcinomas with the same technique to ensure a sensitivity adequate to detect single base-pair genetic alterations. These five nonmedullary carcinomas included those also studied for EBV latent infection by in situ hybridization. Therefore, we microdissected and genetically analyzed 18 carcinomas (13 medullary and five nonmedullary. in this study. Each of the 18 carcinomas to be genetically analyzed were microdissected from unstained 10-μm sections cut from archived paraffin blocks. The microdissected tissue was deparaffinized, digested with proteinase K, purified by phenol-chloroform extraction, and precipitated, as previously described.9Hruban RH van Mansfeld ADM Offerhaus GJ van Weering DHJ Allison DC Goodman SN Kensler TW Bose KK Cameron JL Bos JL K-ras oncogene activation in adenocarcinoma of the human pancreas: a study of 82 carcinomas using a combination of mutant-enriched polymerase chain reaction analysis and allele-specific oligonucleotide hybridization.Am J Pathol. 1993; 143: 545-554PubMed Google Scholar, 17Slebos RJC Boerrigter L Evers SG Wisman P Mooi WJ Rodenhuis S A rapid and simple procedure for the detection of ras mutations in formalin-fixed, paraffin-embedded tissue.Diagn Mol Pathol. 1992; 1: 136-141Crossref PubMed Google Scholar The final product was reconstituted in 50 μl LoTE (3 mmol/L Tris, 0.1 mmol/L ethylenediaminetetraacetic acid, pH 7.5) and stored at −20°C. Because of the solid growth pattern of medullary carcinomas and microdissection with avoidance of lymphocytes, relatively pure samples of carcinoma were obtained. Samples containing at least 200 neoplastic cells were used for each polymerase chain reaction (PCR) below. Both the medullary and adenosquamous components of the mixed medullary carcinoma were separately microdissected. Portions of exons 1 and 2 (encompassing codons 12, 13, and 61) of the K-ras gene (KRAS2) were amplified by PCR, as previously described.9Hruban RH van Mansfeld ADM Offerhaus GJ van Weering DHJ Allison DC Goodman SN Kensler TW Bose KK Cameron JL Bos JL K-ras oncogene activation in adenocarcinoma of the human pancreas: a study of 82 carcinomas using a combination of mutant-enriched polymerase chain reaction analysis and allele-specific oligonucleotide hybridization.Am J Pathol. 1993; 143: 545-554PubMed Google Scholar Direct cycle sequencing (SequiTherm Excel II, Epicentre Technologies, Madison, WI) was performed using internal primers at an annealing temperature of 60°C. Products were separated on a 6% polyacrylamide gel and subjected to autoradiography. Unusual K-ras genotypes were confirmed by direct sequencing of an independent PCR product and by sequencing of cloned PCR products (TOPO TA, Invitrogen, Carlsbad, CA). The N- and H-ras genes were not studied in this series, as we found previously that mutations in these two genes do not occur in pancreatic carcinomas, including medullary carcinomas.18Wilentz RE Kern SE No H-ras gene mutations in pancreatic adenocarcinomas.NOGO. 1999; 3: 22Google Scholar, 19Wilentz RE Kern SE No N-ras gene mutations in pancreatic adenocarcinomas.NOGO. 1999; 3: 23Google Scholar Evidence of MSI was sought in each of the 18 microdissected samples by: 1) length analysis of BAT 25 and BAT 26 markers; and by 2) direct sequencing of the polythymidine tract of the TGFBR2 gene.4Goggins M Offerhaus GJ Hilgers W Griffin CA Shekher M Tang D Sohn TA Yeo CJ Kern SE Hruban RH Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar, 20Hahn SA Seymour AB Hoque ATMS Schutte M da Costa LT Redston MS Caldas C Weinstein CL Fischer A Yeo CJ Hruban RH Kern SE Allelotype of pancreatic carcinoma using xenograft enrichment.Cancer Res. 1995; 55: 4670-4675PubMed Google Scholar, 21Goggins M Shekher M Turnacioglu K Yeo CJ Hruban RH Kern SE Genetic alterations of the transforming growth factor beta receptor genes in pancreatic and biliary adenocarcinomas.Cancer Res. 1998; 58: 5329-5332PubMed Google Scholar, 22Boland CR Thibodeau SN Hamilton SR Sidransky D Eshleman JR Burt RW Meltzer SJ Rodriguez-Bigas MA Fodde R Ranzani GN Srivastava S A National Cancer Institute workshop on microsatellite instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer.Cancer Res. 1998; 58: 5248-5257PubMed Google Scholar BAT 25 and BAT 26 sequences were amplified by PCR incorporating [α]-32P-dCTP, and the products were resolved with 6% denaturing polyacrylamide gels, as previously described.4Goggins M Offerhaus GJ Hilgers W Griffin CA Shekher M Tang D Sohn TA Yeo CJ Kern SE Hruban RH Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar A portion of the TGFBR2 gene was amplified by PCR, and the products were cycle sequenced and similarly resolved.4Goggins M Offerhaus GJ Hilgers W Griffin CA Shekher M Tang D Sohn TA Yeo CJ Kern SE Hruban RH Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar The three MSI carcinomas from the previous study of Goggins et al4Goggins M Offerhaus GJ Hilgers W Griffin CA Shekher M Tang D Sohn TA Yeo CJ Kern SE Hruban RH Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar were used as positive controls. For each of these three loci, gain or loss of at least one nucleotide was regarded as a shift. Each carcinoma tested was found to have either shifts in at least two of the three loci (designated MSI), or shifts at no locus (designated microsatellite stable, MSS). Because carcinomas lacking a mononucleotide shift in at least one of these three markers generally do not have shifts in additional dinucleotide markers, additional markers were not used.4Goggins M Offerhaus GJ Hilgers W Griffin CA Shekher M Tang D Sohn TA Yeo CJ Kern SE Hruban RH Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar, 23Perucho M Correspondence.Cancer Res. 1999; 59: 249-255PubMed Google Scholar Immunohistochemical labeling for the products of the MLH1 and MSH2 genes was performed on the current series of 13 medullary and five nonmedullary carcinomas, as well as on three MSI medullary carcinomas identified in the previous series collected by Goggins et al.4Goggins M Offerhaus GJ Hilgers W Griffin CA Shekher M Tang D Sohn TA Yeo CJ Kern SE Hruban RH Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar Unstained 6-μm sections were deparaffinized and rehydrated with xylene and graded alcohols. The slides were treated with sodium citrate buffer (10 mmol/L, pH 6.0) and then submitted to microwave antigen retrieval. Endogenous peroxidase was blocked by 20% hydrogen peroxide, and nonspecific binding was blocked by 20% Protein Blocker (Signet Laboratories, Dedham, MA) in buffered saline. After cooling for 5 minutes, the slides were labeled with mouse monoclonal antibodies to Mlh1 (1:50 dilution, clone G168–728; PharMingen, San Diego, CA) or to Msh2 (1:100 dilution, clone FE11; Oncogene Research Products, Cambridge, MA) and incubated overnight at room temperature. Each primary antibody was detected by biotinylated secondary antibodies, avidin-biotin complex, and 3,3′-diaminobenzidine. Sections were counterstained with hematoxylin.24Marcus VA Madlensky L Gryfe R et al.Immunohistochemistry for hMLH1 and hMLH2: a practical test for DNA mismatch repair deficient tumors.Am J Surg Pathol. 1999; 23: 1248-1255Crossref PubMed Scopus (248) Google Scholar Cancers interpreted to lack Mlh1 or Msh2 expression required a complete absence of labeling in the nuclei of neoplastic cells. Labeling of nonneoplastic epithelium, stromal cells, or lymphocytes served as an internal positive control in each of the sections. One patient in this study had synchronous pancreatic and colonic carcinomas. This patient's pancreatic carcinoma had a medullary phenotype with microglandular features and MSI, and his colonic carcinoma was an adenocarcinoma that also had MSI. In addition, as will be discussed later, neither carcinoma had immunodetectable MLH1 gene product. Therefore, informed consent was obtained for germline MLH1 analysis under a protocol approved by the institutional review board of The Johns Hopkins Hospital. Conformation-sensitive gel electrophoresis was performed under clinical laboratory conditions at the University of Pennsylvania (Philadelphia, PA).25Ganguly A Williams C Detection of mutations in multi-exon genes: comparison of conformation sensitive gel electrophoresis and sequencing strategies with respect to cost and time for finding mutations.Hum Mutat. 1997; 9: 339-343Crossref PubMed Scopus (23) Google Scholar, 26Ganguly A Prockop DJ Detection of mismatched bases in double stranded DNA by gel electrophoresis.Electrophoresis. 1995; 16: 1830-1835Crossref PubMed Scopus (32) Google Scholar, 27Ganguly A Rock MJ Prockop DJ Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments: evidence for solvent-induced bends in DNA heteroduplexes.Proc Natl Acad Sci USA. 1993; 90: 10325-10329Crossref PubMed Scopus (619) Google Scholar, 28Korkko J Annunen S Pihlajamaa T Prockop DJ Ala-Kokko L Conformation sensitive gel electrophoresis for simple and accurate detection of mutations: comparison with denaturing gradient gel electrophoresis and nucleotide sequencing.Proc Natl Acad Sci USA. 1998; 95: 1681-1685Crossref PubMed Scopus (187) Google Scholar The results of the analysis were discussed with the patient, and he is currently undergoing genetic counseling. Clinical and pathological data for each of the patients were obtained from various sources, including the medical records of The Johns Hopkins Hospital and the Academic Medical Center, the surgical pathology databases of The Johns Hopkins Hospital and Academic Medical Center, and The Johns Hopkins Oncology Center's clinical information system. Specifically, family history was obtained from extensive review of these sources. Fourteen (78%) of the 18 patients with medullary carcinomas and 69 (90%) of the 77 patients with nonmedullary carcinomas had familial cancer pedigrees available. Data collected included tumor size, presence of lymph node metastases at presentation, age, gender, race, smoking history, alcohol history, comorbidities, presenting symptoms, and family history of cancer in a first-degree relative. Comorbidities included a history of myocardial infarction, peptic ulcer disease, peripheral vascular disease, hypertension, chronic or acute pancreatitis, and inflammatory bowel disease. Presenting symptoms included weight loss, abdominal pain, jaundice, nausea/vomiting, and fever/chills. Data from the 18 microdissected tumors first described in this study and 77 xenografted tumors, many previously studied by Goggins et al4Goggins M Offerhaus GJ Hilgers W Griffin CA Shekher M Tang D Sohn TA Yeo CJ Kern SE Hruban RH Pancreatic adenocarcinomas with DNA replication errors (RER+) are associated with wild-type K-ras and characteristic histopathology. Poor differentiation, a syncytial growth pattern, and pushing borders suggest RER+.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar and by others in our laboratory,9Hruban RH van Mansfeld ADM Offerhaus
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