Molecular Analysis of Microdissected Tumors and Preneoplastic Intraductal Lesions in Pancreatic Carcinoma
2000; Elsevier BV; Volume: 157; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)64520-8
ISSN1525-2191
AutoresErnst Heinmöller, Wolfgang Dietmaier, Hubert Zirngibl, Petra Heinmöller, William Scaringe, K. W. Jauch, F Hofstädter, Josef Rüschoff,
Tópico(s)Cholangiocarcinoma and Gallbladder Cancer Studies
ResumoLittle or no data exist concerning the inactivation of tumor suppressor genes in intraductal lesions surrounding invasive ductal pancreatic carcinomas. Using a novel improved primer extension and preamplification polymerase chain reaction, we analyzed microdissected paraffin-embedded specimens of pancreatic carcinoma (n = 29) and their corresponding pancreatic intraductal lesions (PIL, n = 331) for loss of heterozygosity (LOH) of p16INK4, DPC4, and p53 by microsatellite analysis and for p53 protein by immunohistochemistry. LOH at thep16INK4 locus (9p21) was found in nine of 22 informative tumors (41%), in 15 of 25 tumors (60%) at theDPC4 locus (18q21.1), and in 22 of 27 tumors (81%) at the p53 locus (17p13). Homozygous deletions ofp16INK4 and DPC4 were found in eight of 22 (36%) and four of 25 tumors (16%), respectively. Furthermore, 24 of 29 tumors (83%) revealed considerable intratumoral genetic heterogeneity. In 165 of 277 PILs (60%) having suitable DNA for microsatellite analysis, alterations in at least one tumor suppressor gene were found. In individual PILs, up to three alterations were detected, and p53 LOH occurred even in morphologically normal-appearing ductal epithelium near the tumor. Although deletions of all three tumor suppressor genes were found in PILs without nuclear atypia, there was a tendency toward earlier LOH of p16INK4 compared to DPC4 and p53 in these lesions. LOH in tumors accompanied positive p53 immunohistochemistry in 81% but only in 38% in PILs. Little or no data exist concerning the inactivation of tumor suppressor genes in intraductal lesions surrounding invasive ductal pancreatic carcinomas. Using a novel improved primer extension and preamplification polymerase chain reaction, we analyzed microdissected paraffin-embedded specimens of pancreatic carcinoma (n = 29) and their corresponding pancreatic intraductal lesions (PIL, n = 331) for loss of heterozygosity (LOH) of p16INK4, DPC4, and p53 by microsatellite analysis and for p53 protein by immunohistochemistry. LOH at thep16INK4 locus (9p21) was found in nine of 22 informative tumors (41%), in 15 of 25 tumors (60%) at theDPC4 locus (18q21.1), and in 22 of 27 tumors (81%) at the p53 locus (17p13). Homozygous deletions ofp16INK4 and DPC4 were found in eight of 22 (36%) and four of 25 tumors (16%), respectively. Furthermore, 24 of 29 tumors (83%) revealed considerable intratumoral genetic heterogeneity. In 165 of 277 PILs (60%) having suitable DNA for microsatellite analysis, alterations in at least one tumor suppressor gene were found. In individual PILs, up to three alterations were detected, and p53 LOH occurred even in morphologically normal-appearing ductal epithelium near the tumor. Although deletions of all three tumor suppressor genes were found in PILs without nuclear atypia, there was a tendency toward earlier LOH of p16INK4 compared to DPC4 and p53 in these lesions. LOH in tumors accompanied positive p53 immunohistochemistry in 81% but only in 38% in PILs. Ductal pancreatic carcinoma shows a growing incidence in the Western world, representing the fifth leading cause of cancer death of either sex.1Parker SL Tong T Bolden S Wingo PA Cancer statistics.CA Cancer J Clin. 1996; 46: 5-27Crossref PubMed Scopus (1971) Google Scholar Because of its aggressive growth and early metastatic dissemination, only 20% of the patients can be treated by surgery with curative intent at the time of diagnosis. The overall 5-year survival rate of 95% of pancreatic carcinomas.3Schutte M Hruban RH Geradts J Maynard R Hilgers W Rabindran SK Moskaluk CA Hahn SA Schwarte-Waldhoff I Schmiegel W Baylin SB Kern SE Herman JG Abrogation of the Rb/p16 tumor-suppressive pathway in virtually all pancreatic carcinomas.Cancer Res. 1997; 57: 3126-3130PubMed Google Scholar, 4Redston MS Caldas C Seymour AB Hruban RH Da Costa L Yeo CJ Kern SE p53 mutations in pancreatic carcinoma and evidence of common involvement of homocopolymer tracts in DNA microdeletions.Cancer Res. 1994; 54: 3025-3033PubMed Google ScholarDPC4 and p53 are deleted in 53% and 76%, respectively,5Rozenblum E Schutte M Goggins M Hahn SA Panzer S Zahurak M Goodman SN Sohn TA Hruban RH Yeo CJ Kern SE Tumor-suppressive pathways in pancreatic carcinoma.Cancer Res. 1997; 57: 1731-1734PubMed Google Scholar and many more genetic alterations have been described.6Gorunova L Höglund M Andren-Sandberg A Dawiskiba S Jin Y Mitelman F Johansson B Cytogenetic analysis of pancreatic carcinomas: intratumor heterogeneity and nonrandom pattern of chromosome aberrations.Genes Chromosom Cancer. 1998; 23: 81-99Crossref PubMed Scopus (97) Google Scholar, 7Gress TM Müller-Pillasch F Geng M Zimmerhackl F Zehetner G Friess H Büchler M Adler G Lehrach H A pancreatic cancer-specific expression profile.Oncogene. 1997; 13: 1819-1830Google Scholar, 8Solinas-Toldo S Wallrap C Muller-Pillasch F Bentz M Gress T Lichter P Mapping of chromosomal imbalances in pancreatic carcinoma by comparative genomic hybridization.Cancer Res. 1997; 56: 3803-3807Google Scholar Although much is known about the invasive tumors, little is known about the genetic alterations in progenitor lesions. K-ras mutations have been found in preneoplastic intraductal lesions (PILs) and appear to be early events in pancreatic carcinogenesis.9Lemoine NR Jain S Hughes CM Staddon SL Maillet B Hall PA Klöppel G Ki-ras oncogene activation in preinvasive pancreatic cancer.Gastroenterology. 1992; 102: 230-236Abstract PubMed Google Scholar, 10Caldas C Hahn SA Hruban RH Redston MS Yeo CJ Kern SE Detection of K-ras mutations in the stool of patients with pancreatic adenocarcinoma and pancreatic ductal hyperplasia.Cancer Res. 1994; 54: 3568-3573PubMed Google Scholar, 11Sugio K Molberg K Albores-Saavedra J Virmani A Kishimoto Y Gazdar AF K-ras mutations and allelic loss at 5q and 18q in the development of human pancreatic cancers.Int J Pancreatol. 1997; 1: 205-217Crossref Scopus (38) Google Scholar To date, analysis of thep53 gene in PILs has only been performed by immunohistochemistry and often without a precise morphological definition of preneoplastic lesions according to the World Health Organization International Histological Classification of Tumors.12Klöppel G Solcia E Longnecker DS Capela C Sobin LH Histological typing of tumours of the exocrine pancreas.WHO International Histological Classification of Tumours. ed 2. Springer, Heidelberg, New York, Berlin1996Google Scholar The available data are controversial and suggest mutational inactivation of p53 at either an early13Boschman CR Stryker S Reddy JK Rao MS Expression of p53 protein in precursor lesions and adenocarcinoma of human pancreas.Am J Pathol. 1994; 145: 1291-1295PubMed Google Scholar or late stage of pancreatic carcinogenesis.14van den Berg FM Polak M Baas IO Offerhaus GJA Detection of p53 overexpression in routinely paraffin embedded tissue of human carcinomas using a novel target unmasking fluid.Am J Pathol. 1993; 142: 381-385PubMed Google Scholar, 15di Guiseppe JA Hruban RH Goodman SN Polak M van den Berg FM Allison DC Cameron JL Offerhaus JA Overexpression of p53 protein in adenocarcinoma of the pancreas.Am J Clin Pathol. 1994; 101: 684-688PubMed Google Scholar, 16Zhang SY Ruggeri B Agarwal P Sorling AF Obara T Ura H Namiki M Klein-Szanto AJP Immunohistochemical analysis of p53 expression in human pancreatic carcinomas.Arch Pathol Lab Med. 1994; 118: 150-154PubMed Google Scholar In two recent studies that addressed more precisely the different histological forms of preneoplastic lesions in pancreatic carcinoma, p16INK4 has been found inactivated in both low-grade and high-grade ductal lesions.17Moskaluk CA Hruban RH Kern SE p16 and K-ras gene mutations in the intraductal precursors of human pancreatic adenocarcinoma.Cancer Res. 1997; 57: 2140-2143PubMed Google Scholar, 18Wilentz 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 Alterations of DPC4 in PILs have not yet been described. For analysis of loss of heterozygosity (LOH), a homogeneous population of tumor cells or epithelial cells from PILs is required. This can only be achieved by precise microdissection, usually of ∼50 to 200 cells, which allows few specific (nested) polymerase chain reaction (PCR) amplifications. Because LOH studies need to be done with multiple markers, preamplification of DNA by whole genome amplification could be very helpful. For this purpose, we established a protocol which allows multiple DNA analyses of single cells or small cell groups by conducting whole genome amplification followed by locus-specific PCR of multiple specific sites.19Dietmaier W Hartmann A Wallinger S Heinmöller E Kerner T Endl E Jauch KW Hofstädter F Rüschoff J Multiple mutation analyses in single tumor cells enabled by improved whole genome amplification.Am J Pathol. 1999; 154: 83-95Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar Microsatellite analysis was performed with multiple markers mappingp16INK4, DPC4, andp53. With this technique, we obtained information about allelic deletion of up to three tumor suppressor genes within a single lesion. In addition, p53 immunohistochemistry was correlated with the molecular data. Twenty-nine archival cases of pancreatic adenocarcinoma from the University Clinic of Regensburg were selected for the presence of PILs associated with invasive carcinoma. One case of chronic pancreatitis and one case of nesidioblastosis with PILs were included as controls of nonneoplastic disease. According to the World Health Organization Classification12Klöppel G Solcia E Longnecker DS Capela C Sobin LH Histological typing of tumours of the exocrine pancreas.WHO International Histological Classification of Tumours. ed 2. Springer, Heidelberg, New York, Berlin1996Google Scholar and a recently published study,17Moskaluk CA Hruban RH Kern SE p16 and K-ras gene mutations in the intraductal precursors of human pancreatic adenocarcinoma.Cancer Res. 1997; 57: 2140-2143PubMed Google Scholar PILs were classified on the basis of their growth pattern and on the degree of nuclear atypia and were divided into five groups (Figure 1, Table 1). Normal appearing epithelia with cuboidal to low columnar cells with round-to-oval nuclei without signs of atypia were graded PIL grade 0. Flat focal lesions with uniformly tall columnar mucin-filled cells without signs of nuclear atypia were graded PIL grade 1. Focal lesions graded PIL grade 2 consisted of mucin-filled cells with basally located nuclei without signs of atypia and a papillary growth pattern, the papillary folds typically showing a fibrovascular stalk. If these lesions exhibited mild nuclear atypia like larger sized round or oval nuclei and a more pronounced chromatin structure with clumped or dense hyperchromatic chromatin pattern, the lesion was graded PIL grade 3. When the epithelial lining showed irregular budding and bridging and signs of severe nuclear atypia like prominent nucleoli, hyperchromasia, loss of polarity, irregular size and contours, the lesion was graded PIL grade 4. In all lesions described, signs of invasion into the surrounding tissue were absent. To avoid examination of infiltrating cancers extending into ducts thereby mimicking a PIL, the first and the last sections were stained with H 38 E to ensure proper alignment of serial sections. If in the last section signs of invasive carcinoma were discovered, the sections were excluded from the study. In addition, only tissue blocks were processed for which a distance of at least 10 mm from the invasive carcinoma was described in the pathology report. For each patient, at least one example of nonneoplastic tissue was microdissected as a normal control.Table 1Histomorphological Classifications of Preneoplastic LesionsPanIN*PanIN grading according to a recently proposed new nomenclature for classification of duct lesions in the pancreas. More information is available at the http://www.pathology.jhu.edu/pancreas_panin website.PIL†Classification of pancreatic intraductal lesions (PILs) of Moskaluk et al.17 Given is the gradual change (grades 0–4) of morphological appearance of PILs.Klöppel et al‡Classification scheme reported in Ref. 12.GradeDescriptionGradeDescriptionDescriptionNormalNormal0Normal—1AMucinous hypertrophy without nuclear atypia1Flat PIL without nuclear atypiaTumor-like lesions/mucinous hypertrophy1B(Pseudo)papillary PIL without nuclear atypia2Flat PIL with mild nuclear atypiaTumor-like lesions/adenomatoid hyperplasia2Papillary PIL with mild nuclear atypia3Papillary PIL with mild nuclear atypiaTumor-like lesions/papillary hyperplasia with mild dysplasia3Papillary PIL with severe nuclear atypia/CIS4Papillary PIL with severe nuclear atypia/CISMalignant/severe dysplasia/CISPanIN, pancreatic intra-epithelial neoplasia; CIS, carcinoma in situ.* PanIN grading according to a recently proposed new nomenclature for classification of duct lesions in the pancreas. More information is available at the http://www.pathology.jhu.edu/pancreas_panin website.† Classification of pancreatic intraductal lesions (PILs) of Moskaluk et al.17 Given is the gradual change (grades 0–4) of morphological appearance of PILs.‡ Classification scheme reported in 12Klöppel G Solcia E Longnecker DS Capela C Sobin LH Histological typing of tumours of the exocrine pancreas.WHO International Histological Classification of Tumours. ed 2. Springer, Heidelberg, New York, Berlin1996Google Scholar. Open table in a new tab PanIN, pancreatic intra-epithelial neoplasia; CIS, carcinoma in situ. Five-micron serial sections of formalin-fixed, paraffin-embedded tissue were deparaffinized by incubating the slides in xylene for 2 × 15 minutes and rehydrating in 99.9% ethanol for 2 × 10 minutes, in 96% ethanol for 2 × 10 minutes, and in 70% ethanol for 2 × 10 minutes. Methylene blue-stained sections were microdissected (Figure 2) using a joystick hydraulic micromanipulator (Leitz, Wetzlar, Germany). Between 50 and 200 cells were collected with sterile needles (microlance3R; Becton Dickinson, Franklin Lakes, NJ) and transferred into 10 μl of TL-buffer (1 × Taq PCR buffer, from Life Technologies, Eggenstein, Germany, including 4 mg/ml of Proteinase K and 0.5% Tween 20 from Merck, Darmstadt, Germany). In tumors, the microdissected samples were enriched for a neoplastic cellularity of at least 60% to avoid false-negative results in LOH analysis because of contamination by normal stromal cells present in tumors. Cell lysis was performed by incubation for 16 hours at 50°C and a 10-minute inactivation step at 94°C. As a first step, whole genome amplification was performed by using an improved primer extension preamplification (I-PEP)-PCR as described recently19Dietmaier W Hartmann A Wallinger S Heinmöller E Kerner T Endl E Jauch KW Hofstädter F Rüschoff J Multiple mutation analyses in single tumor cells enabled by improved whole genome amplification.Am J Pathol. 1999; 154: 83-95Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar using a MJR PTC200 thermocycler (Biozym, Oldenburg, Germany). Briefly, I-PEP PCR was set up by adding 50 μl I-PEP mix (final concentration: 0.05 mg/ml gelatin, 16 μmol/L (N)15 random primer, 0.1 mmol/L dNTP, 3.6 UTaq Expand High Fidelity polymerase, 2.5 mmol/L MgCl2, in 1× PCR buffer No. 3 from Boehringer Mannheim, Mannheim, Germany) to 10 μl of lysed cells. PCR was run for 50 cycles. Step 1: 92°C for 90 seconds; step 2: 92°C for 40 seconds; step 3: 37°C for 2 minutes; step 4: ramp 0.1°C per 1 second to 55°C; step 5: 55°C for 4 minutes; step 6: 68°C for 30 seconds; step 7: go to step 2, 49 times; step 8: 68°C for 15 minutes; step 9: 4°C. The presence and relative quantity of PCR product was ascertained by resolution on a 2% agarose gel. Specific single-round PCR (0.2 mmol/L dNTP, 0.3 μmol/L primers, 0.5 U Taq Expand High Fidelity polymerase) was done using 3-μl aliquots of the preamplified DNA in a final volume of 20 μl in a MJ Research Thermocycler (PTC100, MJ Research, Watertown, MA) for 50 cycles: 94°C for 1 minute, 50 to 60°C for 1 minute, 72°C for 1 minute, followed by a final extension at 72°C for 8 minutes as described previously.20Dietmaier W Wallinger S Bocker T Kullmann F Fishel R Rüschoff J Diagnostic microsatellite instability: definition and correlation with mismatch repair protein expression.Cancer Res. 1997; 57: 4749-4756PubMed Google Scholar, 21Rüschoff J Dietmaier W Lüttges J Seitz G Bocker T Zirngibl H Schlegel J Schackert HK Jauch KW Hofstädter F Poorly differentiated colonic adenocarcinoma, medullary type.Am J Pathol. 1997; 150: 1815-1825PubMed Google Scholar Primers used are given in Table 2. Amplified microsatellites (3 μl) were analyzed by 6.7% polyacrylamide/50% urea gel electrophoresis (1 hour, 1500V, 50°C) in a SequiGen sequencing gel chamber (BioRad, Hercules, CA) and by silver nitrate staining as described previously.22Schlegel J Bocker T Zirngibl H Hofstädter F Rüschoff J Detection of microsatellite instability in human colorectal carcinomas using a non-radioactive PCR-based screening method.Virchows Arch. 1995; 426: 223-227Crossref PubMed Scopus (52) Google ScholarTable 2Microsatellite PrimerGeneMicrosatellite primerRate of PCR amplification*Data were obtained by amplification of tumor DNA.% Informative*Data were obtained by amplification of tumor DNA.p16INK4D9S1751 (PKY11)30/31 (97%)67D9S94219/31 (61%)58D9S1748 (PKY3)26/31 (84%)58D9S171 (XC3)23/26 (88%)83DPC4D18S46 (CU18007)30/31 (97%)67D18S6330/31 (97%)70p53TP53alk22/23 (97%)77D17Sp5328/29 (97%)72* Data were obtained by amplification of tumor DNA. Open table in a new tab LOH was diagnosed when a significantly lower ratio in the signal intensity ( 30%), low labeling index (>1%, <30%), and no positivity (<1%). The Fisher's exact test24Mehta CR Pantel NR A network algorithm for performing Fisher's exact test in r x c contingency tables.J Am Stat Assoc. 1983; 78: 427-434Google Scholar was used to calculateP values using Stat Xact software (Cytel Corporation, Cambridge, MA). A P value of <0.05 was considered statistically significant. Table 2 gives the informative rate and the rate of amplification that could be achieved by the microsatellite markers. These markers have been found suitable for chromosomal mapping for detection of LOH and homozygous deletions of p16INK425Cairns P Polascik TJ Eby Y Tokino K Califano J Merlo A Mao L Herath J Jenkins R Westra W Rutter JL Buckler A Gabrielson E Tockman M Cho KR Hedrick L Bova GS Isaacs W Koch W Schwab D Sidranski D Frequency of homozygous deletion at p16/CDKN2 in primary human tumours.Nat Genet. 1995; 11: 210-212Crossref PubMed Scopus (656) Google Scholar and DPC4.26Hahn SA Shamsul Hoque ATM Moskaluk CA Da Costa LT Schutte M Rozenblum E Seymour AB Weinstein CL Yeo CJ Hruban RH Kern SE Homozygous deletions map at 18q21.1 in pancreatic cancer.Cancer Res. 1996; 56: 490-494PubMed Google Scholar In our hands, the microsatellite markers D9S1751 and D9S1748 (p16INK4) and CU18007 and D18S63 (DPC4) gave reliable results. For multiplex PCR, D9S1751 was found to be most suitable. p53 markers D17Sp53 and TP53alk showed a 97% rate of amplification and were informative in 77% and 72% of cases, respectively. At least two and up to seven tumor loci were microdissected per case (Table 3). LOH ofp16INK4 was found in nine of 22 cases (41%), and eight of 22 tumors (36%) showed homozygous deletion in multiplex PCR (Figure 3). LOH ofDPC4 was present in 15 out of 25 cases (60%), and homozygous deletion was detected in four out of 25 cases (16%). p53 showed LOH in 22 out of 27 cases (81%).Table 3Microsatellite Alterations in Tumors and p53 ImmunohistochemistryStudy No.Staging (acc. to WHO 1978)p16*For tumor suppressor genes, numbers in parentheses show the number of LOHs detected/number of tumor foci microdissected. n, normal; LOH, loss of heterozygosity; HD, homozygous deletion; n.d., not done; n.i., not informative.DPC4*For tumor suppressor genes, numbers in parentheses show the number of LOHs detected/number of tumor foci microdissected. n, normal; LOH, loss of heterozygosity; HD, homozygous deletion; n.d., not done; n.i., not informative.p53*For tumor suppressor genes, numbers in parentheses show the number of LOHs detected/number of tumor foci microdissected. n, normal; LOH, loss of heterozygosity; HD, homozygous deletion; n.d., not done; n.i., not informative.p53 protein†Immunohistochemical staining of p53 protein was classified from no positivity (−), low labeling (+/++), and high labeling (+++).PC 1pT1a,pN0,pMX,R0,G2HD(3/7)LOH(5/5)LOH(5/7)(+)/(+++)PC 2pT1a,pNX,pMX,R0,G3HD(2/3), LOH(1/3)n(2/2)n(2/2)(−)PC 3pT3,pN1,pMX,G3HD(2/3)HD(2/3)LOH(1/3)n.d.PC 4pT1b,pN0,pMX,R0,G3HD(4/4)LOH(5/5)LOH(2/5)(−)PC 5pT2,pN1,pMX,R1,G1LOH(2/4)n(5/5)n(5/5)(+++)PC 6pT1,pN0,pMX,R0,G1n.i.LOH(2/4)n(3/3)n.d.PC 7pT2,pN0,pMX,R0,G2LOH(3/3)LOH(3/3)LOH(2/2)(++)PC 8pT1b,pN1,pMX,R0,G2n.d.HD(1/2), LOH(1/2)LOH(2/2)n.d.PC 9pT2,pN1,pMX,R0,G2n.d.LOH(2/4)n(3/3)n.d.PC 10pT3,pN0,pMX,R1,G2n.i.LOH(1/2)LOH(1/2)n.d.PC 11pT2,pN1,pMX,R0,G3n(2/2)LOH(3/5)n.i.(+++)PC 12pT2,pN0,pMX,R0,G2n(2/2)n(7/7)LOH(4/7)(−)PC 13pT2,pN1,pMX,R1,G2LOH(1/2)n(2/2)LOH(1/1)(+++)PC 14pT2,pN1,pMX,G2LOH(1/1)n.i.LOH(3/4)(−)/(+++)PC 15pT2,pN1,pM0,R0,G3n.i.n.i.LOH(1/2)(+++)PC 16pT2,pN1,pMX,R0,G3LOH(1/2)n.i.LOH(3/3)(++)PC 17pT2,pN0,pMX,R0,G3HD(3/3)LOH(1/3)LOH(2/3)(+)/(+++)PC 18pT1,pN0,pMX,R0,G1HD(1/1)n(2/2)n(3/3)(−)PC 19pT2,pN1,pMX,R0,G2n.d.HD(1/2)LOH(1/2)n.d.PC 20pT2,pN1,pMX,R0,G3n(2/2)n.i.LOH(2/2)(−)/(+++)PC 21pT2,pN1,pMX,R0,G2n(3/3)n(3/3)LOH(1/3)(++)PC 22pT2,pN1,pMX,R0,G2HD(3/4), LOH(1/4)HD(3/4), LOH(1/4)LOH(4/4)n.d.PC 23pT2,pN1,pMX,R0,G2n(2/2)LOH(2/3)LOH(4/4)(+++)PC 24pT2,pN1,pMX,R0,G2HD(2/3), LOH(1/3)LOH(2/3)LOH(3/3)n.d.PC 25pT2,pN0,pMX,R0,G2n.d.LOH(4/5)LOH(3/5)(+++)PC 26pT2,pN1,pMX,R0,G3n(3/3)LOH(2/4)LOH(3/4)(−)/(++)PC 27pT1a,pN0,pMX,R0,G3n(2/2)LOH(1/3)LOH(1/3)(−)PC 28pT3,pN1,pMX,R1,G3n(2/2)n(2/2)n.i.(+++)PC 29pT2,pN1,pMX,R1,G2LOH(2/2)n(2/2)LOH(2/2)(++)* For tumor suppressor genes, numbers in parentheses show the number of LOHs detected/number of tumor foci microdissected. n, normal; LOH, loss of heterozygosity; HD, homozygous deletion; n.d., not done; n.i., not informative.† Immunohistochemical staining of p53 protein was classified from no positivity (−), low labeling (+/++), and high labeling (+++). Open table in a new tab Microdissection of distinct tumor loci uncovered the presence ofp16INK4 LOH in three out of eight cases (38%) with homozygous deletion of p16INK4. LOH of DPC4 was coincidentally found in two out of four cases (50%) with homozygous deletion of DPC4. In 13 out of 27 cases (48%) with LOH of p53, at least one microdissected sample showed the presence of both alleles. In these cases, contamination of normal cell-derived DNA masking the detection of LOH was excluded by demonstrating LOH at a different locus, eg, p53, DPC4, p16INK4 or Deleted in Colon Carcinoma(DCC), by using the same template DNA (Figure 3). Overall, the loss of all three tumor suppressor genes was found in 7 out of 29 cases (24%), and two of three tumor suppressor genes were lost in 11 out of 29 cases (38%). Only one of 29 carcinomas (4%) showed no alteration by microsatellite analysis at the three loci investigated but this case did show strong staining for the p53 protein in immunohistochemistry, which is in favor of the presence of a mutatedp53 gene in this case. Furthermore, in 24 out of 29 tumors (83%) microdissection revealed the presence of genetic heterogeneity in at least one tumor suppressor gene indicating the presence of different tumor cell subclones. No correlation could be observed between marker loss and either malignancy grade or tumor stage. From a total of 331 microdissected PILs, the DNA of 277 PILs was suitable for microsatellite analysis (Figure 4, Figure 5). In total, 163 of the 277 PILs (59%) showed LOH. Two PILs grade 4 (0.7%) exhibited a homozygous deletion at thep16INK4 locus. In two of 22 PILs (9%) having histologically normal epithelium (grade 0), LOH ofp53 was detected in two different patients; neither stained positively for p53 protein.Figure 5Spectrum of deletions in PILs (n = 277). Microdissected cells were preamplified by I-PEP and one-tenth aliquot was used for microsatellite analysis. Given are the deletions of single tumor suppressor genes in percentage. Normal epithelium (PIL grade 0) shows LOH (9%) only at thep53 locus. The frequency of LOH at thep16INK4 locus is not statistically different (P = 0.38) in PILs without nuclear atypia (PIL grade 1 and 2) compared to PILs showing nuclear atypia (PIL grade 3 and 4). At the p53 andDPC4 locus, LOH is more frequent in PILs with nuclear atypia (PIL grade 3 and 4) compared to PILs without nuclear atypia (PIL grade 0 to 2; p53 P < 0.001; DPC4 P = 0.043).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The majority of the PILs investigated contained LOH of at least one single gene (Figure 6). Deletions of two of the three tumor suppressor genes was first observed in histologically altered epithelium (grade 1), and triple lesions were first observed in PILs having moderate (grade 3) to severe (grade 4) nuclear atypia. Single lesions were found in 15 of 81 PILs (19%) without nuclear atypia compared to 75 of 196 PILs (38%) showing nuclear atypia. Multiple deletions (double or triple) were found in five of 81 PILs (6%) without nuclear atypia and in 17 of 196 PILs (9%) showing nuclear atypia (Figure 6). Of the total 22 PILs with multiple lesions, 17 (77%) were found in PILs showing nuclear atypia. The trend of multiple lesions associated with a PIL with nuclear atypia was found to be not statistically significant (P= 0.538). When the genetic combinations of double lesions in PILs were analyzed, the most frequent combination of LOH was p53 withDPC4 (13 of 20 PILs, 65%) followed by p53 withp16INK4 (6 of 20 PILs, 30%) andp16INK4 with DPC4 (1 of 20 PILs, 5%). Furthermore, we analyzed the presence of mutations in single tumor suppressor genes in relationship to the histological grade (PILs without nuclear atypia versus PILs with low to severe nuclear atypia). In general, LOH ofp16INK4, DPC4, or p53 was found in PILs even without nuclear atypia and the number of alterations of these tumor suppressor genes in one individual PIL grows with the grade of nuclear atypia. For p53 andDPC4, we found LOH more often in PILs showing nuclear atypia (P < 0.001 and P = 0.043, respectively) compared to PILs without nuclear atypia. Forp16INK4, we found an early accumulation of mutations in PILs without nuclear atypia that was not statistically different from the accumulation of mutations in PILs showing nuclear atypia (P = 0.38) (Figure 5). In the two cases with chronic pancreatitis and nesidioblastosis, a total of 21 PILs were microdissected. Seven of these PILs were grade 3. LOH was not detected in a single lesion. Twenty-one of 29 carcinomas were available for p53 immunostaining. Sixteen of the 21 carcinomas (76%) showed moderate to strong expression of p53 protein, 13 (81%) of these had LOH at thep53 locus. In five carcinomas that stained negative for p53, LOH of p53 was detected in three cases (60%). Three of the 16 tumors (19%) with p53 protein stabilization showed no LOH ofp53. A total of 151 PILs were stained for p53 protein stabilization (Table 4). 33 PILs (22%) revealed weak to strong p53 protein staining (Figure 7). In comparison, 42 of 151 PILs (28%) showed LOH of p53. Sixteen (38%) of these PILs accompanied positive p53 immunohistochemistry. Of the PILs without nuclear atypia, only two (both grade 2) of 52 PILs (4%) showed a weak staining for p53 protein. All other PILs with p53 protein stabilization had low to severe signs of nuclear atypia. Five of 52 PILs (10%) without nuclear atypia and absence of p53 protein expression showed p53 LOH. Twenty-one of the 99 PILs (21%) show
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