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00019606-200609000-00007ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2006 Lippincott Williams & Wilkins, Inc.15September 2006 p 162-168Adequacy of Colonoscopic Biopsy Specimens for Molecular Analysis: A Comparative Study With Colectomy TissueOriginal ArticlesZauber, Neil P. MD*; Sabbath-Solitare, Marlene PhD†; Marotta, Stephen PhD†; Perera, Lilani P. MD*; Bishop, David T. PhD‡Departments of *Medicine†Pathology, Saint Barnabas Medical Center, Livingston, NJ 07039‡Genetic Epidemiology Division, Cancer Research UK Clinical Center in Leeds, St James's University Hospital, Leeds LS9 7TF, UKReprints: N. Peter Zauber, MD, 22 Old Short Hills Road, Livingston, NJ 07039 (e-mail: [email protected]).Support by Healthcare Foundation of NJ, Inc, H. Nussbaum Foundation of Saint Barnabas, and Cancer Research, UK.AbstractMolecular analyses of tumors are increasingly useful for prognosis and for guiding therapy. Colonoscopic biopsy provides the first source of tissue for most cases of colorectal carcinoma and therefore might become an important source for molecular analyses. We have addressed the question whether molecular analyses of colonoscopic biopsy yield results similar to the findings from the surgical specimen. Further, we analyzed 2 separate areas of the colectomy specimen to assess tumor heterogeneity. We evaluated 3 samples from each of 67 patients for point mutations in the KRAS gene, loss of heterozygosity (LOH) at the Adenomatous Polyposis Coli (APC) and Deleted in Colon Cancer (DCC) genes and for microsatellite instability (MSI) using polymerase chain reaction based techniques. The average time interval between biopsy and surgery was 2.2±0.15 weeks. Lesions were from all colon segments and all surgical stages. The degree of agreement between the biopsy and surgical sites was high for APC LOH, MSI, and KRAS mutations (κ=0.85, 1.00, and 0.93, respectively) but less so for DCC LOH (κ=0.62). Colonoscopic biopsies are an acceptable source of neoplastic DNA for studies of KRAS, APC LOH, and MSI, but less so for DCC LOH, primarily resulting from technical considerations.Colorectal cancer (CRC) is the second most common cause of cancer-related death in the United States. It is now generally accepted that carcinogenesis in CRC is a multistep process.1,2 At the molecular level, this includes biallelic inactivating mutations in the adenomatous polyposis coli (APC) tumor suppressor gene, activating mutations in the KRAS oncogene, and biallelic inactivating mutation of the P53 gene.3,4 Other genes may also be involved. Another mechanism of colorectal carcinogenesis involves inactivation, primarily through methylation, of mismatch repair genes, with the resulting tumor phenotype of microsatellite instability (MSI).5It is possible the molecular understanding of colorectal carcinoma will eventually have direct prognostic and therapeutic implications. This was recently demonstrated for nonsmall cell lung carcinoma, for which lesions containing specific mutations in the epidermal growth factor receptor gene show responsiveness to the tyrosine kinase inhibitor drug gefitinib.6Colonoscopic biopsy provides the first source of tissue for most cases of CRC. If clinical decision-making is to be based upon the detailed analysis, including molecular analysis, of these biopsies then it is important to document the accuracy of the biopsy specimen results with respect to the larger surgical specimen of the same carcinoma. For example, if patients with biopsy-proven rectal carcinoma were selected to receive external radiation before definitive surgery based on molecular analysis of the biopsy tissue, then it would be imperative to assure that the biopsy changes were truly representative of the entire de novo tumor. Even if sufficient tumor tissue remains in the resected specimen, additional genetic alterations may occur in response to chemotherapy or radiation therapy, or simply because of the tumor evolution over time. Genetic abnormalities detected in the original biopsy may thus be more indicative of the status of the preirradiation tumor.To address this issue, we investigated the accuracy of molecular changes in samples from patients with colon carcinoma who had colonoscopic biopsy and subsequent surgery. Further, we analyzed 2 separate areas of the colectomy specimen to assess tumor heterogeneity. We studied these specimens for point mutations in the KRAS gene, loss of heterozygosity (LOH) at the APC and DCC genes and for MSI (Figs. 1, 2)JOURNAL/dimp/04.03/00019606-200609000-00007/figure1-7/v/2021-02-17T195928Z/r/image-tiff A sample plot or electropherogram showing D18S58 microsatellite analysis of DNA used to determine LOH for the DCC gene. A, DNA from normal colon mucosa indicating the patient's 2 normal alleles. B, DNA from a carcinoma from the transverse colon. There is a significant loss of the intensity of the second band (arrow).JOURNAL/dimp/04.03/00019606-200609000-00007/figure2-7/v/2021-02-17T195928Z/r/image-tiff A sample plot or electropherogram showing the mononucleotide marker BAT-26 used to determine MSI. A, DNA from normal colon mucosa indicating normal peaks. B, DNA from a carcinoma from the transverse colon. The arrow indicates allelic shift (additional, abnormal peaks in tumor DNA compared with normal) consistent with MSI (arrow).MATERIALS AND METHODSPatient SamplesA list of patients was generated using the Powerpath computer database of the Department of Pathology for the years 1998 to 2002. Seventy patients were identified who had a biopsy and surgery both performed at our hospital within a 3-month interval. The hospital institutional review board approved the protocol, and a waiver from HIPAA requirement was obtained as the study did not require protected health information. All tissue samples were processed by coded numbers.Paraffin blocks of tissue samples were obtained from warehouse storage. Slides stained with hematoxylin and eosin were reviewed to guide sample selection. Samples of ulcerated, and/or necrotic areas as well as identifiable nontumor tissue such as muscle or adipose tissue were avoided. In situ lesions were used if the biopsy contained at least 15% to 20% carcinoma by visual estimate. Two samples from the carcinoma removed at surgery were taken from areas widely separated, and one area of normal mucosa for internal comparison. Analysis of each of the 3 samples from a patient was performed without knowledge of the results of the other samples from the same patient.DNA Extraction and PurificationAll tissue specimens were formalin-fixed and paraffin-embedded. The histologically relevant regions from unstained sections were isolated using a blade and transferred to an Eppendorf tube. For biopsy specimens, 6 unstained sections of 10 μm yielded a small but sufficient tissue pellet. Surgical specimens are larger than the biopsy specimens and 2 unstained sections of 10 μm would suffice. The paraffin wax was removed by xylene and ethanol washes. The cellular material was lysed and the DNA was isolated and purified using the Qiagen QIAmp Tissue Kit (Qiagen Inc, Santa Clara, CA). The samples were then diluted in 50 to 150 μL of buffer. One to 2 μL of the sample was used for each PCR reaction; each containing approximately 50 ng of DNA. Genomic DNA was stored at 4°C in 10 mM TE buffer, pH 9.0. Normal colonic mucosa was used as normal control for all reactions. If normal mucosa was available with the colonoscopic biopsy, then this was used for the analyses of the colonoscopic tumor sample; if not, then normal mucosa from the surgical specimen was used.LOH of APC and Deleted in Colon Cancer (DCC) GenesLOH of the APC gene was determined through the PCR amplification of a CA repeat marker within the D5S346 locus of the DP1 gene using the primer set: 5′-ACT CAC TCT AGT GAT AAA TCG GG-3′ (sense) and 5′-AGC AGA TAA GAC AAG TAT TAC TAG TT-3′ (antisense).7 This primer set generates a PCR product of 108 to 136 base pairs. Samples that were homozygous for the D5S346 primer set were analyzed using primer sets for CA repeats within the D5S1965 and/or D5S492 loci.8LOH of the DCC gene was determined by amplification of the CA repeat markers within the D18S58 or D18S61 loci.9 The primers for D18S58 were: 5′-GCT CCC GGC TGG TTT T-3′ (sense) and 5′-GCA GGA AAT CGC AGG AAC TT-3′ (antisense) and for D18S61 were: 5′-ATT TCT AAG AGG ACT CCC AAA CT-3′ (sense) and 5′-ATA TTT TGA AAC TCA GGA GCA T-3′ (antisense). These primers generated PCR products 144 to 164 base pairs and 152 to 184 base pairs, respectively.All PCR reactions were carried out with Applied Biosystems reagents (Roche Molecular Systems, Inc, Branchburg, NJ). Four picomoles of each primer and a 1.5 mM MgCl2 concentration was used in the PCR reactions. All PCR primer sets for microsatellite analysis had a 5′-fluorescence-label on the sense strand and a 5-GTGTCTT tail on the antisense strand. All primer sets were obtained from the Applied Biosystems Custom Oligo Synthesis Service ([email protected]).All reactions were run on a PE 9700 thermal cycler (PE Applied Biosystems, Foster City, CA) under the following conditions: 6 minutes denaturation at 94°C followed by 36 cycles of a 30 seconds denaturation at 94°, 30 seconds annealing at 55°C, and a 50 seconds elongation at 72°C, with a final 30-minute extension at 72°C. PCR products were loaded onto a 5% Long Ranger acrylamide gel (Cambrex, Rockland, ME) containing 6 M urea and analyzed on an ABI Prism 377 DNA sequencer with GeneScan collection software (Applied Biosystems, Foster City, CA).Neoplastic tissue was evaluated simultaneously with normal colonic mucosal tissue from the same patient for all LOH studies. The ratio of the height of the allele band intensities of the neoplastic tissue was divided by the corresponding ratio for the normal tissue. LOH was defined as a resultant ratio of less than or equal to 0.5.10 Our experience with DNA extraction from archival paraffin-embedded samples has consistently indicated that 36 cycles yields a strong signal without over-amplification that might obscure LOH measurements. With fewer cycles (such as 30 to 35) we have found less consistent DNA amplification, whereas the normalized ratios were quite similar, all within 0.05 (data not shown).KRAS MutationsSingle-stranded conformational polymorphism (SSCP) analysis was employed to screen for mutations within the KRAS oncogene. The codon 12/13 region of exon 1 in the KRAS oncogene was amplified using the primer set 5′-CCT GCT GAA AAT GAC TGA AT-3′ (sense) and 5′-TGT TGG ATC ATA TTC GTC CA-3′ (antisense).11 This generates a 115 base pair PCR product. Samples showing KRAS mutation bands by SSCP were sequenced to verify the point mutation. PCR-based DyeDeoxy Terminator sequencing was performed using an ABI Prism 377 DNA Sequencing System (PE Biosystems, Foster City, CA). The sequencing reaction was performed using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and 5 picomoles of the antisense primer. Sequence reaction products were cleaned using AutoSeq G-50 spin columns (Amershan Pharmacia, Piscataway, NJ) and were separated on a 5% Long Ranger acrylamide gel (Cambrex, Rockland, ME).MSITumors were evaluated for MSI through PCR analysis of a panel of 3 microsatellite markers. We primarily used BAT26 to identify MSI. BAT26 has been shown to identify MSI-H neoplasms sufficiently by itself.12 However, the D5S346 and D18S58 (or D18S61) dinucleotide repeats are also quite sensitive to MSI. The markers D5S346 and D18S61 are both on the NCI consensus panel.13 PCR primers for each marker were used to amplify both normal DNA and tumor DNA. The amplified products from the normal and tumor DNA underwent electrophoresis side by side on the ABI 377 DNA Sequencer using GeneScan software. For BAT26, the primers used were: 5′-TGA CTA CTT TTG ACT TCA GCC-3′ (sense) and 5′-AAC CAT TCA ACA TTT TTA ACC C-3′ (antisense). The same conditions were followed as for the microsatellite analysis of APC and DCC. Microsatellite stable specimens produce 1 cluster of peaks centered at 126 base pairs, whereas microsatellite unstable peaks produce an additional cluster of peaks centered at 116 base pairs. MSI-H tumors were defined as having instability demonstrated with at least BAT-26.Statistical MethodsThe premise of this study is to assess the concordance of colonoscopic biopsy for detecting molecular changes, as compared with the changes present in the surgical samples. A biopsy and surgical specimen are considered concordant if a molecular change present in 1 (or both) of 2 surgical sites is also present in the biopsy. The McNemar's χ2 test was used to assess whether the percentage of patients with molecular changes in the biopsy is comparable to the percentage of patients with molecular changes in the surgical specimens. Furthermore, the κ statistic was derived to assess the degree of agreement between the 2 types of samples, with adjustment for chance agreement. A κ greater than 0.8 indicates excellent agreement. Statistics were calculated with Categorical Data Analyis using the SAS System, SAS Institute, Inc, Cary, NC.RESULTSTissue blocks could not be located for 3 patients, and the study group consists of 67 patients with material available from both colonoscopic biopsy and surgical resection. The average time interval between the biopsy and surgery was 2.2±0.15 weeks (CI 1.9-2.5). Thirty-nine patients (58.2%) were male and 28 (41.8%) were female, and the mean age was 73 years. Carcinomas from all segments of the colon were studied: cecum=18, ascending=16, transverse=12, descending=2, sigmoid=15, and rectum=4; 46 (69%) were right-sided and 21 (31%) were left-sided. Eight (12%) of the carcinomas were in situ, with obvious residual adenoma, whereas 59 (88%) were carcinoma only. According to the TNM staging system, the tumors were of the following pathologic stages at the time of surgery: Stage 1=21 (31%); Stage 2=26 (39%); Stage 3=18 (27%); and Stage 4=2 (3%).As is common with colonoscopic biopsies, some of the biopsies contained noncarcinomatous elements, either normal mucosa or adenoma. Among all biopsies 41 (61.2%) contained at least 90% carcinoma cells, whereas 16 (23.9%) contained 51% to 90% carcinoma, 9 (13.4%) contained only 25% to 50% carcinoma, and 1 (1.5%) contained less than 25% carcinoma. Histologic interpretations of the biopsies were described as: well differentiated=10 (14.9%), moderately differentiated=48 (71.6%), and poorly differentiated=9 (13.4%). There was some difference between the colonoscopic biopsy and the surgical specimen with respect to the histology: Histology for the surgical specimen was: well differentiated=6 (9%), moderately differentiated=49 (73.1%), and poorly differentiated=12 (17.9%). The histologic interpretation of the degree of differentiation was concordant for 51 cases (76.1%) between the biopsy and the surgical specimen.LOH of the APC GeneResults of APC LOH for the biopsy and the 2 surgical sites are reported in Table 1. One patient was uninformative for the 2 APC markers. For 1 patient, PCR amplification from the second surgical site failed. Eleven carcinomas demonstrated MSI with the APC markers. The percentage of biopsy specimens with APC LOH (41%) was comparable to that of surgical specimens with APC LOH (48%) (P=0.13) and the degree of agreement between the 2 types of specimens corrected for chance agreement was excellent (κ=0.85 with 95% CI of 0.71-0.99). Two carcinomas showed no LOH for APC on biopsy but showed LOH at both surgical sites. For these 2 cases, the percentage of the biopsy sample that was involved with carcinoma was 60% and 40%. Two other carcinomas demonstrated no APC LOH on either the biopsy or 1 surgical site, but they both demonstrated LOH at the second surgical site. These 4 carcinomas are considered discordant. Only 1 carcinoma showed LOH on the biopsy and LOH at just 1 of the 2 surgical sites. Fifty-one of 54 (94.4%) carcinomas were identical for the 2 surgical sites regarding APC LOH. For just 3 cases, APC LOH was demonstrated at 1 surgical site but not the other.JOURNAL/dimp/04.03/00019606-200609000-00007/table1-7/v/2021-02-17T195928Z/r/image-tiff LOH Results for Biopsy and 2 Surgical Sites for APC and DCC GenesLOH of the DCC geneFour individuals were homozygous for 2 markers for DCC and 11 carcinomas demonstrated MSI with the DCC marker. DNA did not amplify for 2 biopsies, for both surgical sites for 1 carcinoma and for 1 of 2 surgical sites for 2 other carcinomas. The percentage of biopsy specimens with DCC mutated (47%) was lower than that from the surgical samples (66%) (McNemar test P=0.004). The degree of agreement adjusted for that expected just by chance was only moderate (κ=0.62 with 95% CI of 0.42-0.83), Table 1. There were 9 lesions for which the biopsy showed no DCC LOH, but LOH was demonstrated at both surgical sites [8], and at one of two surgical sites [1]. One additional lesion, for which the biopsy revealed no abnormality with the DCC marker, demonstrated MSI at both surgical sites. For these 10 biopsies demonstrating no DCC abnormality, 6 contained at least 90% carcinoma histologically, whereas the other 4 biopsies contained from 25% to 75% carcinoma admixed with normal and adenomatous tissue. There was high agreement between the 2 surgical sites with respect to DCC results. Forty-eight of 49 (98.0%) lesions were concordant, with just 1 lesion demonstrating DCC LOH at 1 surgical site but no LOH at the second surgical site.MSIThe results for MSI were identical for all 67 lesions (Table 2). Twelve (17.9%) lesions demonstrated MSI and 55 (82.1%) were stable. All carcinomas demonstrated MSI at both surgical sites and on the corresponding biopsies. The same consistency was seen with carcinomas showing no MSI. The percentage of biopsy specimens with MSI (82%) was the same as that of surgical specimens with MSI (82%), and there was complete agreement between the 2 types of specimens corrected for chance, with κ=1.0. Eleven of the 12 lesions revealed instability by all 3 markers at all 3 sites, whereas 1 lesion showed instability just with BAT 26 at all 3 sites but showed no instability with the DCC or APC markers at the 3 sites. The 12 carcinomas demonstrating MSI were located in the cecum [3], ascending [6], transverse [1], and sigmoid [2] segments.JOURNAL/dimp/04.03/00019606-200609000-00007/table2-7/v/2021-02-17T195928Z/r/image-tiffKRAS Gene Mutation and MSI Results for Biopsy and 2 Surgical SitesKRAS GeneThe results for KRAS gene mutations were highly consistent (Table 2). Among the biopsies, 46 (68.7%) showed no mutations, whereas 21 (31.3%) contained mutations. The mutations found were: GGT to AGT [1]; GGT to GAT [9]; GGT to GTT [4]; GGT to TGT [2]; and 5 were mutations of codon 13: GGC to GAC. One biopsy with a codon 13 mutation also had a simultaneous mutation at codon 8: GTA to GTG, and this second mutation was also seen at both surgical sites.The 2 surgical sites yielded identical results: 44 (65.7%) lesions showed no mutations at both surgical sites, whereas 23 (34.3%) showed a KRAS mutation at both surgical sites. The specific types of KRAS mutation were identical for each pair of 2 surgical sites. The biopsy site demonstrated KRAS results identical to the surgical sites for 65 of 67 (97.0%) lesions. The percentage of biopsy specimens with KRAS mutated (31%) was comparable to that of surgical specimens with KRAS mutated (31%) (McNemar's P=0.50). The degree of agreement between the 2 types of specimens corrected for chance agreement was excellent (κ=0.93 with 95% CI of 0.84-1.00). One carcinoma showed a codon 12 mutation (GGT to GCT) at both surgical sites but no mutation was demonstrated on biopsy; the other carcinoma had a codon 13 mutation (GGC to GAC) at both surgical sites but no mutation seen on the biopsy. The 2 biopsies failing to show a KRAS mutation present in the surgical sites had only 25% and 40% carcinoma in the biopsy, respectively.DISCUSSIONMolecular changes in colorectal carcinoma have been used recently for primary diagnosis through the analysis of exfoliated colonocytes found in stool samples. There are clearly inherit difficulties with this new diagnostic testing concept, but the underlying premise is that superficial tumor cells obtained in stool samples have the critical molecular genetic changes of the lesion. Molecular changes in APC and KRAS genes, as well as MSI, have been evaluated in several initial screening studies of exfoliated cells.14,15 A colonoscopic biopsy is similar because it is a sample of superficial cells of the neoplasm. It is valid to question the accuracy of these superficial cells to reflect the molecular changes present in the deeper levels of the tumor.There is growing interest in the use of molecular changes in colorectal carcinoma for prognosis. For instance, MSI of colon tumors has been reported to be associated with a better prognosis than lesions that are microsatellite stable.16 Thus far, the reported prognostic significance for colorectal carcinomas with mutations in the P53 gene,17,18KRAS gene,19,20 and LOH of APC and DCC genes21,22 has been inconsistent. It had been hypothesized that a major reason for the inconsistent results is the practice of limiting molecular analysis to just 1 sample site, which may not be representative of the entire lesion. Genetic heterogeneity within tumors has been documented in CRC.23,24 One study documented significant intratumor heterogeneity for LOH with regard to the APC (67%) and DCC (58%) loci, with less heterogeneity documented for point mutations of either the KAS gene or the P53 gene (20%).25 In a further analysis, these authors reported that heterogeneity for point mutations is more pronounced in the early, than in the advanced, stages of colorectal carcinoma. They further showed a relative stability of intratumoral heterogeneity for allelic loss as the tumor progresses. They postulated that this may be a consequence of the high level of chromosomal instability without a sufficient compensation by the clonal selection. They concluded that most subclones disappear during the clonal selection process that occurs during tumor progression.26 Similarly, we have reported that KAS gene mutations are not a uniformly consistent finding with respect both to the presence of mutation and to the specific mutation between paired adenomas and carcinomas7 and between the benign and malignant portions of an in situ carcinoma.10 This study evaluates 2 surgical sites and also expands comparative analysis for the first time to the colonoscopic biopsy.LOH is by definition only partial loss, involving just 1 of 2 alleles of a segment of DNA harvested from neoplastic cells. It is possible that not all neoplastic cells from 1 neoplasm will have LOH simultaneously. Additionally, malignant cells may become hyperdiploid, thereby potentially masking any LOH. Further, becuase normal cells are usually diploid, admixture of neoplastic DNA with normal cellular DNA will dilute the neoplastic DNA and thereby render the determination of LOH more difficult. Adenomatous tissue may also vary from carcinomatous areas with respect to molecular changes, and colonoscopic biopsies frequently contain a mixture of both carcinoma and residual adenoma. Although we made every effort to sample areas of biopsy that contained only carcinoma cells, we did not use laser capture technology. Routine use of microdissection techniques may lead to higher accuracy in LOH determinations. Furthermore, even those biopsies showing at least 90% carcinoma have a scattering of connective tissue cells, lymphocytes, and inflammatory cells. Those biopsies that were less than 90% carcinoma were, therefore, further diluted. Finally, LOH depends on the degree of loss used to define this biologic process. We used a conservative ratio of 0.50, but others have used 0.7, which therefore increases the number of lesions defined as demonstrating LOH.Two of our cases showing no APC LOH on the biopsy but LOH at both surgical sites each had limited carcinoma cells on the biopsy. However, 3 of the lesions were discrepant between the two surgical sites for APC LOH, clearly indicating molecular heterogeneity within tumors. We found that accuracy of the biopsy for DCC LOH compared with the results for the surgical sample was only 80.8%. A total of 9 carcinomas were discrepant between the biopsy and the surgical sites. There were 8 carcinomas demonstrating no LOH on the biopsy, but LOH on both surgical samples. For each, the ratio of the allele band intensity (carcinoma over normal tissue) for the surgical sites was less than 0.5, whereas the ratio for the biopsy sites was between 0.66 and 0.86. Similar results for these 9 carcinomas were found when the samples were rerun (data not shown). Of note, for 2 of these 8 carcinomas, LOH was also demonstrated for the APC gene, and the ratios for biopsy and surgical sites for the APC gene were lower than that seen for LOH of the DCC gene. We believe that dilution of the biopsy sample with normal cellular elements has a greater impact in detecting LOH of the DCC markers than of the APC markers. One carcinoma with no LOH for the DCC gene in the biopsy showed LOH for the DCC gene for 1 surgical site but not the second, again suggesting heterogeneity within carcinomas. Differences in histologic assessment of cellular differentiation between the biopsy and surgical samples did not mirror the discrepant DCC LOH results. For the 9 discrepant lesions, 6 had the same level of differentiation, whereas 1 showed more and 2 showed less differentiation on the biopsy than the surgical samples.Many colorectal carcinomas develop from an existing benign adenoma through the accumulation of multiple mutations, and collectively these genetic events provide the affected cells with self-sufficiency for growth. Mutations in the APC gene have been described as an early change in the adenoma-carcinoma progression, followed by KRAS mutations, with DCC a later change in the progressing adenoma.27 There is less information indicating when MSI first develops. However, differences in the molecular profile of cells from separate areas of 1 colorectal lesion have been shown.26Our data indicate a high degree of consistency between the colonoscopic biopsy and the surgical sample, despite the fact that most biopsies contain some adenomatous areas. We believe our colonoscopic biopsies contained sufficient carcinomatous cells for the detection of those molecular changes also present in the invasive components from the surgical sample. However, it is also possible that any adenomatous cells admixed in the colonoscopic biopsies contained the same molecular changes as the carcinomatous cells.10In conclusion, our results indicate that colonoscopic biopsies demonstrate 100% specificity for molecular genetic changes found within the larger tumor. This implies that a carcinoma free of these particular molecular changes will not demonstrate such changes on the biopsy. Further, KRAS mutations, APC LOH, and MSI are accurately assessed on biopsy. However, our results indicate a limitation of colonoscopic biopsy as a source of DNA to assay the DCC gene for LOH. We, therefore, suggest that colonoscopic biopsies are an acceptable routine source of neoplastic DNA for the molecular studies of KRAS mutation, APC LOH, and MSI; but less so for DCC LOH with currently used markers.ACKNOWLEDGMENTSThe authors thank Dr Errol Berman for review of histologic slides and Dr Ann Zauber for statistical assistance.REFERENCES1. Bodmer WF. The somatic evolution of cancer. The Harveian oration of 1996. T Royal Coll Physicians London. 1996;31:82–89.[Context Link]2. Farber E. Cancer development and its natural history. A cancer prevention perspective. Cancer. 1988;62:1676–1679.[Context Link][CrossRef][Medline Link]3. Chung DC. The genetic basis of colorectal cancer: insight into critical pathways of tumorigenesis. Gastroenterology. 2000;119:854–865.[Context Link][CrossRef][Medline Link]4. Vogelstein B, Fearon ER, Hamilton SR, et al. Genetic alterations during colorectal-tumor development. N Engl J Med. 1988;319:525–532.[Context Link][CrossRef][Medline Link]5. 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Cell. 1990;61:759–767.[Context Link][CrossRef][Medline Link]colorectal carcinoma; colonoscopic biopsy; molecular markers; APC; KRAS; MSI00019606-200609000-0000700002808_1999_86_31_zauber_heterozygosity_|00019606-200609000-00007#xpointer(id(citation_FROM_JRF_ID_d2379e1051_citationRF_FLOATING))|11065213||ovftdb|SL000028081999863111065213citation_FROM_JRF_ID_d2379e1051_citationRF_FLOATING[CrossRef]10.1002%2F%28SICI%291097-0142%2819990701%2986%3A1%3C31%3A%3AAID-CNCR6%3E3.0.CO%3B2-O00019606-200609000-0000700002808_1999_86_31_zauber_heterozygosity_|00019606-200609000-00007#xpointer(id(citation_FROM_JRF_ID_d2379e1051_citationRF_FLOATING))|11065405||ovftdb|SL000028081999863111065405citation_FROM_JRF_ID_d2379e1051_citationRF_FLOATING[Medline 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A, DNA from normal colon mucosa indicating the patient's 2 normal alleles. B, DNA from a carcinoma from the transverse colon. There is a significant loss of the intensity of the second band (arrow). A sample plot or electropherogram showing the mononucleotide marker BAT-26 used to determine MSI. A, DNA from normal colon mucosa indicating normal peaks. B, DNA from a carcinoma from the transverse colon. The arrow indicates allelic shift (additional, abnormal peaks in tumor DNA compared with normal) consistent with MSI (arrow). LOH Results for Biopsy and 2 Surgical Sites for APC and DCC GenesKRAS Gene Mutation and MSI Results for Biopsy and 2 Surgical SitesAdequacy of Colonoscopic Biopsy Specimens for Molecular Analysis: A Comparative Study With Colectomy TissueZauber Neil P. MD; Sabbath-Solitare, Marlene PhD; Marotta, Stephen PhD; Perera, Lilani P. MD; Bishop, David T. PhDOriginal ArticlesOriginal Articles315p 162-168