00019606-200309000-00006ArticleDiagnostic Molecular PathologyDiagnostic Molecular Pathology© 2003 Lippincott Williams & Wilkins, Inc.12September 2003
p 151-159Microsatellite Analysis of Synchronous and Metachronous TumorsA Tool For Double Primary Tumor And Metastasis AssessmentArticleTang, Moying BS; Pires, Yumai MD; Schultz, Marcela MD; Duarte, Ignacio MD; Gallegos, Marcela MD; Wistuba, Ignacio I. MDFrom the Department of Anatomic Pathology, Pontificia Universidad Catolica de Chile, Santiago, Chile.Manuscript received March 27, 2003; revision accepted March 28, 2003.Supported in part by grant FONDECYT (Fondo Nacional de Desarrollo Cientifico y Tecnologico) 1020960 (I. I. W.).Address correspondence and reprint requests to Dr. Ignacio I. Wistuba, Department of Anatomic Pathology, Pontificia Universidad Catolica de Chile, Marcoleta 367, PO Box 114-D Santiago, Chile (e-mail:
[email protected]).AbstractDespite well-established histopathological features and the development of immunostaining of human neoplasms, there are a number of cases in which surgical pathologists cannot assure the origin of synchronous and metachronous tumors. In many cases, the classification of these lesions as either two separate primary tumors or as a single primary tumor with a metastasis has significant implications with respect to patient prognosis and recommendations for therapy. To establish the origin of tumors, we assessed tumor cell clonality using PCR-based microsatellite analysis on microdissected archival tissues for loss of heterozygosity (LOH) and microsatellite instability (MSI) in a series of 19 paired synchronous and metachronous tumors from several organs. As a control group, 15 autopsy cases with an unequivocally recognizable primary tumor and associated metastases were also examined. Based on LOH and MSI findings, and using a panel of 4 to 12 (median 7) microsatellite markers, we were able to establish the clonal pattern of microsatellite changes in 17 out of 19 (89%) biopsy cases and thus determine if they were either double primary tumors (41%) or metastases (59%). Of interest, identical or similar pattern of microsatellite abnormalities were detected in 15 primary tumors and corresponding metastasis from autopsies. Our results indicate that microsatellite analysis for LOH and MSI, as an expression of clonality, provides a useful tool to distinguish double primary neoplasms and metastases in synchronous and metachronous tumors.Despite well-established histopathological features and the development of immunostaining of human neoplasms, there are a number of cases in which surgical pathologists cannot assure the origin of synchronous and metachronous tumors. 1–7 In many cases, the classification of these lesions as either two separate primary tumors, or as a single primary tumor with metastasis, has significant implications with respect to patient prognosis and recommendations for therapy. Multiple synchronous and metachronous tumors have been described in a variety of anatomic sites, including the lung, 2,5 colorectum, 8,9 stomach, 10,11 endometrium and ovary, 1,3,4 urothelial epithelium, 12 and abdomen (specifically, ovarian and appendiceal pseudomyxoma peritonei). 3 The clinicopathologic distinction between a single tumor with metastasis from two independent primary tumors may represent a diagnostic conundrum.Currently, clonal derivation of cells is the hallmark of neoplasia and strongly implicates acquired somatic mutations resulting in a survival advantage to a clonal cell population. 13 Thus, clonality analysis could be a powerful tool in determining if two populations of cells (such as cells from similar or different tumors of the same patient) are genetically similar or different, and the information thus obtained may be of great clinical use. There are several tests to assess clonality in neoplasms, including methylation-related inactivation of one X chromosome in females and the analysis of polymorphic DNA regions from patients heterozygous for a given marker. 14 X-linked clonality examination can be used only for analysis in females, does not assess tumor heterogeneity, and does not provide any data on the precise genetic alteration responsible for clonal proliferation. 14 In contrast, the analysis of polymorphic repetitive DNA sequences (microsatellites) provides a useful tool for clonal assessment of tumor cells, as well as complementary information related to the pathway of neoplastic transformation. 1,2,4–7,14Microsatellites are highly polymorphic short tandem repeat DNA sequences. 15 Several of these microsatellite markers have been used extensively for DNA fingerprinting and are very useful in genetic linkage analysis. The allelic loss or loss of heterozygosity (LOH) of a given microsatellite marker could be linked to loss of tumor suppressor gene (TSG) by DNA deletions, which would also contribute to the multistep carcinogenesis process selecting cells with growth advantages. 16,17 In addition to those specific genetic changes, other evidence indicates that genetic instability occurs in human cancers. This evidence includes changes in the length of microsatellite markers, frequently present in a wide variety of cancer types, and is known as microsatellite instability (MSI). 18The development of methodologies for PCR-based assays in small number of cells from formalin-fixed, paraffin-embedded tissues, and of precise microdissection techniques have facilitated the analyses of genetic abnormalities (including LOH and MSI) in tissues processed using conventional histopathological techniques. 19 Few studies have reported the use microsatellite analysis in determining clonality in synchronous and metachronous tumors, particularly in the lung, 2,5 colorectum, 8,9 stomach 10,11 endometrium and ovary, 1,3,4,20 ovary and appendix, 3 N and urinary bladder. 12 In addition, case reports showing the application of microsatellite analysis to establish the origin of synchronous tumors have been published. 7,21–24 However, to the best of our knowledge, no study of a series of cases from general surgical pathology practice, including tumors from various organs, has been reported. In this study, we performed a PCR-based microsatellite analysis for LOH and MSI of 19 synchronous and metachronous tumors from several organs to address clonality and to establish the origin of the tumors. We used a control group of 15 autopsy cases having neoplastic disease with unequivocally recognizable primary tumors and metastases.MATERIAL AND METHODSSelection of casesCases from patients with synchronous and metachronous tumors in which clinicopathologic distinction between a single tumor with metastasis from two independent primary tumors presented a diagnostic conundrum were selected. The histopathological features were reviewed by at least two surgical pathologists. They were classified as synchronous (diagnosis of both tumors at the same time) or metachronous (diagnosis of the second tumor at least 6 months after the first operation).We studied 19 cases of double neoplasms from various locations comprised of 12 synchronous and 7 metachronous tumors. To examine if our molecular analysis was a reliable marker for clonality and metastasis, we used a control group of 15 autopsy cases harboring neoplastic disease with easily recognizable primary tumor and metastases. The clinicopathologic features of the control group and biopsy cases studied are shown in Table 1 and 2, respectively.JOURNAL/dimp/04.03/00019606-200309000-00006/table1-6/v/2021-02-17T195847Z/r/image-tiffClinicopathologic features of primary tumors and metastases from autopsiesJOURNAL/dimp/04.03/00019606-200309000-00006/table2-6/v/2021-02-17T195847Z/r/image-tiffClinicopathologic features of synchronous (S) and metachronous (M) tumors analyzedMicrodissection and DNA extractionSerial 5-μm sections from each tumor were cut from archival, formalin-fixed, paraffin-embedded tissues. The slides were stained with H&E, and one of the slides was cover-slipped. The cover-slipped slide was used as a reference to localize regions of interest for microdissection on the other slides. Precise microdissection from archival paraffin embedded tissues was performed under microscopic visualization using a micromanipulator. DNA extraction was performed using proteinase K, as previously described. 25 Briefly, the DNA was extracted by digesting the cells in buffer consisting of tromethamine (pH, 8.3), 50 mmol/L; ethylenediaminetetraacetic acid, 1 mmol/L; 0.5% Tween 20; and proteinase K, 200 μg/mL at 37°C for 24 to 36 hours, followed by 10 minutes of incubation at 100°C to destroy any remaining proteinase K activity. After DNA extraction, 5 μL of the proteinase K–digested samples containing DNA from at least 200 cells were used directly for each PCR reaction.Microsatellite PCR-based analysisTo evaluate microsatellite abnormalities such as LOH and microsatellite instabilityMSI, we used a total of 24 primers flanking dinucleotide and multinucleotide microsatellite repeat polymorphisms located at 10 chromosomal arms having regions with frequent allelic loss in many human tumors. The microsatellite markers used and the chromosomal arm location were as following: 3p (D3S2432, D3S1029, D3S1234, D3S4623, D3S1274, D3S1511, D3S1076), 5q (LNSCA), 6q (D6S300, D6S262, D6S246); 8p (D8S264, NEFL, D8S136), 9p (IFNA, D9S1748), 10q (D10S2491), 11q (PYGM, FGF-3), 13q (RB-CA), 17p (TP53-CA, TP53-penta, D17S969), and 22q (D22S1150). The markers were selected from the Genome Database (http://www.gdb.org/). For each case, 4 to 12 (median 7) microsatellite markers were examined.A two-round PCR (using the same set of primers in two consecutive amplifications) methods was used. Multiplex PCR was performed during the first amplification, followed by uniplex PCR for individual markers. In the multiplex PCR, four to six markers were amplified during the same reaction. Volumes of 50 μL were used for each multiplex reaction, containing 20 mmol/L Tris (pH 8.3), 50 mmol/L KCl, 2.0 mmol/L MgCl2, 400 mmol/L of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP), 0.5 mmol/L of each forward and reverse primer, and 3.5 units of AmpliTaq Gold (Perkin Elmer, Foster City, CA). The first PCR product was diluted 1:10 in double distilled sterile water and used for the second PCR reaction, which was performed in a 10 mL reaction volume containing 20 mmol/L Tris (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, 100 mmol/L of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP), 0.5 mmol/L of forward and reverse primer, 0.25 units of Taq polymerase (GIBCO-BRL, Alameda, CA), 0.25 mL of [alfa-32P]dCTP (3000 Ci/mol, 10 mCi/mL, Amersham LIFE SCIENCE, Arlington Heights, IL), and 1 to 2 mL of diluted first PCR product. For both PCR reactions, a “touch-down” PCR was performed. After initial denaturation of 94°C for 12 minutes, 11 cycles each consisting of denaturation at 95°C for 20 seconds, annealing at 65° to 56°C for 55 seconds, and extension at 72°C for 20 seconds were performed, followed by an additional 24 cycles that included denaturation at 90°C for 20 seconds, annealing at 55°C for 20 seconds, and extension at 72°C for 20 seconds. Then, the second PCR reaction product was diluted 1:3 with loading buffer, heat denatured, and separated by electrophoresis on a denaturing 6% polyacrylamide gel containing urea.For each case, constitutional heterozygosity was determined by examination of non-malignant tissue. LOH was scored by visual detection of complete absence of one tumor allele in heterozygous (ie, “informative”) cases. MSI were detected by shift in the mobility of one allele, irrespective of whether it was accompanied by LOH.DNA quality was assessed evaluating the microsatellite amplification rate. In all samples examined, the amplification rate was higher than 93% (230 out of 247 attempts), indicating that DNA was suitable for the microsatellite analyses proposed. Because artifacts resulting from PCR amplification may be mistaken for LOH, especially when minute amounts of input DNA are used, 62 out of 89 (70%) examples of LOH and MSI were repeated for confirmation. Of those, the same microsatellite abnormality was detected in virtually all (59 out of 62, 95%) replicate experiments.RESULTSMicrosatellite analysis on primary tumors and metastases from autopsiesTo determine if microsatellite abnormalities (pattern of LOH and MSI) can be used as reliable marker to distinguish between the same and different tumors, we analyzed 15 cases of paired primary tumors and metastases obtained from autopsies (referred to as control group) (Table 1). In all autopsy cases selected, primary tumors and their corresponding metastasis were established using unequivocal gross examination and histopathological features. Cases of this control group were selected randomly from a large series of autopsy archival cases available to us. All tumors were carcinomas from various organs (Table 1).Using a panel of 3 to 9 (median 7) microsatellite markers, all paired primary tumors and metastasis demonstrated either LOH and/or MSI. In all cases the microsatellite abnormality pattern in the metastases indicated a clonal relationship with the pattern detected in the primary tumor (Tables 1 and 3). Five representative examples of the detailed pattern of microsatellite abnormalities detected in some of the paired cases are shown in Figure 1. The clonally related patterns were either “identical,” when the same combination of parental alleles were lost or the same shifted band shifts as an indicator of microsatellite instability were detected, or “similar,” when the metastases exhibited additional microsatellite abnormalities to those present in the primary tumor (Fig. 1). The latter phenomenon was seen in 10 out of 15 (67%) autopsy cases analyzed and was regarded as a phenomenon related to tumor progression.JOURNAL/dimp/04.03/00019606-200309000-00006/table3-6/v/2021-02-17T195847Z/r/image-tiffSummary of clonality assessment of double primary tumors and metastases, using PCR-based microsatellite analysis for LOH and MSIJOURNAL/dimp/04.03/00019606-200309000-00006/figure1-6/v/2021-02-17T195847Z/r/image-png
Five examples of microsatellite analysis for clonality in primary tumors and metastases from autopsies. For each case, histopathology, microsatellite abnormality pattern (T1 = primary tumor; T2 = metastasis), and representative autoradiographs for loss of heterozygosity (LOH, closed arrowhead) and microsatellite abnormality (MSI, opened arrowhead) are shown. All cases showed identical or similar pattern of microsatellite abnormalities between primary tumor and metastasis indicating clonal relationship. There are examples, case nos. 12 (markers D6S300, D6S262 and D8S264) and 14 (D3S1234), in which additional genetic MSI changes are present in metastasis compared with primary tumor, related to a tumor progression phenomenon. Horizontal bars on the left of the autoradiographs indicate the main allelic bands.Both identical and similar patterns of microsatellite abnormalities detected in paired primary tumors and their corresponding metastasis were based mostly on microsatellite instability changes (10 out of 15 paired cases, 67%;Table 3). The same combination of parental allele lost as indication of an identical or similar clonal pattern was found in 5 out of 15 cases (33%). The number of microsatellite markers needed to establish a clonally related pattern between primary tumors and metastases ranged between 2 to 6.Microsatellite analysis synchronous and metachronous tumors from biopsiesNineteen synchronous and metachronous tumors were examined for microsatellite abnormalities to determine if they were clonally related, using the pattern of allelic loss and microsatellite instability (Table 2). Of those, two cases (11%) did not demonstrate any microsatellite abnormality in all polymorphic markers examined; therefore, no conclusion regarding their clonal relationship could be established. Both cases (no. 14 and no. 18, Table 2) were analyzed for 10 and 7 microsatellite markers, respectively.Using a panel of 4 to 12 (median 7) microsatellite marker, any microsatellite abnormality was detected in 17 (89%) cases of synchronous and metachronous tumors examined (Table 3). Based on those changes an identical or similar pattern of microsatellite changes indicating a clonal relationship was established in 10 cases (59%), suggesting that one tumor was a metastasis from the other (Table 2). In the remaining 7 cases (41%), the pattern of microsatellite changes was not clonally related, indicating the presence of double primary tumors. Five representative examples of the detailed pattern of microsatellite abnormalities detected in some paired primary/metastasis and double primary tumors cases are shown in Figure 2.JOURNAL/dimp/04.03/00019606-200309000-00006/figure2-6/v/2021-02-17T195847Z/r/image-png
Five examples of microsatellite analysis for clonality in synchronous and metachronous tumors. For each case, histopathology, microsatellite abnormality pattern (T1 = primary tumor; T2 = second primary or metastasis), and representative autoradiographs for loss of heterozygosity (LOH, closed arrowhead) and microsatellite abnormality (MSI, opened arrowhead) are shown. Case nos. 2, 4, and 16 were considered double primary tumors (DPT) based on their different pattern of microsatellite abnormalities. Case nos. 8 and 17 were considered metastases (MET) based on identical or similar pattern of microsatellite abnormalities between both tumors indicating a clonal relationship. Horizontal bars on the left of the autoradiographs indicate the main allelic bands.The determination of clonal relationship between metachronous and synchronous tumors was based on patterns of MSI in 9 out of 17 (53%) cases, and on patterns of parental allele lost (ie, LOH) in the remaining 8 cases (47%;Table 3). The number of microsatellite markers necessary to establish a clonal-related pattern between primary tumors and metastases in biopsies ranged from 1 to 5.Although the purpose of this study was to test the microsatellite analysis to distinguish double primary tumor from metastasis, in a subset of biopsy cases (#5, 8, and 16, Table 2) the molecular findings were correlated with the immunohistochemical profile. For example, in case #5, tumor cells from both the malignant pancreatic islet cell tumor and its corresponding synchronous neuroendocrine carcinoma cells in the tonsil, demonstrated cytoplasmic immunolabeling with neuroendocrine markers chromogranin, synaptophy sin and neuron-specific enolase. Neoplastic cells at both sites did not label with insulin, glucagon and vasoactive inhibitor peptide (VIP). These findings correlate with microsatellite data which indicated that tonsil neuroendocrine carcinoma was most likely a metastasis from the malignant islet cell pancreatic tumor. In contrast, in case #8, the primary thyroid follicular carcinoma cells were strongly positive for thyroglobulin; however, osseous metastases were negative. Thus, in this case, it was the molecular analysis which clearly indicated that bone tumor was a metastasis from the thyroid carcinoma. Similarly, in case #16, immunohistochemical markers such as estrogen and progesterone receptors and BRST-2 antigen expressed by the primary breast cancer cells were not detected in the lymph node metastasis. In this case the microsatellite abnormalities were the only indicators available to exclude a metastasis.DISCUSSIONOur results indicate that microsatellite analysis for LOH and MSI, as an expression of clonality, provides a useful tool to distinguish double primary neoplasms from metastases in synchronous and metachronous tumors. In 17 out of 19 cases, using a panel of 4 to 12 (median 7) microsatellite markers, and based on LOH and MSI findings, we were able to establish the clonal pattern of microsatellite changes and thus determine if they were either double primary tumors (41%) or metastasis (59%). The finding of similar and identical patterns of microsatellite abnormalities in 15 primary tumors and corresponding metastasis from autopsies in which the origin of both synchronous tumors was unequivocal, confirms our conclusion.Relatively few studies reporting microsatellite analysis to determine clonality in synchronous and metachronous tumors have been published. Those studies have been mainly focused to understand the pathogenesis of multiple tumors of lung, 2,5 gastrointestinal tract, 8–11 female genital organs, 1,3,4,20 and urinary bladder. 12 In addition, few case reports showing the application of microsatellite analysis to establish the origin of synchronous tumors have been published. 7,21–24 Thus, to the best of our knowledge, this is the first study in which microsatellite analysis methodology has been applied to distinguish double primary tumors or metastases in a series of tumors from several organs taken from surgical pathology practice.The paired cases analyzed in this study corresponded to a variety of tumor types from different organs (Table 2). Of those, seven cases (nos. 1, 2, 4, 6, 7, 13, and 19) involved synchronous or metachronous neoplasms arising in the female genital tract, and four of them were synchronous ovarian cancers with colonic, gallbladder, or endometrial adenocarcinomas. While the presence of simultaneous carcinomas involving both ovary and endometrium is a relatively common phenomenon, 1,4 ovarian metastasis from gastrointestinal tract tumors, 26 particularly colon, are relatively infrequent. 27 Six paired cases (nos. 1, 4, 5, 6, 9, and 11) analyzed included gastrointestinal, pancreatic, and extrahepatic bile duct tumors. Of those, one case was a pancreatic adenocarcinoma with distant metastasis, and two were gallbladder adenocarcinomas with synchronous, independent choledochal and ovarian adenocarcinomas. The other pancreatic tumor examined (case no. 5) was an islet cell tumor with malignant histopathological features, including venous and perineural invasion, paired with a corresponding synchronous neuroendocrine carcinoma invading the tonsil. Strikingly, both tumors demonstrated an identical pattern of microsatellite markers, including two examples of MSI, indicating that the neuroendocrine carcinoma invading the tonsil was most likely metastasis from the pancreatic neoplasm. The presence of an identical neuroendocrine immunohistochemical profile in both tumors further supported this conclusion. Four cases examined (nos. 7, 9, 15, and 18) involved the lung, and in all but one, the lung lesion was a metachronous neoplasm. One case (no. 15) corresponded to a synchronous bilateral bronchioalveolar adenocarcinoma, which has been described as relatively frequent multifocal tumor. 28 Recent microsatellite analysis of multiple synchronous and metachronous lung cancers data suggests that molecular analysis can help fingerprint tumors and has the potential to significantly impact management and prognosis of patients. 5Although tumor cells are considered clonal in origin, 29 genetic tumor cell heterogeneity representing subclonal drifts has been well characterized. 30 Tumor cell heterogeneity is related to the phenomenon of genetic instability associated with tumor progression. Examples include the presence of oncogene and tumor suppressor gene mutations, which provide proliferation advantages or apoptotic dysregulation. 31 Our finding of similar or identical patterns of LOH and MSI comparing primary tumor and metastases from autopsy cases supports the concept of clonal expansion of tumors and their metastasis. In the same group, more genetic changes (67% of cases) were detected in metastases compared with their corresponding primary tumor, suggesting the accumulation of genetic changes related to tumor progression. This phenomenon was also detected in biopsy cases that were considered a metastasis based on their comparative microsatellite profile.The microsatellite analysis approach for clonality is based in the identification of LOH and/or MSI. Several allelotyping analyses of cancers have demonstrated that allelic loss is a widespread phenomenon in neoplasms. 32–35 Although there is a great deal of overlap in chromosomal regions with frequent deletions between tumor types, many cancers are associated with site-specific chromosomal losses. 32–35 The microsatellite marker selection used to address clonality of tumors was performed based on the tumor type examined. In our study, a subset (median 7; range 4-12) of microsatellites were selected from a panel of 24 highly polymorphic markers located on chromosomal regions (on arms 3p, 5q, 6q, 8p, 9p, 10q, 11q, 13q, 17p, and 22q) frequently deleted in cancers, and harboring several known and putative tumor suppressor genes, such as 3p22-24 (RARβ gene), 3p14.2 (FHIT gene), 3p12 (DUTT1 gene), 5q22 (APC-MMC genes region), 9p21 (CDKNA2 gene), 10q22 (PTEN gene), 13q12 (Rb gene), and 17p13 (TP53 gene). For clonality assessment, LOH was scored by comparing the loss or retention of identical or different parental allele in the same microsatellite marker between synchronous and metachronous tumors. The odds of loosing the same parental allele for synchronous and metachronous tumors by chance alone is high (50%) and decreases with increasing number of markers examined demonstrating loss of the same parental allele. The presence of different parental alleles lost at the same microsatellite marker between synchronous and metachronous tumors are an indicator of no clonal relationship. The same or different pattern of losses, involving identical or different parental alleles, allowed us to establish clonality in a subset (33%) of metastases from autopsy cases and a larger proportion (47%) of biopsy cases.In addition to those specific genetic changes, other evidence indicates that genetic instability occurs in human cancers. This evidence includes changes in the length of microsatellite markers, frequently present in a wide variety of cancer types, and known as microsatellite instability (MSI). 18 Although MSI abnormalities have been associated with defects in mismatch repair genes, their pathogenesis is not fully understood in all human neoplasms. 18 In any case, MSI changes represent random alterations in the length of repetitive DNA sequences and are a reliable marker for metastasis when the same MSI pattern is present in both synchronous and metachronous tumors. 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Link]1118204400019606-200309000-0000600004347_1999_18_320_krebs_adenocarcinomas_|00019606-200309000-00006#xpointer(id(citation_FROM_JRF_ID_d2108e918_citationRF_FLOATING))|11065404||ovftdb|SL0000434719991832011065404citation_FROM_JRF_ID_d2108e918_citationRF_FLOATING[Full Text]00004347-199910000-0000500019606-200309000-0000600004347_1999_18_320_krebs_adenocarcinomas_|00019606-200309000-00006#xpointer(id(citation_FROM_JRF_ID_d2108e918_citationRF_FLOATING))|11065213||ovftdb|SL0000434719991832011065213citation_FROM_JRF_ID_d2108e918_citationRF_FLOATING[CrossRef]10.1097%2F00004347-199910000-0000500019606-200309000-0000600004347_1999_18_320_krebs_adenocarcinomas_|00019606-200309000-00006#xpointer(id(citation_FROM_JRF_ID_d2108e918_citationRF_FLOATING))|11065405||ovftdb|SL0000434719991832011065405citation_FROM_JRF_ID_d2108e918_citationRF_FLOATING[Medline Link]10542939Clinicopathologic features of primary tumors and metastases from autopsiesClinicopathologic features of synchronous (S) and metachronous (M) tumors analyzedSummary of clonality assessment of double primary tumors and metastases, using PCR-based microsatellite analysis for LOH and MSI
Five examples of microsatellite analysis for clonality in primary tumors and metastases from autopsies. For each case, histopathology, microsatellite abnormality pattern (T1 = primary tumor; T2 = metastasis), and representative autoradiographs for loss of heterozygosity (LOH, closed arrowhead) and microsatellite abnormality (MSI, opened arrowhead) are shown. All cases showed identical or similar pattern of microsatellite abnormalities between primary tumor and metastasis indicating clonal relationship. There are examples, case nos. 12 (markers D6S300, D6S262 and D8S264) and 14 (D3S1234), in which additional genetic MSI changes are present in metastasis compared with primary tumor, related to a tumor progression phenomenon. Horizontal bars on the left of the autoradiographs indicate the main allelic bands.
Five examples of microsatellite analysis for clonality in synchronous and metachronous tumors. For each case, histopathology, microsatellite abnormality pattern (T1 = primary tumor; T2 = second primary or metastasis), and representative autoradiographs for loss of heterozygosity (LOH, closed arrowhead) and microsatellite abnormality (MSI, opened arrowhead) are shown. Case nos. 2, 4, and 16 were considered double primary tumors (DPT) based on their different pattern of microsatellite abnormalities. Case nos. 8 and 17 were considered metastases (MET) based on identical or similar pattern of microsatellite abnormalities between both tumors indicating a clonal relationship. Horizontal bars on the left of the autoradiographs indicate the main allelic bands.Microsatellite Analysis of Synchronous and Metachronous Tumors: A Tool For Double Primary Tumor And Metastasis AssessmentTang Moying BS; Pires, Yumai MD; Schultz, Marcela MD; Duarte, Ignacio MD; Gallegos, Marcela MD; Wistuba, Ignacio I. MDArticleArticle312p 151-159
