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Loss of heterozygosity in clonal evolution with genetic progression and divergence in spindle cell carcinoma of the gallbladder
Loss of Heterozygosity in Clonal Evolution With Genetic Progression and Divergence in Spindle Cell Carcinoma of the Gallbladder ATSUSHI ARAKAWA, MD, HIROAKI FUJII, MD, TOSHIHARU MATSUMOTO, MD, SHU HIRAI, MD, AND KOICHI SUDA, MD Spindle cell carcinoma (SpCC) of the gallbladder is a rare neoplasm that shows carcinoma with a variable component of sarcomatoid spindle cells. The clinical and pathological features of this neoplasm have been well documented, but the histogenesis has long been a matter of speculation. In an attempt to clarify the clonality and genetic relationships involved in the evolution of this neoplasm, we microdissected a total of 18 carcinomatous and sarcomatous foci from 2 gallbladder SpCCs and analyzed the allelic status with 42 microsatellite markers on chromosomal arms 1p, 1q, 3p, 4q, 5q, 6q, 8p, 9p, 10q, 11p, 11q, 13q, 16q, 17p, 17q, 18q, and 22q. The 2 cases examined had a polypoid tumor in the gallbladder, in which both adenocarcinomatous and sarcomatoid spindle cell components were identified histologically. In both SpCCs, homogenous allelic losses were identified in both the carcinomatous and sarcomatous components; 17p, 18q, and 5q in case 1 and 17p and 11q in case 2. These indicated that both SpCCs had a single clonal origin. In case 1, additional loss of heterozygosity (LOH; 6q) consisting of genetic progression occurred in both the carcinomatous and sarcomatoid components. In case 2, there was additional LOH (9p) in the carcinomatous components and additional microsatellite instability at
D5S644 in both the carcinomatous and sarcomatoid components, indicating a monoclonal neoplasm with genetic progression and divergence. In the 2 cases, the genetic changes indicated that an original clone of a pure adenocarcinoma apparently acquired sarcomatoid spindle cell phenotype by successive genetic changes. On the other hand, we saw no evidence of tumors in which a sarcomatoid spindle cell appeared to give rise to a carcinomatous subclone in the examined cases. In conclusion, the current study includes the first LOH analyses of SpCC of the gallbladder. Our data support the concept that gallbladder SpCC is derived from a single clone originating from a carcinoma. Furthermore, we showed genetic heterogeneity accompanying the phenotypic divergence, with patterns of genetic alterations that are consistent with both the progression and divergence within the individual tumors. HUM PATHOL 35:418-423. © 2004 Elsevier Inc. All rights reserved. Key words: spindle cell carcinoma, gallbladder, loss of heterozygosity, single clone, genetics, progression, divergence. Abbreviations: SpCC, spindle cell carcinoma; CS, carcinosarcoma; LOH, loss of heterozygosity; PCR, polymerase chain reaction; H&E, hematoxylin and eosin.
Spindle cell carcinoma (SpCC) of the gallbladder is an uncommon neoplasm that is characterized by components admixed with carcinoma cells and sarcomatoid spindle cells.1,2 The carcinomatous components are usually adenocarcinoma and rarely include squamous cell carcinomas.2 SpCC has been called by other names, including sarcomatoid carcinoma, pseudosarcoma, and carcinosarcoma (CS).2 The clinical and pathological features of SpCC of the gallbladder have been well documented, but the histogenesis has long been a matter of speculation. Histological studies including immunostaining showed the epithelial nature of sarcomatoid spindle cells and the transition between the carcinoma cells and spindle cells in SpCC of the gallbladder.2 These findings led to the speculation that SpCC of the gallbladder is derived from a carcinomatous component.2 Although our previous studies on loss of heterozygosity (LOH) indicated that LOH analysis is useful to deter-
mine the pathogenesis of the tumors with complex histologic patterns,3,4 LOH studies of SpCC of the gallbladder have not been published. We microdissected multiple neoplastic foci and performed LOH analyses on 2 cases with SpCC of the gallbladder. Our LOH analyses offered new information on the pathogenesis and genetic changes in SpCC of the gallbladder.
From the First Department of Pathology and Second Department of Pathology, Juntendo University, School of Medicine, Tokyo, Japan. Accepted for publication October 22, 2003. Address correspondence and reprint requests to Atsushi Arakawa, MD, First Department of Pathology, Juntendo University, School of Medicine, Hongo 2-1-1, Bunkyo-ku, 113-8421, Tokyo, Japan. 0046-8177/$—see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2003.10.026
MATERIALS AND METHODS Case Selection Samples for the present genetic analysis were taken from surgical materials that were obtained from 8 patients who each had SpCC of the gallbladder. The DNA quality of each case was assessed for adequate and reproducible polymerase chain reaction (PCR) amplification, and 2 of the 8 cases were selected for the present molecular genetic study. The 2 cases were of women 63 and 71 years of age. The carcinomatous and sarcomatoid spindle cell components were evaluated by using hematoxylin and eosin (H&E), periodic acid–Schiff, and alcian blue stains, as well as immunohistochemical stains. The antibodies used for the immunohistochemical staining consisted of cytokeratins, including CAM5.2 (Becton Dickinson, San Jose, CA; 1:800) and AE1/ AE3 (Dakopatts, Glostrup, Denmark; 1:100), epithelial membrane antigen (E29, Dakopatts; 1:100), vimentin (V9, Dakopatts; 1:200), muscle actin (HHF35, Dakopatts; 1:100), smooth muscle actin (1A4, Dakopatts; 1:200), desmin (D33, Dakopatts; 1:400), S-100 protein (Dakopatts; 1:400), and carcinoembryonic antigen (CEA; Dakopatts; 1:200). Enzyme
418
LOH IN CLONAL EVOLUTION IN GALLBLADDER SPCC (Arakawa et al)
(proteinase K, 10 minutes) antigen retrieval was used for CAM 5.2 and AE1/AE3 immunostains.
Microdissection and DNA Extraction Serial 8-m sections were cut, deparaffinized, stained with H&E, visualized with an inverted microscope, and microdissected with a 27-gauge needle. Eight to 10 carcinomatous and sarcomatoid spindle cell foci were individually microdissected from each gallbladder tumor (Fig 1A). A total of 18 foci were microdissected. In addition, reference control tissue was dissected from the adjacent nonmalignant stroma, epithelium, or inflammatory infiltrate. The microdissected tissues were digested overnight at 50°C in buffer containing 0.5% Nonidet P-40, 50 mmol/L Tris-HCl pH 8.0, 1 mmol/L ethylenediamine tetraacetic acid, and 200 g/mL of proteinase K. The lysates were heated at 95°C for 10 minutes and were stored at ⫺20°C until subjected to PCR.
Detection of LOH The PCR reactions contained 1 L of DNA lysate, 0.4 mol/L [␥-32P] ATP-radiolabeled microsatellite primers, 0.2 mmol/L deoxyribonucleoside triphosphate, 10 mmol/L TrisHCl pH 8.3, 1.5 mmol/L MgCl2, 50 mmol/L KCl, and 0.4 U Taq polymerase, in a total reaction volume of 10 L. Taq polymerase was added to the reactions prewarmed to 94°C (hot-start PCR), and the samples were amplified with 35 cycles of PCR amplification. The PCR products were separated on a 5% denaturing polyacrylamide-urea-formamide gel, and LOH was determined based on a ⬎75% reduction of the relative intensity in 1 of the 2 alleles, compared with those in the reference control. Elimination of PCR artifacts was performed as described previously.5 When only a portion of the microdissected foci showed LOH, PCR reactions were repeated at least 3 times to confirm the LOH and exclude spurious PCR reactions. Other informative microsatellite markers located on the same chromosomal arm also confirmed LOH. Microdissection was repeated as required. LOH is detected either homogenously or heterogenously in the microdissected foci of each chromosomal locus. When homogenous LOH of several chromosomal arms are detected throughout the microdissected foci, the lesion is considered to be a single clonal process. Heterogenous LOH is detected as genetic progression or genetic divergence. In the genetic progression, in addition to early and homogenous LOH events, LOH of some alleles are detected only in some of the microdissected tumor foci, indicating a linear genetic progression of the neoplastic clone. In the genetic divergence, in addition to early homogenous LOH events, different LOH patterns for several chromosomal loci are detected in different parts of the tumor. These findings indicate that the tumor starts as a single clone, and thereafter the neoplastic clone diverges in 2 or more directions like the branches of a tree.
Microsatellite Markers All of the PCR primers for the microsatellite markers were purchased from Research Genetics (Huntsville, AL). The microsatellite markers were selected to cover chromosomal regions commonly deleted in gallbladder carcinomas and various types of human carcinomas. The following primers were used: 1p and 1q (D1S500, D1S228, D1S158, and D1S318), 3p (D3S1286, and D3S1293), 4q (D4S424, D4S415, and D4S413), 5q (D5S2072, D5S421, and D5S1956), 6q (D6S264, D6S473, and D6S255), 8p (D8S255, D8S264, and D8S261), 9p (D9S1748, and D9S1749), 10q (D10S221,
D10S219, and D10S574), 11p (D11S1324), 11q (D11S29, and Int2), 13q (D13S166, D13S168, and D13S171), 16q (D16S265, D16S541, and D16S261), 17p (TP53, CHRNB1, and D17S786), 17q (D17S588, and D17S579), 18q (D18S46, D18S55, D18S474, and D18S487), and 22q (D22S270). These chromosomal arms harbor the following known tumor suppressor genes: FHIT, hMLH1, and VHL genes at 3p; APC at 5q; p16 and p15 at 9p; PTEN at 10q; MEN1 and ATM genes at 11q; RB1, ING1, and BRCA2 at 13q; TP53 at 17p; and DCC and DPC4 at 18q.
RESULTS Pathologic Features In the examined cases, the resected gallbladders had a polypoid tumor (Fig 1A) that was surrounded with hemorrhagic, erosive, and ulcerative areas. The maximal diameter of the polypoid tumor was 4 cm (case 1) and 3 cm (case 2). Histologically, both adenocarcinomatous and sarcomatoid spindle cell components, associated with the areas of admixture (cases 1 and 2) and transition (case 2) of the 2 phenotypes, were identified in the polypoid tumors (Fig 1C-D). In the areas surrounding the polypoid tumors, widespread adenocarcinoma in situ associated with invasive adenocarcinoma was present (Fig 1B). The adenocarcinoma cells presented as tubular and solid structures. None of the heterogenous components, such as osteosarcoma and chondrosarcoma, as well as squamous cell carcinomatous components were identified. Metastases to the lymph nodes and liver were seen in case 2, with metastases of the sarcomatoid cells alone. The examined cases were classified as pT2 according to the UICC TNM clinical classification.6 Immunostaining of epithelial markers, including cytokeratins and epithelial membrane antigen, showed positivity in both the carcinomatous and sarcomatoid components (Fig 1E and F). In the sarcomatoid components, immunohistochemical positivity for vimentin in 1 case (case 2) and S-100 protein positivity in both cases was noted. On the other hand, CEA positivity was noted in the carcinoma cells alone. Muscular markers, including muscle actin, smooth muscle actin, and desmin, were negative in each component. Loss of Heterozygosity In 2 SpCC cases, we succeeded in amplifying DNA from an 8-m H&E section; however, in 6 other cases we failed to amplify DNA. In both SpCCs, homogenous allelic losses were identified in both the carcinomatous and sarcomatous components: 17p, 18q, and 5q in case 1, 17p and 11q in case 2 (Figs 2 through 5). These findings indicated that both SpCCs were monoclonal neoplasms. In case 1, additional LOH (6q) consisting of genetic progression occurred in both the carcinomatous and sarcomatoid components (Figs 2 and 3) In case 2, there were additional LOH (9p) in the carcinomatous components and additional microsatellite instability at D5S644 in both the carcinomatous and sarcomatoid components, indicating a monoclonal
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FIGURE 1. (A) Low-power view of the section showing examples of microdissected foci from the polypoid tumor in case 1. L#, microdissected foci in case 1 (see Fig. 2). (B) Low-power view of adenocarcinoma in situ and invasive adenocarcinoma. (C, D) High-power view of spindle cell carcinoma of gallbladder. Note an admixture of adenocarcinoma and sarcomatoid spindle cells (C) and transition zone between adenocarcinoma and spindle cell carcinoma (D). (E) Immunostain for cytokeratin shows immunopositivity in both adenocarcinoma cells and sarcomatoid spindle cells. (F) High-power view of cytokeratin positivity in sarcomatoid spindle cells. (A-D, H&E stain; E-F, CAM5.2 immunostain; original magnification: (A) ⫻1, (B) ⫻40, (C and D) ⫻100, (E) ⫻40, (F) ⫻200.)
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FIGURE 2. Schematic gallbladder specimen and genetic progression of case 1. L#, microdissected foci.
FIGURE 3. Loss of heterozygosity in case 1. N, reference cell DNA; L#, microdissected neoplastic foci; *, reference alleles; arrowheads, LOH of all foci (D17S786, D5S2072); arrows, LOH of L2 to L6 only (D6S292, D6S383).
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FIGURE 4. Schematic gallbladder specimen and genetic-phenotypic relationship of case 2. L#, microdissected foci.
neoplasm with genetic progression and divergence (Figs 4 and 5). DISCUSSION Our previous LOH study of 17 gynecologic CSs is the largest number of cases with CSs to be subjected to LOH analysis.3 In the previous gynecologic CSs study, 16 tumors (94%) were monoclonal neoplasms, in which homogenous allelic losses were detected in different histological components.3 In the present study, in an attempt to clarify the clonality in the evolution of SpCC of the gallbladder, we examined numerous individually microdissected carcinomatous and sarcomatous foci for LOH by using microsatellite markers. Subsequently, the 2 cases we examined showed allelic losses shared by all the tumor foci, consistent with the monoclonal origin of these tumors. Although we examined only 2 cases in the current study, the demonstration of monoclonal evolution in the examined tumors in addition to the very high incidence of monoclonal neoplasms in gynecologic CSs3 led to the speculation that most SpCC of the gallbladder are monoclonal neoplasms. Nishihara and Tsuneyoshi2 performed an immunohistochemical study of 11 cases with SpCC of the gallbladder and reported that this neoplasm was epithelial in nature. They described the possibility that both adenocarcinomatous and squamous cell carcinomatous clones may be of epithelial origin.2 The genetic changes observed in our cases indicated that an original clone of a pure adenocarcinoma apparently acquired a sarcomatoid spindle cell phenotype by succes-
sive genetic changes. On the other hand, we saw no evidence of tumors in which sarcomatoid spindle cells appeared to give rise to a carcinomatous subclone in the examined cases. These genetic results support the concept that gallbladder SpCC is of epithelial origin. Moreover, we showed 2 cases of gallbladder SpCCs derived from a single clone originating from adenocarcinoma. In the current study, we noted evidence of genetic progression or genetic divergence in the examined tumors. A high frequency of genetic progression or divergence was reported in gynecologic,3 pulmonary,7 and urinary bladder CSs.8 Because genetic progression, divergence, or genetic bifurcations all lead to mosaics of neoplastic subclones, it is reasonable to assume that this heterogeneity may parallel the phenotypes of neoplasms with divergent histologic features like CSs and SpCC. Concerning the LOH in gallbladder CS, Hidaka and colleagues9 reported that LOH on 17p may play an important role in the evolution of gallbladder CS from a relatively early phase and that LOH on 9p and 18q may play roles in progression. Chang and colleagues10 reported that LOH on 5q is an early change in gallbladder carcinogenesis and that LOH on 3p and 9p is related to the progression of gallbladder CS. Furthermore, they described that LOH on 17p not only occurs in dysplasia but also increases during the subsequent stage.10 We found homogenous LOH on 17p, a chromosomal arm that includes the p53 locus, in the examined cases. Because 17p LOH occurs concordantly in sarcomatous and carcinomatous components, we sus-
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FIGURE 5. Representative microsatellite amplifications of case 2. N, reference cell DNA; L#, microdissected neoplastic foci; *, reference alleles; arrowheads, LOH of all foci (D11S29); thin arrows, LOH of L1 and L2 only (D9S1748); thick arrows, microsatellite instability (sporadic) of L3 to L8 (D5S644).
pect that p53 is affected very early in the development of SpCC as well as in CS of the gallbladder. In conclusion, the current study involves the first LOH analyses of SpCC of the gallbladder. Our data support the concept that gallbladder SpCC is derived from a single clone originating from a CS. Furthermore, we showed genetic heterogeneity accompanying the phenotypic divergence, with patterns of genetic alterations that are consistent with both the progression and divergence within the individual tumors. Acknowledgment. The authors thank Mr. D. Mrozek (Medical English Service, Kyoto, Japan) for the English revision.
REFERENCES 1. Guo K-J, Yamaguchi K, Enjoji M: Undifferentiated carcinoma of the gallbladder. A clinicopathologic, histochemical, and immunohistochemical study of 21 patients with a poor prognosis. Cancer 61:1872-1879, 1988 2. Nishihara K, Tsuneyoshi M: Undifferentiated spindle cell carcinoma of the gallbladder: A clinicopathologic, immunohistochemi-
cal, and flow cytometric study of 11 cases. HUM PATHOL 24:1298-1305, 1993 3. Fujii H, Yoshida M, Gong ZX, et al: Frequent genetic heterogeneity in the clonal evolution of gynecological carcinosarcoma and its influence on phenotypic diversity. Cancer Res 60:114-120, 2000 4. Fujii H, Zhu XG, Matsumoto T, et al: Genetic classification of combined hepatocellular-cholangiocarcinoma. HUM PATHOL 31:10111017, 2000 5. Yamano M, Fujii H, Takagaki T, et al: Genetic progression and divergence in pancreatic carcinoma. Am J Pathol 156:2123-2133, 2000 6. Hermanek P, Hutter RVP, Sobin LH, et al (eds): UICC TNM Atlas: Illustrated Guide to the TNM/pTNM Classification of Malignant Tumours, 4th ed. Berlin, Germany, Springer-Verlag, 1997 7. Dacic S, Finkelstein SD, Sasatomi E, et al: Molecular pathogenesis of pulmonary carcinosarcoma as determined by microdissection-based allelotyping. Am J Surg Pathol 26:510-516, 2002 8. Halachmi S, DeMarzo AM, Chow N-H, et al: Genetic alterations in urinary bladder carcinosarcoma: Evidence of a common clonal origin. Eur Urol 37:350-357, 2000 9. Hidaka E, Yanagisawa A, Sakai Y, et al: Losses of heterozygosity on chromosomes 17p and 9p/18q may play important roles in early and advanced phases of gallbladder carcinogenesis. J Cancer Res Clin Oncol 125:439-443, 1999 10. Chang HJ, Kim SW, Kim Y-T, et al: Loss of heterozygosity in dysplasia and carcinoma of the gallbladder. Mod Pathol 12:763-769, 1999
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D5S644 in both the carcinomatous and sarcomatoid components, indicating a monoclonal neoplasm with genetic progression and divergence. In the 2 cases, the genetic changes indicated that an original clone of a pure adenocarcinoma apparently acquired sarcomatoid spindle cell phenotype by successive genetic changes. On the other hand, we saw no evidence of tumors in which a sarcomatoid spindle cell appeared to give rise to a carcinomatous subclone in the examined cases. In conclusion, the current study includes the first LOH analyses of SpCC of the gallbladder. Our data support the concept that gallbladder SpCC is derived from a single clone originating from a carcinoma. Furthermore, we showed genetic heterogeneity accompanying the phenotypic divergence, with patterns of genetic alterations that are consistent with both the progression and divergence within the individual tumors. HUM PATHOL 35:418-423. © 2004 Elsevier Inc. All rights reserved. Key words: spindle cell carcinoma, gallbladder, loss of heterozygosity, single clone, genetics, progression, divergence. Abbreviations: SpCC, spindle cell carcinoma; CS, carcinosarcoma; LOH, loss of heterozygosity; PCR, polymerase chain reaction; H&E, hematoxylin and eosin.
Spindle cell carcinoma (SpCC) of the gallbladder is an uncommon neoplasm that is characterized by components admixed with carcinoma cells and sarcomatoid spindle cells.1,2 The carcinomatous components are usually adenocarcinoma and rarely include squamous cell carcinomas.2 SpCC has been called by other names, including sarcomatoid carcinoma, pseudosarcoma, and carcinosarcoma (CS).2 The clinical and pathological features of SpCC of the gallbladder have been well documented, but the histogenesis has long been a matter of speculation. Histological studies including immunostaining showed the epithelial nature of sarcomatoid spindle cells and the transition between the carcinoma cells and spindle cells in SpCC of the gallbladder.2 These findings led to the speculation that SpCC of the gallbladder is derived from a carcinomatous component.2 Although our previous studies on loss of heterozygosity (LOH) indicated that LOH analysis is useful to deter-
mine the pathogenesis of the tumors with complex histologic patterns,3,4 LOH studies of SpCC of the gallbladder have not been published. We microdissected multiple neoplastic foci and performed LOH analyses on 2 cases with SpCC of the gallbladder. Our LOH analyses offered new information on the pathogenesis and genetic changes in SpCC of the gallbladder.
From the First Department of Pathology and Second Department of Pathology, Juntendo University, School of Medicine, Tokyo, Japan. Accepted for publication October 22, 2003. Address correspondence and reprint requests to Atsushi Arakawa, MD, First Department of Pathology, Juntendo University, School of Medicine, Hongo 2-1-1, Bunkyo-ku, 113-8421, Tokyo, Japan. 0046-8177/$—see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2003.10.026
MATERIALS AND METHODS Case Selection Samples for the present genetic analysis were taken from surgical materials that were obtained from 8 patients who each had SpCC of the gallbladder. The DNA quality of each case was assessed for adequate and reproducible polymerase chain reaction (PCR) amplification, and 2 of the 8 cases were selected for the present molecular genetic study. The 2 cases were of women 63 and 71 years of age. The carcinomatous and sarcomatoid spindle cell components were evaluated by using hematoxylin and eosin (H&E), periodic acid–Schiff, and alcian blue stains, as well as immunohistochemical stains. The antibodies used for the immunohistochemical staining consisted of cytokeratins, including CAM5.2 (Becton Dickinson, San Jose, CA; 1:800) and AE1/ AE3 (Dakopatts, Glostrup, Denmark; 1:100), epithelial membrane antigen (E29, Dakopatts; 1:100), vimentin (V9, Dakopatts; 1:200), muscle actin (HHF35, Dakopatts; 1:100), smooth muscle actin (1A4, Dakopatts; 1:200), desmin (D33, Dakopatts; 1:400), S-100 protein (Dakopatts; 1:400), and carcinoembryonic antigen (CEA; Dakopatts; 1:200). Enzyme
418
LOH IN CLONAL EVOLUTION IN GALLBLADDER SPCC (Arakawa et al)
(proteinase K, 10 minutes) antigen retrieval was used for CAM 5.2 and AE1/AE3 immunostains.
Microdissection and DNA Extraction Serial 8-m sections were cut, deparaffinized, stained with H&E, visualized with an inverted microscope, and microdissected with a 27-gauge needle. Eight to 10 carcinomatous and sarcomatoid spindle cell foci were individually microdissected from each gallbladder tumor (Fig 1A). A total of 18 foci were microdissected. In addition, reference control tissue was dissected from the adjacent nonmalignant stroma, epithelium, or inflammatory infiltrate. The microdissected tissues were digested overnight at 50°C in buffer containing 0.5% Nonidet P-40, 50 mmol/L Tris-HCl pH 8.0, 1 mmol/L ethylenediamine tetraacetic acid, and 200 g/mL of proteinase K. The lysates were heated at 95°C for 10 minutes and were stored at ⫺20°C until subjected to PCR.
Detection of LOH The PCR reactions contained 1 L of DNA lysate, 0.4 mol/L [␥-32P] ATP-radiolabeled microsatellite primers, 0.2 mmol/L deoxyribonucleoside triphosphate, 10 mmol/L TrisHCl pH 8.3, 1.5 mmol/L MgCl2, 50 mmol/L KCl, and 0.4 U Taq polymerase, in a total reaction volume of 10 L. Taq polymerase was added to the reactions prewarmed to 94°C (hot-start PCR), and the samples were amplified with 35 cycles of PCR amplification. The PCR products were separated on a 5% denaturing polyacrylamide-urea-formamide gel, and LOH was determined based on a ⬎75% reduction of the relative intensity in 1 of the 2 alleles, compared with those in the reference control. Elimination of PCR artifacts was performed as described previously.5 When only a portion of the microdissected foci showed LOH, PCR reactions were repeated at least 3 times to confirm the LOH and exclude spurious PCR reactions. Other informative microsatellite markers located on the same chromosomal arm also confirmed LOH. Microdissection was repeated as required. LOH is detected either homogenously or heterogenously in the microdissected foci of each chromosomal locus. When homogenous LOH of several chromosomal arms are detected throughout the microdissected foci, the lesion is considered to be a single clonal process. Heterogenous LOH is detected as genetic progression or genetic divergence. In the genetic progression, in addition to early and homogenous LOH events, LOH of some alleles are detected only in some of the microdissected tumor foci, indicating a linear genetic progression of the neoplastic clone. In the genetic divergence, in addition to early homogenous LOH events, different LOH patterns for several chromosomal loci are detected in different parts of the tumor. These findings indicate that the tumor starts as a single clone, and thereafter the neoplastic clone diverges in 2 or more directions like the branches of a tree.
Microsatellite Markers All of the PCR primers for the microsatellite markers were purchased from Research Genetics (Huntsville, AL). The microsatellite markers were selected to cover chromosomal regions commonly deleted in gallbladder carcinomas and various types of human carcinomas. The following primers were used: 1p and 1q (D1S500, D1S228, D1S158, and D1S318), 3p (D3S1286, and D3S1293), 4q (D4S424, D4S415, and D4S413), 5q (D5S2072, D5S421, and D5S1956), 6q (D6S264, D6S473, and D6S255), 8p (D8S255, D8S264, and D8S261), 9p (D9S1748, and D9S1749), 10q (D10S221,
D10S219, and D10S574), 11p (D11S1324), 11q (D11S29, and Int2), 13q (D13S166, D13S168, and D13S171), 16q (D16S265, D16S541, and D16S261), 17p (TP53, CHRNB1, and D17S786), 17q (D17S588, and D17S579), 18q (D18S46, D18S55, D18S474, and D18S487), and 22q (D22S270). These chromosomal arms harbor the following known tumor suppressor genes: FHIT, hMLH1, and VHL genes at 3p; APC at 5q; p16 and p15 at 9p; PTEN at 10q; MEN1 and ATM genes at 11q; RB1, ING1, and BRCA2 at 13q; TP53 at 17p; and DCC and DPC4 at 18q.
RESULTS Pathologic Features In the examined cases, the resected gallbladders had a polypoid tumor (Fig 1A) that was surrounded with hemorrhagic, erosive, and ulcerative areas. The maximal diameter of the polypoid tumor was 4 cm (case 1) and 3 cm (case 2). Histologically, both adenocarcinomatous and sarcomatoid spindle cell components, associated with the areas of admixture (cases 1 and 2) and transition (case 2) of the 2 phenotypes, were identified in the polypoid tumors (Fig 1C-D). In the areas surrounding the polypoid tumors, widespread adenocarcinoma in situ associated with invasive adenocarcinoma was present (Fig 1B). The adenocarcinoma cells presented as tubular and solid structures. None of the heterogenous components, such as osteosarcoma and chondrosarcoma, as well as squamous cell carcinomatous components were identified. Metastases to the lymph nodes and liver were seen in case 2, with metastases of the sarcomatoid cells alone. The examined cases were classified as pT2 according to the UICC TNM clinical classification.6 Immunostaining of epithelial markers, including cytokeratins and epithelial membrane antigen, showed positivity in both the carcinomatous and sarcomatoid components (Fig 1E and F). In the sarcomatoid components, immunohistochemical positivity for vimentin in 1 case (case 2) and S-100 protein positivity in both cases was noted. On the other hand, CEA positivity was noted in the carcinoma cells alone. Muscular markers, including muscle actin, smooth muscle actin, and desmin, were negative in each component. Loss of Heterozygosity In 2 SpCC cases, we succeeded in amplifying DNA from an 8-m H&E section; however, in 6 other cases we failed to amplify DNA. In both SpCCs, homogenous allelic losses were identified in both the carcinomatous and sarcomatous components: 17p, 18q, and 5q in case 1, 17p and 11q in case 2 (Figs 2 through 5). These findings indicated that both SpCCs were monoclonal neoplasms. In case 1, additional LOH (6q) consisting of genetic progression occurred in both the carcinomatous and sarcomatoid components (Figs 2 and 3) In case 2, there were additional LOH (9p) in the carcinomatous components and additional microsatellite instability at D5S644 in both the carcinomatous and sarcomatoid components, indicating a monoclonal
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FIGURE 1. (A) Low-power view of the section showing examples of microdissected foci from the polypoid tumor in case 1. L#, microdissected foci in case 1 (see Fig. 2). (B) Low-power view of adenocarcinoma in situ and invasive adenocarcinoma. (C, D) High-power view of spindle cell carcinoma of gallbladder. Note an admixture of adenocarcinoma and sarcomatoid spindle cells (C) and transition zone between adenocarcinoma and spindle cell carcinoma (D). (E) Immunostain for cytokeratin shows immunopositivity in both adenocarcinoma cells and sarcomatoid spindle cells. (F) High-power view of cytokeratin positivity in sarcomatoid spindle cells. (A-D, H&E stain; E-F, CAM5.2 immunostain; original magnification: (A) ⫻1, (B) ⫻40, (C and D) ⫻100, (E) ⫻40, (F) ⫻200.)
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FIGURE 2. Schematic gallbladder specimen and genetic progression of case 1. L#, microdissected foci.
FIGURE 3. Loss of heterozygosity in case 1. N, reference cell DNA; L#, microdissected neoplastic foci; *, reference alleles; arrowheads, LOH of all foci (D17S786, D5S2072); arrows, LOH of L2 to L6 only (D6S292, D6S383).
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FIGURE 4. Schematic gallbladder specimen and genetic-phenotypic relationship of case 2. L#, microdissected foci.
neoplasm with genetic progression and divergence (Figs 4 and 5). DISCUSSION Our previous LOH study of 17 gynecologic CSs is the largest number of cases with CSs to be subjected to LOH analysis.3 In the previous gynecologic CSs study, 16 tumors (94%) were monoclonal neoplasms, in which homogenous allelic losses were detected in different histological components.3 In the present study, in an attempt to clarify the clonality in the evolution of SpCC of the gallbladder, we examined numerous individually microdissected carcinomatous and sarcomatous foci for LOH by using microsatellite markers. Subsequently, the 2 cases we examined showed allelic losses shared by all the tumor foci, consistent with the monoclonal origin of these tumors. Although we examined only 2 cases in the current study, the demonstration of monoclonal evolution in the examined tumors in addition to the very high incidence of monoclonal neoplasms in gynecologic CSs3 led to the speculation that most SpCC of the gallbladder are monoclonal neoplasms. Nishihara and Tsuneyoshi2 performed an immunohistochemical study of 11 cases with SpCC of the gallbladder and reported that this neoplasm was epithelial in nature. They described the possibility that both adenocarcinomatous and squamous cell carcinomatous clones may be of epithelial origin.2 The genetic changes observed in our cases indicated that an original clone of a pure adenocarcinoma apparently acquired a sarcomatoid spindle cell phenotype by succes-
sive genetic changes. On the other hand, we saw no evidence of tumors in which sarcomatoid spindle cells appeared to give rise to a carcinomatous subclone in the examined cases. These genetic results support the concept that gallbladder SpCC is of epithelial origin. Moreover, we showed 2 cases of gallbladder SpCCs derived from a single clone originating from adenocarcinoma. In the current study, we noted evidence of genetic progression or genetic divergence in the examined tumors. A high frequency of genetic progression or divergence was reported in gynecologic,3 pulmonary,7 and urinary bladder CSs.8 Because genetic progression, divergence, or genetic bifurcations all lead to mosaics of neoplastic subclones, it is reasonable to assume that this heterogeneity may parallel the phenotypes of neoplasms with divergent histologic features like CSs and SpCC. Concerning the LOH in gallbladder CS, Hidaka and colleagues9 reported that LOH on 17p may play an important role in the evolution of gallbladder CS from a relatively early phase and that LOH on 9p and 18q may play roles in progression. Chang and colleagues10 reported that LOH on 5q is an early change in gallbladder carcinogenesis and that LOH on 3p and 9p is related to the progression of gallbladder CS. Furthermore, they described that LOH on 17p not only occurs in dysplasia but also increases during the subsequent stage.10 We found homogenous LOH on 17p, a chromosomal arm that includes the p53 locus, in the examined cases. Because 17p LOH occurs concordantly in sarcomatous and carcinomatous components, we sus-
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LOH IN CLONAL EVOLUTION IN GALLBLADDER SPCC (Arakawa et al)
FIGURE 5. Representative microsatellite amplifications of case 2. N, reference cell DNA; L#, microdissected neoplastic foci; *, reference alleles; arrowheads, LOH of all foci (D11S29); thin arrows, LOH of L1 and L2 only (D9S1748); thick arrows, microsatellite instability (sporadic) of L3 to L8 (D5S644).
pect that p53 is affected very early in the development of SpCC as well as in CS of the gallbladder. In conclusion, the current study involves the first LOH analyses of SpCC of the gallbladder. Our data support the concept that gallbladder SpCC is derived from a single clone originating from a CS. Furthermore, we showed genetic heterogeneity accompanying the phenotypic divergence, with patterns of genetic alterations that are consistent with both the progression and divergence within the individual tumors. Acknowledgment. The authors thank Mr. D. Mrozek (Medical English Service, Kyoto, Japan) for the English revision.
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cal, and flow cytometric study of 11 cases. HUM PATHOL 24:1298-1305, 1993 3. Fujii H, Yoshida M, Gong ZX, et al: Frequent genetic heterogeneity in the clonal evolution of gynecological carcinosarcoma and its influence on phenotypic diversity. Cancer Res 60:114-120, 2000 4. Fujii H, Zhu XG, Matsumoto T, et al: Genetic classification of combined hepatocellular-cholangiocarcinoma. HUM PATHOL 31:10111017, 2000 5. Yamano M, Fujii H, Takagaki T, et al: Genetic progression and divergence in pancreatic carcinoma. Am J Pathol 156:2123-2133, 2000 6. Hermanek P, Hutter RVP, Sobin LH, et al (eds): UICC TNM Atlas: Illustrated Guide to the TNM/pTNM Classification of Malignant Tumours, 4th ed. Berlin, Germany, Springer-Verlag, 1997 7. Dacic S, Finkelstein SD, Sasatomi E, et al: Molecular pathogenesis of pulmonary carcinosarcoma as determined by microdissection-based allelotyping. Am J Surg Pathol 26:510-516, 2002 8. Halachmi S, DeMarzo AM, Chow N-H, et al: Genetic alterations in urinary bladder carcinosarcoma: Evidence of a common clonal origin. Eur Urol 37:350-357, 2000 9. Hidaka E, Yanagisawa A, Sakai Y, et al: Losses of heterozygosity on chromosomes 17p and 9p/18q may play important roles in early and advanced phases of gallbladder carcinogenesis. J Cancer Res Clin Oncol 125:439-443, 1999 10. Chang HJ, Kim SW, Kim Y-T, et al: Loss of heterozygosity in dysplasia and carcinoma of the gallbladder. Mod Pathol 12:763-769, 1999
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