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Genotypic characterization of a primary mucoepidermoid carcinoma of the parotid gland by cytogenetic, fluorescence in situ hybridization, and DNA ploidy analysis
ELSEVIER
Genotypic Characterization of a Primary Mucoepidermoid Carcinoma of the Parotid Gland By Cytogenetic, Fluorescence In Situ Hybridization, and DNA Ploidy Analysis Adel K. EI-Naggar, Mercedes Lovell, Ann M. Killary, and John G. Batsakis
ABSTRACT: We present a case of mucoepidermoid carcinoma with t(3;12)(q24;p13) and polysomy X by cytogenetic and fluorescence in situ hybridization (FISH) techniques. Flow cytometric DNA analysis of the primary tumor showed DNA aneuploidy and analysis of cultured tumor cells showed DNA diploidy indicating restricted growth of the diploid tumor cells in short-term tissue culture. Interphase cytogenetic analysis of chromosomes 1, 3, 6-12, 16-18, and X in the primary tumor showed polysomy 1, 9, 18, and X, monosomy 8 and 17, and disomy 3, 6, 7, 10-12, and 16. Except for chromosome X, other numerical chromosomal abnormalities were not detected by conventional cytogenetic analysis. Our combined approach allowed for better characterization of the genotypic features of this neoplasm. INTRODUCTION Cytogenetic studies of mucoepidermoid carcinoma are limited because of their infrequent occurrence and the difficulty of in vitro cultivation of these neoplasms [1-8]. Furthermore, although the available conventional cytogenetic results provide critical information regarding the structural and numerical aberrations, it is limited to the analysis of a few dividing cells and does not account for intra-tumoral heterogeneity [9-14]. Applying several analytical approaches to study the genotypic features of tumors has been shown to provide additional information that may allow for better characterization [15]. We applied conventional cytogenetics, fluorescence in situ hybridization (FISH), and DNA flow cytometry on the primary tumor and the cell culture of a high-grade mucoepidermoid carcinoma. The findings are discussed in relation to existing literature to underscore the relevance of such approaches in future studies of these tumors. CASE REPORT A 74-year-old female presented with a history of a three month swelling and tenderness in her right parotid area. From the Departments of Pathology (A. K. E.-N., J. G. B.) and Laboratory Medicine (M. L., A. K.), the University of Texas, M. D. Anderson Cancer Center, Houston, Texas. Address reprint requests to: Adel K. EI-Naggar, M.D., Department of Pathology, The University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 85, 1515 Holcombe Boulevard, Houston, Texas 77030, Received November 17, 1994; accepted March 3, 1995. Cancer Genet Cytogenet 89:38-43 (1996) © Elsevier Science Inc., 1996 655 Avenue of the Americas, N e w York, NY 10010
She denied history of dysphagia, change of voice, weight loss, and anorexia. On examination, a firm and tender right parotid swelling, approximately 2.0 cm in diameter, was identified. She underwent right parotidectomy and right radical neck dissection. The patient recovered uneventfully and was referred to medical oncology for chemotherapy. Pathological examination of the resected specimen revealed a 2.0 cm light tan and firm parotid mass and multiple grossly positive lymph nodes. All twenty-eight neck resected lymph nodes showed metastatic carcinoma. MATERIALS AND METHODS Cytogenetics Tissue for cytogenetic studies was taken under sterile conditions and placed in RPMI 1640 media. Specimens were immediately transported to the cytogenetic laboratory and processed for short-term culture according to the procedure of Kovacs and colleagues [16]. Cultured cells were harvested after two weeks and divided into two parts, one used for chromosome preparation and the other was deep frozen. Properly aged slides (5-7 days old) were subjected to chromosome banding according to Pathak's procedure [17]. Chromosomes were classified according to ISCN [18]. Flow Cytometry Single-cell suspensions from tumor tissue were prepared mechanically by mincing the tissue in flesh RPMI-1640 medium. Cells were stained with acridine orange according to the two-step procedure of Traganos and co-workers
0165-4608/96/$15.00 PII 0165-4608(96)00032-5
Combined FISH and DNA Ploidy Analysis
39
[19]. Samples were analyzed on a Coulter-Profile flow cytometer (Coulter Electronics, Hialeah, FL).
Interphase Cytogenetics Interphase FISH was performed on methanol-acetic acid fixed cells treated with pepsin (0.01 mg/ml, Sigma Chemical Co., St. Louis, MO) ila 0.01 N HC1 for 5 min at 37°C for cytoplasmic removal as described by Pinkel et al. [20]. The biotinylated probes for chromosomes #1, 3, 6, 7, 8, 9, 10, 11, 12, 16, 17, 18, and X (Oncor, Gaithersburg, MD) were identified as previously described [21]. Evaluation was made using a Leitz-D Laborlux fluorescence photomicroscope equipped with filter sets Leitz 12/3 (FITC). The analysis of metaphase and interphase cells was performed according to the criteria described by Hopman et al. [21]. Overlapping nuclei wer,~ excluded from the analysis. The signals in a given nucleus had to be regular in both size and intensity; split signals were counted as one and nonspecific signals were excluded. For monosomy determination, >10% of nuclei with single spot hybridization was used. Polysomy was determined if >4% of nuclei contained three or more hybridization spots. A total of 200 cells were counted per slide preparation.
Chromosome Painting Whole chromosome painting probes (WCP-probes, Imagenetics, Framingham, MA, USA), WCP-12 spectrum green, WCP-X spectrum orange, and WCP-3 mixture of spectrum green and orange were used on unbanded chromosome preparations. The conditions for hybridization and posthybridization washes were in accordance with the recommendations of the manufacturer. All hybridizations were performed for 40-48 hc,urs. The hybridization areas were counter stained with 4'6-diamidine-2-phenyl-indole dihydrochloride (DAPI). Sl!.des were viewed with a Leitz-D Labolux epifluorescence microscope using appropriate filter sets.
RESULTS
Figure I Photomicrographs of the primary lesion showing mucoepidermoid carcinoma, intermediate grade (a) and high grade (b) features.
Histopathology Histologically, the neoplasm manifested poor differentiation with focal areas of moderate (Fig la) differentiation. The poorly differentiated areas and the lymph node metastasis displayed an anaplastic cellular features (Fig lb).
Cytogenetics Short-term culture cell:~ were confirmed morphologically and by reactivity to low-molecular weight cytokeratin to be epithelial. Thirty metaphases were analyzed by G-banding and revealed a normal karyotype (46,XX) in 10 cells, 46,XX,t(3:12)(q24;p13) in four cells, and 47,XX,+X in five cells; and 48,XX,+X,+X in eleven cells (Fig 2a and b). In situ Hybridization Whole chromosome painting probes for chromosomes X, 3, and 12 were evaluated on five unbanded chromosomes from primary culture. Polysomy of chromosome X (Fig 3A) was noted in three metaphases and translocation between chromosomes 3 and 12 and disomy for all probes was observed in one rnetaphase each (Fig 3B). Interphase
FISH for chromosomes #1, 3, 6-12, 16-18, and X revealed monosomy 8 and 17 (Fig 3C) in 15% and 38% of cells, respectively, and polysomy 1 (Fig 3), 9, and 18 in 11 to 13% of cells, and polysomy X in 13% of cells. Other chromosomes showed disomy in 97% of cells.
Flow Cytometry Flow cytometric DNA content analysis of the primary tumor revealed a hyperdiploid cell population with DI of 1.6 in 15% of ceils analyzed (Fig 4A). Analysis of shortterm cultured tumor cells showed DNA diploidy (Fig 4B). This was confirmed by the overlapping of the diploid peaks of normal human lymphocytes tissue culture sampies and after 50/50% mixing (data not shown).
DISCUSSION Cytogenetic studies of mucoepidermoid carcinoma of salivary glands are few and inconclusive. Only 14 cases of pri-
40
A.K. E1-Naggar et al.
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Figure 2 (a) A karyogram of the mucoepidermoid carcinoma displaying translocation between cnr'Jnlosomes 3 and 12 (arrows) in a 46,XX karyotype. (b) A metaphase of a tumor cell showing 47,XX,+X.
mary, recurrent and metastatic lesions have been published [1-8]. Clonal chromosomal abnormalities were detected in all these cases but a common cytogenetic denominator has not been defined. Nordkvist and colleagues [1], however, identified a t(11;19)(q14-21;p12) translocation in three mucoepidermoid carcinomas of a minor salivary gland, among other aberrations, and suggested that this may represent a primary genetic abnormality. Two other tumors with 11q deletion and a recent case with both 11q and 19p rearrangements have also been identified [2, 4, 8]. The
remaining tumors lacked abnorme ,ties in either chromosome 11 or 19 [1, 3, 7]. Recently, FISH, a technique ~.at permits the analysis of numerical and structural chromosomal aberrations in interphase and metaphase cells, has been widely used to complement conventional cytogenetic analysis [14, 2226]. This technique eliminates major steps that hamper cytogenetic analysis and allows for the study of the heterogeneity of tumor subpopulations [14, 27]. However, the scope of such analysis is currently limited to certain
Combined FISH and DNA Ploidy Analysis
41
A
O
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D Figure 3 Chromosome painting of two metaphases showing: (A) trisomy X and disomy of chromosomes 3 and 12; and (B) translocation between chromosomes 3 and 12 (arrow head). Interphase FISH showing monosomy 17 (C) and polysomy 1 {D) in tumor cells.
defined sequences and the availability of probes for the intended target. Studies using this technique in salivary gland tumors are few in :number [28, 29]. Our tumor manifested structural and numerical abnormalities that have not been previously reported in mucoepidermoid carcinomas. A mosaic clonal t(3;12)(q24;p13) and polysomy X were observed by both conventional cytogenetic and in situ techniques. The lack of other structural and/or numerical aberr~tions in different chromosomes may suggest that these changes either represent an early aberration and/or an acquired feature in a subset of dividing cells with growth advantage in tissue culture. The latter reasoning is the most likely explanation. This is supported by the presence in the primary tumor of a hyperdiploid cell population by flow cytometry and polysomy 1, 9, and 18, and monosomy 8 and 17 by FISH and the lack of these findings in cultured tumor cells. The possibility that such discrepancies are caused by intratumoral hetero-
geneity is unlikely because adjacent tissues from a defined area of the primary tumor were used. The multifactorial approach in our study and those of others has shown that both a preferential growth advantage of dividing near-diploid cells and selection against a highly aneuploid population in tissue culture are the underlying causes for the discordance between the cytogenetic and interphase analyses [15, 21, 27, 30]. This further highlights the added advantage of combined analysis in the genotypic characterizations of these and other solid neoplasia. It is more likely that the cytogenetic findings in our tumor represent an early chromosomal abnormality in the diploid stemline [22]. Subsequent acquisition of additional numerical aberrations during tumor progression, however, may have led to the evolution of DNA aneuploidy and other chromosomal abnormalities. We consider that the genetic events attendant to salivary gland
42
A . K . E1-Naggar et al.
(1990): Mucoepidermoid tumor of the parotid gland showing a translocation (3;81)(p21;q12) and a deletion (5)(q22) as sole chromosome abnormalities. Cancer Genet Cytogenet 50:161164. 7. Nordkvist A, Edstrom S, Mark J, Stenman G (1992): Multiple unrelated chromosome abnormalities in a metastatic mucoepidermoid carcinoma of the parotid gland. Cancer Genet Cytogenet 61:158-161. 8. Dahlenfors R, Lindahl L, Mark J (1994): A fourth minor salivary gland mucoepidermoid carcinoma with 11q14-21 and 19p12 rearrangements. Hereditas 120:287-288. 9. E1-Naggar AK, Pathak S (1991): Cytogenetic and corresponding flow cytometric DNA analysis of renal cell neoplasms. Anticancer Res 12:1491-1500. 10. Wolman SR, Camuto PM, Golimbu M, Schinella R (1988): Cytogenetic, flow cytometric and ultrastructural studies of twenty-nine non-familial human renal carcinomas. Cancer Res 48:2690-2897.
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11. Lorenzato M, Doco M, Visseaux-Coletto B, Ferre D, Bellaoui H, Evrard G, Adnet JJ (1993): Discrepancies of DNA content of various solid tumors before and after culture measured by image analysis. Comparison of cytogenetical data. Path Res Pract 189:1161-1168. 12. Shackney SE, Burholt DR, Pollice AA, Smith CA, Pugh RB, Hartsock R] (1990): Discrepancies between flow cytometric and cytogenetic studies in the detection of aneuploidy in human solid tumors. Cytometry 11:94-104.
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Figure 4 (A) A single parameter histogram of cultured cells of the mucoepidermoid carcinoma displaying single G0/G1 peak. (B) Histogram of the primary tumor displaying aneuploid DNA stemline with DI of 1.66. tumors are best explored by a combination of in situ hybridization, DNA flow cytometry, and conventional cytogenetic techniques of a large cohort. We are grateful to Ms. Edith Wong for performing the chromosome painting and Sulema M. Martinez for typing the manuscript.
REFERENCES 1. Nordkvist A, Gustafsson H, Juberg-Ode M, Stenman G (1994): Recurrent rearrangements of 11q14-22 in mucoepidermoid carcinoma. Cancer Genet Cytogenet 74:77-83. 2. Sandros J, Stenman G, Mark J (1990): Cytogenetic and molecular observations in human and experimental salivary gland tumors. Cancer Genet Cytogenet 44:153-167. 3. E1-Naggar AK, Lovell M, Killary A, Batsakis JG (1994): Trisomy 5 as the sole chromosomal abnormality in a primary mucoepidermoid carcinoma of the minor salivary gland. Cancer Genet Cytogenet 76:96-99. 4. Stenman G, Sandros J, Dahlenfors R, Juberg-Ode M, Mark J (1986): 6 q - and loss of the Y chromosome---two common deviations in malignant human salivary gland tumors. Cancer Genet Cytogenet 22:283-293. 5. Mark J, Sandros J, Wedell B, Dahlenfors R, Stenman G (1989): Karyotypic evaluation in a human mucoepidermoid carcinoma. Hereditas 110:75-78. 6. Bullerdiek J, Vollrath M, Wittekind C, Caselitz I, Bartnitzke S
13. Dooley WC, Allison DC, Robertson J (1991): Discrepancies among the metaphase, telophase and the GO/G1 and G2 DNA peaks of heteroploid cell lines. Cytometry 12:99-106. 14. Price CM (1993): Fluorescence in situ hybridization. Blood 7:127-134. 15. Breitkreuz T, Romanakis K, Lutz S, Seitz G, Bonkhoff H, Unteregger G, Zwergel T, Zang KD, Wullich B (1993): Genotypic characterization of prostatic carcinomas: A combined cytogenetic, flow cytometry and in-situ hybridization (FISH). Cancer Genet Cytogenet 62:180-185. 16. Kovacs G, Szucs S, DeRiese W, Baumgartel H (1987): Specific chromosome aberration in human renal cell carcinoma. Int J Cancer 40:171-178. 17. Pathak S (1976): Chromosome banding techniques. J Reprod Med 17:25-28. 18. ISCN (1978): An international system for human cytogenetic nomenclature. Cytogenet Cell Genet 21:309-404. 19. Traganos F, Darzynkiewicz Z, Sharpless T, Melamed MR (1977): Simultaneous staining of ribonucleic and deoxyribonucleic acids in unfixed cells using acridine orange in a flow cytometric system. J Histochem Cytochem 25:46-56. 20. Pinkel D, Straume T, Gray JW (1986): Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 83:2934-2938. 21. Hopman AHN, van Hooren E, van Dekaa CA, Vooijs PGP, Ramaekers FCS (1991): Detection of numerical chromosome aberrations using in situ hybridization in paraffin sections of routinely processed bladder cancers. Modern Pathol 4:504513. 22. Micale MA, Sanford JS, Powell IJ, Sakr WA, Wolman SR (1992): Defining the extent and nature of cytogenetic events in prostate adenocarcinoma: Paraffin FISH versus metaphase analysis. Cancer Genet Cytogenet 69:7-12. 23. Gebhart E, Trautmann U, Reichardt S, Liehr T (1993): Chromosomal heterogeneity of aneuploid leukemic cell populations detected by conventional karyotyping and by fluorescence in-situ hybridization (FISH). Anticancer Research 13:1857-1862.
Combined FISH a n d DNA Ploidy Analysis
24. Ramaekers F, Hopman A, Vooijs P (1992): Advances in the detection of ploidy differences in cancer by in-situ hybridization. Analytical Cellular Pathology 4:337-344. 25. Hopman AHN, Voorter CEM, Ramaekers FCS (1994): Detection of genomic changes in cancer by in-situ hybridization. Molecular Biology Reports 19:13-44. 26. Lucas J, Sachs RK (1993): Using three-color chromosome painting to test chromosome aberrations models. Proc Natl Acad Sci 90:1484-1487. 27. E1-Naggar AK, van Dekken HD, Ensign LG, Pathak S (1994): Interphase cytogenetics in paraffin-embedded sections from renal cortical neoplasms. Correlation with cytogenetic and
43
flow cytometric DNA ploidy analysis. Cancer Genet Cytogene. 73:134-141. 28. Sahlin P, Mark J, Stenman G (1994): Interphase cytogenetic analysis of cultured pleomorphic adenomas with a normal karyotype. Int J Oncol 4:1225-1228. 29. Mark J, Dahlenfors R, Wedell B, Stenman G, Bockman P (1994): Cytogenetical and FISH analysis on a salivary sebaceous lymph adenoma. Oncology Reports 1:561-562. 30. Brothman AR, Patel AM, Peehl DM, Schellhammer PF (1992): Analysis of prostatic tumor cultures using fluorescence in situ hybridization (FISH): Cancer Genet Cytogenet 62:180-185.
Genotypic Characterization of a Primary Mucoepidermoid Carcinoma of the Parotid Gland By Cytogenetic, Fluorescence In Situ Hybridization, and DNA Ploidy Analysis Adel K. EI-Naggar, Mercedes Lovell, Ann M. Killary, and John G. Batsakis
ABSTRACT: We present a case of mucoepidermoid carcinoma with t(3;12)(q24;p13) and polysomy X by cytogenetic and fluorescence in situ hybridization (FISH) techniques. Flow cytometric DNA analysis of the primary tumor showed DNA aneuploidy and analysis of cultured tumor cells showed DNA diploidy indicating restricted growth of the diploid tumor cells in short-term tissue culture. Interphase cytogenetic analysis of chromosomes 1, 3, 6-12, 16-18, and X in the primary tumor showed polysomy 1, 9, 18, and X, monosomy 8 and 17, and disomy 3, 6, 7, 10-12, and 16. Except for chromosome X, other numerical chromosomal abnormalities were not detected by conventional cytogenetic analysis. Our combined approach allowed for better characterization of the genotypic features of this neoplasm. INTRODUCTION Cytogenetic studies of mucoepidermoid carcinoma are limited because of their infrequent occurrence and the difficulty of in vitro cultivation of these neoplasms [1-8]. Furthermore, although the available conventional cytogenetic results provide critical information regarding the structural and numerical aberrations, it is limited to the analysis of a few dividing cells and does not account for intra-tumoral heterogeneity [9-14]. Applying several analytical approaches to study the genotypic features of tumors has been shown to provide additional information that may allow for better characterization [15]. We applied conventional cytogenetics, fluorescence in situ hybridization (FISH), and DNA flow cytometry on the primary tumor and the cell culture of a high-grade mucoepidermoid carcinoma. The findings are discussed in relation to existing literature to underscore the relevance of such approaches in future studies of these tumors. CASE REPORT A 74-year-old female presented with a history of a three month swelling and tenderness in her right parotid area. From the Departments of Pathology (A. K. E.-N., J. G. B.) and Laboratory Medicine (M. L., A. K.), the University of Texas, M. D. Anderson Cancer Center, Houston, Texas. Address reprint requests to: Adel K. EI-Naggar, M.D., Department of Pathology, The University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 85, 1515 Holcombe Boulevard, Houston, Texas 77030, Received November 17, 1994; accepted March 3, 1995. Cancer Genet Cytogenet 89:38-43 (1996) © Elsevier Science Inc., 1996 655 Avenue of the Americas, N e w York, NY 10010
She denied history of dysphagia, change of voice, weight loss, and anorexia. On examination, a firm and tender right parotid swelling, approximately 2.0 cm in diameter, was identified. She underwent right parotidectomy and right radical neck dissection. The patient recovered uneventfully and was referred to medical oncology for chemotherapy. Pathological examination of the resected specimen revealed a 2.0 cm light tan and firm parotid mass and multiple grossly positive lymph nodes. All twenty-eight neck resected lymph nodes showed metastatic carcinoma. MATERIALS AND METHODS Cytogenetics Tissue for cytogenetic studies was taken under sterile conditions and placed in RPMI 1640 media. Specimens were immediately transported to the cytogenetic laboratory and processed for short-term culture according to the procedure of Kovacs and colleagues [16]. Cultured cells were harvested after two weeks and divided into two parts, one used for chromosome preparation and the other was deep frozen. Properly aged slides (5-7 days old) were subjected to chromosome banding according to Pathak's procedure [17]. Chromosomes were classified according to ISCN [18]. Flow Cytometry Single-cell suspensions from tumor tissue were prepared mechanically by mincing the tissue in flesh RPMI-1640 medium. Cells were stained with acridine orange according to the two-step procedure of Traganos and co-workers
0165-4608/96/$15.00 PII 0165-4608(96)00032-5
Combined FISH and DNA Ploidy Analysis
39
[19]. Samples were analyzed on a Coulter-Profile flow cytometer (Coulter Electronics, Hialeah, FL).
Interphase Cytogenetics Interphase FISH was performed on methanol-acetic acid fixed cells treated with pepsin (0.01 mg/ml, Sigma Chemical Co., St. Louis, MO) ila 0.01 N HC1 for 5 min at 37°C for cytoplasmic removal as described by Pinkel et al. [20]. The biotinylated probes for chromosomes #1, 3, 6, 7, 8, 9, 10, 11, 12, 16, 17, 18, and X (Oncor, Gaithersburg, MD) were identified as previously described [21]. Evaluation was made using a Leitz-D Laborlux fluorescence photomicroscope equipped with filter sets Leitz 12/3 (FITC). The analysis of metaphase and interphase cells was performed according to the criteria described by Hopman et al. [21]. Overlapping nuclei wer,~ excluded from the analysis. The signals in a given nucleus had to be regular in both size and intensity; split signals were counted as one and nonspecific signals were excluded. For monosomy determination, >10% of nuclei with single spot hybridization was used. Polysomy was determined if >4% of nuclei contained three or more hybridization spots. A total of 200 cells were counted per slide preparation.
Chromosome Painting Whole chromosome painting probes (WCP-probes, Imagenetics, Framingham, MA, USA), WCP-12 spectrum green, WCP-X spectrum orange, and WCP-3 mixture of spectrum green and orange were used on unbanded chromosome preparations. The conditions for hybridization and posthybridization washes were in accordance with the recommendations of the manufacturer. All hybridizations were performed for 40-48 hc,urs. The hybridization areas were counter stained with 4'6-diamidine-2-phenyl-indole dihydrochloride (DAPI). Sl!.des were viewed with a Leitz-D Labolux epifluorescence microscope using appropriate filter sets.
RESULTS
Figure I Photomicrographs of the primary lesion showing mucoepidermoid carcinoma, intermediate grade (a) and high grade (b) features.
Histopathology Histologically, the neoplasm manifested poor differentiation with focal areas of moderate (Fig la) differentiation. The poorly differentiated areas and the lymph node metastasis displayed an anaplastic cellular features (Fig lb).
Cytogenetics Short-term culture cell:~ were confirmed morphologically and by reactivity to low-molecular weight cytokeratin to be epithelial. Thirty metaphases were analyzed by G-banding and revealed a normal karyotype (46,XX) in 10 cells, 46,XX,t(3:12)(q24;p13) in four cells, and 47,XX,+X in five cells; and 48,XX,+X,+X in eleven cells (Fig 2a and b). In situ Hybridization Whole chromosome painting probes for chromosomes X, 3, and 12 were evaluated on five unbanded chromosomes from primary culture. Polysomy of chromosome X (Fig 3A) was noted in three metaphases and translocation between chromosomes 3 and 12 and disomy for all probes was observed in one rnetaphase each (Fig 3B). Interphase
FISH for chromosomes #1, 3, 6-12, 16-18, and X revealed monosomy 8 and 17 (Fig 3C) in 15% and 38% of cells, respectively, and polysomy 1 (Fig 3), 9, and 18 in 11 to 13% of cells, and polysomy X in 13% of cells. Other chromosomes showed disomy in 97% of cells.
Flow Cytometry Flow cytometric DNA content analysis of the primary tumor revealed a hyperdiploid cell population with DI of 1.6 in 15% of ceils analyzed (Fig 4A). Analysis of shortterm cultured tumor cells showed DNA diploidy (Fig 4B). This was confirmed by the overlapping of the diploid peaks of normal human lymphocytes tissue culture sampies and after 50/50% mixing (data not shown).
DISCUSSION Cytogenetic studies of mucoepidermoid carcinoma of salivary glands are few and inconclusive. Only 14 cases of pri-
40
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Figure 2 (a) A karyogram of the mucoepidermoid carcinoma displaying translocation between cnr'Jnlosomes 3 and 12 (arrows) in a 46,XX karyotype. (b) A metaphase of a tumor cell showing 47,XX,+X.
mary, recurrent and metastatic lesions have been published [1-8]. Clonal chromosomal abnormalities were detected in all these cases but a common cytogenetic denominator has not been defined. Nordkvist and colleagues [1], however, identified a t(11;19)(q14-21;p12) translocation in three mucoepidermoid carcinomas of a minor salivary gland, among other aberrations, and suggested that this may represent a primary genetic abnormality. Two other tumors with 11q deletion and a recent case with both 11q and 19p rearrangements have also been identified [2, 4, 8]. The
remaining tumors lacked abnorme ,ties in either chromosome 11 or 19 [1, 3, 7]. Recently, FISH, a technique ~.at permits the analysis of numerical and structural chromosomal aberrations in interphase and metaphase cells, has been widely used to complement conventional cytogenetic analysis [14, 2226]. This technique eliminates major steps that hamper cytogenetic analysis and allows for the study of the heterogeneity of tumor subpopulations [14, 27]. However, the scope of such analysis is currently limited to certain
Combined FISH and DNA Ploidy Analysis
41
A
O
B
D Figure 3 Chromosome painting of two metaphases showing: (A) trisomy X and disomy of chromosomes 3 and 12; and (B) translocation between chromosomes 3 and 12 (arrow head). Interphase FISH showing monosomy 17 (C) and polysomy 1 {D) in tumor cells.
defined sequences and the availability of probes for the intended target. Studies using this technique in salivary gland tumors are few in :number [28, 29]. Our tumor manifested structural and numerical abnormalities that have not been previously reported in mucoepidermoid carcinomas. A mosaic clonal t(3;12)(q24;p13) and polysomy X were observed by both conventional cytogenetic and in situ techniques. The lack of other structural and/or numerical aberr~tions in different chromosomes may suggest that these changes either represent an early aberration and/or an acquired feature in a subset of dividing cells with growth advantage in tissue culture. The latter reasoning is the most likely explanation. This is supported by the presence in the primary tumor of a hyperdiploid cell population by flow cytometry and polysomy 1, 9, and 18, and monosomy 8 and 17 by FISH and the lack of these findings in cultured tumor cells. The possibility that such discrepancies are caused by intratumoral hetero-
geneity is unlikely because adjacent tissues from a defined area of the primary tumor were used. The multifactorial approach in our study and those of others has shown that both a preferential growth advantage of dividing near-diploid cells and selection against a highly aneuploid population in tissue culture are the underlying causes for the discordance between the cytogenetic and interphase analyses [15, 21, 27, 30]. This further highlights the added advantage of combined analysis in the genotypic characterizations of these and other solid neoplasia. It is more likely that the cytogenetic findings in our tumor represent an early chromosomal abnormality in the diploid stemline [22]. Subsequent acquisition of additional numerical aberrations during tumor progression, however, may have led to the evolution of DNA aneuploidy and other chromosomal abnormalities. We consider that the genetic events attendant to salivary gland
42
A . K . E1-Naggar et al.
(1990): Mucoepidermoid tumor of the parotid gland showing a translocation (3;81)(p21;q12) and a deletion (5)(q22) as sole chromosome abnormalities. Cancer Genet Cytogenet 50:161164. 7. Nordkvist A, Edstrom S, Mark J, Stenman G (1992): Multiple unrelated chromosome abnormalities in a metastatic mucoepidermoid carcinoma of the parotid gland. Cancer Genet Cytogenet 61:158-161. 8. Dahlenfors R, Lindahl L, Mark J (1994): A fourth minor salivary gland mucoepidermoid carcinoma with 11q14-21 and 19p12 rearrangements. Hereditas 120:287-288. 9. E1-Naggar AK, Pathak S (1991): Cytogenetic and corresponding flow cytometric DNA analysis of renal cell neoplasms. Anticancer Res 12:1491-1500. 10. Wolman SR, Camuto PM, Golimbu M, Schinella R (1988): Cytogenetic, flow cytometric and ultrastructural studies of twenty-nine non-familial human renal carcinomas. Cancer Res 48:2690-2897.
~B
11. Lorenzato M, Doco M, Visseaux-Coletto B, Ferre D, Bellaoui H, Evrard G, Adnet JJ (1993): Discrepancies of DNA content of various solid tumors before and after culture measured by image analysis. Comparison of cytogenetical data. Path Res Pract 189:1161-1168. 12. Shackney SE, Burholt DR, Pollice AA, Smith CA, Pugh RB, Hartsock R] (1990): Discrepancies between flow cytometric and cytogenetic studies in the detection of aneuploidy in human solid tumors. Cytometry 11:94-104.
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Figure 4 (A) A single parameter histogram of cultured cells of the mucoepidermoid carcinoma displaying single G0/G1 peak. (B) Histogram of the primary tumor displaying aneuploid DNA stemline with DI of 1.66. tumors are best explored by a combination of in situ hybridization, DNA flow cytometry, and conventional cytogenetic techniques of a large cohort. We are grateful to Ms. Edith Wong for performing the chromosome painting and Sulema M. Martinez for typing the manuscript.
REFERENCES 1. Nordkvist A, Gustafsson H, Juberg-Ode M, Stenman G (1994): Recurrent rearrangements of 11q14-22 in mucoepidermoid carcinoma. Cancer Genet Cytogenet 74:77-83. 2. Sandros J, Stenman G, Mark J (1990): Cytogenetic and molecular observations in human and experimental salivary gland tumors. Cancer Genet Cytogenet 44:153-167. 3. E1-Naggar AK, Lovell M, Killary A, Batsakis JG (1994): Trisomy 5 as the sole chromosomal abnormality in a primary mucoepidermoid carcinoma of the minor salivary gland. Cancer Genet Cytogenet 76:96-99. 4. Stenman G, Sandros J, Dahlenfors R, Juberg-Ode M, Mark J (1986): 6 q - and loss of the Y chromosome---two common deviations in malignant human salivary gland tumors. Cancer Genet Cytogenet 22:283-293. 5. Mark J, Sandros J, Wedell B, Dahlenfors R, Stenman G (1989): Karyotypic evaluation in a human mucoepidermoid carcinoma. Hereditas 110:75-78. 6. Bullerdiek J, Vollrath M, Wittekind C, Caselitz I, Bartnitzke S
13. Dooley WC, Allison DC, Robertson J (1991): Discrepancies among the metaphase, telophase and the GO/G1 and G2 DNA peaks of heteroploid cell lines. Cytometry 12:99-106. 14. Price CM (1993): Fluorescence in situ hybridization. Blood 7:127-134. 15. Breitkreuz T, Romanakis K, Lutz S, Seitz G, Bonkhoff H, Unteregger G, Zwergel T, Zang KD, Wullich B (1993): Genotypic characterization of prostatic carcinomas: A combined cytogenetic, flow cytometry and in-situ hybridization (FISH). Cancer Genet Cytogenet 62:180-185. 16. Kovacs G, Szucs S, DeRiese W, Baumgartel H (1987): Specific chromosome aberration in human renal cell carcinoma. Int J Cancer 40:171-178. 17. Pathak S (1976): Chromosome banding techniques. J Reprod Med 17:25-28. 18. ISCN (1978): An international system for human cytogenetic nomenclature. Cytogenet Cell Genet 21:309-404. 19. Traganos F, Darzynkiewicz Z, Sharpless T, Melamed MR (1977): Simultaneous staining of ribonucleic and deoxyribonucleic acids in unfixed cells using acridine orange in a flow cytometric system. J Histochem Cytochem 25:46-56. 20. Pinkel D, Straume T, Gray JW (1986): Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 83:2934-2938. 21. Hopman AHN, van Hooren E, van Dekaa CA, Vooijs PGP, Ramaekers FCS (1991): Detection of numerical chromosome aberrations using in situ hybridization in paraffin sections of routinely processed bladder cancers. Modern Pathol 4:504513. 22. Micale MA, Sanford JS, Powell IJ, Sakr WA, Wolman SR (1992): Defining the extent and nature of cytogenetic events in prostate adenocarcinoma: Paraffin FISH versus metaphase analysis. Cancer Genet Cytogenet 69:7-12. 23. Gebhart E, Trautmann U, Reichardt S, Liehr T (1993): Chromosomal heterogeneity of aneuploid leukemic cell populations detected by conventional karyotyping and by fluorescence in-situ hybridization (FISH). Anticancer Research 13:1857-1862.
Combined FISH a n d DNA Ploidy Analysis
24. Ramaekers F, Hopman A, Vooijs P (1992): Advances in the detection of ploidy differences in cancer by in-situ hybridization. Analytical Cellular Pathology 4:337-344. 25. Hopman AHN, Voorter CEM, Ramaekers FCS (1994): Detection of genomic changes in cancer by in-situ hybridization. Molecular Biology Reports 19:13-44. 26. Lucas J, Sachs RK (1993): Using three-color chromosome painting to test chromosome aberrations models. Proc Natl Acad Sci 90:1484-1487. 27. E1-Naggar AK, van Dekken HD, Ensign LG, Pathak S (1994): Interphase cytogenetics in paraffin-embedded sections from renal cortical neoplasms. Correlation with cytogenetic and
43
flow cytometric DNA ploidy analysis. Cancer Genet Cytogene. 73:134-141. 28. Sahlin P, Mark J, Stenman G (1994): Interphase cytogenetic analysis of cultured pleomorphic adenomas with a normal karyotype. Int J Oncol 4:1225-1228. 29. Mark J, Dahlenfors R, Wedell B, Stenman G, Bockman P (1994): Cytogenetical and FISH analysis on a salivary sebaceous lymph adenoma. Oncology Reports 1:561-562. 30. Brothman AR, Patel AM, Peehl DM, Schellhammer PF (1992): Analysis of prostatic tumor cultures using fluorescence in situ hybridization (FISH): Cancer Genet Cytogenet 62:180-185.