Genetic Alterations in Acinic Cell Carcinoma of the Parotid Gland Determined by Microsatellite Analysis

Genetic Alterations in Acinic Cell Carcinoma of the Parotid Gland Determined by Microsatellite Analysis

Genetic Alterations in Acinic Cell Carcinoma of the Parotid Gland Determined by Microsatellite Analysis Adel K. El-Naggar, Fadi W. Abdul-Karim, Kennet...

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Genetic Alterations in Acinic Cell Carcinoma of the Parotid Gland Determined by Microsatellite Analysis Adel K. El-Naggar, Fadi W. Abdul-Karim, Kenneth Hurr, David Callender, Mario A. Luna, and John G. Batsakis

ABSTRACT: We investigated, for the first time, the genetic alterations at certain chromosomal loci in 25 primary parotid acinic cell carcinomas to define the most frequently altered chromosomal regions and their association with pathologic features and DNA content analysis. Our results showed that 21 (84.0%) of the tumors had alteration in at least one of the loci tested. In general, chromosomal regions at chromosomes 4p, 5q, 6p, and 17p were more frequently altered than those on chromosomes 1p and 1q, 4q, 5p, and 6q. Certain markers at 4p15–16, 6p25-qter, and 17p11 regions showed the highest incidence of LOH, suggesting the presence of tumor suppressor genes associated with the oncogenesis of these tumors. LOH was significantly associated only with tumor grade. No apparent correlation between LOH and other clinicopathologic and DNA content characteristics was identified. Our study broadly defined the chromosomal arms and loci that may be targeted for further localization of the minimally deleted regions involved in the tumorigenesis of these tumors. © Elsevier Science Inc., 1998

INTRODUCTION Acinic cell carcinoma, an uncommon and distinctive salivary gland neoplasm, is characterized by a protean biological behavior and spectrum of cytomorphologic features that may pose differential diagnostic difficulties [1–3, 4]. Attempts to identify histopathologic and/or prognostic parameters that may assist in their clinical evaluation have been largely unrewarding [4–8]. Recent advances in molecular genetic techniques have allowed for extensive genetic analysis of a wide variety of human solid neoplasms. Such information is important, not only to the understanding of tumor biology but also for the potential development of novel diagnostic and biologic markers for their management [9–11]. Few molecular studies of salivary gland tumors, however, have been published [12–18]. We investigated the molecular alterations in 25 examples of acinic cell carcinoma by PCR-based microsatellite analysis of certain chromosomal loci. The loci selected were

From the Department of Pathology (A. K. E.-N., K. H., M. A. L., J. G. B.) and the Department of Head and Neck Surgery (D. C.), The University of Texas, M. D. Anderson Cancer Center, Houston, Texas, U.S.A.; and the Department of Pathology, Case Western Reserve University (F. W. A.-K.), Cleveland, Ohio, U.S.A. Address reprint requests to: Adel K. El-Naggar, M.D., Ph.D., Department of Pathology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Box 85, Houston, TX 77030. Received March 20, 1997; accepted June 19, 1997. Cancer Genet Cytogenet 102:19–24 (1998)  Elsevier Science Inc., 1998 655 Avenue of the Americas, New York, NY 10010

based on our own experience [13–15] and previous cytogenetic studies of these tumors [19–22]. Findings were correlated with clinicopathologic features and DNA analysis. MATERIALS AND METHODS Twenty-five acinic cell carcinomas with available paraffin-embedded blocks, accessioned between 1975 and 1995, formed the materials for this study. In 17 lesions, separate blocks for normal salivary gland and tumor tissues were found; tumor blocks were selected so as to contain .80% neoplastic tissue. In eight cases, both tumor and surrounding normal salivary gland were microdissected from the same block. Hematoxylin and eosin stained slides of all tumors were available. Histologic pattern and tumor grade were evaluated according to the criteria of Batsakis et al. [1]. Because of the consultative nature of more than two thirds of the cases, the follow-up information was deemed unreliable or unobtainable. Microdissection Microdissection was performed after reviewing the H&E stained section of each selected block. Normal and tumor components were identified, outlined by a marking pin, and labelled. Five 8 mc thick unstained sections were subsequently prepared and marked to match the corresponding H&E stained section. An additional H&E section was prepared and reviewed for further confirmation of the selection process. The microdissected tissue of each histologic component was carefully lifted off the slides, within

0165-4608/98/$19.00 PII S0165-4608(97)00273-2

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its outlined boundaries, and placed in an eppendorf tube. At least 100 cells were present in each sample and used for further DNA extraction. DNA Extraction DNA was extracted as previously described [23]. Microsatellite Markers Twenty microsatellite markers on the short and long arms of chromosomes 1, 4, 5, and 6 and the short arm of 17 were selected based on available cytogenetic information on these tumors and our own experience [13–15, 19–22]. Primers for PCR amplification of microsatellite markers were obtained from Research Genetics (Huntsville, AL). Analysis of LOH and Genetic Instability LOH of the informative microsatellite loci was compared with heterozygosity at the analyzed locus by visually evaluating allele band intensity of normal tissues and corresponding tumor from the same patient. Complete loss or .50% reduction in a single-band intensity in an informative locus was scored as LOH by two independent observers. Genetic instability in microsatellite sequence (MI) was defined as an expansion or reduction in the number of dior tetra-nucleotide repeats in the tumor, compared with normal control DNA in the same locus of the same tumor. To exclude technical and labeling errors, all observed alterations were reproduced by separate DNA extraction and independent PCR amplification. Further verification

and exclusion of specimen mismatching was carried out by comparing banding patterns of concordant alleles in normal tissue and tumor samples at different microsatellite loci. DNA Flow Cytometry Single-cell suspensions from 50 mc thick sections of carefully selected tumor blocks were prepared and DNA analysis was performed on EPICS profile flow cytometer (EPICS Division, Coulter Corp., Hialeah, FL) as previously described [24]. DNA ploidy was determined based on the DNA index into DNA diploidy (DI of 1.0) and DNA aneuploidy (DI . 1.0 or ,0.1). The proliferative index was determined as the percentage of cells in S-phase of the cell cycle after debris subtraction; when necessary, this was based on the method of Baisch et al. [25]. RESULTS Table 1 presents the clinicopathologic and DNA content characteristics as well as the molecular alterations of these tumors. Patients were comprised of 13 males and 12 females who ranged in age from 27 to 96 years, with a mean of 57.5 years. Tumor size (24 with available information) ranged from 0.5 to 5.0 cm, with a mean of 2.5 cm. Morphologic patterns consisted of 12, lobular acinar; three, micropapillary; six, microcystic; two, ductal, and one each clear cell type and macrocystic. Tumor grades were as follows: 11, grade I; 12, grade II; and two, grade III.

Table 1 Clinicopathologic, DNA and LOH characteristics of acinic cell carcinoma Patient no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Gender

Age

Size (cm)

Pattern

Grade

DNA index

PI

LOH >2a

M M M F F M F M F M M F M F F F M F F F M M F M M

59 44 79 64 62 52 91 48 56 41 62 64 49 46 70 60 53 38 96 27 60 54 61 49 52

2.5 1.5 1.5 NI 5.0 2.5 1.5 3.0 1.5 1.3 2.5 2.8 5.0 3.1 3.0 0.5 1.5 5.0 2.3 3.5 2.0 1.2 2.5 3.5 1.8

Lobular acinar Lobular acinar Microcystic Lobular acinar Micropapillary Lobular acinar Lobular acinar Microcystic Lobular acinar Lobular acinar Lobular acinar Lobular acinar Ductal Ductal Lobular acinar Clear cell Lobular acinar Micropapillary Microcystic Microcystic Lobular acinar Macrocystic Microcystic Microcystic Micropapillary

II II II I II I II III I I II I II I I II II I III II II I II I I

1.00 NI 1.00 1.00 1.00 1.00 NI 1.20 NI 1.00 1.00 1.00 1.00 1.20 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.10

6 NI 4 3 4 5 NI 5 NI 2 5 4 8 NI 3 6 2 3 26 4 3 4 3 7 5

3 4 3 0 3 2 2 3 0 0 4 1 3 3 1 5 0 3 4 3 4 3 3 1 3

Abbreviations: PI, proliferative index; LOH, loss of heterozygosity; NI, no information. a

LOH at 2 or more loci.

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Microsatellites in Acinic Parotid Gland Tumors

Figure 1 Composite photomicrograph of different morphologic patterns of acinic cell carcinoma and corresponding microsatellite alterations at selected loci. Cases #2, 3, 8, 14, and 18 show LOH for markers on chromosomes 4, 5, and 17. Case #22 displays extra band in tumor sample indicating instability.

Stage and follow-up information were difficult to verify because of the consultative nature of the materials and were therefore considered unreliable. DNA ploidy analysis by flow cytometry revealed DNA diploidy in 19 (86.4%) and near-diploid aneuploidy in three (13.6%); the DI’s were 1.1, 1.2, and 1.25. The proliferative fraction ranged from 2 to 26% with a mean of 5.4%. Figure 1 displays selected examples of LOH and MI of acinic cell carcinoma at various chromosomal loci examined. Microsatellite analysis showed 21 (84%) tumors with alterations in at least one locus and four tumors (16%) without any alterations (Table 1 patient numbers 4, 9, 10, and 17). Loss of heterozygosity (LOH) was the dominant alteration present; only two tumors displayed concurrent microsatellite instability (MI) and LOH. In one tumor (#22), simultaneous LOH and MI at the same marker D5S820 (Table 2) was found. Overall, alterations at chromosomes 4p, 5q, 6p, and 17p were higher than those at chromosomes 1p and 1q, 4q, 5p, and 6q. The highest incidence of alterations was observed at 4p with 52% and 17p with 50% (Fig. 2). Microsatellite markers D4S2366 on 4p16, D6S477 at 6q25-qter, and D17S799 at 17p11 showed the highest incidence of alterations with 50%, 41%, and 42%, respectively. Other

markers showed alterations ranging from 7 to 31% of the informative loci. Table 3 presents the correlation between clinicopathologic factors and DNA content analysis and the LOH in acinic cell carcinoma. Significant statistical correlation was only found between LOH and tumor grade (P 5 0.03). The majority of grade II and grade III tumors (92.3%) had LOH at >2 loci, in contrast to the six (50%) grade I. No apparent association between age, gender, tumor size, DNA ploidy, and proliferative fraction and LOH was found. DISCUSSION Defining the genetic abnormalities in salivary gland neoplasms may allow for better understanding of their development and biological behavior. Molecular investigations of these neoplasms, however, are few and limited to small numbers of both benign and malignant lesions [12–18, 20]. These, together with the broad histogenetic, phenotypic and biological continuum of these neoplasms [1–3, 6, 26], underscore the need for studies of defined histopathologic subtypes. Previous cytogenetic analysis of a few examples of acinic cell carcinomas have shown structural alterations at chromosomes 1, 4, 5, and 6 [19, 21, 22].

D1S219 D1S464 D1S549 D4S412 D4S2366 D4S398 D4S1541 D4S2367 D5S1492 D5S819 D5S431 D5S1719 D5S820 D6S477 D6S285 D6S980 CHRNB1 D17S969 D17S799 D17S122

1p32-p22 1p32-p22 1q25-q32 4p16-p15 4p16-p15 4q11-q13 4q11-q13 4q12-q13 5p15 5p15 5p11-q11.2 5q11-q13 5q33-q34 6p25-pter 6p22.3-p22.2 6q21-qter 17p12-p11 17p12-p11 17p11 17p11.2

Di Di Tet Di Tet Di Di Tet Tet Tet Di Tet Tet Tet Di Tet Di Tet Di Di

Type

Ho He He LOH He LOH Ho Ho Ho Ho LOH Ho He He Ho He He Ho Ho He

1 He He He N LOH LOH He He LOH He He He He Ho He He He N LOH N

2 He He LOH He He He He He He Ho He N He LOH He N He Ho LOH He

3 Ho Ho He N Ho Ho Ho He He He He Ho He LOH Ho LOH He He LOH Ho

5 He He He He Ho He He He He He He Ho He Ho He He LOH He LOH He

6 Ho He He He He He Ho He He He He Ho He He He He LOH Ho He LOH

7 Ho LOH N N LOH He He Ho He N Ho LOH He N He N N N N N

8 LOH Ho He Ho LOH He He Ho He Ho Ho He LOH He He Ho He Ho LOH N

11 Ho He He N He He He He N Ho He He He He He He He LOH He Ho

12 Ho He Ho Ho LOH N He He N N He Ho LOH He LOH N He Ho He N

13 Ho Ho He He LOH LOH He He Ho He Ho Ho He LOH He Ho He He He He

14

Case No.

He He He N He He He He He Ho LOH He He Ho Ho Ho Ho He He He

15 Ho LOH N N LOH LOH He He He He Ho N He N Ho N LOH He LOH He

16 He He He N LOH He Ho He He Ho He Ho LOH He LOH He He Ho He He

18 LOH He N N Ho N He He Ho He LOH LOH Ho LOH Ho N He Ho Ho N

19 He I LOH Ho LOH Ho Ho He N Ho He He He I Ho He He LOH I N

20

LOH He N Ho LOH N Ho LOH He He He LOH Ho He He Ho Ho Ho Ho He

21

He He I I Ho He He I LOH I Ho He X I LOH He LOH Ho I He

22

Ho He LOH He Ho He He He He LOH He He He He He Ho LOH He He He

23

He He Ho He He LOH Ho Ho Ho Ho Ho He He He He He Ho Ho He He

24

He Ho N He He Ho He LOH He Ho Ho LOH Ho LOH Ho N He He Ho He

25

21 16 25 22 50 32 0 16 13 18 19 31 20 41 19 9 25 18 42 7

MA (%)

Abbreviations: LOH, loss of heterozygosity; He, heterozygous; Ho, homozygous; I, instability; X, loss of heterozygosity and instability; N, no amplification; Di, dinucleotide; Tet, tetranucleotide; MA, microsatellite alteration.

Marker

Chromosome

Microsatellite

Table 2 Allelotyping of acinic cell carcinoma

22 A. K. El-Naggar et al.

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Microsatellites in Acinic Parotid Gland Tumors REFERENCES

1. Batsakis JG, Luna MA, El-Naggar AK (1990): Histopathologic grading of salivary gland neoplasms: II. Acinic cell carcinoma. Ann Otol Rhino Laryngol 99:929–933. 2. Batsakis JG, Regezi JA, Luna MA, El-Naggar AK (1989): Histogenesis of salivary gland neoplasms: a postulate with prognostic implications. J Laryngol Otol 103:939–944. 3. Batsakis JG, Wojniak KD, Regezi JA (1977): Acinous cell carcinoma: a histogenetic hypothesis. J Oral Surg 35:904–906. 4. Fujita S, Takahashi H, Okabe H (1992): Nuclear organizer regions in malignant salivary gland tumors. Acta Pathol Jpn 42:727–733. 5. Birek C, Lui E, Dardick I (1993): c-fos underexpression in salivary gland tumors as measured by in-situ hybridization. Am J Pathol 142:917–923. 6. El-Naggar AK, Batsakis JG, Luna MA, McLemore D, Byers RM (1990): DNA flow cytometry of acinic cell carcinoma of major salivary glands. J Laryngol Otol 104:410–416. 7. Ellis GL, Corio RL (1983): Acinic cell adenocarcinoma. A clinicopathologic analysis of 294 cases. Cancer 52:452–459.

Figure 2 Frequency distribution of microsatellite alterations at each chromosomal arm tested.

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Our results show that 21 (84%) of the 25 neoplasms manifested alterations in at least one marker. The most frequently altered regions were found on 4p and 17p, followed by 5q and 6p (Fig. 2). LOH was the most prevalent alteration in our cases. The high incidence of alteration at markers D4S2366 on 4p16, D17S799 on 17p11, and D6D477 at 6p25-pter loci further suggests that these loci may broadly identify the regions to be targeted for further in-depth molecular studies. Microsatellite instability (MI) was noted in only two tumors concurrently with LOH at different markers; in one tumor, concurrent LOH and MI at the same marker was noted. Such an occurrence has previously been reported in head and neck squamous carcinoma but its significance remains unclear [27]. These findings suggest that the MI plays a minor role in the oncogenesis of these neoplasms. Based on our results and those found in other salivary gland tumors, it appears that variations in the chromosomal alterations at certain regions characterize different neoplastic subtypes [13–18]. The diagnostic and clinical implications of these findings on the future evaluation of these neoplasms are under investigation. In our study, high tumor grade was the only pathologic parameter to significantly correlate with the extent of genetic alteration. No similar correlations between tumor size, high proliferative index, and histologic pattern were found. Future efforts to establish cell lines for functional studies and to further localize the most altered loci for gene identification are needed.

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