Multiple Allelic Deletions and Intratumoral Genetic Heterogeneity in ...

0021-972X/01/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 2001 by The Endocrine Society

Vol. 86, No. 3 Printed in U.S.A.

Multiple Allelic Deletions and Intratumoral Genetic Heterogeneity in MEN1 Pancreatic Tumors* OLA HESSMAN, BRITT SKOGSEID, GUNNAR WESTIN,

AND

¨ RAN ÅKERSTRO ¨M GO

From the Department of Surgical Sciences, Endocrine Unit (O.H., G.W., G.Å.), and Department of Medical Sciences, Endocrine Oncology Unit (B.S.), Uppsala University Hospital, SE-751 85 Uppsala, Sweden ABSTRACT Multiple endocrine neoplasia type 1 (MEN1) is an inherited syndrome with multiple tumors of the endocrine pancreas, the parathyroid, the pituitary, and other tissues. The MEN1 gene at 11q13 is homozygously mutated in the majority of MEN1 tumors. Here we present a genome-wide loss of heterozygosity (LOH) screening of 23 pancreatic lesions, one duodenal tumor, and one thymic carcinoid from 13 MEN1 patients. Multiple allelic deletions were found. Fractional allelic loss varied from 6 –75%, mean 31%. All pancreatic tumors displayed LOH on chromosome 11, whereas the frequency of

T

HE MULTIPLE endocrine neoplasia type 1 (MEN1) syndrome is an autosomal, dominantly inherited disorder that classically consists of multiple tumors of the parathyroid, endocrine pancreas, duodenum, pituitary, and often other tissues as well (1). The endocrine pancreatic and duodenal tumors are often associated with clinical syndromes of hormone excess or may be clinically nonfunctioning. Pancreatic endocrine tumors also occur as nonfamilial lesions without association with the MEN1 syndrome and, in general, have a more indolent behavior than the more common pancreatic adenocarcinomas. A variety of somatic genetic alterations have been detected in pancreatic exocrine tumors (2), whereas knowledge of the genetic events in pancreatic endocrine tumorigenesis is more scarce. The MEN1 gene at chromosome 11q13 encodes the protein menin, which was recently shown to repress JunD transcriptional activity (3, 4). The vast majority of MEN1 probands display heterozygous germline mutations of this gene (5, 6). Tumors of MEN1 patients generally show somatic deletions of the remaining wild-type allele of this locus, strongly indicating a tumor suppressor function of the MEN1 gene (5, 6). In addition, about one-third of nonfamilial pancreatic endocrine tumors display homozygous somatic MEN1 gene mutations (7–9). Deletions on chromosomal arms 1p, 3p, 17p, and 18q have been found in pancreatic endocrine tumors, each of them has been suggested to relate to malignancy in nonfamilial lesions Received February 21, 2000. Revised July 5, 2000. Rerevised October 31, 2000. Accepted November 2, 2000. Address all correspondence and requests for reprints to: Dr. Ola Hessman, Department of Surgical Sciences, Endocrine Unit, Uppsala University Hospital, SE-751 85 Uppsala, Sweden. E-mail: ola.hessman@ kirurgi.uu.se. * This work was supported by Swedish Medical Research Council, Swedish Cancer Society; Lions Fund for Cancer Research; Swedish Society of Medicine; Erik, Karin and Go¨sta Selanders Foundation; and Swedish Society for Medical Research.

losses for chromosomes 3, 6, 8, 10, 18, and 21 was over 30%. Different lesions from individual patients had discrepant patterns of LOH. Intratumoral heterogeneity was revealed, with chromosome 6 and 11 deletions in most tumor cells, whereas other chromosomal loci were deleted in portions of the analyzed tumor. Chromosome 6 deletions were mainly found in lesions from patients with malignant features. Fractional allelic loss did not correlate to malignancy or to tumor size. Our findings indicate that MEN1 pancreatic tumors fail to maintain DNA integrity and demonstrate signs of chromosomal instability. (J Clin Endocrinol Metab 86: 1355–1361, 2001)

(10 –13). Recently, genome-wide allelotyping of sporadic pancreatic endocrine tumors revealed deletions mainly on chromosome arms 3p, 3q, 11p, 11q, 16p, and 22q, but there was no apparent correlation between accumulation of these deletions and malignancy (14). A comparative genomic hybridization (CGH) study on nonpancreatic tumors from a single MEN1 patient revealed deletions on several chromosomes (15), but no study has so far described the extent of genetic alterations in MEN1 pancreatic tumors. In this present study, we performed a genome-wide loss of heterozygosity (LOH) screening on MEN1 tumors to describe the distribution of allelic deletions and the variation of the LOH profile between different lesions in individual patients. Subjects and Methods Subjects and samples The material consisted of 23 endocrine pancreatic tumors, one duodenal tumor, and one thymic carcinoid tumor from 13 MEN1 patients of different kindred. All patients had unequivocal signs of the MEN1 syndrome. In nine patients, evidence of malignant disease were lacking, whereas four patients had obvious malignant disease with distant metastases or locally invasive tumors. Multiple tumors were analyzed in five patients. The tumor size varied between 2 and 90 mm, median 8 mm, in diameter. Clinical characteristics of the individual patients and tumors are summarized in Table 1. All tumors were snap frozen at time of surgery. To avoid gross contamination by nontumor cells, the specimens were microdissected. Tumor DNA was prepared from cryosections with standard proteinase K/SDS digestion and phenol/chloroform extraction. Paired germ-line DNA was extracted from leukocytes. Tumor 387:11 was prepared as follows: The available tumor had a previously cut surface of approximately 3 mm2 with a thickness of 1 mm. Primarily ten 6-␮m cryostat sections were made, the sections were pooled, and DNA was extracted (original tumor DNA preparation). The remaining tumor was cut perpendicular to the already cut surface into four quadrants, from each of which DNA was extracted separately. Informed consent and approval of ethical committee was achieved.

1355

Insulin, gastrin pp, VIP

ZEa Nonfunctioning

ZE Nonfunctioning

Hyperinsulinemia ZEa

Nonfunctioning ZE Nonfunctioninga Nonfunctioning

M4 M5

M6 M7

M8 M9

M10 M11d M12 M13e

Local overgrowth None None None

hCG␣, pp Gastrin, pp, hCG␣ Proinsulin, pp Proinsulin, insulin, pp

3 4.5 5.5 9.5

2 8

4 4

6.5 4

11.5 10.5 8

Follow-up (years)

62 65 33 20

25 53

47 36

46 46

72 54 56

Age at surgery (years)

392 1110 3020 382.22 382.20 3659 5331c 5325 721.2 721.5 5359 5465 5474 5475 5477 6370 387.2 387.11 387.12 5850 702.1 624.3 227.5 227.12 227.15

Tumor no

Pancreas Pancreas Pancreas Pancreas Pancreas Duodenum Thymus Pancreas Pancreas Pancreas Pancreas Pancreas Pancreas Pancreas Pancreas Pancreas Pancreas Pancreas Pancreas Pancreas Pancreas Pancreas Pancreas Pancreas Pancreas

15 9 20 3 3 8 80 9 20 4 5 20 2 3 4 40 15 3 2 90 15 3 9 4 2

Tumor size (mm)

Tumor characteristics Tumor origin

ZE, Zollinger Ellison syndrome; FAL, fraction of allelic loss; pp, pancreatic polypeptide; VIP, vasoactive intestinal polypeptide; hCG␣, human CG subunit ␣. a Patients with adrenal hyperplasia. b Lymph node metastasis diagnosed 9 yrs after surgery. c Recurrent thymic carcinoid. d Belongs to kindred with low rate of malignancy. e Belongs to kindred with high rate of malignancy.

None Lymph node metastasis

None None

None Recurrent thymic carcinoid

None Lymph node metastasisb None

Insulin, proinsulin Gastrin, pp

Gastrin, pp, VIP Gastrin, pp, chromogranin

Gastrin, pp pp, calcitonin, gastrin Gastrin, chromogranin

ZEa ZEa ZEa

M1 M2 M3

Signs of malignancy

Clinical characteristics of MEN1 patients Elevated circulating markers

Hormonal syndrome

Patient ID

TABLE 1. Clinical and tumor characteristics of MEN1 patients and tumors analyzed by genome-wide LOH screening

11 14 26 12 29 9 6 75 69 27 18 24 19 22 70 43 26 62 22 42 61 13 30 41 6

FAL (%)

1356 HESSMAN ET AL. JCE & M • 2001 Vol. 86 • No. 3

FREQUENT LOH IN MEN1 PANCREATIC TUMORS LOH analysis

Results

A genome-wide LOH screening was performed with 98 different microsatellite markers in the Weber set 6 supplied by Nordic Consortium Primer Resource Center at the Department of Clinical Genetics (Uppsala, Sweden) to achieve at least one informative marker per chromosomal arm for the majority of tumors (Table 2). Custom-made primers were used for additional chromosome 11q13, 16p, and 18q microsatellite markers (Applied Biosystems, Inc., Foster City, CA). For acrocentric chromosomes, only the long arm was analyzed. The LOH analyses were performed on ABI 877 (Applied Biosystems, Inc.) with fluorescent-labeled primers included in the PCR amplification reaction. PCR contained 10 ng of DNA, 2 pmol of each primer (one of which was end-labeled with HEX, 6-FAM or TET), 200 ␮mol/L of each deoxynucleotide triphosphate, 1⫻ reaction buffer (Life Technologies, Inc., Gaithersburg, MD), 1.5 mmol/L MgCl2 and 0.2 U Taq DNA polymerase (Life Technologies, Inc.) in a final volume of 5 ␮L. The reactions were performed as follows: denaturation at 95 C for 5 min, followed by 27 cycles of annealing at 55 C for 30 sec, extension at 72 C for 30 sec, and denaturation at 95 C for 30 sec. Lastly, a 10-min final extension at 72 C was carried out. The amplified alleles were resolved on an ABI 310 semiautomated sequencer together with GeneScan 350 TAMRA as size marker, and quantified with GeneScan software (Applied Biosystems, Inc.). The levels of reduction of one of the two alleles present in heterozygous individuals were divided into three groups. A reduction of 50% or more was considered as complete LOH, a reduction of 30 –50% was regarded as partial LOH, whereas a reduction of less than 30% was defined as no LOH.

Statistics Statistical analysis was performed with Statistica for Windows 5.1 (StatSoft, Inc., Tulsa, OK). Student’s t test was used for comparing malignant features and fractional allelic loss (FAL), whereas relationship between tumor size and FAL was determined by Pearson’s correlation analysis. P ⬍ 0.05 was considered significant.

TABLE 2. Microsatellite markers used in genome-wide LOH analysis of MEN1 tumors D1S551 D1S642 D1S1589 D1S549 D2S1384 D2S1356 D2S1363 D2S1649 D2S434 D3S1768 D3S1050 D3S2387 D3S2398 D3S2427 D4S2397 D4S2366 D4S2408 D4S2639 D4S2431 D4S2368 D5S2505 D5S392 GATA7C06 D5S2501 D5S816

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D6S1019 D6S1050 D6S1281 D6S1009 D6S1003 D6S1277

D12S391 D12S372 D12S374 D12S373 D12S1045 D12S395

D7S513 D7S1802 D7S821 D7S1804

D13S325 D13S793 D13S173

D8S1099 D8S592 D8S1179 D8S373 D9S925 D9S1118 D9S302 D10S189 GAAT5F06 D10S1230 D10S1237 D10S1239 D11S1392 D11S1985 PYGM INT2

D14S749 D14S611 D14S118 D14S617 D15S652 D15S642 D16S748 D16S769 D16S403 D16S2624 D16S3031 D16S496 D17S1298 D17S1308 D17S974 D17S1299 D17S809 D17S1290

D18S976 D18S843 D18S851 D18S64 D18S541 D19S247 D19S254 D19S601 D20S95 D20S604 D20S470 D20S1085 D20S481 D21S156 D21S1446 D21S1435 D21S1270 D22S685 D22S445 D22S683

The results from our genome-wide LOH screening of MEN1-associated tumors are shown in Table 3 and Figs. 1, 2, and 3. In total, of 975 examined chromosomal arms, 884 displayed at least one informative marker (91%). A high number of deletions were found in most tumors, with a FAL ranging from 6 –75%, and a mean FAL of 31%. FAL for each tumor is defined as the number of chromosomal arms with LOH divided by the total number of informative arms. Deletions were seen on all the 39 examined chromosomal arms, and similar levels of allele retention were often detected on both arms of affected chromosomes. Allelic loss on chromosome 7 and 14 were rare and when present consisted only of partial LOH (see Fig. 2). All pancreatic tumors had complete LOH on chromosome 11. This pattern was not true for the duodenal tumor (no. 3659) and the thymic carcinoid (no. 5331), both retaining the wild-type allele for the MEN1 locus. These latter tumors displayed a low number of deletions overall (9 and 6%, respectively). In more than one third of all tumors, deletions were seen on both arms for chromosomes 3, 6, 8, 10, 18, and 21. Of the eleven tumors with deletions on chromosome 6, nine displayed complete LOH on both arms. In five patients, multiple primary tumors were analyzed. We found a considerable intertumoral discrepancy of LOH patterns for these multiple tumors. Furthermore, different levels of allele retention for different chromosomes were frequently found at analyses of individual tumor specimens. To address the possibility of intratumoral genetic heterogeneity more directly, tumor 387:11 was divided into four quadrants, which did not differ in architectural or stromal components in conventional Hematoxylin-Eosin staining (Fig. 4, A–D). The four fractions displayed heterogeneous LOH patterns and different FAL, but all four had complete loss of markers on chromosome 6 and 11 (Table 4). All tumors from two of three patients with malignant pancreatic disease (M9 and M10) and two of three tumors from a patient belonging to a kindred with frequent malignancies (M13) displayed complete LOH on both arms of chromosome 6. Furthermore, two of three pancreatic tumors from patient M5 with a malignant thymic carcinoid also had complete chromosome 6 deletions. These tumors with chromosome 6 losses varied in size from 2–90 mm. No association between malignant features and number of allelic deletions was revealed. Neither did tumors size and FAL correlate (r ⫽ 0.006, N.S.). No intraindividual variations of allele sizes were found. Thus, we did not record any signs of microsatellite instability. Discussion

Our genome-wide LOH screening revealed multiple allelic deletions in all MEN1 pancreatic endocrine lesions, with losses found on most chromosomes and often encompassing both arms. The mean FAL of 31% is similar to what has been shown previously for sporadic pancreatic endocrine tumors as well as for many exocrine tumors (14, 16). Discrepant levels of allele retention for different chromosomes within one specific tumor were commonly seen. Repeated examination with additional microsatellite markers for the same chromosomal arms confirmed the accuracy of

11

FAL(%)

14

▫ ▫ ▫ ▫ 䡲 Œ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ NI 䡲 ▫ ▫ 䡲 ▫ NI 䡲 ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ NI ▫ ▫ ▫ ▫ ▫ 12

䡲 ▫ ▫ ▫ ▫ ▫ ▫ ▫ NI ▫ ▫ ▫ Œ ▫ ▫ Œ ▫ ▫

䡲 ▫ ▫ Œ Œ ▫ ▫ ▫ NI ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫

26

▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ NI ▫ ▫ ▫ 䡲

▫ ▫ NI ▫ ▫

▫ ▫ NI ▫ ▫ ▫ Œ Œ ▫ Œ ▫ ▫ Œ Œ ▫ NI ▫ Œ ▫ ▫

29

Œ 䡲 䡲 ▫ ▫ ▫ 䡲 䡲 NI ▫ ▫ ▫ ▫ ▫ ▫ Œ ▫ 䡲

▫ ▫ NI ▫ ▫ ▫ Œ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ NI ▫ Œ ▫ Œ

9

▫ ▫ ▫ ▫ NI ▫ ▫ ▫ ▫ Œ Œ ▫ NI ▫ NI ▫ NI ▫ ▫ ▫

▫ ▫ ▫

▫ ▫ ▫ Œ ▫ NI ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫

6

▫ Œ Œ ▫ ▫ ▫ ▫ NI ▫ ▫ ▫ ▫ ▫ ▫ NI ▫ ▫ ▫ ▫ ▫ ▫ NI ▫ ▫ ▫ ▫

▫ ▫ ▫ ▫

▫ ▫ ▫ ▫ ▫ ▫ ▫

a

M6

M7

M8

M9

M10

M11

M12

M13

75

▫ ▫ 䡲 ▫ 䡲 䡲 䡲 䡲 Œ ▫ 䡲 䡲 ▫ ▫ 䡲 䡲 Œ Œ 䡲 䡲 NI 䡲 Œ Œ ▫ ▫ Œ NI 䡲 ▫ 䡲 Œ Œ 䡲 NI Œ 䡲 䡲 䡲 69

NI 䡲 NI 䡲 Œ Œ 䡲 NI Œ Œ Œ Œ

▫ 䡲 䡲 ▫ ▫ 䡲 䡲 NI 䡲 ▫ ▫ ▫ ▫

▫ Œ 䡲 䡲

▫ ▫ 䡲 䡲 䡲 Œ 䡲

27

▫ ▫ ▫ ▫ ▫ ▫ ▫ NI 䡲 ▫ ▫ Œ ▫ ▫ NI ▫ ▫ 䡲 ▫ ▫ Œ NI ▫ ▫ Œ ▫

Œ Œ ▫ ▫

▫ ▫ NI ▫ 䡲 Œ ▫

18

NI ▫ ▫ ▫ ▫ Œ ▫ NI ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ NI ▫ ▫ 䡲 䡲 ▫ ▫ NI ▫ ▫ ▫ ▫ ▫ ▫ 䡲 䡲 NI ▫ ▫ NI Œ ▫ 24

▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ Œ 䡲 䡲 ▫ ▫ 䡲 ▫ ▫ ▫ ▫ ▫ ▫ 䡲 䡲 ▫ ▫ NI Œ 䡲 ▫

Œ ▫ ▫ ▫ ▫ ▫ ▫

19

▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ Œ 䡲 䡲 ▫ 䡲 ▫ ▫ Œ ▫ ▫ ▫ ▫ 䡲 䡲 ▫ ▫ NI ▫ ▫ ▫

▫ ▫ ▫ ▫ ▫ ▫ ▫

22

▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ Œ 䡲 䡲 ▫ 䡲 ▫ ▫ ▫ ▫ ▫ ▫ ▫ 䡲 䡲 䡲 䡲 NI ▫ ▫ ▫

▫ ▫ ▫ ▫ ▫ ▫ NI

70

䡲 䡲 Œ Œ ▫ ▫ 䡲 䡲 Œ Œ Œ 䡲 䡲 䡲 Œ Œ 䡲 Œ ▫ Œ Œ Œ ▫ 䡲 䡲 ▫ ▫ NI ▫ ▫ ▫

▫ ▫ Œ Œ Œ Œ 䡲

43

䡲 Œ ▫ Œ Œ Œ ▫ ▫ ▫ NI 䡲 Œ 䡲 䡲 ▫ ▫ ▫ ▫ Œ ▫ ▫ ▫ ▫ Œ 䡲 ▫ NI 䡲 ▫ 䡲 ▫

NI ▫ ▫ ▫ ▫ ▫ Œ

26

䡲 䡲 ▫ ▫ Œ ▫ 䡲 䡲 ▫ ▫ ▫ ▫ ▫ Œ ▫ ▫ 䡲 䡲 ▫ Œ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ 䡲 ▫

▫ ▫ ▫

62

▫ ▫ 䡲 䡲 Œ Œ Œ Œ Œ ▫ Œ Œ Œ 䡲 ▫ NI ▫ Œ ▫ Œ Œ ▫ ▫ Œ Œ ▫ Œ ▫ ▫ Œ Œ

▫ ▫ Œ Œ Œ Œ ▫

22

▫ ▫ 䡲 䡲 ▫ ▫ ▫ ▫ 䡲 ▫ ▫ ▫ 䡲 䡲 ▫ NI Œ ▫ ▫ ▫ ▫ ▫ ▫ ▫ Œ ▫ ▫ ▫ ▫ ▫ ▫

▫ ▫ ▫ Œ ▫ ▫ ▫

42

▫ ▫ 䡲 䡲 ▫ ▫ ▫ ▫ ▫ ▫ 䡲 䡲 ▫ ▫ Œ Œ ▫ ▫ NI ▫ 䡲 䡲 ▫ ▫ 䡲 ▫ ▫ 䡲 䡲 䡲 䡲 䡲 ▫ ▫ ▫ ▫ ▫ 䡲 䡲 61

䡲 䡲 NI 䡲 䡲 䡲 ▫ NI ▫ ▫ 䡲 䡲 ▫ ▫ NI 䡲 䡲 䡲 䡲 䡲 䡲 䡲 ▫ ▫ 䡲 ▫ 䡲 䡲 䡲 ▫ ▫ 䡲 䡲 ▫ ▫ ▫ ▫ 䡲 䡲 13

Œ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫

▫ NI NI ▫ NI ▫ ▫ NI ▫ ▫ ▫ ▫ ▫ ▫ NI ▫ ▫ ▫ Œ ▫ 䡲 䡲 ▫ ▫ ▫ ▫ ▫ NI

30

Œ ▫ 䡲 䡲 ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ 䡲 䡲 ▫ ▫ ▫ NI ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ Œ ▫ Œ ▫

Œ ▫ ▫ Œ 䡲 䡲 ▫

41

▫ ▫ 䡲 䡲 ▫ ▫ 䡲 䡲 ▫ ▫ Œ 䡲 䡲 䡲 ▫ Œ 䡲 NI ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ Œ ▫ Œ ▫

Œ ▫ ▫ ▫ 䡲 䡲 ▫

6

䡲 ▫ ▫ ▫ NI ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫

▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫

▫ ▫ ▫ Œ ▫ ▫ ▫

5325 721.2 721.5 5359 5465 5474 5475 5477 6370 387.2 387.11 387.12 5850 702.1 624.3 227.5 227.12 227.15

M5

17 4 26 38 42 48 25 22 24 20 40 44 14 12 33 41 20 26 43 50 94 92 12 32 33 14 17 25 33 19 17 48 44 27 5 25 21 56 24

%b

HESSMAN ET AL.

䡲, Loss of heterozygosity; Œ, 50 –70% retention; ▫, retention of heterozygosity; NI, non-informative; FAL, fractional allelic loss. a Tumor 5331 is a thymic carcinoid. b Frequency of allelic loss.

▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ NI ▫ ▫ ▫ ▫ ▫ ▫ NI 䡲 䡲 ▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ NI NI ▫ 䡲 ▫ ▫ ▫ 䡲 ▫

1p 1q 2p 2q 3p 3q 4p 4q 5p 5q 6p 6q 7p 7q 8p 8q 9p 9q 10p 10q 11p 11q 12p 12q 13q 14q 15q 16p 16q 17p 17q 18p 18q 19p 19q 20p 20q 21q 22q

M4

392 1110 3020 382.22 382.20 3659 5331

M3

Tumor no

M2

M1

Patient ID

TABLE 3. Results of genome-wide LOH screening in MEN1-associated pancreatic endocrine tumors

1358 JCE & M • 2001 Vol. 86 • No. 3

FREQUENT LOH IN MEN1 PANCREATIC TUMORS

the results (data not shown). Furthermore, similar levels of retention were often found for both arms of affected chromosomes. Markers showing no LOH in a specific sample had

FIG. 1. FAL values for 25 individual tumors from 13 patients.

FIG. 2. Frequency of LOH on each chromosomal arm for all analyzed tumors.

FIG. 3. A–C, Examples of different LOH categories in tumor 5325 of patient M5. A, Complete LOH of marker D6S1281 and D8S373 at chromosome 6p and 8q, respectively. B, Partial LOH (42% reduction) of marker D12S395 at chromosome 12q. C, Retention of marker D14S617 at chromosome 14q.

1359

most often levels close to 100% and were distinctly separated from markers, indicating a loss in that particular specimen (Fig. 3). Such consistent results seem to exclude PCR artifacts as the cause of intermediate levels of retention (partial LOH). Moreover, preferentially PCR amplification of one allele is likely to involve the shorter one, but in this study both shorter and longer alleles were partially lost. Intermediate levels of allele retention could theoretically also be due to a variable degree of intratumoral gene amplification of one chromosomal copy. However, no gain of genetic material was revealed in MEN1 tumors in a recently published CGH study (15). The frequent finding of complete LOH for chromosome 11 (retention levels of 15–34%) and partial LOH on others within a single tumor, indicates that the DNA preparations lacked significant contamination from nontumor cells and that subclones of tumor cells had acquired additional genetic alterations. This variation of levels of allele retention implies true intratumoral genetic heterogeneity. The extended analyses of tumor 387:11, which was divided in four portions before additional DNA preparations, revealed discrepant percentage of allele retention. Chromosomes with partial LOH in the original tumor sample such as number 3 and 10, showed complete loss in some of the tumor subdivisions, whereas chromosomes 6 and 11 were completely lost in all four portions of the tumor (Table 4). These results further illustrate the intratumoral genetic heterogeneity. Previously X-chromosome inactivation studies of sporadic pancreatic endocrine tumors (17), as well as MEN1 associated parathyroid lesions (18), have revealed multiple clones or oligoclonality in 50% of the specimens. Whether this heterogeneity could be due to hyperplastic growth or by coalescence of multiple neoplastic clones as proposed in parathyroid lesions (18) is yet unclear. However, an accumulation of chromosome alterations in different subclones that all originate from the same neoplastic tumor cell clone is indicated in our tumors and would be demonstrated by a uniform pattern of X-chromosome inactivation. We found complete LOH of chromosome 11 in all pancreatic tumors. Because allelic losses on chromosome 11 were apparently present in most tumor cells of a tumor, they are likely to represent early genetic events. This result supports the tumor suppressor activity ascribed to the MEN1 gene. Several tumors displayed complete LOH on chromosome

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JCE & M • 2001 Vol. 86 • No. 3

FIG. 4. A–D, Quadrant subdivisions of tumor 387:11. The frozen sections were stained with Hematoxylin-Eosin. Magnification, ⫻123.

6, including all tumors from two of three patients with malignant disease (M9 and M10) and two of three tumors from a patient belonging to kindred with frequent malignancies (M13). Two additional tumors with incomplete LOH on chromosome 6 showed no obvious signs of a malignant potential. All but one tumor with alteration on chromosome 6 had deletions on both arms; this single tumor with partial LOH on 6q retained 6p markers. In a transgenic mouse model developing multiple insulinomas, LOH was found on mouse chromosome 9 (corresponding to human 3q, 3p21, 6q, and 15q24) and chromosome 16 (human 3q and 22q) (19). The chromosome 9 deletion, in a region corresponding to human 6q, seemed to occur with progression from an angiogenic stage to solid tumor formation. A study of nonpancreatic tumors from a single MEN1 patient by comparative genomic hybridization also revealed chromosome 6 deletions (15), and another recent study showed obvious overrepresentation of 6q losses in metastatic sporadic pancreatic endocrine tumors (20). Loss of 6q and 10q in melanomas, as well as high FAL, are associated with poorer clinical outcome (21). Chromosome 6 deletions detected in our lesions had low levels of retention and were therefore believed to be present in the majority of cells of each MEN1 tumor with such losses. The reason for such a distribution could be a growth advantage to those cells that had acquired deletions on chromosome 6. These data support the hypothesis that, besides the MEN1

gene on chromosome 11, additional candidate tumor suppressor gene(s) located on chromosome 6 may be of importance in MEN1 pancreatic tumorigenesis. Association between various chromosomal loci and malignancy has been suggested (10 –13), but the one genomewide LOH screening study of sporadic pancreatic endocrine tumors could not verify a correlation between number of deletions and malignancy (14). Our data confirm that this lack of correlation between malignancy and FAL as well as tumor size and FAL is also true for the familial pancreatic endocrine tumors. Also, lesions smaller than 5 millimeters had high FAL. The lack of association to tumor progression is in contrast to the reported increased frequency of chromosomal alterations in late stage of colorectal cancer and melanomas (21, 22). This discrepancy may be explained by our ignorance of defined stages in the tumorigenic process of pancreatic endocrine tumors, including the stage when these tumors acquire potential for malignant growth. Genetic instability at the nucleotide level is found in a small subset of cancers, sometimes resulting in microsatellite instability with altered allelic size (16). Such instability has previously only been found in one of 16 sporadic neuroendocrine gastrointestinal tumors (23). None of our tumors displayed signs of microsatellite instability for any of the examined markers. In most other cancers, the genetic instability is at the chromosome level with losses or gains of large

FREQUENT LOH IN MEN1 PANCREATIC TUMORS

tation is associated with inability to maintain DNA integrity or whether this is caused by secondary molecular alterations.

TABLE 4. Genome-wide LOH in four portions of MEN1associated pancreatic endocrine tumor 387.11 Original tumor DNA preparation

1p 1q 2p 2q 3p 3q 4p 4q 5p 5q 6p 6q 7p 7q 8p 8q 9p 9q 10p 10q 11p 11q 12p 12q 13q 14q 15q 16p 16q 17p 17q 18p 18q 19p 19q 20p 20q 21q 22q FAL(%)

▫ ▫ Œ Œ Œ Œ ▫ NI ▫ ▫ 䡲 䡲 Œ Œ Œ Œ Œ ▫ Œ Œ Œ 䡲 ▫ NI ▫ Œ ▫ Œ Œ ▫ ▫ Œ Œ ▫ Œ ▫ ▫ Œ Œ 62

1361

DNA preparation after tumor quadrant subdivision A

B

C

D

▫ ▫ ▫ Œ 䡲 Œ ▫ NI ▫ ▫ 䡲 䡲 ▫ ▫ ▫ ▫ Œ ▫ Œ ▫

▫ Œ Œ Œ Œ Œ ▫ NI ▫ ▫ 䡲 䡲 Œ Œ Œ Œ Œ ▫ Œ Œ

Œ ▫ Œ Œ Œ 䡲 ▫ NI ▫ ▫ 䡲 䡲 Œ Œ Œ Œ Œ ▫ 䡲 Œ

䡲 ▫ NI ▫ ▫ ▫ ▫

䡲 ▫ NI ▫ Œ Œ Œ

▫ ▫ ▫ ▫ ▫ Œ ▫ NI ▫ ▫ 䡲 䡲 ▫ ▫ ▫ ▫ Œ Œ ▫ Œ 䡲 䡲 ▫ NI ▫ ▫ ▫ Œ

䡲 ▫ NI ▫ Œ Œ Œ

▫ ▫ ▫ ▫ ▫ ▫ ▫ ▫ Œ ▫

▫ ▫ ▫ ▫ ▫ Œ ▫ ▫ 䡲 Œ

▫ ▫ ▫ Œ ▫ ▫ ▫ ▫ ▫ ▫

▫ ▫ Œ Œ ▫ Œ ▫ ▫ Œ 䡲

26

60

28

66

䡲, Loss of heterozygosity; Œ, 50 –70% retention; ▫, Retention of heterozygosity; NI, noninformative; and FAL, fractional allelic loss.

portions of chromosomes. Chromosomal instability may be caused by errors in the processes involved in replication and segregation of chromosomes during mitosis (16). Premature centromere division, which has been suggested to manifest as chromosomal instability, has been found in lymphocytes of patients with verified MEN1 gene mutation (24), but cytogenetical investigation have not revealed a significantly elevated number of chromosomal breakage in MEN1 patients (25). A recent CGH study reported a higher frequency of CGH alterations in sporadic parathyroid lesions with somatic MEN1 mutations and/or LOH at 11q13 compared with those without MEN1 involvement (26). Our findings indicate a possible presence of chromosomal instability in MEN1 pancreatic tumors. Future studies with other methodologies are needed to elucidate whether the inborn MEN1 gene mu-

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