Neoplasia Type 2 Syndromes Pheochromocytomas ...

Consistent Association of 1p Loss of Heterozygosity with Pheochromocytomas from Patients with Multiple Endocrine Neoplasia Type 2 Syndromes Jeffrey F. Moley, Michele B. Brother, Chin-To Fong, et al. Cancer Res 1992;52:770-774. Published online February 1, 1992.

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[CANCER RESEARCH 52, 770-774, February 15, 1992)

Consistent Association of Ip Loss of Heterozygosity with Pheochromocytomas Patients with Multiple Endocrine Neoplasia Type 2 Syndromes1

from

Jeffrey F. Moley,2 Michele B. Brother, Chin-To Fong,3 Peter S. White, Stephen B. Baylin, Barry Nelkin, Samuel A. Wells, and Garrett M. Brodeur Departments of Surgery [J. F. M., M. B. B., S. A. W.], and Pediatrics fC-T. F., P. S. W.. G. M. B.]. Washington University School of Medicine, St. Louis, Missouri 63110, and the Department of Oncology fS. B. B., B. Ã‘.Â¡,Johns Hopkins Oncology Center, Baltimore, Maryland 21231

the MEN-2 syndromes to the pericentromeric region of chro mosome 10 (5, 6); neurofibromatosis to 17qll (7); and vHL syndrome to 3p (8). MTCs can occur in MEN types 2A and 2B, as well as a distinct familial MTC syndrome that maps to chromosome 10 also, but is not associated with other features of the MEN-2 phenotype (9, 10). The genetic abnormalities underlying these tumors are not well understood. Deletion or LOH in the region of the predis posing locus on chromosome 10 is found rarely in pheochrom ocytomas and MTCs. Recent reports have noted allelic loss on chromosome 10 in tumors of only 4 of 86 informative cases (11-13). This contrasts with the situation in hereditary retinoblastoma, in which tumors that occur in predisposed individuals achieve homozygosity for a defective RB-1 alÃ-elein the majority of cases (14, 15). It is possible that malignant transformation in the MEN-2 syndromes is caused by other mechanisms, such as oncogene activation or the inactivation of a tumor suppressor gene at a different chromosomal locus. Evidence of oncogene activation in these tumors has not been found as yet (16, 17). We (17) and others (18-21) have noted chromosome l LOH in hereditary and sporadic pheochromocytomas and medullary thyroid cancers, but extensive mapping of the deleted region has not been previously reported. In the studies reported here, we used a battery of 15 chro mosome Ip probes to analyze 18 pheochromocytomas and 6 chromosome l p probes to analyze 27 medullary thyroid cancers from patients with sporadic and familial disease to define the locus on Ip that is consistently deleted in these tumors and to determine its relationship to the genetic background of the patient. We found LOH of l p in all pheochromocytomas from patients with MEN-2A and -2B and in a minority of patients with sporadic pheochromocytomas. LOH of Ip was found in only 3 of the 24 informative MTCs studied, and all occurred in patients with MEN-2A.

ABSTRACT Pheochromocytomas

and medullary thyroid cancers (MTCs) are neu-

roendocrine tumors which arise sporadically or as part of the multiple endocrine neoplasia type 2 (MEN-2) hereditary syndromes. The most consistent molecular genetic abnormality which has been described in these tumors is loss of heterozygosity (LOH) of the short arm of chromosome 1 (Ip). This finding is particularly interesting because the predisposition gene for the hereditary form of these tumors has been mapped to chromosome 10, but LOH on chromosome 10 in MEN-2 tumors is found rarely. We have used a battery of Ip DNA probes to elucidate the region of loss of Ip in 18 pheochromocytomas and 27 MTCs. Using restriction fragment length polymorphism analysis, we identified loss of all or a portion of l p in 12 of 18 pheochromocytomas. Ip LOH was identified in nine of nine pheochromocytomas in MEN-2A and -2B patients, compared with only two of seven sporadic pheochrom ocytomas. We also found 1p LOH in one of two von Hippel-Lindau patients. LOH on Ip was noted in only three of 24 informative MTCs, and these were from patients with MEN-2A. In most of the pheochrom ocytomas, the entire short arm of chromosome l p appears to have been lost; however, in three of the non-MEN pheochromocytomas and in three MEN-2A MTCs, the region of loss is smaller, allowing estimation of the smallest region of overlap. The combined data for MTCs and pheochrom ocytomas suggest that the smallest region of overlap of LOH is bounded by D1S15 (Ipter-p22) and D1Z2 (1P36.3), excluding a region around MYCL (Ip32). Although other regions of Ip should not be completely ruled out, the data suggest that this region may harbor a tumor suppressor gene or genes w hose inactivation is important in the development of these tumors. Eurthermore, the strong association between 1p LOH and the MEN-2 syndromes, especially in pheochromocytomas, suggests a rela tionship between the predisposition gene on chromosome 10 and the loss of the suppressor gene on Ip. Alternatively, other loci may be more important in sporadic disease.

INTRODUCTION Pheochromocytomas and MTCs4 are neuroendocrine tumors which originate from adrenal chromaffin cells and thyroid Ccells, respectively (1-4). These tumors occur in both sporadic and familial forms and are heterogeneous in their clinical manifestations. Pheochromocytomas are a common finding in patients with MEN-2 syndromes, but these tumors also occur in patients with other hereditary syndromes, such as neurofibromatosis type 1 and vHL disease. Interestingly, each of these genetic diseases has been mapped to a different chromosome:

MATERIALS AND METHODS Tumor and Blood Samples. In each case, tumor tissue was obtained in the operating room at the time of therapeutic surgery. The tissue was immediately frozen and stored at -80Â°C. LCLs were established

Received 8/19/91; accepted 11/22/91. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was supported by a Veterans Administration grant. Research Advisory Group 0001 (J. F. M.), and N. I. H. Grant CA-39771 (G. M. B.). 2To whom requests for reprints should be addressed, at Department of Surgery, Box 8109, Washington University School of Medicine, 4960 Audubon Avenue, St. Louis, MO 63110. 3 Present address: Department of Pediatrics, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. 4 The abbreviations used are: LCL, lymphoblastoid cell line; LOH; loss of heterozygosity; MEN-2, multiple endocrine neoplasia type 2; MTC, medullary thyroid carcinoma; RFLP, restriction fragment length polymorphism; vHL, von Hippel-Lindau.

(22) from 10 ml of blood obtained from the patients at the time of blood drawing. These studies have been reviewed and approved by a human studies internal review committee for Washington University. DNA Studies. DNA was prepared from tumors and LCLs by standard detergent-proteinase K lysis, followed by organic extraction and exten sive dialysis (23, 24). DNA was quantitated by a very sensitive fluori metrie assay (25, 26). Equal amounts of normal genomic and tumor DNA (5 Â»ig) from individual patients were digested with an appropriate restriction enzyme, and DNA fragments were separated by continuous agarose gel electrophoresis. Then, DNA was transferred to a nylon membrane by a modification of the procedure of Southern (27). Filters were baked for 2 h at 80Â°C,equilibrated in prehybridization solution, and then hybridized overnight with either a plasmili labeled by nick translation (28) or with an isolated DNA fragment labeled by the random hexamer primer (29) technique. Filters were washed and ex-

770


Ip DELETIONS IN TUMORS OF MEN-2 PATIENTS

were examined. These were a patient with MEN-2 A (pheochro mocytoma Case 9, MTC Case 8) and a patient with MEN-2B (pheochromocytoma Case 11, MTC Case 23) (Figs. 2 and 3). In both patients, LOH was observed in the pheochromocytoma but not in the MTC.

posed to autoradiography film for 1 to 5 days, and then the film was developed. Probes. We used 16 DNA probes that identified RFLPs and have been regionally mapped to chromosome 1. These probes include pronatriodilatin (Ip36), alkaline phosphatase (lp36.1-34), a c-src-related protooncogene sequence (c-src-2/c-fgr, or FGR) (Ip36.2-36.1), MYCL (also known as L-myc) (Ip32), N-ras (Ipl3), nerve growth factor (0NGF) (Ipl3), renin (Iq32 or q42) and nine random polymorphic DNA sequences: D1S13 (Ip22-pl3); DIS 15-16 (Ipter-p22); D1S17 (Ipter-p32); D1S18-19 (Ipter-p22); D1S21 (lpter-22); D1S57 (Ipter31); and D1Z2 (Ip36.3) (Fig. 2) (30-32). Analysis of Alidie Loss. Pairs of normal and tumor DNA from each patient were analyzed using probe-enzyme combinations that are known to identify RFLPs in a substantial number of individuals. Normal DNA samples which were polymorphic at a given locus were considered to be informative, and if the tumor DNA demonstrated a loss or marked reduction in the intensity of one of the allelic bands, the tumor was considered to have LOH at that alÃ-ele.Tumor DNA samples contained varying amounts of normal stromal cells, as confirmed by histology. Therefore, in most cases with LOH at a given alÃ-ele,there was a faint residual band in the position of the lost alÃ-eleon the autoradiogram (see Fig. 1). In these cases, we performed densitometry using a com puterized gel scanner (Apple Scanner) and densitometry software (cour tesy of Dr. Hershel Ginsberg, Amoco Technology). For samples which were informative, results were adjusted for DNA content and back ground in the following manner. The residual density of the deleted band in the tumor DNA was normalized against the density of the retained band. The band in the constitutional DNA corresponding to the deleted band was normalized against the corresponding retained band, and the ratio of normalized, tumor-band density to the normal ized corresponding constitutional band was calculated (20). If this value was less than 0.5, indicating a greater than 50% reduction in signal intensity in the tumor DNA band, this was considered to represent LOH.

DISCUSSION Knudson et al. (15) initially postulated a two-mutational model of tumorigenesis to explain the development of heredi tary and sporadic types of certain tumors. The applicability of this theory to the MEN-2A and -2B syndromes comes from the fact that tumor development is preceded by characteristic hyperplasia of the thyroid C-cell and adrenal chromaffin cell populations (3, 4) and that MTC and pheochromocytomas develop as multifocal clonal tumors within this cellular back ground (33, 34). Furthermore, Jackson et al. (35) demonstrated that the average age of onset was 36 yr for hereditary cases of MTC and 52 yr for sporadic MTC. The MEN-2A and -2B syndromes differ from hereditary retinoblastoma (14) in that the predisposition locus has been mapped to chromosome 10 (5, 6), but deletion or LOH at that locus rarely occurs in the pheochromocytomas and MTCs which develop (11-13). A small deletion, rearrangement, or mutation of the normal alÃ-elecannot be completely ruled out; however, these data suggest that malignant transformation in MEN-2A patients is probably caused by some mechanism other than homozygous loss or mutation of the predisposition locus. We have demonstrated a high frequency of LOH for Ip in pheochromocytomas from patients with both hereditary and sporadic disease. This consistent abnormality probably indi cates the presence of a suppressor gene which is lost or inacti vated in these tumors. The finding of frequent LOH at chro-

RESULTS Representative RFLP studies for three pheochromocytomas (Tumors 2, 8, and 9) are shown in Fig. 1. As can be seen in this figure, individuals with LOH can be identified readily, despite some residual signal from contaminating normal cells in the tumor samples. LOH on Ip was found in all pheochromocyto mas from patients with MEN-2A and -2B (see Fig. 2). LOH on l p was also noted in two of seven patients with sporadic disease, and in one of two patients with vHL syndrome. We have not noted a characteristic pattern of LOH in any specific family or kindred with MEN-2A. In most tumors, the loss was extensive, involving most of the chromosomal arm. In Case 16, however, the region of LOH is bounded proximally by D1S15 (Ipter-p22) (30) and distally by D1Z2 (Ip36.3) (31). D1Z2 was also found to flank the deleted region distally in Cases 4 and 19. In general, there were less tumor tissue and DNA available for the MTCs, because many were detected by clinical screening studies before they were large enough to produce symptoms. Twenty-four of 27 MTCs (14 MEN-2A, 5 MEN-2B, 5 sporadic) studied so far were informative for at least one of 6 loci (D1Z2, D1S17, D1S2I, D1S57, MYCL, NRAS). Of these, three dem onstrated LOH for loci on Ip (Fig. 3). In Case 21, LOH occurred at D1S17 ( l pter-p32) (30) and N-ros (l p 13) (30), with retention of heterozygosity at D1Z2 (Ip36.3). In Case 32, LOH occurred at D1S21 (Ipter-p22) (30), MYCL (Ip32) (30), and D1S57 (Ipter-p31) (32); and in Case 13, LOH was found at D1S17 and D1Z2, but not at MYCL. All of these patients had MEN-2A. In two individuals, both MTC and pheochromocytoma tissue

2.3Kb-

DIZ2 (Eco RI) 1.1Kb

DIS57 (Toq I)

4.6Kb3.5Kb-

MYCL (Eco RI) DIS2I (Toq I)

1.6Kb 1.3Kb-

Fig. 1. Analysis of LOH on IP by RFLp studies. Three representative pheo chromocytomas are shown. Cases 8 and 9 are patients with MEN-2A, and Case 2 is a patient with sporadic disease. In each case, DNA from a normal source (N) and from the tumor (7") was digested with a restriction enzyme, electrophored, transferred to a nylon filter, and probed with a radiolabeled DNA probe (see left of figure). For each case, probe-enzyme combination was considered informative if the normal DNA demonstrated more than one polymorphic band. Case 2 demonstrates no LOH at three informative loci: D1Z2; D1S57; and DIS21 (MYCL is uninformative). Case 8 demonstrates LOH at D1Z2. MYCL, and D1S21. Case 9 demonstrated LOH at DIZ2, D1S57, and D1S21. Kb, kilobases.

771


Ip DELETIONS

IN TUMORS OF MEN-2 PATIENTS

We have found that the most consistently deleted region in pheochromocytomas is bounded by the D1Z2 locus (Ip36.3) distally and proximally by D1S15 (Ipter-p22). This region 0â€¢ contains several known oncogenes and other structural genes. â€¢¿â€¢ Unfortunately, none of these appear to be attractive candidates for the suppressor gene that presumably is the target of this â€¢¿0 0o genetic rearrangement. Furthermore, no evidence of re â€¢¿â€¢ arrangement or amplification was found for any of the probes â€¢¿0 studied in normal or tumor tissues, including those tumors with LOH (data not shown). Although the region that is consistently .â€¢*â€¢â€¢â€¢.â€¢â€¢.â€¢*.â€¢.â€¢â€¢.â€¢â€¢â€¢*â€¢â€¢â€¢â€¢,â€¢'0iOOâ€¢:â€¢â€¢,.â€¢â€¢0.â€¢oo,â€¢.0 lost in our tumors potentially overlaps the Ip36 locus that is 0 consistently deleted in neuroblastomas (39), it extends to a large region of the short arm, suggesting that a more proximal Iq 23-25 |Â«""|o|oH-MÃ¶Fl[Ã¶nfÃ¶|o|o|o|o|o| l FF1 locus may be involved. In the MTCs we studied, one MEN-2A Fig. 2. Analysis for LOH at various chromosome 1 loci in human pheochromtumor (Case 13) with LOH at Dl SI 7 and D1Z2 demonstrated ocytomas. The probes used and their relative locations on Ip (30) are listed at retention of heterozygosity at MYCL. If this finding is com the left of the box. To the right are the specific results obtained in the pheochrombined with our data on pheochromocytomas, it suggests a ocytomas. Informative combinations are indicated (iv. LOH; m, no LOH). Q, noninformative combinations. Shaded areas indicate the minimum area presumed potential suppressor locus between MYCL (Ip32) and D1S15 to be deleted, as defined by probes showing LOH. LOH was seen in 7 of 7 (Ipter-p22) or between MYCL and D1Z2 (Ip36.3). This is pheochromocytomas from patients with MEN-2A, in 2 of 2 MEN-2B, in 2 of 7 consistent with the results of Yang et al. (19) whose data suggest sporadic, and in one of 2 pheochromocytomas from patients with vHL. Note that the smallest region of overlap appears to be between D1S1S and D1Z2. a common region of deletion in pheochromocytomas in the middle of the short arm (Ip22 to Ip36). The only known structural gene which has been mapped to the region between mosomal locations other than predisposition loci is not unprec MYCL and D1S15 is a human glucose transporter (40). We edented. In colon cancers, the predisposition locus for familial have not found any evidence of abnormality of this gene at the polyposis coli maps to the long arm of chromosome 5, whereas DNA level (data not shown) in tumors with LOH of Ip nor LOH for 17p and 18q is predominant in frank colon cancers have we found any abnormality of several structural genes which (36, 37). map to the region between MYCL and D1Z2, including pronOther groups have noted chromosome 1 deletions in MENatriodilatin, alkaline phosphatase, and a c-irc-related protoon 2A tumors, using fewer probes than in the present study (18cogene (FGR). 21). Mathew et al. (18) noted LOH using a single Ip probe, Ip LOH has been demonstrated in other tumors of neuroecD1S7 (lp35-33) in 7 of 14 pheochromocytomas and MTCs from patients with MEN-2A. Yang et al. (19) found LOH on todermal origin, such as neuroblastoma (39) and melanoma (41, 42), as well as other tumor types, such as breast cancer Ip in 5 of 8 pheochromocytomas and MTCs, and Khosla et al. (43, 44), colon cancer (45), and liver cancer (46). It is not clear (20) reported LOH on chromosome Ip in 42% of pheochrom if a single gene or locus is involved in all these cases, or if there ocytomas and in 9% of MTCs. LOH has also been described in are two or more loci on the short arm of chromosome 1 that a small percentage of pheochromocytomas on chromosomes are involved in different tumor types. There are clear evidence 3p, 17p, and 22q (20, 38). for two distinct suppressor loci on the short arm of chromosome In addition to identifying 1p LOH in the largest series of 11 (47-49) and mounting evidence for two loci on the short hereditary tumors reported to date, our studies indicate heter arm of chromosome 17 (50, 51), so it is reasonable to assume ogeneity in the frequency of LOH for Ip in pheochromocyto that more than one locus on Ip may be involved in these diverse mas, depending on the genetic background of the patient. The tumor types. Further investigation should determine if there is frequency of LOH for Ip in pheochromocytomas was 100% (9 a common region on Ip that is involved in tumors of neuroecof 9) in patients with the MEN-2A and -2B syndromes versus todermal origin. 29% (2 of 7) in patients with sporadic disease (P < 0.001, Fisher's exact test). This difference may reflect alternative LOH FOR CHROMOSOME IP IN PHEOCHROMOCVTOMAS MEN2A

it

Sporadic

vu L

D1Z27~~ 'F-

,MD00D'0'0â€¢â€¢â€¢â€¢.â€¢â€¢*â€¢â€¢â€¢"*â€¢â€¢â€¢â€¢â€¢â€¢â€¢â€¢â€¢â€¢â€¢â€¢.*â€¢â€¢.â€¢â€¢â€¢.â€¢.â€¢â€¢â€¢â€¢â€¢.

molecular mechanisms at work in the development of these tumors. These findings also suggest an interaction between the predisposition locus and the loss of a putative suppressor gene (or genes) on Ip in hereditary pheochromocytomas. Alterna tively, other chromosomal loci (such as 3p, 17p, or 22q) may be important in the pathogenesis of sporadic pheochromocytomas. Another striking finding was the difference in the frequency of Ip LOH between pheochromocytomas and MTCs. We only found 3 of 27 MTCs with 1p LOH. Even among the 19 patients with MEN-2A or -2B, MTCs showed a low frequency of LOH on Ip (16%). In two individuals, one with MEN-2A and one with MEN-2B, both pheochromocytoma and MTC tissue were examined. LOH was found in the pheochromocytoma tissue but not in the MTCs. This genetic heterogeneity may reflect differences in pathogenesis of these two tumor types, which may be related to clinical presentation, natural history, and histolÃ³gica! features of pheochromocytomas and MTCs.

LOH FOR CHROMOSOME IP IN MEDULLARV THYROID CANCERS SPORADIC1:21SS7VCL1SZ1~^7c MEN 2A

OOCo

oooooo

MEN 2B

C0â€¢o

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oo0oo o0o o0oo o0o00o0oo

â€¢¿ â€¢¿oo0Co o5â€¢

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0o

â€¢¿ o oOâ€¢¿ o oo

Fig. 3. Analysis for LOH at various chromosome 1 loci in MTCs. The probes used and their relative locations on Ip (30) are listed at the left of the box. To the rialti are the specific results obtained in the 24 informative MTCs. Informative combinations are indicated as in Fig. 2. Shaded areas indicate the minimum area presumed to be deleted, as defined by probes showing LOH. Case 13 demonstrated LOH at D1S17 and D1Z2 but not at MYCL. Case 21 demonstrated LOH at D1S17 and NRAS, but not at D1Z2, and Case 32 demonstrated LOH at DIS21, MYCL, and D1S57. but retained heterozygosity at D1Z2. In Cases 13 and 21, more probe-enzyme combinations were not done because of lack of tumor DNA (small tumor size). 772



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