0021-972X/90/7102-0293$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 71, No. 2 Printed in U.S.A.
Genetic Abnormalities in Sporadic Parathyroid Adenomas EITAN FRIEDMAN, ALLEN E. BALE, STEPHEN J. MARX, JEFFREY A. NORTON, ANDREW ARNOLD*, THOMAS TU, GERALD D. AURBACH, AND ALLEN M. SPIEGEL Molecular Pathophysiology (E.F., T.T., A.M.S.) and Metabolic Diseases (S.J.M., G.D.A.) Branches, National Institute of Diabetes, Digestive and Kidney Diseases, and the Division of Cancer Treatment, Surgery Branch, National Cancer Institute (J.A.N.), National Institutes of Health, Bethesda, Maryland 20892; the Department of Human Genetics, Yale University School of Medicine (A.E.B.), New Haven, Connecticutt 06510; and Endocrine Unit, Massachusetts General Hospital and Harvard Medical School (A.A.), Boston, Massachusetts 02114
ABSTRACT. We analyzed genomic DNA from 43 sporadic benign parathyroid adenomas for rearrangements of the PTH gene, and for point mutations of the H-ras (codons 12, 13, and 61), N-ras (codons 12,13, and 61), and K-ras (codons 12 and 13) genes. One of 43 parathyroid adenomas showed a chromosome 11 rearrangement involving both the PTH gene on the short arm of chromosome 11 (at band pl5) and a locus on the long arm (Ilql3). This rearrangement was indistinguishable from
one that was previously described in a parathyroid adenoma by Arnold et al., indicating that this may be an important contributor to tumorigenesis in a small subset of patients with parathyroid adenoma. H-ras, K-ras, and N-ras oncogene activation by point mutation at codons 12, 13, or 61, known to occur in many tumors, could not be detected in any parathyroid adenoma. (J Clin Endocrinol Metab 7 1 : 293-297, 1990)
T
UMOR development is a complex multistep process that is still poorly characterized and understood. Genetic alterations have been found in many tumors; Philadelphia chromosome in chronic myelogenous leukemia (1) and the chromosomal translocations in Burkitt's lymphoma (2) are two well documented examples. These genetic abnormalities have given important insight into the potential mechanisms of tumor initiation, development, and progression. Imaging of chromosomes was the first method employed to demonstrate gross tumor-specific DNA anomalies. Many solid tumors, however, have not been amenable to this approach (3). With the advent of molecular biological techniques, equivalent or even subtler genetic alterations could be detected in tumors by these methods. Vogelstein et al. (4) have demonstrated a spectrum of genetic anomalies in colorectal tumors and the possible relation of these anomalies to the tumor's histological features and biological behavior. The majority of genetic alterations have been identified in malignant or prema-
lignant tumors (4, 5) and only a handful in truly benign tumors (6-8). Primary hyperparathyroidism is generally a sporadic (i.e. nonfamilial) disorder. A single benign hyperfunctioning parathyroid tumor, termed parathyroid adenoma, is the most common histopathological finding (9). Recently, through molecular biological techniques, two types of genetic anomalies have been described in benign parathyroid adenomas: a novel gene rearrangement involving the PTH gene on chromosome 11 (10-12) and allelic loss from at least two regions in chromosome 11 (13, 14). Activation of ras protooncogenes, generally by point mutation altering amino acids at position 12, 13, or 61, has been detected in many human tumors, including benign thyroid adenomas, but has not been studied in parathyroid tumors. In this report we present data from the analysis of genomic DNA from sporadic parathyroid adenomas. This study was aimed at defining the frequency of PTH gene rearrangement in our patient population and the possible occurrence of ras oncogene point mutations in benign parathyroid tumors.
Received November 30, 1989. Address all correspondence and requests for reprints to: Eitan Friedman, National Institutes of Health, Building 10, Room 8D17, Bethesda, Maryland 20892. * Dr. Arnold is the recipient of a junior faculty research award from the American Cancer Society.
Materials and Methods Patients and tumors We analyzed DNA from 43 patients with primary hyperparathyroidism, shown by surgery at the NIH to be caused by 293
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parathyroid adenoma, for PTH gene rearrangements. This patient population includes 34 patients whose adenomas were previously analyzed for allelic losses (13). The criteria for classifying parathyroid tumor as sporadic adenoma were the following: 1) one parathyroid gland judged to be abnormal by intraoperative evaluation of gland's size and cellularity, 2) postoperative histological evaluation indicating hypercellularity in that gland, 3) at least one other parathyroid gland judged to be normal by similar evaluation at the current operation or at previous parathyroid surgery, 4) no family history of hyperparathyroidism, and 5) remission of hypercalcemia lasting at least 3 months postoperatively. Eighteen tumors were from cryopreserved tissue (15). The other 25 tumors were evaluated immediately after the operation. Ras oncogene point mutations were evaluated in the 43 patients described above and in tumors from 15 additional patients with adenoma, 25 tumors from patients with familial multiple endocrine neoplasia type I (FMEN-I), 2 tumors from patients with familial parathyroid carcinoma, and 3 tumors from patients with uremic hyperparathyroidism.
JCE & M • 1990 Vol 71 • No 2
Bam HI
Bam HI Kb
PB
PB T
3' PTH PROBE B
T
Kb
5' PTH PROBE
(Breakpoint) B
H
1 !
B B
Southern blot analysis Tumor and leukocyte DNA extractions, restriction enzyme digests, DNA separation on 0.8% agarose gels, and transfer to nylon filters were performed as previously described (13). Similarly, probe labeling technique, prehybridization, hybridization, washing conditions and autoradiography were performed as previously described (12, 13). The probes used were 5' and 3' PTH gene probes (10), and three probes from the D11S287 chromosomal locus. The latter were derived from the normal unrearranged version of a DNA fragment originating from Ilql3 that was found to be spliced into the first intron of the PTH gene in one parathyroid adenoma (12) (Fig. IB).
Probe C
Probe D
-3.8 kb17 kb PROBE A
PROBE C
PROBE D
Bam HI Hind M
Hind III
Hindlll
PB T
PB T
PB T
PB T —17
12-
•—17 '—10.5
—17
Ras oncogene point mutations Sequences for H-ras, N-ras, and K-ras spanning 110-120 bases across the codon 12, 13 region and the codon 61 region were amplified using two different sets of flanking 20-mer oligonucleotide primers (Clontech Laboratories, Inc., PaloAlto, CA).1 One microgram of tumor DNA and 20 pmol of each of the two primers were added to a 50-/uL reaction mixture containing 2.5 U Thermus acquaticus DNA polymerase {Taq polymerase), as previously described (16). Forty cycles of denaturation (96 C; 30 s), annealing (56-58 C; 15 s), and extension (74 C; 30 s) were carried out with an automated DNA thermal cycler (Perkin-Elmer DNA, Norwalk, CT.). Fifteen percent of amplified DNA was analyzed on 4% agarose gel (to ascertain achievement of specific amplification, i.e. a single band after ethidium bromide stain). The rest of the amplified product (1525% of the polymerase chain reaction-amplified product/blot) was dot-blotted onto nylon filters (Oncor, Inc., Gaithersburg, MD) and screened with a panel of 20-mer oligonucleotides. These included an oligonucleotide complementary to the nonmutated wild-type allele (referred to as wild-type oligonucleotide); in addition, we tested five or six additional oligonu-
1
For complete nucleotide sequences of primers used for amplification and screening, see Clontech Laboratories catalog.
3.8—
Afe
FIG. 1. A, Autoradiograms of Southern blots from the parathyroid adenoma displaying PTH gene rearrangement. DNA from the patient's tumor (T) and peripheral blood leukocytes (PB) were digested with fiamHI restriction endonuclease and probed with radiolabeled DNA fragments from the 5' or 3' part of the PTH gene (see Materials and Methods). Tumor-specific bands are marked with arrows, while bands representing normal alleles are marked with dashes. B, Partial genomic restriction map of the D11S287 region (from Ilql3) in its normal (unrearranged) form. B denotes BamHI, and H denotes Hindlll restriction sites. Heavy dashed line indicates areas not drawn to scale. The position at which the D11S287 locus is broken in the patient with PTH gene rearrangement is marked with an arrow. The probes (A, C, and D) cloned from this locus and used in Southern blot analysis (see C below) are indicated beneath the restiction map. At the bottom are shown the restriction fragments (3.8-kb fiomHI and 17-kb Hindlll) to be visualized on Southern blot analysis with the indicated probes [reproduced, with modification, from J Clin Invest 1989;83:2034-40 (Fig. 3) by copyright permission of the American Society for Clinical Investigation). C, The same Southern blots as in A, rehybridized with the indicated D11S287 probes (see Materials and Methods and Fig. IB). Tumor-specific rearranged bands are marked with arrows, while bands representing normal (unrearranged) alleles are marked with dashes.
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GENETIC ABNORMALITIES IN PARATHYROID ADENOMAS cleotides, each containing a different, single base change corresponding to a different encoded amino acid (mutated oligonucleotides). The oligonucleotides were end labeled with T4 polynucleotide kinase (Stratagene, La Jolla, CA). Prehybridization, hybridization, and high stringency washes with 3 M tetramethylammoniumchloride were performed as previously described (17). Filters were exposed to x-ray films (XAR-5, Eastman Kodak, Rochester, NY) for 1 h at room temperature.
Results
N-ras; CODON 13
ASPARTIC ACID
GLYCINE (WILD TYPEI
B. N-ias; HL-60 CELLS
PTH gene rearrangement We used 5' and 3' PTH gene probes to analyze BamHI-digested tumor and leukocyte DNA in all 43 tumors, to screen for PTH gene rearrangement as described by Arnold et al. (12). In 1 of the 43 adenomas, novel tumor-specific bands were clearly seen: a 12-kilobase (kb; kilobase pairs) BamHl band with the 3' probe and an 8-kb JBamHI band with the 5' probe (Fig. 1A). To clarify further the nature of this rearrangement and to ascertain similarity with the rearrangement previously characterized in detail, we used 3 probes (arbitrarily termed A, C, and D) cloned from D11S287, a non-PTH region on chromosome 11 that was rearranged by recombination with the PTH gene in the described parathyroid adenoma (12) (Fig. IB). As shown in Fig. 1C, these probes detected abnormal tumor-specific bands on Southern blot analysis. These bands are remarkably similar to those from the previously studied tumor (12), suggesting that the rearrangement breakpoints in the PTH gene (on Ilpl5) and in D11S287 (on Ilql3) are similar or even identical for both tumors. This patient's leukocyte DNA displayed heterozygosity with 4 different chromosome 11 probes: INS, PTH, D11S149, and D11S146. Retention of both alleles was evident with each of these probes (data not shown). Ras-oncogene point mutations Parathyroid tumor DNA at regions spanning codons 12 and 13, was successfully amplified for H-ras, N-ras, and K-ras (located on chromosomes 11, 1, and 12, respectively). Successful amplification for codon 61 was achieved only for H-ras and N-ras. At high stringency hybridization conditions (i.e. washing at 61 C in the presence of 3 M tetramethylammoniumchloride) only a perfectly matched oligonucleotide forms a stable duplex with the amplified tumor DNA blotted onto the filter, whereas an oligonucleotide mismatched at even a single basepair does not and, hence, is washed off. Using this so-called allele-specific hybridization technique, we screened for ras oncogene point mutations at codons known to be sites for such mutations in other tumors (18-22). Hybridization with the wild-type oligonucleotide showed a strong signal for each tumor. In no case was a
295
— CODONS 12, 13 #
—CODON 61
0% ' *
FIG. 2. A, Dot blot analysis for mutations of human N-ras at codon 13 in 88 parathyroid tumors (58 sporadic parathyroid adenomas, 25 parathyroid tumors from patients with FMEN-I, 2 familial parathyroid carcinoma, and 3 parathyroid tumors from patients with chronic renal failure). Each dot contains amplified DNA from a different tumor. The names of amino acids under each blot represent the encoded product of the point mutation specifically tested for at codon 13. B, DNA from HL-60 cells amplified for N-ras at codons 12 and 13 region and codon 61 region, and probed with the mutated oligonucleotide detecting a mutation encoding leucine at codon 61. Not shown are the results of probing with the wild-type oligonucleotides of codons 12, 13, and 61 of N-ras. HRAS; CODON 12
I :.
PARATHYROID ADENOMAS EJ CELL r LINE L
,
mt m
# GLYCINE (WILD TYPE)
VALINE
FlG. 3. Dot blot analysis for mutations of human H-ras at codon 12 in 24 sporadic parathyroid adenomas and in the EJ bladder cancer cell line. Each dot contains amplified DNA from a different tumor. Blots shown were probed with oligonucleotides encoding either the wild-type gene product (glycine) or the mutant with valine at position 12.
signal of similar intensity produced using any of the other mutated oligonucleotides (Figs. 2 and 3). To test whether our analysis with these mutated oligonucleotides can actually detect a known mutation, we used HL-60 cells, a promyelocytic leukemia cell line known to contain an N-ras point mutation [A to T mutation (changing Gin to Leu), at codon 61], (23). Hybridization of the appropriate oligonucleotide to the codon 61 amplified DNA of N-ras gave a signal of intensity similar to that of the wild-type oligonucleotide (Fig. 2B), reflecting one normal and one mutated allele. A similar analysis performed on the EJ bladder cancer cell line, which contains one normal allele and one mutated
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FRIEDMAN ET AL.
296
allele at H-ras position 12 (changing wild-type Gly to Val) indicated hybridization of both the wild-type and mutant-encoding oligonucleotides, whereas in DNA from parathyroid adenomas, only the wild-type oligonucleotide hybridized (Fig. 3).
Discussion Recent studies have begun to offer insights into the molecular genetic basis for benign parathyroid neoplasia. Allelic loss (sometimes called loss of heterozygosity) from chromosome 11 was found in the majority of parathyroid tumors from patients with the inherited disorder, FMEN-I (13). Allelic loss from chromosome 11 was also found in 11 of 43 benign sporadic parathyroid adenomas (13, 24) (E. Friedman, unpublished data). This frequency of allelic loss has several implications. First, it confirms the monoclonal origin of many sporadic parathyroid adenomas (10). Second, it indicates that inactivation of a tumor suppressor gene may contribute to initiation or evolution of benign parathyroid tumors. Another genetic abnormality implicated in parathyroid neoplasia is a DNA rearrangement involving the PTH gene. The latter abnormality was originally noted in 2 of 43 parathyroid adenomas (10), and the molecular structures of the loci involved have been further characterized (11, 12). One of these DNA rearrangements was shown to juxtapose the first intron of the PTH gene on the short arm of chromosome 11 to band ql3 on the long arm. This may be due to inversion of a large chromosomal fragment about the centromere in 1 copy of chromosome 11. In the present study only 1 of 43 parathyroid tumors analyzed showed a PTH gene rearrangement, and this was remarkably similar to that previously described in detail (12). By Southern blot analysis using the cloned breakpoint region non-PTH DNA probes (D11S287) (12), the rearrangement in our case appears indistinguishable from that previously reported. Based on previous (10, 12) and present data, it seems that PTH gene rearrangement occurs uncommonly (total of 3 cases of 86 tumors). The most unique clinical feature shared by all 3 patients thus far reported to display this PTH gene rearrangement is the association of a very large parathyroid adenoma (6-8 g) with PTH levels that were only moderately elevated. This is unusual in primary hyperparathyroidism and may suggest dissociation of gland size from degree of PTH hypersecretion. There are several potential explanations of how this rearrangement might relate to parathyroid tumorigenesis. The rearranged DNA at Ilql3 may contain a direct acting oncogene, which in its new configuration is activated by PTH gene regulatory elements. Recent evidence indicates that a previously unidentified gene is present at this location and is indeed overexpressed in parathy-
JCE & M • 1990 Vol71«No2
roid tissue as a result of this rearrangement (25). Alternatively, the rearrangement could have disrupted and inactivated a gene on Ilql3 that normally functions as a tumor suppressor gene (such as the MEN-I gene) (13) or could have led to activation of a different oncogene at Ilql3 by unidentified regulatory elements within the first intron of the PTH gene. We did not find point mutations leading to activation of ras oncogenes at codons with recurring ras gene point mutations in other tumors (18). With few exceptions (20, 21), human tumors that display ras oncogene activation are malignant or premalignant (18, 19, 22, 23). Lemoine and co-workers (21), using the polymerase chain reaction and allele-specific hybridization techniques, demonstrated ras oncogene point mutations (predominantly Nras and H-ras at codon 61) in 33% of benign thyroid
adenomas. A similar high frequency of ras point mutation was detected in malignant thyroid tumors (21). These researchers suggested, therefore, that ras oncogene activation is an early event in thyroid tumor development, possibly associated with tumor initiation. Furthermore, Baumer and Loeb (26) detected K-ras point mutations in histologically normal colonic mucosa in regions adjacent to colonic carcinoma. They suggest that a field of genetically abnormal mucosa may exist in patients predisposed to the development of colonic neoplasia and support the notion that ras oncogene activation is an early event in colonic tumor development. Recently, Corominas et al. (20) have shown H-ras point mutations and subsequent activation in 30% of keratoacanthomas, which are benign self-regressing skin tumors. These researchers speculated that activation of H-ras may have a role in tumor regression associated with tumor cell differentiation. Nakagawa et al. (27) transfected thyroid C-cells with activated v-Ha-ras with subsequent differentiation and loss of neoplastic phenotype. The absence of demonstrable mutated ras oncogenes in benign parathyroid adenomas may be an indication that such an occurence is confined to tumors associated with either dedifferentiation (i.e. malignant, premalignant) or differentiaiton (i.e. self-regressing). Further studies in parathyroid tumors would be necessary to exclude other mechanisms of ras oncogene activation, such as overexpression. In summary, two types of genetic alterations have been detected in benign sporadic parathryoid adenomas: 1) allelic loss, which is chromosome 11 specific, suggesting a possible role for inactivation of a tumor suppressor gene(s) on chromosome 11 in at least 25% of patients; and 2) PTH gene rearrangement, which seems important in tumor development in a smaller subset of patients. Ras oncogene activation by point mutation occurs rarely, if ever, in benign parathyroid tumors. It seems likely that underlying molecular mechanisms in benign spo-
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GENETIC ABNORMALITIES IN PARATHYROID ADENOMAS
radic parathyroid neoplasia are heterogeneous, and that additional genetic defects will be identified with further studies.
13.
Acknowledgments
14.
We wish to thank Dr. H. Kronenberg from the Endocrine Unit, Massachusetts General Hospital, for providing the PTH gene probes and for fruitful discussion of this manuscript; Drs. E. Streeten, L. Weinstein, M. Zimering, and J. Merendino for patient evaluation and care, and Drs. A. Papapgeorge and R. Collins for assistance in studies of RAS point mutations. We are also grateful to Mrs. Sonia M. Akar for excellent secretarial assistance.
15. 16. 17.
References 1. Kurzrock R, Gutterman JU, Talpaz M. The molecular genetics of Philadelphia chromosome-positive leukemias. N Engl J Med. l988;319:990-8. 2. Croce CM, Nowell PC. Molecular basis of human B cell neoplasia. Blood. 1985;65:l-7. 3. Sandberg AA, Turc-Carel C. The cytogenetics of solid tumors. Cancer. 1987;59:387-95. 4. Vogelstein B, Fearon ER, Hamilton SR, et al. Genetic alterations during colorectal-tumor development. N Engl J Med. 1988;319:52532. 5. Kovacs G, Erlandsson R, Boldog F, et al. Consistent chromosome 3p deletion and loss of heterozygosity in renal cell carcinoma. Proc Natl Acad Sci USA. 1988;85:1571-5. 6. Seizinger BR, de la Monte S, Atkins L, Gusella JF, Martuza RL. Molecular genetic approach to human meningioma: loss of genes on chromosome 22. Proc Natl Acad Sci USA. 1987;84:5419-23. 7. Rouleau GA, Wertelecki W, Haines JL, et al. Genetic linkage of bilateral acoustic neurofibromatosis to a DNA marker on chromosome 22. Nature. 1987;329:246-8. 8. Landis CA, Masters SB, Spada A, Pace AM, Bourne HR, Vallar
18. 19. 20.
21. 22. 23.
24.
L. GTPase inhibiting mutations activate the chain of Gs and
9. 10. 11. 12.
stimulate adenylyl cyclase in human pituitary tumours. Nature. 1989;340:692-6. Wang CA. Surgical management of primary hyperparathyroidism. Curr Prob Surg. 1985;22:l-50. Arnold A, Staunton CE, Kim HG, Gaz RD, Kronenberg HM. Monoclonality and abnormal parathyroid hormone genes in parathyroid adenomas. N Engl J Med. l988;318:658-62. Rosenberg CL, Shows TB, Kronenberg HM, Arnold A. Intrachromosomal recombination in two parathyroid adenomas involving the PTH gene and loci on llq [Abstract]. Clin Res. 1989;37:861A. Arnold A, Kim HG, Gaz RD, et al. Molecular cloning and chro-
25.
26. 27.
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mosomal mapping of DNA rearranged with the parathyroid hormone gene in a parathyroid adenoma. J Clin Invest. 1989;83:203440. Friedman E, Sakaguchi K, Bale AE, et al. Clonality of parathyroid tumors in familial multiple endocrine neoplasia type 1. N Engl J Med. 1989;321:213-8. Arnold A, Kim HG. Clonal loss of one chromosome 11 in a parathyroid adenoma. J Clin Endocrinol Metab. 1989;69:496-9. Norton JA, Aurbach GD, Marx SJ, Doppman JL. Surgical management of hyperparathyroidism. In: DeGroot LJ, ed. Endocrinology, 2nd ed. Philadelphia: Saunders; 1989;2:1013-31. Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988;239:487-91. Verlaan-de Vries M, Bogaard ME, van der Elst H, van Boom JH, van der Eb AJ, Bos JL. A dot-blot screening procedure for mutated ras oncogenes using synthetic oligodeoxynucleotides. Gene. 1986;5O:313-2O. Bos JL. Ras oncogenes in human cancer: a review. Cancer Res. l989;49:4682-9. Bos JL, Fearon ER, Hamilton SR, et al. Prevalence of ras gene mutations in human colorectal cancers. Nature (Lond). 1987;327:293-7. Corominas M, Kamino H, Leon J, Pellicer A. Oncogene activation in human benign tumors of the skin (keratoacanthomas): is H-ras involved in differentiation as well as proliferation? Proc Natl Acad Sci USA. 1989;86:6372-6. Lemoine NR, Mayall ES, Wyllie FS, et al. High frequency of ras oncogene activation in all stages of human thyroid tumorigenesis. Oncogene. 1989,4:159-64. Bos JL, Toksoz D, Marshall CJ, et al. Amino acid substitutions at codon 13 of the N-ras oncogene in human acute myeloid leukaemia. Nature (Lond). 1985;315:726-30. Farr CJ, Saiki RK, Erlich HA, McCormick F, Marshall CJ. Analysis of ras gene mutations in acute myeloid leukaemia by polymerase chain reaction and oligonucleotide probes. Proc Natl Acad Sci USA. 1988;85:1629-33. Thakker RV, Bouloux P, Wooding C, et al. Association of parathyroid tumors in multiple endocrine neoplasia type 1 with loss of alleles on chromosome 11. N Engl J Med. 1989;321:60-8. Arnold A, Kim HG, Gaz RD, Kronenberg HM. DNA rearranged adjacent to the PTH gene in a parathyroid adenoma encodes an abnormally expressed gene [Abstract]. J Bone Mineral Res. 1989;4(Suppl 1):5262. Baumer GC, Loeb LA. Mutations in the K-ras2 oncogene during progressive stages of human colon carcinoma. Proc Natl Acad Sci USA. 1989;86:2403-7. Nakagawa T, Mabry M, de Bustros A, Ihle JN, Nelkin BD, Baylin SB. Introduction of v-Ha-ras oncogene induces differentiation of cultured human medullary thyroid carcinoma cells. Proc Natl Acad Sci USA. 1987;84:5923-7.
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Vol. 71, No. 2 Printed in U.S.A.
Genetic Abnormalities in Sporadic Parathyroid Adenomas EITAN FRIEDMAN, ALLEN E. BALE, STEPHEN J. MARX, JEFFREY A. NORTON, ANDREW ARNOLD*, THOMAS TU, GERALD D. AURBACH, AND ALLEN M. SPIEGEL Molecular Pathophysiology (E.F., T.T., A.M.S.) and Metabolic Diseases (S.J.M., G.D.A.) Branches, National Institute of Diabetes, Digestive and Kidney Diseases, and the Division of Cancer Treatment, Surgery Branch, National Cancer Institute (J.A.N.), National Institutes of Health, Bethesda, Maryland 20892; the Department of Human Genetics, Yale University School of Medicine (A.E.B.), New Haven, Connecticutt 06510; and Endocrine Unit, Massachusetts General Hospital and Harvard Medical School (A.A.), Boston, Massachusetts 02114
ABSTRACT. We analyzed genomic DNA from 43 sporadic benign parathyroid adenomas for rearrangements of the PTH gene, and for point mutations of the H-ras (codons 12, 13, and 61), N-ras (codons 12,13, and 61), and K-ras (codons 12 and 13) genes. One of 43 parathyroid adenomas showed a chromosome 11 rearrangement involving both the PTH gene on the short arm of chromosome 11 (at band pl5) and a locus on the long arm (Ilql3). This rearrangement was indistinguishable from
one that was previously described in a parathyroid adenoma by Arnold et al., indicating that this may be an important contributor to tumorigenesis in a small subset of patients with parathyroid adenoma. H-ras, K-ras, and N-ras oncogene activation by point mutation at codons 12, 13, or 61, known to occur in many tumors, could not be detected in any parathyroid adenoma. (J Clin Endocrinol Metab 7 1 : 293-297, 1990)
T
UMOR development is a complex multistep process that is still poorly characterized and understood. Genetic alterations have been found in many tumors; Philadelphia chromosome in chronic myelogenous leukemia (1) and the chromosomal translocations in Burkitt's lymphoma (2) are two well documented examples. These genetic abnormalities have given important insight into the potential mechanisms of tumor initiation, development, and progression. Imaging of chromosomes was the first method employed to demonstrate gross tumor-specific DNA anomalies. Many solid tumors, however, have not been amenable to this approach (3). With the advent of molecular biological techniques, equivalent or even subtler genetic alterations could be detected in tumors by these methods. Vogelstein et al. (4) have demonstrated a spectrum of genetic anomalies in colorectal tumors and the possible relation of these anomalies to the tumor's histological features and biological behavior. The majority of genetic alterations have been identified in malignant or prema-
lignant tumors (4, 5) and only a handful in truly benign tumors (6-8). Primary hyperparathyroidism is generally a sporadic (i.e. nonfamilial) disorder. A single benign hyperfunctioning parathyroid tumor, termed parathyroid adenoma, is the most common histopathological finding (9). Recently, through molecular biological techniques, two types of genetic anomalies have been described in benign parathyroid adenomas: a novel gene rearrangement involving the PTH gene on chromosome 11 (10-12) and allelic loss from at least two regions in chromosome 11 (13, 14). Activation of ras protooncogenes, generally by point mutation altering amino acids at position 12, 13, or 61, has been detected in many human tumors, including benign thyroid adenomas, but has not been studied in parathyroid tumors. In this report we present data from the analysis of genomic DNA from sporadic parathyroid adenomas. This study was aimed at defining the frequency of PTH gene rearrangement in our patient population and the possible occurrence of ras oncogene point mutations in benign parathyroid tumors.
Received November 30, 1989. Address all correspondence and requests for reprints to: Eitan Friedman, National Institutes of Health, Building 10, Room 8D17, Bethesda, Maryland 20892. * Dr. Arnold is the recipient of a junior faculty research award from the American Cancer Society.
Materials and Methods Patients and tumors We analyzed DNA from 43 patients with primary hyperparathyroidism, shown by surgery at the NIH to be caused by 293
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parathyroid adenoma, for PTH gene rearrangements. This patient population includes 34 patients whose adenomas were previously analyzed for allelic losses (13). The criteria for classifying parathyroid tumor as sporadic adenoma were the following: 1) one parathyroid gland judged to be abnormal by intraoperative evaluation of gland's size and cellularity, 2) postoperative histological evaluation indicating hypercellularity in that gland, 3) at least one other parathyroid gland judged to be normal by similar evaluation at the current operation or at previous parathyroid surgery, 4) no family history of hyperparathyroidism, and 5) remission of hypercalcemia lasting at least 3 months postoperatively. Eighteen tumors were from cryopreserved tissue (15). The other 25 tumors were evaluated immediately after the operation. Ras oncogene point mutations were evaluated in the 43 patients described above and in tumors from 15 additional patients with adenoma, 25 tumors from patients with familial multiple endocrine neoplasia type I (FMEN-I), 2 tumors from patients with familial parathyroid carcinoma, and 3 tumors from patients with uremic hyperparathyroidism.
JCE & M • 1990 Vol 71 • No 2
Bam HI
Bam HI Kb
PB
PB T
3' PTH PROBE B
T
Kb
5' PTH PROBE
(Breakpoint) B
H
1 !
B B
Southern blot analysis Tumor and leukocyte DNA extractions, restriction enzyme digests, DNA separation on 0.8% agarose gels, and transfer to nylon filters were performed as previously described (13). Similarly, probe labeling technique, prehybridization, hybridization, washing conditions and autoradiography were performed as previously described (12, 13). The probes used were 5' and 3' PTH gene probes (10), and three probes from the D11S287 chromosomal locus. The latter were derived from the normal unrearranged version of a DNA fragment originating from Ilql3 that was found to be spliced into the first intron of the PTH gene in one parathyroid adenoma (12) (Fig. IB).
Probe C
Probe D
-3.8 kb17 kb PROBE A
PROBE C
PROBE D
Bam HI Hind M
Hind III
Hindlll
PB T
PB T
PB T
PB T —17
12-
•—17 '—10.5
—17
Ras oncogene point mutations Sequences for H-ras, N-ras, and K-ras spanning 110-120 bases across the codon 12, 13 region and the codon 61 region were amplified using two different sets of flanking 20-mer oligonucleotide primers (Clontech Laboratories, Inc., PaloAlto, CA).1 One microgram of tumor DNA and 20 pmol of each of the two primers were added to a 50-/uL reaction mixture containing 2.5 U Thermus acquaticus DNA polymerase {Taq polymerase), as previously described (16). Forty cycles of denaturation (96 C; 30 s), annealing (56-58 C; 15 s), and extension (74 C; 30 s) were carried out with an automated DNA thermal cycler (Perkin-Elmer DNA, Norwalk, CT.). Fifteen percent of amplified DNA was analyzed on 4% agarose gel (to ascertain achievement of specific amplification, i.e. a single band after ethidium bromide stain). The rest of the amplified product (1525% of the polymerase chain reaction-amplified product/blot) was dot-blotted onto nylon filters (Oncor, Inc., Gaithersburg, MD) and screened with a panel of 20-mer oligonucleotides. These included an oligonucleotide complementary to the nonmutated wild-type allele (referred to as wild-type oligonucleotide); in addition, we tested five or six additional oligonu-
1
For complete nucleotide sequences of primers used for amplification and screening, see Clontech Laboratories catalog.
3.8—
Afe
FIG. 1. A, Autoradiograms of Southern blots from the parathyroid adenoma displaying PTH gene rearrangement. DNA from the patient's tumor (T) and peripheral blood leukocytes (PB) were digested with fiamHI restriction endonuclease and probed with radiolabeled DNA fragments from the 5' or 3' part of the PTH gene (see Materials and Methods). Tumor-specific bands are marked with arrows, while bands representing normal alleles are marked with dashes. B, Partial genomic restriction map of the D11S287 region (from Ilql3) in its normal (unrearranged) form. B denotes BamHI, and H denotes Hindlll restriction sites. Heavy dashed line indicates areas not drawn to scale. The position at which the D11S287 locus is broken in the patient with PTH gene rearrangement is marked with an arrow. The probes (A, C, and D) cloned from this locus and used in Southern blot analysis (see C below) are indicated beneath the restiction map. At the bottom are shown the restriction fragments (3.8-kb fiomHI and 17-kb Hindlll) to be visualized on Southern blot analysis with the indicated probes [reproduced, with modification, from J Clin Invest 1989;83:2034-40 (Fig. 3) by copyright permission of the American Society for Clinical Investigation). C, The same Southern blots as in A, rehybridized with the indicated D11S287 probes (see Materials and Methods and Fig. IB). Tumor-specific rearranged bands are marked with arrows, while bands representing normal (unrearranged) alleles are marked with dashes.
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GENETIC ABNORMALITIES IN PARATHYROID ADENOMAS cleotides, each containing a different, single base change corresponding to a different encoded amino acid (mutated oligonucleotides). The oligonucleotides were end labeled with T4 polynucleotide kinase (Stratagene, La Jolla, CA). Prehybridization, hybridization, and high stringency washes with 3 M tetramethylammoniumchloride were performed as previously described (17). Filters were exposed to x-ray films (XAR-5, Eastman Kodak, Rochester, NY) for 1 h at room temperature.
Results
N-ras; CODON 13
ASPARTIC ACID
GLYCINE (WILD TYPEI
B. N-ias; HL-60 CELLS
PTH gene rearrangement We used 5' and 3' PTH gene probes to analyze BamHI-digested tumor and leukocyte DNA in all 43 tumors, to screen for PTH gene rearrangement as described by Arnold et al. (12). In 1 of the 43 adenomas, novel tumor-specific bands were clearly seen: a 12-kilobase (kb; kilobase pairs) BamHl band with the 3' probe and an 8-kb JBamHI band with the 5' probe (Fig. 1A). To clarify further the nature of this rearrangement and to ascertain similarity with the rearrangement previously characterized in detail, we used 3 probes (arbitrarily termed A, C, and D) cloned from D11S287, a non-PTH region on chromosome 11 that was rearranged by recombination with the PTH gene in the described parathyroid adenoma (12) (Fig. IB). As shown in Fig. 1C, these probes detected abnormal tumor-specific bands on Southern blot analysis. These bands are remarkably similar to those from the previously studied tumor (12), suggesting that the rearrangement breakpoints in the PTH gene (on Ilpl5) and in D11S287 (on Ilql3) are similar or even identical for both tumors. This patient's leukocyte DNA displayed heterozygosity with 4 different chromosome 11 probes: INS, PTH, D11S149, and D11S146. Retention of both alleles was evident with each of these probes (data not shown). Ras-oncogene point mutations Parathyroid tumor DNA at regions spanning codons 12 and 13, was successfully amplified for H-ras, N-ras, and K-ras (located on chromosomes 11, 1, and 12, respectively). Successful amplification for codon 61 was achieved only for H-ras and N-ras. At high stringency hybridization conditions (i.e. washing at 61 C in the presence of 3 M tetramethylammoniumchloride) only a perfectly matched oligonucleotide forms a stable duplex with the amplified tumor DNA blotted onto the filter, whereas an oligonucleotide mismatched at even a single basepair does not and, hence, is washed off. Using this so-called allele-specific hybridization technique, we screened for ras oncogene point mutations at codons known to be sites for such mutations in other tumors (18-22). Hybridization with the wild-type oligonucleotide showed a strong signal for each tumor. In no case was a
295
— CODONS 12, 13 #
—CODON 61
0% ' *
FIG. 2. A, Dot blot analysis for mutations of human N-ras at codon 13 in 88 parathyroid tumors (58 sporadic parathyroid adenomas, 25 parathyroid tumors from patients with FMEN-I, 2 familial parathyroid carcinoma, and 3 parathyroid tumors from patients with chronic renal failure). Each dot contains amplified DNA from a different tumor. The names of amino acids under each blot represent the encoded product of the point mutation specifically tested for at codon 13. B, DNA from HL-60 cells amplified for N-ras at codons 12 and 13 region and codon 61 region, and probed with the mutated oligonucleotide detecting a mutation encoding leucine at codon 61. Not shown are the results of probing with the wild-type oligonucleotides of codons 12, 13, and 61 of N-ras. HRAS; CODON 12
I :.
PARATHYROID ADENOMAS EJ CELL r LINE L
,
mt m
# GLYCINE (WILD TYPE)
VALINE
FlG. 3. Dot blot analysis for mutations of human H-ras at codon 12 in 24 sporadic parathyroid adenomas and in the EJ bladder cancer cell line. Each dot contains amplified DNA from a different tumor. Blots shown were probed with oligonucleotides encoding either the wild-type gene product (glycine) or the mutant with valine at position 12.
signal of similar intensity produced using any of the other mutated oligonucleotides (Figs. 2 and 3). To test whether our analysis with these mutated oligonucleotides can actually detect a known mutation, we used HL-60 cells, a promyelocytic leukemia cell line known to contain an N-ras point mutation [A to T mutation (changing Gin to Leu), at codon 61], (23). Hybridization of the appropriate oligonucleotide to the codon 61 amplified DNA of N-ras gave a signal of intensity similar to that of the wild-type oligonucleotide (Fig. 2B), reflecting one normal and one mutated allele. A similar analysis performed on the EJ bladder cancer cell line, which contains one normal allele and one mutated
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FRIEDMAN ET AL.
296
allele at H-ras position 12 (changing wild-type Gly to Val) indicated hybridization of both the wild-type and mutant-encoding oligonucleotides, whereas in DNA from parathyroid adenomas, only the wild-type oligonucleotide hybridized (Fig. 3).
Discussion Recent studies have begun to offer insights into the molecular genetic basis for benign parathyroid neoplasia. Allelic loss (sometimes called loss of heterozygosity) from chromosome 11 was found in the majority of parathyroid tumors from patients with the inherited disorder, FMEN-I (13). Allelic loss from chromosome 11 was also found in 11 of 43 benign sporadic parathyroid adenomas (13, 24) (E. Friedman, unpublished data). This frequency of allelic loss has several implications. First, it confirms the monoclonal origin of many sporadic parathyroid adenomas (10). Second, it indicates that inactivation of a tumor suppressor gene may contribute to initiation or evolution of benign parathyroid tumors. Another genetic abnormality implicated in parathyroid neoplasia is a DNA rearrangement involving the PTH gene. The latter abnormality was originally noted in 2 of 43 parathyroid adenomas (10), and the molecular structures of the loci involved have been further characterized (11, 12). One of these DNA rearrangements was shown to juxtapose the first intron of the PTH gene on the short arm of chromosome 11 to band ql3 on the long arm. This may be due to inversion of a large chromosomal fragment about the centromere in 1 copy of chromosome 11. In the present study only 1 of 43 parathyroid tumors analyzed showed a PTH gene rearrangement, and this was remarkably similar to that previously described in detail (12). By Southern blot analysis using the cloned breakpoint region non-PTH DNA probes (D11S287) (12), the rearrangement in our case appears indistinguishable from that previously reported. Based on previous (10, 12) and present data, it seems that PTH gene rearrangement occurs uncommonly (total of 3 cases of 86 tumors). The most unique clinical feature shared by all 3 patients thus far reported to display this PTH gene rearrangement is the association of a very large parathyroid adenoma (6-8 g) with PTH levels that were only moderately elevated. This is unusual in primary hyperparathyroidism and may suggest dissociation of gland size from degree of PTH hypersecretion. There are several potential explanations of how this rearrangement might relate to parathyroid tumorigenesis. The rearranged DNA at Ilql3 may contain a direct acting oncogene, which in its new configuration is activated by PTH gene regulatory elements. Recent evidence indicates that a previously unidentified gene is present at this location and is indeed overexpressed in parathy-
JCE & M • 1990 Vol71«No2
roid tissue as a result of this rearrangement (25). Alternatively, the rearrangement could have disrupted and inactivated a gene on Ilql3 that normally functions as a tumor suppressor gene (such as the MEN-I gene) (13) or could have led to activation of a different oncogene at Ilql3 by unidentified regulatory elements within the first intron of the PTH gene. We did not find point mutations leading to activation of ras oncogenes at codons with recurring ras gene point mutations in other tumors (18). With few exceptions (20, 21), human tumors that display ras oncogene activation are malignant or premalignant (18, 19, 22, 23). Lemoine and co-workers (21), using the polymerase chain reaction and allele-specific hybridization techniques, demonstrated ras oncogene point mutations (predominantly Nras and H-ras at codon 61) in 33% of benign thyroid
adenomas. A similar high frequency of ras point mutation was detected in malignant thyroid tumors (21). These researchers suggested, therefore, that ras oncogene activation is an early event in thyroid tumor development, possibly associated with tumor initiation. Furthermore, Baumer and Loeb (26) detected K-ras point mutations in histologically normal colonic mucosa in regions adjacent to colonic carcinoma. They suggest that a field of genetically abnormal mucosa may exist in patients predisposed to the development of colonic neoplasia and support the notion that ras oncogene activation is an early event in colonic tumor development. Recently, Corominas et al. (20) have shown H-ras point mutations and subsequent activation in 30% of keratoacanthomas, which are benign self-regressing skin tumors. These researchers speculated that activation of H-ras may have a role in tumor regression associated with tumor cell differentiation. Nakagawa et al. (27) transfected thyroid C-cells with activated v-Ha-ras with subsequent differentiation and loss of neoplastic phenotype. The absence of demonstrable mutated ras oncogenes in benign parathyroid adenomas may be an indication that such an occurence is confined to tumors associated with either dedifferentiation (i.e. malignant, premalignant) or differentiaiton (i.e. self-regressing). Further studies in parathyroid tumors would be necessary to exclude other mechanisms of ras oncogene activation, such as overexpression. In summary, two types of genetic alterations have been detected in benign sporadic parathryoid adenomas: 1) allelic loss, which is chromosome 11 specific, suggesting a possible role for inactivation of a tumor suppressor gene(s) on chromosome 11 in at least 25% of patients; and 2) PTH gene rearrangement, which seems important in tumor development in a smaller subset of patients. Ras oncogene activation by point mutation occurs rarely, if ever, in benign parathyroid tumors. It seems likely that underlying molecular mechanisms in benign spo-
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GENETIC ABNORMALITIES IN PARATHYROID ADENOMAS
radic parathyroid neoplasia are heterogeneous, and that additional genetic defects will be identified with further studies.
13.
Acknowledgments
14.
We wish to thank Dr. H. Kronenberg from the Endocrine Unit, Massachusetts General Hospital, for providing the PTH gene probes and for fruitful discussion of this manuscript; Drs. E. Streeten, L. Weinstein, M. Zimering, and J. Merendino for patient evaluation and care, and Drs. A. Papapgeorge and R. Collins for assistance in studies of RAS point mutations. We are also grateful to Mrs. Sonia M. Akar for excellent secretarial assistance.
15. 16. 17.
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stimulate adenylyl cyclase in human pituitary tumours. Nature. 1989;340:692-6. Wang CA. Surgical management of primary hyperparathyroidism. Curr Prob Surg. 1985;22:l-50. Arnold A, Staunton CE, Kim HG, Gaz RD, Kronenberg HM. Monoclonality and abnormal parathyroid hormone genes in parathyroid adenomas. N Engl J Med. l988;318:658-62. Rosenberg CL, Shows TB, Kronenberg HM, Arnold A. Intrachromosomal recombination in two parathyroid adenomas involving the PTH gene and loci on llq [Abstract]. Clin Res. 1989;37:861A. Arnold A, Kim HG, Gaz RD, et al. Molecular cloning and chro-
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mosomal mapping of DNA rearranged with the parathyroid hormone gene in a parathyroid adenoma. J Clin Invest. 1989;83:203440. Friedman E, Sakaguchi K, Bale AE, et al. Clonality of parathyroid tumors in familial multiple endocrine neoplasia type 1. N Engl J Med. 1989;321:213-8. Arnold A, Kim HG. Clonal loss of one chromosome 11 in a parathyroid adenoma. J Clin Endocrinol Metab. 1989;69:496-9. Norton JA, Aurbach GD, Marx SJ, Doppman JL. Surgical management of hyperparathyroidism. In: DeGroot LJ, ed. Endocrinology, 2nd ed. Philadelphia: Saunders; 1989;2:1013-31. Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988;239:487-91. Verlaan-de Vries M, Bogaard ME, van der Elst H, van Boom JH, van der Eb AJ, Bos JL. A dot-blot screening procedure for mutated ras oncogenes using synthetic oligodeoxynucleotides. Gene. 1986;5O:313-2O. Bos JL. Ras oncogenes in human cancer: a review. Cancer Res. l989;49:4682-9. Bos JL, Fearon ER, Hamilton SR, et al. Prevalence of ras gene mutations in human colorectal cancers. Nature (Lond). 1987;327:293-7. Corominas M, Kamino H, Leon J, Pellicer A. Oncogene activation in human benign tumors of the skin (keratoacanthomas): is H-ras involved in differentiation as well as proliferation? Proc Natl Acad Sci USA. 1989;86:6372-6. Lemoine NR, Mayall ES, Wyllie FS, et al. High frequency of ras oncogene activation in all stages of human thyroid tumorigenesis. Oncogene. 1989,4:159-64. Bos JL, Toksoz D, Marshall CJ, et al. Amino acid substitutions at codon 13 of the N-ras oncogene in human acute myeloid leukaemia. Nature (Lond). 1985;315:726-30. Farr CJ, Saiki RK, Erlich HA, McCormick F, Marshall CJ. Analysis of ras gene mutations in acute myeloid leukaemia by polymerase chain reaction and oligonucleotide probes. Proc Natl Acad Sci USA. 1988;85:1629-33. Thakker RV, Bouloux P, Wooding C, et al. Association of parathyroid tumors in multiple endocrine neoplasia type 1 with loss of alleles on chromosome 11. N Engl J Med. 1989;321:60-8. Arnold A, Kim HG, Gaz RD, Kronenberg HM. DNA rearranged adjacent to the PTH gene in a parathyroid adenoma encodes an abnormally expressed gene [Abstract]. J Bone Mineral Res. 1989;4(Suppl 1):5262. Baumer GC, Loeb LA. Mutations in the K-ras2 oncogene during progressive stages of human colon carcinoma. Proc Natl Acad Sci USA. 1989;86:2403-7. Nakagawa T, Mabry M, de Bustros A, Ihle JN, Nelkin BD, Baylin SB. Introduction of v-Ha-ras oncogene induces differentiation of cultured human medullary thyroid carcinoma cells. Proc Natl Acad Sci USA. 1987;84:5923-7.
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