High Frequency of Germline p53 Mutations in Childhood Adrenocortical Cancer
Josephine Wagner, Carol Portwine, Karen Rabin, Jean-Marie Leclerc, Steven A. Narod, David Malkin*
Background: Adrenocortical carcino- ma (ADCC) is a rare childhood cancer, affecting three of 1 million children younger than 16 years old in the United States. ADCC may be found in associa- tion with the Li-Fraumeni and Beck- with-Wiedemann syndromes. Children with ADCC are also at substantially in- creased risk of second primary can- cers. Because of these associations, it is believed that the genetic basis for ADCC is stronger than for most child- hood malignancies. Germline muta- tions of the TP53 tumor suppressor gene are associated with cancer pre- disposition in families with the Li- Fraumeni syndrome as well as in individuals with sporadically occurring component tumors of the syndrome. Purpose: We investigated the pos- sibility that germline TP53 gene altera- tions existed in children with ADCC. Methods: Sixteen children with ADCC under the age of 18 were identified from searches of medical oncology records at three Canadian hospitals. Eleven of these 16 patients identified were alive. The mean age at diagnosis was 4.8 years (range, 1-17 years). Family histories were obtained for 11 unselected children with ADCC (six girls and five boys). Pathologic confir- mation of tumor diagnosis was ob- tained from the medical records. Using single-strand conformational polymor- phism analysis followed by single- strand DNA sequencing, genomic DNA extracted from whole blood was analyzed for the presence of TP53 mutations for six living ADCC patients. Results: Three of six (50%) children were found to carry germline TP53 mutations in exons 5, 6, and 7, respec- tively. Both wild-type and mutant al-
leles were identified in all three TP53 sequences, indicating that the patients were heterozygous for germline TP53 mutations. None of these children was from a family with the Li-Fraumeni syndrome. The mutation in one child was shown to be inherited from the mother, who subsequently developed breast cancer. A striking excess of can- cer was found in one family of a patient carrying wild-type TP53. Conclusions: Our observation of a high frequency of germline TP53 mutations in children with sporadic ADCC suggests that these children may represent probands with which to ascertain Li-Fraumeni syndrome families. It may be reason- able for children with adrenocortical carcinoma to be candidates for germ- line TP53 analysis. In light of the wealth of information in the Li- Fraumeni literature that associates germline TP53 mutations with a variety of malignancies, this testing may have important consequences for risk assessment for other close rela- tives, including early-onset breast can- cer in the mothers. [J Natl Cancer Inst 86:1707-1710, 1994]
Adrenocortical carcinoma (ADCC) is a rare pediatric tumor, affecting only three children per 1 million under the age of 16 years in the United States (1). Adrenocor- tical cancer and other embryonic tumors are also found in excess in children with the Beckwith-Wiedemann syndrome and among children with isolated hemihy- pertrophy (2). The risk of a second primary cancer in survivors of childhood ADCC is increased (3). ADCC is found more than 100 times as often as expected in families with the Li-Fraumeni syn- drome. Families adhering to the “classic” definition of this syndrome include one individual, the proband, diagnosed with sarcoma before 45 years of age, a first-de- gree relative (parents, siblings, and off- spring) with cancer before 45 years of age, and another first- or second-degree relative (one generation removed from proband: grandparents, grandchildren, and first cousins) in the same lineage with any cancer diagnosed under 45 years of age or with sarcoma occurring at any age (4). In a survey of 24 families with the Li-Fraumeni syndrome, four of 151 af- fected family members with cancer had
ADCC (4). All patients were less than 14 years of age at the time of diagnosis. In- herited germline mutations of the TP53 tumor suppressor gene have been iden- tified in several families with the Li- Fraumeni syndrome (5-7) and have been described in some patients with second neoplasms (8), multifocal osteosarcoma (9), multifocal glioma (10), sarcomas (11), breast cancer (12,13), and osteosar- coma (14) in the absence of a positive family history of cancer. The TP53 tumor suppressor gene encodes a 53-kd nuclear phosphoprotein that appears to function as a negative regulator of cell growth and proliferation (15). It is thought that TP53 regulates transcriptional activation, ena- bling suppression of abnormal cell prolif- eration by acting as a checkpoint control factor in the presence of DNA damage (16-19). Alterations of the TP53 gene and its encoded protein are the most frequent somatic genetic abnormalities in human cancer (20-22).
On the basis of the significant role that TP53 plays in the Li-Fraumeni syndrome and in many other forms of human can- cer, we investigated the possibility that germline TP53 alterations existed in children with ADCC, a rare component tumor of this familial cancer syndrome.
Patients and Methods
Sixteen children under the age of 18 years who were diagnosed with ADCC were identified from the records of the Divisions of Oncology of The Hospital for Sick Children, Toronto, Canada, and the Hôpital St. Justine and the Montreal Children’s Hospital, Montreal, Canada. At that time, 11 of the patients were still alive. The mean age at diagnosis was 4.8 years (range, 1-17 years). One child had been diagnosed with Beckwith-Wiedemann syn- drome. We were able to obtain family medical his- tories for 11 unselected children with ADCC (six girls and five boys), all of whom contributed to the
*Affiliations of authors: J. Wagner, S. A. Narod, Department of Human Genetics, McGill University, Montreal, Quebec, Canada.
C. Portwine, K. Rabin, D. Malkin, Division of Oncology, The Hospital for Sick Children, Toronto, Ontario, Canada.
J .- M. Leclerc, Department of Oncology, Hôpital St. Justine, Montreal.
Correspondence to: David Malkin, M.D., Division of Oncology, The Hospital for Sick Children, 555 University Ave., Toronto, ON, Canada M5G 1X8.
See “Notes” section following “References.”
Downloaded from http://jnci.oxfordjournals.org/ at The University of British Colombia Library on July 5, 2015
family history analysis. Of these 11 children, we were able to obtain constitutional DNA from six for TP53 testing. The patient with Beckwith-Wiede- mann syndrome was not included in the molecular analysıs. Pathologic confirmation of tumor diagno- sis was obtained from the medical records.
Peripheral blood leukocytes were isolated using a standard phenol-chloroform extraction method. The cells were then resuspended in high TE buffer (100 mM Tris and 40 mM EDTA; pH 8.0) and lysed by the addition of an equal volume of high TE buffer containing 0.2% sodium dodecyl sulfate. After two extractions with TE-saturated phenol and one with chloroform-isoamyl alcohol (25:1), DNA was pre- cipitated with 1/10 volume of 4 M ammonium ace- tate and an equal volume of isopropanol. The DNA was redissolved in 10 mM Tris (Ph 7.5) and 1 mM EDTA and stored at 4 ℃ at a concentration of 50 ng/uL.
Nine sets of primers were generated to amplify DNA fragments encompassing exon 2 and exons 4 through 11 of the TP53 gene to screen for the presence of point mutations by single-strand confor- mational polymorphism (SSCP) analysis. The primer sequences and corresponding fragment lengths have been published previously (23,24). The polymerase chain reaction (PCR) was performed with the use of 250 ng of template DNA in 50 mM Tris-HCI (pH 8.6), with 1.5 mM MgCl2, 0.2 mM of each deoxynucleoside triphosphate, 250 ng of each primer, 1 uL of [32P]deoxycytidine triphosphate (3000 Ci/mmol) diluted 1:10, and 2.5 U Taq polymerase (AmpliTaq; Chiron Therapeutics, Emeryville, Calif.) in a 50-uL total reaction volume. Reaction conditions for the Perkin-Elmer 480 Ther- mocycler were as follows: 94 ℃ (45 seconds), 55 ℃ (45 seconds), and 72 °℃ (45 seconds) for 35 cycles. The reaction was initiated with a 6-minute “hot-start” incubation at 85 ℃ and was completed with a 7- minute extension at 72 ℃, followed by 3 minutes at 94 °C. Five microliters of the PCR product was added to 5 uL of loading buffer (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol). The samples were denatured for 5 minutes at 85 ‘C and loaded immediately onto a polyacrylamide-TBE nondenaturing gel containing 4.5%-9.0% acrylamide and 2.0%-10.0% glycerol. Electrophoresis was conducted at room temperature at 25 W for 6-7 hours or at 10 W for 15-17 hours. The concentration of glycerol or acrylamide and the duration of electrophoresis were varied according to conditions previously established for each respective fragment. Both positive and negative controls were applied to each gel and were consistently discern- ible. The gel was blotted onto 3M filter paper, dried, and exposed to x-ray film (Kodak) with an inten- sifying screen at -70 ℃ for 4-72 hours.
DNA samples determined to be abnormal by multiple SSCP analyses were amplified with the SSCP primers encompassing the abnormal region. Fragments were directly subcloned into a T-tailed pBSK vector and sequenced by the Sanger dideoxy- nucleotide method with a Sequenase 2.0 kit (US Biochemical) to determine the precise nature of the sequence alteration. At least six individual clones were sequenced for each sample. In addition, where a base-pair alteration was identified, duplicate PCR reactions were performed to rule out the possibility of a PCR-generated artifact.
Results
Although the diagnosis of cancer in relatives could not always be confirmed, cancers in relatives were excluded if the proband expressed uncertainty of the diagnosis. The family history included all first- and second-degree relatives, with respect to current age, age at death, and age and site of any cancer. The expected numbers are based on Canadian cancer incidence data. The family history was obtained in all cases by face-to-face inter- views by a genetic counselor (J. Wagner) or pediatric oncologist (D. Malkin). A total of 17 cancers were identified among the 157 relatives. Based on Canadian cancer incidence data, the expected number was 16.4. Eight of these cancers were diag- nosed before the age of 55 (expected, 6.6). There were no additional cases of ADCC or of childhood cancer of any site in family members. Cancers found in ex- cess included those of the breast (five ob- served and 2.7 expected; P = . 14) and colon (three observed and 1.2 expected; P = . 18). Two of the colon cancers were diagnosed before age 55 (0.16 expected; P = . 01). Other cancers included lung cancer (three patients), cancer of the larynx (one), ovarian cancer (one), pros-
tatic cancer (one); leukemia (one); and cancer of unknown site (two).
Three of the six patients with ADCC were found to carry germline mutations in the highly conserved regions of the TP53 gene. In this study, no SSCP gels with demonstrable band shifts yielded normal TP53 sequence. Details of the mutations are provided in Table 1 and in Figs. 1 and 2. Both wild-type and mutant alleles were identified in all three sequen- ces, indicating that the patients were heterozygous for germline TP53 muta- tions. In patient 1, a transition from C to T at the second position of codon 152 in exon 5 resulted in a conservative amino acid change from proline to leucine. Patient 3 harbored a C to T transition at the first position of codon 219 in exon 6, yielding an amino acid change from proline to serine. Patient 6 exhibited an A to G transition in the first position of codon 235 in exon 7, changing the en- coded amino acid from asparagine to aspartic acid. Band shifts could not be demonstrated by SSCP with the use of multiple electrophoretic conditions for patients 2, 4, or 5. Previous studies (24,25) indicated that the sensitivity of our assay is high, with more than 85% of known mutations being discernible and
| Patient No. | Exon No. | Codon No. | Sequence change | Amino acid switch |
|---|---|---|---|---|
| 1 | 5 | 152 | CCG -> CTG | Proline -> leucine |
| 3 | 6 | 219 | CCC -> TCC | Proline -> serine |
| 6 | 7 | 235 | AAC -> GAC | Asparagine -> aspartic acid |
EXON 5
EXON 6
EXON 7
W.T.
W.T.
P.T. 1
W.T.
W.T.
PT. 3
W.T.
MUT.
PT. 6
PT. 1
C
PT. 3
T
PT. 6
T
AGCT ÅGCT
C
ÅGCT AGCT
À
C
T
ÅGCT
ÅGCT
A
C
G
C
A →G
C→T
C
C
C→T
A
C
C
G
T
W.T.
MUT.
C
T
À
C
W.T.
MUT.
G
W.T.
MUT.
C
suggesting a low false-negative rate for mutation detection. Twelve years after the appearance of ADCC in her son, the mother of patient 1 developed breast can- cer at the age of 46. She harbors the iden- tical germline TP53 mutation at codon 152. The parents of the other two children with TP53 mutations have not been tested. Although their pedigrees are un- remarkable for cancer, it cannot at this point be determined whether the germline TP53 mutations in the probands occurred de novo or were inherited. Among the relatives of the three TP53-positive patients, there were four cases of cancer observed, compared with 3.4 expected. The family of one child carrying wild- type TP53 showed a remarkable excess of cancer (Fig. 3). The mother of the proband died of colon cancer at the age of 35; there were four other early-onset tumors of the type associated with the Li- Fraumeni syndrome.
Discussion
To date, most germline p53 mutations have been found in families with strong histories of cancer. A survey of six Li- Fraumeni-type cancer families, each of which had at least one case of ADCC, revealed two germline TP53 mutations (26). The proportion of germline TP53 mutations associated with other tumor types is low; germline mutations have been identified in one of four children with multifocal osteosarcoma (9), in seven of 235 children with osteosarcoma (14), and in four of 59 children and young adults with multiple primary neoplasms (8). It appears, therefore, that ADCC is the childhood cancer associated with the highest frequency of germline TP53 mu- tations. If we include the three children with germline TP53 mutations, the one
patient with a strong family history of cancer, and the child with Beckwith- Wiedemann syndrome, we could estab- lish a hereditary component for five (45%) of the 11 patients in our small series. Among childhood cancers, only retinoblastoma has a heritable fraction of this magnitude (27).
The sites of the mutations in this study are unique as germline mutations. How- ever, they have all been described in sporadic malignancies (21). They are all found within the most highly conserved regions of the TP53 gene where more than 90% of previously identified muta- tions have been reported. In this series, children with ADCC who were not found to harbor germline TP53 mutations may yet have an increased risk of developing another malignancy or have relatives at increased risk of developing cancer for several reasons. It is possible that not all TP53 mutations were detected because of the less than 100% sensitivity of the SSCP technique. On the other hand, some patients may carry wild-type TP53, yet they may inherit a predisposition for can- cer development through a post-transla- tional mechanism that inactivates the
wild-type protein. Such a scenario has been recently suggested in a patient from a Li-Fraumeni syndrome family who lacked detectable germline TP53 muta- tions but who overexpressed wild-type TP53 in constitutional cells, indicating the likelihood of abnormal stabilization of the protein (28); it is also suggested by the observations that not all “classic” Li- Fraumeni syndrome families harbor detectable germline p53 mutations (29). Although not previously described in the germline, it is possible that amplification of the mdm-2 gene or a similar gene whose product stabilizes wild-type TP53 protein may inhibit the normal function of the protein. Of course, children with ADCC may also develop their disease as a consequence of alterations of other can- cer-associated genes.
Our observation of a high frequency of germline TP53 mutations in children with sporadic ADCC suggests that these chil- dren may represent probands with which to ascertain Li-Fraumeni syndrome fami- lies. It may be reasonable for children with ADCC to be candidates for germline TP53 analysis. In light of the wealth of information in the Li-Fraumeni literature
ADCC Pedigree: Wild-Type p53 Genotype
Bone, 30’s
Breast, 40
Leukemia, 33
Colon, 35
Brain, 32
ADCC, 4
that associates germline TP53 mutations with a variety of malignancies, this test- ing may have important consequences for risk assessment for other close relatives, including early-onset breast cancer in the mothers.
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Notes
Present address. J. Wagner, Fox Chase Cancer Center, Philadelphia, Pa.
Supported in part by the Medical Research Coun- cil of Canada; and by the National Cancer Institute of Canada.
We thank Drs. M. Whitehead, E. Colle, and M. L. Greenberg for providing access to patients and Elisabeth Sexsmith, Jodi Lees, and Dr. P. Tonin for excellent technical support.
Manuscript received April 13, 1994; revised August 10, 1994; accepted August 23, 1994.
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