RAPID COMMUNICATION
Claudia Pötzsch · Theda Voigtländer · Michael Lübbert p53 Germline mutation in a patient with Li-Fraumeni Syndrome and three metachronous malignancies
Received: 28 December 2001 / Accepted: 14 May 2002 / Published online: 10 August 2002 @ Springer-Verlag 2002
Abstract Purpose: Germline mutations of the p53 coding region are present in ~50-70% of patients with Li-Fraumeni Syndrome (LFS), a rare hereditary dis- order of familial and intraindividual clustering of dif- ferent malignancies such as sarcoma (index tumor), breast cancer, brain tumors, leukemias, and adreno- cortical carcinomas, the latter usually in young chil- dren. Both onset and spectrum of malignancies in individuals with LFS are thus heterogenous and may, less frequently, also include other epithelial and mes- enchymal tumors. A 32-year-old female presented for genetic counseling with a history of leiomyosarcoma at age 22, malignant melanoma (a rare component of LFS) at age 26, and breast cancer at age 30. All three tumors had been treated surgically. Astrocytoma and breast cancer, respectively, had been diagnosed in her brother and mother before age 30. Other malignancies diagnosed early in life in relatives of the mother were: prostate cancer, stomach cancer, and carcinoma of the larynx. Methods: Upon written informed consent, DNA was extracted from peripheral blood mononu- clear cells of the proband, and p53 exons 4-8 analyzed for mutations by SSCP and DNA sequencing. Results: A G:C to A:T mutation at codon 175 of p53 resulting in an arginine → histidine substitution was detected, confirming the clinical diagnosis of LFS. Conclusions: The patient and her family are being followed further, but testing of her children for the presence of this mutation is currently being withheld. The difficulties in the management and treatment of
C. Pötzsch · M. Lübbert
Div. Hematology/Oncology, University of Freiburg Medical Center,
Hugstetter Str. 55, 79106 Freiburg i. Brsg., Germany
E-mail: Luebbert@mm11.ukl.uni-freiburg.de
Tel .: +49-761-2703279
Fax: +49-761-2703697
T. Voigtländer Institute for Human Genetics, University of Heidelberg, Im Neuenheimer Feld 344A, 69120 Heidelberg, Germany
patients with this clinically heterogenous disorder are discussed.
Keywords Tumor suppressor gene . Hereditary cancer . Second primary neoplasm · Melanoma
Introduction
A striking accumulation of cancers in a family or in an individual is suggestive of an underlying genetic predis- position. Heterozygous germline mutations of several tumor suppressor genes are associated with predisposi- tion to cancer (Knudson 1971). In 1969, Li and Fraumeni first described a familial clustering of tumors (soft tissue sarcomas, breast cancer, acute leukemias, lung cancer, adrenocortical carcinomas, and brain tu- mors) (Li and Fraumeni 1969). The rigorous criteria for this “Li-Fraumeni Syndrome” (LFS) are defined as follows (Li et al. 1988): a proband aged less than 45 years with a sarcoma having both a first-degree rel- ative aged less than 45 years with any cancer and an additional first- or second-degree relative aged less than 45 years in the same lineage with any cancer or a sar- coma at any age. Families that do not fulfill the strong criteria of classical LFS, but show striking similarities, have been described as Li-Fraumeni-like families (LFL) according to the definition of Birch et al. (Birch et al. 1994 a): a patient with a childhood cancer or sarcoma, brain tumor or adrenocortical carcinoma at age under 45 years with a first- or second-degree relative with a typical LFS tumor at any age and an additional relative with a cancer under 60 years of age.
Germline mutations of the p53 gene were described in six families with LFS in 1990 (Malkin et al. 1990; Srivastava et al. 1990). Recently, a heterozygous germ- line human checkpoint kinase 2 (hCHK2) mutation was also shown to be associated with LFS in a family with rhabdomyosarcoma, brain tumors, and multiple cases of early onset and bilateral breast cancer in which no p53 germline mutation could be found (Bell et al. 2000).
Wild-type p53 is a tumor-suppressor gene located on chromosome 17p13. Via sequence-specific DNA binding sites it has important roles in DNA repair (Ford and Hanawalt 1995) and apoptosis (Yonish-Rouach et al. 1991). Currently the p53 gene is known as the most frequently altered gene in human tumors (Hollstein et al. 1996).
Thus far, more than 100 families with classic LFS or Li-Fraumeni-like Syndrome with a p53 germline muta- tion have been described (Kleihues et al. 1997; Varley et al. 1997a; Akashi and Koeffler 1998). However, germline p53 mutations within the coding region of the gene are only found in up to 70% of LFS-families (Frébourg et al. 1995; Varley et al. 1997a). On the other hand, germline p53 mutations have only infrequently been detected in patients with tumors typical of LFS whose family histories are not indicative of the LFS or LFL; for example, in patients with second malig- nant neoplasms (Malkin et al. 1992), with sarcoma (Toguchida et al. 1992; McIntyre et al. 1994), with breast cancer (Borresen et al. 1992) or glioma (Kyritsis et al. 1994), and among children with adrenocortical carci- noma (Sameshima et al. 1992).
We describe a 33-year-old female patient in whom a leiomyosarcoma was diagnosed and surgically removed at age 22. Four years later a malignant melanoma was excised. Again four years later breast cancer was diag- nosed during pregnancy. The family history of the pa- tient was fully compatible with LFS, and molecular analysis revealed a previously described germline muta- tion of the p53 gene at codon 175, resulting in an arginine → histidine substitution. The clinical implications of di- agnosis and treatment of malignancies in individuals with LFS, as well as genetic counseling, are discussed.
Materials and methods
After informed consent was obtained, peripheral blood mononu- clear cells were isolated from heparinized venous blood by Ficoll gradient. Extraction of DNA and single-stranded conformational polymorphism (SSCP) analysis was performed as described previ- ously (Pötzsch et al. 1999). The cell lines used as positive controls for p53 mutations were: U-266 (myeloma, mutation in exon 5, codon 161), Jurkat (T-lymphoblasts, exon 6, codon 196), CEM (T-lymphoid, exon 5, codon 175; exon 7, codon 248), HEK 1B (endometrial carcinoma, exon 7, codon 248), BT 474 (exon 8, codon 285), and A 431 (epithelial carcinoma, exon 8), which were kindly provided by Carl Miller, Cedars-Sinai Medical Center/ UCLA School of Medicine, Los Angeles, California, and Drs. Marion Kiechle and Hans Ikenberg, Dept. of Gynecology, University of Freiburg. Sequence alterations as visualized by the PCR/SSCP analysis were confirmed by direct sequencing of PCR products with an Applied Biosystems model 373A DNA sequenc- ing system (Applied Biosystems, Warrington, UK). PCR products were purified with the Qiaquick PCR purification Kit (Qiagen, Hilden, Germany) and sequenced in the forward and reverse directions by the dideoxy termination method using the same primers as for the PCR/SSCP analysis [exon 5 sense: 5’-TCT GTT CAC TTG TGC CCT GAC TTT C-3’ and exon 5 antisense 5’-ACC CTG GGC AAC CAG TCC CTG TCG TC-3’ as used by Paquette et al. (Paquette et al. 1993)] and a PRISM Dye Termi- nator Cycle Sequencing Kit (Perkin Elmer, Weiterstadt, Germany).
Results and discussion
The patient presented for genetic counseling at 32 years of age because of a history of three metachronous ma- lignancies: cutaneous leiomyosarcoma of the left thigh (pTlpNOpM0, G 2) at age 22, malignant melanoma of the left foot (superficial spreading, pT1N0M0) at age 26 and breast cancer (multifocal, multicentric ductal carci- noma in situ of the right breast) during pregnancy at age 30. All three tumors were treated surgically (without adjuvant radio-/chemotherapy being indicated) with the patient being in continuous complete remission at age 33. Family history revealed that her mother had died of breast cancer at age 26 and an astrocytoma was diag- nosed in her only brother at age 21. Other malignancies diagnosed early in her mother’s relatives were: prostate cancer, stomach cancer, and carcinoma of the larynx. The patient has three healthy children and has had two miscarriages. A pedigree of the family is shown in Fig. 1A. The patient’s history was fully compatible with Li-Fraumeni Syndrome. Exons 4-8 of the p53 gene were analyzed by SSCP. A band shift was detected in exon 5 (Fig. 1B). Exon 5 was sequenced and a G:C to A:T mutation at codon 175 of p53 was revealed (Fig. 1C), resulting in an arginine-histidine substitution.
Main component cancers associated with LFS are soft-tissue sarcomas, osteosarcomas, breast cancer, brain tumors, leukemias, and adrenocortical carcinomas (Birch 1994). Other, less frequent component tumors are melanomas (Hartley et al. 1987) or germ cell tumors (Hartley et al. 1989). Sarcoma and breast carcinoma, especially premenopausal breast carcinoma in early adulthood, are the most frequent tumors in LFS and LFL families. Breast cancer in carriers of a p53 germline mutation occurs in over 30% under the age of 30 and in nearly 100% under the age of 50 (Birch 1994). Brain tumors are part of LFS, especially brain tumors in children and young adults. Apart from LFS, p53 mu- tations were detectable in 2-18% of patients with glioma at young age or with an unusual personal or family history of cancer or multifocal disease (Kyritsis et al. 1994; Chung et al. 1991; Chen et al. 1995; Felix et al. 1995; Li et al. 1995; Metzger et al. 1991).
Although studies in patients with multiple primary tumors showed a possible involvement of a p53 germline mutation (Malkin et al. 1992; Russo et al. 1994), the frequency of three or four metachronous tumors in a patient with LFS is only about 4% and 2%, respectively (Hisada et al. 1998). This may be due to the high mor- tality rate of these patients with early-onset malignant diseases.
Malignant melanoma is a rare component tumor of LFS. Nevertheless there are some reports about malig- nant melanomas in LFS families (Birch et al. 1994 a; Malkin et al. 1992). The mutation of the p53 gene found in these families was located in exon 7, codon 248. Therefore, there is no evidence for an association be- tween the rare phenotype of a melanoma and a mutation
a
Neck cancer AO/AD nk
Stomach cancer AD 50
Breast cancer AD 26
y
Leiomyosarcoma, AO 22 Malignant melanoma, AO 26 Breast cancer, AO 30
Astrocytoma AO 21
Prostate cancer AO/AD nk
1989
1990
1992
12 3 4 5 6 7 8 9
Codon
174 175 176 177
A
Wild type AGG CGC TGC CCC
G TTG TG AGG C CTG C CCC ACC.
160
170
b
C
of codon 175 of the p53 gene. Whether melanoma is really part of LFS with lower penetrance or a coinci- dentally arising tumor is not clear.
The missense mutation at codon 175 we detected in this patient has been described in other LFS families before (Birch et al. 1994 a; Varley et al. 1997a, Frébourg et al. 1995; McIntyre et al. 1994). This mutation is sit- uated in the highly conserved region III. Most of the germline and somatic mutations are found in the highly conserved regions II-V in the central part of the protein. They encode the DNA binding region and build loop motifs. Mutations at codon 175 do not affect residues that directly contact DNA but residues that stabilize the DNA structure by connecting two of the loops (Cho et al. 1994). For the 175 His-mutation a gain of function with a growth advantage has been demon- strated (Dittmer et al. 1993).
In the patient described all three malignomas were detected in an early stage, and she could be treated surgically with curative intent without additional radi- ation or chemotherapy being indicated.
This is important to note, because an increased sus- ceptibility to ionizing radiation and other mutagenic agents in LFS families has to be assumed. For instance, in the four families first described by Li and Fraumeni, four soft-tissue sarcomas within the field of previous radiotherapy occurred over a 12-year-period. This fact must also be considered with regard to further screening and diagnostic procedures, where clinical examination,
ultrasound, and magnetic resonance imaging are clearly preferable to mammography and computer tomography whenever possible. Other reliable screening strategies will have to be established concerning methods and target organs in order to survey such cancer families. The diversity of phenotypes among patients with a germline p53 mutation complicates an effective screening strategy and requires their care in specialised centers.
Testing of the patient’s children for the presence of the p53 germline mutation is presently being withheld. So far, the children do not suffer from any tumor. As the p53 germline mutation cannot be cured, predictive testing for p53 mutations should be guided by ethical principles: a person chosen for testing should be given current, relevant information on the test to make an informed voluntary decision. Furthermore, these per- sons must be provided with the highest quality of in- formation and counseling available, including psychosocial care (Li et al. 1992). As minors are not yet able to make such a voluntary decision themselves, which can stigmatize them for a lifetime, testing of children is not recommended routinely (Varley et al. 1997b). Nevertheless, some authors prefer testing chil- dren as young as possible with the goal of reducing cancer morbidity and mortality (Li et al. 1992). In LFS families, the risk of childhood tumors is estimated to be in the order of 20% (Lustbader et al. 1992), and an early cancer detection can substantially improve the likeli- hood of cure. The children of our proband are regularly followed and examined and the girls will have a regular gynecological examination when they reach puberty. Being of age, they may make a decision regarding a predictive test for themselves.
With regard to the rarity of germline p53 mutations and the diversity of their clinical manifestations, therapy and counseling in LFS families should be administered on the basis of clinical studies involving experts in on- cology, genetic counseling, medical ethics, psychology, and medical and molecular genetics.
Acknowledgements This study was supported by Deutsche Krebshilfe (W48/92/Lü1)
References
Akashi M and Koeffler HP (1998) Li-Fraumeni Syndrome and the role of the p53 tumor suppressor gene in cancer susceptibility. Clin Obstet Gynecol 41:172-199
Bell DW, Varley JM, Szydlo TE, Kang DH, Wahrer DCR, Shannon KE, Lubratovich M, Verselis SJ, Isselbacher KJ, Fraumeni JF, Birch JM, Li FP, Garber JE, Haber DA (2000) Heterozygous germline hCHK2 mutations in Li-Fraumeni syndrome. Science 286:2528-2531
Birch JM (1994) Li-Fraumeni syndrome. Eur J Cancer 30A:1935- 41
Birch JM, Hartley AL, Tricker KJ, Prosser J, Condie A, Kelsey AM, Harris M, Morris Jones PH, Binchy A, Crowther D, Craft AW, Eden OB, Evans DGR, Thompson E, Mann JR, Martin J, Mitchell ELD, Santibanez-Koref MF (1994a) Prevalence and diversity of constitutional mutations in the p53 gene among 21 Li-Fraumeni Families. Cancer Res 54:1298-1304
Borresen AL, Andersen TI, Garber J, Barbier-Piraux Nthorlacius S, Eyfjord J, Ottestad L, Smith-Sorensen B, Hovig E, Malkin D, Friend SH (1992) Screening for germline TP53 mutations in breast cancer patients. Cancer Res 52:3234-3236
Chen P, Iavarone A, Fick J, Edwards M, Prados M, Israel MA (1995) Constitutional p53 mutations associated with brain tu- mors in young adults. Cancer Genet Cytogenet 82:106-115
Cho Y, Gorina S, Jeffrey PD, Pavletich NP (1994) Crystal structure of a p53 tumor suppressor-DNA complex: understanding tu- morigenic mutations. Science 265:346-355
Chung R, Whaley J, Kley N, Anderson K, Louis D, Menon A, Hettlich C, Freiman R, Hedley-White ET, Martuza R, Jenkins R, Yandell D, Seizinger BR (1991) TP53 gene mutations and 17p deletions in human astrocytomas. Genes Chrom Cancer 3:323-331
Dittmer D, Pati S, Zambetti G, Chu S, Teresky AK, Moore M, Finlay C, Levine AJ (1993) Gain of function mutations in p53. Nature Genet 4:42-46
Félix CA, Slave I, Dunn M, Strauss EA, Philips PC, Rorke LB, Sutton L, Bunin GR, Biegel JA (1995) P53 gene mutations in pediatric brain tumors. Med Pediat Oncol 25:431-436
Ford JM, Hanawalt PC (1995) Li-Fraumeni syndrome fibroblasts homozygous for p53 mutations are deficient in global DNA repair but exhibit normal transcription-coupled repair and en- hanced UV-resistance. Proc Natl Acad Sci USA 92:8876-8880 Frebourg T, Barbier N, Yan Y, Garber JE, Dreyfus M, Fraumeni J, Li FP, Friend SH (1995) Germ-line p53 muta- tions in 15 families with Li-Fraumeni syndrome. Am J Hum Genet 56:608-615
Garber JE, Goldstein AM, Kantor AF, Dreyfus MG, Fraumeni JF, Li FP (1991) Follow-up study of twenty-four families with Li-Fraumeni syndrome. Cancer Res 51:6094-6097
Hartley AL, Birch JM, Marsden HB, Harris M (1987) Malignant melanoma in families of children with osteosarcoma, chon- drosarcoma, and adrenal cortical carcinoma. J Med Genet 24:664-668
Hartley AL, Birch JM, Kelsey AM, Marsden HB, Harris M, Teare MD (1989) Are germ cell tumors part of the Li-Fraumeni syndrome? Cancer Genet Cytogenet 42:221-226
Hisada M, Garber JE, Fung CY, Fraumeni JF Jr, Li FP (1998) Multiple primary cancers in families with Li-Fraumeni syn- drome. J Natl Cancer Inst 90:606-611
Hollstein M, Shomer B, Greenblatt M, Soussi T, Hovig E, Montesano R, Harris CC (1996) Somatic point mutations in the p53 gene of human tumors and cell lines: updated compilation. Nucl Acids Res 24:141-146
Kleihues P, Schäuble B, zur Hausen A, Estève J, Ohgaki H (1997) Tumors associated with p53 germline mutations. Am J Path 150:1-13
Knudson AG Jr (1971) Mutation and cancer. Statistical analysis of retinoblastoma. Proc Natl Acad Sci USA 68:820-823
Kyritsis AP, Bondy ML, Xiao M, Berman EL, Cunningham JE, Lee PS, Levin VA, Saya H (1994) Germline p53 mutations in subsets of glioma patients. J Natl Cancer Inst 86:344-349
Li FP, Fraumeni JF Jr (1969) Soft-tissue sarcomas, breast cancer, and other neoplasms. A familial syndrome? Ann Int Med 71:747-752
Li FP, Fraumeni JF Jr, Mulvihill JJ, Blattner WA, Dreyfus MG, Tucker MA, Miller RW (1988) A cancer familiy syndrome in twenty-four kindreds. Cancer Res 48:5358-5362
Li FP, Garber JE, Friend SH, Strong LC, Patenaude AF, Juengst ET, Reilly PR, Correa P, Fraumeni JF Jr (1992) Recommen- dations on predictive testing for germ line p53 mutations among cancer-prone individuals. J Natl Cancer Inst 84:1156-1160
Li Y-J, Sanson M, Hoang-Xuang K, Delattre J-Y, Poisson M, Thomas G, Hamelin R (1995) Incidence of germ-line p53 mu- tations in patients with gliomas. Int J Cancer 64:383-387
Lustbader ED, Williams WR, Bondy ML, Strom S, Strong LC (1992) Segregation analysis of cancer in families of childhood soft tissue sarcoma patients. Am J Hum Genet 51:344-356
Malkin D, Li FP, Strong LC, Fraumeni JF Jr, Nelson CE, Kim DH, Kassel J, Gryka MA, Bischoff FZ, Tainsky MA, Friend
SH (1990) Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 250:1233-1238
Malkin D, Jolly KW, et al (1992) Germline mutations of the p53 tumor-suppressor gene in children and young adults with sec- ond malignant neoplasms. N Engl J Med 326:1309-1315
McIntyre JF, Smith-Sorensen B, Friend SH, Kassell J, Borresen AL, Yan YX, Russo C, Sato J, Barbier N, Miser J, Malkin D, Gebhardt MC (1994) Germline mutations of the p53 tumor suppressor gene in children with osteosarcoma. J Clin Oncol 12:925-930
Metzger AK, Sheffield VC, Duyk G, Daneshvar L, Edwards MSB, Cogen PH (1991) Identification of a germ-line mutation in the p53 gene in a patient with an intracranial ependymoma. Proc Natl Acad Sci USA 88:7825-7829
Paquette RL, Lee YY, Wilczynski SP, Karmakar A, Kizaki M, Miller CW, Koeffler HP (1993) Mutations of p53 and human papillomavirus infection in cervical carcinoma. Cancer 72:1272-1280
Pötzsch C, Schaefer H-E, Lübbert M (1999) Familial and meta- chronous malignant lymphoma: absence of constitutional p53 mutations. Am J Hemat 62:144-149
Russo CL, McIntyre J, Goorin AM, Link MP, Gebhardt MC, Friend SH (1994) Secondary breast cancer in patients present- ing with osteosarcoma: possible involvement of germline p53 mutations. Med Pediat Oncol 23:354-358
Sameshima Y, Tsunematsu Y, Watanabe S, Tsukamoto T, Kawa-ha K, Hirata Y, Mizoguchi H, Sugimura T, Terada M, Yokota J (1992) Detection of novel germ-line p53 mutations in diverse cancer-prone families identified by screening patients with childhood adrenocortical carcinoma. J Natl Cancer Inst 84:703-707
Srivastava S, Zou ZQ, Pirollo K, Blattner W, Chang EH (1990) Germ-line transmission of a mutated p53 gene in a cancer- prone family with Li-Fraumeni syndrome. Nature 348:747-749 Toguchida J, Yamaguchi T, Dayton SH, Beauchamp RL, Herrera GE, Ishizaki K, Yamamuro T, Meyers PA, Little JB, Sasaki MS, Weichselbaum RR, Yandell DW (1992) Prevalence and spectrum of germline mutations of the p53 gene among patients with sarcoma. N Engl J Med 326:1301-1308
Varley JM, McGown G, Thorncroft M, Santibanez-Koref MF, Kelsey AM, Tricker KJ, Gareth D, Evans R, Birch JM (1997a) Germ-line mutations of TP53 in Li-Fraumeni families: an ex- tended study of 39 families. Cancer Res 57:3245-3252
Varley JM, Evans DGR, Birch JM (1997b) Li-Fraumeni syndrome B a molecular and clinical review. Br J Cancer 76:1-14
Yonish-Rouach E, Resnitzky D, Lotem J, Sachs L, Kimchi A, Oren M (1991) Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6. Nature 352:345-347