ANTICANCER RESEARCH 28: 1341-1348 (2008)

Review Inherited Cancer Predisposition Syndromes in Greece

ANGELA APESSOS1, EIRINI PAPADOPOULOU1, IOULIA BELOGIANNI1, SOTIRIS BARATSIS2, JOHN K. TRIANTAFILLIDIS3, PARIS KOSMIDIS3, EIRINI KARYDAS4, EVANGELOS BRIASOULIS5, CHRISTOS PISIOTIS6, KOSTAS PAPAZISIS7 and GEORGE NASIOULAS1,3

1Department of Molecular Biology, 4Breast Cancer Center, 6DTCA HYGEIA SA, Kifissias Av. & 4 Er. Stavrou str, 151 23 Maroussi, Athens; 2First Surgical Department, ‘Evangelismos' General Hospital of Athens, 45-47 Ipsilantou Street, Kolonaki, Athens; 3Hellenic Cooperative Oncology Group; 5Department of Medical Oncology, University Hospital of Ioannina, Medical School, University of Ioannina, Ioannina; 7“Theagenion” Cancer Hospital, Al. Simeonides str. 2, 54007 Thessaloniki, Greece

Abstract. Hereditary cancer syndromes comprise Hereditary cancer syndromes comprise approximately 5-10% approximately 5-10% of diagnosed carcinomas. They are of diagnosed carcinomas. They are caused by mutations in caused by mutations in specific genes. Carriers of mutations in specific genes. Carriers of such mutations are at an increased these genes are at an increased risk of developing cancer at a risk of developing cancer at an early age. Today, there are a young age. When there is a suspicion of a hereditary cancer number of options available for the prevention and treatment predisposition syndrome a detailed family tree of the patient of inherited cancer predisposition syndrome for patients and requesting screening is constructed. DNA is isolated from all their families. Furthermore, advances in molecular biology available members of the family. Mutation detection is carried and in vitro fertilization techniques have made possible out on DNA from an affected family member. If a mutation is preimplantation genetic diagnosis (PGD) for the prevention found the remaining family is screened. The genetic basis of a of the birth of children with the syndrome. large number of inherited cancer predisposition syndromes is The majority of diagnosed cancer cases are sporadic, known. In this paper the focus is on mutations in genes meaning there is no obvious family history. However, there responsible for colorectal cancer, meaning adenomatous is a small percentage of patients with an inherited cancer polyposis coli (APC), which is involved in familial predisposition syndrome (1, 2). With the completion of the adenomatous polyposis and homo sapiens mutL homolog 1 human genome project and the massive technological (hMLH1) and homo sapiens mutS homolog 2 (hMSH2), advances in the field of molecular diagnosis the number of involved in hereditary non-polyposis colorectal cancer. In known genes implicated in inherited cancer predisposition addition, the genes responsible for inherited breast and/or syndromes is increasing rapidly (3, 4). The types of inherited ovarian cancer, breast cancer genes 1 and 2 (BRCA1 and cancer syndromes known to date are summarized in Table I. BRCA2), and the rearranged during transfection proto- When there is a suspicion of an inherited cancer oncogene RET which is responsible for multiple endocrine predisposition syndrome it is necessary for a clinical geneticist neoplasia type 2 are discussed. In all cases emphasis is given to to obtain a complete family history, when possible, confirmed the data available on the Greek population. by medical records. The details are recorded in the form of a pedigree comprising of more than three generations, when possible. Individuals who meet one or more of the following characteristics are candidates for investigation of an inherited Correspondence to: G. Nasioulas, Ph.D., Head, Molecular Biology cancer predisposition syndrome (1): first degree affected Research Center HYGEIA “Antonis Papayiannis”, Kifissias Ave. relatives; two or more relatives with a cancer diagnosis; & Erythrou Stavrou 4 Str, GR151-23 Maroussi, Athens, Greece. cancer diagnosis in a family member before the age of 50; the Tel: +30 210 686 7932, Fax: +30 210 686 7933, e-mail: same type of cancer diagnosed in more than one relative; [email protected] more than one type of cancer diagnosed in the same Key Words: Inherited cancer syndrome, adenomatous polyposis coli, individual and a very rare type of cancer identified in one or HNPCC, BRCA1, BRCA2, MEN 2, review. more members of the same family.

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Table 1. Inherited cancer predisposition syndromes.

Syndrome Type of cancer Gene(s)

Inherited breast and / or ovarian cancer syndrome Mainly breast and ovaries, in males BRCA1, BRCA2, prostate and pancreas CHEK2 Li-Fraumeni Sarcoma, breast, brain, leukemia p53, CHEK2 Cowden Syndrome Breast, thyroid, endometrium, etc PTEN Ataxia telangiectasia Leukemia, lymphoma ATM HNPCC Mainly CRC, endometrium, ovaries, hMLH1, hMSH2, urinary tract, stomach, pancreas hMSH6, hPMS1 FAP Mainly CRC, more rarely brain, thyroid and liver APC, MYH Hereditary gastric cancer Stomach CDH1 Peutz-Jeghers Syndrome Carcinomas of the gastrointestinal tract, breast, testicular cancer, STK11 gynecological malignancies Juvenile Polyposis CRC, stomach SMAD4, BMPR1A Neurofibromatosis 1 Neurofibrosarcomas, pheochromocytoma, NF1 optical glioma, meningioma Neurofibromatosis 2 Vestibular neuromas NF2 Xeroderma pigmentosum Skin cancer, melanoma, leukemia XPA, B, C, D, E, F, G POLH Von Hippel-Lindau Hemangioblastoma of the retina VHL Wilms Syndrome Wilms tumors WT1 Retinoblastoma Retinoblastoma, osteosarcoma RB1 Multiple endocrine neoplasia type 1 Adenomas of the pituitary glands and parathyroid parathyroid MEN1 adenoma, pituitary adenoma, pancreatic islet cell, carcinoid Multiple endocrine neoplasia type 2 Myeloid carcinoma of the thyroid, pheochromocytoma, RET hyperplasias of the parathyroid

HNPCC: hereditary non-polyposis colorectal cancer, FAP: familial adenomatous polyposis, CRC: colorectal cancer.

In this review the focus is on the syndromes of inherited MRC-Holland, The Netherlands) (12, 18). If a large genomic colorectal cancer (CRC), breast and/or ovarian cancer and rearrangement is identified, the exact nature is characterized multiple endocrine neoplasia type 2 (MEN2) in the Greek by additional methods such as long-PCR, fluorescent in situ population. hybridization (FISH) restriction mapping and Southern blotting. Characterization of the mutation helps in Method confirming its pathogenicity and in developing an accurate diagnostic test for relatives of the proband (8, 12, 18). When investigating inherited disorders the first step is the compilation of a detailed family tree of the patients and Inherited Colorectal Cancer collection of specimens of whole blood. In our laboratory DNA is extracted from the whole blood by standard Familial adenomatous polyposis (FAP). CRC is the third methods. Mutation detection for point mutations and small most common type of cancer. The majority of cases are insertions / deletions is carried out by cycle sequencing using sporadic. Approximately 10% of all CRC cases are the BigDye Terminator Cycle Sequencing kit (Applied inherited, comprising a series of rare syndromes, which are Biosystems, Foster City, USA). Analysis of the sequencing mostly transmitted in an autosomal dominant manner. products is carried out by electrophoresis on a fluorescent The best known type of inherited CRC FAP with a ® automated DNA Sequencer (ABI Prism 310, Applied frequency of ~1:8,000, corresponding to 1% of all CRC Biosystems, Foster City, USA) (5, 6, 12, 18). Sequences diagnosed (5-8). FAP is a premalignant clinical entity obtained are aligned, using Sequencher® PC software (Gene characterized by the development of hundreds to thousands Codes, USA), with normal sequences from Genbank and of adenomatous polyps in the colorectum during the second examined for the presence of mutations. If no point and third decade of life. Approximately 80% of FAP patients mutations or small insertions / deletions are identified, harbour truncating germ-line mutations in the APC screening is carried out for the detection of large genomic (adenomatous polyposis coli) tumor suppressor gene. FAP has rearrangements using the recently described method of a very high penetrance that reaches 90%. If polyps are left Multiplex Ligation-dependent Probe Amplification (MLPA, untreated 100% of the patients develop colorectal cancer.

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MYH is a recently identified gene, which encodes for a protein responsible for repairing oxidative damage in the DNA. It is believed to be involved in 1.4% of all cases of adenomatous polyposis and approximately 20% of APC- negative FAP cases. MYH-associated FAP is inherited in an autosomal recessive manner, i.e. mutations in both copies of the gene are necessary in order to cause the disease (11). As far as the Greek population is concerned, a search of the PubMed revealed only the results of our group by which 185 patients (62 families) with the suspicion of FAP have been analyzed. A pathogenic APC mutation has been identified in 26 families (41.9%) including a 250 Kb deletion starting from intron 5 and extending beyond exon 15 (5-8). No founder mutations were found in the Greek population. In addition, 28 families have been investigated for in MYH mutations and in twelve patients from five families, a heterozygous mutation has been identified in the MYH gene. In one of the families that were analyzed, two mutations were identified. The affected individuals of the family had a classical FAP phenotype and were all shown to be compound heterozygotes for the two mutations. Individuals with a single mutation did not have a FAP phenotype.

Hereditary non-polyposis colorectal cancer (HNPCC). HNPCC is the most common inherited syndrome predisposing to CRC, accounting for 5-10% of the total CRC (12, 13). HNPCC segregates in an autosomal dominant manner and it is caused by germline mutations in a group of genes encoding proteins involved in the DNA mismatch repair Figure 1. A. Pedigree of a FAP family. Black symbols indicate affected (MMR) pathway. At least five genes of the pathway, namely individuals. N inside a symbol indicates individual tested and found to be normal for the family mutation. B. Pedigree of a FAP family indicating the homo sapiens mutL homologue 1 (hMLH1) and mutS absence of the identified mutation in the parents of the affected child. The homologues 2 and 6 (hMSH2 and hMSH6) and the mutation identified (1309del5) is usually associated with a very early onset postmeiotic segregation increased genes 1 and 2 (hPMS1 and of the disease. In this case the child developed cancer at the age of 7. hPMS2), have been implicated in HNPCC (14-16). However, the majority of mutations (~90%) have been identified in hMLH1 (~50%) and hMSH2 (~40%). In HNPCC families, Frequently, patients with FAP develop other tumors affected individuals have inherited a germline mutation, including brain, thyroid and liver tumors. Other exocolonic which leads to loss of function of one of the MMR genes. manifestations of FAP include desmoid tumors (Gardner Malignant transformation is the result of a second, somatic syndrome), cysts in the sebaceous glands and the jawbones mutation in the patient’s cells. HNPCC is characterized by and congenital hypertrophy of the retinal pigment epithelium early-onset CRC (mean age at diagnosis ~45 years) and an (CHRPE) (9). Despite its strong selective disadvantage, the increased incidence of cancer in other organs such as the incidence of FAP is maintained by the frequency of de novo endometrium, stomach, small bowel, ovary, hepatobiliary mutations, which account for about 30% of all cases. tract, renal pelvis and ureter (13, 17). An inherited predisposition to CRC may also be caused As was the case for FAP, the only available data on the by other rarer syndromes such as juvenile polyposis, caused Greek population has originated from our group (12). We by mutations in the homologue of mothers against have carried out genetic analysis of the MMR genes hMLH1 decapentaplegic drosophila, 4 (SMAD4) and the gene and hMSH2 in 140 patients from 37 families with a clinical encoding for bone morphogenetic protein receptor-1A diagnosis of HNPCC (Figure 2). A pathogenic mutation has (BMPR1A), Peutz-Jeghers syndrome (Serine/threonine been identified in 10 (27%) of the families including a 2.2 kb protein kinase 11 – STK11 gene) (10), and MutY human deletion encompassing exon 3 of the hMSH2 gene. In contrast homologue - associated FAP transmitted in an autosomal to other populations (16) the majority of mutations have been recessive manner (11). identified in the hMSH2 gene (60%). Furthermore, five of the

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Hereditary Breast and Ovarian Cancer

Breast cancer is the most common malignancy in women and a major health problem in developed countries. It has been shown that the majority of hereditary breast / ovarian carcinomas can be attributed to mutations in two genes, breast cancer genes 1 and 2 (BRCA1 and BRCA2). The lifetime risk of developing breast or ovarian cancer for mutation carriers is 60-80% and 20-40%, respectively. The average age of breast cancer onset for mutation carriers is 42 years old, that is 20 years earlier than the average female population in the USA and W. Europe (18, 19). BRCA1 encodes for a protein, which seems to act as a transcription regulator and also have a role in the maintenance of genomic integrity. The major role of the protein encoded by BRCA2 seems to be in DNA damage repair. It is estimated that 6-7% of breast cancer and 10% of ovarian cancer in the general population is caused by mutations in one of the two genes. Our group and that of Yannoukakos's laboratory (20-22) have been carrying out mutation screening for the BRCA 1 and 2 genes in the Greek population for the past ten years. In our group a pathogenic mutation has been identified in 15.7% of the 70 families examined, eight families with a BRCA1 mutation including a deletion that encompasses 3.2 kb and three families with a BRCA2 mutation (18) (Figure 3). These results are in agreement with those of Yannoukakos et al. where deleterious mutations in one of the BRCA genes were identified in 20% of the families tested (20-22).

Multiple Endocrine Neoplasia Type 2 (MEN 2)

MEN 2 is an autosomal dominant inherited syndrome whose main characteristic is a strong predisposition to the development of endocrine tumors. Based on the affected tissue MEN 2 has been subdivided to three groups: MEN 2A - 69-90% of MEN 2 cases; MEN 2B - 5% and Familial medullary thyroid cancer - FMTC (5-35%). In all three Figure 2. HNPCC family with a Q158X (474C>T) point mutation in groups medullary thyroid cancer (MTC) is the main hMSH2. A. Family pedigree. Diagonal line across symbol indicates characteristic (23, 24). deceased individual. Arrow points to the index patient. A black dot inside MEN 2A is characterized by MTC (95% of cases), a symbol indicates carrier of the mutation, N inside symbol indicates individual tested and found not to carry the mutation. B. Denaturing high pheochromocytoma (50%) and hyperparathyroidism (20- performance liquid chromatography (dHPLC) analysis of exon 3 of the 30%). MEN 2B is characterized by MTC (100%), hMSH2. Control DNA of a normal individual was used for comparison. pheochromocytoma (50%), muciferous neuroma, intestinal C. Sequencing analysis of DNA from patient III:2 (Figure 2A). The ganglioneuroma and Marfan-like constitution. FMTC is mutation changes a glutamine for a stop codon (included in the box). characterized by a strong predisposition to MTC without associated problems of the parathyroid or the adrenal medulla. MEN 2 is caused by mutations in the rearranged identified mutations have not been previously described in the during transfection proto-oncogene (RET), which encodes literature (12). for a tyrosine kinase transmembrane receptor. The Our center has become the reference center for inherited syndrome is characterized by high penetrance (92%). CRC genetic diagnosis in Greece and has made an enormous Inherited MTC accounts for 20-25% of all MTC cases, the effort to record all Greek CRC families (5, 6, 9, 12). remainder being sporadic (23, 24).

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Figure 3. Pedigree of a family in which the mutation 5382insC has been identified in the BRCA1 gene.

As far as the Greek population is concerned there have before embryonic implantation. Couples requesting PGD have been only two reports of familial analysis of the RET proto- to go through routine in vitro fertilization (IVF) procedures, oncogene (23, 24). In the first report published in 1998 (23) although they are usually fertile, in order to generate a large a pathogenic mutation was identified in 66.67% (8/12 number of embryos for genetic testing. Genetic analysis is families). In the second report the RET proto-oncogene was performed within 24 hours allowing the selection of unaffected examined in 43 patients from 17 families. A mutation in embryos for transfer to the mother's uterus within the same exon 10 of the gene was identified in seven families and one IVF cycle (25). In theory, PGD could be offered for any in exon 11 in two families (Figure 4) (24). disorder of which the underlying molecular basis is known. In fact, in the period between January 1997 and May 2001, more Preimplantation Genetic Diagnosis than 1,500 clinical PGD cycles were carried out in 25 European and Australian centers, resulting in 215 pregnancies and 117 Individuals who are affected by a genetic disease or carry a babies born (26). These cycles cover a wide spectrum of mutation are at an increased risk of transmitting this to disorders, including chromosomal abnormalities and a number their offspring. With increased understanding of the of late onset disorders, such as inherited predisposition to underlying basis of inherited disorders, the ability to screen cancer (FAP, Von Hippel-Lindau syndrome, retinoblastoma, individuals for a specific disorder has become possible. This Li-Fraumeni syndrome, neurofibromatosis types I and II and identifies mutation carriers who can then receive effective familial posterior fossa brain tumour) (27-29) or non-life genetic counseling and management. threatening, but nevertheless incapacitating disorders such as Despite the obvious advantages to couples at risk of having fragile X and Crouzon syndrome (30-33). an affected child, prenatal diagnosis of an established pregnancy suffers from a major drawback, namely the need for Conclusion terminating a fetus diagnosed as affected. For some couples termination of an established pregnancy is unacceptable on The results of genetic testing for inherited cancer moral or religious grounds. This is especially true for non life- predisposition syndromes in the Greek population have threatening or late onset disorders. This led to the advent of revealed that a combination of methods capable of detecting PGD, a very early form of prenatal diagnosis carried out both single point substitutions and small insertions/deletions

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a common founder mutation in the BRCA1 gene, 5382insC, accounts for 45% of the mutations identified in Greek breast/ovarian cancer families (22). Today, the genetic basis of a large portion of inherited cancer predisposition syndromes is known. In such cases, patients can benefit from genetic testing and genetic counseling. For confirmed carriers closer inspection is required while there is no need for clinical examinations for non-carriers. Early close inspection of carriers may significantly improve their life quality and expectancy. Furthermore, carriers can opt for genetic testing of their fetus (prenatal diagnosis) or embryos (PGD) in order to prevent transmission of the disorder to their offspring.

Acknowledgements

The recording of the Greek CRC families this has been possible through a grant obtained from the Stavros Niarchos Foundation and HYGEIA hospital.

References

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