Molecular basis of the Li-Fraumeni syndrome: an update from the French LFS families.

We have performed an extensive analysis of TP53 in 474 French families suggestive of Li-Fraumeni syndrome (LFS), including 232 families fulfilling the Chompret criteria. We identified a germline alteration of TP53 in 82 families (17%), in 67/232 of the families fulfilling the Chompret criteria (29%) and in 15/242 which did not fulfil these criteria (6%). Most of the alterations corresponded to missense mutations (67%), and we identified in four families genomic deletions removing the entire TP53 locus, the promoter and the non-coding exon 1, or exons 2-10. These results represent a definitive argument demonstrating that LFS results from TP53 haplodeficiency. The mean ages of tumour onset were significantly different between patients harbouring TP53 missense mutations and other types of alterations, missense mutations being associated with a 9 year earlier tumour onset. These results confirm that missense mutations not only inactivate p53 but also have an additional oncogenic effect. Germline alterations of TP53 that lead exclusively to loss of function are therefore associated with a later age of tumour onset and the presence of such mutations should be considered in atypical LFS families with tumours diagnosed after 40 years.

The contribution of large genomic deletions at the CDKN2A locus to the burden of familial melanoma.

Mutations in two genes encoding cell cycle regulatory proteins have been shown to cause familial cutaneous malignant melanoma (CMM). About 20% of melanoma-prone families bear a point mutation in the CDKN2A locus at 9p21, which encodes two unrelated proteins, p16(INK4a) and p14(ARF). Rare mutations in CDK4 have also been linked to the disease. Although the CDKN2A gene has been shown to be the major melanoma predisposing gene, there remains a significant proportion of melanoma kindreds linked to 9p21 in which germline mutations of CDKN2A have not been identified through direct exon sequencing. The purpose of this study was to assess the contribution of large rearrangements in CDKN2A to the disease in melanoma-prone families using multiplex ligation-dependent probe amplification. We examined 214 patients from independent pedigrees with at least two CMM cases. All had been tested for CDKN2A and CDK4 point mutation, and 47 were found positive. Among the remaining 167 negative patients, one carried a novel genomic deletion of CDKN2A exon 2. Overall, genomic deletions represented 2.1% of total mutations in this series (1 of 48), confirming that they explain a very small proportion of CMM susceptibility. In addition, we excluded a new gene on 9p21, KLHL9, as being a major CMM gene.

[Prophylactic mastectomies and immediate breast reconstruction in patients at very high genetic risk: our experience with 14 cases]. / Mastectomies prophylactiques et reconstruction immédiate chez les patientes à très haut risque génétique : notre expérience à propos de 14 cas.

AIMS: Prophylactic mastectomy has been the subject of major publications by international groups. Its oncology benefit is undisputed in patients with a genetic mutation. Nevertheless, its impact on quality of life, its psychological, esthetic, sexual, functional and pain repercussions are such that it should not and must never be programmed in an emergency situation. We report our experience with 14 patients at very high genetic risk having undergone a bilateral prophylactic mastectomy with immediate breast reconstructive surgery. METHODS: From 1 March 2001 to 31 March 2006, 14 patients at very high genetic risk, seven of whom had a history of breast cancer, underwent immediate breast reconstruction with skin sparing mastectomy and preservation of the nipple-areolar complex. RESULTS: In 71.5% of the cases, we obtained a definitive conservation of the nipple-areolar complex, with esthetic results judged very satisfying. CONCLUSION: Immediate bilateral breast reconstruction by provisional or definitive implant with conservation of the skin flap and the nipple-areolar complex may constitute a positive radical issue for requesting and motivated patients at high genetic risk, managed by a multidisciplinary team.

Impact of the MDM2 SNP309 and p53 Arg72Pro polymorphism on age of tumour onset in Li-Fraumeni syndrome.

Li-Fraumeni syndrome, resulting from p53 (TP53) germline mutations, represents one of the most devastating genetic predispositions to cancer. Recently, the MDM2 SNP309 (T-->G variation) was shown to be associated with accelerated tumour formation in p53 mutation carriers. The impact of the common p53 codon 72 polymorphism on cancer risk remains controversial. We therefore investigated the effect of these two polymorphisms in 61 French carriers of the p53 germline mutation. The mean age of tumour onset in MDMD2 SNP309 G allele carriers (19.6 years) was significantly different from that observed in patients homozygous for the T allele (29.9 years, p<0.05). For the p53 codon 72 polymorphism, the mean age of tumour onset in Arg allele carriers (21.8 years) was also different from that of Pro/Pro patients (34.4 years, p<0.05). We observed a cumulative effect of both polymorphisms because the mean ages of tumour onset in carriers of the MDM2G and p53Arg alleles (16.9 years) and those with the MDM2T/T and p53Pro/Pro genotypes (43 years) were clearly different (p<0.02). Therefore, our results confirm the impact of the MDM2 SNP309 G allele on the age of tumour onset in germline p53 mutation carriers, and suggest that this effect may be amplified by the p53 72Arg allele. Polymorphisms affecting p53 degradation therefore represent one of the rare examples of modifier genetic factors identified to date in mendelian predispositions to cancer.

Comprehensive analysis of CDKN2A (p16INK4A/p14ARF) and CDKN2B genes in 53 melanoma index cases considered to be at heightened risk of melanoma.

OBJECTIVE: Comprehensive analysis of the 9p21 locus including the CDKN2A, ARF, and CDKN2B genes in 53 individuals from melanoma index cases considered to be at heightened risk of melanoma. METHODS AND RESULTS: Using a combination of DNA sequencing, gene copy number by real time quantitative PCR, linkage analysis, and transcript analysis in haploid somatic cell hybrids, we found no evidence for germline alteration in either coding or non-coding domains of CDKN2A and CDKN2B. However, we identified a p14ARF exon 1beta missense germline mutation (G16D) in a melanoma-neural system tumour syndrome (CMM+NST) family and a 8474 bp germline deletion from 196 bp upstream of p14ARF exon 1beta initiation codon to 11233 bp upstream of exon 1alpha of p16(INK4A) in a family with five melanoma cases. For three out of 10 families with at least three melanoma cases, the disease gene was unlinked to the 9p21 region, while linkage analysis was not fully conclusive for seven families. CONCLUSIONS: These data reinforce the hypothesis that ARF is a melanoma susceptibility gene and suggest that germline deletions specifically affecting p14ARF may not be solely responsible for NST susceptibility. Predisposition to CMM+NST could either be due to complete disruption of the CDKN2A locus or be the result of more complex genetic inheritance. In addition, the absence of any genetic alteration in 50 melanoma prone families or patients suggests the presence of additional tumour suppressor genes possibly in the 9p21 region, and on other chromosomes.

The contribution of germline rearrangements to the spectrum of BRCA2 mutations.

BACKGROUND: Few germline BRCA2 rearrangements have been described compared with the large number of germline rearrangements reported in the BRCA1 gene. However, some BRCA2 rearrangements have been reported in families that included at least one case of male breast cancer. OBJECTIVE: To estimate the contribution of large genomic rearrangements to the spectrum of BRCA2 defects. METHODS: Quantitative multiplex PCR of short fluorescent fragments (QMPSF) was used to screen the BRCA2 gene for germline rearrangements in highly selected families. QMPSF was previously used to detect heterozygous deletions/duplications in many genes including BRCA1 and BRCA2. RESULTS: We selected a subgroup of 194 high risk families with four or more breast cancers with an average age at diagnosis of < or = 50 years, who were recruited through 14 genetic counselling centres in France and one centre in Switzerland. BRCA2 mutations were detected in 18.6% (36 index cases) and BRCA1 mutations in 12.4% (24 index cases) of these families. Of the 134 BRCA1/2 negative index cases in this subgroup, 120 were screened for large rearrangements of BRCA2 using QMPSF. Novel and distinct BRCA2 deletions were detected in three families and their boundaries were determined. We found that genomic rearrangements represent 7.7% (95% confidence interval 0% to 16%) of the BRCA2 mutation spectrum. CONCLUSION: The molecular diagnosis of breast cancer predisposition should include screening for BRCA2 rearrangements, at least in families with a high probability of BRCA2 defects.

[Clinical and genetic study in 22 patients with basal cell nevus syndrome]. / Etude clinique et recherche de mutations germinales du gène PTCH 1 dans le syndrome des hamartomes basocellulaires.

BACKGROUND: Nevoid basal cell carcinoma syndrome is an autosomal dominant disorder characterized by developmental abnormalities and cancer predisposition. The PTCH 1 gene, the human homolog of the Drosophila segment polarity gene patched, has been shown to be involved in the development of nevoid basal cell carcinoma syndrome. PTCH 1 is mapped to chromosome 9q22.3. The aim of the present study was to report on clinical and genetic characteristics in patients followed for nevoid basal cell carcinoma syndrome and to compare them to the data in the literature. PATIENTS AND METHODS: Screening for PTCH 1 mutations was done in 22 patients followed between 1981 and 2003 for clinical suspicion of nevoid basal cell carcinoma syndrome. Clinical and radiological data were reviewed retrospectively from records. Genetic analysis was performed using blood samples after patient informed consent was obtained. When possible, DNA was also analyzed from the parents of patients in whom PTCH 1 mutations were found. RESULTS: All patients had developed basal cell carcinomas: 45% palmar and plantar pitting, 62% jaw cysts and 66% calcification of falx cerebri. Medulloblastomas and meningiomas were the most common associated tumors. PTCH 1 mutations were identified in 13 patients: 6 familial cases, 3 sporadic cases and for 4 patients, it was not possible to conclude. Nine different new germ-line mutations were identified. DISCUSSION: Genetic analysis allows molecular confirmation of diagnosis in about half of all patients. Early diagnosis is essential for detection of clinical and radiological manifestations in young patients and for provision of advice concerning protection of the skin from the sunlight.

Screening for germ line p53 mutations in children with malignant tumors and a family history of cancer.

We have undertaken a routine investigation of the p53 status for all the children treated at our institution either affected by multiple tumors or whose family displays at least one second degree relative or less, affected by cancer before the age of 45 years. We report here on the first set of ten such families, eight of which were identified through a proband with sarcoma. p53 exons 5 to 8 have been sequenced following polymerase chain reaction amplification performed on DNA isolated from total blood. A missense mutation affecting codons 248, 273, and 282 was identified in three families. The mutation was inherited in these three families and was detected in unaffected members. In seven families no mutation was detected in exons 5 to 8.

Germ-line exclusion of a single p53 allele by premature termination of translation in a Li-Fraumeni syndrome family.

Germline p53 mutations have been detected in approximately half of the families affected by the Li-Fraumeni syndrome (LFS), in which they are believed to represent the genetic status predisposing to multiple cancers. Failure to detect mutations in the other half of LFS families suggests that sequence analysis, which has been limited to the p53 gene coding region, have overlooked other genetic events lying outside of this region or/and that alterations in other gene(s) than p53 may also lead to the syndrome. In this report, we present the evidence that a single base pair deletion in the p53 coding sequence, leading to premature signal termination of translation, generates a null allele by preventing transport of mutant allele mRNAs into the cytoplasm. This allelic exclusion which confers a status of unizygote vis-à-vis the wild-type p53 gene to individuals who carry the mutant allele, leads to predisposition to multiple cancers in a Li-Fraumeni family. Thus, the loss of the wild-type p53 allele appears as the rate limiting step in tumor induction.

Low-dose radiation therapy and reduced chemotherapy in childhood Hodgkin's disease: the experience of the French Society of Pediatric Oncology.

PURPOSE: With the aim of decreasing undesirable side effects of therapy, we investigated the reduction of both chemotherapy and radiation therapy (RT) in children with Hodgkin's disease, and compared Adriamycin (doxorubicin; Farmitalia Carlo Erba, Rueil-Malmaison, France), bleomycin, vinblastine, and dacarbazine (ABVD) alone to mechlorethamine, vincristine, procarbazine, and prednisone (MOPP) and ABVD in favorable cases and assessed the effectiveness of low-dose RT (20 Gy) after good response to chemotherapy. PATIENTS AND METHODS: A French national study began in 1982 that included 238 pediatric patients with Hodgkin's disease. Initial staging was clinical and without laparotomy. In patients with localized disease (IA-IIA), an equivalence trial compared the effectiveness of four cycles of ABVD with two cycles of ABVD that were alternated with two cycles of MOPP. Patients with more advanced disease (IB-IIB-III-IV) received three courses of MOPP that was alternated with three courses of ABVD. All of the patients who achieved a good remission after chemotherapy were administered 20 Gy RT, which was limited to the initially involved areas for localized disease, and encompassed the paraaortic nodes and the spleen as well for more advanced stages. When a good remission was not obtained, 40 Gy RT was administered. RESULTS: At the completion of chemotherapy, 227 patients (97%) were considered good responders, whereas 11 did not achieve a good remission. With a median follow-up of 6 years, the 6-year actuarial survival was 92% and the disease-free survival was 86%. The relapse-free survival in favorable stages was 90% in the ABVD arm and was 87% in the MOPP and ABVD arm. In June 1987, inclusion of stage IV patients was discontinued because of poor results. CONCLUSIONS: Present findings indicate that (1) in favorable stages, ABVD alone and alternating MOPP and ABVD are equivalent, and (2) chemotherapy followed by 20 Gy RT represents a valid therapeutic approach in the vast majority of children with Hodgkin's disease.

Testing genomic imprinting in Wilm's tumor.

Data from 511 cases of Wilms' tumor in France (including 12 familial cases) and 8 pedigrees from the literature were analyzed to test three modifications of Knudson's classical bimutational theory, based on genomic imprinting in Wilms' tumor carcinogenesis. Analysis of data of age at diagnosis and segregation analysis were performed to determine the number of independent events for Wilms' tumor development and to search for a differential role of paternal and maternal alleles. Unexpectedly, we show that only one rare event is required for tumor development in isolated unilateral cases which are considered to be mainly nonhereditary. In familial cases, we observe no effect of the sex of the transmitting parent on either hge at diagnosis or segregation ratio. We show that this could be explained by models of genomic imprinting which assume two nonindependent events, or only one rare genetic event. In bilateral cases we show a bimodality for age at diagnosis which could be due to a mixture of hereditary and nonhereditary cases. This result completely questions the classical assumption according to which all bilateral cases would be hereditary. These findings support the hypothesis that this childhood cancer arises from a variety of etiological pathways and might be useful to find strategies for further molecular investigations.

Cancers in relatives of children with non-Hodgkin's lymphoma.

We undertook a family study of children treated at the Institute Gustave-Roussy in France to investigate a familial aggregation of cancer in the families of children with non-Hodgkin's lymphoma (NHL). We obtained family dat for 284 children with NHL. Using the Standardized Incidence Ratio, we compared the observed and expected number of families with at least one proband relative affected by cancer at a young age (before 46 years). We found a small but non-significant excess of all tumors in first-degree relatives (SIR = 1.3, 95% CI = 0.7-2.3) explained by a small but non-significant excess of hematological malignancies (SIR = 1.5, 95% CI = 0.2-5.5), particularly Hodgkin's disease and leukemia, and of osteosarcoma (SIR = 7.5, 95% CI = 0.1-41.4). This is probably a lower bound of the SIR, because the expected number of families was estimated from cancer incidence in France between 1978 and 1982, whereas most cancers occurred before this period. Other tumors were not in excess in first-degree relatives.

Patterns of familial aggregation of three melanoma risk factors: great number of naevi, light phototype and high degree of sun exposure.

BACKGROUND: Besides melanoma susceptibility genes and shared environmental exposures, part of the familial clustering of cutaneous malignant melanoma (CMM) might be due to familial aggregation of melanoma-associated phenotypes. Our goal was to assess the patterns of familial aggregation of three melanoma risk factors: great number of naevi (GNN), light phototype (LP) and high degree of sun exposure (HDSE). METHODS: Familial aggregation of GNN, LP and HDSE was investigated in 66 French families with at least two CMM cases and was measured by the asssociation of the relatives' traits with the probands' traits, using the generalized estimating equations approach. The probands were the melanoma cases leading to ascertainment of the families, subdivided into cases (with the trait studied) and controls (without the trait). RESULTS: We found significant evidence for familial aggregation of GNN only among sibs (OR = 3.7, 95% CI : 1.4-10.5, P = 0.01), of LP among blood relatives (OR = 3.8, 95% CI : 1.8-8.0, P = 0.004) and of HDSE among blood relatives (OR = 4.5, 95% CI : 2.1-9.9, P < 0.001) and spouses (OR = 44.3, 95% CI : 5.1-382.2, P < 10(-3)). These results suggest that genetic factors might account for the clustering of GNN and LP and shared environment for the aggregation of HDSE. The GNN clustering was lower in families with increasing numbers of CMM (>/=3 cases) or presence of p16 mutations, the opposite being observed for LP and HDSE. Moreover, the familial aggregation of LP was significantly lower in families with highly sun-exposed members. CONCLUSION: Melanoma might not only result from specific genetic and environmental factors but also from those underlying melanoma-associated traits involving complex gene-gene and gene-environment interactions.

Comparison between familial and nonfamilial melanoma in France.

BACKGROUND AND DESIGN: Five percent to 10% of cutaneous malignant melanomas (CMMs) occur in a familial setting. Clinical, epidemiologic, and genetic studies of familial CMM in different regions of the world have led to various results. To assess the characteristics of familial CMM in France, the clinical, histologic, and epidemiologic characteristics of 295 patients with CMM were recorded, and a comprehensive familial investigation was performed for each case. Patients with a family history of CMM were compared with nonfamilial cases. RESULTS: Cutaneous malignant melanoma occurred as a familial cancer in 22 (8%) of 295 patients. Among the multiple variables studied, those significantly associated with the familial occurrence of CMM were red hair, inability to tan, and presence of clinically atypical moles. When these variables were considered together in a multivariate analysis, only the association with red hair (P = .001) and atypical moles (P < .05) remained significant. In addition, the patients with familial melanoma exhibited the following tendencies: a younger age at diagnosis, a higher number of moles, and the development of multiple primary melanomas, but these results did not reach statistical significance. Factors relating to UV light exposure, histologic features of CMM, course of the disease, and occurrence of nonmelanoma cancers showed a similar distribution between familial and nonfamilial cases. CONCLUSION: A familial investigation should be performed for each patient with CMM in France, particularly when he or she exhibits phenotypic risk factors for CMM such as red hair and atypical moles.

Association between germ cell tumours, large numbers of naevi, atypical naevi and melanoma.

Identifying groups of subjects at high risk for the development of melanoma is crucial for the early diagnosis of curable tumours. In the present study, we performed a skin examination in a group of 63 patients followed up after treatment of germ cell tumours (GCTs) who were referred to the dermatologist for multiple pigmented cutaneous spots. Forty-nine patients bearing a great number of naevi or atypical naevi were included in the study. Two thin cutaneous melanomas were discovered in two patients. In addition, a third patient had had a conjunctival melanoma since treatment of the GCT. Our study confirms the presence of atypical naevi in a subgroup of GCT patients, who are shown to be at high risk of developing melanoma. Patients harbouring multiple pigmented spots should be referred for a skin examination aimed at early detection of curable melanomas, and should be advised to protect themselves from sun exposure.

Surgical procedure in patients with ovarian cancer diagnosed at the time of prophylactic oophorectomy. Analysis of two cases, literature review and surgical implications.

The aim of this study was to discuss the surgical management for ovarian or tubal cancers diagnosed at the time of prophylactic oophorectomy. Two patients with BRCA1 & BRCA2 mutations carriers had ovarian cancer diagnosed during laparoscopic oophorectomy. Conversion to laparotomy was performed in order to complete surgery (hysterectomy, multiple peritoneal biopsies, omentectomy, pelvic and para-aortic lymphadenectomy). These two patients were upstaged on the basis of para-aortic lymphadenectomy and had massive nodal spread into para-aortic area. One of them had no intra-peritoneal disease and the other one had minor peritoneal disease (only one positive pelvic biopsy in the Douglas pouch). These two patients are alive, one of them with 3.5 years of follow-up after the end of adjuvant treatment. In order to ensure the exact spread of the disease. Lymphadenectomy should be performed in patients with ovarian cancer diagnosed at the time of prophylactic surgery.

[A French genetic and epidemiologic study on nephroblastoma: preliminary results]. / Etude génétique et épidémiologique française sur le néphroblastome: résultats préliminaires.

Preliminary results of genetic and epidemiological study of Wilms' tumor in France are given in this paper. The higher age at diagnosis in unilateral than in bilateral cases supports the bimutational theory of Knudson. The excess of cancer deaths found in relatives of patients might be due to genetic non specific factors of susceptibility to cancer.

[Li-Fraumeni syndrome: update, new data and guidelines for clinical management]. / Le syndrome de Li-Fraumeni: mise au point, données nouvelles et recommandations pour la prise en charge.

The Li-Fraumeni syndrome (LFS) is an inherited form of cancer, affecting children and young adults, and characterized by a wide spectrum of tumors, including soft-tissue and bone sarcomas, brain tumours, adenocortical tumours and premenopausal breast cancers. In most of the families, LFS results from germline mutations of the tumor suppressor TP53 gene encoding a transcriptional factor able to regulate cell cycle and apoptosis when DNA damage occurs. Recently, germline mutations of hCHK2 encoding a kinase, regulating cell cycle via Cdc25C and TP53, were identified in affected families. The LFS working group recommendations are the following: (i) positive testing (screening for a germline TP53 mutation in a patient with a tumor) can be offered both to children and adults in the context of genetic counseling associated to psychological support, to confirm the diagnosis of LFS on a molecular basis. This will allow to offer to the patient a regular clinical review in order to avoid a delay to the diagnosis of another tumor; (ii) the 3 indications for positive testing are: a proband with a tumor belonging to the narrow LFS spectrum and developed before age 36 and, at least, first- or second-degree relative with a LFS spectrum tumor, before age 46, or a patient with multiple primary tumors, 2 of which belonging to the narrow LFS spectrum, the first being developed before 36 or a child with an adenocortical tumour; (iii) presymptomatic testing must be restricted to adults; (iv) the young age of onset of the LFS tumors the prognosis of some tumors, the impossibility to ensure an efficient early detection and the risk for mutation carriers to develop multiple primary tumors justify that prenatal diagnosis might be considered in affected families.

[Clinical and molecular diagnosis of inherited breast-ovarian cancer]. / Diagnostic génétique du cancer du sein et de l'ovaire héréditaire.

OBJECTIVE: The aim of this work was to pinpoint familial breast and/or ovarian cancer risk. Clinical cancer genetics include: diagnostic cancer genetics, cancer genetic counseling and management of women at high risk of developing breast and/or ovarian cancer. MATERIAL AND METHODS: An update of documented data was performed in order to assess our current knowledge about inherited breast-ovarian cancer. RESULTS: Most breast cancers (BC) are sporadic while 5-10% are estimated to be due to an inherited predisposition. Rare autosomal dominant alteration in two genes, BRCA1 and BRCA2, which confer a high risk of developing BC and ovarian cancer (OC), are likely to account for most families with multiple cases of early-onset BC and OC, but only 3-4% of all BC. The estimations of their prevalence and penetrance vary greatly, depending on the population studied, the study design, statistical methods and the sensitivity of technical methods for detection of mutations. Penetrance can be very high, but incomplete (maximum 80%). Penetrance and age at onset of the same mutation show great variability within and between BC families. This could be due to the effects of other genetic as well as non-genetic factors that are being studied. The heterogeneity of families makes the identification of other high-risk genes difficult. Additive effects of several susceptibility genes could explain much more BC in the general population (polygenic models). Molecular diagnoses have become feasible. Specific consultations in oncogenetics help clinicians and patients understand hereditary components, establish molecular diagnoses, provide guidelines for a better surveillance of people at high risk of developing BC, OC or both and reassure the mutation non-carriers. The strategies of management and treatment of BC and OC in mutation carriers remain under discussion and it is is difficult to choose preventive options. CONCLUSION: Inherited BC or OC due to a mutation in BRCA1 or BRCA2 are rare in the general population. However, women and men have the right to be informed of the possibilities concerning presymptomatic testing, risk assessment, surveillance, prevention and psychological support with respect to the potential benefits and limitations. Other large prospective studies are needed to validate rapidly the methods of surveillance and prevention in this group at "high risk" in order to adapt them to very large populations. However, predictive medicine should be used with care.