Tesi sul tema "Syndrome de Li-Fraumeni (LFS)"

Le syndrome de Li-Fraumeni (LFS) prédispose les porteurs de variations pathogènes de TP53 à un large spectre de tumeurs dès l’enfance. La variabilité phénotypique du LFS complique la prise en charge des patients et peut s’expliquer en partie par le type de variations de TP53, mais également par l'influence de facteurs modificateurs génétiques. Afin d’évaluer ces facteurs modificateurs, il est nécessaire de mettre au point des tests fonctionnels adaptés.L’activité des isoformes de p53 suggère qu’elles pourraient agir comme facteurs modificateurs du LFS. C’est pourquoi nous avons mis au point des techniques pour l’analyse des transcrits alternatifs, présentées dans la première partie de ces travaux de thèse. Si nos résultats ont montré que ces techniques n’étaient pas adaptées pour répondre à cette hypothèse, elles nous ont néanmoins permis d’identifier un nouveau transcrit physiologique, non décrit dans la littérature, augmenté chez une patiente porteuse d’une variation située sur le site accepteur d’épissage du dernier exon de TP53. Ce transcrit démontre l’existence d’un épissage alternatif du dernier exon de TP53 avec un exon terminal alternatif situé à plus de 2 kb en aval.Afin d’aider à la classification des variations de TP53, notre laboratoire évalue l’activité transcriptionnelle de p53 dans le contexte génétique du patient. Ce test ne permet donc pas de s’affranchir de l’influence potentielle des facteurs modificateurs génétiques individuels. C’est pourquoi, dans la deuxième partie de ces travaux de thèse, nous avons mis au point un modèle cellulaire de cellules souches pluripotentes induites humaines permettant d’étudier les variations de TP53 insérées par CRISPR-Cas9 dans un fond génétique commun. Ces travaux soulignent l’importance de l’expression de TP53, notamment pour l’étude des variations dont la pénétrance est moindre par rapport aux variations « hot-spot ». Par ailleurs, nous montrons que les variations en phase exercent un impact différentiel sur l’activité fonctionnelle de p53, selon le domaine protéique dans lequel elles se situent. L’avantage de notre modèle réside également sur son hétérozygotie pour PEX4 sur lequel nous avons pu insérer une seconde variation, ici le polymorphisme p.(Pro47Ser) inséré en trans d’une variation pathogène. Nos résultats soulignent l’importance du contexte génétique dans l’analyse des variations de TP53. L’ensemble de ces travaux de thèse mettent en lumière la nécessité d’étudier l’activité transcriptionnelle de p53 dans un contexte physiologique, sans surexpression, dans le but de mieux comprendre ce syndrome et d’optimiser la prise en charge des patients LFS
Li-Fraumeni Syndrome (LFS) predisposes carriers of pathogenic TP53 variants to a wide spectrum of cancers throughout life. The phenotypic variability of LFS complicates patient management and can be partly attributed to the type of TP53 variant, as well as the influence of genetic modifier factors. To evaluate these modifier factors, it is essential to develop suitable functional tests.The activity of p53 isoforms suggests that they may act as modifier factors in LFS. Consequently, we developed assays for analyzing alternative transcripts, as presented in the first part of this work. While our results demonstrated that these assays were not well-suited to addressing this specific hypothesis, they nevertheless led us to the discovery of a novel physiological transcript not previously described in the literature. This transcript was found to be increased in a patient carrying a variant located at the splice acceptor site of TP53’s last exon, revealing an alternative splicing event involving TP53’s final exon and an alternative terminal exon located more than 2 kb downstream.To facilitate the classification of TP53 variants, our laboratory evaluates p53’s transcriptional activity in the patient’s specific genetic context. However, this approach does not allow us to fully disentangle the potential influence of individual genetic modifier factors. Therefore, in the second part of this work, we developed a human-induced pluripotent stem cell model to study TP53 variants introduced by CRISPR-Cas9 within a standardized genetic background. Our findings highlight the importance of physiological TP53 expression, particularly for studying variants with lower penetrance compared to "hot-spot" variants. Additionally, we show that in-frame variants exert differential impacts on p53’s functional activity, depending on the protein domain in which they are located. The advantage of our model also lies in its heterozygosity for PEX4, into which we were able to insert a second variant, in this case, the p.(Pro47Ser) polymorphism, inserted in trans with a pathogenic variant. Our results highlight the importance of the genetic context in the analysis of TP53 variants. This thesis work emphasizes the necessity of studying p53 transcriptional activity in a physiological context, without overexpression, with the aim of improving our understanding of this syndrome and optimizing the management of LFS patients

This thesis presents various clinical, genetic and biological aspects of a cancer-prone family. Two probands were studied at the initiation of the study: a 36-year-old female with infiltrating ductal breast carcinoma and a recent history of adrenocortical carcinoma (ACC), and her 21/2-year-old son, also with ACC. The occurrence of the latter, an extremely rare tumour, in two members of the one family prompted speculation that these two individuals may belong to a Li-Fraumeni cancer syndrome (LFS) family. ACC is a hallmark tumour of this particular cancer family syndrome, which also includes tumours affecting such diverse tissues as breast, soft-tissue, bone, brain and haematopoietic cells. A pedigree spanning five generations was compiled using family recollections and medical records of cancer. A comparison of the pedigree for this family with the clinical requirements for LFS and a genetic analysis confirming that the cancer predisposition was inherited in an autosomal dominant fashion showed that this was indeed an LFS cancer family. Because the predisposing lesion for LFS had not yet been discovered at the initiation of this study. examination of the cancer inheritance in this family was of interest. In order to provide material for long-term studies and to enable a preliminary characterisation of tumourigenesis in the probands, attempts were made to grow tumour and constitutional cells from both probands in culture. The primary breast carcinoma grew for a brief period only, before stromal fibroblast-like cells took over the culture. A bony metastasis of the same tumour, however, was found to grow as non—adherent multicellular spheroids which persisted longer in culture, probably indicative of the progressive changes that occurred in vivo. The stromal fibroblasts were found to be normal and thence served as a constitutional control for the breast carcinoma. The boy's adrenocortical carcinoma recurred twice before his death at age 6 years and it was from the first and second recurrences of his tumour that cells were cultured. The cells which grew were heterogeneous in morphology and were not immortalised. There were also morphological and immunocytochemical differences between the cell strains derived from the first and second relapses. These differences were thought to indicate both the heterogeneous nature of the adrenocortical carcinoma and to represent progressive changes in tumourigenesis. Normal abdominal skin fibroblasts were cultured as constitutional controls. In order to investigate the predisposing lesion for the cancer predisposition, the p53 tumour suppressor gene was selected as a candidate, based on its frequent involvement in sporadic tumours of the same type as those found in LFS patients and also the finding that transgenic mice expressing a mutant p53 allele showed a higher frequency of malignancy than normal controls. The involvement of the p53 gene was examined by Northern and Southern blot analysis, 81 nuclease mapping, and genomic DNA and cDNA PCR/sequencing. An additional larger transcript was found in both tumour and constitutional RNA from the probands, and in constitutional RNA from some obligate carriers, that was not present in controls and unaffected individuals. This was thought to be indicative of an underlying germline alteration to the p53 gene in those individuals with the larger transcript. The size discrepancy in the transcripts was not caused by a genomic rearrangement but was found to be due to the retention of all or most of intron 4 in the mature mRNA because of a germline point mutation in the intron 4 5' splice donor site (at the last base o fexon 4) of one p53 allele. The frequent occurrence of stop codons in intron 4 ensured that if a protein could be produced from the larger transcript. it would be truncated and the p53 immunoprecipitations gave some evidence for the existence of a truncated protein product. Interestingly. the mutation, a G to A transition, appeared to produce only a partial disruption to splicing in Northern and cDNA PCR/sequencing analysis, with a proportion of the transcripts produced from the mutant allele being normally spliced. The mutation was in the last base of exon 4 which is also the last base of codon 125. Because of coding degeneracy, it was found that these normally-spliced mutant transcripts would not result in an amino acid substitution when translated implying that this mutation, rather than abolishing the functionality of the mutant allele could result in a reduction in p53 levels. Thus the mutation is unusual in that it is potentially weaker in its effect than most other sporadic and germ-line p53 mutations. This is the first report of a germ-line splicing mutation in the p53 gene and appears to be the most 5' of the LFS germ-line mutations reported. The secondary events in tumourigenesis in both of the probands. in particular the fate of the remaining p53 allele, were investigated. Evidence from Northern and PCR/sequence analysis suggested that the second p53 allele had been lost during development of both the breast carcinoma and the ACC in the probands, with retention of the mutant allele. Both tumours showed extensive karyotypic abnormalities including aneuploidy, with translocations, deletions and both consistent and random chromosome losses. Some of the consistent chromosomal losses were thought to be linked to the loss of tumour suppressor genes involved in the tumoun'genic process. Other genetic events included the over-expression of the dominantly-acting nuclear oncogene c-myc in the breast carcinoma, which is linked to a highly proliferative tumour and a poor prognosis, and the over-expression of Rb in the ACC. The over-expression of Rb was carried through to over-production of an apparently wild-type pr, probably due to disruption of the Rb functional pathway. The over-expression of Rb was an unusual finding as this gene is normally mutated or lost in tumours. This gene over-expression and protein overproduction in the ACC could also have been influenced by the lack of sufficient functional p53, which would normally down-regulate the expression of Rb. In contrast to the reports of others, the constitutional fibroblasts of both probands did not show an increased rate of spontaneous immortalisation nor did they show karyotypic abnormalities, perhaps because of the weak nature of the germ-line mutation uncovered in this family. Thus, in this project a cancer-prone family was identified, and constitutional and tumour cells from two probands were cultured for analysis. A putative predisposing lesion for the cancer-predisposition in an LFS cancer family was discovered in the p53 gene. The mutation was novel in both its location (in exon 4 rather than the hotspot for LFS mutations, exon 7) and type (a splicing mutation rather than the more common missense mutation). The tumourigenic process in the two probands was investigated and some of the secondary genetic events in the tumours of the probands were uncovered, including the loss of the remaining allele of the p53 gene, overexpression of C-myc and Rb. Please note that the literature reviewed in this thesis is current up to February 1993.

Si le syndrome de prédisposition héréditaire au cancer du sein et de l’ovaire constitue uneentité reconnue et supportée par l’identification de variations délétères sur les gènes BRCA1,BRCA2, PALB2, RAD51C et RAD51D, et si le cancer du sein de la femme jeune (avant 31 ans) estintégré dans le spectre du syndrome de Li-Fraumeni lié aux altérations de TP53, une large fractiondes patientes adressées en consultation d’oncogénétique pour ce motif demeure orpheline dediagnostic moléculaire. La connaissance du génome humain et l’avènement du séquençage denouvelle génération ont permis des avancées considérables, notamment dans l’observation de latrès grande variabilité du génome et de la survenue de variations de novo.Nous avons ainsi appliqué ces outils et ces concepts au cancer du sein de la femmejeune, afin de tenter d’identifier de nouveaux déterminants moléculaires constitutionnels. Dansune première approche basée sur la réalisation d’exomes soustractifs pour des trios parents- enfant, nous avons recherché des variants de novo délétères et effectivement identifié un variantrare de novo et délétère sur le gène INHBA dans le contexte d’un cancer de l’ovaire chez unejeune femme. Cette approche n’a cependant pu être reproduite dans le contexte d’un cancer dusein précoce. Nous avons également tenté une approche par exomes comparatifs dans unefamille remarquable avec survenue de cancers du sein précoces sur trois générations, sansvariation délétère identifiée commune à ces individus. Dans une seconde approche basée surun panel de 201 gènes impliqués dans la cancérogenèse, nous avons tenté d’identifier desvariants délétères ou des enrichissements en variants rares dans une cohorte de cancers du seinprécoces. Nous avons identifié une variation en mosaïque de TP53, sans autre détection devariations formellement délétères parmi 30 patientes atteintes de cancers du sein avant 31 ans.Un enrichissement non significatif en variants rares affectant les voies de la réparation de l’ADN aété néanmoins mis en évidence, suggérant des études plus larges ciblant ces voies. Enfin, nousavons recherché spécifiquement des variants de novo en mosaïque de TP53 dans lecontexte du cancer du sein de la femme jeune ou de cancers pédiatriques, et démontré ainsi laprévalence relativement importante de ces évènements. Ces observations supportent la nécessitéd’user d’un séquençage de forte profondeur et de ne pas restreindre les indications d’analyses deTP53 aux seules situations familiales évocatrices
Despite previous identifications of deleterious variants on BRCA1, BRCA2, PALB2,RAD51C and RAD51D supporting the hereditary breast and ovarian cancer syndrom, and thecontribution of TP53 mutations in very early-onset breast carcinomas, a large fraction of patientssuggestive of Medelian disease remains without molecular diagnosis. In the past years,sequencing of the Human genome and next-generation sequencing offered major advances, inparticular in the field of genome variability and de novo variants.We applied these new tools and concepts in the context of very early-onset breastcarcinomas, in order to identify new molecular germline determinants. First, we dealt withsoustractive exomes, in parents - child trios, and succeed in the identification of a deleterious denovo variant in the INHBA gene, in the context of very early-onset of ovarian cancer. However, wehave failed with this approach in a second trio with an index affected by early-onset breastcarcinoma. We also tried a comparative exome sequencing approach in a remarkable pedigreewith multiple probands affected by early-onset breast carcinomas, without identification of ashared deleterious variant. Secondly, we used a home-made 201 genes panel assuming thatgenes somatically affected in cancers might be altered in inherited conditions. We analyzed acohort of very early-onset breast carcinomas, and identified a mosaic TP53 variation. Moreover,we identified some interesting candidate variants and observed a non-significant trend of rarevariants enrichment in the DNA repair pathway. Finally, we designed a specific TP53 gene capturein order to detect mosaic variants in pediatric cancers and very early-onset breast carcinomas.We confirmed the clinically significant prevalence of these alterations, which support TP53analysis in these conditions even in sporadic presentations

Introdução: As síndromes de Li-Fraumeni (LFS) e Li-Fraumeni Like (LFL) são síndromes hereditárias de predisposição a câncer frequentemente associadas à mutações germinativas no gene TP53. Devido à importância de p53 e diversidade de processos celulares que ela regula, várias vias de sinalização podem ser afetadas pela doença. Nesse estudo discutimos o impacto da mutação p.R337H na proliferação, senescência, autofagia, população e funcionalidade mitocondrial. Métodos: As taxas de proliferação foram avaliadas pelo ensaio de Population Doubling. Os experimentos de senescência, autofagia, massa total e funcionalidade mitocondrial foram realizados por citometria de fluxo. Resultados: As células contendo a mutação proliferaram mais do que as células controle. Além disso, as células mutadas não ativaram autofagia sob tratamento de Rapamicina nem senescência sob tratamento de Doxorubicina ou Cisplatina e exibiram maior população mitocondrial, mas com funcionalidade inalterada após os tratamentos. Conclusão: os dados sugerem que a mutação p.R337H em TP53 afeta a indução de senescência realizada por p53 e suas funções pró-autofágicas, bem como seu controle. As células mutadas proliferam mais do que células sem mutação em TP53 e exibiram maior massa mitocondrial sem perda de funcionalidade após o tratamento com Doxorubicina.
Background: Li-Fraumeni (LFS) and Li-Fraumeni Like (LFL) syndromes are hereditary cancer predisposition syndromes frequently associated with germline mutation in TP53. Due to the importance of the protein p53 and its regulation of several important cellular processes, impairment in some pathways can be implicated. Here we discuss the impact of p.R337H TP53 mutation on proliferation, senescence, autophagy, mitochondrial population and functionality. Methods: Growth rates were assayed with Population Doubling assay. Senescence and autophagy were assessed through flow cytometry and functionality and total population of mitochondria were also analyzed through flow cytometry. Results: mutated cells proliferated more than control cells. TP53 mutated cells didn’t build up autophagy under Rapamycin treatmend nor senescence under Doxorubicin or Cisplatin treatments and showed more mitochondrial mass, but no alterations in mitochondrial functionality after Doxorubicin treatment. Conclusion:data suggests that p.R337H TP53 mutation affect senescence induction by p53 and pro-autophagic actions of p53. Mutated cells proliferate more than control cells and exhibited larger mitochondrial mass without effects in their functionality in response to Doxorubicin treatment.

TP53 est un gène suppresseur de tumeur muté dans la moitié des cancers humains. La plupart des mutations de TP53 affectent le domaine de liaison à l’ADN de la protéine, et sont situées au niveau de sept « hotspot » mutationnels. Parmi elles, la mutation Y220C est retrouvée dans 100 000 nouveaux cas de cancer par an et dans 10 familles Li-Fraumeni de prédisposition au cancer. Pour étudier cette mutation in vivo, j’ai créé au cours de ma thèse le modèle murin exprimant une protéine p53 portant la mutation Y217C équivalente à la Y220C humaine. J’ai pu démontrer que non seulement la mutation inactive la fonction de la protéine mais qu’elle semble également conférer de nouvelles fonctions oncogéniques. Plus intéressant encore, ce modèle constitue un très bon outil pour le test d’une nouvelle stratégie thérapeutique spécifique de cette mutation. Par ailleurs, le modèle murin p53∆31/∆31 a été créé dans notre laboratoire et a permis de démontrer in vivo le rôle de régulateur négatif du domaine C-terminal de p53 sur son activité. Au cours de mes travaux de recherche, j’ai pu montrer que p53 joue un rôle dans la régulation de nouvelles voies cellulaires chez la souris et chez l’Homme, et établi le mécanisme en jeu dans cette régulation. De façon surprenante, ces fonctions insoupçonnées sont impliquées dans la promotion de l’instabilité génétique et une activité accrue de p53 peut entraîner des maladies d’insuffisance médullaire prédisposant au cancer. Nos observations ont donc des retombées fondamentales mais également cliniques pour le diagnostic de ces maladies ainsi que pour les stratégies anti-tumorales selon le statut de p53
TP53 is a tumor suppressor gene mutated in half of human cancers. Most of TP53 mutations affect the core DNA binding domain of the protein, and are located on seven « hotspot » residues. Among them, the Y220C mutation is found in 100 000 new cancer cases per year and in 10 Li-Fraumeni families of cancer predisposition. To study this mutation in vivo, I created a mouse model carrying the Y217C mutation, equivalent to the human Y220C. I showed that not only this mutation inactivates the function of the protein, but that it also confers new oncogenic functions. Moreover, this model is a great tool to evaluate the efficiency of a novel anti-tumoral strategy in vivo. Furthermore, the mouse p53∆31/∆31 model was created in our team and allowed us to demonstrate the negative regulatory role of the C-terminus of p53 in vivo. I used this model to discover the implication of p53 in the regulation of unsuspected cellular pathways in mice but also in humans, and to establish the mechanism of regulation. Surprisingly, these functions are responsible for the promotion of the genomic instability and p53’s high activity can cause bone marrow failure syndromes that predispose to cancer. Our observations have both fundamental and clinical impact for the diagnosis of these diseases and for the therapeutic anti-tumoral strategies according to p53’s status

TP53 est un gène suppresseur de tumeur muté dans la moitié des cancers humains. La plupart des mutations de TP53 affectent le domaine de liaison à l’ADN de la protéine, et sont situées au niveau de sept « hotspot » mutationnels. Parmi elles, la mutation Y220C est retrouvée dans 100 000 nouveaux cas de cancer par an et dans 10 familles Li-Fraumeni de prédisposition au cancer. Pour étudier cette mutation in vivo, j’ai créé au cours de ma thèse le modèle murin exprimant une protéine p53 portant la mutation Y217C équivalente à la Y220C humaine. J’ai pu démontrer que non seulement la mutation inactive la fonction de la protéine mais qu’elle semble également conférer de nouvelles fonctions oncogéniques. Plus intéressant encore, ce modèle constitue un très bon outil pour le test d’une nouvelle stratégie thérapeutique spécifique de cette mutation. Par ailleurs, le modèle murin p53∆31/∆31 a été créé dans notre laboratoire et a permis de démontrer in vivo le rôle de régulateur négatif du domaine C-terminal de p53 sur son activité. Au cours de mes travaux de recherche, j’ai pu montrer que p53 joue un rôle dans la régulation de nouvelles voies cellulaires chez la souris et chez l’Homme, et établi le mécanisme en jeu dans cette régulation. De façon surprenante, ces fonctions insoupçonnées sont impliquées dans la promotion de l’instabilité génétique et une activité accrue de p53 peut entraîner des maladies d’insuffisance médullaire prédisposant au cancer. Nos observations ont donc des retombées fondamentales mais également cliniques pour le diagnostic de ces maladies ainsi que pour les stratégies anti-tumorales selon le statut de p53
TP53 is a tumor suppressor gene mutated in half of human cancers. Most of TP53 mutations affect the core DNA binding domain of the protein, and are located on seven « hotspot » residues. Among them, the Y220C mutation is found in 100 000 new cancer cases per year and in 10 Li-Fraumeni families of cancer predisposition. To study this mutation in vivo, I created a mouse model carrying the Y217C mutation, equivalent to the human Y220C. I showed that not only this mutation inactivates the function of the protein, but that it also confers new oncogenic functions. Moreover, this model is a great tool to evaluate the efficiency of a novel anti-tumoral strategy in vivo. Furthermore, the mouse p53∆31/∆31 model was created in our team and allowed us to demonstrate the negative regulatory role of the C-terminus of p53 in vivo. I used this model to discover the implication of p53 in the regulation of unsuspected cellular pathways in mice but also in humans, and to establish the mechanism of regulation. Surprisingly, these functions are responsible for the promotion of the genomic instability and p53’s high activity can cause bone marrow failure syndromes that predispose to cancer. Our observations have both fundamental and clinical impact for the diagnosis of these diseases and for the therapeutic anti-tumoral strategies according to p53’s status

Abstract In the present study, mutations in BRCA1 and BRCA2, the two major genes predisposing individuals to hereditary breast and ovarian cancer, were screened in Finnish and Turkish cancer families. Germline BRCA1 mutations were found in 7% (6/88) and BRCA2 mutations in 6% (5/88) of the Finnish families studied in Oulu. Two distinct BRCA1 (3745delT, 4216nt-2A→G) and three BRCA2 (999delTCAAA, 6503delTT, 9346nt-2A→G) mutations were identified, all of which are recurrently found in Finland. In the 15 Turkish cancer families studied, 5382insC and 5622C→T were detected in BRCA1, and 3414delTCAG in BRCA2. The novel 3414del4 mutation was found in a family with a case of male breast cancer. In order to determine their ages and origin, 9 recurrent Finnish BRCA1 and BRCA2 mutations were studied further as regards haplotype conservation. Common origins approximately 18–80 generations (400–1600 years) ago were demonstrated for all studied mutations by partial haplotype sharing. The majority of the mutations showed geographical clustering, supporting the theory of regional founder effects. Four of the nine mutations are unique for Finland, whereas five have also been seen elsewhere. Mutations in the 5' end of BRCA1 tend to predispose individuals to ovarian cancer and those found in the 3' end to breast cancer. The age of ovarian cancer onset was significantly lower for BRCA1 (51 years) than for BRCA2 mutation carriers (61 years). Germline TP53 mutations were sought in the Finnish breast cancer families found to be negative after BRCA1 and BRCA2 screening but who exhibited some phenotypic features of the Li-Fraumeni syndrome. The Asn235Ser was found in a family displaying Li-Fraumeni syndrome phenotype and the Tyr220Cys in a family with a milder Li-Fraumeni-like phenotype. The nature of both mutations as cancer-predisposing alterations was supported by means of loss of heterozygosity (LOH) and p53 immunohistochemistry studies. Regional clustering of BRCA1 and BRCA2 founder mutations enables targeted genetic tests including especially those mutations characteristic of the birthplace of each patient. Additional genes are likely to explain a large proportion of the inherited susceptibility to breast cancer in particular. Germline TP53 mutations are expected to be found in the breast cancer families with other clinical features seen in the Li-Fraumeni syndrome.

Objetivo: Até 10% dos casos de câncer de mama estão associados com uma síndrome genética de predisposição ao câncer. A identificação de possíveis portadores dessas síndromes e o consequente encaminhamento para aconselhamento genético permitem a adoção de estratégias direcionadas de prevenção e rastreamento capazes de diminuir morbidade e mortalidade. O objetivo do presente estudo foi avaliar a proporção de pacientes com câncer de mama atendidos no Hospital Universitário de Santa Maria (HUSM) que necessitariam ser encaminhados para avaliação genética. Métodos: Pacientes com câncer de mama que iniciaram tratamento oncológico no HUSM durante o ano de 2014 foram considerados elegíveis. Uma entrevista foi conduzida com cada paciente para coleta de dados e exame físico dirigido. O questionário FSH-7 (Family Story Screening 7) e os critérios do NCCN (National Comprehensive Cancer Network) foram utilizados para identificar os pacientes que deveriam ser encaminhados para avaliação genética. Estes pacientes foram então avaliados quanto à indicação de teste genético - de acordo com as recomendações do NCCN para teste genético – e à probabilidade de mutações nos genes BRCA1 e BRCA2 através de modelos de predição de risco (BOADICEA, Penn II, sistema de escore de Manchester e tabelas da Myriad). Resultados: Dentre os 114 participantes do estudo, 65 (57%) preenchiam critérios de encaminhamento para avaliação genética de acordo com as diretrizes do NCCN. O questionário FHS-7 apresentou uma sensibilidade de 90% para identificar estes pacientes, com uma especificidade de 85%. A presença de história pessoal ou familiar de câncer de mama antes dos 50 anos foi o critério mais comum para indicar avaliação genética. Em relação aos testes genéticos, 52 pacientes (45%) deveriam ser testados para mutações nos genes BRCA1 e BRCA2 e 4 pacientes (3,5%) possuíam indicação de teste para mutações em TP53, de acordo com as recomendações do NCCN. Utilizando os modelos de predição de risco, 10,2% a 57,1% dos pacientes apresentavam uma probabilidade ≥ 10% de mutações em BRCA1 ou BRCA2. Conclusão: Este estudo revelou que a maioria dos pacientes com câncer de mama atendidos no HUSM possui indicação de encaminhamento para avaliação genética. A utilização de um questionário simples e rápido poderia identificar 90% destes pacientes.
Objective: Up to 10% of breast cancers are associated with a hereditary cancer syndrome. The identification of possible carriers of these syndromes and the subsequent referral for genetic counselling allow the adoption of tailored screening and prevention strategies capable of reducing morbidity and mortality. The aim of this study is to assess the proportion of patients with breast cancer treated at the University Hospital of Santa Maria (HUSM) that would need to be referred for genetic evaluation. Methods: Breast cancer patients who began cancer treatment at HUSM during the year 2014 were eligible. An interview was conducted with each patient for data collection and targeted physical examination. The FSH-7 (Family Story Screening 7) questionnaire and the NCCN (National Comprehensive Cancer Network) criteria were used to identify patients who should be referred for genetic evaluation. Then these patients were assessed for genetic testing criteria - according to the NCCN recommendations for genetic testing - and the likelihood of BRCA1 and BRCA2 mutations through risk prediction models (BOADICEA, Penn II, Manchester score system and Myriad tables). Results: Among the 114 study participants, 65 (57%) meet referral criteria for genetic evaluation according to the NCCN guidelines. The FHS-7 questionnaire showed a sensitivity of 90% to identify such patients with a specificity of 85%. The presence of personal or family history of breast cancer before age 50 was the most common criteria to indicate genetic evaluation. With respect to genetic testing, 52 patients (45%) should be tested for BRCA1 and BRCA2 mutations and 4 patients (3.5%) had test indication for TP53 mutations in accordance with the recommendations of the NCCN. Using risk prediction models, 10.2% to 57.1% of patients had a BRCA1 or BRCA2 mutations probability ≥ 10%. Conclusion: This study showed that most of the patients with breast cancer treated at HUSM have referral indication for genetic evaluation. The use of a fast and simple questionnaire could identify 90% of these patients.

Le gène TP53 occupe la première place parmi les gènes fréquemment mutés dans les cancers humains. Il est responsable de la production du facteur de transcription p53, également appelée “gardien du génome”, avec des fonctions cellulaires diverses et des régulations complexes, largement reconnu aujourd'hui pour son rôle dans la suppression tumorale. Ma thèse s'est concentrée sur l’étude et la caractérisation de la mutation p53Y217C chez la souris (correspondant à p53Y220C chez l’homme). La plupart des mutations de TP53 affectent le domaine de liaison à l'ADN de la protéine et se trouvent généralement au niveau de sept “hotspot” mutationnels. Parmi celles-ci, la mutation Y220C est particulièrement intéressante, étant présente dans 100 000 nouveaux cas de cancer par an et dans 15 familles Li-Fraumeni prédisposées au cancer. Pour étudier cette mutation in vivo, on a développé un modèle murin exprimant une protéine p53 avec la mutation Y217C, équivalente à la Y220C chez l'homme. La caractérisation phénotypique de cette souris a révélé que la mutation non seulement désactive la fonction de la protéine, mais semble également conférer de nouvelles fonctions tératogénique et oncogénique
The TP53 gene is the most frequently mutated gene in human cancers. It is responsible for the production of the transcription factor p53, also known as the 'guardian of the genome', with diverse cellular functions and complex regulations, widely recognized today for its role in tumor suppression. My thesis focused on the study and characterization of the p53Y217C mutation in mice (corresponding to p53Y220C in humans). Most TP53 mutations affect the DNA-binding domain of the protein and are generally found at seven mutational hotspots. Of these, the Y220C mutation is of particular interest, being present in 100,000 new cases of cancer per year and in 15 cancer-prone Li-Fraumeni families. To study this mutation in vivo, we developed a mouse model expressing a p53 protein with the Y217C mutation, equivalent to Y220C in humans. Phenotypic characterization of this mouse revealed that the mutation not only deactivates the function of the protein, but also appears to confer new teratogenic and oncogenic functions

A síndrome de Li-Fraumeni (LFS) e sua variante like (LFL) são associadas a mutações germinativas no gene TP53 e predispõe ao alto risco para múltiplos tumores em idade jovem. Analisamos 91 famílias LFS/LFL do sul/sudeste do Brasil para mutações germinativas e haplótipos de TP53 (PIN2, PIN3 e PEX4) e MDM2 (309T-G). A mutação R337H ocorreu em 44,4% das famílias avaliadas. Em 750 controles da região a freqüência populacional da mutação foi 0,3%. A genotipagem de oito indivíduos não relacionados R337H-positivos para 29 TAG SNPs intragênicos demonstrou o mesmo haplótipo raro estabelecendo efeito fundador para R337H. O alelo duplicado no PIN3 apresenta impacto modificador e retardo de 17,1 anos na ocorrência de tumores em famílias com mutação no TP53, enquanto o SNP309 MDM2 modula a idade dos sarcomas de partes moles.
Li-Fraumeni syndrome (LFS) and its variant like (LFL) are associated with germline mutations in the TP53 gene and predispose to a variety of cancers at an earlier age. We analyzed 91 LFS/LFL families from southern Brazil for germline mutations in TP53 and polymorphisms in TP53 (PIN2, PIN3, PEX4) and MDM2 (309T-G). The germline TP53 mutation R337H was found in 44.4% of all families included. In 750 controls from the same region, mutation prevalence was 0.3%. Genotyping of eight unrelated R337H-positive individuals for 29 intragenic TAG SNPs showed that they all shared the same rare haplotype confirming the founder effect for the mutation. Duplication of PIN3 had a modifier effect on the age of tumor onset (delay of 17.1 years) in TP53 mutation carriers whereas MDM2 SNP309 modulated age of onset for soft-tissue sarcomas.

Introdução: Estudos prévios têm indicado que uma mutação germinativa específica no gene TP53, p.R337H está associada a efeito fundador no Brasil e é muito frequente entre certos tipos tumorais. Objetivos e Metodologia: O objetivo deste estudo foi verificar a prevalência da mutação germinativa TP53 p.R337H em indivíduos com tumores do espectro da Síndrome de Li-Fraumeni (SLF) e/ou Li- Fraumeni-Like (LFL) divididos em diferentes grupos: (1) mulheres com câncer de mama (CM) subdivididas em 2 subgrupos: (a) com critérios para síndromes de predisposição hereditária ao câncer, exceto para SLF/LFL; (b) não selecionadas para história familiar (HF), diagnosticadas antes dos 45 e após 55 anos de idade em diferentes regiões do país; (2) mulheres com tumores phyllodes da mama, diagnosticadas em diferentes idades e não selecionadas para HF da doença nas regiões Sul e Sudeste; (3) crianças com tumores do espectro da SLF/LFL, não selecionadas para HF e atendidas em um Serviço de Oncologia Pediátrica do Sul do país. Adicionalmente, foi investigada a presença do haplótipo fundador brasileiro nos portadores da mutação TP53 p.R337H. No grupo 3 foi ainda avaliada a prevalência de critérios para SLF/LFL e a presença de outras mutações germinativas no gene TP53. Resultados: No subgrupo 1A foram incluídas 59 mulheres das quais 2 eram portadoras da mutação TP53 p.R337H, ambas com HF exclusivamente para a Síndrome de Prediposição Hereditária ao Câncer de Mama e Ovário no momento do recrutamento. No subrupo 1B foram incluídas 815 mulheres (403 com CM N45 anos e 412 casos com CM O 55 anos) provenientes de 25 diferentes Estados do Brasil. A mutação foi encontrada em 8,6% (70/815) dos casos, sendo observada diferença significativa na frequência de acordo com o centro de recrutamento. Em todos centros, a mutação foi mais frequente em mulheres com CM antes dos 45 anos. Análise do tecido tumoral de 15 portadoras evidenciou perda de heterozigosidade (LOH) em apenas 2 casos. Os tumores de mama de mulheres portadoras apresentavam mais frequentemente alguma expressão de HER2 (1+, 2+ ou 3+). No grupo 2, foram incluídas 148 mulheres com tumores phyllodes da mama e 8 (5,4%) apresentaram a mutação, sendo mais frequente entre os tumores malignos (3/13; 23%) quando comparada aos benignos (5/128; 3.4%). No grupo 3 foram incluídas 292 crianças diagnosticadas com tumores do espectro da SLF/LFL, das quais 25,3% tinham critérios para SLF/LFL. O sequenciamento completo e pesquisa de rearranjos gênicos de TP53 em 48 das crianças com câncer e critérios estritos para LFL evidenciou apenas um caso com uma mutação clássica – p.G245S. A mutação TP53 p.R337H foi identificada somente entre os casos com carcinoma adrenocortical (9/11; 81,8%) e de plexo coróide (2/2; 100%), correspondendo a 3,8% da amostra total. Um dos casos de carcinoma adrenocortical apresentou a mutação TP53 p.R337H em homozigose. Todos tumores de portadores analisados (n=8) apresentaram LOH. Conclusão: Conclui-se que a mutação germinativa TP53 p.R337H ocorre em mulheres brasileiras com CM em diferentes faixas etárias, independente da HF de câncer. Em mulheres com fenótipo de CM hereditário sem critérios para SLF/LFL a mesma também foi observada. A freqüência da mutação foi variável de acordo com o centro de recrutamento, sendo identificada em todas as regiões brasileiras. Os tumores de mama de pacientes portadoras da mutação têm um perfil peculiar, com baixa frequência de LOH e presença de alguma expressão de HER2. A mutação p.R337H ocorre em tumores phyllodes da mama benignos e especialmente malignos, confirmando a hipótese prévia de associação de mutações germinativas em TP53 com estes tumores. Entre crianças com tumores do espectro SLF/LFL de um centro de referência no Sul do Brasil, um percentual significativo tem indicação para teste de mutações em TP53, no entanto, na maioria destes casos não se identifica mutação germinativa no gene. A mutação TP53 p.R337H ocorre em elevada frequência na linhagem germinativa de crianças com carcinomas adrenocortical e de plexo coróide, como descrito em outras regiões brasileiras. Por fim, o haplótipo fundador foi identificado em todos portadores da mutação analisados, incluindo 3 casos das regiões Norte, Nordeste e Centro-Oeste do país. A mutação TP53 p.R337H está associada a efeito fundador no Brasil e tem importante contribuição na gênese de tumores de mama, bem como de carcinomas adrenocortical e de plexo coróide no Sul do país.
Introduction: Previous studies have reported that a specific germline mutation in TP53, p.R337H, is associated with a founder effect and is common among certain tumor types. Objectives and Methodology: Assess the prevalence of TP53 p.R337H germline mutation in subjects with tumors of the Li-Fraumeni/ Li-Fraumeni- Like (LFS/LFL) spectrum divided into different groups: (1) women diagnosed with breast cancer (BC) subdivided into 2 subgroups: (a) with criteria for hereditary predisposition syndromes, except for LFS/LFL (b) unselected for family history (FH) diagnosed at or before 45 years and at or after 55 years recruited in different regions of the country; (2) women with phyllodes breast tumors, diagnosed at different ages and not selected for FH of the disease from the South and Southeast of Brazil; (3) children with tumors of the LFS/LFL spectrum and not selected for FH of the disease from a Pediatric Oncology Service in South Brazil. In addition, presence of the founder Brazilian haplotype in mutation carriers identified in groups 1, 2 and 3 was assessed. Finally, in group 3, prevalence of LFS/LFL criteria and presence of other germline TP53 mutations were investigated. Results: In subgroup 1A, 59 women were included and two of these were mutation carriers, both had a FH consistent only with Hereditary Breast and Ovarian Cancer Syndrome at recruitment. In subgroup 1B, 815 women (403 cases diagnosed N 45 years and 412 cases, O 55 years) were included from 25 different Brazilian States. The TP53 p.R377H mutation was found in 8.6% (70/815), and significant differences in mutation frequency were observed according to recruitment center. In all centers the mutation was more frequent in women diagnosed with BC at or before 45 years. Analysis of tumor tissue of 15 carriers showed loss of heterozygosity (LOH) in only 2 cases. Breast tumors of carriers showed, more frequently, some expression of HER2. In group 2 were included 148 women with breast phyllodes tumors and 8 (5.4%) were p.R337H carriers, which was most common among malignant phyllodes tumors (3/13, 23%) when compared to benign (5/128, 3.4%). In the group 3, 292 children with tumors of the LFS/LFL spectrum were included, and 25.3% of these had criteria for LFS/LFL. TP53 gene sequencing and rearrangement testing in 48 children with the more strict criteria for the syndrome identified a classic germline mutation, p.G245S, in only one proband. The TP53 p.R337H mutation was identified only among patients with adrenocortical carcinoma (9/11, 81.8%) and choroid plexus carcinoma (2/2, 100%), corresponding to 3.8% of the total sample. One of the patients with adrenocortical carcinoma was homozygous mutant. All tumors of TP53 p.R37H carriers analyzed (n=8) showed LOH. Conclusion: We conclude that the TP53 p.R337H germline mutation occurs in Brazilian women with BC in different age groups, regardless of BC FH. In women with hereditary BC without criteria for LFS/LFL criteria, it was also observed. The mutation frequency varied according to the recruiting center and carriers were identified in all Brazilian regions. Breast tumors of carriers have a peculiar profile, with low frequency of LOH and the presence of HER2 expression. The mutation was present, also, in benign and especially malignant phyllodes tumors, confirming the previous hypothesis of an association between germline mutations in the TP53 gene and these specific tumors. Among children with tumors of the LFS/LFL spectrum from a Oncopediatrics referral center in South Brazil, a significant percentage fulfills criteria for TP53 mutation testing, however, in most of these cases, no germline mutations were identified. The TP53 p.R337H mutation occurs in high frequency in the germline of children with adrenocortical and choroid plexus carcinomas, as described in other Brazilian regions. Finally the founder haplotype was identified in all mutation carriers analyzed, including 3 cases from the Northern, Northeastern and Midwestern Brazilian regions. The TP53 p.R337H mutation is associated with a founder effect in Brazil and has an important contribution in the genesis of breast tumors in the country, as well as adrenocortical and choroid plexus carcinomas in the Southern region of the country.

Le syndrome de Li-Fraumeni (LFS) représente l’une des formes mendéliennes de cancer les plus graves, caractérisée par une forte hétérogénéité phénotypique tant au niveau du spectre tumoral que de l’âge de survenue des tumeurs et définie au niveau moléculaire par une altération constitutionnelle du gène suppresseur de tumeurs TP53, détectée dans 15 % des familles évocatrices du LFS. Les objectifs de cette thèse étaient doubles. D’une part, afin de comprendre les bases moléculaires de l'hétérogénéité phénotypique du LFS, nous avons étudié les conséquences fonctionnelles des différentes classes de mutations de TP53, dans le contexte génétique des patients via le développement d’un essai fonctionnel ex vivo de la voie p53 basé sur la caractérisation du profil transcriptomique des lymphocytes immortalisés de patients porteurs d’une mutation hétérozygote de TP53 en réponse à un stress génotoxique. Dans les lymphocytes de 3 témoins, nous avons identifié 173 gènes dont l'expression est induite plus de 2 fois, dont 46 gènes cibles connus de p53. Dans les lymphocytes de 3 patients atteints du LFS, porteurs de mutations faux-sens canoniques (p. Arg175His, p. Arg248Trp, p. Arg273His), le nombre de gènes induits et le niveau d’induction des gènes cibles connus de p53 ont été fortement réduits par rapport aux 3 témoins et à 3 patients porteurs de mutations nulles. Ces résultats montrent que certaines mutations constitutionnelles faux-sens de TP53 associées à un effet transdominant-négatif modifient considérablement la réponse aux lésions de l’ADN, ce qui explique probablement pourquoi ces mutations sont retrouvées de façon majoritaire dans le LFS. D’autre part, nous avons étudié l'hétérogénéité génétique des patients évocateurs du LFS sans altération détectable de TP53 en recherchant de nouvelles bases moléculaires via le développement d’une puce à ADN pangénomique, hautement résolutive dans des régions génomiques d’intérêt, afin d’évaluer la contribution de variations rares du nombre de copies d’ADN (CNV) dans le déterminisme génétique du LFS. Nous avons détecté chez 15 patients, 20 nouveaux CNV touchant des régions codantes et absents chez 600 témoins. De façon remarquable, chez 4 de ces patients ayant développé une tumeur cérébrale, les CNV détectés touchent des gènes impliqués dans le remodelage de la chromatine (KDM1A, MTA3, TRRAP ou SIRT3). Nous avons focalisé nos analyses sur la duplication touchant SIRT3 et montré qu’elle entraîne une surexpression de SIRT3 chez les patients et que, in vitro, cette surexpression protège de l’apoptose, augmente la phase G2/M du cycle cellulaire et provoque un défaut d’induction de gènes induits en réponse à un stress génotoxique et une hyperméthylation de gènes impliqués dans le cancer. Ces résultats sont en faveur du rôle délétère d’altérations constitutionnelles touchant des gènes impliqués dans le remodelage de la chromatine dans les prédispositions génétiques au cancer et en particulier aux tumeurs cérébrales

Le syndrome de Li-Fraumeni (LFS), qui résulte d’altérations constitutionnelles hétérozygotes du gène TP53, représente l’une des formes héréditaires de cancer les plus graves. Afin de déterminer les bases moléculaires du gradient de sévérité clinique des mutations de TP53, nous avons étudié les conséquences fonctionnelles des différentes classes de mutation de TP53 dans le contexte génétique des patients LFS, et nous avons montré que les mutations faux-sens de TP53 associées à un effet dominant négatif altèrent drastiquement la réponse de p53 aux lésions de l’ADN en comparaison avec les mutations nulles, dans les cellules non tumorales des patients LFS, définissant ainsi un véritable endophénotype de gravité. Grâce au développement d’une version simplifiée de l’essai fonctionnel, nous avons pu confirmer ces observations. L’analyse de ChIP-Seq, que nous avons mise au point, nous a permis de montrer que l’altération drastique de la réponse transcriptionnelle de p53 aux lésions de l’ADN dans les lymphocytes de patients LFS porteurs d’une mutation faux-sens à effet dominant négatif résulte d’une altération de fixation de p53 sur l’ADN. Afin de montrer la contribution des chimiothérapies dans la survenue des cancers primitifs multiples dans le LFS, nous avons développé un nouveau test de génotoxicité nommé p53 genotoxicity assay, et nous avons montré que toutes les classes de chimiothérapies que nous avons testées, à l’exception des poisons du fuseau mitotique, sont fortement génotoxiques. Ainsi, l’ensemble de nos résultats montrent que les mutations de TP53 agissent comme des mutations permissives permettant aux stimuli oncogéniques de conduire à une transformation maligne
Li-Fraumeni Syndrome (LFS), resulting from heterozygous germline mutations of TP53, is one of the most severe hereditary cancer syndromes. In order to determine the molecular basis of the clinical gradient of germline TP53 mutations, we studied the functional consequences of the different types of TP53 mutations in the genetic context of the patients, and we showed that TP53 missense mutations with dominant-negative effect alter the p53 transcriptional response to DNA damage more drastically than null mutations. These results indicate that the impact of the mutations on p53 transcriptional response to DNA damage in LFS lymphocytes can be considered as an endophenotype of the clinical severity of germline TP53 mutations. The use of the simple p53 functional assay allowed us to confirm these observations on a large number of mutations. ChIP-Seq analysis performed on lymphocytes derived from TP53 wild-type control subject and LFS patient with TP53 dominant-negative missense, showed that the drastic alteration of p53 transcriptional response to DNA in LFS lymphocytes harboring dominant negative missense mutations, is explained by a massive and global alteration of p53 DNA binding. In order to determine the causative role of chemotherapies in the appearance of secondary tumours in LFS, we developed a new genotoxicity assay, named the p53 genotoxicity assay. This assay allowed us to show that most of the drugs commonly used in cancer treatment, except the microtubule poisons, are highly genotoxic. Thus, in TP53 mutation carriers, germline TP53 mutations represent a genetic permissive context facilitating the malignant transformation of cells in which DNA damage has occurred

Le gène suppresseur de tumeurs TP53 exprime plusieurs isoformes, dont Δ40p53 (perte du domaine de transactivation) et Δ133p53 (perte du domaine de transactivation et d’une partie du domaine de liaison à l’ADN). Ces isoformes inhibent l’activité suppressive de p53 et seraient sur-exprimées dans les cancers (sein et mélanome). Dans les cancers faiblement associés à une mutation TP53, ces isoformes seraient de bons candidats pour inactiver p53. Il convient de comprendre les mécanismes transcriptionnels qui régulent leurs expressions. Δ133p53 est produite par un promoteur alternatif P3 localisé dans TP53. Nous avons montré que Δ133p53 est un gène cible de p53, qui transactive le promoteur P3 par fixation sur un élément de réponse présent dans l’exon 4. L’expression de Δ133p53 est corrélée à celle d’autres gènes cibles de p53 en réponse à un stress génotoxique. De plus, elle réprime la suppression de la prolifération induite par p53 en inhibant ses capacités de liaison à l’ADN. Δ40p53 est produite par épissage alternatif, dont la rétention de l’intron 2 favorise sa traduction et empêche celle de p53. Nous avons montré que des structures de type G-quadruplexes présentes dans l’intron 3 régulent l’exclusion de l’intron 2. Ces structures comprennent le polymorphisme TP53PIN3 (duplication de 16pb), qui change leur localisation et affecte l’expression des ARNm codant p53 et Δ40p53. De plus, nous avons montré que ce polymorphisme est associé à une accélération de la cancérogenèse dans le syndrome Li-Fraumeni, caractérisé par la présence d’une mutation germinale TP53 (effet modificateur: 19 ans de différence à l’âge moyen du premier diagnostique entre les deux variants). L’expression des isoformes de p53 dépend de mécanismes transcriptionnels différents, indiquant des rôles différents dans la modulation des fonctions suppressives de p53. En plus d’inactiver p53 dans les cancers, ces isoformes pourraient être à l’origine des effets modificateurs des polymorphismes de TP53 sur les mutants p53
The TP53 tumour suppressor gene expresses several isoforms, of which Δ40p53 (lack of transactivation domain) and Δ133p53 (lack of both transactivation and part of DNA-binding domains). These isoforms inhibit p53 suppressive activity and have been shown to be over-expressed in cancers (breat and melanoma). In cancers associated with low TP53 mutation rate, these isoforms could be great candidates to inactivate p53. It seems important to understand the transcriptional mechanisms that regulate their expression. Δ133p53 is produced by an alternative P3 promoter within TP53. We showed that Δ133p53 is a p53 target gene. p53 transactivates the P3 promoter and interact with a response element within exon 4. Δ133p53 expression is correlated to other p53 target genes in response to genotoxic stress. In addition, Δ133p53 inhibits p53-dependent suppression of proliferation by inhibiting p53 DNA-binding activity. Δ40p53 is produced by alternative splicing: retention of intron 2 favours its translation while it avoid the one of p53. We showed that G-quadruplex structures are formed in intron 3 and regulate retention of intron 2. The TP53PIN3 polymorphism (16 bp duplication) is embedded within these structures and affects their locations leading to variation of mRNA expression of p53 and Δ40p53. In addition, we showed that this polymorphism is associated with acceleration of carcinogenesis in Li-Fraumeni syndrome, characterized by germline TP53 mutation (genetic modifier effect: difference of 19 years in mean age at first diagnosis of cancer between the two variants). The expression of p53 isoforms depends on different transcriptional mechanisms, suggesting different roles in the modulation of p53 suppressive functions. In addition to inactivate p53 in cancers, these isoforms could be the mediators of modifier effects observed for TP53 polymorphisms on mutant p53

Il existe dans la littérature une grande quantité de données épidémiologiques concernant l'effet des radiations ionisantes sur la santé humaine en particulier sur le développement de tumeurs solides. Des études ont notamment porté sur la fréquence et la nature des tumeurs secondaires est généralement très faible (environ 1%) mais il peut augmenter en cas de prédisposition génétique à certains cancers pour les patients porteurs d'une mutation germinale inactivante d'un des allèles par la radiothérapie. Le risque de développer une tumeur dans le champ d'irradiation est très grand. A côté des données épidémiologiques et cliniques, il existe toutefois peu de données cytogénétiques et moléculaires précises mettant en relation l'effet des radiations et le développement de tumeurs humaines. Les caryotypes de quelques cancers apparus après radiothérapie sont généralement très réarranges avec de nombreuses délétions et des pertes de chromosomes. Jusqu’à présent, aucune caractéristique moléculaire spécifique des tumeurs radio-induites n’a été mise en évidence. Afin de pouvoir disposer de tumeurs dont la nature radio-induite était bien établie, nous nous sommes intéressés à des tumeurs apparues après le traitement d’enfants atteints de rétinoblastome bilatéral. En nous plaçant dans un contexte génétique de prédisposition précis nous espérons définir des voies et des mécanismes spécifiques de la radio-induction. L’étude a été menée sur 7 tumeurs radio-induites. L’analyse cytogénétique et moléculaire de ces tumeurs a permis de définir un certain nombre de caractéristiques. Le génome est pulvérisé de nombreux chromosomes. De plus, on observe plus de pertes que de gains de matériel génétique. Par ailleurs, les séquences microsatellites et minisatellites sont stables. Enfin, les deux allèles des gènes TP53 et RB1 sont inactives dans toutes les tumeurs analysées. Pour chacun des gènes, un allèle est perdu avec une partie ou la totalité du chromosome qui le porte, tandis que l’autre allèle porte la mutation germinale (RB1) ou une mutation somatique (TP53). Les 7 mutations (somatiques) de TP53 observées dans cette série de tumeurs radio-induites apparaissent inhabituelles lorsqu’elles sont comparées à celles retrouvées dans des tumeurs spontanées. Ainsi, les substitutions et les délétions d’une seule base représentent la quasi-totalité (95%) des mutations observées dans les cancers spontanés. Bien que notre échantillon soit petit, le fait d’observer 3 délétions qui touchent plus d’une base sur 7 mutations est très inhabituel. Ceci suggère fortement qu’un mécanisme particulier de mutagenèse soit intervenu. Le statut d’une série de gènes impliqués dans la réparation non-homologue des cassures double-brin de l’ADN a également été analysé dans 5 des 7 tumeurs radio-induites. Des pertes alléliques récurrentes ont été mises en évidence pour 7 des 8 gènes analysés (KU70, KU80, XRCC4, LigaseIV, MRE11, RAD50, NBS1) mais pas pour DNA-PKcs. Aucune mutation de l’autre allèle de chacun de ces gènes n’a été retrouvée. L’haplo-insuffisance dans ces gènes de éparation suggère qu’une diminution de la capacité à réparer ces tumeurs radio-induites fortement réarrangées.

Le gène TP53 est extrêmement polymorphique avec 85 polymorphismes décrits. Certains de ces polymorphismes sont associés à une augmentation du risque de cancer, par exemple rs10425222 peut moduler les fonctions de p53. Cependant, pour d’autres, comme le rs17878362 qui est le polymorphisme intronique le plus étudié, leur association avec une augmentation du riques au cancer est controversée.Pour analyser l’association entre le polymorphisme rs17878362 et la susceptibilité au cancer, nous avons analysé son rôle dans des contextes de cancers sporadiques et familiaux. Les résultats obtenus pour le polymorphisme rs17878362 sont paradoxaux avec une augmentation des cancers sporadiques associée avec le génotype A2A2 alors que l’allèle A2 est associé avec un effet « protectif » chez les patients atteints du syndrome de Li-Fraumeni porteurs d’une mutation germinale de TP53 situé sur l’haplotype A1. Ces observations suggèrent que des haplotypes spécifiques de TP53 pourraient moduler les capacités suppressives de p53. Une hypothèse possible est que les différents haplotypes de TP53 présenteraienrt des mutations somatiques à des fréquences différentes dans la population.De plus, le gène TP53 exprime différentes isoformes, comme le D40p53, inhibant l’activité suppressive de p53. Le D40p53 peut être produite par le maintien de l’intron 2 par épissage alternatif. Nous avons montré que les G-quadruplexes, des structures tridimensionnelles formées dans des régions riches en G, sont formés dans l’intron 3 et régulent la rétention de l’intron 2 et la formation du transcrit p53I2. Nous avons aussi observé que le polymorphisme rs1652785 (localisé dans l’intron 2) semble réguler la stabilité du p53I2. Ces résultats suggèrent que les polymorphismes de TP53 localisés dans une région de 412 pb située entre l’exon 2 et l’exon 4 régulent l’expression des isoformes de p53 dans une séquence temporelle d’évènements en modulant la formation des pré-ARNm (rs17878362), la stabilité des ARNm (rs1642785) et les fonctions protéiques (rs10425222).L’expression des isoformes de p53 est donc finement régulée par des mécanismes impliquant les polymorphismes de TP53 qui sont aussi associés avec une altération dans la susceptibilité au cancer
The TP53 gene is a highly polymorphic gene with 85 polymorphisms described. Some of these have been associated with an increase of cancer susceptibility, for example rs10425222 that can modulate certain p53 activities. However for others such as rs17878362, the most studied intronic polymorphism, the association with cancer risk is more controversial. To investigate the influence of rs17878362 on cancer susceptibility, we analysed its role in sporadic and familial contexts. The results are paradoxical with an increase of sporadic cancer associated with the rs17878362 A2A2 genotype whereas the rs17878362 A2 allele is associated with a “protective” effect in the context of Li-Fraumeni patients carrying a TP53 germline mutation on an A1 haplotype. These observations suggest that specific TP53 haplotypes could modulate p53’s tumour suppression capacities. A possible hypothesis to explain this could be that somatic mutations are carried on different haplotypes of TP53 present at different allele frequencies in the population. In addition, TP53 is expressed as several protein isoforms, such as D40p53, which inhibits p53’s suppressive activity. D40p53 can be produced from an alternative spliced transcript that retains intron 2. We have shown that G-quadruplexes, tri-dimensional structures formed in G-rich sequences, are formed in intron 3 and regulate the retention of intron 2 and the formation of the p53I2 transcript. We also observed that rs1642785 (located in intron 2) could regulate p53I2’s stability. These results suggest that the TP53 polymorphisms located in a 412 bp region located between exon 2 and exon 4 regulate the expression of p53 isoforms in a temporal sequence of events by modulating the pre-mRNA formation (rs17878362), mRNA stability (rs1642785) and protein functions (rs1042522).p53 isoforms’ expression is thus finely regulated by mechanisms involving TP53 polymorphisms, which are also associated with altered cancer susceptibility

L’identification des mutations constitutionnelles à l’origine d’une prédisposition génétique au cancer est essentielle à la prise en charge médicale des patients et de leurs familles. Depuis l’implémentation des technologies de séquençage à haut-débit dans les laboratoires diagnostiques, le principal défi n’est plus la détection des variations génétiques mais leur interprétation et leur classification. La question de l’interprétation de la variation est particulièrement cruciale lorsqu’elle conditionne la stratégie thérapeutique. Ainsi, il est essentiel de disposer de tests simples adaptables en routine diagnostique pour faciliter l’interprétation des variations génétiques. Dans ce contexte, nous avons utilisé un test fonctionnel développé par notre équipe pour classer des variations dans le gène TP53 à l’origine du syndrome de Li-Fraumeni et pour appréhender la corrélation génotype - phénotype chez les patients LFS. Dans un deuxième temps, nous avons évalué la pertinence d’une approche multi-omique (RNA-Seq et métabolomique) pour discriminer les cellules sauvages des cellules avec mutation hétérozygote du gène TP53 ou des gènes BRCA impliqués dans la prédisposition génétique aux cancers du sein et de l’ovaire. Sur la base des données de transcriptome, un modèle mathématique a été développé pour détecter les variants correspondant à des mutations délétères. Nous avons ensuite sélectionné les biomarqueurs les plus discriminants pour les intégrer dans un test fonctionnel de RT-MLPA dédié à la voie p53. Nous avons enfin adapté cet essai pour qu’il soit réalisable sur une simple prise de sang, sans immortalisation des lymphocytes du patient
The identification of the constitutional mutation responsible for a genetic predisposition to cancer is essential to the clinical management of the patient and its relatives. With the implementation of high-throughput sequencing to the diagnostic routine of these pathologies, the challenge no longer lies within the detection of alterations but in their biological and clinical interpretation. While specific treatments are emerging, simple functional assays to help with the interpretation of the detected variants are needed. In this context, we used a functional test developed by our team to classify variations in the TP53 gene responsible for Li-Fraumeni syndrome and to understand the genotype-phenotype correlation in LFS patients. On the other hand, we assessed the relevance of a multi-omic approach (RNA-Seq and metabolomics) to discriminate wild-type cells from cells with a deleterious heterozygous mutation in TP53 or in the BRCA genes implicated in genetic predisposition to breast and ovarian cancers. Based on the transcriptomic data, a mathematical model has been developed to detect variants corresponding to deleterious mutations. Then we selected the most discriminating biomarkers and integrated them into a RT-MLPA functional assay dedicated to the p53 pathway. We finally adapted this test to be feasible on a simple blood test, without immortalization of the patient's lymphocytes

Li-Fraumeni Syndrome (LFS) is a rare cancer predisposition syndrome, typically involving germline mutations in the TP53 gene. Despite the high penetrance of TP53 mutations, LFS patients display striking phenotypic differences, suggesting the presence of secondary risk loci. To date, all genetic modifiers in LFS have been shown to map to either TP53 or its principal negative regulator, Mdm2. Given this strong association, we set out to interrogate the contribution of a recently-described miRNA regulator of the p53-MDM2 loop, called miR-605. We hypothesized that, if functional, the miR-605 gene and its variant (rs2043556) could impact cancer risk in TP53 mutation carriers. Consistent with this proposition, the variant allele of miR-605 was associated with a significant acceleration in tumor onset and caused a decrease in the processing efficiency of its host miRNA. We also demonstrate that miR-605 overexpression activates the MAPK pathway and leads to tumor suppression in TP53 mutant cell lines.

Copy number variations (CNVs) are a major source of inter-individual genetic difference, accounting for a greater proportion of the human genome than other forms of variation. Recently, the identification of benign and pathogenic CNVs has improved due to arrays with increased coverage. Nevertheless, most CNVs have not been studied with great precision and questions persist regarding their exact breakpoint, gene content, frequency and functional impact. This is especially true in cancer, in which a role for CNVs as risk factors is under-explored. Li-Fraumeni syndrome (LFS) is a dominantly inherited disorder with an increased risk of early-onset breast cancer, sarcomas, brain tumors and other neoplasms in individuals harboring germline TP53 mutations. Known genetic determinants of LFS do not fully explain its clinical phenotype. In this thesis we describe the association between CNVs and LFS. First, by examining DNA from a healthy population and an LFS cohort using oligonucleotide arrays, we show that the number of CNVs per genome is well conserved in the healthy population, but remarkably enriched in these cancer-prone individuals. We found a significant increase in CNVs among carriers of germline TP53 mutations with a familial cancer history. Second, we find that ii specific CNVs at 17p13.1 are associated with LFS or developmental delay, depending on the exact breakpoint with respect to TP53. Using a purpose built array with 93.75% accuracy, we fine-mapped these microdeletions and find that they arise by Alu-mediated non-allelic homologous recombination, and contain common genes, whose under-expression distinguishes the two phenotypes. Third, we explore somatic CNVs in choroid plexus carcinoma tumor genomes. We show that this tumor is over-represented in LFS, and the number of somatic CNVs is associated with TP53 mutations and disease progression. These studies represent the first genomic analyses of LFS, and suggest a more generalized association between CNVs and cancer.

Indiana University-Purdue University Indianapolis (IUPUI)
Although rare, classification of over 200 hereditary cancer susceptibility syndromes accounting for ~5-10% of cancer incidence has enabled the discovery and understanding of cancer predisposition genes that are also frequently mutated in sporadic cancers. The need to prevent or delay invasive cancer can partly be addressed by characterization of cells derived from healthy individuals predisposed to cancer due to inherited "single-hits" in genes in order to develop patient-derived samples as preclinical models for mechanistic in vitro studies. Here, we present microarray-based transcriptome profiling of Li-Fraumeni syndrome (LFS) patient-derived unaffected breast epithelial cells and their phenotypic characterization as in vitro three-dimensional (3D) models to test pharmacological agents. In this study, the epithelial cells derived from the unaffected breast tissue of a LFS patient were cultured and progressed from non-neoplastic to a malignant stage by successive immortalization and transformation steps followed by growth in athymic mice. These cell lines exhibited distinct transcriptomic profiles and were readily distinguishable based upon their gene expression patterns, growth characteristics in monolayer and in vitro 3D cultures. Transcriptional changes in the epithelial-to-mesenchymal transition gene signature contributed to the unique phenotypes observed in 3D culture for each cell line of the progression series; the fully transformed LFS cells exhibited invasive processes in 3D culture with disorganized morphologies due to cell-cell miscommunication, as seen in breast cancer. Bioinformatics analysis of the deregulated genes and pathways showed inherent differences between these cell lines and targets for pharmacological agents. After treatment with small molecule APR-246 that restores normal function to mutant p53, we observed that the neoplastic LFS cells had reduced malignant invasive structure formation from 73% to 9%, as well as an observance of an increase in formation of well-organized structures in 3D culture (from 27% to 91%) by stereomicroscopy and confocal microscopy. Therefore, the use of well-characterized and physiologically relevant preclinical models in conjunction with transcriptomic profiling of high-risk patient derived samples as a renewable laboratory resource can potentially guide the development of safer and more effective chemopreventive approaches.

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014
Multiple and distinct primary intracranial tumors are known to occur in several well-defined hereditary tumor syndromes. In the absence of a tumor syndrome or previous irradiation, this situation is rare. We present the case of a patient with a secondary glioblastoma (SGBM) and a pituitary adenoma (PA). There are few reports of associated glioma and PA and no specific information over SGBMs. Some authors suggest these events might be a coincidence but we need more evidence to exclude any genetic association, especially in the presence of family history.
Tumores múltiplos e primários intracranianos ocorrem em síndromes hereditárias bem-definidas. Na ausência de síndrome tumoral ou irradiação prévia, a sua ocorrência é rara. É apresentado o caso clínico de uma doente com a associação de um glioblastoma secundário e de um adenoma pituitário. Existem poucas publicações que relatam a coexistência de glioma e adenoma pituitário e não existe informação específica sobre casos que incluam o subtipo glioblastoma secundário. Alguns autores admitem que estes fenómenos podem representar uma coincidência mas é necessária evidência para excluir qualquer associação genética, sobretudo na presença de história familiar.