Assessment of the p53 transcriptional activity : a way to predict the clinical and therapeutic responses in superficial transitional cell carcinoma of the bladder and in head and neck squamous cell carcinoma

La protéine suppresseur de tumeur p53 est une nucléo-phosphoprotéine essentielle à la stabilité de l’ADN. Elle agit principalement comme un facteur transcriptionnel, capable de trans-activer ou –réprimer l’expression de nombreux gènes cibles, ceux-ci étant impliqués dans des voies métaboliques variées, dont notamment l’arrêt du cycle cellulaire et l’apoptose. Actuellement, on estime qu’environ la moitié des cancers humains présente un p53 muté. Suite à ces mutations, la protéine p53 perd en général sa capacité à se fixer sur l’ADN, ce qui conduit à une résistance accrue à la radio- ou chimiothérapie. En conséquence, de nombreuses études ont essayé de déterminer la valeur prédictive clinique des mutations de p53 et/ou de sa surexpression. Cependant, étant donné le nombre de discordances rapportées dans la littérature ces dix dernières années, des études supplémentaires tentant d’établir une meilleure corrélation entre l’état du gène p53, les voies de réponse p53-dépendantes et l’évolution thérapeutique ou clinique semblent nécessaires. Le premier objectif de notre étude a été de déterminé l’état de la protéine p53 par une analyse fonctionnelle en levure (FASAY : functional analysis of separated alleles in yeast), ainsi que par une approche conventionnelle, l’immunohistochimie. Ces données ont ensuite été comparées et corrélées aux caractéristiques clinico-pathologiques et l’évolution clinique de patients présentant des tumeurs vésicales, ainsi qu’aux réponses cellulaires radio- et/ou chimio-induites de lignées cellulaires de la sphère ORL. Sur base de nos études ex vivo et in vitro, nous concluons que le FASAY présente de nombreux avantages, comparé à l’immunohistochimie ou autre analyse génotype. Le FASAY est une méthode de sélection rapide et reproductible. Elle permet une détection sensible et spécifique de mutants p53 transcriptionnellement inactifs tout en excluant les polymorphismes silencieux. De plus, un p53 non fonctionnel tel que déterminé par le FASAY a semblé être le meilleur marqueur prédictif de récurrence dans les tumeurs de la vessie, y compris dans le sous-groupe des tumeurs de stade a-1/grade 3, surpassant le stade ou grade tumoral ainsi que le marquage immunohistochimique. Un p53 non fonctionnel s’est également révélé être un marqueur prédictif fiable des réponses radio- et/ou chimio-induites dans les lignées cellulaires ORL. De façon intéressante, un effet antagoniste a été observé entre le docetaxel et l’irradiation pour chaque lignée cellulaire. Cet antagonisme semblait être influencé par l’état fonctionnel de p53, la lignée p53-sauvage montrant la plus importante réduction apoptotique. Il est à noter que cette combinaison pourrait avoir des conséquences nuisibles sur l’efficacité des thérapies cliniques. Le FASAY semble donc être un puissant test pour déterminer l’état de p53, lequel semble alors être une bonne valeur prédictive aussi bien pour l’évolution d’autres techniques, tels que les microdamiers à ADN qui permettront d’identifier des marqueurs biologiques prédictifs additionnels ainsi que d’investiguer les mécanismes moléculaires radiochimio-induits.Tumor suppressor p53 is a nuclear phosphoprotein essential for the DNA stability. It primarily acts as a transcriptional factor, able to trans-activate or -repress expression of multiple target genes. These downstream genes are implicated in various pathways, including the best-described cell-cycle arrest and apoptotic cell death. Currently, it is estimated than around half of all human cancers carry p53 mutations. These mutations generally induce a loss of binding to DNA and thus increased radiotherapy or chemotherapy resistance. Accordingly, multiple studies have attempted to assess the predictive value of detecting p53 mutations and/or overexpression in clinical settings. However, a large discrepancy reported over the last ten years justified repeated calls for a better correlation study between the p53 genetic status, p53 pathway and the therapeutic or clinical outcome. The first aim of our study was to determine the functional status of the p53 protein by a functional analysis of separated alleles in yeast (FASAY), as well as by a conventional approach, immunohistochemistry. These data were then compared and correlated with clinicopathologic features and the clinical outcome of bladder tumor, and also with radio- and/or chemo-induced cellular responses (cell cycle, apoptosis, gene expression) of head and neck squamous cell carcinoma (HNSCC) cell lines. Based on our ex vivo and in vitro studies, we conclude that there are many advantages of FASAY compared to immunohistochemistry or genotypic analysis. FASAY is a rapid, reproducible and robust screening assay. It permits a sensitive and specific detection of transcriptionally inactive p53 mutants while ruling out biologically silent polymorphisms. Moreover, a nonfunctional p53 determined by FASAY seemed to be the strongest predictor of recurrence in bladder tumor, including the subgroup of stage a-1/grade 3, superseding tumor grade and stage as well as p53 immunohistochemistry staining. Nonfunctional p53 also seemed to be a reliable predictor of radio- and/or chemo-induced cellular responses of HNSCC cell lines. Interestingly, an antagonistic effect between docetaxel and radiation was found within each cell line. This antagonism appeared to be influenced by a functional p53, the p53 wild-type cell line showing the higher apoptotic decrease. Note that such a combination may have deleterious consequences on the clinical therapeutic efficiency. FASAY thus seems to be a powerful assay to determine the p53 status, which by this method appeared the best predictive value so far of the clinical outcome and therapeutic responses. It may now be associated to other techniques, such as DNA microarrays that will allow to identify additional predictive biological markers and to further investigate the radiochemotherapeutic-induced molecular mechanisms.Thèse de doctorat en sciences biomédicales (oncologie)(ONCO) -- UCL, 200

Rapid combined genotyping of factor V, prothrombin and methylenetetrahydrofolate reductase single nucleotide polymorphisms using minor groove binding DNA oligonucleotides (MGB probes) and real-time polymerase chain reaction.

Risk factors for cardiovascular diseases and venous thromboembolism involve both acquired and hereditary conditions. Among the latter, mutations in genes coding for coagulation factors (factor V Leiden [Arg506Gly], G20210A in the 3'-untranslated region of factor II ) and variant C677T of the methylenetetrahydrofolate reductase (MTHFR ) are often involved and co-inherited. These three factors were genotyped simultaneously in the same 96-well plate, using a real-time polymerase chain reaction (PCR) Taqman assay and minor groove binding DNA oligonucleotides (MGB probes). While primers and MGB probes matched their corresponding single nucleotide polymorphism (SNP), the real-time MGB program was identical for each target gene. Homozygous wild-type (WT; -/-), heterozygous (+/-) or homozygous (+/+) variants (n = 362) were selected for factor V (n = 115, with -/-, 40; +/-, 40; +/+, 35), factor II (n = 122, with -/-, 60; +/-, 60; +/+, 2), and MTHFR (n = 120, with -/-, 40; +/-, 40; +/+, 40), according to the results of conventional PCR-restriction fragment length polymorphism (PCR-RFLP), but the allelic discrimination was performed blind. Results of the real-time MGB and PCR-RFLP assays were identical. This new assay was easy and fast with high throughput, without risk of molecular carryover, and cost-effective for laboratories utilizing the Taqman or related fluorescence reading methods. These advantages make it particularly suitable for large-scale combined genotyping of several polymorphisms in the routine setting

Evaluation of the correlation between KRAS mutated allele frequency and pathologist tumorous nuclei percentage assessment in colorectal cancer suggests a role for zygosity status.

Evaluation of molecular tumour heterogeneity relies on the tumorous nuclei percentage (TNP) assessment by a pathologist, which has been criticised for being inaccurate and suffering from interobserver variability. Based on the 'Big Bang theory' which states that KRAS mutation in colorectal cancer is mostly homogeneous, we investigated this issue by performing a critical analysis of the correlation of the KRAS mutant allele fraction with the TNP in 99 colorectal tumour samples with a positive KRAS mutation status as determined by next-generation sequencing. Our results yield indirect evidence that the KRAS zygosity status influences the correlation between these parameters and we show that a well-trained pathologist is indeed capable of accurately assessing TNP. Our findings indicate that tumour zygosity, a feature which has largely been neglected until now, should be taken into account in future studies on (colorectal) molecular tumour heterogeneity.status: publishe

Evaluation of the correlation between KRAS mutated allele frequency and pathologist tumorous nuclei percentage assessment in colorectal cancer suggests a role for zygosity status

Evaluation of molecular tumour heterogeneity relies on the tumorous nuclei percentage (TNP) assessment by a pathologist, which has been criticised for being inaccurate and suffering from interobserver variability. Based on the 'Big Bang theory' which states that KRAS mutation in colorectal cancer is mostly homogeneous, we investigated this issue by performing a critical analysis of the correlation of the KRAS mutant allele fraction with the TNP in 99 colorectal tumour samples with a positive KRAS mutation status as determined by next-generation sequencing. Our results yield indirect evidence that the KRAS zygosity status influences the correlation between these parameters and we show that a well-trained pathologist is indeed capable of accurately assessing TNP. Our findings indicate that tumour zygosity, a feature which has largely been neglected until now, should be taken into account in future studies on (colorectal) molecular tumour heterogeneit

Identification of a transcriptionally inactive p53 mutant by functional analysis of separated alleles in yeasts (FASAY) in a child osteosarcoma tumor: a case report.

Tumor suppressor gene p53 is one of the most specific genetic alterations occurring in osteosarcoma pathogenesis. It is thought to be an early and key step in the tumorigenesis of osteosarcoma. However, whether the p53 status is a marker predictive of response to therapy and a marker of prognostic value remains controversial. The choice of p53 status detection method certainly account for discrepancies. The authors used a simple functional assay (functional analysis of separated alleles in yeast) on the tumor sample of an 8-year-old girl presenting with an osteosarcoma of the tibia. While making it possible to exclude the presence of a germline mutation, FASAY indicated the presence of a somatic p53 mutation lacking transcriptional activity on p21 and bax target genes. FASAY also strongly suggested a loss of heterozygosity p53, which was confirmed by cytogenetic analysis. Sequencing of cDNA extracted from yeast colonies containing mutated p53 identified a 213 stop mutation in exon 6. Despite these p53 alterations, the child is still in complete remission after a follow-up of 48 months

Assessment of the transcriptional activity of p53 improves the prediction of recurrence in superficial transitional cell carcinoma of the bladder.

PURPOSE: To investigate the value of p53 functional analysis of separated alleles in yeast (FASAY) as a witness of p53/p21 pathway alteration and as a predictor of recurrence in superficial transitional cell carcinomas. EXPERIMENTAL DESIGN: p53 transcriptional activity was prospectively analyzed in 52 newly diagnosed transitional cell carcinoma using FASAY competent for the transactivation of p21 and bax promoters. TP53 and p21 gene expression was quantified by real-time PCR, and expression of corresponding proteins was assessed by immunohistochemistry. In addition to tumor stage and grade, the predictive value of FASAY, real-time PCR, and immunohistochemistry for tumor recurrence was assessed by Cox survival analysis. RESULTS: A total (p21 and bax) or partial (bax only) loss of transcriptional activity was observed in 15 of 52 (29%) and 4 of 52 (7.7%) cases, respectively, a partial loss being consistently associated with R283H mutation. p53 nuclear overexpression grossly overestimated (approximately 40%) or underestimated (approximately 10%) the true incidence of p53 transcriptional abnormalities, especially in Ta-T1 grade 1 to 2 tumors. Loss of p21 transactivation significantly correlated with decreased p21 gene expression and lack of expression of p21 (P = 0.001). FASAY had a better predictive value for recurrence than p53 immunohistochemistry (Cox hazard ratio, 6.57 versus 3.95; P = 0.0002 versus 0.019, respectively), whereas neither p21 immunohistochemistry (hazard ratio, 1.9; P = 0.29) nor TP53 or p21 gene expression were significant predictors of recurrence. The prognostic difference between FASAY and p53 immunohistochemistry was maintained in the subgroup of Ta-T1 grade 3 tumors. CONCLUSIONS: FASAY is a valuable surrogate marker for assessing p53/p21 pathway alteration and predicts transitional cell carcinoma recurrence better than p53 immunohistochemistry

Preservation of RNA for functional analysis of separated alleles in yeast: comparison of snap-frozen and RNALater solid tissue storage methods.

BACKGROUND: The aim of the present study was to compare RNALater with the usual method of liquid nitrogen snap freezing as a surrogate mRNA preservation method for functional analysis of separated alleles in yeast (FASAY). METHODS: A total of 81 patients with transitional cell carcinoma of the bladder underwent fresh tissue biopsies directly transferred into RNALater and stored at room temperature or at 4 degrees C for increasing time intervals until RNA processing. From this cohort of patients, 53 paired snap-frozen and RNALater preservative-suspended tissues were obtained. Samples immediately frozen in liquid nitrogen were further stored at -80 degrees C. RESULTS: Of the 81 RNALater samples, 14 were not processed for FASAY because of RNA degradation. Of the remaining 67 samples, 15 (22%) were FASAY-positive. Identical FASAY results were found for 50 of 53 (94.4%) paired samples and the percentage of red yeast colonies was highly correlated (Cohen's kappa<0.82; p<0.00001). A single p53 missense mutation was found in each of the three discordant positive FASAY and was identical in each concordant positive sample (10/53). Storing samples in RNALater at room temperature for 3 days and at 4 degrees C for less than 1 month provided high-quality mRNA suitable for FASAY. CONCLUSIONS: Our results demonstrate that RNALater is a suitable and flexible alternative to snap freezing for FASAY analysis

Long-lasting benefit on multimodal treatment combining osimertinib and stereotaxic radiotherapy for metastatic non-small cell lung cancer with the EGFR exon 20 insertion 773-774 HVdelinsLM: a case report.

A non-small-cell-lung-cancer patient with cerebral metastasis presenting an atypical exon 20 mutation in the EGFR gene had a long-lasting tumor cotrol on mulimodal treatment with osimertinib and stereotaxic radiotherapy on oligoprogressing lesions. Most exon-20 mutations are resistant to first, second and third generation EGFR-directed TKI. This case was discussed on our molecular tumour board. As the more specific exon-20 targeted therapies were not yet available and as sporadic short responses on the third generation EGFR-directed TKI, osimertinib had been described, the patient started osimertinib. She had a prolonged tumoral response on Osimertinib. The patient is still asymptomatic up to 32 months after initiating the medication. This case confirms that not all exon20 EGFR mutations are equal to osimertinib and that the localization of the exon 20 insertion mutation is probably important to consider when treating EGFR mutated NSCLC. The long-term clinical benefit can be maintained through stereotactic radiotherapy on focal progressive lesions

Assessment of p53 functional activity in tumor cells and histologically normal mucosa from patients with head and neck squamous cell carcinoma.

BACKGROUND: The purpose of this study was to investigate the value of p53 functional analysis of separated alleles in yeast (FASAY) as a witness of p53/p21 pathway alteration in head and neck squamous cell carcinoma (HNSCC). METHODS: The p53 transcriptional activity was prospectively analyzed in 82 newly diagnosed patients with HNSCC. FASAY and p53 immunostaining were carried out on paired tumoral and histologically normal tissues. The predictive value of FASAY for locoregional recurrence was assessed by Cox survival analysis. RESULTS: Loss of p53/p21 transcriptional activity was encountered in 88% tumoral and 18% histologically normal samples, associated with mutations (79%) and insertions/deletions (21%). The p53 overexpression underestimated p53 transcriptional abnormalities. FASAY-positive histologically normal mucosa was significantly associated with locoregional recurrence. CONCLUSION: FASAY positivity indicates field cancerization in a subgroup of patients with HNSCC, in which nonfunctional p53 was significantly associated with locoregional recurrence. This prompted us to pursue the study on the p53 functional status of normal mucosa in patients with HNSCC. © 2011 Wiley Periodicals, Inc. Head Neck, 2011

Using a constraint-based regression method for relative quantification of somatic mutations in pyrosequencing signals: a case for NRAS analysis.

BACKGROUND: Pyrosequencing Allele Quantification (AQ) is a cost-effective DNA sequencing method that can be used for detecting somatic mutations in formalin-fixed paraffin-embedded (FFPE) samples. The method displays a low turnaround time and a high sensitivity. Pyrosequencing suffers however from two main drawbacks including (i) low specificity and (ii) difficult signal interpretation when multiple mutations are reported in a hotspot genomic region. RESULTS: Using a constraint-based regression method, the new AdvISER-PYRO-SMQ algorithm was developed in the current study and implemented into an R package. As a proof-of-concept, AdvISER-PYRO-SMQ was used to identify a set of 9 distinct point mutations affecting codon 61 of the NRAS oncogene. In parallel, a pyrosequencing assay using the Qiagen software and its AQ module was used to assess selectively the presence of a single point mutation (NRAS[Formula: see text] - Q61R-1) among the set of codon 61 mutations, and to analyze related pyrosequencing signals. AdvISER-PYRO-SMQ produced a lower limit of blank (0 %) than the AQ module of Qiagen software (5.1 %) and similar limit of detection were obtained for both software (5.6 vs 4.8 %). AdvISER-PYRO-SMQ was able to screen for the presence of 9 distinct mutations with a single pyrosequencing reaction whereas the AQ module was limited to screen a single mutation per reaction. CONCLUSION: Using a constraint-based regression method enables to analyze pyrosequencing signal and to detect multiple mutations within a hotspot genomic region with an optimal compromise between sensitivity and specificity. The AdvISER-PYRO-SMQ R package provides a generic tool which can be applied on a wide range of somatic mutations. Its implementation in a Shiny web interactive application (available at https://ucl-irec-ctma.shinyapps.io/Pyrosequencing-NRAS-61/) enables its use in research or clinical routine applications