Dissertations / Theses on the topic 'Chemotherapy biomarker'

New treatments such as anti-angiogenic therapy, chemoembolisation, radio-embolisation cause tumour stabilisation which may render conventional size based imaging invalid for monitoring early response to therapy. The aim of this thesis was to develop an alternative index which can predict response for patients with liver tumours earlier, at 2 weeks, than the contemporary gold standard response evaluation criteria at 3 months. This technique was based on the use of microbubble ultrasound as an alternative method for detection of hepatic arterialisation. In this thesis, I evaluated the microbubble ultrasound derived liver blood flow parameters to reflect global liver blood flow in phantom as well as in patients. I also evaluated the impact of several variable factors affecting the liver blood flow and studied the potential role of the blood flow parameters and proposed an alternative index -the contrast enhanced hepatic perfusion index (CE-HPI) - in prediction of early response to treatment for liver tumours. We showed the ability of this technique to reflect global liver blood flow parameters in vitro as well as in patients. Subsequently the index was applied to determine the response assessment in patients receiving treatments. The results from these experiments present the first demonstration in humans that microbubble ultrasound derived liver blood flow parameters including CE-HPI can be helpful in early identification of non-responders. I propose that the microbubble ultrasound technique developed in this thesis can be used to further probe and investigate response assessment in randomized and multicentre studies.

Introduction The prognosis in oesophago-gastric cancer is poor with less than 15% patients surviving beyond 5 years after diagnosis. The addition of neoadjuvant therapy has been shown to increase survival in patients suitable for curative surgery. However, the additional gains are modest and the majority of patients do not respond sufficiently from therapy to gain any benefit. There is an urgent need to identify markers that can predict response to neoadjuvant therapy in order provide safer, more effective, individualised treatment regimes. Methods A prospective, multi-centre, collaborative study was undertaken in patients with oesophago-gastric cancer undergoing neoadjuvant therapy and potentially curative surgery. Levels of circulating biomarkers M2-Pyruvate kinase, alkaline phosphatase, CA19-9, CEA and CA 72-4 were measured in patients before and after administering the first cycle of chemotherapy. Binary logistic regression analysis was performed to assess the ability of biomarkers to predict histological response to therapy. Results 165 patients were recruited to the main study. 105 patients had complete histopathological data for analysis. There were 27 responders and 78 non-responders to neoadjuvant therapy. There were no differences in pre-therapy demographic, pathological or treatment factors between the two groups. Responders had less post-operative lymphovascular invasion (P= 0.004) and higher R0 resection rates (P=0.03). Pre-therapy M2-Pyruvate kinase levels were lower in responders compared to non-responders (P=0.037) and levels were able to predict response with each unit increase in the biomarker level being associated with a 4.1% decrease in the likelihood of response (P=0.027). M2-PK levels were not associated with any pre-operative demographic, clinical or pathological factors. Conclusions Pre-therapy dimeric M2-PK levels can predict response to neoadjuvant therapy in patients with oesophago-gastric cancer. The test could be of clinical value for 1 in every 8 patients undergoing the test.

Introduction: Glucose-regulated protein 78-kDa (GRP78) is an endoplasmic reticulum (ER)-resident molecular chaperone that is essential for correct protein folding and assembly in the ER lumen. Micro-environmental stress and a requirement for increased protein synthesis, typical of solid tumours, leads to a disruption of ER homeostasis, and accumulation of misfolded proteins. The ability of GRP78 to dissociate from several important ER-resident transmembrane proteins under conditions of ER stress leads to a cascade of signal transduction pathways, known as the unfolded protein response (UPR), that modulate cell survival or, if the stress is significantly severe, apoptosis. GRP78 has been found to be overexpressed in a variety of cancers compared with benign tissue and has been associated with poor outcome. In-vitro data indicate that GRP78 expression is often associated with aggressive phenotype and drug resistance. Thus, GRP78 has potential as a biomarker for tumour behaviour and treatment response. For stage III colorectal cancer, there is overwhelming evidence to recommend the use of fluoropyrimidine-based adjuvant chemotherapy. Unfortunately, a large proportion of patients do not benefit from adjuvant chemotherapy, and biomarkers that can determine the likelihood of response to chemotherapy remain elusive. The benefit of chemotherapy in stage II disease is less certain and markers that could reliably predict benefit would be particularly useful in this population. This study explores a potential mechanistic relationship between GRP78 and 5-FU sensitivity using both siRNA transfection and treatment with an engineered fusion protein, epidermal growth factor (EGF)-SubA, which has been demonstrated to cause highly selective cleavage of GRP78 at a single amino acid point. It was then examined whether GRP78 may have prognostic or predictive value in the context of colorectal cancer patients treated with fluoropyrimidine-based chemotherapy. The potential therapeutic value of targeting GRP78 in vitro using EGF-SubA is also examined. Methods: Colon cancer cell lines were used to examine response to 5-FU upon modulation of endogenous GRP78 using siRNA technology and EGF-SubA. Apoptosis and cell cycle progression were assessed using flow cytometry. Immunohistochemistry was used to characterise GRP78 expression in a large cohort of colorectal cancers on tissue microarrays and the results were correlated with clinicopathological parameters and with 5-year survival for the whole cohort and those treated with fluoropyrimidine-based (5-FU) adjuvant chemotherapy. The action of EGF-SubA upon colon cancer cells was examined using western blotting, MTT assay and flow cytometry. Results: GRP78 promotes apoptosis in response to 5-FU. Better overall 5-year survival was associated with high GRP78 expression (P=0.036). Stage III patients with high GRP78 showed significant benefit from adjuvant chemotherapy (P=0.026), whereas patients with low GRP78 failed to benefit (P=0.805). Low GRP78 was an independent poor prognostic indicator of overall 5-year survival (P=0.005; HR=1.536; 95%CI 1.139-2.122). Colon cancer cells expressing EGFR were highly sensitive to EGF-SubA, demonstrating reduced proliferation and cell cycle arrest. However, EGF-SubA did not induce significant apoptosis and reduced the effectiveness of 5-FU in vitro. Conclusion: This study demonstrates a mechanistic relationship between GRP78 expression and response to 5-FU. GRP78 expression may provide a useful additional risk stratification to inform the adjuvant treatment of colorectal cancer. EGF-SubA does not have therapeutic value in colorectal cancer but is a useful tool for studying GRP78 and the UPR.

Background: Mutation and loss of TP53 function is one of the most frequent genetic abnormalities in ovarian cancer. TP53 genomic and functional status have been shown to provide potentially prognostic and predictive value in ovarian cancer; however, the results are controversial and evaluation in the context of a controlled clinical trial with single agent treatment have been lacking. Reactivation of p53 using MDM2-p53 antagonists is a promising therapeutic target for most patients with type I epithelial ovarian cancer and those left from type II harbouring wild-type TP53. BRCA1/2 mutations are present in 70-85% of germline mutations in patients with inherited ovarian cancer, and deficiencies in homologous recombination repair (HRR) account for up to 50% of epithelial ovarian cancer, indicating the possible sensitivity of ovarian cancer patients to PARP inhibitors. MDM2-p53 antagonists and PARP inhibitors are now undergoing clinical trials as targeted therapy for different types of cancer. The effect of RG7388 on its own and in combination with cisplatin, and combined treatment between MDM2-p53 antagonists and PARP inhibitors have not been investigated in ovarian cancer. Hypotheses: 1) Different genomic and functional status of p53 and some of its downstream targets such as p21WAF1, MDM2 and WIP1 can be used as prognostic and predictive biomarkers for the outcome of chemotherapy and overall survival in ovarian cancer. 2) Reactivation of p53 by inhibition of its negative regulator MDM2, using the MDM2-p53 antagonists Nutlin-3 and RG7388, will result in p53-mediated growth arrest and apoptosis in wild-type TP53 ovarian cancer cells, and combination of them with current therapeutic agents or rucaparib increases growth inhibition and/or apoptosis in ovarian cancer cell lines compared to either agent alone. Methods: TP53 was sequenced in 260 ovarian cancer samples from the ICON3 trial using Sanger sequencing and Next Generation Sequencing (NGS) methods. The prognostic value of the expression levels of p53, p21WAF1, MDM2 and WIP1 was investigated using immunohistochemistry (IHC). The effect of MDM2-p53 antagonists, Nutlin-3/RG7112/RG7388, and PARP inhibitor, rucaparib, as single agents and in combination with cisplatin or together were investigated on a panel of ovarian cancer cell lines. Sensitivity was measured by growth inhibition, clonogenic cell survival assay, apoptosis assays including caspase 3/7 activity and flow cytometry. The effect on the p53 molecular pathway and p53-regulated candidate gene expression were investigated by western blotting and Quantitative Reverse-Transcription Polymerase Chain Reaction (qRT-PCR) respectively. Results: Patients from the ICON3 clinical trial treated with carboplatin whose tumours harbour wild-type TP53 had a significantly better overall survival based on both univariate and multivariate analysis compared to those with mutant TP53 regardless of sequencing method. Adding paclitaxel to the platinum-based treatment showed a trend in favour of greater benefit for those with mutant TP53, although this failed to reach statistical significance (p > 0.05). Overexpression of p53 has potential prognostic value for overall survival of ovarian cancer patients. Ovarian cancer cell lines with wild-type TP53 were sensitive to MDM2-p53 antagonists, Nutlin-3/RG7112/RG7388, while those with mutant TP53 were resistant to MDM2 inhibitors. Among the individual cell lines, A2780 and MDAH-2774 were sensitive and other cell lines (IGROV-1, OAW42, CP70, MLH1-corrected CP70+ and SKOV-3) were resistant to rucaparib regardless of BRCA1/BRCA2 status or deficiencies in HRR reported for these cell lines. Combination of Nutlin-3/RG7388 with cisplatin or rucaparib has synergistic and/or dose reduction potential dependent on cell genotype and the type of MDM2-p53 antagonist. Combined treatments using Nutlin-3/RG7388 and cisplatin led to greater levels of p53 stabilisation and upregulation of p21WAF1 and MDM2, and higher expression of p21WAF1 was associated with a greater synergistic effect for growth inhibition. In combination treatment with rucaparib and Nutlin-3/RG7388, rucaparib showed no increase in the effect of MDM2 inhibitors on the p53 pathway, indicating that the mechanism of observed synergy does not involve enhancement of p53 pathway activation by MDM2 inhibitors. Nutlin-3/RG7388 in combination with cisplatin or rucaparib resulted in changes in cell cycle distribution, SubG1 events and caspase 3/7 activity in a cell type, time and compound-dependent manner. The fold changes in expression of candidate genes in response to MDM2 inhibitors were less in A2780 cells than IGROV-1 and OAW42. The balance of activity between growth inhibitory/pro-survival and pro-apoptotic genes dominates a small increase in the expression of several DNA repair genes as an explanation for the synergy observed for treatment with cisplatin and MDM2 inhibitors. Conclusions: The genomic and functional status of TP53 have potentially important prognostic and predictive values in ovarian cancer. Targeting the interaction between MDM2 and p53 using MDM2-p53 antagonists is a promising therapeutic strategy for ovarian cancer patients with wild-type TP53 tumours, and combination treatment with them and cisplatin or rucaparib.

Le glioblastome est la forme la plus agressive des tumeurs du système nerveux central. Le traitement de référence consiste en l'exérèse chirurgicale, suivie d'une radiothérapie associée à une chimiothérapie concomitante et adjuvante par le témozolomide. Son bénéfice est démontré par une médiane de survie entre 12 et 14 mois. Le glioblastome est caractérisé par une population cellulaire hétérogène hautement infiltrante, angiogénique et résistante à la chimiothérapie. Dans le but d'optimiser l'effet des molécules thérapeutiques, un suivi de leur pharmacocinétique ainsi qu'une bonne caractérisation tumorale sont nécessaires. L'imagerie par désorption laser assistée par matrice en spectrométrie de masse (IMS MALDI) a été utilisée pour l'identification de marqueurs diagnostiques, pronostiques et prédictifs de réponse aux traitements. Elle a aussi permis de suivre la pharmacocinétique in situ des chimiothérapies.L'identification de protéines directement sur tissu par fragmentation en source a permis la mise en évidence de différents isotypes de tubuline, une des cibles majeures en thérapie anticancéreuse. Le couplage de cette stratégie d'identification à l'imagerie MALDI a permis d'identifier et de localiser dans des zones tumorales, des protéines impliquées dans la tumorigenèse. La distribution intra-tissulaire du bévacizumab et du témozolomide a été étudiée pour la première fois.Des marqueurs de réponse aux traitements ont ensuite été identifiés par comparaison des profils d'expression protéique de tumeurs avec et sans traitement. Ces résultats montrent l'intérêt de l'imagerie MALDI pour l'étude des chimiothérapies et permettent d'envisager son utilisation clinique future
Glioblastoma is the most aggressive of the gliomas, a collection of tumors arising from glia or their precursors within the central nervous system. The current standard of care, comprised of surgical resection followed by radiation and the chemotherapeutic agent temozolomide, only provides patients with a 12-14 months survival period post-diagnosis. The glioblastoma is characterized by a heterogeneous population of cells that are highly infiltrative, angiogenic and resistant to chemotherapy. In order to optimize the therapy effect, a pharmacokinetic monitoring and a better understanding and characterization of tumor biology are needed. For this purpose, matrix assisted laser desorption/ionization imaging mass spectrometry imaging mass spectrometry (MALDI IMS) technology was applied to identify diagnostic, prognostic and predictive markers of therapy response; and to understand/follow the pharmacokinetic of chemotherapies. The top-down in-source decay strategy was used for protein identification directly on tissue. This strategy allowed tubulin protein isoforms distinction and identification, which is one of the main targets in cancer therapy. MALDI imaging coupled to ISD identified tumorigenesis proteins within tumor structures. Bevacizumab and temozolmide distribution was followed within brain tissue sections. For the first time a monoclonal antibody was deciphered on tissue. Finally, markers that predict therapy response were demonstrated by a comparison between protein expression profiles from tumors with and without chemotherapy treatment. These results highlight the interest of MALDI imaging for chemotherapy improvement and open the way for its use in the clinics

L’hypothèse prédominante de cette thèse est que les changements métaboliques tumoraux mesurés par FDG-PET/CT sous l’influence des traitements anticancéreux, apparaissent plus précocement et parfois exclusivement par rapport aux modalités d’imagerie morphologique classique. L’imagerie multimodale, en combinant les avantages de chacune des techniques, dépasse leur limitations et pourrait permettre (i) une évaluation du bénéfice du traitement plus rapide et plus adéquate ;(ii) de modifier les algorithmes thérapeutiques à différents stades de cancer colorectal et (iii) d’améliorer la compréhension des mécanismes d’échappement aux traitements anticancéreux. Pour évaluer l’apport de l’imagerie multimodale dans l’évaluation de la réponse au traitement des cancers colorectaux (CCR), nous avons poursuivi 3 séries d’expérimentation cliniques.

1) Le premier projet explore l’imagerie multimodale comme un outil d’individualisation pour la radio-embolisation (microsphères chargées en 90Yttrium) chez des patients porteurs d’un CCR métastatique au niveau du foie, pour laquelle l’imagerie morphologique classique est incapable de mesurer l’effet thérapeutique. Nous montrons que l’usage non sélectif de la radio-embolisation améliore l’histoire clinique de ces patients, bien que certains d’entre eux ne semblent pas en bénéficier. Ensuite, par une analyse multimodale lésion par lésion intégrant angiographie-CT Scan, FDG-PET/CT et scintigraphie aux macro-agrégats d’albumine marqués au 99mTechnetium, nous démontrons que la distribution pré-thérapeutique des macro-agrégats d’albumine est hétérogène entre les différentes lésions des patients et prédictive de la réponse métabolique au sein de ces lésions, permettant le développement d’un outil de prédiction et de planification pour la radio-embolisation.

2) Le deuxième projet explore le domaine du CCR métastatique traité par chimiothérapie palliative. (i) Nous démontrons d’abord que la réponse métabolique (RM) tumorale après une cure de chimiothérapie cytolytique prédit plus vite et plus adéquatement que l’imagerie morphologique basée sur les critères RECIST les bénéfices cliniques du traitement. La RM précoce a une excellente valeur prédictive négative sur l’absence de réponse morphologique et met en évidence une variabilité de réponse inter-lésionnelle chez une proportion importante des patients. (ii) L’étude SoMore explore ensuite des patients présentant un CCR avancé et réfractaire, traités par capecitabine et sorafenib, et confirme l’importance pronostique des RM mixtes, suggérant une méthodologie de classification clinique basée sur la consistance de la RM. (iii) Cette classification cherche confirmation dans l’étude RegARd-C, encore en cours, évaluant les effets du regorafenib, et explorant également la signification génomique et épigénétique de la variabilité de RM.

3) Le troisième projet cherche à utiliser les propriétés de l’imagerie métabolique pour modifier l’algorithme de traitement adjuvant des patients porteurs d’un cancer du côlon de stade III. Ce projet, encore en cours, fait l’hypothèse que l’absence de RM de la lésion primitive après une cure de chimiothérapie prédit l’absence de bénéfice du traitement adjuvant complet. Une analyse intérimaire en démontre la faisabilité et confirme la présence de 40% de tumeurs présentant des caractéristiques métaboliques de chimio-résistance.

En conclusion, pour des patients porteurs d’un CCR, l’imagerie multimodale comprenant une évaluation du métabolisme tumoral permet une évaluation plus précoce et plus adéquate du bénéfice au traitement anticancéreux pour différentes modalités thérapeutiques comme la radio-embolisation, la chimiothérapie cytotoxique et les agents biologiques. L’imagerie multimodale permet de prédire et planifier les radio-embolisations et se révèle très prometteuse pour les traitements chimiothérapiques cytotoxiques ou combinés à des biologiques en situation adjuvante ou métastatique. Elle démontre par ailleurs une importante variabilité de réponse métabolique inter-lésionnelle qui représente un axe de recherche majeur sur les mécanismes moléculaires d’hétérogénéité génomique tumorale et de résistance aux traitements anti-cancéreux.

Doctorat en Sciences médicales
info:eu-repo/semantics/nonPublished

Schlafen 11 (SLFN11) est une ADN/ARN hélicase décrite pour la première fois pour son rôle dans le développement et la différenciation des thymocytes chez la souris. Elle fait partie d'une famille de protéines présentant divers degrés d'homologie entre les espèces, mais qu’est présente de façon constante chez les mammifères. Le rôle de cette ADN/ARN hélicase, SLFN11, a été associé de façon causale à la sensibilité de réponse aux différents agents alkylants (agents endommageant l'ADN, les inhibiteurs de topo-isomérase I et II) dans le NCI-60. Dans la première étude, nous avons développé un protocole d’immunohistochimie (IHC) sur des biopsies paraffinées de carcinome séreux de l'ovaire de haut grade (HGSOC), afin de valider un anticorps (Ab) anti-SLFN11 et d’en déterminer l'expression. En IHC, nous avons testé et validé un Ab anti-SLFN11, en choisissant entre deux anti-SLFN11 Ab utilisés normalement pour le Western Blot. Premièrement, il a été développé dans une culture cellulaire (CCB) de HGSOC et, successivement, dans une série indépendante de micro-array (TMA) de HGSOC. Pour chaque cas, nous avons évalué soit le score d'intensité (IS) que le score de distribution (DS) en évaluant au moins 300 cellules. Un score histologique (HS) a été obtenu comme suit : HS=IS x DS. Successivement, nous avons appliqué notre protocole à une plus large série d'échantillons de HGSOC pour confirmer nos résultats préliminaires. Nous avons trouvé un anticorps fiable dans les séries CCB et TMA permettant de déterminer l'expression IHC de SLFN11. Ces résultats ont été confirmés dans notre plus large série de HGSOC. Brièvement, comme pour les séries indépendantes de TMA, nous avons constaté que la HS de l'expression de SLFN11 est présente dans environ 60%. En parallèle, le SLFN11 n'a pas été exprimé dans 40 % des cas qui, cliniquement, correspondent, dans environ 60 % de ces cas (16/27), aux patients résistant aux sels de platine. Une faible expression de SLFN11 en IHC pourrait être corrélée à la réponse à la chimiothérapie(CT) à base de platine. Dans la deuxième étude, nous étudions l’état transcriptionnel du SLFN11 dans le cancer du sein en effectuant une méta-analyse de plus de 7000 cas à partir de 35 étudies publiquement disponibles. Par l’analyse de corrélation, nous avons identifié 537 transcrits qui corrèle, au-delà du 95e percentile selon le coefficient de Pearson, avec l’expression de SLFN11. En particulier, voie l’analyse par “Gene Ontology” SLFN11 est lié au transcrits impliqués dans le système immunitaire : "réponse immunitaire", "l’activation lymphocytaire" et "l’activation des lymphocytes T". En outre, voie le “likehood lasso regression ”, nous avons signalé une très forte association entre le SLFN11 et les signatures immunitaires dans le cancer du sein. Enfin, grâce à la “multiple corresponded analysis ”, nous avons découvert un sous-groupe de patients, défini "SLFN11-Hot cluster", caractérisé par une expression élevé de SLFN11, récepteurs d'œstrogènes(ER) négatives, un phénotype basal, un jeune âge, une signature élevée de CD3D et de STAT1. En utilisant la "Cox proportional hazard regression", l’expression élevé de SLFN11, l’indice de prolifération élevé et le ER négative sont des paramètres indépendants lié à la survie sans maladie chez les patients soumises à la CT. Notre deuxième travail decrit un rôle spécifique pour le SLFN11 dans le cancer du sein probablement en relation avec la modulation du système immunitaire et une forte corrélation entre l’expression de SFLN11 et un sous-type moléculaire spécifique de cancer du sein (récepteurs négatifs aux œstrogènes, phénotype de type basal). Autres études devront être réalisées afin de: 1) mieux comprendre la fonction du SLFN11 dans les cellules cancéreuses, 2) valider un protocole IHC fiable et standardisé pour évaluer l’expression de SLFN11, 3) utiliser SFLN11 comme biomarqueur prédictif de réponse aux DDA et PARP inhibiteurs et 4) établir sa relation avec le système immunitaire
Schlafen 11 (SLFN11) is a putative DNA/RNA helicase, first described for its role in thymocyte development and differentiation in mouse models. SLFN11 is part of a family of proteins with various degree of homology across species, but intriguingly being consistently present only in vertebrates and especially in mammals. Recently, the role of this putative DNA/RNA helicase, SLFN11, was causally associated with sensitivity to DNA damaging agents, such as platinum salts, topoisomerase I and II inhibitors, and other alkylators in the NCI-60 panel of cancer cell lines. In the first study, we validate an anti-SLFN11 antibody in formalin-fixed paraffin-embedded (FFPE) high-grade serous ovarian carcinoma (HGSOC) samples, developing an immunohistochemistry (IHC) protocol in order to determinate the expression of SLFN11 in our series of HGSOC. Indeed, we tested and validated a reliable SLFN 11 antibody (Ab) in IHC choosing between two anti-SLFN11 Ab used normally for Western Blot (WB) in culture cell block (CCB) of ovarian carcinoma and in an independent series of HGSOCs tissue micro-array (TMA). For each case, we evaluated both the Intensity Score (IS) and the Distribution Score (DS) evaluating at least 300 cells. A Histological Score (HS)was obtained as follow: HS=IS x DS. Successively, we applied our protocol to a large case series of HGSOC samples to confirm our preliminary results. We found one antibody to be reliable in CCB and TMA series allowing to determinate clearly IHC expression of SLFN11. These results were confirmed in our large case series of FFPE HGSOC samples. Briefly, as for TMA independent series, we found that the HS for SLFN11 expression presents a normal distribution with a prevalent (≈ 60%) intermediate expression. Parallel SLFN11 was not expressed in practically 40% of cases that clinically corresponded to the platinum resistant patients in about 60% of cases (16/27). So, we believe that low IHC expression of SLFN 11 should be correlated to response to the platinum-based chemotherapy. In the second study, we investigate the transcriptional landscape of SLFN11 in breast cancer performing a gene expression microarray meta-analysis of more than 7000 cases from 35 publicly available data sets. By correlation analysis, we identified 537 transcripts in the top 95th percentile of Pearson’s coefficients with SLFN11 identifying “immune response”, “lymphocyte activation” and “T cell activation” as top Gene Ontology enriched processes. Furthermore, we reported very strong association of SLFN11 with immune signatures in breast cancer through penalized maximum likelihood lasso regression. Finally, through multiple corresponded analysis we discovered a subgroup of patients, defined “SLF11-hot cluster”, characterized by high SLFN11 levels, estrogen receptor(ER) negativity, basal-like phenotype, elevated CD3D, STAT1 signature, and young age. Using Cox proportional hazard regression, we characterized that SLFN11 high levels, high proliferation index, and ER negativity are independent parameters for longer disease-free interval in patients undergoing chemotherapy. We believe that our second work supports proof of concept that: i) A clear and specific role for SLFN11 in breast cancer, in likely connection with the immune system modulation in such disease entity, ii) a strong correlation between high SFLN 11 and specific molecular subtype of breast cancer (estrogen receptor negativity, basal-like phenotype). Further studies will be performed to confirm our hypothesis in order to: 1) better understand the function of SLFN 11 in cancer cell, 2) validate an easy, reliable and standardized IHC protocol to assessment SLFN11, 3) use SFLN11expression as a predictive biomarker of response to DDA and PARP inhibitors and 4) determinate the relationship with immune system

In advanced lung cancer, careful selection of systemic anticancer therapy (SACT) is of vital importance. Companion biomarkers can optimise treatment selection, such as with the use of EGFR tyrosine kinase inhibitors (EGFR TKi) in patients with EGFRmut+ve adenocarcinoma of the lung. There is increasing interest in mutation detection and monitoring, in circulating cell free tumour DNA (ctDNA). This thesis reports that Next Generation Sequencing (NGS) with software VarScan with Annovar, can detect mutations at a 10-fold lower alternate allele frequency compared to alternative software available through Ion Torrent, but with a greater number of low level ‘false positive’ genetic variants. Droplet digital PCR (ddPCR) is more sensitive than NGS, successfully detecting mutations as low as 0.1% alternate allele frequency. Lung cancer mutations were successfully detected in small, formalin-fixed, paraffin embedded (FFPE) tumour tissue samples, and ctDNA, from lung cancer patients, using the same NGS technique, with a commercially available, targeted 50 gene cancer hotspot panel. Results are compared to a custom 22-gene panel. The kinetics of mutation levels in serial ctDNA samples is reported in a case series. In EGFRmut+ve lung adenocarcinoma patients treated with EGFR TKi, decreases in levels of mutant EGFR in ctDNA were observed. Levels remained undetectable during periods of disease control/stability, and increases in mutant EGFR in ctDNA were seen several weeks before the diagnosis of clinical or radiological disease progression. NGS of ctDNA during disease progression revealed novel genetic mutations that were not detected in the original tumour biopsy, and may inform subsequent treatment options. Similar ctDNA kinetics was seen in advanced SCLC patients treated with SACT. The levelof mutated ctDNA, at diagnosis may be an independent prognostic biomarker, using a cut-off of 44.3% alternate allele frequency. SCLC patients who experienced a greater absolute decrease in mutant ctDNA had a poorer prognosis.

Les môles hydatiformes sont une prolifération placentaire prétumorale pouvant évolueren tumeur alors traitée par chimiothérapie. Afin de réduire la mortalité et d’optimiser laprise en charge thérapeutique, l’objectif de cette thèse est d’identifier les gènespermettant de prédire la transformation en tumeur post môlaire et la chimiorésistance.Concernant la prédiction de la transformation, l’analyse de l’expression de gènescandidatssur tissu molaire décrit la relocalisation apicale de la Syncytine-1 en cas detransformation maligne, sans modification de transcription de ses récepteurs ni de deuxautres enveloppes rétrovirales placentaires. L’analyse sans à priori du transcriptome par3 méthodes différentes n’a pas permis d’identifier de gène différentiellement expriméselon la transformation. Cela suggère que la variabilité interindividuelle et les diverscritères utilisés pour le diagnostic de tumeur nuisent à l’identification de biomarqueursrobustes.Concernant la prédiction de la chimiorésistance, une approche transcriptomique largespectre sur tissu tumoral de choriocarcinome identifie une réduction de transcriptiond’HLA-G en cas de monochimiorésistance, confirmée au niveau protéique par immunohistochimie. L’analyse en réseaux de l’ensemble des gènes différentiellementexprimés suggère que la monochimiorésistance est associée à une altération de ladifférenciation des lymphocytes T alors que la polychimiorésistance est associée à unealtération de la prolifération des cellules sanguines.In fine, l’objectivation de l’expression trophoblastique du point de contrôle PD-L1 aconduit à évaluer l’efficacité d’un anti PD-L1 chez les patientes chimiorésistantes. Lesrésultats encourageants de cet essai et la possibilité de stratifier les patientes à l’aidedes marqueurs HLA-G et Syncytine-1 incitent à évaluer la place de l’anti PD-L1 associéà une monochimiothérapie en première ligne de traitement des tumeurstrophoblastiques
Hydatidiform moles are a pretumoral placental proliferation which can turn into a tumorrequiring chemotherapy. In order to reduce mortality and propose an optimal therapeuticmanagement, the aim of this thesis is to identify genes which are predictive of postmolartumor transformation and chemoresistance.Concerning the prediction of transformation, the expression analysis of candidate-geneson molar tissue shows a relocalization of Syncytin-1 at the syncytiotrophoblast apicalborder in moles followed by malignant transformation, without modification oftranscription of its receptors and two other retroviral placental envelopes. A wholetranscriptomeapproach using 3 different microarrays-based methods did not identify anydifferentially expressed gene according to the post molar evolution. This may reflect thatinter-individual variability and the different criteria used for tumor diagnosis impede theidentification of robust biomarkers.Concerning the prediction of chemoresistance, a broad-spectrum transcriptomicapproach on choriocarcinoma tumor tissue identifies a down regulation of HLA-G in case of monochemoresistance, confirmed at the protein level by immunohistochemistry.Pathway analysis of the differentially expressed genes suggests thatmonochemoresistance is associated with impaired T-cell differentiation, whereaspolychemoresistance is associated with impaired proliferation of blood cells.Ultimately, the evidence of trophoblastic ubiquitous expression of the PD-L1 immunecheckpoint led us to the evaluation of the efficacy of PD-L1 blockade in chemoresistantpatients. The encouraging results of this trial and the possibility of stratifying patientswith HLA-G and Syncytin-1 markers encourages the assessment of PD-L1 blockadecombined with monochemotherapy as a first line treatment for trophoblastic tumors

Background:Chemotherapy resistance is a major obstacle in effective neoadjuvant treatment for locally advanced breast cancer. The ability to predict tumour response would allow chemotherapy administration to be directed towards only those patients who would benefit, thus maximising treatment efficiency. This project aimed to identify predictive protein biomarkers associated with chemotherapy resistance, using proteomic analysis of fresh breast cancer tissue samples. Materials and Methods:Chemotherapy-sensitive (CS) and chemotherapy-resistant (CR) tumour samples were collected from breast cancer patients who received neoadjuvant therapy consisting of epirubicin with cyclophosphamide followed by docetaxel. Comparative proteomic analysis was performed, to identify differentially expressed proteins (DEPs) between CS and CR invasive ductal carcinoma samples, using 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with MALDI-TOF/TOF mass spectrometry and antibody microarray analysis. DEPs were submitted to Ingenuity Pathway Analysis (IPA) to identify any canonical pathway links, confirmed using western blotting and clinically validated in a pilot series of archival breast cancer samples, from patients treated with neoadjuvant chemotherapy. Results:Five datasets were generated by antibody microarray analysis, revealing 38 targets. Of these, 7 DEPs were identified in at least 2 datasets and these included 14-3-3 theta/tau, BID and Bcl-xL. Three datasets were generated using 2D-PAGE with MALDI-TOF/TOF MS, containing 132 unique DEPs. These included several isoforms of 14-3-3 proteins. The differential expression of 14-3-3, BID and Bcl-xL was confirmed by immunoblotting in samples used for the discovery phase. Clinical validation using immunohistochemical analysis of archival breast cancers revealed 14-3-3 theta/tau and tBID to be significantly associated with chemotherapy resistance. Discussion:The use of comparative proteomic techniques using fresh clinical tumour samples, for the search for putative biomarkers of chemotherapy resistance has been successful. Two DEPs; 14-3-3 theta/tau and tBID have passed through all stages of the biomarker discovery pipeline, and present themselves as putative predictive biomarkers of neoadjuvant chemotherapy resistance in breast cancer.

Background: Neoadjuvant chemotherapy is a standard treatment for locally advanced breast cancer however chemoresistance can be a major obstacle in ER+ cancers. Using comparative proteomic approaches (antibody microarray/AbMA and 2D-PAGE with MALDI-TOF/TOF MS) to investigate a pilot series of breast cancer samples our research group recently identified 14-3-3 theta/tau, tBID and BcL-XL as putative biomarkers of response to neoadjuvant chemotherapy (Hodgkinson et al J Prot 2012, 75:1276-1283 and 75:2745-2752). Here we aimed to analyse further samples using the AbMA approach and to re-analyse the combined data. Methods: Samples from chemoresistant and chemosensitive breast cancers were selected following anthracycline-taxane chemotherapy and 4 experiments were performed using ductal ER+ tumours. Differential protein expression was compared between chemoresistant and chemosensitive samples using the Panorama XPRESS Profiler725 AbMA kit. The combined data from 9 AbMA assays and 3 2D-PAGE/MS experiments was then analysed using Ingenuity Pathway Analysis (IPA; Ingenuity Systems). A pilot series of archival samples was used for clinical validation of putative predictive biomarkers. Results: 89 differentially expressed proteins (DEPs) were seen in the 4 further AbMA experiments. In the combined dataset (12 experiments from 2 proteomic platforms), 8 DEPs were seen in at least 3 experiments. These were 14-3-3 theta, 14-3-3 epsilon, 14-3-3 gamma, Bcl-xl, Bid, Phosphokinase B, Vimentin and FAK. 121 DEPs from the combined data were analysed using IPA; 13 DEPs were mapped onto the PI3K/AKT pathway. Clinical validation in a pilot series of archival samples revealed AkT-1 Ser473 and FAKY397 alongside the previously identified and validated 14-3-3 theta/tau, and tBID to be significantly associated with chemotherapy resistance. Conclusion: We have now identified at least 8 proteins which could play a role in breast chemoresistance. We propose a potential role for AkT-1, FAK, 14-3-3 theta/tau and tBID as predictive biomarkers of neoadjuvant chemotherapy resistance in breast cancer. Further validation in a larger sample series is now required.

Aims: Identification of neurophysiological or skin innervation biomarkers which can be used to assess and monitor progression of diabetic sensory polyneuropathy (DPN) and chemotherapy-induced neuropathy (CIPN). Sensitive and robust measures are needed to detect changes in the relatively short duration of clinical trials aimed to modify progression of neuropathy. Methods: 40 patients with DPN were studied longitudinally over 1 year, and 33 patients with CIPN in a cross-sectional study. Clinical assessments, questionnaires, quantitative sensory testing, histamine-induced skin flare, nerve conduction studies and contact heat evoked potentials were measured. Repeat skin biopsies were performed at a 6 month interval to quantify intra- (IENF) and sub-epidermal (SENF) nerve fibres immunoreactive for PGP 9.5 (pan-neuronal marker), TRPV1 (heat and capsaicin receptor) and GAP-43 (marker of regenerating fibres) in the DPN group, and at baseline in the CIPN group. Results: There was no change in symptoms and sensory tests in the DPN group. However, there was a significant reduction in IENF and SENF for both PGP 9.5 and TRPV1 fibres in the second DPN skin biopsy (n = 29 had repeat biopsy). GAP- 43 fibres were present in the dermis and remained unchanged. Patients in the CIPN group had less painful neuropathy, but similar abnormalities on examination and sensory tests. Despite this, a preserved number of IENF and SENF were seen in the CIPN group, with abnormal morphology. This has not been reported previously. Conclusion: PGP 9.5 and TRPV1-immunoreactive nerve fibres in sequential skin biopsies provide objective markers of progression of neuropathy, while the preserved GAP 43-immunoreactive fibres may detect enhanced regeneration. Novel findings in the CIPN group suggest prevention of degeneration and restoration of function should be the treatment strategy, rather than enhancing regeneration.

Significant advances in the efficacy of cancer therapy have been accompanied by an escalation of side effects that result from therapy-induced injury to normal tissues. Patients with high grade cancer or metastasis are often treated with chemotherapy, 50% of which are associated with reactive oxygen species generation and cellular oxidative stress. Heart is the normal tissue most susceptible to chemotherapy-induced oxidative stress and heart disease is the most common leading cause of death in cancer survivors. However, early and sensitive biomarkers to identify heart disease are still lacking. Extracellular vesicles (EVs) are released from cells during oxidative stress and send oxidized proteins into the circulation as a compensatory mechanism that prevents cellular proteotoxicity. Thus, the protein contents of EVs released during the pre-degeneration stage reveal that oxidative stress is occurring early in the damaged tissue. Using a mouse model of doxorubicin (DOX)-induced cardiac injury, we demonstrated that EVs can be used as an early diagnostic tool for tissue injury as they are oxidatively modified with 4-hydroxynonenal and contain tissue specific proteins—glycogen phosphorylase brain/heart, muscle, and liver isoforms—that indicate their origins. These biomarkers increased early, before the changes of conventional biomarkers occurred. EVs also mediate intercellular communication by transferring bioactive molecules between cells. In the cell culture system, EVs play an important role in oxidative stress response by inducing macrophage polarization. EVs from cardiomyocytes promoted both proinflammatory (M1) and anti-inflammatory (M2) macrophage polarization evidenced by higher pro- and anti-inflammatory cytokines and nitric oxide generation, as well as mitochondrial oxidative phosphorylation suppression and glycolysis enhancement. In contrast, EVs from the hepatocytes supported anti-inflammatory macrophage (M2) by enhancing oxidative phosphorylation and anti-oxidant proteins. DOX promoted the immunostimulatory effects of cardiomyocyte EVs but not hepatocyte EVs. The differential functions of EVs on macrophage phenotype switching are due to their different effects on Thioredoxin 1 redox state, which regulates activities of redox sensitive transcription factors NFκB and Nrf-2. Our findings shed light on the role of EVs as a redox active mediator of immune response during chemotherapy.

Chemotherapy and radiotherapy are widely accepted as common forms of treatment for cancers. The majority of cancer patients receive chemotherapy alone or in combination with radiotherapy. Most chemotherapeutic drugs cause DNA damage to the rapidly dividing cancer cells but normal cells are also damaged in the process. Therefore DNA repair levels in tumour and normal cells may determine the success of the treatment. The aim of this work was to evaluate the use of DNA repair biomarkers for assessing responses to chemotherapeutic drugs. The novel technique of imaging flow cytometry was employed to analyse the induction and resolution of γ-H2AX and RAD51 DNA repair biomarkers in DNA repair normal cell lines MRC5-SV1 and NB1-HTERT, an ATM-deficient cell line AT5BIVA (derived from an Ataxia Telangiectasia patient) and an XPF-deficient cell line GM08437B. Two cell lines were also developed, MRC5-SV1R and NB1-HTERTR which had been made resistant to HN2. A range of chemotherapeutic drugs, Adriamycin, Cisplatin and Nitrogen Mustard which have different modes of action were examined in this work. We have demonstrated distinct differences in γ-H2AX and RAD51 foci induction and resolution between the two DNA repair normal cell lines following exposure to different chemotherapeutic drugs. Additionally, it was demonstrated that both the resistant and sensitive cell lines have elevated γ-H2AX and RAD51 expression profiles in comparison to the parental cell lines over a 48 hour period post treatment with the cross-linking agent HN2. It is concluded that while both the γ-H2AX and Rad51 biomarkers may be useful for determining chemotherapeutic response, a larger cohort of cell lines and tumour samples is required for further analysis.

El càncer de mama associat a l’embaràs (PABC) és definit com càncer de mama diagnosticat durant l’embaràs o un any després del part. La quimioteràpia durant l’embaràs pot incrementar la producció d’estrès oxidatiu i inflamació elevant el risc de complicacions obstètriques. Per tant, vam examinar biomarcadors d’estrès oxidatiu, inflamació i de defensa antioxidant en dones amb PABC (N=17) abans del tractament, abans de cada cicle (antraciclines i paclitaxel) i al part. També es va obtenir sang de pacients amb càncer de mama sense embaràs (N=10) abans i després d’ antraciclines i dones embarassades sanes (N=16). Paral·lelament, vam analitzar sang de cordó umbilical (N=13) i d’orina (N=5) 24 h de vida de nounats que van rebre quimioteràpia en l’úter. Mostres de cordó (N=14) i d’orina (N=6) de nadons nascuts de mares sanes es van utilitzar com a controls. En general, les pacients amb PABC presentaven nivells semblants d’estrès oxidatiu i d’antioxidants abans de la quimioteràpia que les controls . Això no obstant, les pacients amb PABC mostraven un augment en l’activitat chitotriosidasa (P=0.004) en comparació amb les controls, indicant inflamació. A conseqüència de la quimioteràpia, vam observar: l’activitat chitotriosidasa va ser reduïa després del tractament amb paclitaxel en comparació amb les controls (P=0.018); l’activitat de YKL-40 va disminuir respecte els nivells d’abans de la quimioteràpia (P=0.047) i els grups de proteïnes-SH van ser significativament reduïts després d’antraciclines per tornar a incrementar amb paclitaxel en comparació amb les controls (P=0.010 i P=0.012 respectivament). L’efecte acumulatiu es va analitzar comparant els nivells basals amb els obtinguts al part. El nostre estudi va observar un augment de dany a l’ADN i de proteïnes a les pacients amb PABC segons l’elevació de les ràtios 8-OHdG/2dG i o-Tyr/Phe (P=0.031; P=0.032 respectivament). A més, vam observar nivells significativament elevats en tres biomarcadors de peroxidació lipídica: 5-F2t-IsoP+5-epi-5-F2t-IsoP (P=0.031), 15-epi-2,3-dinor-15-F2t-IsoP + 2,3-dinor-11-PGF2α + 2,3-dinor-15-F2α-IsoP (P=3.72e-03) i 10-F4t-NeuroP (P=0.031), així com PGF2α (P=0.026) marcador d’inflamació i la ràtio GSH/GSSG (P=4.79e-03) marcador antioxidant en comparació amb els controls al part. No es van observar diferencies en els biomarcadors de dany a l’ADN i de proteïnes Les pacients amb càncer de mama però no embarassades, presentaven nivells elevats dels biomarcadors de dany a l’ADN i de proteïnes (8-OH-dG/2dG ràtio; P=1.52e-03 i m-Tyr/Phe ràtio, P=7.88e-04 respectivament), però el biomarcador de defensa antioxidant (GSH/GSSG ràtio, P=5.67e-06) estava reduït en comparació amb les pacients amb PABC. D’altra banda, els nounats exposats a quimioteràpia a l’úter presenten generalment nivells més baixos d’estrès oxidatiu i marcadors d’inflamació que els nounats controls. Excepcionalment, vam detectar un increment en els nivells plasmàtics de 15-F2T-IsoP (P=8.05e-03) i els nivells urinaris de GSA (P=0.016). Per últim, vam observar una correlació positiva entre els nivells de 15-epi-15-F2t-IsoP y 15-epi-2,3-dinor-15-F2t-IsoP + 2,3-dinor-11-PGF2α + 2,3-dinor-15-F2α-IsoP (R=-0.55, P=0.049 i R=-0.68, P=0.011 respectivament) mesurats en sang de cordó de nounats exposats a quimioteràpia en l’úter amb les seves mares corresponents al part. En resum, el nostre estudi mostra que la quimioterapia durant l’embaràs està significativament associada amb una alteració de l’estat redox. No obstant això, les pacients amb PABC mostraven una alta capacitat antioxant mentre que els nounats no van ser afectats negativament. Per tant, les complicacions obstètriques reportades a l’estudi no van ser causades per un augment de l’estrès oxidatiu i inflamació, però de ser-hi, van ser eficientment contrarestats.
El cáncer de mama asociado al embarazo (PABC en inglés) es definido como cáncer de mama diagnosticado durante el embarazo o un año después del parto. La quimioterapia administrada durante el embarazo puede aumentar la producción de estrés oxidativo e inflamación elevando el riesgo de problemas obstétricos. Para ello, examinamos diferentes marcadores de estrés oxidativo, inflamación y de defensa antioxidante en mujeres con PABC (N=17) antes del tratamiento, antes de cada ciclo (antraciclinas y paclitaxel) y al parto. También se obtuvo sangre de pacientes con cáncer de mama sin embarazo (N=10) antes y después de antraciclinas y mujeres embarazadas sanas (N = 16). Paralelamente, analizamos sangre de cordón umbilical (N=13) y de orina (N=5) 24h de vida de recién nacidos que recibieron quimioterapia en el útero. Muestras de cordón (N=14) y de orina (N=6) de bebés nacidos de madres sanas se utilizaron como controles. En general, las pacientes con PABC exhibían niveles similares de estrés oxidativo y de antioxidantes antes de la quimioterapia que las controles. Sin embargo, las pacientes con PABC tenían aumentada la actividad chitotriosidasa (P=0.004) en comparación con las controles, indicando inflamación. A consecuencia de la quimioterapia, observamos: la actividad chitotriosidasa fue reducida después del tratamiento con paclitaxel en comparación con las controles (P=0.018); la actividad de YKL-40 disminuyó también en comparación con los niveles de antes de la quimioterapia (P=0.047) y los grupos de proteínas-SH fueron significativamente reducidos después del tratamiento con antraciclinas para volver a incrementar seguidamente con paclitaxel en comparación con las controles. (P=0.010 y P=0.012 respectivamente). El efecto acumulativo de la quimioterapia se analizó comparando los niveles basales con los del parto. Encontramos un aumento de daño al ADN y de proteínas en las pacientes con PABC tal como se aprecia en los ratios 8-OHdG/2dG y o-Tyr/Phe (P=0.031; P=0.032 respectivamente). Además, observamos niveles significativamente elevados en tres metabolitos de peroxidación lipídica: 5-F2t-IsoP+5-epi-5-F2t-IsoP (P=0.031), 15-epi-2,3-dinor-15-F2t-IsoP + 2,3-dinor-11-PGF2α + 2,3-dinor-15-F2α-IsoP (P=3.72e-03) y 10-F4t-NeuroP (P=0.031), así como el biomarcador de inflamación, PGF2α (P=0.026) y el ratio antioxidante, GSH/GSSG (P=4.79e-03) en comparación con las controles. No se observó estas diferencias en los biomarcadores de daño al ADN y proteínas. Las mujeres con cáncer de mama no embarazadas, mostraron niveles elevados de daño en el ADN y proteínas (ratios 8-OHdG/ 2dG ratio; P =1.52e-03 y m-Tyr/Phe ratio; P=7.88e-04 respectivamente), pero una capacidad antioxidante reducida (GSH/GSSG ratio, P=5.67e-06) en comparación con las pacientes con PABC. Por otro lado, los recién nacidos expuestos a quimioterapia en el útero presentaron generalmente niveles más bajos de estrés oxidativo e inflamación que los controles. Excepcionalmente, encontramos incrementados los niveles plasmáticos de 15-F2T-isoP (P=8.05e-03) y los niveles urinarios de GSA (P =0.016). Por último, encontramos una correlación positiva entre los niveles de 15-epi-15-F2t-IsoP y 15-epi-2,3-dinor-15-F2t-IsoP + 2,3-dinor-11-PGF2α + 2,3-dinor-15-F2α-IsoP (R=-0.55, P=0.049 y R=-0.68, P=0.011 respectivamente) medidos en sangre de cordón de recién nacidos expuestos a quimioterapia en el útero con sus correspondientes madres al parto. En resumen, la quimioterapia durante el embarazo está significativamente asociado a una alteración del estado redox. Aun así las pacientes con PABC mostraron una alta capacidad antioxidante mientras que los neonatos no se vieron negativamente afectados. Por tanto, las complicaciones obstétricas reportadas en el estudio no fueron causadas por un aumento del estrés oxidativo e inflamación o si lo hubo, fueron eficientemente contrarrestados.
Pregnancy-associated breast cancer (PABC) is defined as breast cancer diagnosed during pregnancy or one-year following postpartum. Chemotherapy treatment in pregnancy may aumentate the production of oxidative stress and inflammation increasing risk of obstetrics complications. For this purpose, we examined multiple oxidative stress, inflammation and antioxidant defence biomarkers in blood samples from PABC patients (N=17) prior to treatment, before each chemotherapy cycle (anthracyclines and paclitaxel) and at labour. Furthermore, the study also included blood samples from non-PABC patients (N=10) before and after treatment with anthracyclines and healthy pregnant women (N=16) as groups of control. We also assessed chemotherapy-induced oxidative stress and inflammation of neonates exposed to chemotherapy in utero using cord blood (N=13) and urine (N=5) samples. Additionally we also collected cord blood (N=14) and urine (N=6) samples from neonates born to healthy pregnant women. Overall, our data showed that PABC patients exhibited similar levels of oxidative stress and antioxidant defence markers as compared to pregnant controls before treatment with chemotherapy. However, we observed that PABC women have increase levels of chitotriosidase (P=0.004), a marker of inflammation. Following chemotherapy, some changes in oxidative stress markers and inflammation markers were found: chitotriosidase was reduced after paclitaxel as compared to healthy pregnant women (P=0.018); YKL-40 was decreased after paclitaxel as compared before treatment (P=0.047) and protein-SH groups were reduced as compared to healthy pregnant women following anthracyclines but then were again increased after paclitaxel (P=0.010 and P=0.012 respectively).The accumulative effect of chemotherapy treatment was analysed by comparing the baseline levels with those obtained at delivery, and our findings demonstrated that chemotherapy exposure during pregnancy increased the DNA and protein damage in PABC patients, as shown by an increase in 8-OHdG/2dG ratio and o-Tyr/Phe ratio, respectively (P=0.031; P=0.032). In addition, we found significant increased levels of three metabolites involved in lipid peroxidation: 55-F2t-IsoP+5-epi-5-F2t-IsoP (P=0.031), 15-epi-2,3-dinor-15-F2t-IsoP + 2,3-dinor-11-PGF2α + 2,3-dinor-15-F2α-IsoP (P=3.72e-03) and 10-F4t-NeuroP (P=0.031); as well as, PGF2α (P=0.026) metabolite of inflammation and antioxidant defence GSH/GSSG ratio (P=4.79e-03) biomarkers compared to controls at delivery. However, these differences were not observed regarding DNA and protein damage.Regarding non-PABC patients, they displayed elevated levels of DNA and protein damage markers (8-OH-dG/2dG ratio; P=1.52e-03 and m-Tyr/Phe ratio, P=7.88e-04 respectively) but reduced antioxidant capacity (GSH/GSSG ratio, P=5.67e-06) compared to PABC patients. On the other hand, neonates exposed to chemotherapy in utero generally displayed lower levels of oxidative stress and inflammation markers than neonates born to healthy women. Exceptionally, we found increased plasma levels of 15-F2f-IsoP (P=8.05e-03) and urine levels of GSA (P=0.016) compounds. Lastly, we found a positive correlation between 15-epi-15-F2t-IsoP and 15-epi-2,3-dinor-15-F2t-IsoP + 2,3-dinor-11-PGF2α + 2,3-dinor-15-F2α-IsoP (R=-0.55, P=0.049 y R=-0.68, P=0.011 respectively) in neonates with intrauterine exposure to chemotherapy and the corresponding mother. In summary, our data show that the administration of chemotherapy during pregnancy is significantly associated with a disruption of redox balance. However, PABC patients showed an elevated antioxidant capacity while the neonates were not negatively affected. Therefore, the obstetric outcomes reported in the study were not caused by an overproduction of oxidative stress and inflammation, or if increased, they were counteracted by the redox system.
Universitat Autònoma de Barcelona. Programa de Doctorat en Bioquímica, Biologia Molecular i Biomedicina

Background: Systemic chemotherapy and radiotherapy play an important role in the treatment of colorectal cancer. Tumour perfusion and oxygenation is known to influence radiosensitivity and chemosensitivity. In this thesis, I propose that the evaluation of changes in tumour perfusion using perfusion CT (pCT) and dynamic contrast-enhanced (Dce) MRI can guide the rational sequencing of drugs and radiation. Methods: Dce-MRI and pCT scans were incorporated into a clinical trial of hypofractionated pelvic radiotherapy and nelfinavir in 10 patients with rectal cancer. Toxicity and tissue biomarkers (tumour cell density, microvessel density, CAIX, HIF1-alpha, phospho-Akt and phospho-PRAS40) were evaluated. pCT liver scans were incorporated into an imaging study in patients with colorectal liver metastases randomised to receive either oxaliplatin/ 5FU chemotherapy or oxaliplatin/ 5FU chemotherapy plus selective internal radiotherapy. Results: After 7 days of nelfinavir concurrent with hypo-fractionated pelvic radiotherapy, there was a mean 42% increase in median K^trans (P=0.03, paired t test) on Dce-MRI and a median 30% increase in mean blood flow on pCT (P=0.028, Wilcoxon Rank Sum), although no statistically significant changes in perfusion parameters were demonstrated after 7 days of nelfinavir prior to radiotherapy. The feasibility of evaluating tumour cell density in rectal biopsies before and after radiotherapy and a radiosensitising drug as an early endpoint of response was demonstrated. In patients with colorectal liver metastases who received oxaliplatin and modified de Gramont chemotherapy alone, after 4 cycles of chemotherapy, a 28% decrease in the mean hepatic arterial fraction was observed (P=0.018, paired t test). Between pCT scans 2 days before SIRT and 39-47 days following SIRT and continued 2-weekly chemotherapy, there was a mean 62% (P=0.009) reduction in Blood Flow and 61% (P=0.006) reduction in Blood Volume (paired t test). Conclusions This research does not support the hypothesis that nelfinavir before radiotherapy improves blood flow to human rectal cancer. Increases in rectal tumour perfusion during radiotherapy and concurrent nelfinavir are likely to be primarily explained by the acute biological effects of radiation. Four or more cycles of oxaliplatin and modified de Gramont chemotherapy may result in changes in tumour perfusion of colorectal liver metastases which would be detrimental to subsequent radiotherapy. Selective internal radiotherapy resulted in substantial reductions in tumour perfusion 39-47 days after the treatment. Perfusion imaging can be used to detect changes in tumour perfusion in response to radiotherapy and systemic therapy which have implications for the sequencing of therapies.

The survival for high-grade glioma patients is poor and the treatment may cause severe side effects. A common obstacle in the treatment is chemoresistance. To improve the quality of life and prolong survival for these patients prognostic biomarkers and new approaches for chemotherapy are needed. To this end, a strategy to evade chemoresistance was evaluated by combining chemotherapeutic drugs with agents inhibiting resistance mechanisms identified by a bioinformatic analysis (paper I). The prognostic value of 13 different proteins was analyzed in this thesis (papers II-IV). Two of them, p38 mitogen-activated protein kinase (MAPK) and protein tyrosine phosphatase non-receptor type 6 (PTPN6, also known as SHP1) were analyzed for their potential as targets in combination chemotherapy (in paper III and IV, respectively).   We found that: PTPN6 expression and methylation status may be important for survival of anaplastic glioma patients, p38 MAPK phosphorylation may be a potential negative prognostic biomarker for high-grade glioma patients and FGF2 expression may be a potential negative prognostic biomarker for proneural glioma patients. PTPN6 may be a useful target for combination chemotherapy with cisplatin, melphalan or bortezomib in high-grade gliomas. The following drug combinations; camptothecin combined with an EGFR or RAC1 inhibitor, imatinib combined with a Notch or RAC1 inhibitor, temozolomide combined with an EGFR or FAK inhibitor and vandetanib combined with a p38 MAPK inhibitor may be useful combination chemotherapy for high-grade gliomas.

L’objectif général de cette thèse a été de mettre en évidence la contribution de l’approche transcriptomique dans la physiopathologie et le traitement des hémopathies malignes. En particulier, comment la technologie des microarrays nous a aidée à étudier diverses problématiques en onco-hématologie.Dans la première partie, notre objectif était d’étudier les cellules Natural killer (Nk) chez les patients atteints de leucémie aiguë myéloïde (LAM). Nous avons comparé la signature transcriptomique des cellules Nk de patients LAM à celle des cellules Nk de sujet sains et suggéré que le facteur de transcription ETS-1 est un bon candidat capable de réguler les récepteurs activateurs NCR (natural cytotoxicity receptors) dont les gènes sont situés sur deux chromosomes différents, même si leur expression reste fortement cordonnée.Dans la seconde partie, nous nous sommes intéressés à la prédiction du sepsis en utilisant une approche transcriptomique dans le cadre de l’autogreffe de cellules souches hématopoïétiques (auto-CSH). En utilisant le même modèle, dans la troisième partie, nous avons mis en évidence l’effet du melphalan en tant que chimiothérapie de conditionnement sur les cellules mononuclées du sang périphérique et identifié un marqueur potentiel de rechute précoce chez les patients atteints de myélome dans le cas de l’auto-CSH. Enfin, dans la dernière partie, notre objectif a été d’analyser le profil d’expression génique des lymphomes B diffus à grandes cellules liés à l’infection par le VIH afin de vérifier ou pas l’existence des sous-types décrits chez les patients immunocompétents
The aim of this research is to demonstrate transcriptomic approach contribution in the physiopathology and treatment of hematological malignancies. In particular, how microarrays technology is used to study several oncohematology difficulties; which remain deaths-related infection, as well as the failure to obtain remission and death related relapse. In the first part, our focus was to study natural killer cells (Nks) in patients affected with acute myeloid leukemia (AML). We compared transcriptomic AML-NKs signature with healthy donors-NKs signature and suggested that ETS-1 transcription factor is a good candidate able to regulate the natural cytotoxicity receptors (NCRs), whose coding genes, are located on two different chromosomes even if their expression remain strongly coordinated.Our second part, aimed to predict sepsis using a transcriptomic approach in the case of autologous stem cell transplantation (auto-HSCT). Using the same model, in the third part, we highlighted the melphalan high-dose chemotherapy effect on peripheral blood mononuclear cells and identified a potential good biomarker of early relapse in patients affected by myeloma in the case of auto-HSCT.Our final focus was to analyze gene expression profile of HIV-related large diffuse B-cell lymphoma type in order to verify the existence of subgroups described in immune-competent patients

Introduction: les progrès thérapeutiques pour la prise en charge du carcinome urothélial de la vessie, ou tumeur de vessie infiltrant le muscle (TVIM), sont faibles depuis deux décennies, notamment sur le plan de la chimiothérapie, tant dans les formes localisées que métastatiques. L'identification de nouveaux biomarqueurs pouvant améliorer la prise en charge et le pronostic des patients apparait indispensable, notamment pour prédire la réponse aux traitements systémiques. Notre travail a cherché : (i) à identifier des facteurs biologiques prédictifs de rechute après les chimiothérapies adjuvantes (CA) utilisées actuellement, (ii) à déterminer si les altérations moléculaires, cibles de nouvelles thérapies, identifiées dans la tumeur vésicale sont conservées dans les métastases.Objectifs:(i) Rechercher une association entre la rechute après CA et l'expression de six biomarqueurs, ERCC1, Emmprin, Survivin, MDR1, p53 et topoisomérase IIα, dans la tumeur vésicale et/ou une métastase ganglionnaire(ii) Etudier l'hétérogénéité des mutations de FGFR3 dans les TVIM localement avancéesMatériels et Méthodes: Une cohorte rétrospective de 226 patients traités par cystectomie et CA, et une collection tumorale d'échantillons représentatifs de la tumeur primitive et d'une métastase ganglionnaire ont été constituées. L'expression des six biomarqueurs a été déterminée en immunohistochimie sur ces échantillons. Le statut mutationnel de FGFR3 a été déterminé en SNaPshot sur 84 paires tumeur primitive/métastase ganglionnaire.Résultats: Les survies observées dans la cohorte étaient proches de celles rapportées dans la littérature pour des patients de mêmes stades. La collection tumorale étudiée semble donc représentative des TVIM traitées en France. Aucun des six biomarqueurs n'était retrouvé comme prédictif de rechute après traitement. Une mutation de FGFR3 était retrouvée dans 4 (4,7%) des 84 échantillons appariés sans aucune discordance entre tumeur primitive et métastase ganglionnaire.Conclusions: L'expression de ces six biomarqueurs étudiés en immunohistochimie n'est pas associée à un risque de rechute après CA. Le statut mutationnel de FGFR3 est conservé au cours de la dissémination métastatique, et ses mutations activatrices sont présentes dans près de 5% des TVIM localement avancées. Leur conservation dans le processus métastatique renforce l'intérêt de l'évaluation de nouvelles thérapies ciblant le récepteur FGFR3 muté. Enfin, il est possible de rechercher cette mutation sur la tumeur vésicale ou une métastase avec la même pertinence
Introduction: In muscle invasive urothelial carcinoma of the bladder (MI UCB), few progresses have been made in the last two decades, particularly in chemotherapy, both in localized and metastatic setting. The identification of new biomarkers that can improve the management and prognosis of patients appears essential. Such biomarkers will be clinically relevant if they can predict the response to systemic treatments. In this thesis, we studied biomarkers in two ways: (i) to identify predictive factors of relapse after cisplatin-based adjuvant chemotherapy (AC), (ii) to confirm that the molecular alterations targeted by new therapies and identified in bladder tumor are present in metastases.Objectives:(i) To examine the relationship between relapse after AC and expression of six biomarkers, ERCC1, Emmprin, Survivin, MDR1 p53 and topoisomerase IIα, in bladder tumor and / or lymph node metastases.(ii) To study the heterogeneity of FGFR3 mutations in locally advanced MI UCBMaterials and Methods: A retrospective cohort of 226 patients treated with cystectomy and AC, and tumor collection of representative samples of the primary tumor and lymph node metastases were constituted. The expression of six biomarkers was determined by immunohistochemistry on these samples and determined by morphometry. FGFR3 mutation status was determined by SNaPshot in 84 paired primary tumors and positive lymph nodes.Results: Survivals observed in the cohort were similar to those reported in the literature for patients of same stages, with tumor collection being representative of the MI UCB treated in France. None of the six biomarkers was found as an independent predictive factor of relapse after AC. FGFR3 mutation was found in 4 (4.7%) of the 84 paired samples with complete concordance between primary tumor and lymph node metastases.Conclusions: The expression of the six studied biomarkers by immunohistochemistry is not associated with risk of relapse following AC. FGFR3 mutation status is maintained during metastases process, and FGFR3 activating mutations are present in about 5% of locally advanced MI UCB. FGFR3 mutation remains a promising therapeutic target in a low subset of patients, and the evaluation of new therapies targeting the mutated FGFR3 receptor are needed. Finally, search for these mutations either in bladder tumor or in metastasis is relevant

Dans le cancer du sein, la recherche de nouveaux biomarqueurs, permettant de prédire la réponse thérapeutique et de déterminer le pronostic d’une patiente, est importante pour adapter au mieux les traitements mais aussi pour améliorer la compréhension des phénomènes physiopathologiques. Nous nous sommes particulièrement intéressés aux cancers du sein traités par chimiothérapie néoadjuvante. Nous avons étudié, dans des biopsies tumorales réalisées avant traitement et dans des résidus tumoraux, à la fois les paramètres télomériques et la réparation des lésions d’ADN puisque ces 2 mécanismes, lorsqu’ils sont dysfonctionnels, sont à l’origine d’une forte instabilité génomique. Nous avons corrélés ces paramètres à la réponse à la chimiothérapie néoadjuvante et à la survie des patientes. Dans un 1er temps, nous avons montré que des télomères courts, une surexpression de la télomérase (TERT) et une sous-expression d’une protéine impliquée dans différents mécanismes de réparation de l’ADN, ERCC1, sont des marqueurs de mauvais pronostic. La dysfonction simultanée des télomères et des mécanismes de réparation de l’ADN peut contribuer de façon synergique à la progression tumorale et à la résistance thérapeutique. Nous avons ensuite étudié une population de tumeurs du sein triple négatives. Nous avons montré que des télomères courts sont associés aux tumeurs les plus agressives et les plus résistantes. De plus, une résistance thérapeutique est retrouvée associée à une instabilité génomique plus importante. Nous avons enfin analysé les altérations génomiques caractéristiques permettant d’identifier le statut BRCA1-like. Le profil BRCA1-like est corrélé à une résistance thérapeutique et à une forte instabilité génomique.Enfin, nous avons réalisé une étude plus fondamentale visant à identifier les mécanismes à l’origine de la réactivation de la télomérase dans le cancer du sein. Nous avons démontré que la surexpression de TERT, dans le cancer du sein, n’est pas liée à la présence de mutations somatiques activatrices mais plutôt à celle de gain du locus TERT. Ces gains sont associés à une résistance thérapeutique et un risque de rechute plus important. Enfin, la présence de gain de TERT combinée à la surexpression de MYC, permet de définir un sous groupe de très mauvais pronostique et pourrait être utilisé pour évaluer le risque de récidives. Les paramètres télomériques et l’instabilité génomique semblent donc être des biomarqueurs prédictifs de la réponse à la chimiothérapie néoadjuvante dans le cancer du sein triple négatif. Ces paramètres ont également une forte valeur pronostique dans le cancer du sein en général et pourraient être utilisés cliniquement comme biomarqueurs utiles au choix des traitements
In breast cancer, discovering new biomarkers, that can predict therapeutic response and prognosis, is important to find the best therapeutic options and improve our understanding of physiopathology. Our particular interest is in breast cancer treated by neoadjuvant chemotherapy (NCT). In pre-NCT biopsies and post-NCT tumors, we studied both telomeric parameters and DNA damage repair (DDR), because when these are dysfunctional, both result in high genomic instability. We correlated these parameters to neoadjuvant chemotherapy response and patient outcomes. First, we demonstrated that short telomeres, high telomerase (TERT) expression and low expression of ERCC1 (a protein involved in a number of DNA repair mechanisms) are markers of poor prognosis. Telomere and DDR dysfunction can contribute synergistically to tumor progression and chemoresistance. We then studied a triple negative breast cancer population. We demonstrated that short telomeres were associated with tumor aggressiveness and chemoresistance. Chemoresistance was also associated with high genomic instability. We analyzed genomic alterations specific to BRCA1-like status and demonstrated that BRCA1-like profile correlated with chemoresistance and high genomic instability. Finally, we performed a comprehensive study of telomerase reactivation in breast cancer. We demonstrated that high TERT expression in breast cancer is not associated with somatic enhancer mutations but more probably to TERT locus gains. These gains were correlated to chemoresistance and increased risk of relapse. TERT gain, combined with high MYC expression, was able to isolate a subgroup with a very poor prognosis, and this could be used to evaluate risk of relapse. Telomeric parameters and genomic instability seem to be predictive biomarkers for neoadjuvant chemotherapy response in triple negative breast cancer. These parameters also have strong prognostic value in breast cancer and could be used clinically as biomarkers for tailoring treatment

Les cancers MSI (Microsatellite Instable) sont caractérisés par un niveau élevé d'instabilité des séquences répétées de l'ADN, les microsatellites, suite à l'inactivation du système MMR (Mismatch Repair). L'étude de la carcinogenèse MSI a révélé des mutations somatiques (délétion/insertion) de nombreux gènes sur des microsatellites codants, qui décalent le cadre de lecture et engendrent la production de protéines tronquées dont la fonction est le plus souvent perdue. Ces altérations affectent des gènes suppresseurs de tumeurs ou apparentés agissant au niveau de voies de signalisation en rapport avec l'oncogenèse, et sont sélectionnées lorsqu'elles promeuvent le développement tumoral. Ces travaux font état de la découverte de la première mutation d'une chaperonne dans une pathologie tumorale, affectant l'oncogène codant pour HSP110 (Heat Shock Protein) sur un microsatellite non-codant intronique situé dans un site accepteur d'épissage. Cette mutation, conduisant à un saut d'exon, est inéluctable et bi-allélique dans l'ADN tumoral, et entraîne la perte des activités oncogéniques d'HSP110 dans les cellules cancéreuses (effet pro-apoptotique, chimio-sensibilisant, antiprolifératif et de ralentissement de la croissance tumorale). Ces résultats remettent en question les mécanismes de l'oncogenèse MSI et le caractère oncogénique de l'instabilité microsatellitaire. Au niveau physiopathologique et clinique, ils pointent HSP110 comme cible pronostique (facteur prédictif de réponse à la chimiothérapie) et thérapeutique. Je propose une approche de traitement pour les patients avec une tumeur MSI, visant à exacerber le caractère délétère de cette mutation dans les cellules tumorales
MSI cancers (MicroSatellite Instability) are characterized by widespread instability of DNA repeated sequences, known as microsatellites, due to MMR system (Mismatch Repair) deficiency. Since the detection of this tumor phenotype, most of the oncogenic events reported in these tumors are somatic mutations (1-2 bp insertions or deletions) that affect coding DNA repeats, resulting in frameshifts and inactivation of the corresponding proteins. They accumulate in tumor cells due to positive selection during the MSI-driven tumorigenic process when they promote tumor development by inactivating genes with tumor suppressor-related functions. This work reports the first somatic mutation of a chaperone protein in a cancer so far, i.e. HSP110 (Heat Shock Protein) in MSI colorectal cancer. This mutational event consists in the somatic deletion of the intronic microsatellite, located in the splice acceptor site of HSP110. We demonstrate that it is almost systematic and bi-allelic in these cancers, leading to inactivation of the oncogenic functions of the HSP110 chaperone (pro-apoptotic and anti-proliferative impact leading to chemosensitization of tumor cells and tumor growth decrease). Our findings support an unexpected and paradoxical anticancer impact of the microsatellite instability-driven pathway in mismatch repair-deficient colon cancer. From a pathophysiological and clinical point of view, they highlight HSP110 as a putative relevant prognostic marker (improvement of patients’ response to chemotherapy) and therapeutic target. According to these findings, I propose a therapeutic strategy targeting HSP110 and its mutant for personalized medicine of MSI colon cancer patients

La métabolomique propose une approche basée sur l’étude d’une réponse métabolique globale et dynamique d’un organisme à des stimuli biologiques ou à des mutations génétiques et ainsi constitue un outil de recherche translationnel à fort potentiel en oncologie. Nous avons appliqué l’approche métabonomique par RMN à haut champ à l’analyse d’échantillons sanguins issus d’études cliniques coordonnées par le Centre Léon Bérard et pour la cohorte prospective E3N (Etude Epidémiologique auprès de femmes de la MGEN) dans le but d’identifier de marqueurs systémiques du cancer à partir de fluides biologiques humains. Tout d’abord, l’analyse statistique des profils métaboliques de sérums de patientes atteintes d’un cancer du sein a permis de mettre en évidence une signature métabolique discriminant les patients ayant un cancer localisé des patientes métastatiques et de révéler des biomarqueurs associés à la sévérité de ce cancer. D’autre part, cette approche permet des investigations sur les effets de la chimiothérapie. Ainsi, lors de l’étude de sérums de patients atteints d’un cancer du rein métastatique traités par une chimiothérapie expérimentale, une signature métabolique caractéristique de la modification plus rapide du métabolisme de l’hôte a été mise en évidence pour ce traitement en comparaison à deux thérapies conventionnelles. Enfin, l’analyse de plasmas sanguins issus de la cohorte E3N s’intéresse à l’identification de biomarqueurs associés à la survenue du cancer du sein et de son étiologie. Après identification et quantification des sources de variations systématiques impactant les profils métaboliques obtenus, une analyse stratifiée de la cohorte « cas prospectifs »-contrôles est présentée dans cette thèse
Metabolomics offers an approach based on the study of a comprehensive and dynamic metabolic response of an organism to biological stimuli or genetic mutations and thus constitutes a tool for translational research with a strong potential in oncology. We applied the metabonomic approach by high field NMR for the analysis of blood samples from clinical studies coordinated by the Centre Léon Bérard and for the prospective E3N cohort ( Etude Epidémiologique auprès de femmes de la MGEN ) in order to identify systemic cancer markers from human biological fluids . First, the statistical analysis of metabolic serum profiles from patients with breast cancer has highlighted a metabolic signature discriminating patients with localized cancer and metastatic patients and reveal biomarkers associated with severity of this cancer. Furthermore , this approach allows investigations on the effects of chemotherapy. Thus, during the study of sera from patients with metastatic renal cell carcinoma treated with experimental chemotherapy, a characteristic metabolic signature of faster modification of the host metabolism has been demonstrated for this treatment in comparison to two standard therapies. Finally, analysis of blood plasma from the E3N cohort interests the identification of biomarkers associated with the development of breast cancer and its etiology . After identification and quantification of sources of systematic variations affecting the metabolic profiles obtained, a stratified analysis of the "prospective case "- controls cohort is presented in this thesis

Neutrophils are prominent immune components of solid tumors, which can protect against the onset of cancer (N1) or have protumor activity (N2). Circulating neutrophils, divided into high density neutrophils (HDN) and low density neutrophils (LDN), functionally mirror those N1 and N2 cells, respectively. LDN, practically absent in non-pathological conditions, have been extensively studied in cancer, due to their increased frequency in this disease and their protumor phenotype. However, this has been mainly demonstrated in animal models and proper validation in humans is an urgent need. In this thesis, we enlightened the clinical impact of LDN in breast cancer (BC) patients. We observed that LDN were practically absent in healthy donors’ blood, while significantly increased in the blood of BC patients, particularly with metastatic disease. Within the population of non metastatic patients, LDN were more prevalent in patients with poor response to neoadjuvant chemotherapy than in patients with good response. The association of a higher incidence of circulating LDN and the worse prognosis of BC patients could be explained by the protumor features exhibited by these cells. Namely, there are more LDN expressing the immunosuppressive markers PD-L1 and CCR4, than HDN. Additionally, LDN also showed increased expression of activation markers; robust formation of neutrophil extracellular traps; augmented phagocytic activity and higher capacity to release reactive oxygen species, which altogether contribute for tumor development and metastization. Moreover, the percentage of LDN in BC patients’ blood was positively correlated with the immunosuppressive CCR4+ regulatory T cells and negatively correlated with activated cytotoxic T lymphocytes, corroborating the impairment on the antitumor immune responses by LDN, which was further demonstrated ex vivo. Hence, this thesis reveals the potential of LDN as a clinical meaningful biomarker of BC response to treatment and opens new avenues for developing targeted immunotherapies.

Colorectal cancer (CRC) is a major public health problem worldwide. Despite improvements in the diagnostic process and advancement in the treatment methods, the prognosis remains poor. To improve survival rates, it is important to identify people with the predisposition for CRC and to detect the potentially curable early stage of the disease. Furthermore, identifying those who would have an adverse clinical outcome associated with a particular chemotherapy would help to avoid redundant chemotherapy burden in patients and contribute to enhanced therapeutic efficacy, while minimizing treatment-related toxicity. The aim of the Thesis was to search for novel promising diagnostic, prognostic and predictive DNA-based biomarkers of sporadic form of CRC. As each patient is genetically unique, these biomarkers would aid clinicians in better diagnosis and/or in the selection of an optimal type of therapy for an individual CRC patient based on their molecular profile. In order to explore this issue, we investigated several candidate genes in healthy individuals as well as in newly diagnosed cancer patients. The major outcomes of this PhD study, which were fully reported in seven publications included in the present Thesis, are 1) The observation of several candidate single nucleotide polymorphisms in microRNA...

碩士
國立臺灣大學
臨床醫學研究所
102
According to the 2011 annual cancer registry report in Taiwan, epithelial ovarian cancer (EOC) ranked eighth in cancer-related death and 1240 women was diagnosed in 2011 with an incidence 8.3 per 100000 women. Ovarian cancer, especially epithelial ovarian carcinoma (EOC), has become an extremely important disease in recent years because it has the highest mortality rate of all gynecological malignancies [2,3,21]. with an overall 5-year survival rate of only 20-30% [2]. It became a more and more important disease in recent years [18,27] and the incidence of ovarian cancer also increased in recent year in Taiwan. Initial evaluation for these patients includes a thorough history and physical examination, imaging studies, assessment of tumor markers (CA-125, CA-199, HE4), and/or colonoscopy and upper panendoscopy [23]. The lack of symptoms, difficulties in early diagnosis, insufficient accurate tumor markers, and lack of information about ovarian tumor biology contribute to the poor prognosis in ovarian cancer patients [9]. The prognostic parameters for ovarian carcinomas are tumor stage, histologic subtype, degree of malignancy, and residual tumor after surgical treatment [24,29]. These factors present an incomplete picture of the tumor biology of ovarian cancer and are frequently interrelated [26]. For these reasons, many patients may be delay diagnosed. Thus, the identification of new biologic factors predictive of individual disease course and prognosis would be extremely useful. From the above-mentioned data, ovarian cancer indeed is a disease that should be respected it in Taiwan. We used immunohistochemical staining combined with tissue microarray for evaluation of gene expression;combined with bioformatics with clinicopathological data in search of biomarkers related to chemotherapy response and disease prognosis

Platinbasierte Chemotherapie ist die effektivste Chemotherapie für das fortgeschrittene Nebennierenrindenkarzinom (ACC). Excision repair cross complementing group 1 (ERCC1) spielt eine entscheidende Rolle bei der Reparatur durch Platin entstandener DNA-Schäden. Zwei Studien die die Rolle von ERCC1 per Immunhistochemie als prädiktiver Marker für das Ansprechen auf platinbasierte Chemotherapie beim ACC untersuchten kamen zu sich widersprechenden Ergebnissen. Beide Studien nutzten den ERCC1-Antikörper Klon 8F1 der sich später als nicht spezifisch herausstellte. Das Ziel der Doktorarbeit war die Evaluation von ERCC1 mit einem neuen hoch spezifischen Antikörper in einer großen Kohorte von Patienten mit ACC. Material und Methoden: 146 Patienten mit verfügbaren FFPE-Schnitten wurden eingeschlossen. Alle Patienten erhielten eine platinbasierte Chemotherapie im Median für 6 Zyklen, nämlich Cisplatin (n=131) oder Carboplatin (n=15), in den meisten Fällen in Kombination mit Etoposid (n=144 , Doxorubicin (n=131) und Mitotane (n=131). Die Immunhistochemische Färbung wurde mit dem neuen Antikörper Klon 4F9 durchgeführt. Der Zusammenhang zwischen ERCC1-Expression und klinischen Parametern, Therapieansprechen, progressionsfreiem Überleben und Gesamtüberleben wurde analysiert. Ergebnisse: Eine hohe ERCC1-Expression wurde in 66% der Tumorproben beobachtet. Während der Chemotherapie zeigte sich bei 43 Pateinten ein Therapieansprechen (29,5%), bei 49 Patienten eine Stabilisierung der Erkrankung (33,6%), bei 8 Patienten ein gemischtes Ansprechen (5,5%) und bei 46 Pateinten ein Krankheitsprogress (31,5%), ohne Zusammenhang zur ERCC1-Expression. Auch zeigte sich kein signifikanter Zusammenhanf zwischen der ERCC1-Expression und dem progressionsfreien Überleben (Median 6.5 vs. 6 Monate, P=0.33, HR=1.23, 95%CI=0.82-2.0) oder dem Gesamtüberleben. Zusammenfassung: Es besteht kein Zusammenhang zwischen der ERCC1-Expression und der Platinsensitivität beim Nebennierenrindenkarzinom. Somit werden andere Biomarker zur Therapieentscheidung benötigt
Objective: Platinum-based chemotherapy (PBC) is the most effective cytotoxic treatment for advanced adrenocortical carcinoma (ACC). Excision repair cross complementing group 1 (ERCC1) plays a critical role in the repair of platinum-induced DNA damage. Two studies investigating the role of ERCC1 immunostaining as a predictive marker for the response to PBC in ACC had reported conflicting results. Both studies used the ERCC1-antibody clone 8F1 that later turned out to be not specific. The aim of this study was to evaluate the predictive role of ERCC1 with the new specific antibody in a larger series of ACC. Design and methods: 146 ACC patients with available FFPE slides were investigated. All patients underwent PBC (median cycles=6), including cisplatin (n=131) or carboplatin (n=15), in most cases combined with etoposide (n=144), doxorubicin (n=131) and mitotane (n=131). Immunostaining was performed with the novel ERCC1-antibody clone 4F9. The relationship between ERCC1 expression and clinico-pathological parameters, as well as best objective response to therapy and progression-free survival (PFS) during PBC was evaluated. Results: High ERCC1 expression was observed in 66% of ACC samples. During PBC, 43 patients experienced objective response (29.5%), 49 stable disease (33.6%), 8 mixed response (5.5%) and 46 progressive disease (31,5%) without any relationship with the ERCC1 immunostaining. No significant correlation was also found between ERCC1 expression and progression-free survival (median 6.5 vs 6 months, P=0.33, HR=1.23, 95%CI=0.82-2.0). Conclusion: ERCC1 expression is not directly associated with sensitivity to PBC in ACC. Thus, other predictive biomarkers are required to support treatment decisions in patients with ACC

Chemotherapy is one of the basic therapeutic procedures of breast cancer (BC) which may precede and/or follow the surgical resection of a tumor as a part of neoadjuvant or adjuvant therapy. However, the selective pressure of chemotherapy on tumor cells may change their molecular and expression profile and thus also their chemosensitivity. The aim of our work was to document the expression changes of selected markers in BC after neoadjuvant chemotherapy, which may contribute to the understanding of the role of these proteins and genes in tumor response to chemotherapy and the development of chemoresistance. Immunohistochemical analysis of expression of standard BC markers [estrogen (ER) and progesterone receptors (PR), HER2 and proliferation activity (Ki67)] and intercellular junction proteins (claudin 1, 3 and 4, E- and N-cadherin) before and after neoadjuvant chemotherapy revealed a decrease of PR, Ki67 and claudin 3 expression and an increase of claudin 1 expression. The expression of ER, HER2, claudin 4, E- and N-cadherin proved to be stable. Assessment of standard BC markers is performed routinely during a bioptic investigation as a necessary factor for therapy indication. Our results support the current recommendations for the re-examination before indication of adjuvant chemotherapy. Claudins...

Dissertação de Mestrado em Farmacologia Aplicada apresentada à Faculdade de Farmácia
Lung cancer worldwide is a major cause of mortality. Pathologically, lung cancer is a heterogeneous disease, classically divided into two main categories, namely NSCLC (non-small cell lung cancer) and SCLC (small cell lung cancer). NSCLC represent 85% of malignant lung cancer and are subdivided into three categories: adenocarcinoma, squamous cell carcinoma and large cell carcinoma. The therapeutic option chosen for the treatment of any type of cancer is always dependent on the stage of the tumour at the diagnosis and on its biological characteristics. In lung cancer, this decision is a substantial challenge, since most of the patients are diagnosed at advanced stages, with a 5-years survival estimate. Thus, constant research in an area of intervention as important as cancer is unquestionably necessary. One of these areas is metabolomics, defined as the study of biological processes involving low molecular weight compounds (metabolites), present in cells or organisms, which participate in the maintenance of cellular functions. The main objective of this research project is to understand the altered metabolism of NSCLC cell lines, using metabolomics. Thus, it is intended to study changes in the metabolomic profile after different treatment regimens, namely after exposure to drugs regularly used in clinical practice within classic QT regimens (carboplatin/cisplatin) in which these are used in combination with pemetrexed and gemcitabine. In immunotherapy, namely ICPIs (immune checkpoint inhibitors) such as nivolumab and pembrolizumab, to reveal potential biomarkers.The first part of this work included the characterization of two lung cancer tumour cell lines (A549 and H1299) in terms of morphological and immunohistochemical characterization.Subsequently, the effect of different drugs and their combinations was evaluated on cell proliferation, using the study metabolic activity, through MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The second part of this work aimed to quantify intra or intercellular metabolites in view of the exposure of cells to different therapies using nuclear magnetic resonance spectroscopy. Finally, to support the study of metabolomics, cell viability and the profile of cell death caused by the different therapeutic approaches studied were evaluated, using double labelling with annexin V and propidium iodide by flow cytometry. The characterization of the cell lines showed that H1299 cell line is representative of large cell carcinoma and A549 cell line is representative of adenocarcinoma. Both cell lines do not express the PD-L1 receptor (Programmed death ligand 1), so studies with ICPIs, namely nivolumab and pembrolizumab, were not followed up, as their mode of action is dependent on this receptor. The drugs and combinations, which showed greater anti-proliferative activity in the H1299 cell line, were cisplatin associated with gemcitabine. In the A549 cell line, it can be concluded that cisplatin administered alone showed greater anti-proliferative activity. Thus, the association between cisplatin and gemcitabine is seen as an excellent candidate for combination therapy, as it is associated with moderate side effects and toxicity is dependent on the dose administered. The study of cell viability did not verify that there was only a decrease of approximately 10% of viable cells, without compromising the metabolomic analysis. The technique of spectroscopy by nuclear magnetic resonance allows quantifying several metabolites and obtaining the metabolic profile of cells after exposure to different chemotherapeutic treatment regimens. In addition to the existing constraints due to the pandemic, technical issues associated with a failure in the nuclear magnetic resonance equipment in which these samples would be analyzed hampered the obtaining of the metabolomics results. Thus, the continuity of this work comprises the quantification of the metabolites present in the samples of the cell lines under study and subsequent identification of possible biomarkers in vitro and the translate this preclinical study into a clinical study, whose main objective is to validate the identification of these biomarkers in biofluids, in order to understand if they could be considered specific biomarkers for lung cancer.
O cancro do pulmão, a nível mundial, constitui uma das principais causas de mortalidade. Em termos patológicos o cancro do pulmão é uma doença heterogénea, classicamente dividida em duas categorias principais, os NSCLC (do inglês, non-small cell lung cancer) e os SCLC (do inglês, small cell lung carcinoma). Os NSCLC, representam cerca de 85% das neoplasias malignas do pulmão e subdividem-se em três subcategorias: adenocarcinoma, carcinoma de células escamosas e carcinoma de grandes células. A opção terapêutica recomendada para o tratamento de qualquer tipo de cancro é sempre condicionada pelo estádio em que se encontra o tumor no momento do diagnóstico e pelas características biológicas do mesmo. No cancro do pulmão esta tomada de decisão torna-se um desafio ainda mais substancial, já que a maioria dos doentes são diagnosticados em estádios avançados, estimando-se uma sobrevida de 5 anos. A investigação constante, numa área de intervenção tão importante como o cancro, é inquestionavelmente necessária. Uma dessas áreas é a metabolómica, definida como o estudo de processos biológicos envolvendo compostos de baixo peso molecular (metabolitos), presentes em células ou organismos, que participam na manutenção das funções celulares.O principal objetivo deste projeto de investigação foi compreender as alterações do metabolismo de células de linhas de cancro do pulmão do tipo não pequenas células, com recurso à metabolómica. Pretendemos estudar as alterações no perfil metabolómico após diferentes regimes de tratamento, nomeadamente após exposição a fármacos utilizados regularmente na prática clínica nos regimes clássicos de quimioterapia (QT) (carboplatina/cisplatina) em que estes são usados em combinação com o pemetrexede e a gemcitabina. Na imunoterapia, nomeadamente os ICPIs (do inglês immune checkpoint inhibitors), tais como o nivolumab e o pembrolizumab, foram igualmente alvo de estudo, a fim de tentar revelar potenciais biomarcadores.A primeira parte do trabalho consistiu na caracterização de duas linhas celulares tumorais de NSCLC (A549 e H1299) em termos de morfologia e de imunocitoquímica. Posteriormente, avaliou-se o efeito dos diferentes fármacos e suas combinações, na proliferação celular, recorrendo ao estudo da atividade metabólica, por espetrofotometria, através do ensaio MTT ( do inglês, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).A segunda parte deste trabalho teve como objetivo quantificar os metabolitos intra ou intercelulares, face à exposição das células às diferentes terapias, com recurso à espetroscopia por ressonância magnética nuclear. Por último, para apoiar o estudo da metabolómica, foram avaliadas a viabilidade celular e o perfil de morte celular provocadas pelos diferentes regimes terapêuticos, com recurso à dupla marcação com anexina V e iodeto de propídeo, através da técnica de citometria de fluxo.A caracterização das linhas celulares mostrou que a linha celular H1299 é representativa do carcinoma de grandes células e a linha celular A549 é representativa de adenocarcinoma. Ambas as linhas celulares não expressam o recetor PD-L1 (do inglês, Programmed death ligand 1) pelo que não foi dado seguimento aos estudos com os ICPIs, nivolumab e pembrolizumab, visto o seu mecanismo de ação estar na dependência deste recetor. Os fármacos e as combinações que revelaram maior atividade anti-proliferativa na linha celular H1299 foram a cisplatina associada à gemcitabina. Por outro lado, na linha celular A549, concluiu-se que a cisplatina administrada isoladamente foi a que apresentou maior atividade anti-proliferativa. Assim, a associação entre a cisplatina e a gemcitabina é um excelente candidato à terapia combinada, pois está associada a efeitos adversos moderados e a toxicidade está dependente da concentração administrada.O estudo da viabilidade celular permitiu-nos verificar que apenas houve uma diminuição de aproximadamente 15% de células viáveis, não comprometendo a análise metabolómica.A técnica de espetroscopia por ressonância magnética permite quantificar diversos metabolitos e obter o perfil metabolómico das células após exposição a diferentes regimes de tratamento quimioterapêuticos. Além dos constrangimentos existentes devido à pandemia, questões técnicas associadas a uma avaria no equipamento de ressonância magnética nuclear, no qual estas amostras seriam analisadas, impediram a obtenção dos resultados da análise metabolómica. Assim, a continuidade deste trabalho prende-se com a quantificação dos metabolitos presentes nas amostras das linhas celulares em estudo e posterior identificação de possíveis biomarcadores in vitro, como também a translação deste estudo pré-clínico para um estudo clínico, cujo principal objetivo consiste na validação da identificação desses biomarcadores em biofluídos, de forma a perceber se poderão efetivamente ser considerados biomarcadores específicos para o cancro do pulmão.

Background: Breast cancer is a complex disease, both phenotypically and etiologically. Accordingly, the responses to various treatments in the adjuvant setting among individuals vary considerably. There is a demand for tools that can distinguish patients who may benefit or may suffer from particular systemic treatments. We hypothesized that combination of data on genetic biomarkers with data from traditional clinical and pathophysiological (clinicopathologic) factors using traditional Cox model or Support Vector Machine (SVM) method, a new machine learning method, may provide a better tool for prediction of benefits to chemotherapy for the treatment of early breast cancer than using either biomarker or clinicopathologic data alone. Methods: This project included 531 patients from NCIC-CTG MA.5 trial who had data on both clinicopathologic factors, such as age, tumor size, ER status, type of surgery, tumor grade and lymph node involvement, and biomarkers assayed on tissue microarrays (TMAs), including HER2, p53, CA9, MEP21, clusterin, pAKT, COX2 and TOP2A. The Cox model and SVM methods were used to develop prognostic indices for relapse-free or overall survival with either data from TMAs and clinicopathologic assessments alone or their combination. The prognostic indices developed were then examined for their value as predictive classifiers for benefits from CEF treatment. The power of the predictive classifiers derived was evaluated and compared using the bootstrap approach. Results: None of the prognostic indices developed were found to have significant predictive value, although the prognostic index developed using SVM method based on only biomarkers yielded a marginal significant p-value (p=0.0527) for the interaction between classifier and treatment. In accordance with results published previously, the interaction between the classifier developed based on HER2 or TOP2A and treatment was significant (p=0.02 and 0.04 respectively). Comparisons based on the bootstrap approach indicate classifiers developed based on SVM performed better than those based on the Cox model method. Conclusions: Combination of data using biomarkers and clinical-pathological factors, and using either the traditional COX model method or the new machine learning method was not shown to perform better than two single previously known biomarkers in prediction of response to CEF treatment for early breast cancer.
Thesis (Master, Community Health & Epidemiology) -- Queen's University, 2010-04-27 10:16:11.811

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia
O estágio curricular é a última etapa de uma formação de 5 anos conferida pelo Mestrado Integrado em Ciências Farmacêuticas na Faculdade de Farmácia da Universidade de Coimbra. É a ligação entre a formação teórica adquirida até então e a realidade prática. Este relatório pretende explicar a minha experiência do estágio realizado na Farmácia Feliz em Mangualde sob a forma de uma análise SWOT. A análise SWOT divide as experiências vividas em Pontos fortes (Strenghts); Pontos fracos (Weaknesses); Oportunidades(Opportunities); e Ameaças (Threats).O farmacêutico não está só presente na farmácia comunitária, mas em todas as etapas do medicamento e portanto é importante que a formação incida em todas essas áreas. Tive a oportunidade de realizar um estágio em indústria farmacêutica além do estágio em farmácia comunitária, único que é obrigatório no plano curricular do MICF. Este relatório pretende explicar a minha experiência do estágio realizado nos laboratórios Basi, em Mortágua, sob a forma de uma análise SWOT. A análise SWOT divide as experiências vividas em Pontos fortes (Strenghts); Pontos fracos (Weaknesses); Oportunidades (Opportunities); e Ameaças (Threats).A epigenética pode ser definida como mecanismos que modulam a expressão genéticas em alterar a sequência primária de DNA. Os três principais grupos de alterações epigenéticas que podemos enumerar são a modificação química das bases do DNA,nomeadamente a metilação do DNA por adição de um grupo metilo ao quinto carbono de uma citosina, reação esta que é catalisada pelas DNMT’s; modificações pós-translacionais de histonas e RNAs não codificantes, sendo os mais relevantes os miRNAs. O epigenoma de cada indivíduo não é, assim, constante ao longo da nossa vida, podendo ser alterado pela dieta, pelo metabolismo, ambiente, xenobióticos e, também por doença, como acontece no cancro. Com base nesse conhecimento foram desenvolvidos fármacos que atuam na maquinaria epigenética, modulando o perfil epigenético (o epigenoma) da célula como, por exemplo, DNMTi, HDACi, inibidores de acetiltransferases, demetilases e metiltrasferasesdas histonas e ainda fármacos que atuam nas proteínas reconhecedoras de marcas epigenéticas. Uma das maiores causas de insucesso no tratamento do cancro é o desenvolvimento de resistência à quimioterapia e as alterações epigenéticas podem estar na base de alguns casos. Fármacos epigenéticos estão a ser associados às quimioterapias mais clássicas para contornar este problema e aumentar e eficiência do tratamento. Por outro lado, o diagnóstico precoce é mais uma ferramenta que pode aumentar a taxa de sucesso do tratamento do cancro por isso é importante o estudo de biomarcadores que sejam específicos, precoces e o menos invasivos possível e aqui, mais uma vez, a epigenética releva a sua importância nas futuras direções da ciência. Neste trabalho discutem-se novas estratégias biomédicas que utilizam a modificação do epigenoma com vista à terapia do cancro.
The curricular internship is the last stage of a 5-year training conferred by theIntegrated Master's Degree in Pharmaceutical Sciences at the Faculty of Pharmacy of theUniversity of Coimbra. It is the link between the theoretical training acquired up until thenand the practical reality. This report is meant to explain my experience from the stage heldat Farmácia Feliz in Mangualde in the form of a SWOT analysis. The SWOT analysis dividesthe lived experiences in Strenghts; Weaknesses; Opportunities and Threats.The pharmacist is not only present in the community pharmacy, but in all drug stagesand therefore it is important that the training focuses on all these areas. I had theopportunity to undertake an internship in the pharmaceutical industry beyond the internshipin community pharmacy, which is the only mandatory in the curricular plan of the MICF. Thisreport is meant to explain my experience from the stage held at Laboratórios Basi in Mortáguain the form of a SWOT analysis. The SWOT analysis divides the lived experiences inStrenghts; Weaknesses; Opportunities and Threats.Epigenetics can be defined as the mechanisms that modulate gene expression but don’talter the primary DNA sequence. The three major groups of epigenetic changes that can beenumerated are the chemical modifications of DNA bases, namely DNA methylation, byaddition of a methyl group to the fifth carbon position of a cytosine, reaction catalyzed byDNMT’s; posttranslational changes of histones and non-coding RNAs, the most relevantbeing miRNAs. The epigenome of each isn’t constant throughout life, it can be altered, byseveral factors, including diet, environment, xenobiotics, metabolism and by disease states.Based on that knowledge, new drugs that act in the epigenetic machinery have beendeveloped to modulate the epigenome, such as DNMTi, HDACi, acetyltransferases,demethylases and methyltransferases inhibitors as well and drugs that act on epigeneticreader proteins. One of the biggest causes of failure in the cancer treatments is theresistance to chemotherapy and epigenetic alterations may be one of the underlying causes.Epigenetic drugs are being associated with more classical chemotherapy drugs to circumventthis problem and increase the efficiency of the treatment. The early diagnosis is an importanttool to increase the rate of success in the cancer treatment and because of this is importantthe study of biomarkers that are specific and less invasive as possible, and here again, theepigenetics reveals its importance in the future directions of science. In this work wesummarize these new avenues of biomedical research with emphasis in the epigeneticsapproaches to cancer therapy.

Trabalho de Projeto do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina
Objectives: Analyse the results of the treatment of Testicular Germ Cell Tumours (TGCT) patients orchiectomized at Centro Hospitalar e Universitário de Coimbra (CHUC) over a period of seven years, comparing the results with international data and previous studies (1990-2009) regarding CHUC. Identify new factors that justify the increase in worldwide incidence values described in the literature and new prognostic approaches based on tumoural biomarkers.Methods: 77 clinical registries were analysed, corresponding to the patients orchiectomized due to suspect of testicular cancer, diagnosed with TGCT, between January 2009 and November 2016 at CHUC. For comparison of diagnosis, treatment and follow-up, the European Association of Urology’s 2016 guidelines were followed.Results: An average of 11 surgeries per year were performed, with a mean age of 32.9 years (17-58). The seminomas had a mean age of 35.8 years (29-51) and non-seminomatous (NSGCT) 31 years (17-58). Testicular prosthesis was inserted in 48 (62.3%) cases. Regarding the histopathological diagnosis, 47 (61%) were classified as NSGCT and 30 (39%) as seminoma. Metastasis were detected in 24 (31.2%) of the cases, 20 (83.3%) of them corresponding to NSGCT. According to the 2009 TNM classification, 67.5% were staged as stage I, 14.3% stage II and 18.2% as stage III. Stage I seminomas were treated mainly with a single cycle of Carboplatin (11 patients, 40.1%) as well as active surveillance (also 40.1%). In stage II and III seminomas and NSGCT stage I, II and III the most frequent first adjuvant therapy was chemotherapy with BEP. One seminoma (3.7%) and four (8.5%) NSGCT required second-line chemotherapy. A statistically significant correlation between survival and post-orchiectomy values, absolute and percentual variation of β-HCG was found (respectively p=0.01, ρ=0.384; p=0.003, ρ=0.355; p=0.004, ρ=0.337). Complete remission was achieved in 79.2% of the sample, with relapse in six (7.8%) patients and death after refractory disease in seven (9.1%) patients. Disease free survival was on average 33.9 months All patients treated with second-line chemotherapy died, with a mean survival time of 10.8 months (7-17). Survival at the of the first year was achieved in 96% of the patients and 90% at the end of third and fifth (Seminoma:100%, 96%, 96%; NSGCT: 93%, 85%, 85%).Discussion and Conclusions: There was an average of 11 cases per year (comparatively to 4.5 from 1990 e 2009). The sample mean age increased, from 30.1 to 32.9 years, due to the increase in NSGCT mean age (27.1 to 31.0). There was an increase in the adhesion to the testicular prosthesis (42.2% to 62.3%). Compared to the previous study, we found a higher rate of diagnosis at earlier stages (NSGCT stage III decreased from 42.9% to 27.7%; stage I seminomas increased from 79.3% to 90%). Based on the statistically significant correlation between the variation β-HCG and survival, we propose that, when interpreted by itself, a decrease in β-HCG may be considered a predictor of patient’s survival. When compared to previous studies, a higher rate of survival was attained but still inferior the internationally published data. In localized and regional disease, the 5-year survival of 100% was superior to the described in the literature.
Objetivos: Analisar os resultados do tratamento de Tumores de Células Germinativas do Testículo (TCGT) orquidectomizados no Centro Hospitalar e Universitário de Coimbra (CHUC) num período de sete anos comparando-os com o panorama internacional e estudos anteriores (1990-2009) desenvolvidos no CHUC. Identificar novos fatores que justifiquem o aumento da incidência mundial descrito na literatura e novas abordagens prognósticas a partir dos biomarcadores tumorais.Métodos: Foram analisados 77 processos clínicos, correspondentes aos doentes orquidectomizados por suspeita de tumor testicular, diagnosticados com TCGT entre janeiro de 2009 e novembro de 2016 no CHUC. Para comparação do diagnóstico, tratamento e seguimento foram seguidas as recomendações de 2016 da Associação Europeia de Urologia.Resultados: Foram realizadas, em média, 11 cirurgias por ano, com uma média de idades de 32,9 anos (17-58). Os seminomas apresentaram uma média de idades de 35,8 anos (29-51) e os Não Seminomatosos (TCGNS) 31 anos (17-58). Foi colocada prótese testicular em 48 (62,3%) casos. No diagnóstico histopatológico, 47 (61%) foram classificados como TCGNS e 30 (39%) como seminoma. Foi detetada doença metastática em 24 (31,2%) casos, correspondendo 20 (83,3%) a TCGNS. De acordo com a classificação TNM 2009, 67,5% dos tumores foram estadiados como estadio I, 14,3% estadio II e 18,2% estadio III. No tratamento dos seminomas estadio I, as estratégias mais usadas foram um ciclo de Carboplatina em 11 (40,1%) casos e vigilância ativa no mesmo número (40,1%). Nos seminomas estadio II e III e TCGNS estadio I, II e III a primeira terapêutica adjuvante mais frequente foi quimioterapia com BEP. Um (3,7%) seminoma e quatro (8,5%) TCGNS necessitaram de terapêuticas de segunda linha. Observou-se uma correlação estatisticamente significativa entre a sobrevivência e a variação absoluta, percentual e o valor β-HCG pós-orquidectomia (respetivamente p=0,003, ρ=0,355; p=0,004, ρ=0,337; p=0,01, ρ=0,384). Foi alcançada remissão completa em 79,2% da amostra estudada, ocorrendo recidiva em seis (7,8%) e óbito após doença refratária ao tratamento em sete (9,1%) doentes. Registámos uma sobrevivência média livre de doença de 33,9 meses. Todos os doentes que necessitaram de quimioterapia de segunda linha faleceram, com sobrevivência média de 10,8 meses (7-17). Observámos sobrevivência ao fim do primeiro ano de 96% e 90% ao fim de três e cinco anos (Seminomas: 100%, 96%, 96%; TCGNS: 93%, 85%, 85%).Discussão e conclusão: Registámos, em média, 11 casos por ano (comparativamente a 4,5 entre 1990 e 2009). A idade média da amostra aumentou, de 30,1 para 32,9 anos, à custa do aumento da idade nos TCGNS (27,1 para 31,0). Também na adesão à prótese testicular encontrámos um aumento (42,2% para 62,3%). Verificou-se uma maior taxa de diagnósticos em estadios mais precoces (TCGNS estadio III: 42,9% para 27,7%; seminomas estadio I: 79,3% para 90%). Com base na correlação estatisticamente significativa entre a variação da β-HCG e a sobrevivência, propomos que, quando interpretada isoladamente, uma diminuição da β-HCG possa ser considerada um preditor da sobrevivência do doente. Registou-se uma taxa de sobrevivência superior aos estudos anteriores, mas ainda inferior aos dados publicados internacionalmente. Na doença localizada e regional, a sobrevivência de 100% a 5 anos registada, supera o descrito na literatura.

Introduction: Les mutations du gène RAS sont présentes dans plusieurs types de cancers et ont une influence sur la réponse à la chimiothérapie. Excision repair cross- complementation group 1 (ERCC1) est un gène impliqué dans la réparation de l’acide désoxyribonucléique (ADN), et son polymorphisme au codon 118 est également associé à la réponse au traitement. Le peu d’études pronostiques portant sur ces deux gènes dans les cancers oto-rhino-laryngologiques (ORL) ne permet de tirer des conclusions claires. Objectifs: Déterminer l’influence des mutations de K-RAS codons 12 et 13 et du polymorphisme de ERCC1 codon 118 dans le traitement des cancers épidermoïdes avancés tête et cou traités par chimioradiothérapie concomitante à base de sels de platine. Méthode: Extraction de l’ADN provenant de spécimens de biopsie de patients traités par chimioradiothérapie concomitante pour des cancers avancés tête et cou, et ayant un suivi prospectif d’au moins deux ans. Identification des mutations de K-RAS codons 12 et 13 et du polymorphisme de ERCC1 au codon 118 dans les spécimens et corrélation de ces marqueurs avec la réponse au traitement. Résultats: Les mutations de K-RAS codon 12 sont associées à un moins bon contrôle loco-régional par rapport aux tumeurs ne démontrant pas la mutation (32% vs 83% p=0.03), sans affecter pour autant la survie globale. Aucune mutation de K-RAS codon 13 n’a été identifiée. Les différents polymorphismes de ERCC1 n’ont pas eu d’impact sur la réponse au traitement. Conclusion: Les mutations de K-RAS codons 12 et 13 et le polymorphisme de ERCC1 au codon 118 ne semblent pas mettre en évidence les patients qui bénéficieraient d’une autre modalité thérapeutique.
Background: RAS gene mutations have been shown to occur in certain malignancies and have an impact treatment response and overall prognosis. Excision repair cross- complementation group 1 (ERCC1) is a gene implicated in deoxyribonucleic acid (DNA) repair, whose polymorphism at codon 118 has been linked to treatment response. Studies of these two genes in head and neck oncology literature have shown inconsistent results. Objectives: Determine the influence of K-RAS mutations (codons 12 and 13) and the polymorphism of ERCC1 codon 118 in patients with locally advanced head and neck cancer treated with concomitant platinum-based chemoradiation therapy. Methods: DNA extraction from paraffin-embedded biopsy specimens of patients with advanced head and neck squamous cell carcinoma treated with concomitant chemoradiation and followed prospectively for at least two years. Identification of K- RAS mutations (codons 12 and 13) and ERCC1 codon 118 polymorphism in the extracted DNA. Correlation of these markers with treatment response. Results: K-RAS codon 12 mutations were associated with a worse locoregional control than tumors without any mutations (32% vs 83% p=0.03); however, mutational status did not influence overall survival. No K-RAS codon 13 mutation was identified in our specimens. The different ERCC1 polymorphisms did not have an impact on treatment response. Conclusion: K-RAS mutational status (codon 12 and 13) and ERCC1 codon 118 polymorphism does not seem to discriminate between patients for whom another treatment option should be sought in patients with locally advanced head and neck squamous cell carcinoma.