Identificación de biomarcadores de cáncer de laringe en pacientes candidatos a terapias de preservación de órgano.

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! UNIVERSIDAD DE SEVILLA Programa de Biología Molecular, Biomedicina e Investigación Biomédica Identificación de biomarcadores de cáncer de laringe en pacientes candidatos a terapias de preservación de órgano ___ Identification of novel laryngeal cancer biomarkers in patients treated with organ preservation approaches. TESIS DOCTORAL María José de Miguel Luken Director: Amancio Carnero Moya Trabajo realizado en el Hospital Universitario Virgen del Rocío en colaboración con el Instituto de Biomedicina de Sevilla (IBIS). ! ! ! ! ! ! ! ! ! A mi madre, de quien he aprendido a levantarme tras cada caída, y me ha acompañado en cada etapa del Camino ! ! INDEX - ÍNDICE ACKNOWLEDGMENTS - AGRADECIMIENTOS ............................................................. 10 SUMMARY ................................................................................................................................ 13 RESUMEN ................................................................................................................................. 15 INTRODUCTION ..................................................................................................................... 18 1.1 LARYNX CANCER EPIDEMIOLOGY ........................................................................... 19 1.2 ANATOMY AND HISTOLOGY OF THE LARYNX. PATTERNS OF SPREAD. TNM STAGING ................................................................................................................................ 22 1.2.1. General Anatomy, histology and pathology ............................................................... 22 1.2.2. Patterns of spread ...................................................................................................... 26 1.2.3. AJCC staging and survival ......................................................................................... 28 1.3 RISK AND PROGNOSTIC FACTORS FOR LARYNGEAL CANCER .......................... 30 1.3.1. Risk factors of laryngeal cancer ................................................................................. 30 1.3.2. Clinical and pathological prognostic factors ............................................................. 34 1.4 MOLECULAR BIOLOGY OF LARYNGEAL CANCER ................................................ 34 1.4.1. Genetic susceptibility ................................................................................................. 34 1.4.2. Cytogenetic alterations .............................................................................................. 35 1.4.3. TP53 ........................................................................................................................... 36 1.4.4. Notch .......................................................................................................................... 36 1.4.5. EGFR/EGFRvIII ......................................................................................................... 36 1.4.6. DNA damage repair biomarkers. MAP17 and pH2AX .............................................. 37 1.4.7. Vaccinia-related kinase-1 (VRK1) protein ................................................................. 38 1.4.8. STAT3 pathway .......................................................................................................... 38 1.4.9. Micro RNAS ................................................................................................................ 39 1.4.10. Tumour microenvironment. Immune-phenotypes ..................................................... 40 1.5 LARYNGEAL CANCER TREATMENT ......................................................................... 41 1.5.1. Preservation approaches ............................................................................................ 41 1.5.2 Metastatic disease (stage IVC) .................................................................................... 43 HYPOTHESIS, OBJECTIVE AND ENDPOINTS OF THE STUDY .................................. 45 2.1. HYPOTHESIS .................................................................................................................. 45 2.2. GOAL ................................................................................................................................ 46 2.3. ENDPOINTS ..................................................................................................................... 46 2.3.1 Primary endpoint ......................................................................................................... 46 ! ! 2.3.2 Secondary endpoints ................................................................................................... 46 MATERIALS AND METHODS .............................................................................................. 47 3.1 PATIENT´S CHARACTERISTICS AND TREATMENT ................................................ 48 3.2 TISSUE ACQUIREMENT AND PREPARATION ........................................................... 49 3.2.1 Immunohistochemistry ................................................................................................ 49 3.3. CELL CULTURE FOR IN VIVO RADIATION TREATMENT ..................................... 49 3.4. STATISTICAL ANALYSIS AND DEFINITIONS .......................................................... 50 4.1. MAP17 (PDZKIP1) AS A NOVEL PROGNOSTIC BIOMARKER FOR LARYNGEAL CANCER ................................................................................................................................. 53 4.1.1. Clinical cohort description ......................................................................................... 53 4.1.1. MAP17 expression in larynx tumour samples ............................................................ 53 4.1.2. SGLT1 overexpression in human larynx tumours correlates with MAP17 levels ...... 56 4.1.3. MAP17 as predictive biomarker for laryngeal cancer ............................................... 57 4.1.4. Tumour cells overexpressing MAP17 are more sensitive to radiation ...................... 58 4.2. PHOSPHORYLATION OF GH2AX AS A NOVEL PROGNOSTIC BIOMARKER FOR LARYNGOESOPHAGEAL DYSFUNCTION-FREE SURVIVAL ....................................... 61 4.2.1. Clinical cohort description ......................................................................................... 61 4.2.2. Cisplatin and radiotherapy as prognostic markers in larynx cancer ........................ 61 4.2.3. pH2AX in larynx tumour samples .............................................................................. 63 4.2.4. pH2AX relationship with cisplatin and radiotherapy ................................................ 65 4.2.5. Correlation of pH2AX with p53 and KI67 ................................................................. 66 4.2.6. Correlation of pH2AX and MAP17 ............................................................................ 67 DISCUSSION ............................................................................................................................. 70 CONCLUSIONS ........................................................................................................................ 75 CONCLUSIONES ..................................................................................................................... 77 REFERENCES ........................................................................................................................... 79 ARTICLES ................................................................................................................................. 92 ! ! ! 16! inmunohistoquímica de los microarrays. Se han estudiado marcadores de proliferación como Ki67 y la forma activada de ERK, de apoptosis como la mutación de p53 o la forma activada de AKT y finalmente marcadores implicados en los mecanismos de reparación del daño de ADN (RDA) como MAP17, SGLT y pH2AX. Posteriormente se llevó a cabo el estudio estadístico tomando en consideración tanto los factores clínicos como histopatológicos. Respecto a los factores clínicos, la extensión del tumor primario T4 y aquellos pacientes que precisaron de traqueotomía previa al tratamiento obtuvieron una peor supervivencia con laringe funcionante (SLF) y parecen no beneficiarse de tratamientos de preservación de órgano. Por otro lado, recibir una dosis óptima de platino durante el tratamiento influyó en la SLF ya que aquellos que no lo completaron tuvieron un peor pronóstico. Respecto a los factores moleculares, las marcadores relacionados con la RDA se asociaron a supervivencia. Los mecanismos RDA conforman una red de diagnóstico, señalización y reparación de las lesiones producidas en el ADN. MAP17 es una proteína de membrana altamente expresada en carcinomas. Dicha expresión se asocia a un aumento de las especies de oxígeno reactivo (EOR) dependiente de SGLT. Mientras que incrementos moderados de EOR activan cascadas de señalización que activan los procesos tumorigénicos, un aumento más significativo conlleva a un ambiente celular tóxico dando lugar a la muerte celular programada. Por otro lado, γH2AX es un componente del octámero de la histona del nucleosoma encargado de reclutar proteínas de reparación de ADN en respuesta al daño de la doble cadena de ADN. Por tanto, su aumento se relaciona con daño de ADN. Ya que ROS produce daño de ADN, pH2AX debería aumentar en este contexto. La hipótesis es que aquellos tumores laríngeos con niveles altos de ROS producidos por MAP17 y SGLT, y con expresión de pH2AX podrían beneficiarse de terapias como el cisplatino y la radioterapia, que aumentan el estrés oxidativo y dar lugar a la muerte celular. El análisis confirmó que existe una relación entre MAP17 y el aumento de la supervivencia global (SG), control locoregional (CLR) y SLF. Niveles altos de MAP17 se asociaron a un aumento de SG estadísticamente significativo comparado con niveles bajos (67 meses vs. 31.7 meses, IC 95%; p<0.001). La combinación de niveles altos tanto de MAP17 como de SGLT obtuvieron mejor SG que MAP17 solo. Por otro lado, pH2AX se correlacionó con SLF (alto-pH2AX HR 0.26, p = 0.02). El análisis conjunto de pH2AX junto con una dosis óptima de platino también se correlacionó con SG y SLF, demostrando resultados similares el análisis de pH2AX junto con alto-MAP17. Por tanto, niveles altos de MAP17 inducen EOR dando lugar a un aumento del daño de ADN y de los procesos de RDA, medido indirectamente por el aumento de pH2AX. En este tipo de tumores con alto estrés oxidativo, el sucesivo daño producido por terapias basadas en platino y la radiación tienen una mayor sensibilidad a la apoptosis. Por tanto, los biomarcadores implicados en los mecanismos de RDA MAP17 y pH2AX son marcadores pronósticos para aquellos pacientes con cáncer de laringe candidatos a terapias de preservación de órgano. ! ! ! 17! ! ! ! 18! INTRODUCTION ! ! ! 19! 1.1 LARYNX CANCER EPIDEMIOLOGY Around 157.000 new cases of laryngeal cancer were diagnosed worldwide in 2012, which represents approximately 1% of the total of malignancies. The estimated general incidence agestandardised rate (ASR) is 4.4 per 100.000 persons, with a broad difference between males and females (8.8 versus –vs0.8). In Europe, laryngeal cancer is the 20th most common tumour, with 39.900 new cases diagnosed in 2012; in contrast, the United States (US) registered 12.300 new cases in the same period. Worldwide, the highest incidence rate is found in the Caribbean area and are lowest in Western Africa, but this may just reflect varying data quality worldwide though (1). On the other hand, estimated general mortality ASR is 2.1 per 100.000 persons; 4.3 males and 0.3 women. Worldwide, the highest ASR mortality rates for laryngeal cancer are in Hungary for men and Albania for women (1). In contrast, the lowest rates are found in Iceland for both men and women. Mortality differs significantly between European countries. Males have lowest mortality rates in Northern Europe (ASR 1.9) followed by Southern Europe (ASR 4.3) and finally Eastern Europe (ASR 7.0) (Figure 1). Spain has similar mortality rates to the rest of Southern Europe countries, with a male ASR of 4.3 and female ASR of 0.3; however, those numbers are still far away from Northern countries (2) (Table 1). Larynx cancer in Spain Incidence Mortality 5-year prevalence Total (%) ASR Total (%) ASR Total (%) ASR All population 3.182 1,5 4,1 1.321 1,3 1,5 11.200 1,9 28,3 Male 2.914 2,3 7,8 1.235 1,9 2,9 10.246 3,1 52,7 Female 268 0,3 0,7 86 0,2 0,2 954 0,4 4,7 ASR: age-standarized rate. Table 1. Estimated Incidence, Mortality and 5-year prevalence in Spain, 2012. Source: Spanish Society of Medical Oncology annual report (6). The incidence of laryngeal cancer is falling in developed countries, because of the success of risk factors prevention campaigns. However, population growth and increased aging still results still in a larger number of total annual cases. During the last twenty years, larynx cancer has decreased globally by a 25% (3); for example, in the US the rate of decreasing is about 2% to 3% per year (4) (Figure 2). In Spain, the incidence in 2012 was 3182 cases, and the prediction of the World Health Organization (WHO) for 2020 is of 3735 new cases. However, this slight increase in total numbers translates a decreasing rate of 19.4% since 2003-07 (5). Therefore, the incidence of laryngeal carcinoma shows a decreasing trend for for both sexes in our country similar to the rest ! ! ! 20! of Europe and the US. But also within Spain, there is a significant variability. In the period 20032007, male incidence varied from 8.9 per 100.000 habitants in Albacete to 13.1 in the Basque Country. For females, Cuenca was found to be the region with a lower incidence and the Basque Country the highest rate (0.1 vs 0.8/100.000). Therefore, the Basque Country is the autonomous community with a higher incidence of laryngeal cancer. This variability is influenced by the differences in the prevalence of alcohol and tobacco consumption between regions (5). Figure 1. Worldwide laryngeal cancer incidence and mortality. Source: International Agency for research on cancer - World Health Organization. GLOBOCAN 2012. ! ! ! 21! However, the Surveillance, Epidemiology, and End Results (SEER) program of the National Cancer Institute in the US, has established that 5-year relative survival has slightly dropped during the last decades. In numbers, the 5-year survival decreased by a 3.3% from 1985 until 2008. Although this fact has coincided in time with the implantation of preservation approaches, they were not broadly used and standardized by then and major conclusions may not be extrapolated (4). Interestingly, in a recent publication it was found that in the US, uninsured patients or Medicaid patients (joint federal-state program that provides health coverage) had a decreased relative survival when compared to privately insured individuals (7), being one of the facts that could modify general survival. On the other hand, in the United Kingdom (UK) statistics show a trend towards improved survival over the last decades. A 5.5% increasing of the 5-year relative survival has been observed from 1990 till 2011 (69.8% survival in the period 2010-2011) (8). In Spain, 5year relative survival was around 60% survival in the period of 2000-2007, so in the range of developed countries (6). Figure 2. Number of new cases, deaths and 5-year relative survival in US. Source: SEER Cancer Statistics. Median age at presentation is 65 years, where it is most frequently diagnosed among people aged 55-64, in a 30.9% of the cases (4). This is similar for both developed and emerging countries, as some series in India showed maximum incidence between the 60-69 years (31.93%) (9). In the US, at the time of diagnosis most patients have locoregional disease (77%); of them, 55% localized disease and 22% regional disease. As metastatic disease is found as a later event in laryngeal cancer, only 19% were found to have them (4). In conclusion, laryngeal cancer is more prevalent in males than females, aged around the sixth decade of life, and frequently diagnosed as localized or locoregional disease. There are significant incidence variations worldwide that depends mainly on risk factors habits. In Europe, laryngeal cancer rates are dropping but there are still differences between North, Central and South regions. Finally, Spain behaves similar to other Mediterranean countries, but there are areas where the incidence is still high. ! ! ! 22! 1.2 ANATOMY AND HISTOLOGY OF THE LARYNX. PATTERNS OF SPREAD. TNM STAGING ! 1.2.1. General Anatomy, histology and pathology General Anatomy The larynx is an air passage and an organ of phonation that extends from the tongue to the trachea. It projects ventrally between the great vessels of the neck and is covered anteriorly by skin, fasciae and the infrahyoid strap muscles that lower the hyoid bone and the larynx. Above, it opens into the laryngopharynx and forms its anterior wall; below, it continues into the trachea. Until puberty, male and female larynges are similar in size. After puberty, the male larynx enlarges considerably in comparison with the female. Skeletal framework. The skeletal framework of the larynx is formed by cartilages interconnected by ligaments and fibrous membranes. The laryngeal cartilages are the single thyroid, cricoid and epiglottic cartilages, and the paired arytenoid, cuneiform and corniculate cartilages (Figure 3). The hyoid bone is not part of the larynx but provides the muscular attachments from above that aid in laryngeal motion. Larynx cartilages are joined to surrounding structures by extrinsic membranes. It is also interconnected by intrinsic ligaments and fibrobroelastic membranes, of which the thyrohyoid and quadrangular membranes, together with the conus elasticus, are the most significant. The thyrohyoid membrane is external to the larynx, whereas the paired quadrangular membranes and conus elasticus are internal. The named ligaments are the anterior cricothyroid ligament, the hyoepiglottic and thyroepiglottic ligaments, and the cricotracheal ligament. Figure 3. General laryngeal anatomy. Version adapted from the Human Anatomy Atlas, Netter 2nd Edition. ! ! ! 23! Laryngeal cavity. The walls of the cavity are formed of fibroelastic membranes and lined with mucous membrane that folds over the free edges of these membranes within the larynx. On either side, the continuity of the fibroelastic membrane is interrupted between the upper vestibular and lower true vocal folds. The folds project into the lumen of the cavity and divide it into upper and lower parts, separated by a middle portion between the two sets of folds that leads into the laryngeal ventricle. The upper folds are the vestibular (ventricular or false vocal) folds; the median aperture between them is the rima vestibuli. The lower pair is the (true) vocal folds, primary source of phonation. In tumor staging the supraglottis refers to all those parts of the larynx that lie above the glottis, meanwhile the glottis is defined as the anterior and inferior surfaces of the true vocal folds and the anterior and posterior commissures. Finally, the subglottis is the region below the glottis that extends to the inferior border of the cricoid cartilage. Muscles. The muscles of the larynx may be divided into extrinsic and intrinsic groups. The extrinsic muscles connect the larynx to neighbouring structures and are responsible for moving it vertically during phonation and swallowing, thus opposite to the infrahyoid muscles that lower the larynx. They include the infrahyoid strap muscles, thyrohyoid, sternothyroid and sternohyoid, and the inferior constrictor muscle of the pharynx. The extrinsic muscles can affect the pitch and the quality of the voice by raising or lowering the larynx, and geniohyoid elevates and anteriorly displaces the larynx, particularly during deglutition. The intrinsic laryngeal muscles may be placed in three groups according to their main actions. The posterior and lateral cricoarytenoids and oblique and transverse arytenoids vary the degree of abduction and adduction of the vocal folds and thus the dimensions and the degree of opening of the rima glottidis. The cricothyroids, posterior cricoarytenoids, thyroarytenoids and vocalis regulate the length and tension of the vocal folds. The third group of muscles is the oblique arytenoids, aryepiglottic and thyroepiglottic muscles, which modify the laryngeal inlet. Vascular supply and inervation. The blood supply of the larynx is derived mainly from the superior and inferior laryngeal arteries. Rich anastomoses exist between the corresponding contralateral laryngeal arteries and between the ipsilateral laryngeal arteries. The superior laryngeal arteries supply the greater part of the tissues of the larynx, from the epiglottis down to the level of the vocal cords, including the majority of the laryngeal musculature. The inferior laryngeal artery supplies the region around cricothyroid, while its posterior laryngeal branch supplies the tissue around posterior cricoarytenoid. The larynx is innervated by the internal and external branches of the superior laryngeal nerve, the recurrent laryngeal nerve and sympathetic nerves. Conventionally, the internal laryngeal nerve is described as sensory, the external laryngeal ! ! ! 24! nerve as motor, and the recurrent laryngeal nerve as mixed. Lymphatic drainage. The upper deep cervical lymph nodes act as pathways for the spread of malignant tumours of the supraglottic larynx. Up to 40% of these tumours will have undergone such spread at the time of clinical presentation, mainly along the superior laryngeal artery draining to the deep cervical lymph nodes at the bifurcation of the common carotid artery. On the contrary, the glottis is very poorly endowed with lymphatic vessels; some 95% of malignant tumours confined to the glottis will present with no spread to adjacent lymph nodes. Tumours of the subglottic larynx will often spread to the paratracheal lymph node chain prior to clinical presentation. The paratracheal lymph nodes occupy a deep-seated position in the root of the neck (Figure 4) and so their enlargement may remain occult (10-16). Figure 4. Cervical lymph node chains. Laryngeal histology The laryngeal mucosa is continuous with that of the pharynx above and the trachea below. Over the vocal folds, it is thinner and is firmly attached to the underlying vocal ligaments. The laryngeal epithelium is mainly a ciliated, pseudostratified respiratory epithelium that covers the inner aspects of the larynx, and it provides a mucociliary clearance mechanism shared with most of the respiratory tract. The vocal folds, however, are covered by non-keratinized, stratified squamous epithelium; this important variation protects the tissue from the effects of the considerable mechanical stresses that act on the surfaces of the vocal folds. The exterior surfaces of the larynx, which merge with the laryngopharynx and oropharynx, are subject to the abrasive effects of swallowed food, and are therefore also covered by non-keratinized, stratified squamous epithelium (Figure 5). The laryngeal mucosa has numerous mucous glands, especially over the epiglottis, and along the margins of the aryepiglottic folds anterior to the arytenoid cartilages. (11, 17). ! ! ! 25! Figure 5. Laryngeal cavity histology subtypes. Laryngeal pathology The vast majority of all laryngeal malignancies (95%) are conventional squamous cell carcinomas (SCC). They vary according to their degree of differentiation to well, moderate and poor carcinomas. Glottic cancers are generally well differentiated and have a less aggressive behaviour in comparison with carcinomas at the other sites of the larynx. SCC often arises in a background of mucosal squamous dysplasia or carcinoma in situ and typically presents islands, tongues and clusters of atypical cells invading the laryngeal stroma. Features of squamous differentiation also comprise individual cell keratinisation, intercellular bridges and keratin pearls (17). Recognition of the less common variants is significant because their biological behaviour is often different from conventional SCC. Verrucous SCC: comprises approximately 1% to 4% of laryngeal malignancies. It presents as a locally invasive fungating mass that can be confused with a benign process and characteristically does not metastasize to regional lymph nodes. Histologically, it appears broad pegs of highly differentiated squamous cells invading the laryngeal stroma in a pushing pattern, and mitoses rarely found. Surgical excision is the cornerstone of therapy since radiotherapy is associated to poor results and with the possibility of anaplastic transformation of the tumour (18). ! ! ! 32! intensity of smoking. Particularly in laryngeal cancer, it has been seen that risk of a second primary tumour was proportional to the level of exposure (37, 38). Data also suggest that smoking may have an adverse effect on the response to medical treatment. In a prospective trial with more than one hundred patients it was seen that patients that stopped smoking during trial had better results than those who did not (75% of complete tumour responses vs 45%) (35). Also in terms of toxicities and comorbidities, patients that continue smoking have more treatment-related complications. Mucositis for example takes a longer time to heal in smoker patients than in nonsmokers during radiotherapy treatment (39). Moreover, population with HNC has also an increased risk of deep venous thrombosis, pulmonary embolism, and impaired wound healing following surgery which compromise treatments and outcomes (40, 41). Taking into account the risk of cancer development, cardiovascular disease and other related diseases to tobacco, it is estimated that smoking kills nearly 6 million people each year worldwide, more than HIV/AIDS, tuberculosis and malaria combined. Tobacco consumption imposes a considerable cost upon society, including direct healthcare costs and indirect costs due to absenteeism and premature death. In the European Union for the year 2000, cost was estimated to be approximately €363 billion (42). Alcohol is the second major risk factor for laryngeal cancer, which potentiates the effects of tobacco. The association between alcohol drinking and laryngeal cancer was first reported in a case-control study in the 1950s (43). In 1987, the International Agency for Research on Cancer working group classified alcoholic beverages as ‘‘carcinogenic to humans” and concluded that laryngeal cancers are causally related to the consumption of alcoholic beverages (44), a conclusion that was reaffirmed in a more recent evaluation in 2009 (45). The risk is proportional to the amount of ethanol drunk per day, as a meta-analysis reported in 2001. Laryngeal cancer relative risk (RR) adjusted by smoking status was 1.29 (1.23–1.36), 1.68 (1.53–1.84), and 2.72 (2.36–3.30) for drinking 25, 50, and 100 g of ethanol per day, respectively (46). However, in another meta-analysis, significant association was not found for light alcohol drinking (≤1 drink/day), RR = 0.88; 95% CI: 0.71–1.08), whereas moderate drinking (>1 to <4 drinks/day) was associated with a 1.5-fold increase in risk (RR = 1.47; 95% CI: 1.25–1.72) and heavy drinking (≥4 drinks/day) with a 2.5fold increased risk (RR = 2.62; 95% CI: 2.13–3.23). Overall, alcohol drinking versus nondrinking was associated with an approximately 2-fold increase in risk of laryngeal cancer. In this meta-analysis, subgroup analyses for studies that adjusted for main potential confounding factors (age, sex, and tobacco use) and several further subgroup analyses showed similar results, which suggest the robustness of the results (47). Human papillomavirus (HPV) is emerging as an important HNC factor in developed countries, especially in oropharyngeal tumours. HPV-induced HNC has distinct epidemiology and biology, and a more favourable prognosis compared to HPV negative tumours. In patients with oropharyngeal carcinoma treated with chemoradiotherapy, HPV-positive had better 3-year ! ! ! 33! overall survival rates than patients with HPV-negative tumours (82.4%, vs. 57.1%; P<0.001) (48). In another trial with more than five thousand patients with HNC it was observed that the hazard ratio for death in patients HPV positive was 0.42 and that they had a better response to therapy. Positivity for HVP was found in 22% of patients, most of them having the HPV 16 positive genotype (49,50). However, it is known that there is a low rate of HPV-positive tumours among the laryngeal cancer population, which is estimated in around 1.6-6.5%. Therefore, although HPV is emerging as an important risk factor that correlates with prognoses in HNC, the role in laryngeal cancer it is probably less relevant and it is not yet stablished (51-53). A recent meta-analysis supports the association of exposure to asbestos with an increased risk of laryngeal cancer mortality among male workers, with a standardized mortality rate of 1.69 (54). It has been also suggested that there is an additive joint effect between asbestos exposure and alcohol consumption, and a more than additive joint effect between asbestos exposure and tobacco consumption, as well as between the three of them together (55). In a multicentre case-control study conducted in four European countries it was evaluated the role of occupational exposures and risk of laryngeal and hypopharyngeal cancer. Elevated risks for ever exposure to coal dust were found for both hypopharyngeal (OR= 4.19, 95% CI: 1.18, 14.89) and laryngeal cancer (OR= 1.81, 95% CI: 0.94, 3.47), with clear dose-response patterns. Laryngeal cancer was significantly associated with exposure to hard-alloys dust (OR = 2.23, 95% CI: 1.08, 4.57) and chlorinated solvents (OR = 2.18, 95% CI: 1.03, 4.61), without dose-response relations. A possible link between high formaldehyde exposure and laryngeal cancer was also suggested but further studies are required (56). Gastroesophageal reflux is another risk factor for laryngeal cancer as it was shown is a systematic review where the pooled odds ratio (OR) was 2.21 (95%, CI= 1.53-3.19), regardless of age, gender, tobacco and alcohol consumption (57). Genetic susceptibility has been implicated in HNC. Emerging phenotypic and genotypic data support the idea of genetic susceptibility for HNC. In an historical cohort study the RR for HNC was 7.89 (CI-1.50 to 41.6) in first degree relatives of patients with multiple primary head and neck cancer (58). In a more recent pooled analysis with 8.967 HNC cases and 13.627 controls, having a family history of HNC in first-degree relative increased the risk of HNC 1.7, which was higher when the affected relative was a sibling (OR= 2.2, 95% CI 1.6-3.1) rather than a parent (OR=1.5, CI 1.1-1.8) and for more distal HNC anatomic sites (hypopharynx and larynx) (59). Instead, vegetable, fruits, and vitamin C intake might reduce the risk of laryngeal cancer. Higher intakes of vitamin C were inversely related to laryngeal cancers (OR = 0.52, 95% CI: 0.40-0.68) in a pooled analysis in the International Head and Neck Cancer Epidemiology Consortium (60). In another study, total vegetable and fruit consumption was inversely associated with risk of HNC ! ! ! 34! (OR=0.61, CI 0.44-0.85) and all HNC subtypes, with the strongest associations for oropharyngeal cancer (61). 1.3.2. Clinical and pathological prognostic factors For laryngeal cancer, the main prognostic factor for overall survival (OS) is tumour staging. Pathologic tumour volume was found to be an independent predictive factor for distant metastasis, overall survival, disease-free survival and locoregional recurrence (62). T4 primary extension and more than 2 cm tumoral invasion of the base of the tongue increased salvage laryngectomy in the Veterans study (63). Moreover, lymph node invasion and extracapsular extension are also independent predictive factors for survival (62). Location of the primary tumour also plays a significant role in prognosis, as supra and subglottic cancers have worse prognosis than glottis cancers, probably in relation to earlier detection, and later node extension (27,28). Other prognostic factors are patient’s comorbidity and performance status-ECOG (PS) (64), most probably because frail patients are not suitable candidates for aggressive treatment management. Finally, surgical resection margins affection and pretreatment need for a tracheotomy have been related to poor disease-free survival (65,66). However, none of those factors have been validated for treatment decision-making so far. 1.4 MOLECULAR BIOLOGY OF LARYNGEAL CANCER 1.4.1. Genetic susceptibility Genetic polymorphisms variants in tobacco carcinogen and alcohol metabolism genes may increase HNC risk. GSTM1 null genotype appears to confer increased HNC and particularly laryngeal increased risk (OR=1.22, 95% CI 1.1-1.36) (67, 68). The variant Val allele of the CYP1A1 Ile462Val polymorphism is another consistent susceptibility maker for HNC, with a 35% increased risk in a meta-analysis of 12 studies (68). Presenting fast metabolizing alleles for alcohol dehydrogenase (ADH), ADH1B and ALDH2 genes, resulted in increased acetyladehyde levels and associated with HNC significantly interacting with alcohol consumption (69). In a case-control study single nucleotide polymorphisms (SNPs) in nucleotide excision repair (NER) genes such as ERCC5, ERCC6 and RAD23B could modify laryngeal cancer risk. In particular, ERCC6 showed a decreased risk, while ERCC5 and RAD23B increased it (70). Furthermore, XPD and ERCC1 may be associated to poor disease free survival (DFS) in HNC, suggesting a significant role of NER in these tumours (71). ! ! ! 35! 1.4.2. Cytogenetic alterations Malignant progression may be associated to particular chromosomal alterations. For instance, early changes at 3p, 4q, 8p, 9p, 11q, 13q and 17p have been observed in leukoplakias while loss of heterozygosity (LOH) of 9p21 is a common genetic event in oral premalignancies (72,73). Califano et at described a model of carcinogenesis in 1996, but it seems to be more the accumulation rather than the order of genetic events what determines tumoral progression (Figure 8) (74). Amplifications/loss of heterozygosity Amplification in a variety of key genes have been described in HNC, such as of 3q, 3q24-qter, 5p, 8q23-24, 11q13, 11q14-22, 18p, 18q11.2, and 19q among others (75, 76). The critical protooncogene Cyclin D1 is amplificated within the 11q13 region and may be a marker of progression in primary HNC (77). Cyclin D1 overexpression has been reported in laryngeal carcinoma and might be implicated in regulating cell proliferation by the critical G1/S checkpoint (78). P63 is a p53 homologue and a potential oncogene in squamous cell cancer that has been found in the distal arm of 3q (79). Figure 8. Frequent cytogenetic alterations in head and neck cancer. Loss of chromosomes 3p, 5q, 8p, 9p, 18q and 21q are commonly identified as well, where loss of 18q could indicate poor prognosis tumours (80). The most commonly deleted region in HNC is located at chromosome 9p21-22 (81). It occurs in the majority of invasive tumours and is present at a high frequency in early premalignant lesions, including dysplasia and carcinoma in situ (82). P16 or cyclin-dependent kinase inhibitor 2 (CDKN2-) is the tumour suppressor gene contained ! ! ! 36! within this critically deleted region and is a potent inhibitor of cyclin D1/CDK4 (83). However, p16 amplification is independent of cyclin D1 inactivation in HNC (84). It is possible that a second tumour suppressor gene resides at 9p21. In the same INK4 loci, an alternative reading frame codifies for a very different unrelated protein, p14ARF, which regulates MDM2 and therefore p53. Introduction of p16 or p14ARF into head and neck cancer cell lines results in potent growth suppression (85). Loss of heterozygosity (LOH) at the Retinoblastoma (Rb) locus in 13q14 has been found in around 14% of HNC, leading to Rb protein inactivation and tumour progression (86). Rb LOH is correlated with altered pRB expression in endometrial and oesophageal cancer (87, 88) but same results could not be found for laryngeal cancer (89). 1.4.3. TP53 Mutations in P53 are one of the most frequent abnormalities in HNC and can be observed in severe dysplasia. TP53 mutations are found in 39-53% of HNC tumours and in 56.7% of laryngeal carcinomas (90, 91). P53 mutations have been demonstrated to be related to poor survival in different publications by using microarray technology (90), immunohistochemistry (IHC) (92) or singlestrand conformational polymorphism (SSCP) analysis followed by DNA sequencing. However, there is not necessarily correlation between IHC and SSCP results (91); moreover, decrease in survival can vary if the mutation is disruptive or non-disruptive (90). These controversies may explain differences as a prognostic factor found so far. 1.4.4. Notch Inactivating mutations of Notch1 have been found in 10-15% of HNC, being the second most frequently mutated gene after TP53 (93,94). Notch signalling pathway has been linked to multiple biological functions, such as regulation of self-renewal capacity, cell cycle exit, and survival. In HNC, several of the Notch family mutations encode inactivating mutations, suggesting a tumour suppressor function (95). In a study that included 289 patients with laryngeal carcinoma Notch3 was associated with unfavourable disease-free survival and overall survival (96). 1.4.5. EGFR/EGFRvIII The EGFR pathway is involved in cell transformation through autocrine overproduction of epidermal growth factor/transforming growth factor alpha (EGF/TGFá) and overexpression of EGFR by gene amplification or altered transcriptional mechanisms (97). Production of TGFá and EGFR mRNA has been found in normal mucosa of patients at risk for a primary or secondary HNC suggesting these changes to be early markers of carcinogenesis (98). ! ! ! 37! In up to 90% of HNC, increased expression of EGFR is observed which is associated with advanced stage, poor survival and resistance to treatments (99, 100). Similarly, EGFR overexpression in laryngeal carcinoma has also been linked to poor survival (101) and also as a predictive biomarker for radiotherapy treatment (102). In a phase III trial comparing cisplatin with or without the monoclonal antibody cetuximab for metastatic/recurrent HNC patients, it was seen that doses of cetuximab may need to be adjusted for patients with very high levels of EGFR to achieve better response (103). The EGFRvIII mutant variant has been found in up to 40% of HNC and it seems to contribute to cancer growth and resistance to EGFR targeting. EGFRvIII is seen in cells that overexpress wild-type EGFR suggesting that mutations are a later event caused by rapid proliferation induced by EGFR overexpression (104). 1.4.6. DNA damage repair biomarkers. MAP17 and pH2AX As laryngeal cancer responds to treatments based on platinum and radiotherapy that result in DNA damage, biomarkers implicated in the nucleotide excision repair (NER) and the double strand breaks repair might have a significant role. DNA Double break streams (DBS) can be originated by drugs and ionizing radiation but also by increasing the levels of reactive oxygen species (ROS) (105) through MAP17 activation. MAP17 is a small 17 Kda non-glycosylated membrane protein overexpressed in carcinomas. It has been found present in adenoma and benign tumours, and is highly expressed in metastatic carcinoma. The expression is mainly driven at a transcriptional level either by promoter activation or demethylation. Expression of MAP17 in primary cells triggers senescence through p38, but in tumoral cells enhances the malignant capabilities of these cells, increasing proliferation, migration, resistance to apoptosis, etc. MAP17 expression increases the levels of ROS in cells which may account for some of the increased tumoral properties. In turn, a further increase of ROS might switch the balance towards apoptosis. Thus, MAP17 may increase the efficacy of therapies increasing ROS and therefore constitute a biomarker for better prognosis of these tumours. In cervix tumours treated with cisplatin and radiotherapy, high levels of MAP17 mark good survival of the patients. Therefore, MAP17 is not only a marker for stage and malignant status but also may be a marker of prognosis and response to therapies involving oxidative stress (106-108). DNA DBS lead to activation of three kinases, ataxia telangiectasia mutated (ATM), ATM-Rad3-related (ATR) and DNA-PK which phosphorylate γH2AX, a component of the histone octamer in nucleosomes. PH2AX is involved in recruiting DNA repair proteins in response to double-strand breaks (DSB) (108). Therefore, it is considered as a biomarker of DNA damage, as the presence and magnitude of pH2AX is an indication of persistent, unrepaired DNA damage. pH2AX induction appears within minutes in cells after DNA damage and reaches maximum levels after 30 minutes. The repair process includes the phosphorylation of hundreds to thousands γH2AX surrounding the DSB site in order to form a ! ! ! 38! focus that open the chromatin structure and serve as a platform for the accumulation of factors involved in the DNA damage response (109). The NER pathway guard the integrity of the genome by recognizing and removing DNA cross-links caused by cisplatin or radiation mainly driven by the excision repair cross-complementation group 1 (ERCC1) (110). In locally advanced HNC low expression of ERCC1 was an independent predictor factor for prolonged in patients treated with cisplatin-based CCRT; however just 18% of patients had laryngeal carcinoma (111). Furthermore, in a case-control study focused on laryngeal cancer, ERCC1 rs11615 and ERCC5 rs17655 polymorphisms were associated with increased risk of developing laryngeal cancer (112). 1.4.7. Vaccinia-related kinase-1 (VRK1) protein VRK1 protein belongs to a family of three protein kinases implicated in regulation of cell proliferation by phosphorylation of p53 and cooperation with c-Jun and ATF2. VRK1 expression is activated by E2F and inhibited by p16 and Rb. In HNC, VRK1 could be a significant control mechanism of the cell cycle, particularly in G1-S phase (113). Figure 9. Mechanism of response to DNA damage in laryngeal cancer. IR: ionizing radiation. 1.4.8. STAT3 pathway Stat3 (signal transducer and activator of transcription 3) is a transcription factor that responds to cytokines and growth factor receptor activation (114). Constitutive activation of the pathway in response to deregulate upstream signals is commonly observed in diverse cancers including dead and neck and laryngeal tumours (115-117). This constitutive activation of the pathway has been involved in proliferation and survival of tumours as well as resistance to chemo and radiotherapy ! ! ! 39! (116,117). Recent works indicate that STAT signalling also contributes to therapy resistance by modulating also the microenvironment (118). ERp57 (GRP58) is a chaperone that regulated proper folding of glycoproteins (119). ERp57 is associated with tumour progression and has been described to modulate STAT 3 activity, thus regulating radioresistance in laryngeal cancer (120). Accordingly, ERp57 has been described as a poor prognosis factor. 1.4.9. Micro RNAS MicroRNAs (miRs) play important roles in many pathological alterations regulating important cellular and physiological processes such as cell proliferation, differentiation, metabolism, apoptosis, autophagy and intercellular communications (121, 122). It has been considered that miRs regulate around 60% of genes in the human genome. The analysis of miR expression variation in Laryngeal cancer by a variety of techniques including broadly used miR microarrays and massive sequencing have shown a large variety of miRs deregulated in laryngeal cancer, many of them with diagnosis or prognosis value (reviewed in (123)) (124,125) (Table 4). These miRs behave as oncogenes (oncomirs) or tumour suppressors in laryngeal cancer according the effect of the target. However, this simplistic analysis is commonly more complicated since one miR can target several genes and several miRs can target the same gene thus providing some synergistic effects. Interestingly, several miRs have been associated with therapeutic resistance in laryngeal cancer, thus providing worse prognosis. MIRNAS TARGET PROGNOSIS UPREGULATED miR-16 Zyxin ND miR-19a TIMP2 Poor survival, Lymph node metastasis miR-21 BTG2 Poor survival, poor differentiation and Lymph node metastasis miR-27a PLK2 ND miR-106b RUNX3 Poor survival, poor differentiation and Lymph node metastasis miR-129-5p APC ND miR-155 SOCS1, STAT3 Poor differentiation and TNM stage miR-1297 PTEN ND DOWNREGULATED miR-1 FN1 ND miR-24 S100A8 ND ! ! ! 40! miR-144-3p ETS-1 Poor prognosis miR-34a Survivin Good prognosis hsa-miR34c C-Met ND miR-126 Camsap1 ND miR-139 CXCR4 ND miR-203 ASAP1 Good survival , inverse to TNM and grade of differentiation. miR-206 VEGF Good survival , inverse to TNM and clinical stage. miR-299-3p hTERT ND miR-370 Fox-M1 ND miR-519a HuR, COX2 ND miR-874 HDAC1 ND Table 4. Summary of miRNAs relevant in laryngeal cancer. Adapted from Yu and Li, 2015 [10]. ND: not determined 1.4.10. Tumour microenvironment. Immune-phenotypes Most tumour cells express antigens that can mediate recognition by host CD8+ T cells. Cancers that are detected clinically must have evaded antitumor immune responses to grow progressively. HNC analysis of tumour microenvironment has revealed the presence of two major subsets of tumours with distinct mechanisms of resistance to immune-mediated destruction. The inflamed (and mesenchymal) phenotype present in a group of HNC tumours show prominent tumour infiltration by CD8(+) lymphocytes and a broad chemokine profile. This is independent of HPV status. Immune resistance occurs after T-cell migration into the tumour site, implicating the effect of negative immune regulators. On the other hand, the non-inflamed tumours are devoid of T cells and other indicators of innate immunity, such as chemokines. Immune failure is attributed to poor effector T-cell trafficking, as required factors are absent. These two major phenotypes of tumour microenvironment may require distinct immunotherapeutic interventions for maximal therapeutic effect (126). ! ! ! 41! 1.5 LARYNGEAL CANCER TREATMENT ! 1.5.1. Preservation approaches Organ preservation treatments for laryngeal cancer patients depend on whether the tumour is presented in early stages (I and II) or advanced locoregional disease (stage III/IV). In general, early stages are treated with either primary surgery or definitive radiotherapy (RT), while advanced stages require a multimodal approach. Early stage disease (Stages I and II) Total laryngectomy was the gold standard treatment by the 1980s with the subsequent loss of speech and airway patency. Because of the significant quality of life decreasing of these patients, partial laryngectomies and endoscopic laser surgeries were initiated in the 90´s in order to preserve laryngeal function. Furthermore, 95% local control rates have been described in patients with T3 and T4 glottic and supraglottic tumors who underwent supracricoid larynguectomy, with an associated improvement of quality of life (127,128). Later, radiotherapy was given to early stages. Although surgery and radiotherapy have never been compared in a randomized trial, both have been accepted to have similar effectiveness. This is based on one-arm prospective trials and prospective case-control series. In one of these series published in 2002, 31% locoregional control was reported for T3 laryngeal cancer with an organ preservation rate of approximately 50% (129). Alternative radiotherapy schedules were studied such as the hyperfractionated or the accelerated radiotherapies. Both techniques showed improved laryngectomy free-survival, and increased locoregional control compared to standard radiotherapy without increasing toxicity. However, none of them could show a benefit in terms of survival. The RTOG 90-03 phase III trial confirmed those results and demonstrates a better local control rate with hyperfractionated and accelerated radiotherapy compared to standard treatment (55% vs 45%) (130). Locally advanced (Stages III and IVA/B) Although functional organ sparing approaches permit larynx preservation, they do not provide a survival advantage over total laryngectomy (131). Three sparing approaches are accepted: RT, bio or chemotherapy with concomitant radiotherapy (B/CT RT) and induction CT (ICT) followed by RT with or without B/CT. CTRT with concurrent cisplatin showed higher preservation rates compared to other two arms with RT alone or induction cisplatin plus fluorouracil followed by RT (88% vs 70% and 75%, respectively) with similar two and five-year survival (132). Later, a 10-year follow-up publication ! ! ! 48! 3.1 PATIENT´S CHARACTERISTICS AND TREATMENT We evaluated 65 patients with larynx cancer from August 2005 to February 2014. However, out of the 65 tumoral samples, only 53 of them could be studied. All samples were obtained from diagnostic biopsies before treatment. All patients completed the informed consent form and the project was approved by the local ethical committee at the Hospital Universitario Virgen del Rocío (HUVR) (PI13/059). Patients received treatment in our institution but tumour samples were obtained from four different national hospitals where the diagnosis was made. Eligibility criteria for treatment preservation include patients with stage II-IV laryngeal tumours that had no contraindication for CT or RT, significant cartilage destruction, or more than 2 cm tumoral invasion of the base of the tongue. TNM Staging System (7th ed., 2010) was used for tumour classification (27). Patients were mainly male (94%) with squamous cell carcinoma and good general condition. Tumours were more frequently localized in the supraglottic (60%) and 75.5% were stage III. Approximately one third of patients required pre-treatment tracheotomy. Selected organ preservation therapies were B/CTRT (75%), RT (14%), or ICT-B/CTRT (9%). Cisplatin 100 mg per square meter (m2) on days 1, 22, and 43 (74%) was most commonly used, followed by weekly cisplatin 40 mg/m2 (11%), and monoclonal antibody cetuximab (11%) (Table 6). ! ! ! ! ! Table 6. Population characteristics and treatment. ! ! ! 49! 3.2 TISSUE ACQUIREMENT AND PREPARATION Formalin-fixed, paraffin-embedded tissue sections from 65 laryngeal carcinomas were selected with the collaboration of the Andalusian Health Care Biological Resource Centre. Histological characterization of all samples was done by Hematoxylin and Eosin staining, followed by immunohistochemistry (IHQ) analysis of tissue microarrays (TMA). 3.2.1 Immunohistochemistry Three-micrometer slices were sectioned from the TMA block and applied to coated, immunochemistry slides (DAKO, Glostrup, Denmark). The slides were baked overnight in a 56°C oven, deparafinized in xylene for 20 min, rehydrated through a graded ethanol series and washed with PBS. A heat-induced epitope retrieval step was performed by heating a slide in a solution of sodium citrate buffer pH 6.5 for 2 min in a conventional pressure cooker. After heating, the slides were incubated with proteinase K for 10 min and rinsed in cool running water for 5 min. Endogenous peroxide activity was quenched with 1.5% hydrogen peroxide (DAKO) in methanol for 10 minutes, and incubation with the primary antibodies was performed for 40 min. Selected antibodies were: anti-gamma H2A.X (phospho S139) antibody (ab11174 from Abcam), anti-p53: p53 FL 393 (sc-6243 from Santa Cruz), anti-MAP17 (1:4) [29-33], anti-SGLT1 (Abcam #14685), ki67 (clone MIB-1; DAKO, Agilent technologies, United States), AKT-p (phosphorylated S473AKT1, Epitomics), and p44/p42 MAPK (Erk1/2) Rabbit mAb 1:1000 (Cell Signaling 137F5). After incubation, immunodetection was performed with the EnVision (DAKO, Glostrup, Denmark) visualization system using diaminobenzidinechromogen as the substrate, according to the manufacturer’s instructions. Immunostaining was performed in a TechMate 500 automatic immunostaining device (DAKO) and measured through a doubleblind visual assessment using microscopic observation according to the anatomopathological experience of pathologists. Sample scoring was performed by semiquantitative microscopic analysis, considering the number of stained cells (percentage of positive cells) and signal intensity (levels 1, 2, or 3). 3.3. CELL CULTURE FOR IN VIVO RADIATION TREATMENT Hela malignant cervical tumour cells were obtained from the European Collection of Cell Cultures (ECACC) human cell line repository and maintained in Dulbeccós modi ed Eaglés medium (Sigma) containing 10% fetal bovine serum (Sigma), penicillin, streptomycin and fungizone. MAP17 full-length cDNA was cloned into pBabepuro and mass culture generated by stable gene transfer in Hela cells. After selection with 2 ìg/ML puromycin, mass cultures were used for the study. As a control, Hela cells were transfected with pBabepuro alone and selected. Cells were irradiated using Costar 24 well cell culture plates (Corning Incorporated, NY USA). ! ! ! 50! To simulate actual radiobiological experimental conditions, each well was filled with culture medium. The plate dimensions were 12.5 x 8.5 cm. The inner diameter of the well was 16 mm and the distance between the centers of two neighboring wells was 20 mm. Plates were positioned inside a water-equivalent device, specifically designed to fit the plate. This device measures 16 x 16 x 2 cm, and is placed inside the IBA BodyPhantom (IBA Dosimetry GbmH, Schwarzenbruck, Germany) at a depth of 6 cm. Simulation was performed using a Toshiba Aquilion CT scanner (Toshiba Corporation, Japan). CT images were exported to the treatment planning system Philips Pinnacle V9.2 (Philips Radiation Oncology Systems, Madison, WI). Five plans were designed to deliver uniform doses of 0.1 Gray (Gy), 0.3 Gy, 1 Gy, 3 Gy and 10 Gy using static beams of 24 x18 cm. To verify the dose within every well, we delineated 24 regions of interest (ROI) that had a diameter of 16 mm. The ROI was estimated at the bottom of the wells and 5mm upwards. The dose delivered to the cells was verified with the IBA Compass system (IBA Dosimetry GbmH, Schwarzenbruck, Germany). Differences between the prescribed dose and the dose received were within 3%. The irradiation was delivered using 6 megaelectronvolts (MV) photon beams from an Elekta Synergy Linac (Elekta Oncology System, Ltd, Crawley, UK) with a dose rate of 500 mu/min. 3.4. STATISTICAL ANALYSIS AND DEFINITIONS Kaplan-Meier method was used for survival analysis, using Cox Proportional Hazards model to adjust for the explanatory variables, obtain the p-values and estimate the hazard ratios (HR). Multivariate logistic regression was used to obtain odds ratio (OR) and CI 95%. Pearson’s correlation measured dependence between quantitative variables. A receiver operating characteristic (ROC) curve was performed to assess the biomarker cut-off point (two-year OS for pH2AX and three-year OS for MAP17), which was confirmed using the optimal Youden indexbased point. In addition, the log-rank test was used to compare survival distributions. Categorical data were studied with contingency tables that included Chi-square statistics. Calculations were performed using SPSS 15.0 software. OS has been defined as the length of time from diagnosis until the last medical record. Locoregional control (LRC) was measured as length of time from diagnosis until the relapse or last medical record, in those patients who did not develop distant metastases or died due to different causes than the tumour. For laryngoesophageal dysfunctionfree survival (LDS) we adopted Lefebvre Larynx Preservation Consensus Panel that included as endpoint events: death, local relapse, total or partial laryngectomy, tracheotomy at two or more years, or the presence of a feeding tube at two or more years (150). ! ! ! 51! Figure 10. Example of ROC curve to determine the cut-off point for pH2AX. ! ! ! 52! RESULTS ! ! ! 53! 4.1. MAP17 (PDZKIP1) AS A NOVEL PROGNOSTIC BIOMARKER FOR LARYNGEAL CANCER MAP17 is a small 17 Kda membrane protein present in carcinoma, but also in adenoma and benign tumours, and is highly expressed in metastatic carcinoma. Its expression correlates with staging and malignant status of the tumour. MAP17 expression is associated with an SGLTdependent ROS increase that acts as a second messenger enhancing tumorigenesis. While a mild increase in ROS has been shown to activate signalling cascades that upregulate tumorigenic processes, further ROS increases lead to a potentially toxic cellular environment and programmed cell death. The hypothesis is that tumours expressing high levels of ROS producing MAP17 and SGLT1 proteins can benefit from therapies such as cisplatin or radiotherapy that increase oxidative stress and could sensitize them to cell death. In this work we have explored the relevance of the presence of MAP17 in larynx tumours where primary response is mainly achieved by treatments with radiotherapy and platinum compounds or other radiosensitizers. 4.1.1. Clinical cohort description Lymph node metastases were significantly associated with decreased OS (N0 63.1 m, N1 38.6 and N2 22.2 m, p=0.019) while tumour local extension impacted LDS negatively (T4 extension 7.3 m vs. 47.1m non T4 extension, p=0.003). Besides, patients who required pretreatment tracheotomy had significantly worse LDS (54.3 vs. 18.9 months, p=0.001). The two-year cumulative proportion of patients with larynx preservation and OS were 57% and 76% respectively. Besides, locoregional control rate at two years was 60%, so similar to previously reported in the literature (section 1.5.1). 4.1.1. MAP17 expression in larynx tumour samples Out of 65 samples, only 58 were analysed for MAP17 expression, either due to technical problems or because they did not contain any tumour cellularity. Out of the 58 samples, 46 (79%) were positives for MAP17 expression (Figure 11 A, B and C) and there was a trend showing higher levels of MAP17 in advanced grades of the tumour (Figure 11 D), although in this case, probably due to the low number of cases, it was not statistically significant. Surrounding normal tissue did not express MAP17 or expressed very low levels. We also analysed other markers for proliferation such as KI67 or the activated form of ERK (phosphorylated ERK, ERK-p), or apoptosis such as mutant p53 or activated AKT (phosphorylated AKT, AKT-p). Our cohort showed a percentage of samples positive for KI67, mutant p53, ERK-p or AKT-p, but these groups did not show correlation with MAP17 levels (Figure 12). However, KI67 positivity ! ! ! 54! showed statistically significant correlation with OS (Table 7). No correlation of MAP17 was observed with clinical parameters such as tumour localization, smoking habit, alcohol consumption, tumoral stage, pre-treatment tracheotomy and development of acute toxicities during chemoradiotherapy. Figure 11. MAP17 overexpression in larynx tumours. A) Representative images of MAP17 immunostaining are shown for different larynx tumours. B) High magnification of M17 positive and negative tumours. The picture shows a magnification of the inset of figure A. C) A graph is shown representing the percentage of laryngeal tumours with dichotomous MAP17 levels. The score for positive tumours were >62. D) The distribution of the MAP17 expression levels among different grades of larynx tumours is shown. The MAP17 levels (score) refers to maximum levels (0–2) scored by the percentage of cells (0–100). The normalized levels were obtained by multiplying the percentage of cells by the level of intensity observed. Anova test was performed to establish the statistical association between MAP17 protein levels and the grade of the tumour (p<0,05). ! ! ! 55! Table 7. Laryngoesophageal dysfunction-free survival (LDs) and overall survival (Os) multivariate analysis of laryngeal cancer patients treated with preservation approaches. Pret. Tracheo.: pretreatment tracheotomy required. PS: ECOG-performance status. B/CTRT: bio/chemoradiotherapy; ICT-B/CTRT: induction chemotherapy followed by bio/chemoradiotherapy. Figure 12. p53, Ki67, p-ErK or p-AKt do not show correlation with MAP17 expression in larynx tumours. A) Representative images of p53, Ki67, p-ERK or p-AKT immunostainings are shown for larynx tumours. B) Graphs showing lack of correlation between these proliferative or antiapoptotic markers and MAP17 expression. ! ! ! 56! 4.1.2. SGLT1 overexpression in human larynx tumours correlates with MAP17 levels Previous results indicated that MAP17-dependent tumorigenic properties depend on the indirect activation of ROS by SGLT1 transport and that there is a correlation between the expressions of both markers in cervix tumours (107). Therefore, we measured SGLT1 expression levels in the same cohort of larynx tumour samples. We found that some tumours showed positive SLGT1 staining, with approximately 40% tumours being positive for SLGT1 (Figure 13 A, B, C and D). However, only a few samples showed very high staining levels. The distribution of the SGLT1positive tumours among the different larynx tumours showed a clear correlation with MAP17 expression (Figure 13 E, F and G). Pearson indicator expressed a positive significant correlation between MAP17 and SGLT (P=0.3, p=0.022). Figure 13. SGLT1 overexpression in larynx tumours. A) Representative images are shown of SGLT1 immunostaining of different larynx tumours. B) High magnification of SGLT1 positive and C) Negative tumours. D) Graph representing the percentage of larynx tumours positive or negative for SGLT1 expression. E) Graph representing the correlation between MAP17 and SGLT1 expression in each tumour. The statistical analysis was performed by Pearson correlation (p=0,0022). F) Samples from one patient showing clear correlation between the expression of MAP17 and SGLT1. G) High magnification of samples from one patient showing clear correlation between the expression of MAP17 and SGLT1. ! ! ! 57! 4.1.3. MAP17 as predictive biomarker for laryngeal cancer The high MAP17 group correlated in this study with better OS, LDS and LRC. When MAP17 was measured as a continuous variable, multivariate Cox model demonstrated that higher rates of MAP17 levels correlated with improved OS (HR 0.98, p= 0.001). Nevertheless, this could not be confirmed for LDS (HR 0.99, p=0.8), probably due to the limited number of cases. In order to distinguish a cut-off point for MAP17 levels a ROC curve was performed and punctuation of 62 score chosen. When measured as a dichotomous variable, high-MAP17 was related with increased OS, LDS, and LRC. A difference of 35.3 months was observed between high-MAP17 levels (67 months) and low-MAP17 levels (31.7 m) in the KaplanMeier model (IC 95%; p<0.001) (Figure 14) and the HR estimator for high-MAP17 was 0.78, p=0.002 in the multivariate analysis. Regarding LDS, high-MAP17 showed a survival benefit of 13.1m (47.6 m vs. 34.5 m, p=0.002) with a HR 0.14, p=0.003 in multivariate analysis. The effect of MAP17 high levels on the improved survival was significant after controlling for other variables: P53, Ki67, SGLT, PS, TNM, pretreatment tracheotomy and treatment received as shown in table 7. Regarding LRC, patients with high-MAP17 showed to have better outcomes than low-MAP17 (53.9 m vs 44.5 m, p=0.016) and the results were confirmed in the multivariate model (p=0.045). However, although MAP17 correlated with SGLT in the Pearson model (Figure 13E), SGLT by itself did not show statistically significant correlation with OS or LDS. Moreover, the association of high-MAP17 and high-SGLT show improved OS than MAP17 alone (72.4 m vs 42m, p=0,028) (Figure 14D). These data confirm that MAP17 alone, or preferably combined with SGLT1, is a good prognostic marker for survival in patients with larynx cancer treated with B/ CTRT. ! ! ! 64! Figure 18. A. and B. No differences were found for radiotherapy delivered within less than 9 weeks or ≥9 weeks in terms of LDS or OS. C and D. Same results for a cut-off of 10 weeks. Table 8. LDS and OS multivariate analysis. PT: pretreatment tracheotomy. N: pathological lymph nodes. ! ! ! 65! Figure 19. A. Positive pH2AX expression, considered as nuclei staining was shown in 46 (86.8%) samples. B. Levels of pH2AX are equally distributed among tumour stages. C., D. and E. high-pH2AX show a trend towards better OS, LDS and LRC not statistically significant in the Kaplan-Meier analysis. 5.25 as indicated by the ROC curve were used as cut-off for defining high and low expression of pH2AX for survival analysis. 4.2.4. pH2AX relationship with cisplatin and radiotherapy The total dose of cisplatin was not associated with pH2AX levels (p=0.4). We created a variable with two categories from pH2AX and cisplatin, in which one had a potential favourable prognosis (high-pH2AX levels, and optimal dose of cisplatin, ≥200 mg/m2), and the other unfavourable prognosis (low-pH2AX levels, and/or suboptimal dose of cisplatin <200mg/m2 or other radiosensitizers due to the low number of patients). The favourable prognosis group correlated with increased OS, LDS (OS: 72 m vs 38.6 m, p=0.03; LDS 66.9 m vs 27 m, p=0.019). LRC was not statistically significant (p=0.17) although there was a trend towards better outcomes in the good prognostic subgroup (69.9 m vs 35.1 m) (Figures 20A, B and C). Moreover, the unfavourable prognosis group correlated with worse OS (HR= 3.66, p=0.044), and LDS (HR= 3.38, p=0.028). LRC has a not statistically significant HR (HR=2.4, p=0.188). We also tried to ! ! ! 66! stablish whether high-pH2AX and no radiotherapy delays could impact on survival but no differences were found for both OS and LDS. 4.2.5. Correlation of pH2AX with p53 and KI67 We also analysed other markers for proliferation such as KI67 or the activated form of ERK (phosphorylated ERK, ERK-p), and apoptosis such as mutant (m) p53 or activated AKT (phosphorylated AKT, AKT-p). Our cohort showed a percentage of positive samples for ERK-p or AKT-p, but these groups did not show correlation with pH2AX expression (data not shown). KI67 in combination with pH2AX was not significant in any combination (data not shown), being pH2AX also independent of the proliferative capability of the tumour. Our results showed no correlation between p53 and pH2AX although there was a relation towards increased pH2AX with negative P53 (<5% positive nuclei) that was not statistically significant (p=0.33). However, in our cohort p53 samples positive (measured as >5% positive nuclei) (Figure 21A, + p53) correlated with worse OS (- p53=50 vs + p53=35.6 m, p=0.05) (Figure 21B) consistent with previous literature (151). P53 and pH2AX were combined into a new variable with the following categories: potential good prognosis phenotype (negative p53 and high-pH2AX) and unfavourable prognosis phenotype (positive p53 and low-pH2AX). Although there was an apparent relation towards better outcomes in the good prognosis phenotype, this was not significant for both OS and LDS (OS: 48.6 m vs 39 m, p=0.39; LDS: 38.8 m vs 24.4 m, p=0.068) (Figures 21C and D). Figure 20. A., B. and C. pH2AX and dose of concomitant cisplatin were combined in a new variable where high-pH2AX and cisplatin (Cpt) ≥200 mg/m2 was considered as good prognosis phenotype category. The results show improved OS and LDS in this subgroup, and a trend towards better LRC. 5.25 as indicated by the ROC curve were used as cut-off for defining high and low expression of pH2AX for survival analysis. ! ! ! 67! Figure 21. A. P53 was measured as >5% positive nuclei, as shown in the picture. B. positive P53 (+ P53) correlates with worse OS in our cohort. C. and D. results of the combination of P53 and pH2AX in a new variable. Although there was a trend towards better outcomes in the good prognosis phenotype which included negative P53 (-P53) and high-pH2AX, this was not statistically significant for OS and LDS. 4.2.6. Correlation of pH2AX and MAP17 As discussed before, MAP17 increases endogenous ROS, which is a well-known mediator of DNA damage. Therefore, we measured whether pH2AX correlated with MAP17 expression and if the combination of both markers could strength the predictability of responses. We found that patients with high levels of MAP17 and subject to optimal doses of cisplatin had better LDS (58.6 m vs 32.6 m, p=0.053) and OS (76.2 m vs 40.9 m, p=0.005) than patients with low MAP17 or not subject to optimal doses of cisplatin (Figures 22A and 7B). Furthermore, patients with high levels of MAP17 and high-pH2AX, denoting higher structural DNA-damage, conformed the group of better prognosis after therapy (Figures 22C and 7D). Moreover, patients with high-MAP17, high-pH2AX and optimal dose of cisplatin had better OS and LDS than the rest of the population (Figures 22E and F), and also when compared with patients having a poor prognosis phenotype (low-MAP17, low-pH2AX and suboptimal cisplatin dose) (Figures 22G and H). ! ! ! 68! In summary, these results show that pH2AX has a prognostic role in patients with laryngeal cancer. pH2AX was related to LDS (HighpH2AX HR 0.26, p = 0.02) in a cohort of 53 patients with larynx cancer. When analysed together pH2AX expression and dose of cisplatin received during radical treatment, there is a significant correlation with survival (high-pH2AX and optimal dose of cisplatin 72 vs 38.6 m, p = 0.03) and LDS (high-pH2AX and optimal dose of cisplatin 66.9 vs 27 m, p = 0.019). Also, patients with high-MAP17 and high-pH2AX showed to have better OS and LDS. Our data also show the importance of performing optimal cisplatin treatment for tumour response. However, the fact that unexpected radiotherapy delays and interruptions did not affect survival in our cohort could be explained to dose compensations. Radiobiological-based calculations were performed in those patients in order to achieve an equivalent biological effectiveness by adding some more fractions to the overall treatment. Our data suggest that inherent DDR pathway activation (measured by the end-point of phosphorylation of H2AX) is a valuable prognostic marker in patients with laryngeal carcinoma who received organ preservation approaches. ! ! ! 69! Figure 22. A. and B. the combination of high-MAP17 and optimal doses of cisplatin (Cpt) showed better OS and LDS. C. and D. patients with high-MAP17 and high-pH2AX with higher structural DNA-damage showed to have better OS and LDS. E. and F. survival for patients with high-pH2AX, high-MAP17 and optimal dose of cisplatin was statistically better. G. and H. the subgroup of patients with high-pH2AX, high-MAP17 and cisplatin optimal dose patients was compared to the patients that had low-pH2AX, lowMAP17 and did not complete cisplatin. Although limited in numbers, none of the patients with poor prognosis phenotype reached more than 2-years LDS or more than 3-years OS. ! ! ! 70! DISCUSSION ! ! ! 71! In this work novel biomarkers for patients diagnosed of laryngeal cancer who were candidates for organ preservation treatments have been identified. In general, accepted organ sparing approaches include radiotherapy, bio or chemotherapy with concomitant radiotherapy and induction chemotherapy followed by radiotherapy with or without bio/chemotherapy. To date, there are no biomarkers validated that could predict survival or response with these preservation approaches, and a number of patients have significant late toxicities or finally relapse. In those cases, salvage surgery can sometimes be performed. Identifying predictive biomarkers in this setting could avoid unnecessary toxicities and could potentially improve survival because of the better selection of patients. Pathological, clinical and therapeutic features of our population were studied in order to determine whether they could be related to survival. In this regard, patients with T4 primary tumour and those who needed a pre-treatment tracheotomy had worse LDS and seemed not to benefit of preservation treatments. Moreover, receiving optimal platinum total dose can determine LDS, as patients unable to complete treatment have a worse prognosis. A panel of different markers were studied in our population; proliferation markers such as Ki67 or p-ERK, apoptosis such as p53 and p-AKT. However, DNA damage response biomarkers such as MAP17, SGLT and pH2AX were the ones implicated with survival in our cohort. MAP17 is a membrane-associated protein known to increase endogenous ROS through SGLT1 in cancer cells (152,153). ROS are well known mediators of DNA damage. In a previous study, expression of both MAP17 and SGLT1 was associated with survival in a cohort of patients with squamous cervical cancer treated with radiotherapy and cisplatin (107), thus having some similarities to our population. Those results suggested that patients expressing MAP17 and SGLT1 had better response to treatments that boost oxidative stress. In a more recent study, high MAP17 levels correlated with a poor prognosis and a higher grade of sarcoma. In this case, it was a heterogeneous group of different sarcoma histologies, most of them with metastatic disease. As patients were candidates for systemic therapies and not for radiotherapy, oxidative stress may not be implicated in this population (154). Our analysis in laryngeal cancer showed that MAP17 increased not only OS but also LRC and LDS in laryngeal cancer. On the other hand, SGLT-1 expression was significantly related to MAP17. However, just a portion of tumour samples was positive (40%) and therefore survival could not be related to SGLT1 by itself. Nevertheless, the combination of high MAP17 and SGLT improved OS better than MAP17 alone. γH2AX phosphorylation has been studied as prognostic biomarker in early operable non-small cell lung cancer (NSCLC) and endometrial carcinomas. In the NSCLC study, low levels of pH2AX correlated with better survival outcomes. The combination of wild type p53 and lowpH2AX phenotype showed also better survival. In the endometrial trial, pH2AX positively ! ! ! 72! correlated with p53 levels although the relation with survival could not be proved. However, patients in both studies were treated with surgery and not with radiotherapy (155, 156). Tumour cells from clinical specimens show constitutive activation of DNA damage signalling as demonstrated by the presence of gH2AX phosphorylation and other DDR signalling proteins (157,158, 159). This DDR activation was found to peak at early stage tumours, persisting further among malignant tumours mostly by inactivating p53 gatekeeper (159). It has been proposed that the DDR-network may serve as an inducible barrier to control the initial steps of tumour development by inducing p53-dependent senescence or apoptosis (157,158, 159). Further ongoing chronic DDR activation favours the outgrowth of malignant clones with genetic or epigenetic defects in DNA-repair mechanism such as those involved in the DDR pathway (159). Our samples, from already malignant tumours (stages II-IV), in which only a subset of them showed mutant p53, correlated with worse onset of the disease. It is likely DNA-damage defects inducing DDR activation have been carried through the malignant process and it is possible that other proteins are mutated in the process avoiding the requirement for p53 inactivation. pH2AH is a broad DNA damage marker that appears under different physiological conditions. Senescent cells display molecular characteristics of DNA damage (160,161,162). These markers include nuclear foci of phosphorylated histone H2AX, the localization at double-strand break sites of DNA-repair and DNA-damage checkpoint factors, such as 53BP1, MDC1 and NBS1 (163, 164, 165). Senescent cells also contain activated forms of the DNA-damage checkpoint kinases Chk1 and Chk2. During replicative senescence, markers of a DNA damage response localize at telomeres (164, 166), indicating that the DNA damage response is triggered by telomere shortening (167). Similarly, the redox potential also results in DNA damage and senescence (168). Very interestingly, oncogene-induced senescence has been found to induce DNA-damage due to an excess of replication forks. This oncogenic-induced hyper-replication signal, or replication stress, is associated with persistent DNA-damage (169, 170) inducing senescence (171). Therefore, not only senescence is viewed as a response to DNA-damage, but DNA-damage as a marker of senescence. In that sense, high pH2AX appeared in early stage tumors and is a marker of good prognosis (172,173). However, in our cohort, pH2AX levels are increased in advanced stages of tumors, and contrarily to this hypothesis, are a marker of bad prognosis, indicating that our pH2AX observations are not due to cellular senescence, neither by continuous proliferation nor by replication stress. In that line, phosphorylation of H2AX is not always a marker of DNA damage. It also can be a marker of activated mTOR, eliciting replicative stress and a pseudo DNA-damage in senescent cells (170, 174-177). The dynamics of senescence exhibit 2 different steps: cell cycle arrest and further acquisition of senescence features, which includes permanent arrest, termed geroconversion (170, 178-180). If geroconversion is not activated, cells are only transiently arrested with the possibility of ! ! ! 73! resuming growth once the proliferation constraints have been eliminated (163, 180). It has also been shown that if mTOR is activated under conditions of proliferative arrest, then arrest becomes permanent and the cell undergoes senescence (178, 179, 181). Under these conditions of cell cycle arrest and mTOR activation, the phosphorylation of H2AX is launched, becoming a marker of cellular senescence (165, 171, 174, 181). In fact, rapamycin treatment, which inhibits mTOR, can divert senescence into quiescence, allowing the cell to resume growth once conditions are more favourable (182-185). Since mTOR is the master regulator of protein synthesis (186), it has been proposed that this contribution is due to the function of mTOR as a sensor of cellular nutrients and energy status as well as growth factor signals (187, 188). However, it has also been reported that mTOR activation in the context of growth arrest is perceived by the cells as an unwanted oncogenic signal, activating the replicative stress and pseudo DNA-damage signalling (165, 171, 174, 181). In any case, high levels of pH2AX as marker of cellular senescence should be associated to better prognosis, and to some extent to early stage tumours. However, it will be of interest to correlate the levels of pH2AH with those of mTOR activation in laryngeal tumours to provide a more accurate hypothesis of the pH2AH inducers. Our data show that high levels of pH2AX correlate with better prognosis after treatment with DNA-damage agents such as cisplatin and radiotherapy, especially if cisplatin is given at optimal doses. These data are suggestive of a collaboration of DDR pathway activation, perhaps as an indicator of low DNA-repair ability and DNA-damaging agents in tumour therapy. The fact that doses of cisplatin are important for survival seems to confirm this hypothesis. In line with this, wt-P53 with high levels of pH2AX conforms a subgroup of good prognosis suggesting that P53 activity is essential to drive physiological response to apoptosis (or senescence) of DNA-damage agents in tumours with DDR activated. These data are opposite to the found in early operable non-small cell lung cancer (NSCLC). In this study, low levels of pH2AX correlated with better survival outcomes. The combination of wild type p53 and low-phosphorylated ãH2AX phenotype showed also better survival. However, NSCLC patients were treated with surgery and not with radiotherapy (155). This lack of treatment with radiotherapy could be the cause of the different behaviour respect the pH2AX. Radiotherapy increases oxidative stress and reactive oxygen species that in combination with pre-existing DNA damage can increase cell damage above threshold inducing increased tumour efficacy. Our data support this hypothesis since combination with another ROS-inducing agent such as cisplatin is essential to gain better survival in these patients. Furthermore, the combination of MAP17 also supports the essential role of radiotherapy in this response. Therefore, this work suggests that high levels of MAP17 induced ROS that in turn increases DNA-damage and DDR signalling. Upon further DNA-damage and further increase in ROS molecules induced by cisplatin and RT treatment, tumours with higher oxidative stress (higher ! ! ! 80! 1. Ferlay J, Soerjomataram I, Ervik M, et al. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase. Accessed: http://globocan.iarc.fr (link is external), accessed December 2013. 2. Ferlay J, Steliarova-Foucher E, Lortet-Tieulent J, et al. Cancer incidence and mortality patterns in Europe: Estimates for 40 countries in 2012. European Journal of Cancer (2013) 49, 1374-1403. 3. Fitzmaurice C, Dicker D, Pain A, et al. 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