Identification of novel laryngeal cancer biomarkers in patients treated with organ preservation.

El cáncer de laringe representa aproximadamente el 1% del total de las neoplasias, con una incidencia estimada estandarizada por edad de 4.4 cada 100.000 habitantes. Es más frecuente en varones durante la sexta década de vida. Anatómicamente, la laringe se extiende desde la lengua hasta la tráquea y conduce el aire siendo el órgano responsable de la fonación. Se compone fundamentalmente de dos tipos histológicos: epitelio respiratorio pseudoestratificado en la cara interna, y el epitelio no-queratinizado que envuelve las cuerdas vocales y la cara externa. La gran mayoría de los tumores laríngeos son carcinomas epidermoides, aunque existen otros subtipos menos frecuentes. Los cánceres de cabeza y cuello (CCyC) se consideran el estadio final de un complejo proceso que incluye pérdidas de heterocigosidad, amplificaciones, deleciones y alteraciones de la regulación de oncogenes y genes supresores de tumores. La causa más fundamental del CCyC es el tabaco, donde hasta un bajo consumo del mismo se ha asociado a un aumento del riesgo de cáncer. El segundo factor es el alcohol, que potencia el efecto del tabaco. El sistema de clasificación más frecuentemente utilizado es el TNM por sus siglas en inglés “tumour, node, metastasis” elaborado por la American Joint Committee on Cancer (AJCC). El tratamiento de los pacientes no metastásicos depende de si la enfermedad se ha presentado en un estadio inicial o como enfermedad locoregional. En general, los estadios iniciales se benefician de cirugía o radioterapia, mientras que los avanzados requieren de un abordaje multidisciplinar. Tres estrategias terapéuticas están aceptadas: radioterapia, bio o quimioterapia concomitante con radioterapia y quimioterapia de inducción seguida por radioterapia con o sin bio/quimioterapia. Aunque las terapias de preservación de órgano permiten conservar la laringe, no han demostrado aumentar la supervivencia en comparación con cirugía. Además, estos tratamientos conllevan importantes toxicidades y en un 30-40% los pacientes sufren recidivas o pierden la funcionalidad de la laringe. Disponer de biomarcadores predictivos facilitaría la identificación de aquellos pacientes que no se van a beneficiar de tratamiento basado en la radioterapia. Derivando estos pacientes directamente a cirugía, el control local de la enfermedad y potencialmente la supervivencia podrían optimizarse y evitar toxicidades innecesarias. Con este estudio se pretende identificar biomarcadores pronósticos que puedan llegar a ser predictivos para la selección de pacientes. Se han evaluaron 65 pacientes con cáncer de laringe tratados en el Hospital Universitario Virgen de Rocío entre agosto de 2005 y febrero de 2014. Los criterios de selección para la preservación de órgano incluyeron estadios II a IV de cáncer de laringe, sin contraindicación para quimio o radioterapia, ni destrucción significativa de cartílago o invasión mayor de 2 cm de la base de la lengua. Los pacientes fueron mayoritariamente varones con carcinomas epidermoides estadio III y buen estado general. Se procedió a la caracterización histológica de todas las muestras mediante tinción de hematoxilina-eosina seguido de 5 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.Laryngeal cancer represents approximately 1% of the total of malignancies with an estimated incidence age-standardised rate of 4.4 per 100.000 persons. It is most commonly presented in males at their sixth decade of life. Anatomically, the larynx is an air passage and an organ of phonation that extends from the tongue to the trachea. Two different histologies are escribed in the larynx: the pseudostratified respiratory epithelium that covers the inner aspects of the larynx, and the non-keratinized, stratified squamous epithelium that covers the vocal folds and the exterior surfaces of the larynx. The vast majority of tumours arising the larynx are conventional squamous cell carcinomas (SCC), although there are other less frequent subtypes. SCC of the head and neck is considered now to be the final stage of a multi-step process in which loss of heterozygosity, amplifications, deletions, up and down-regulation oncogenes or tumoursuppressor genes take part. The primary cause for head and neck cancer is tobacco use, where even a low quantity of cigarette smoking has been associated with an increased risk. Alcohol consumption is the second major risk factor, which potentiates the effects of tobacco. The most extended cancer staging system is the tumour node metastasis (TNM) system by the American Joint Committee on Cancer (AJCC). For non-metastatic patients, treatment options depend on whether the tumour is presented at early stages, or as locoregional disease. In general, early stages are treated with either surgery or definitive radiotherapy, while advanced stages require a multimodal approach. Three sparing approaches are accepted: radiotherapy, bio or chemotherapy with concomitant radiotherapy and induction chemotherapy followed by radiotherapy with or without bio/chemotherapy. Although functional organ sparing approaches allow larynx preservation, they do not provide a survival advantage over surgery. Moreover, those treatments confer significant toxicities and approximately 30-40% of patients will relapse or lead to an incompetent larynx. Predictive biomarkers would facilitate pre-treatment identification of patients who are unlikely going to be cured by radiation-based therapy. Managing these patients with surgery rather than with preservation approaches, local disease control and survival could be potentially optimized and unnecessary treatment related morbidities from unsuccessful larynx treatments avoided. With this study we aim to find biomarkers that could be related with prognosis and being used as predictive biomarkers for pre-treatment patient selection. We evaluated 65 patients with larynx cancer treated at the Hospital Universitario Virgen del Rocío from August 2005 until February 2014. Eligibility criteria for treatment preservation include patients with stage II-IV laryngeal tumours that had no contraindication for hemotherapy and/or radiotherapy, significant cartilage destruction, or more than 2 cm tumoral invasion of the base of the tongue. Patients were mainly male with stage III supraglottic SCC and with a good general condition. Histological characterization of all samples was done by hematoxylin and eosin staining, followed by immunohistochemistry analysis of tissue microarrays. Analysed markers included proliferation markers such as Ki67 or the activated form of ERK, apoptosis such as mutant p53 or activated AKT, and finally markers involved in the DNA-damage response (DDR) pathway such as MAP17, SGLT and pH2AX. Subsequent statistical analysis was performed, taking into consideration clinical and pathological factors. Concerning clinical factors, T4 primary tumour extension and patients who needed a pre-treatment tracheotomy had worse laryngoesophageal dysfunction-free survival (LDS) and seemed not to benefit of preservation treatments. Moreover, receiving an optimal platinum dose determined LDS, as patients unable to complete treatment had worse prognosis. In what respects to pathological factors, markers related with the DDR were associated with survival. DDR is a network of cellular pathways that sense, signal and repair DNA lesions. MAP17 is a small membrane protein highly expressed in metastatic carcinoma. Its expression is associated with a SGLT-dependent reactive oxygen species (ROS) incease. While a mild increase in ROS activates signalling cascades that upregulate tumorigenic processes, further ROS increases lead to toxic cellular environment and to programmed cell death. Moreover, γH2AX is a component of the histone octamer in nucleosomes involved in recruiting DNA repair proteins in response to the presence of DNA double-strand breaks. Hence, it´s increasing is related to structural DNA damage. Therefore, DNA-damage produced by ROS would lead to pH2AX increasing. Laryngeal cancer tumours with high levels of ROS producing MAP17 and SGLT proteins and with high pH2AX expression should benefit from therapies such as cisplatin or radiotherapy that increase oxidative stress and could sensitize them to cell death. Our analysis confirmed a significant relationship between high MAP17 protein expression and increased overall survival (OS), locoregional control (LRC) and LDS. In fact, high MAP17 levels demonstrated better OS than low levels (67 months vs. 31.7 months, IC 95%; p<0.001). In addition, high-MAP17 and high-SGLT showed improved OS, better than MAP17 alone. In our cohort, pH2AX was related to LDS (high-pH2AX HR 0.26, p = 0.02). When analysed together pH2AX expression and dose of cisplatin received during radical treatment, there was a significant correlation with survival and LDS. Also, patients with high- MAP17 and high-pH2AX showed to have better OS and LDS. Therefore, this work suggests that high levels of MAP17 induced ROS that in turn increases DNA-damage and DDR signalling, measured as high-pH2AX. Upon further DNA-damage and further increase in ROS molecules induced by cisplatin and RT treatment, tumours with higher oxidative stress, are more suitable to undergo apoptosis. The inherent DDR pathway activation biomarkers MAP17 and pH2AX are valuable prognostic markers in patients with laryngeal carcinoma who received organ preservation approaches

MAP17 (PDZKIP1) as a novel prognostic biomarker for laryngeal cancer

Larynx cancer organ preservation treatments with chemo and radiotherapy have substantially improved laryngoesophageal dysfunction-free survival. However, both of them lead to a high incidence of acute and chronic toxicities and a significant number of patients relapse. To date, there is no evidence available to establish the group of patients that may benefit from preservation approaches and clinical criteria such as primary tumor extension or pretreatment tracheotomy are not validated. MAP17 is a small non-glycosylated membrane protein overexpressed in carcinomas. The tumoral behavior induced by MAP17 is associated with reactive oxygen species production in which SGLT1 seems involved. In this study we found that the levels of MAP17 were related to clinical findings and survival in a cohort of 58 patients with larynx cancer. MAP17 expression is associated with overall survival (p < 0.001) and laryngoesophageal dysfunction-free survival (p=0.002). Locoregional control in patients with high MAP17 showed better outcomes than those with low MAP17 (p=0.016). Besides, a positive correlation was observed between MAP17 expression and SGLT (p=0.022) and the combination of high levels of MAP17/SGLT also led to an increased overall survival (p=0,028). These findings suggest that MAP17, alone or in combination with SGLT1, may become a novel predictive biomarker for laryngeal carcinoma.This work was supported by grants from the Spanish Ministry of Economy and Competitivity, ISCIII (Fis: PI12/00137, RTICC: RD12/0036/0028) co-funded by FEDER from Regional Development European Funds (European Union), Consejeria de Ciencia e Innovacion (CTS-1848) and Consejeria de Salud of the Junta de Andalucia (PI-0306-2012). This work has been also possible thanks to the Grant PIE13/0004 co-funded by the ISCIII and FEDER funds.Peer Reviewe

Phosphorylation of gH2AX as a novel prognostic biomarker for laryngoesophageal dysfunction-free survival

Current larynx preservation treatments have achieved an improvement of laryngoesophageal dysfunction-free survival (LDS) but lead to significant toxicities and recurrences. At present, there is no evidence to select the group of patients that may benefit from preservation approaches instead of surgery. Therefore, laryngeal biomarkers could facilitate pretreatment identification of patients who could respond to chemoradiation-based therapy. In this study, we evaluated retrospectively 53 patients with larynx cancer to determine whether gH2AX phosphorylation (pH2AX) alone or in combination with the membrane protein MAP17 (PDZK1IP1) could be used as prognostic biomarkers. We also evaluated whether the completion of cisplatin treatment and radiotherapy could predict survival in combination with pH2AX. We found that the dose of cisplatin received but not the length of the radiotherapy influenced LDS. High-pH2AX expression was associated with prolonged LDS (HR 0.26, p = 0.02) while MAP17 correlated with overall survival (OS) (HR 0.98, p = 0.05). High-MAP17 and high-pH2AX combined analysis showed improved LDS (with 61.35 months vs 32.2 months, p = 0.05) and OS (with 66.6 months vs 39.8 months, p = 0.01). Furthermore, the subgroup of high-pH2AX and optimal dose of cisplatin was also associated with OS (72 months vs 38.6 months, p = 0.03) and LDS (66.9 months vs 27 months, p = 0.017). These findings suggest that pH2AX alone or better in combination with MAP17 may become a novel and valuable prognostic biomarker for patients with laryngeal carcinoma treated with preservation approaches.Junta de Andalucia ISCIII-Red de Biobancos RD12/0036/0017.Unión Europea, Ministerio de Economía y Competitividad PI12/00137, PI15/00045, RTICC: RD12/0036/0028Junta de Andalucía CTS-6844; CTS-1848Junta de Andalucía PI-0135-2010; PI-0306-2012Instituto Carlos III (ISCIII) PIE13/0004

combined pik3ca and fgfr inhibition with alpelisib and infigratinib in patients with pik3ca mutant solid tumors with or without fgfr alterations

PURPOSE Concurrent PIK3CA mutations and fibroblast growth factor receptor (FGFR) alterations occur in multiple cancer types, including estrogen receptor–positive breast cancer, bladder cancer, and endometrial cancer. In this first-in-human combination trial, we explored safety and preliminary efficacy of combining the PI3Kα selective inhibitor alpelisib with the FGFR1-4 selective inhibitor infigratinib. PATIENTS AND METHODS Patients with PIK3CA-mutant advanced solid tumors, with or without FGFR1-3 alterations, were enrolled in the dose escalation or one of three molecular-defined dose-expansion cohorts. The primary end point was the maximum tolerated dose. Secondary end points included safety, pharmacokinetics, and response. Archival tumor samples were sequenced to explore genomic correlates of response. RESULTS In combination, both agents were escalated to full, single-agent recommended doses (alpelisib, 300 mg per day continuously; infigratinib, 125 mg per day 3 weeks on followed by 1 week off). The toxicity profile of the combination was consistent with the established safety profile of each agent, although 71% of all patients required at least one treatment interruption or dose reduction. Molecularly selected dose expansions in breast cancer and other solid tumors harboring PIK3CA mutations, alone or in combination with FGFR alterations, identified sporadic responses, predominately in tumor types and genotypes previously defined to have sensitivity to these agents. CONCLUSION The combination of alpelisib and infigratinib can be administered at full single-agent doses, although the high rate of dose interruption or reduction suggests long-term tolerability may be challenging. In exploratory signal-seeking cohorts of patients harboring dual PIK3CA and FGFR1-3 alterations, no clear evidence of synergistic activity was observed

A genetic view of laryngeal cancer heterogeneity.

During the recent decades significant improvements in the understanding of laryngeal molecular biology allowed a better characterization of the tumor. However, despite increased molecular knowledge and clinical efforts, survival of patients with laryngeal cancer remains the same as 30 years ago. Although this result may not make major conclusions as preservation approaches were not broadly used until the time of database collection, it seems to be clear that there is still window for improvement. Although the cornerstone for laryngeal cancer eradication is to implement smoking cessation programs, survival progresses will be hopefully seen in the future. Introducing molecular biomarkers as predictive factors to determine which patients will benefit of preservation treatments may become one of the next steps to improve survival. Furthermore, the development of new therapeutic modalities joint to biomarkers to selectively apply such new therapy in these patients may help to define new modalities with improved survival. New inhibitors against Notch pathway, EGFR, VRK1 or DNA damage repair may become gold standard if we are able to identify patients that may benefit from them, either on survival or functional larynx preservation. It is the moment for an inflexion point on the way laryngeal cancer is clinically managed

Phosphorylation of gH2AX as a novel prognostic biomarker for laryngoesophageal dysfunction-free survival

Current larynx preservation treatments have achieved an improvement of laryngoesophageal dysfunction-free survival (LDS) but lead to significant toxicities and recurrences. At present, there is no evidence to select the group of patients that may benefit from preservation approaches instead of surgery. Therefore, laryngeal biomarkers could facilitate pretreatment identification of patients who could respond to chemoradiation-based therapy. In this study, we evaluated retrospectively 53 patients with larynx cancer to determine whether gH2AX phosphorylation (pH2AX) alone or in combination with the membrane protein MAP17 (PDZK1IP1) could be used as prognostic biomarkers. We also evaluated whether the completion of cisplatin treatment and radiotherapy could predict survival in combination with pH2AX. We found that the dose of cisplatin received but not the length of the radiotherapy influenced LDS. High-pH2AX expression was associated with prolonged LDS (HR 0.26, p = 0.02) while MAP17 correlated with overall survival (OS) (HR 0.98, p = 0.05). High-MAP17 and high-pH2AX combined analysis showed improved LDS (with 61.35 months vs 32.2 months, p = 0.05) and OS (with 66.6 months vs 39.8 months, p = 0.01). Furthermore, the subgroup of high-pH2AX and optimal dose of cisplatin was also associated with OS (72 months vs 38.6 months, p = 0.03) and LDS (66.9 months vs 27 months, p = 0.017). These findings suggest that pH2AX alone or better in combination with MAP17 may become a novel and valuable prognostic biomarker for patients with laryngeal carcinoma treated with preservation approaches.The authors thank the donors and the Andalusian Public Health System Biobank (ISCIII-Red de Biobancos RD12/0036/0017) for the human specimens used in this study. This work was supported by grants to from the Spanish Ministry of Economy and Competitivity, Plan Nacional de I+D+I 2008-2011, Plan Estatal de I+D+I 2013-2016, ISCIII (Fis: PI12/00137, PI15/00045, RTICC: RD12/0036/0028) co-funded by FEDER from Regional Development European Funds (European Union), Consejeria de Ciencia e Innovacion (CTS-6844 and CTS-1848) and Consejeria de Salud of the Junta de Andalucia (PI-0135-2010 and PI-0306-2012). This work has been also possible thanks to the Plan Estatal de I+D+i 2013-2016, Grant PIE13/0004 co-funded by the ISCIII and FEDER funds.Peer reviewe