Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease, and several attempts have been made to classify these tumors based on anatomic location, clinical stage and tumor phenotype. Once these characteristics are individualized, the appropriate treatment is chosen. For instance, HPV-positive HNSCCs have been defined as a different entity from HPV-negative based on its onset and treatment response. HPV-positive tumors usually respond better to standard treatment choices, while overall survival of these patients is improved. Still, a subset of these patients will present tumor recurrence within the first two years, presenting also reduced overall survival. There is a need to find new markers to serve as molecular targets in HNSCCs. Several studies are ongoing, in order to understand the molecular bases underneath this differential response in HPV-positive patients. Up to date, no common ground has been reached, the main issue being that there are no standard guidelines to classify HPV-positive patients. To improve stratification, many studies investigate HPV DNA, HPV mRNA and p16 overexpression. However, HPV might be present as a transient infection, and not active in the tumors, p16 expression is not always observed in HPV-positive tumors and HPV mRNA levels could be too low to be detected. In this study, similar to other studies, classical markers alone, were not sufficient to stratify HPV-positive HNSCC patients for disease recurrence. Nevertheless, the combination of classical markers with serological markers resulted a strong indicator of patient prognosis, particularly for oropharyngeal squamous cell carcinoma (OPSCC). HPV16 E7 oncoprotein presence in serum at the time of diagnosis correlated with disease recurrence and overall survival. Research for E7 oncoproteins in serum may be useful as a non-invasive procedure for patient stratification and follow-up, helping identify patients at risk for tumor recurrence, giving a tool for clinicians for candidate patients selection to de-escalate treatment. Furthermore, since HPV mRNA is necessary to promoter carcinogenesis and the long control region (LCR) of the virus plays a fundamental role in the control of HPV gene expression, we studied how variation or epigenetic control of this region could affect viral gene expression and patient prognosis. While methylation of the LCR plays a fundamental role in the control of HPV mRNA, the importance of regulation by other cellular transcription factors emerges, showing the importance HPV LCR sequence variations have for transcription factor binding and how they can greatly affect viral gene expression. Sequence variations within the YY1 transcription factor binding site, were correlated to improved patient survival, while patients carrying the reference sequence relapsed more frequently. Finally, my thesis discusses a set of genes dysregulated in HNSCC in correlation to the disease subgroups, and patient prognosis. Differences were significant for EGFR, p16, c-Jun and RARB gene expression between HPV-positive and negative subgroups; these markers may help create an expression profile in order to stratify HNSCC patients at diagnosis, and select the best treatment choice. Furthermore, diminished levels of IRF6 correlated to recurrence in HPV-negative patients. The findings presented in my thesis show molecular differences between HPV-positive and negative tumors and shed light on fundamental aspects of the HPV biology, that had been poorly studied in HNSCCs so far; first, the importance that HPV16 sequence has on the behavior of the virus and its pathogenic potential; and second, the presence of circulating viral oncoproteins in HPV-positive patients in serum. These aspects turned to be equally important related to patient’s progression free survival, and should be studied in more depth as candidate markers for patient stratification in the future.

Il carcinoma a cellule squamose testa-collo (HNSCC) è una malattia eterogenea. La classificazione dei HNSCC si basa sulla posizione anatomica, lo stadio clinico, il fenotipo e la presenza del virus HPV, ed è importante per la scelta del trattamento appropriato. I tumori positivi all'HPV costituiscono un'entità diversa da quelli negativi all'HPV sia per il sito di insorgenza che la risposta al trattamento. Infatti, i tumori HPV-positivi di solito rispondono meglio ai trattamenti standard, e i pazienti presentano una sopravvivenza migliore. Tuttavia, un sottogruppo di questi pazienti presenta recidiva entro i primi due anni dalla diagnosi, e ridotta sopravvivenza. Perciò è necessaria la ricerca di nuovi marcatori che fungano da bersagli molecolari negli HNSCC, per poter aumentare la soppravivenza globale in tutti i gruppi. Diversi studi per comprendere le basi molecolari alla base della risposta differenziale nei pazienti HPV-positivi sono in corso, ma non è stata raggiunta una conclusione. Il problema principale è che non esistono linee guida standard per classificare i pazienti HPV-positivi. Molti studi indagano sul DNA dell'HPV, l'mRNA del virus e la sovraespressione di p16. Tuttavia, l'HPV potrebbe essere presente come infezione transitoria, l'espressione di p16 non è sempre osservata nei tumori HPV-positivi, e i livelli di mRNA dell'HPV potrebbere essere troppo bassi per essere rilevati. Pertanto, la rilevazione di marcatori classici non è sufficiente per stratificare i pazienti HNSCC-HPV positivi per la recidiva. In questo mio studio, ho verificato che la combinazione di marcatori classici con marcatori sierologici è risultata un forte indicatore prognostico. In particolare, per il carcinoma a cellule squamose dell'orofaringe (OPSCC) la presenza della oncoproteina E7 nel siero al momento della diagnosi era correlata alla recidiva della malattia e alla sopravvivenza globale. La ricerca di E7 nel siero può essere utile come procedura non invasiva per la stratificazione dei pazienti HPV-positivi e il follow-up, aiutando a identificare i pazienti a rischio di recidiva e fornendo uno strumento per la selezione dei pazienti candidati alla riduzione dei trattamenti chemio/radioterapici. Inoltre, poiché l'mRNA dell'HPV è necessario per promuovere la carcinogenesi e la long control region (LCR) del virus gioca un ruolo fondamentale nel controllo dell'espressione genica dell'HPV, abbiamo studiato come la variazione nucleotidica o la metilazione di questa regione influenzi l'espressione genica virale e la prognosi. Mentre la metilazione dell'LCR gioca un ruolo fondamentale nel controllo dell'mRNA virale, le variazioni della sequenza dell'HPV LCR resultano rilevanti per il legame con i fattori di trascrizione cellulari, influenzando l'espressione genica virale. Nello specifico, variazioni di sequenza all'interno del sito di legame del fattore di trascrizione YY1 erano correlate a una migliore sopravvivenza. Infine, la mia tesi discute un insieme di geni disregolati nell'HNSCC in correlazione ai sottogruppi di malattia e alla prognosi dei pazienti. Abbiamo trovato significative differenze per l'espressione genica di EGFR, p16, c-Jun e RARB tra i sottogruppi HPV-positivi e negativi; questi marcatori possono aiutare a creare un profilo di espressione al fine di stratificare i pazienti HNSCC alla diagnosi e selezionare il trattamento adeguato. Inoltre, diminuzioni nei livelli di IRF6 sono stati correlati a recidiva in pazienti HPV negativi. Nell’insieme i risultati presentati mostrano differenze molecolari tra tumori HPV-positivi e negativi, facendo luce su aspetti fondamentali della biologia dell'HPV e scarsamente studiati finora. Questi aspetti, sono ugualmente importanti per la sopravvivenza libera da progressione, e dovrebbero essere studiati in modo più approfondito in futuro come marcatori candidati per la stratificazione dei pazienti.

New biomarkers for human papillomavirus positive-head and neck squamous cell carcinomas

OTON GONZALEZ, Lucia
2021

Abstract

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease, and several attempts have been made to classify these tumors based on anatomic location, clinical stage and tumor phenotype. Once these characteristics are individualized, the appropriate treatment is chosen. For instance, HPV-positive HNSCCs have been defined as a different entity from HPV-negative based on its onset and treatment response. HPV-positive tumors usually respond better to standard treatment choices, while overall survival of these patients is improved. Still, a subset of these patients will present tumor recurrence within the first two years, presenting also reduced overall survival. There is a need to find new markers to serve as molecular targets in HNSCCs. Several studies are ongoing, in order to understand the molecular bases underneath this differential response in HPV-positive patients. Up to date, no common ground has been reached, the main issue being that there are no standard guidelines to classify HPV-positive patients. To improve stratification, many studies investigate HPV DNA, HPV mRNA and p16 overexpression. However, HPV might be present as a transient infection, and not active in the tumors, p16 expression is not always observed in HPV-positive tumors and HPV mRNA levels could be too low to be detected. In this study, similar to other studies, classical markers alone, were not sufficient to stratify HPV-positive HNSCC patients for disease recurrence. Nevertheless, the combination of classical markers with serological markers resulted a strong indicator of patient prognosis, particularly for oropharyngeal squamous cell carcinoma (OPSCC). HPV16 E7 oncoprotein presence in serum at the time of diagnosis correlated with disease recurrence and overall survival. Research for E7 oncoproteins in serum may be useful as a non-invasive procedure for patient stratification and follow-up, helping identify patients at risk for tumor recurrence, giving a tool for clinicians for candidate patients selection to de-escalate treatment. Furthermore, since HPV mRNA is necessary to promoter carcinogenesis and the long control region (LCR) of the virus plays a fundamental role in the control of HPV gene expression, we studied how variation or epigenetic control of this region could affect viral gene expression and patient prognosis. While methylation of the LCR plays a fundamental role in the control of HPV mRNA, the importance of regulation by other cellular transcription factors emerges, showing the importance HPV LCR sequence variations have for transcription factor binding and how they can greatly affect viral gene expression. Sequence variations within the YY1 transcription factor binding site, were correlated to improved patient survival, while patients carrying the reference sequence relapsed more frequently. Finally, my thesis discusses a set of genes dysregulated in HNSCC in correlation to the disease subgroups, and patient prognosis. Differences were significant for EGFR, p16, c-Jun and RARB gene expression between HPV-positive and negative subgroups; these markers may help create an expression profile in order to stratify HNSCC patients at diagnosis, and select the best treatment choice. Furthermore, diminished levels of IRF6 correlated to recurrence in HPV-negative patients. The findings presented in my thesis show molecular differences between HPV-positive and negative tumors and shed light on fundamental aspects of the HPV biology, that had been poorly studied in HNSCCs so far; first, the importance that HPV16 sequence has on the behavior of the virus and its pathogenic potential; and second, the presence of circulating viral oncoproteins in HPV-positive patients in serum. These aspects turned to be equally important related to patient’s progression free survival, and should be studied in more depth as candidate markers for patient stratification in the future.
TOGNON, Mauro
PINTON, Paolo
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Descrizione: 2. Firmato Tognon 03.06.21_Definitive_PhD Thesis_Lucia Oton Gonzalez copia firmata
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2478834
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