Valproic acid combined with cisplatin-based chemoradiation in locally advanced head and neck squamous cell carcinoma patients and associated biomarkers

Milena Perez Mak, Fatima Solange Pasini, Lixia Diao, Fabyane O Teixeira Garcia, Tiago Kenji Takahashi, Denyei Nakazato, Renata Eiras Martins, Cristiane Maria Almeida, Marco Aurelio Vamondes Kulcsar, Valdelania Aparecida Lamounier, Emily Montosa Nunes, Isabela Cristina de Souza, Marcio Ricardo Taveira Garcia, Alex Vieira Amadio, Sheila Aparecida C Siqueira, Igor Moysés Longo Snitcovsky, Laura Sichero, Jing Wang, Gilberto de Castro Jr, Milena Perez Mak, Fatima Solange Pasini, Lixia Diao, Fabyane O Teixeira Garcia, Tiago Kenji Takahashi, Denyei Nakazato, Renata Eiras Martins, Cristiane Maria Almeida, Marco Aurelio Vamondes Kulcsar, Valdelania Aparecida Lamounier, Emily Montosa Nunes, Isabela Cristina de Souza, Marcio Ricardo Taveira Garcia, Alex Vieira Amadio, Sheila Aparecida C Siqueira, Igor Moysés Longo Snitcovsky, Laura Sichero, Jing Wang, Gilberto de Castro Jr

Abstract

Background: Cisplatin-based chemoradiation (CCRT) offers locally advanced head and neck squamous cell carcinoma (LAHNSCC) patients high local control rate, however, relapses are frequent. Our goal was to evaluate if association of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, with CCRT improved response rate (RR) and associated biomarkers.

Methods: This phase II trial included patients with unresectable locally advanced (LA) oropharynx (OP) squamous cell carcinoma. CCRT began after 2 weeks of VPA (P1). Primary goal was RR at 8 weeks after chemoradiation (CRT)+VPA (P2). Biomarkers included microRNA (miR) polymerase chain reaction (PCR)-array profiling in plasma compared to healthy controls by two-sample t-test. Distribution of p-values was analysed by beta-uniform mixture. Findings were validated by real-time PCR quantitative polymerase chain reaction (qPCR) for selected miRs in plasma and saliva. p16, HDAC2 and RAD23 Homolog B, Nucleotide Excision Repair Protein (HR23B) tumour immunohistochemistry were evaluated.

Results: Given significant toxicities, accrual was interrupted after inclusion of ten LA p16 negative OP patients. All were male, smokers/ex-smokers, aged 41-65 and with previous moderate/high alcohol intake. Nine evaluable patients yielded a RR of 88%. At false discovery rate of 5%, 169 miRs were differentially expressed between patients and controls, including lower expression of tumour suppressors (TSs) such as miR-31, -222, -let-7a/b/e and -145. miR-let-7a/e expression was validated by qPCR using saliva. A HDAC2 H-score above 170 was 90% accurate in predicting 6-month disease-free survival.

Conclusions: VPA and CRT offered high RR; however, with prohibitive toxicities, which led to early trial termination. Patients and controls had a distinct pattern of miR expression, mainly with low levels of TS miRs targeting Tumor protein P53 (TP53). miR-let-7a/e levels were lower in patients compared to controls, which reinforces the aggressive nature of such tumours (NCT01695122).

Keywords: chemoradiation; epigenetics; head and neck cancer; microRNA; valproic acid.

© the authors; licensee ecancermedicalscience.

Figures

Supplementary Figure 1.. Baseline versus end-of-treatment variation…
Supplementary Figure 1.. Baseline versus end-of-treatment variation of the overall quality of life score. The majority of patients had an improvement in quality of life at the moment of response evaluation (>5 points). EORTC QLC-30.
Supplementary Figure 2.. ROC curve analysis of…
Supplementary Figure 2.. ROC curve analysis of HDAC2 H-score. Values over 170 correlated with DFS at 6 months with a sensitivity of 83% and specificity of 100%.
Supplementary Figure 3.. Pathway analysis of differentially…
Supplementary Figure 3.. Pathway analysis of differentially expressed miRs between responders and non-responders at baseline. Diana miRPath version 3 KEGG pathways of 19 differentially expressed miR targets.
Figure 1.. Consort diagram. Consort diagram of…
Figure 1.. Consort diagram. Consort diagram of patients included in the study from 2012 to 2014. One patient was withdrawn due to performance status deterioration before CCRT.
Figure 2.. miR expression differs between controls…
Figure 2.. miR expression differs between controls and patients at baseline. (a): BUM analysis showed that a great number of miR were differentially expressed between patients and HV. At an FDR of 5%, 169 miRs were found to be relevant, p cutoff of 0.065. (b): Heat map of top differentially expressed miRs by two-way hierarchical clustering (miRs correlation by Pearson). Cancer patients underexpressed several miRs compared to HV. (c): Top miRs which were underexpressed in patients compared to volunteers are TS miRs.
Figure 3.. Heat map of hierarchical clustering…
Figure 3.. Heat map of hierarchical clustering analysis of miR expression (Pearson correlation) of responders and non-responders.
Figure 4.. Functional assays using EVs. (a):…
Figure 4.. Functional assays using EVs. (a): Cisplatin IC50 was determined for three HNSCC cell lines (SCC-4, SCC-25 and SCC-9). Control samples had a statistically significant decrease in cell viability with cisplatin in 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidefor (MTT) assays. The addition of HV, responders and non-responders EVs did not alter significantly cisplatin cell sensitivity. Values are means ± SD of three separate experiments. (b): Scratch migration assay with SCC-9. Assays with EV from HV, responders at P0, P1 and P3 (hashed lines) and non-responders at P0, P1 and P3 (straight lines) were compared to controls. EV from non-responders led to a significant increase in SCC-9 migration rate compared to control (p < 0.001). Values are means ± SD of at least two separate experiments.

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