Increased radiosensitivity of HPV-positive head and neck cancers: Molecular basis and therapeutic perspectives

Cancer Treatment Reviews xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Cancer Treatment Reviews journal homepage: www.elsevierhealth.com/journals/ctrv

Anti-Tumour Treatment

Increased radiosensitivity of HPV-positive head and neck cancers: Molecular basis and therapeutic perspectives Haïtham Mirghani a,⇑, Furrat Amen b, Yungan Tao c, Eric Deutsch c,d,e, Antonin Levy c,d,e a

Department of Otolaryngology – Head and Neck Surgery, Gustave Roussy Cancer Campus, 114 rue Edouard Vaillant, Villejuif, France Department of Otolaryngology, Peterborough City Hospital and Addenbrooke’s Hospital, Cambridge, UK c Department of Radiation Oncology, Gustave Roussy Cancer Campus, 114 rue Edouard Vaillant, Villejuif, France d Université Paris Sud, Faculté de Médecine, Kremlin Bicêtre 94270, France e INSERM U1030 Molecular Radiotherapy, Cancer Research Institute, Villejuif, France b

a r t i c l e

i n f o

Article history: Received 30 June 2015 Received in revised form 22 September 2015 Accepted 4 October 2015 Available online xxxx Keywords: Oropharyngeal Oropharynx Head and neck cancer Human papillomavirus 16 Radiation Treatment

a b s t r a c t Human papillomavirus driven head and neck squamous cell carcinoma (HNSCC), particularly oropharyngeal squamous cell carcinoma (OPSCC), are characterized by a significant survival advantage over their HPV-negative counterparts. Although the reasons behind this are still not fully elucidated, it is widely accepted that these tumors have a higher response to ionizing radiation that might explain their favorable outcomes. Potential underlying intrinsic mechanisms include impaired DNA repair abilities, differences in activated repopulation-signaling pathways and cell cycle control mechanisms. The role of the microenvironment is increasingly highlighted, particularly tumor oxygenation and the immune response. Recent studies have shown a distinct pattern of intratumoral immune cell infiltrates, according to HPV status, and have suggested that an increased cytotoxic T-cell based antitumor immune response is involved in improved prognosis of patients with HPV-positive OPSCC. These significant milestones, in the understanding of HPV-induced HNSCC, pave the way to new therapeutic opportunities. This article reviews the current evidence on the biological basis of increased radiosensitivity in HPV-positive HNSCC and discusses potential therapeutic implications. Ó 2015 Elsevier Ltd. All rights reserved.

Contents Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A higher cellular radiosensitivity? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HPV-status and intrinsic radiosensitivity in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cellular mechanisms supporting HPV-positive cancers radio-sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Arguments for an altered DNA repair in HPV-positive tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Differences in activated repopulation-signaling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Downregulated cell cycle control mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tumoral micro-environment influence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tissue oxygenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Radiotherapy and antitumoral immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Questions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Is there still a place for surgery to treat HPV-driven OPSCC? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Is radiation alone sufficient to treat advanced stage HPV-driven OPSCCs? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Targeting the altered DNA repair capacity of HPV-positive OPSCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potentiate immunity to optimize radiation effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disrupting proliferation pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⇑ Corresponding author. Tel.: +33 1 42 11 44 23; fax: +33 1 42 11 70 10. E-mail address: [email protected] (H. Mirghani). http://dx.doi.org/10.1016/j.ctrv.2015.10.001 0305-7372/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Mirghani H et al. Increased radiosensitivity of HPV-positive head and neck cancers: Molecular basis and therapeutic perspectives. Cancer Treat Rev (2015), http://dx.doi.org/10.1016/j.ctrv.2015.10.001

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Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conflict of interest statement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Introduction

A higher cellular radiosensitivity?

Human papilloma virus (HPV)-driven oropharyngeal squamous cell carcinomas (OPSCCs) represent a distinct disease from other HNSCCs, which are traditionally induced by excessive tobacco and alcohol consumption [1]. They represent up to 80% of OPSCCs and, despite a more aggressive clinical presentation, they are characterized by a significant survival advantage over their HPVnegative counterparts. Indeed, numerous studies have reported a reduction in the mortality risk from 28% to 58% [2,3]. Patients with HPV-positive OPSCCs smoke significantly less so they have less tobacco related comorbidities and second primaries cancers caused by field carcinogenesis that are two main causes of death in patients with HNSCCs [4]. However, this is not enough to account for the survival difference between these two conditions and the underlying reasons for their better prognosis remain largely unknown. Indeed, HPV-status positivity is a marker of good outcome, independent of treatment choice as long as it conforms to current standards of care, but is also predictive of a better response to ionizing radiation (IR) [2,5,6]. Over the last few years, many papers, some conflicting, have been published about the improved radiosensitivity of HPVdriven OPSCC and some of the potential underlying mechanisms have been unraveled [7–14]. These are particularly important as treatment of HPV-driven OPSCC is currently under intensive investigation in order to optimize its therapeutic index [6]. Understanding the molecular basis of this improved radiosensitivity may provide new therapeutic options. From a radiobiological point of view, four parameters (the ‘‘4R”) are classically related to the efficiency of IR: Repair (DNA damage repair systems), Redistribution (cell redistribution within cell cycle), Repopulation (tumor cell repopulation), and Reoxygenation (the level of intra-tumoral hypoxia) [15]. More recently, interactions between tumor cells and the stromal microenvironment, including immune cells, have been shown to significantly influence response to radiation therapy [16]. In this article, we review the current understanding of the biological basis of response to IR in HPV-positive HNSCC and highlight therapeutic perspectives in the context of emerging novel therapies.

HPV-status and intrinsic radiosensitivity in vitro To examine the effect of radiation therapy on HPV-positive and negative HNSCCs, several authors have focused on in-vitro models [7–14]. Published results, for the few HPV-positive HNSCC cell lines investigated to date, are conflicting with some data suggesting enhanced sensitivity to radiation and some suggesting reduced sensitivity or no effect (Table 1). The most recent studies have reported that HPV-positive HNSCC cell lines are, on average, significantly more radiosensitive than their HPV-negative counterparts [7–11]. Kimple et al. [7] have observed that the mean surviving fraction (SF) after 2 Gy was 22% for HPV-positive versus 59% for HPV-negative cells (p < 0.0001). Similarly, Rieckman et al. [8] have reported a significant increase in radiation sensitivity in HPVpositive compared to HPV-negative cells over a range of radiation doses (SF after 3 Gy = 28.3% vs. 44.6% (p = 0.028) and SF after 6 Gy = 4.2% vs. 14.7% (p = 0.048)). Conversely, others authors have suggested that an increased sensitivity of HPV-positive cancer cells to radiation therapy is unlikely [12–14]. Spanos et al. [12] performed clonogenic survival assays with increasing radiation doses in vitro for several HPVpositive and HPV-negative HNSCC lines. The HPV-positive cell lines were slightly more resistant to increasing doses of radiation compared with the HPV-negative cells. In the same study, they also treated HPV-positive and negative transformed mouse tonsil epithelial cells (MTEC) with graded radiation doses. The HPVpositive MTECs exhibited greater survival in vitro than did the HPV-negative MTECs, mirroring the human cell lines. Nagel et al. [14] have compared the radiosensitivity of 4 HPV-positive cell lines to 14 HPV-negative cell lines and they found that the response to radiation did not differ significantly according to HPV-status. Several factors might explain these discrepancies. First of all, methodological biases related to the variability of the cellular models that were assessed and the lack of recognized comparator are obvious sources of inconsistency. Second, it should be emphasize that the HPV status of the assumed HPV-positive cell lines was not systematically confirmed. Only some of the above-mentioned studies [7–9,11] have provided evidence for the presence of HPV

Table 1 Radiosensitivity of HNSCC cell-lines according to HPV status. Authors

Outcomes (HPV+ vs. HPV )

HPV-positive cell-lines

HPV-negative cell-lines

Spanos et al. [12] Nagel et al. [14]

UMSCC-47, UPCI-SCC90 UD-SCC-2, UM-SCC-47, UPCI-SCC090,VU-SCC-147

HPV+ less radiosensitive No difference according to HPV status

Arenz et al.* [9]

UM-SCC-47, UM-SCC-104, UPCI-SCC-152, 93-VU-147T SCC-154, SCC-090 93-VU-147T, UD-SCC-2, UM-SCC-47, UT-SCC-45, UPCI-SCC-154, UD-SCC2, UM-SCC47, UPCI-SCC90, 93-VU-147T

UMSCC-1, 19, 84 VU-SCC-1131, VU-SCC-1365 FaDu, UM-SCC-6, UM-SCC-11B, UM-SCC-14C, UM-SCC- 22A, UM-SCC-22B, UM-SCC-38, VU-SCC- 040, VU-SCC-096, VU-SCC-120, VU-SCC-017, VU-SCC-OE UD-SCC-1, UM-SCC-6, UM-SCC-11b, UT-SCC-33 SQD9, SC263, CAL 27 HSC4, Cal 33, SAT, UT-SCC-5, FaDu

HPV+ more radiosensitive HPV+ more radiosensitive

UM-SCC1, UM-SCC6, UM-SCC22B, and SCC-1483

HPV+ more radiosensitive

Dok et al. [7] Rieckmann et al.* [8] Kimple et al.* [9] *

Investigated HNSCC cell-lines

HPV+ more radiosensitive

Studies providing evidence of the presence of HPV DNA and/or E6/E7 mRNA in the assumed HPV-positive cell-lines. For the other studies, this was not mentioned.

Please cite this article in press as: Mirghani H et al. Increased radiosensitivity of HPV-positive head and neck cancers: Molecular basis and therapeutic perspectives. Cancer Treat Rev (2015), http://dx.doi.org/10.1016/j.ctrv.2015.10.001

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DNA and E6/E7 mRNA which might add to the confusion. Third, some studies have also highlighted a wide range of radiosensitivities across HPV-positive and negative cell-lines resulting in overlapping ranges of survival. This overlap between the less radiosensitive HPV-positive cells and the most radiosensitive HPV-negative cells may also explain some of these contradictory results. Cellular mechanisms supporting HPV-positive cancers radio-sensitivity Arguments for an altered DNA repair in HPV-positive tumors The biological efficacy of IR depends on the accumulation of unrepaired DNA lesions, mainly double-strand breaks (DSBs) which eventually lead to cell death. One of the main mechanisms of cell-death after IR is p53-mediated apoptosis. Although p53 is downregulated by the HPV E6-oncoprotein, HPV-positive tumors generally express a low but functional level of wild-type p53 when compared with HPV-negative tumor in which p53 is altered by genetic anomalies [1]. Kimple et al. [7] have suggested that increased TP53-induced apoptosis following radiation exposure plays a key role in the increased radiosensitivity of HPV-positive OPSCCs. In their study, apoptosis was assessed by 2 assays based respectively on caspase activity examination, and identification of plasma membrane alteration by flow cytometry. On average, 24 h after a single 4 Gy dose of radiation HPV-negative cells showed no increased in caspase activity whereas HPV-positive cells showed an 85% increase (p < 0.002). This significant increase in apoptosis in HPV-positive cells was confirmed by flow cytometry. Additionally, they used a genomewide microarray to compare gene expression between HPVpositive and HPV-negative cell lines, 24 h following irradiation. Multiple genes in the p53 pathway were upregulated in HPVpositive cells. The authors hypothesized that despite the degradation of p53 by HPV E6-oncoprotein, the remaining wild-type p53 was able to induce apoptosis following radiation. To support this hypothesis they show that when p53 was totally inhibited by RNA interference (through siRNA) in HPV-positive cell lines or in human tonsillar epithelial cells transfected by E6, cells became less responsive to IR. Rieckman et al. [8] demonstrated that impaired DNA repair capacity is responsible for the increased response to IR of HPV-positive cells. Using immunofluorescent staining with anti c-H2AX and anti 53BP1 antibodies (early signals of cellular response to DSB), they found that residual DSBs following irradiation were significantly more frequent in HPV-positive cells. Moreover, they noted a significant association between residual DSBs and cellular radio-sensitivity for HPV-positive HNSCC cell lines. Indeed, unrepaired DSBs lead to cell death by mitotic catastrophe. The mechanisms behind this altered DNA repair capacity are still under investigation. Applebaum et al. [17] showed that there was no relationship between XRCC1, a major protein implicated in DSBs repair, and HPV-related HNSCC. Park et al. [11] have suggested a direct or indirect role for the E7 viral oncoprotein. In HPV-positive head and neck cancer-derived cell-lines and HPV16 E7-transgenic mice, E7 decreased sublethal DNA damage repair. The work of Dok et al. [10] is particularly interesting as they show that p16 over-expression, which is characteristic of HPV-induced OPSCCs, impairs the recruitment of RAD51 to the site of DNA damage and therefore decreases the homologous recombination (HR)-mediated DNA repair pathways. This effect on radiosensitivity was independent of p16’s ability to inhibit CDK4/6 but was induced by an unexpected function of p16. Indeed, p16 inhibits the recruitment of RAD51 to the sites of DNA damage via cyclin D1 downregulation. As RAD51 is an essential factor for homologous recombination-mediated DNA repair pathway, Dok

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et al. [10] suggested that p16 over-expression results in an increase in the non-homologous end-joining pathway (NHEJ), which commonly leads to misrepair of DNA DSBs. In support of this hypothesis, they showed an increased frequency of micro nucleated cells in p16-positive cells after radiation compared with p16-depleted cells, which is characteristic of NHEJ misrepair. This finding is particularly interesting as, if it is confirmed it might partly explain the positive impact of p16 over-expression on prognosis independently of HPV status. Although the literature is still limited and sometimes conflicting, several recent studies have highlighted that HPV-negative/p16-positive cancers have a better prognosis than their HPV-negative/p16-negative counterparts even outside the oropharynx [18]. Differences in activated repopulation-signaling pathways Tumor cell repopulation is generally mediated through specific survival pathways. EGFR pathway is the most frequently upregulated mitogenic signaling pathway in HNSCC and correlates with resistance to therapy and poor prognosis [19]. Although data regarding the expression levels of EGFR in HPV-positive HNSCC are conflicting, several studies have demonstrated an inverse relationship between HPV and EGFR expression levels in OPSCC [20]. In a study on three cell lines (two HPV-positive and one HPVnegative), Gupta et al. [13] showed that the HPV negative cell line, that was the most radioresistant, had an increased amount of phosphorylated EGFR. Moreover, activated EGFR resulted in active signaling through Akt, which may activate the oncogene mTOR (Fig. 1). Interestingly, the 2 HPV-positive EGFR-negative HNSCC cell lines used in this study [13] had differential Akt expression level according to their PTEN status. The presence of PTEN expression also seems more frequently observed in HPV-positive tonsillar cancers, as compared with HPV-negative cancer [21]. Such data remain anyway to be further confirmed. In next generation sequencing-based analysis of 252 HNSCC samples, the most common genes with genetic alterations were PIK3CA and PTEN in HPV-positive tumors (confirmed by HPV DNA sequencing) [22]. Genetic alterations also exist in HPVnegative cases and are generally even more frequent in patients that smoke [23]. Seiwert et al. [24] performed a parallel sequencing of 617 cancer-associated genes in 120 matched tumor/normal samples (42.5% HPV-positive). Enrichment for mutations in TP53, CDKN2A, MLL2, CUL3, NSD1, PIK3CA, and NOTCH genes were described in HPV negative HNSCC. Others unique mutations (DDX3X, FGFR2/3) and aberrations (KRAS, MLL2/3, and NOTCH1) were enriched in HPV-positive tumors [24]. FGFR signaling is also dominant or co-dominant with EGFR in HNSCC lines [25]. Notch signaling has been implicated in cellular senescence checkpoint and is discussed hereafter. In an analysis with a reverse-phase protein array in 29 HPV-positive and 13 HPV-negative prospectively collected OPSCCs, all activating PIK3CA mutations occurring in HPV-positive tumors had lower mean levels of activated AKT and downstream AKT target phosphorylation [26]. HPVE6/ E7-oncoproteins overexpression moreover inhibited AKT phosphorylation in HPV-negative cells [29]. Greater radiosensitivity may be explained by decreased repopulation signaling pathways in HPV-positive tumors. Downregulated cell cycle control mechanisms Redistribution in the cell cycle refers to the fact that cells’ radiosensitivity changes as they traverse the division cycle. It is admitted that mitotic cells are particularly sensitive to IR, while those in late S-phase would be most resistant [27,28]. The family of checkpoint kinases 1/2 (Chk1/2 kinases) is activated after IR-induced DNA damages and arrest the cell cycle through ATM and ATR to allow DNA repair [28] (Fig. 2).

Please cite this article in press as: Mirghani H et al. Increased radiosensitivity of HPV-positive head and neck cancers: Molecular basis and therapeutic perspectives. Cancer Treat Rev (2015), http://dx.doi.org/10.1016/j.ctrv.2015.10.001

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Fig. 1. Decreased activated repopulation-signaling pathways in HPV-positive HNSCC. Interactions exist between FGFR (fibroblast growth factor receptors), Phosphatidylinositol 3-kinases-AKT-mTOR (PI3K) and RAS pathways, resulting in cell proliferation, growth and survival. FGFR signaling is also dominant or co-dominant with EGFR (epithelial growth factor receptor). HPV-positive HNSCC lines have higher PTEN, and lower EGFR or AKT expression as compared with HPV negative HNSCC. Tumor infiltrating lymphocytes, also present in HPV-positive HNSCC, secrete IFN-gamma that induces programmed death ligand-1 (PD-L1) expression. Engagement of the PD1 receptor on activated T cells by its ligand PD-L1 results in negative T-cells regulation, which protects cancer cells from immune elimination.

Fig. 2. Potential mechanisms explaining an increased radiosensitivity in HPV-positive HNSCC: Altered DNA repair and downregulated cell cycle control mechanisms. The family of checkpoint kinases 1/2 (Chk1/2 kinases) is activated through ATM and ATR, after ionizing radiation (IR)-induced DNA damages, and arrest the cell cycle to allow DNA repair. One of the main mechanisms of cell-death after IR is p53-mediated apoptosis. Although p53 is downregulated by the HPV E6-oncoprotein, HPV-positive tumors generally express a low but functional level of wild-type p53. p16 over-expression impairs the recruitment of RAD51 to the site of DNA damage (DSB: double strand break) and therefore decreases DNA repair pathways. Strategies such as Chek1 or PARP inhibitors may potentiate lethal effects of IR.

In HPV infected cells, E6 and E7 respectively downregulate p53 and pRb [29]. p53 and pRb normally ensure the G1/G2 cycle arrest through checkpoints kinase in order to assess for DNA damage and to repair this damage prior to entry into mitosis (M phase). Increased p16INK4a expression is normally induced by cellular stress

response mechanisms resulting in cell cycle arrest via the CDK4/6cyclin D1-pRb tumor suppressor pathway. As shown earlier hereafter, p16INK4a expression significantly correlated with decreased cyclin D1 expression [10]. In HPV-driven cancers, however, simultaneous inactivation of the tumor suppressor protein pRb by E7

Please cite this article in press as: Mirghani H et al. Increased radiosensitivity of HPV-positive head and neck cancers: Molecular basis and therapeutic perspectives. Cancer Treat Rev (2015), http://dx.doi.org/10.1016/j.ctrv.2015.10.001

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prevents the cell from undergoing p16INK4a-induced cell cycle arrest [29]. Without these regulations, the cell cycle continues unchecked leading to an accumulation of radio-induced DNA error that lead to cell death by mitotic catastrophe [30]. Arenz et al. [9] compared cell cycle redistribution in four HPVpositive and four HPV-negative cell lines. They showed that irradiated HPV-positive cell lines progressed faster through S-phase and showed a more distinct accumulation in G2/M. Kimple et al. [7] and Rieckman et al. [8] also described an extensive G2 arrest in HPV-positive cells [8]. Phospho-ATM and phospho-ATR do not seem to be modified in HPV-positive cells but a role of checkpoint kinase Chk1 after irradiation has been identified [11]. In bone marrow derived mesenchymal stem cells transformed stepwise by retroviral transfection with HPV16 E6/E7-oncogenes, both Chk1 and Chk2 phosphoprotein levels increased after irradiation compared to unirradiated cells but only Chk1 knockdown radiosensitized all cell lines [31]. This result was specifically confirmed in HPV-positive cells as the selective inhibitor PF-00477736 eliminate the radiation-induced G2-arrest that went along with radiosensitization [32]. Although the effect of Chk1 inhibition had been shown to be dependant on p53 status [33], a recent study showed that radiosensitizing effect of Chk1 inhibition by a selective Chk1 inhibitor (Chir-124) was relatively similar between p53( / ) and p53sufficient wild type cancer cells [34]. The role of inhibition of Chk1 and/or Chk2 needs to be further explored in HPV-positive HNSCC regarding its decreased level of wild-type p53 status. Tumoral micro-environment influence Although there is a growing body of data supporting the assumption that HPV-related HNSCC cell-lines have a higher sensitivity to radiation than their HPV-negative counterparts, it is difficult to attribute their improved prognosis only to these intrinsic features. Indeed, the observed improved response of HPV-positive tumors to radiotherapy may involve other factors and particularly those related to the tumor micro-environment. Tissue oxygenation Hypoxia is a common feature of locally advanced solid tumors including HNSCC. Indeed, newly formed micro vessels in most solid tumors exhibit a series of severe structural and functional abnormalities, which impair effective tissue oxygenation and contribute to resistance to radiation [35–37]. In a retrospective analyze of the DAHANCA 5 trial, Lassen et al. [38] have addressed the question of whether the enhanced radiosensitivity observed for HPV-positive tumors could result from a better tumoral oxygenation. In this study, p16-protein expression was used as a surrogate marker to define HPV-status (p16-positive tumors being considered as HPV-positive). They observed that patients (n = 175) treated with nimorazole, a hypoxic cell radio sensitizer, had significantly better locoregional control than did those (n = 156) given placebo (hazard ratio (HR) 0.70 [95% CI 0.52–0.93]). However, this improvement was only significant in p16-negative tumors. Although cautious interpretation of the results is necessary due to the use of a surrogate marker to define HPV-status, and the limited number of patients and events in the p16-positive OPSCC subgroup, the authors suggested that p16-positive tumors might be less hypoxic. This could contribute to the superior prognosis observed in these patients when treated with radiotherapy. To support this assumption, they further correlated the level of plasma osteopontin, a marker of hypoxia, with p16 status in 252 patients. They found

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that patients with p16-negative tumors were significantly more likely to have high plasma concentrations of osteopontin (78/190, 41%) and thus were more hypoxic than patients with p16-positive tumors (10/62, 16%; p < 0.0001). However, many other studies showed no significant difference in hypoxia between HPV-positive and HPV-negative tumors. In a prospective study, Mortensen et al. [39] have performed PET/CT imaging with a hypoxia specific tracer (F-fluoroazomycin arabinoside) in 40 patients with HNSCC receiving radiotherapy. They found no correlation between HPV-status and hypoxia as the distribution of hypoxia among the HPV-positive (12/16) and negative (13/24) tumors was not significantly different. Toustrup et al. [40] have recently generated a hypoxia gene expression signature that can classify patients with HNSCC into more or less hypoxic group. Using this classifier on a cohort of 323 patients including 84 p16positve cases, they observed the same frequency of hypoxia between p16-positive and negative cases. Kong et al. [41] have explored the relationship between HPV and tumor oxygenation in a cohort of 99 HNSCC including 33 HPV-positive OPSCC. They found no significant association between HPV-status and tumor hypoxia as detected by either pO2 measurements or immunohistochemical staining for carbonic anhydrase-IX that is a hypoxiainduced protein. Although, there is currently no standardized and validated biomarker for hypoxia, it is interesting to observe that all these studies are concordant and that tumor oxygenation is probably not the explanation to the higher response of HPV-positive OPSCCs to radiotherapy. Radiotherapy and antitumoral immunity Increasing evidence points to the fact that radiotherapy harnesses the host’s immune system to indirectly eradicate tumor cells [42–44]. This process, known as immunogenic cell-death, is based on the emission of signals by dying tumor cells that trigger an immune response [42–44]. It is therefore possible that the higher radio-sensitivity of HPV-positive OPSCC might be, at least partly, due to a more effective immune response following radiation. HPV-driven OPSCCs naturally induce an adaptive immune response directed against the viral antigens displayed by tumor cells [45]. Indeed, these tumors express higher levels of immune response-related genes [17,46,47] (particularly those related to CD8+ T-cell effector function) and are significantly more infiltrated by T cells in comparison with their HPV-negative counterparts [48–50]. These tumor-infiltrating lymphocytes are reactive to HPV-oncoproteins [51] and their abundance, assessed on pretherapeutic samples, is predictive of favorable clinical outcomes [48,49]. They could play an active role in tumor eradication. However, despite this anti-tumor immune response, a significant proportion of HPV-driven OPSCCs are able to persist and progress suggesting that tolerance or immune escape mechanisms are involved. Radiation, and its physiological consequences, might tilt the balance from a tolerance to a tumoricidal environment and boost this pre-existing immune response. In an in vivo murine model, Spanos et al. [12] showed that immune-competent mice with HPV-positive tumors were more sensitive to radiation and exhibited complete clearance at 20 Gy, compared to their HPV-negative counterparts, which showed persistent growth. Conversely, radiation therapy was not able to cure immune-deficient mice with HPV-positive or negative tumors. These findings are interesting, however in vivo animal models don’t adequately address the impact of the human immune system on these tumors. Although the underlying mechanisms are not clear, Andersen et al. [52] have recently proposed a model, in which tumoral cell injury and inflammation induced by radiation lead to the release

Please cite this article in press as: Mirghani H et al. Increased radiosensitivity of HPV-positive head and neck cancers: Molecular basis and therapeutic perspectives. Cancer Treat Rev (2015), http://dx.doi.org/10.1016/j.ctrv.2015.10.001

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of tumor antigens and danger-associated-molecular-pattern molecules such as high-mobility group box protein 1 (HMGB1). HMGB1 induced recruitment and maturation of dendritic cells via Toll-likereceptor 4. Once activated dendritic cells take up tumor antigens including HPV-proteins and migrate to the lymph nodes where they stimulate naïve T-cells and induce an anti-tumoral immune response which might be particularly efficient as the viral proteins are foreign molecules that are highly immunogenic to the host. Questions and perspectives Is there still a place for surgery to treat HPV-driven OPSCC? As HPV-driven OPSCCs are more radiosensitive than their HPVnegative counterparts, surgery may not be a relevant therapeutic approach. Indeed, surgery and particularly trans-mandibular approaches can cause significant morbidities that are best avoided. On the other hand, the long-term toxicity of chemoradiation, which is the standard of care for advanced stage OPSCC in numerous institutions, have a considerable negative impact on quality of life [53] especially in patients that have a high likelihood of surviving their disease. Therefore, less morbid surgical approaches such as transoral robotic surgery might be interesting especially if post-operative radiotherapy is not necessary (clear margins, pN01 disease) or if the radiation dose following surgery can be significantly reduced [54]. Indeed, traditional pathologic risk features may not be as meaningful in the selection of adjuvant therapy regimens and doses in HPV-initiated disease [55]. To answer to this question, several clinical trials based on up front transoral surgery followed, or not, by postoperative adjuvant therapy are ongoing [54]. It is however important to highlight that these ‘‘surgical strategies” lose their potential benefits if an adjuvant (full dose of radiation or chemoradiation) is needed. This might be the case if the pathological outcomes are not favorable (positive surgical margins, pN2-3 disease). In this situation, toxicity will be significantly increased. Therefore, an appropriate patients’ selection receiving surgery is crucial. Is radiation alone sufficient to treat advanced stage HPV-driven OPSCCs? Non-surgical treatments of OPSCC usually consist in radiation alone for tumors classified as stage I/II and chemoradiation for stage III/IV. As HPV-positive OPSCCs are more radiosensitive, is it possible that radiation alone could be sufficient for selected advanced HPV-positive OPSCCs, instead of chemoradiation, in order to decrease toxicity. Very few studies, with important methodological limitation have reported on that issue. However, they suggest that radiation alone might be effective in selected cases. Retrospective analysis of prospectively collected data from the DAHANCA 5 trial indicates that HPV-positive OPSCCs respond extremely well to conventionally fractionated radiotherapy with a pronounced proportion of complete responders and long-term survivors. Indeed, Lassen et al. [56] have shown that tumorpositivity for p16 (a surrogate marker for HPV) was significantly correlated with improved locoregional tumor control (5-year actuarial values 58% vs. 28%; p = 0.0005), improved disease-specific survival (72% vs. 34%; p = 0.0006), and improved overall survival (62% vs. 26%; p = 0.0003). However, it must be highlighted that only 35 out of the 156 HNSCC included in that study, from the original cohort, were p16-positive. Additionally, although p16 and HPV status are well correlated, there is a risk that some patients were misclassified. Similarly Chen et al. [57] have also noted very good outcomes in a series of 19 HPV-positive OPSCCs treated exclusively by

radiation, including 17 (74%) patients with stage III/IV and 18 (79%) non-smokers (<100 cigarettes in a lifetime). The 3-year overall survival and locoregional control rates for patients with stage III/IV disease were 81% and 88%, respectively. Among the 18 HPV-positive patients who were never-smokers, the 3-year rates of overall survival and locoregional control were 100% for both. Although limited by their retrospective nature and the small number of patients, these results, suggest the feasibility and efficacy of treating strictly selected patients with advanced HPV-positive OPSCC with radiation therapy alone. To date there is no ongoing prospective trial comparing radiation therapy alone to chemoradiation for advanced stage HPV-positive OPSCC [54]. This might be explained by the desire to avoid any potential risk to patients likely to have a good prognosis. However, such a study seems to make sense especially in non-smokers HPV-positive OPSCC and subgroups least likely to develop distant metastases (T1-3, N0-2C). As the risk of distant metastases is more pronounced in patients with T4 and/or N3 disease, the combination of a systemic therapy to radiation seems preferable for this subset of patients. Targeting the altered DNA repair capacity of HPV-positive OPSCC Activation of PARP (Poly ADP Ribose Polymerase) is one of the early DNA damage responses, among other DNA sensing molecules, such as p53 [58,59]. PARP is more specifically involved in the elimination of DNA single-strand breaks and base damage through base excision repair (BER) [58,59]. PARP inhibition, with subsequent impairment of the BER mechanism, may enhance the cytotoxicity of agents that generate single-strand breaks in DNA, such as IR. In addition, PARP inhibitors may induce death through ‘‘synthetic lethality” if the DNA repair mechanisms that rescue BER-deficient cells are themselves impaired, as in HPV positive cells. Guster et al. [60] demonstrated an increased radosensitivity after the adjunction of the PARP inhibitor Olaparib in five HPV/ p16-positive HNSCC cell lines. Moreover, they also observed a further enhancement of the radiation-induced G2-arrest upon addition of Olaparib. As the use of Chk1 inhibitors eliminate the radiation-induced G2-arrest and induce radiosensitization [61] (Fig. 2), the combination of both inhibitors was investigated. Thus, the use of PARP and Chk1 inhibitors resulted in a substantial reduction of cell survival after irradiation as compared to their usage individually [60]. This remains to be tested in early clinical trial. Potentiate immunity to optimize radiation effects As radiotherapy efficacy relies partially on its ability to trigger an immune response against tumor cells, new strategies based on this concept are being developed. Breaking cancer-induced immune tolerance in order to restore an efficient anti-tumoral immunity and the use of Toll-like-receptor agonists to stimulate the antitumor immune response may potentiate radiotherapy [42–44]. Indeed, such synergistic effects have been shown in mouse tumor models [73] and in patients with melanoma [74] and non-small cell lung cancer [75] treated with combinations of radiotherapy together with diverse immune therapies such as injections of Flt-3 ligand or anti-CTLA4 monoclonal antibodies. Local irradiation was recently associated with a Shiga Toxin B (STxB)-based human HPV vaccination with interesting antitumor activity in vivo. Moreover, the combined treatment induced high levels of tumor-infiltrating, antigen-specific CD8+ T cells and enhanced intratumor vascular permeability, which were required to trigger the antitumor activity [76]. The programmed death-1/programmed death ligand-1 (PD-1/ PD-L1) immune checkpoint belongs to inhibitory pathways

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hardwired into the immune system that are crucial for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses in peripheral tissues in order to minimize collateral tissue damage. This immune checkpoint plays a pivotal role in the ability of tumor cells to evade the host’s immune response and particularly T-cells that are specific for tumor antigens [77]. Several authors have shown that PD-L1 expression is common in HPV-positive OPSCC. Lyford-Pike et al. [78] found its expression in 70% (14/20) and Ukpo et al. [79] in 49% (68/138) of HPV-positive tumors compared to 29% (2/7) and 34% (14/41) in HPV-negative tumors, respectively. Additionally, Pai et al. [80] have proposed a model in which tumor infiltrating lymphocytes, present in HPVinduced OPSCC, secrete IFN-gamma that induce PD-L1 expression on tumor cells localized on tumor periphery at the interface with the inflammatory stroma. Engagement of the PD1 receptor on activated T cells by its ligand PD-L1 results in negative T-cells regulation, which protects cancer cells from immune elimination. Although these findings need to be confirmed they may provide a rationale to develop strategies that combine RT with immune checkpoint antibodies. However, these new approaches raise several issues including questions related to the optimal dose and schedule of radiation. Two ongoing phase I studies [81,82] assessing anti PD-L1/PD-1 monotherapy in metastatic/recurrent HNSCC have shown that the toxicity profile was acceptable. Although the number of patients enrolled in these trials is small, they have reported encouraging antitumor activity in both HPV-positive and negative HNSCC. Studies combining radiotherapy with the anti-CTLA4 ipilimumab, (NCT01935921, NCT01860430), or the anti PD-1 pembrolizumab (NCT02289209) are ongoing.

Disrupting proliferation pathways Whether the activity of anti-EGFR treatment in combination with radiation is affected by HPV status is of great interest and is currently being assessed as a de-escalation strategy in HPVpositive OPSCC [6,20,54]. Recent trials did not report better outcomes in this population, at a recurrent/metastatic stage, with concurrent cetuximab [62] or panitumumab [63]. However, the impact of anti-EGFR therapy in recurrent and ‘‘de novo” disease may be different. The absence of radiosensitizing effect of cetuximab was also highlighted in various HPV-positive HNSCC cell lines [60]. In experiments on non-HNSCC cells, the endogenous NOTCH1 tumor-suppressor action was reversed by and E6/E7 oncogene. Through NOTCH inhibition, E6/E7 promoted xenograft tumor growth [64]. The gamma-secretase/Notch signaling pathway inhibitor RO4929097, that significantly sensitized glioblastoma xenografts [65], is currently assessed in the HNSCC metastatic setting. Since mutations of the PI3K-AKT-mTOR axis, regulator (PTEN), or activator (Ras mutations activate the Raf/MEK/ERK pathway and the PI3K pathway), have been detected in HNSCC, several inhibitors are currently being evaluated. Nefilavir, an Akt inhibitor, radiosensitized HPV-positive and negative cell lines [13]. The inhibition of RAS with lonafarnib also induced radiosensitization in HNSCC cell-lines [66]. The combination of radiotherapy and everolimus, an oral mTOR inhibitor, has been evaluated in phase I trial with promising results [67,68] (Fig. 1). Besides, the authors suggested there that mTORC1-mediated activation of eIF4E may constitute a surrogate biomarker of tumor aggressiveness, but this has not been specifically addressed in HPV-positive OPSCC. Finally, disturbance of other signaling pathways may be promising, including HER2 and Cmyc [69,70]. Antiviral agents such as cidofovir may also synergize with radiation or with anti-EGFR agents in HPV positive tumors [71,72].

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Conclusion In light of the available data, HPV-positive OPSCCs are more radiosensitive than their HPV-negative counterparts. This is likely to be due to impaired DNA repair capacity and the efficient immune response induced by radiation. Additional research is necessary to take advantage of these mechanisms in order to develop more tailored therapeutic strategies.

Conflict of interest statement Nothing to declare.

References [1] Gillison ML, Shah KV. Human papillomavirus-associated head and neck squamous cell carcinoma: mounting evidence for an etiologic role for human papillomavirus in a subset of head and neck cancers. Curr Opin Oncol 2001;13:183–8. [2] Ang KK, Harris J, Wheeler R, et al. Human Papillomavirus and survival of patients with oropharyngeal cancer. N Engl J Med 2010;363:24–35. [3] Fakhry C, Westra WH, Li S, et al. Improved survival of patients with human papillomavirus-positive head and neck squamous cell carcinoma in a prospective clinical trial. J Natl Cancer Inst 2008;100:261–9. [4] Rose BS. Population-based study of competing mortality in head and neck cancer. J Clin Oncol 2011;29:3503–9. [5] Worden FP, Ha H. Controversies in the management of oropharynx cancer. J Natl Compr Canc Netw 2008;6:707–14. [6] Mirghani H, Amen F, Blanchard P, Moreau F, Guigay J, Hartl DM, et al. Treatment de-escalation in HPV-positive oropharyngeal carcinoma: ongoing trials, critical issues and perspectives. Int J Cancer 2015;136:1494–503. [7] Kimple RJ, Smith MA, Blitzer GC, Torres AD, Martin JA, Yang RZ, et al. Enhanced radiation sensitivity in HPV-positive head and neck cancer. Cancer Res 2013;73:4791–800. [8] Rieckmann T, Tribius S, Grob TJ, Meyer F, Busch CJ, Petersen C, et al. HNSCC cell lines positive for HPV and p16 possess higher cellular radiosensitivity due to an impaired DSB repair capacity. Radiother Oncol 2013;107:242–6. [9] Arenz A, Ziemann F, Mayer C, Wittig A, Dreffke K, Preising S, et al. Increased radiosensitivity of HPV-positive head and neck cancer cell lines due to cell cycle dysregulation and induction of apoptosis. Strahlenther Onkol 2014;190:839–46. [10] Dok R, Kalev P, Van Limbergen EJ, Asbagh LA, Vázquez I, Hauben E, et al. P16INK4a impairs homologous recombination-mediated DNA repair in human papillomavirus-positive head and neck tumors. Cancer Res 2014;74:1739–51. [11] Park JW, Nickel KP, Torres AD, Lee D, Lambert PF, Kimple RJ. Human papillomavirus type 16 E7 oncoprotein causes a delay in repair of DNA damage. Radiother Oncol 2014;113:337–44. [12] Spanos WC, Nowicki P, Lee DW, Hoover A, Hostager B, Gupta A, et al. Immune response during therapy with cisplatin or radiation for human papillomavirusrelated head and neck cancer. Arch Otolaryngol Head Neck Surg 2009;135:1137–46. [13] Gupta AK, Lee JH, Wilke WW, Quon H, Smith G, Maity A, et al. Radiation response in two HPV-infected head-and-neck cancer cell lines in comparison to a non-HPV-infected cell line and relationship to signaling through AKT. Int J Radiat Oncol Biol Phys 2009;74:928–33. [14] Nagel R, Martens-de Kemp SR, Buijze M, Jacobs G, Braakhuis BJ, Brakenhoff RH. Treatment response of HPV-positive and HPV-negative head and neck squamous cell carcinoma cell lines. Oral Oncol 2013;49:560–6. [15] Steel GG, McMillan TJ, Peacock JH. The 5Rs of radiobiology. Int J Radiat Biol 1989;56:1045–8. [16] Levy A, Chargari C, Cheminant M, Simon N, Bourgier C, Deutsch E. Radiation therapy and immunotherapy: implications for a combined cancer treatment. Crit Rev Oncol Hematol 2013;85:278–87. [17] Applebaum KM, McClean MD, Nelson HH, Marsit CJ, Christensen BC, Kelsey KT. Smoking modifies the relationship between XRCC1 haplotypes and HPV16negative head and neck squamous cell carcinoma. Int J Cancer 2009;124:2690–6. [18] Chung CH, Zhang Q, Kong CS, Harris J, Fertig EJ, Harari PM, et al. P16 protein expression and human papillomavirus status as prognostic biomarkers of nonoropharyngeal head and neck squamous cell carcinoma. J Clin Oncol 2014;32(35):3930–8. [19] Temam S, Kawaguchi H, El-Naggar AK, Jelinek J, Tang H, Liu DD, et al. Epidermal growth factor receptor copy number alterations correlate with poor clinical outcome in patients with head and neck squamous cancer. J Clin Oncol 2007;25:2164. [20] Mirghani H, Amen F, Moreau F, Guigay J, Hartl DM, Lacau St Guily J. Oropharyngeal cancers: relationship between epidermal growth factor receptor alterations and human papillomavirus status. Eur J Cancer 2014;50:1100–11.

Please cite this article in press as: Mirghani H et al. Increased radiosensitivity of HPV-positive head and neck cancers: Molecular basis and therapeutic perspectives. Cancer Treat Rev (2015), http://dx.doi.org/10.1016/j.ctrv.2015.10.001

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H. Mirghani et al. / Cancer Treatment Reviews xxx (2015) xxx–xxx

[21] Chun SH, Jung CK, Won HS, Kang JH, Kim YS, Kim MS, et al. Divergence of P53, PTEN, PI3K, Akt and mTOR expression in tonsillar cancer. Head Neck 2015;37:636–43. [22] Chung CH, Guthrie VB, Masica DL, Tokheim C, Kang H, Richmon J, et al. Genomic alterations in head and neck squamous cell carcinoma determined by cancer gene-targeted sequencing. Ann Oncol 2015;26:1216–23. [23] Agrawal N, Frederick MJ, Pickering CR, Bettegowda C, Chang K, Li RJ, et al. Exome sequencing of head and neck squamous cell carcinoma reveals inactivating mutations in NOTCH1. Science 2011;333:1154–7. [24] Seiwert TY, Zuo Z, Keck MK, Khattri A, Pedamallu CS, Stricker T, et al. Integrative and comparative genomic analysis of HPV-positive and HPVnegative head and neck squamous cell carcinomas. Clin Cancer Res 2015;21:632–41. [25] Marshall ME, Hinz TK, Kono SA, Singleton KR, Bichon B, Ware KE, et al. Fibroblast growth factor receptors are components of autocrine signaling networks in head and neck squamous cell carcinoma cells. Clin Cancer Res 2011;17:5016–25. [26] Sewell A, Brown B, Biktasova A, Mills GB, Lu Y, Tyson DR, et al. Reverse-phase protein array profiling of oropharyngeal cancer and significance of PIK3CA mutations in HPV-associated head and neck cancer. Clin Cancer Res 2014;20:2300–11. [27] Levy A, Albiges-Sauvin L, Massard C, Soria JC, Deutsch E. Cell cycle, mitosis and therapeutic applications. Bull Cancer 2011;98(9):1037–45. [28] Pawlik TM, Keyomarsi K. Role of cell cycle in mediating sensitivity to radiotherapy. Int J Radiat Oncol Biol Phys 2004;59:928–42. [29] zur Hausen H. Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2002;2(5):342–50. [30] Shay JW, Roninson IB. Hallmarks of senescence in carcinogenesis and cancer therapy. Oncogene 2004;12(23):2919–33. [31] Worku M, Fersht N, Martindale C, Funes JM, Short SC. Sequential transformation of mesenchymal stem cells is associated with increased radiosensitivity and reduced DNA repair capacity. Radiat Res 2013;179:698–706. [32] Busch CJ, Kriegs M, Laban S, Tribius S, Knecht R, Petersen C, et al. HPV-positive HNSCC cell lines but not primary human fibroblasts are radiosensitized by the inhibition of Chk1. Radiother Oncol 2013;108:495–9. [33] Borst GR, McLaughlin M, Kyula JN, Neijenhuis S, Khan A, Good J, et al. Targeted radiosensitization by the Chk1 inhibitor SAR-020106. Int J Radiat Oncol Biol Phys 2013;85(4):1110–8. http://dx.doi.org/10.1016/j.ijrobp.2012.08.006 [Epub 2012 Sep 14]. [34] Tao Y, Leteur C, Yang C, Zhang P, Castedo M, Pierré A, et al. Radiosensitization by Chir-124, a selective CHK1 inhibitor: effects of p53 and cell cycle checkpoints. Cell Cycle 2009;8(8):1196–205. [35] Xia Y, Choi HK, Lee K. Recent advances in hypoxia-inducible factor (HIF)-1 inhibitors. Eur J Med Chem 2012;49:24–40. [36] Vaupel P. The role of hypoxia-induced factors in tumor progression. Oncologist 2004;9:10–7. [37] Vaupel P. Tumor microenvironmental physiology and its implications for radiation oncology. Semin Radiat Oncol 2004;14:198–206. [38] Lassen P, Eriksen JG, Hamilton-Dutoit S, Tramm T, Alsner J, Overgaard J, et al. HPV-associated p16-expression and response to hypoxic modification of radiotherapy in head and neck cancer. Radiother Oncol 2010;94:30–5. [39] Mortensen LS, Johansen J, Kallehauge J, Primdahl H, Busk M, Lassen P, et al. FAZA PET/CT hypoxia imaging in patients with squamous cell carcinoma of the head and neck treated with radiotherapy: results from the DAHANCA 24 trial. Radiother Oncol 2012;105:14–20. [40] Toustrup K, Sørensen BS, Lassen P, Wiuf C, Alsner J, Overgaard J, et al. Gene expression classifier predicts for hypoxic modification of radiotherapy with nimorazole in squamous cell carcinomas of the head and neck. Radiother Oncol 2012;102:122–9. [41] Kong CS, Narasimhan B, Cao H, Kwok S, Erickson JP, Koong A, et al. The relationship between human papillomavirus status and other molecular prognostic markers in head and neck squamous cell carcinomas. Int J Radiat Oncol Biol Phys 2009;74:553–61. [42] Marabelle A, Filatenkov A, Sagiv-Barfi I, Kohrt H. Radiotherapy and toll-like receptor agonists. Semin Radiat Oncol 2015;25:34–9. [43] Apetoh L, Ghiringhelli F, Tesniere A, Obeid M, Ortiz C, Criollo A, et al. Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat Med 2007;13(9):1050–9. [44] Golden EB, Apetoh L. Radiotherapy and immunogenic cell death. Semin Radiat Oncol 2015;25(1):11–7. [45] Kaufmann AM, Nieland J, Schinz M, Nonn M, Gabelsberger J, Meissner H, et al. HPV16 L1E7 chimeric virus-like particles induce specific HLA-restricted T cells in humans after in vitro vaccination. Int J Cancer 2001;92:285–93. [46] Jung AC, Briolat J, Millon R, de Reyniès A, Rickman D, Thomas E, et al. Biological and clinical relevance of transcriptionally active human papillomavirus (HPV) infection in oropharynx squamous cell carcinoma. Int J Cancer 2010;126: 1882–94. [47] Mirghani H, Ugolin N, Ory C, Lefèvre M, Baulande S, Hofman P, et al. A predictive transcriptomic signature of oropharyngeal cancer according to HPV16 status exclusively. Oral Oncol 2014;50:1025–34. [48] Jung AC, Guihard S, Krugell S, Ledrappier S, Brochot A, Dalstein V, et al. CD8alpha T-cell infiltration in human papillomavirus-related oropharyngeal carcinoma correlates with improved patient prognosis. Int J Cancer 2013;132(2):E26–36.

[49] Ward MJ, Thirdborough SM, Mellows T, Riley C, Harris S, Suchak K, et al. Tumour-infiltrating lymphocytes predict for outcome in HPV-positive oropharyngeal cancer. Br J Cancer 2014;110(2):489–500. [50] Partlová S, Boucˇek J, Kloudová K, Lukešová E, Zábrodsky´ M, Grega M, et al. Distinct patterns of intratumoral immune cell infiltrates in patients with HPVassociated compared to non-virally induced head and neck squamous cell carcinoma. Oncoimmunology 2015;4:e965570. [51] Heusinkveld M, Goedemans R, Briet RJ, Gelderblom H, Nortier JW, Gorter A, et al. Systemic and local human papillomavirus 16-specific T-cell immunity in patients with head and neck cancer. Int J Cancer 2012;131:E74–85. [52] Andersen AS, Koldjaer Sølling AS, Ovesen T, Rusan M. The interplay between HPV and host immunity in head and neck squamous cell carcinoma. Int J Cancer 2014;134:2755–63. [53] Machtay M, Moughan J, Trotti A, Garden AS, Weber RS, Cooper JS, et al. Factors associated with severe late toxicity after concurrent chemoradiation for locally advanced head and neck cancer: an RTOG analysis. J Clin Oncol 2008;26:3582–9. [54] Kimple RJ, Harari PM. Is radiation dose reduction the right answer for HPV-positive head and neck cancer? Oral Oncol 2014;50:560–4. [55] Sinha P, Lewis Jr JS, Piccirillo JF, Kallogjeri D, Haughey BH. Extracapsular spread and adjuvant therapy in human papillomavirus-related, p16-positive oropharyngeal carcinoma. Cancer 2012;118:3519–30. [56] Lassen P, Eriksen JG, Hamilton-Dutoit S, Tramm T, Alsner J, Overgaard J. Effect of HPV-associated p16INK4A expression on response to radiotherapy and survival in squamous cell carcinoma of the head and neck. J Clin Oncol 2009;27:1992–8. [57] Chen AM, Zahra T, Daly ME, Farwell DG, Luu Q, Gandour-Edwards R, et al. Definitive radiation therapy without chemotherapy for human papillomavirus-positive head and neck cancer. Head Neck 2013;35:1652–6. [58] Benafif S, Hall M. An update on PARP inhibitors for the treatment of cancer. Onco Targets Ther 2015;26(8):519–28. [59] Michels J, Vitale I, Saparbaev M, Castedo M, Kroemer G. Predictive biomarkers for cancer therapy with PARP inhibitors. Oncogene 2014;33: 3894–907. [60] Güster JD, Weissleder SV, Busch CJ, Kriegs M, Petersen C, Knecht R, et al. The inhibition of PARP but not EGFR results in the radiosensitization of HPV/ p16-positive HNSCC cell lines. Radiother Oncol 2014;113:345–51. [61] Raju U, Riesterer O, Wang ZQ, Molkentine DP, Molkentine JM, Johnson FM, et al. Dasatinib, a multi-kinase inhibitor increased radiation sensitivity by interfering with nuclear localization of epidermal growth factor receptor and by blocking DNA repair pathways. Radiother Oncol 2012;105: 241–9. [62] Ang KK, Zhang Q, Rosenthal DI, et al. Randomized phase III trial of concurrent accelerated radiation plus cisplatin with or without cetuximab for stage III to IV head and neck carcinoma: RTOG 0522. J Clin Oncol 2014;32: 2940–50. [63] Giralt J, Trigo J, Nuyts S, et al. Panitumumab plus radiotherapy versus chemoradiotherapy in patients with unresected, locally advanced squamouscell carcinoma of the head and neck (CONCERT-2): a randomised, controlled, open-label phase 2 trial. Lancet Oncol 2015;16:221–32. [64] Kagawa S, Natsuizaka M, Whelan KA, Facompre N, Naganuma S, Ohashi S, et al. Cellular senescence checkpoint function determines differential Notch1dependent oncogenic and tumor-suppressor activities. Oncogene 2015;34:2347–59. [65] Dantas-Barbosa C, Bergthold G, Daudigeos-Dubus E, Blockus H, Boylan JF, Ferreira C, et al. Inhibition of the NOTCH pathway using c-secretase inhibitor RO4929097 has limited antitumor activity in established glial tumors. Anticancer Drugs 2015;26(3):272–83. [66] Saki M, Toulany M, Rodemann HP. Acquired resistance to cetuximab is associated with the overexpression of Ras family members and the loss of radiosensitization in head and neck cancer cells. Radiother Oncol 2013;108 (3):473–8. [67] Fury MG, Lee NY, Sherman E, Ho AL, Rao S, Heguy A, et al. A phase 1 study of everolimus + weekly cisplatin + intensity modulated radiation therapy in head-and-neck cancer. Int J Radiat Oncol Biol Phys 2013;87:479–86. [68] Deutsch E, Le Péchoux C, Faivre L, Rivera S, Tao Y, Pignon JP, et al. Phase I trial of everolimus in combination with thoracic radiotherapy in non-small-cell lung cancer. Ann Oncol 2015;26:1223–9. [69] Galloway TJ, Wirth LJ, Colevas AD, Gilbert J, Bauman JE, Saba NF, et al. A Phase I study of CUDC-101, a multitarget inhibitor of HDACs, EGFR, and HER2, in combination with chemoradiation in patients with head and neck squamous cell carcinoma. Clin Cancer Res 2015;21:1566–73. [70] Al Moustafa AE, Foulkes WD, Benlimame N, Wong A, Yen L, Bergeron J, et al. E6/E7 proteins of HPV type 16 and ErbB-2 cooperate to induce neoplastic transformation of primary normal oral epithelial cells. Oncogene 2004;23:350–8. [71] Deberne M, Levy A, Mondini M, Dessen P, Vivet S, Supiramaniam A, et al. The combination of the antiviral agent cidofovir and anti-EGFR antibody cetuximab exerts an antiproliferative effect on HPV-positive cervical cancer cell lines’ in-vitro and in-vivo xenografts. Anticancer Drugs 2013;24:599–608. [72] Sirianni N, Wang J, Ferris RL. Antiviral activity of Cidofovir on a naturally human papillomavirus-16 infected squamous cell carcinoma of the head and neck(SCCHN) cell line improves radiation sensitivity. Oral Oncol 2005;41:423–8.

Please cite this article in press as: Mirghani H et al. Increased radiosensitivity of HPV-positive head and neck cancers: Molecular basis and therapeutic perspectives. Cancer Treat Rev (2015), http://dx.doi.org/10.1016/j.ctrv.2015.10.001

H. Mirghani et al. / Cancer Treatment Reviews xxx (2015) xxx–xxx [73] Demaria S, Ng B, Devitt ML, Babb JS, Kawashima N, Liebes L, et al. Ionizing radiation inhibition of distant untreated tumors (abscopal effect) is immune mediated. Int J Radiat Oncol Biol Phys 2004;58(3):862–70. [74] Hiniker SM, Chen DS, Reddy S, Chang DT, Jones JC, Mollick JA, et al. A systemic complete response of metastatic melanoma to local radiation and immunotherapy. Transl Oncol 2012;5:404–7. [75] Golden EB, Demaria S, Schiff PB, Chachoua A, Formenti SC. An abscopal response to radiation and ipilimumab in a patient with metastatic non-small cell lung cancer. Cancer Immunol Res 2013;1:365–72. [76] Mondini M, Nizard M, Tran T, Mauge L, Loi M, Clémenson C, et al. Synergy of radiotherapy and a cancer vaccine for the treatment of HPV-associated head and neck cancer. Mol Cancer Ther 2015 [Epub ahead of print]. [77] Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 2012;12:252–64.

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[78] Lyford-Pike S, Peng S, Young GD, et al. Evidence for a role of the PD-1:PD-L1 pathway in immune resistance of HPV-associated head and neck squamous cell carcinoma. Cancer Res 2013;73:1733–41. [79] Ukpo OC, Thorstad WL, Lewis Jr JS. B7–H1 expression model for immune evasion in human papillomavirus-related oropharyngeal squamous cell carcinoma. Head Neck Pathol 2013;7:113–21. [80] Pai SI. Mission impossible: how HPV-associated head and neck cancers escape a primed immune response. Oral Oncol 2013;49:723–5. [81] NCT01693562 (). [82] Seiwer TY, Burtness B, Weiss J, et al. A phase Ib study of MK-3475 in patients with human papillomavirus (HPV)-associated and non-HPV-associated head and neck (H/N) cancer. J Clin Oncol 2014;32:5s [suppl; abstr 6011].

Please cite this article in press as: Mirghani H et al. Increased radiosensitivity of HPV-positive head and neck cancers: Molecular basis and therapeutic perspectives. Cancer Treat Rev (2015), http://dx.doi.org/10.1016/j.ctrv.2015.10.001