Samuel Boros

Human papillomavirus status and p16INK4A expression in patients with mucosal squamous cell carcinoma of the head and neck in Queensland, Australia

BACKGROUND The last decade has seen changes in the epidemiology of mucosal squamous cell carcinomas of the head and neck (HNSCCs), with increasing numbers of cases attributable to human papillomavirus (HPV) infection. We sought to determine the prevalence of HPV and p16(INK4a) expression in Australian HNSCC patients and to identify predictors of HPV-positivity. METHODS We recruited 248 HNSCC patients with histologically confirmed primary SCC of the oropharynx, oral cavity, hypopharynx or larynx diagnosed between 2004 and 2010. All patients completed a questionnaire. Clinical data were abstracted from medical records. HPV presence in paraffin-embedded tumours was determined by PCR, and expression of p16(INK4a), p21(WAF1), p53, pRB, cyclin D1, and Ki67 by immunohistochemistry. RESULTS Fifty (20%) patients were HPV-positive, 63 (28%) overexpressed p16(INK4a), and 44 (19%) were positive for HPV and p16(INK4a) (high concordance between HPV-positivity and p16(INK4a) status, κ=0.72). HPV-16 was most common (84%), followed by HPV-18 (10%), HPV-33 (4%) and HPV-69 (2%). HPV and p16(INK4a) prevalence was highest for SCCs of the oropharynx, followed by hypopharynx, larynx and oral cavity (HPV and p16(INK4a)p<0.0001). HPV prevalence and p16(INK4a)-overexpression were significantly higher in younger than older patients (HPV p=0.001; p16 (INK4a)p=0.003). Heavy smokers had lower HPV prevalence than non- or moderate smokers (p=0.017). Gender and alcohol consumption were not associated with HPV or p16(INK4a) status. HPV-positive tumours had significantly lower cyclin D1 and higher p21(WAF1) expression than HPV-negative tumours. CONCLUSION HPV prevalence and p16(INK4a)-overexpression were highest in oropharyngeal tumours, younger patients, and non-smokers.

A Novel E2F/Sphingosine Kinase 1 Axis Regulates Anthracycline Response in Squamous Cell Carcinoma

Purpose: Head and neck squamous cell carcinomas (HNSCC) are frequently drug resistant and have a mortality rate of 45%. We have previously shown that E2F7 may contribute to drug resistance in SCC cells. However, the mechanism and pathways involved remain unknown. Experimental Design: We used transcriptomic profiling to identify candidate pathways that may contribute to E2F7-dependent resistance to anthracyclines. We then manipulated the activity/expression of the candidate pathway using overexpression, knockdown, and pharmacological inhibitors in in vitro and in vivo models of SCC to demonstrate causality. In addition, we examined the expression of E2F7 and a downstream effector in a tissue microarray (TMA) generated from HNSCC patient samples. Results: E2F7-deficient keratinocytes were selectively sensitive to doxorubicin and this was reversed by overexpressing E2F7. Transcriptomic profiling identified Sphingosine kinase 1 (Sphk1) as a potential mediator of E2F7-dependent drug resistance. Knockdown and overexpression studies revealed that Sphk1 was a downstream target of E2F7. TMA studies showed that E2F7 overexpression correlated with Sphk1 overexpression in human HNSCC. Moreover, inhibition of Sphk1 by shRNA or the Sphk1-specific inhibitor, SK1-I (BML-EI411), enhanced the sensitivity of SCC cells to doxorubicin in vitro and in vivo. Furthermore, E2F7-induced doxorubicin resistance was mediated via Sphk1-dependent activation of AKT in vitro and in vivo. Conclusion: We identify a novel drugable pathway in which E2F7 directly increases the transcription and activity of the Sphk1/S1P axis resulting in activation of AKT and subsequent drug resistance. Collectively, this novel combinatorial therapy can potentially be trialed in humans using existing agents. Clin Cancer Res; 21(2); 417–27. ©2014 AACR.

RacGAP1 Is a Novel Downstream Effector of E2F7-Dependent Resistance to Doxorubicin and Is Prognostic for Overall Survival in Squamous Cell Carcinoma

We have previously shown that E2F7 contributes to drug resistance in head and neck squamous cell carcinoma (HNSCC) cells. Considering that dysregulation of responses to chemotherapy-induced cytotoxicity is one of the major reasons for treatment failure in HNSCC, identifying the downstream effectors that regulate E2F7-dependent sensitivity to chemotherapeutic agents may have direct clinical impact. We used transcriptomic profiling to identify candidate pathways that contribute to E2F7-dependent resistance to doxorubicin. We then manipulated the expression of the candidate pathway using overexpression and knockdown in in vitro and in vivo models of SCC to demonstrate causality. In addition, we examined the expression of E2F7 and RacGAP1 in a custom tissue microarray (TMA) generated from HNSCC patient samples. Transcriptomic profiling identified RacGAP1 as a potential mediator of E2F7-dependent drug resistance. We validated E2F7-dependent upregulation of RacGAP1 in doxorubicin-insensitive SCC25 cells. Extending this, we found that selective upregulation of RacGAP1 induced doxorubicin resistance in previously sensitive KJDSV40. Similarly, stable knockdown of RacGAP1 in insensitive SCC25 cells induced sensitivity to doxorubicin in vitro and in vivo. RacGAP1 expression was validated in a TMA, and we showed that HNSCCs that overexpress RacGAP1 are associated with a poorer patient overall survival. Furthermore, E2F7-induced doxorubicin resistance was mediated via RacGAP1-dependent activation of AKT. Finally, we show that SCC cells deficient in RacGAP1 grow slower and are sensitized to the cytotoxic actions of doxorubicin in vivo. These findings identify RacGAP1 overexpression as a novel prognostic marker of survival and a potential target to sensitize SCC to doxorubicin. Mol Cancer Ther; 14(8); 1939–50. ©2015 AACR.

Radiation-induced primary cerebral atypical teratoid/rhabdoid tumour in an adult.

Atypical teratoid/rhabdoid tumours (ATRT) of the central nervous system are uncommon embryonal carcinomas that predominantly affect infants and young children, and less commonly adults. We report a 58 year old woman who presented with ATRT involving the right parietal lobe which was treated with surgery and adjuvant radiotherapy. Her history was significant for soft tissue sarcoma of the right ear treated with surgery and adjuvant radiotherapy at age 3, thus raising the possibility of radiation-induced aetiology.

Low prevalence of human papillomavirus in oral cavity squamous cell carcinoma in Queensland, Australia

While human papillomavirus (HPV) is an accepted risk factor for oropharyngeal squamous cell carcinoma (SCC), its aetiological role in oral cavity SCC remains unclear. This study aimed to determine the HPV prevalence in an Australian population.

Hyperdiploid tumor cells increase phenotypic heterogeneity within Glioblastoma tumors

Here we report the identification of a proliferative, viable, and hyperdiploid tumor cell subpopulation present within Glioblastoma (GB) patient tumors. Using xenograft tumor models, we demonstrate that hyperdiploid cell populations are maintained in xenograft tumors and that clonally expanded hyperdiploid cells support tumor formation and progression in vivo. In some patient tumorsphere lines, hyperdiploidy is maintained during long-term culture and in vivo within xenograft tumor models, suggesting that hyperdiploidy can be a stable cell state. In other patient lines hyperdiploid cells display genetic drift in vitro and in vivo, suggesting that in these patients hyperdiploidy is a transient cell state that generates novel phenotypes, potentially facilitating rapid tumor evolution. We show that the hyperdiploid cells are resistant to conventional therapy, in part due to infrequent cell division due to a delay in the G₀/G₁ phase of the cell cycle. Hyperdiploid tumor cells are significantly larger and more metabolically active than euploid cancer cells, and this correlates to an increased sensitivity to the effects of glycolysis inhibition. Together these data identify GB hyperdiploid tumor cells as a potentially important subpopulation of cells that are well positioned to contribute to tumor evolution and disease recurrence in adult brain cancer patients, and suggest tumor metabolism as a promising point of therapeutic intervention against this subpopulation.

Predictors for secondary therapy after surgical resection of nonfunctioning pituitary adenomas

Factors determining recurrence of nonfunctioning pituitary adenomas (NFAs) that require further therapy are unclear as are postoperative follow‐up imaging guidelines. We aimed to identify predictors for secondary therapy after surgical resection of NFAs and use this knowledge to inform postoperative management.

Targeting the XPO1-dependent nuclear export of E2F7 reverses anthracycline resistance in head and neck squamous cell carcinomas

Selective mislocalization of E2F7 in head and neck cancers drives anthracycline resistance and can be reversed. Restoring balance in the nucleus Despite recent advances in cancer treatment, resistance to cancer therapy and resulting mortality remain common in head and neck squamous cell carcinoma. In their search for the causes of treatment resistance, Saenz-Ponce et al. identified a mechanism dependent on the balance of two proteins that regulate transcription and these proteins’ localization within cancer cells. Specifically, the authors discovered that a transcriptional inhibitor called E2F7 is frequently mislocalized to the cytoplasm in these tumors, whereas its transcription-activating counterpart, E2F1, remains in the nucleus and drives transcription of treatment resistance genes. The authors also identified an approved drug that can prevent the export of E2F7 from the nucleus and thereby restore the efficacy of anthracycline chemotherapy in head and neck cancer. Patient mortality rates have remained stubbornly high (40%) for the past 35 years in head and neck squamous cell carcinoma (HNSCC) due to inherent or acquired drug resistance. Thus, a critical issue in advanced SCC is to identify and target the mechanisms that contribute to therapy resistance. We report that the transcriptional inhibitor, E2F7, is mislocalized to the cytoplasm in >80% of human HNSCCs, whereas the transcriptional activator, E2F1, retains localization to the nucleus in SCC. This results in an imbalance in the control of E2F-dependent targets such as SPHK1, which is derepressed and drives resistance to anthracyclines in HNSCC. Specifically, we show that (i) E2F7 is subject to exportin 1 (XPO1)–dependent nuclear export, (ii) E2F7 is selectively mislocalized in most of SCC and multiple other tumor types, (iii) mislocalization of E2F7 in HNSCC causes derepression of Sphk1 and drives anthracycline resistance, and (iv) anthracycline resistance can be reversed with a clinically available inhibitor of XPO1, selinexor, in xenotransplant models of HNSCC. Thus, we have identified a strategy to repurpose anthracyclines for use in SCC. More generally, we provide a strategy to restore the balance of E2F1 (activator) and E2F7 (inhibitor) activity in cancer.

Sexual behaviour, HPV status and p16INK4a expression in oropharyngeal and oral cavity squamous cell carcinomas: a case–case comparison study

A significant proportion of mucosal squamous cell carcinomas of the head and neck (HNSCC; particularly of the oropharynx) are directly attributable to the human papillomavirus (HPV). The increase in the incidence of HPV-related tumours has been postulated to be due to changing sexual practices in the community. We analysed 136 formalin-fixed paraffin-embedded squamous cell carcinomas from the oral cavity (n=40) and oropharynx (n=96) recruited from the Princess Alexandra Hospital (Brisbane, Australia). Samples were analysed for the presence of HPV DNA using a combination of mucosal HPV general primer GP+ PCR and sequencing; p16INK4a expression was assessed by immunohistochemistry. Each patient completed a questionnaire detailing their lifestyle factors, such as tobacco smoking and alcohol consumption, marital status, and sexual behaviour and history. The HPV DNA prevalence was 5 % in the oral cavity cancers and 72 % in the oropharyngeal cancers (P<0.0001). HPV-16 was the most commonly detected HPV type (found in 91 % of all HPV-positive tumours). There was a strong correlation between HPV DNA positivity and positive p16INK4a staining in oropharyngeal tumours (P<0.0001). Having an HPV-related tumour was associated with being married or having been married previously (P=0.046), an increasing number of passionate kissing partners (P=0.046), ever having given oral sex (P=0.0007) and an increasing number of oral sex partners (P=0.0015). This study found a higher prevalence of HPV in oropharyngeal compared to oral cavity tumours, with a strong association being identified between oral sex behaviours and HPV-positive tumours. Further research is needed to establish that vaccines will reduce the transmission and carriage of oropharyngeal HPV infections.