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

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.

Predictors for secondary therapy after surgical resection of nonfunctioning pituitary adenomas.

OBJECTIVE: 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. DESIGN AND PATIENTS: A single-centre retrospective study of surgically resected NFAs in 108 patients followed for up to 15 years. Serial tumour images were analysed for size, location and growth rate (GR) and tissue analysed for hormone cell type and proliferation indices with secondary treatment as outcome measure. RESULTS: Twenty-four of 66 (36%) patients harbouring a postoperative remnant required secondary treatment, all occurring within 10 years. No secondary treatment was required in any of 42 patients with complete tumour resection. Age, gender, remnant volume and tumour histology were not different between patients requiring and not requiring secondary therapy. Remnant GRs in those requiring secondary therapy were more than 10-fold higher (P<.01). Tumours with a GR ≥80 mm3 /y (Hazard Ratio[HR]: 8.1, Confidence Interval [CI]: 2.4-27.3,P<.01) and those located in the suprasellar region (HR: 6.1, CI: 1.1-32, P=.03) had a higher risk for secondary therapy. Tumour GR in the first three postoperative years correlated significantly (r2 =.6, P<.01) with GR during the period of follow-up. CONCLUSION: In surgically resected NFAs further treatment is dependent on the presence of residual tumour, growth rate and location but not tumour histology. Postoperative growth rate of NFAs in the first 3 years of imaging can be used to tailor long-term follow-up to optimize use of health resources.

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

BACKGROUND: 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. METHODS: DNA was extracted from 63 formalin-fixed paraffin-embedded tumour specimens histologically confirmed as SCC of the oral cavity, diagnosed during 2006-2012. Clinical data were extracted from medical records. HPV presence was determined by polymerase chain reaction. Positive samples were typed by sequencing. Immunohistochemistry was used to assess p16INK4A , p53, pRB, Ki67, Cyclin D1 and p21WAF1 expression. RESULTS: Five of the 63 tumours (8%) were positive for HPV DNA (three HPV-16 positive and two HPV-18 positive). Two tumours overexpressed p16INK4A (3%) and one of these was also HPV positive. Overexpression of Cyclin D1 correlated significantly with tumour recurrence (P = 0.029) and death (P = 0.002). CONCLUSIONS: This study has identified a low prevalence of high-risk HPV in Queensland, Australia.

Implementation and evaluation of amyloidosis subtyping by laser-capture microdissection and tandem mass spectrometry.

BACKGROUND: Correct identification of the amyloidosis-causing protein is crucial for clinical management. Recently the Mayo Clinic reported laser-capture microdissection (LCM) with liquid chromatography-coupled tandem mass spectrometry (MS/MS) as a new diagnostic tool for amyloid diagnosis. Here, we report an independent implementation of this proteomic diagnostics method at the Princess Alexandra Hospital Amyloidosis Centre in Brisbane, Australia. RESULTS: From 2010 to 2014, 138 biopsies received from 35 different organ sites were analysed by LCM-MS/MS using Congo Red staining to visualise amyloid deposits. There was insufficient tissue in the block for LCM for 7 cases. An amyloid forming protein was ultimately identified in 121 out of 131 attempted cases (94 %). Of the 121 successful cases, the Mayo Clinic amyloid proteomic signature (at least two of Serum Amyloid P, ApoE and ApoA4) was detected in 92 (76 %). Low levels of additional amyloid forming proteins were frequently identified with the main amyloid forming protein, which may reflect co-deposition of fibrils. Furthermore, vitronectin and clusterin were frequently identified in our samples. Adding vitronectin to the amyloid signature increases the number of positive cases, suggesting a potential 4th protein for the signature. In terms of clinical impact, amyloid typing by immunohistochemistry was attempted in 88 cases, reported as diagnostic in 39, however, 5 were subsequently revealed by proteomic analysis to be incorrect. Overall, the referring clinician's diagnosis of amyloid subtype was altered by proteomic analysis in 24 % of cases. While LCM-MS/MS was highly robust in protein identification, clinical information was still required for subtyping, particularly for systemic versus localized amyloidosis. CONCLUSIONS: This study reports the independent implementation and evaluation of a proteomics-based diagnostic for amyloidosis subtyping. Our results support LCM-MS/MS as a powerful new diagnostic technique for amyloidosis, but also identified some challenges and further development opportunities.

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.

Human papillomavirus status and p16(INK4A) 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.

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.

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.