S. Su

Potentially Novel Candidate Biomarkers for Head and Neck Squamous Cell Carcinoma Identified Using an Integrated Cell Line-based Discovery Strategy

Head and neck squamous cell carcinomas (HNSCC) can arise from the oral cavity, oropharynx, larynx or hypopharynx, and is the sixth leading cancer by incidence worldwide. The 5-year survival rate of HNSCC patients remains static at 40–60%. Hence, biomarkers which can improve detection of HNSCC or early recurrences should improve clinical outcome. Mass spectrometry-based proteomics methods have emerged as promising approaches for biomarker discovery. As one approach, mass-spectrometric identification of proteins shed or secreted from cancer cells can contribute to the identification of potential biomarkers for HNSCC and our understanding of tumor behavior. In the current study, mass spectrometry-based proteomic profiling was performed on the conditioned media (i.e. secretome) of head and neck cancer (HNC) cell lines (FaDu, UTSCC8 and UTSCC42a) in addition to gene expression microarrays to identify over-expressed transcripts in the HNSCC cells in comparison to a normal control cell line. This integrated data set was systematically mined using publicly available resources (Human Protein Atlas and published proteomic/transcriptomic data) to prioritize putative candidates for validation. Subsequently, quantitative real-time PCR (qRT-PCR), Western blotting, immunohistochemistry (IHC), and ELISAs were performed to verify selected markers. Our integrated analyses identified 90 putative protein biomarkers that were secreted or shed to the extracellular space and over-expressed in HNSCC cell lines, relative to controls. Subsequently, the over-expression of five markers was verified in vitro at the transcriptional and translational levels using qRT-PCR and Western blotting, respectively. IHC-based validation conducted in two independent cohorts comprising of 40 and 39 HNSCC biopsies revealed that high tumor expression of PLAU, IGFBP7, MMP14 and THBS1 were associated with inferior disease-free survival, and increased risk of disease progression or relapse. Furthermore, as demonstrated using ELISAs, circulating levels of PLAU and IGFBP7 were significantly higher in the plasma of HNSCC patients compared with healthy individuals.

Salvage surgery for locally recurrent oropharyngeal cancer

There are limited data on whether recurrent human papillomavirus (HPV)‐associated oropharyngeal squamous cell carcinoma (SCC) is associated with higher surgical salvage rates. The purpose of this study was to determine the success rate of salvage surgery for locally recurrent oropharyngeal cancer and factors influencing the outcome, including p16 status.

Integrated Omic Analysis of Oropharyngeal Carcinomas Reveals Human Papillomavirus (HPV)–dependent Regulation of the Activator Protein 1 (AP-1) Pathway

HPV-positive oropharyngeal carcinoma (OPC) patients have superior outcomes relative to HPV-negative patients, but the underlying mechanisms remain poorly understood. We conducted a proteomic investigation of HPV-positive (n = 27) and HPV-negative (n = 26) formalin-fixed paraffin-embedded OPC biopsies to acquire insights into the biological pathways that correlate with clinical behavior. Among the 2,633 proteins identified, 174 were differentially abundant. These were enriched for proteins related to cell cycle, DNA replication, apoptosis, and immune response. The differential abundances of cortactin and methylthioadenosine phosphorylase were validated by immunohistochemistry in an independent cohort of 29 OPC samples (p = 0.023 and p = 0.009, respectively). An additional 1,124 proteins were independently corroborated through comparison to a published proteomic dataset of OPC. Furthermore, utilizing the Cancer Genome Atlas, we conducted an integrated investigation of OPC, attributing mechanisms underlying differential protein abundances to alterations in mutation, copy number, methylation, and mRNA profiles. A key finding of this integration was the identification of elevated cortactin oncoprotein levels in HPV-negative OPCs. These proteins might contribute to reduced survival in these patients via their established role in radiation resistance. Through interrogation of Cancer Genome Atlas data, we demonstrated that activation of the β1-integrin/FAK/cortactin/JNK1 signaling axis and associated differential regulation of activator protein 1 transcription factor target genes are plausible consequences of elevated cortactin protein levels.

Predictors of breast radiotherapy plan modifications: Quality assurance rounds in a large cancer centre

BACKGROUND AND PURPOSE This study describes the process and outcomes of breast radiotherapy (RT) quality assurance (QA) rounds, seeking to identify variables associated with plan modifications. MATERIALS AND METHODS Real-time data were prospectively collected over 2 years. Descriptive statistics determined the proportion of cases requiring no (A), minor (B), or major (C) modifications, which were then subjected to univariate and multivariate analyses. RESULTS A total of 2223 breast cancer QA cases were reviewed; 47 cases (2.1%) underwent a minor, and 52 cases (2.3%) required a major modification. Common changes included boost, volume, seroma, and bolus. On univariate analysis, regional nodal irradiation (RNI), tumour size, and axillary node dissection were significantly associated with major modifications. Upon multivariate analysis, the only independent predictor was RNI (OR 2.12, p=0.0075). For patients with no RNI, <2 cm tumours, no axillary lymph node dissection, and no boosts (n=420); the likelihood of category C was only 1.4%. CONCLUSIONS It is feasible to conduct QA review for all breast cancer cases prior to commencing RT. Patients undergoing RNI had a higher likelihood of plan modifications; a group with low risk of modification was identified, which could direct future re-structuring of QA rounds.

Proteomic Analysis of Cancer-Associated Fibroblasts Reveals a Paracrine Role for MFAP5 in Human Oral Tongue Squamous Cell Carcinoma

Bidirectional communication between cells and their microenvironment is crucial for both normal tissue homeostasis and tumor growth. During the development of oral tongue squamous cell carcinoma (OTSCC), cancer-associated fibroblasts (CAFs) create a supporting niche by maintaining a bidirectional crosstalk with cancer cells, mediated by classically secreted factors and various nanometer-sized vesicles, termed as extracellular vesicles (EVs). To better understand the role of CAFs within the tumor stroma and elucidate the mechanism by which secreted proteins contribute to OTSCC progression, we isolated and characterized patient-derived CAFs from resected tumors with matched adjacent tissue fibroblasts (AFs). Our strategy employed shotgun proteomics to comprehensively characterize the proteomes of these matched fibroblast populations. Our goals were to identify CAF-secreted factors (EVs and soluble) that can functionally modulate OTSCC cells in vitro and to identify novel CAF-associated biomarkers. Comprehensive proteomic analysis identified 4247 proteins, the most detailed description of a pro-tumorigenic stroma to date. We demonstrated functional effects of CAF secretomes (EVs and conditioned media) on OTSCC cell growth and migration. Comparative proteomics identified novel proteins associated with a CAF-like state. Specifically, MFAP5, a protein component of extracellular microfibrils, was enriched in CAF secretomes. Using in vitro assays, we demonstrated that MFAP5 activated OTSCC cell growth and migration via activation of MAPK and AKT pathways. Using a tissue microarray of richly annotated primary human OTSCCs, we demonstrated an association of MFAP5 expression with patient survival. In summary, our proteomics data of patient-derived stromal fibroblasts provide a useful resource for future mechanistic and biomarker studies.

A Comparison of Postoperative MRI Changes between Endoscopic Endonasal and Open Approaches for Olfactory Groove Meningiomas: A Match Paired Analysis

Objective: Olfactory groove meningiomas may be associated with significant brain edema and surgical removal of these tumors may be associated with further injury to the frontal lobes. Endonasal access avoids frontal lobe manipulation, but little objective benefit has been demonstrated. Study Design: Retrospective case-cohort matched pair analysis. Methods: A retrospective review was performed at two institutions to identify patients who had either endonasal or open approach for resection of olfactory groove meningiomas. A matched pair analysis was performed and tumor volume, edema, FLAIR change, and porencephalic cave were volumetrically quantified. Results: Ten matched pairs (20 patients) were identified. The open approach was associated with more postoperative FLAIR change on MRI compared with the endoscopic approach although not statistically significant (13.3 cm3 [SD = 12.0] vs. 6.9 cm3 [SD = 10.0], p = 0.17). The endoscopic approach was also associated with smaller porencephalic cave volumes (1.7 cm3[SD=2.8] vs. 6.9 cm3[SD = 10.0), p = 0.058). In a multivariable model, the endoscopic approach was associated with less postoperative FLAIR change (p = 0.016) and smaller porencephalic cave volumes (p = 0.028). Conclusions: This study provides preliminary evidence that the endoscopic endonasal approach is associated with quantifiable improvements in postoperative brain imaging. Further studies including neurocognitive function are needed to confirm the significance of these findings.

Abstract 666: The effect of comorbidity, smoking and alcohol on survival of head and neck cancer anatomic subsites: A retrospective analysis of 4689 patients

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Increased age and greater comorbidity are independent predictors of worse survival in nasopharyngeal and squamous cell cancers of the head and neck (HNC). The Charlson Comorbidity Index (CCI) provides a quantitative measure of comorbidity that can be abstracted from medical charts. We evaluated the role of anatomic subsite, smoking, alcohol, demographic and other variables on the impact of overall survival (OS) in HNC. Methods: Retrospective chart review was performed for 4689 HNC patients identified by the Princess Margaret Hospital and Ontario Cancer Registries (Toronto, Canada), between 2000 and 2010. Demographic (age, gender, occupation, marital status), and histologic variables were abstracted in addition to smoking and drinking consumption (current, former, never) and quantity (cigarettes: pack-years; alcohol usage: non/light, moderate, and heavy). CCI were categorized as no comorbidities (CCI 0), mild (CCI 1-2), moderate (CCI 3-4), and severe comorbidity (CCI 5+), respectively. HPV status in oropharyngeal tumors was assessed using p16 immunohistochemistry. Results: The population was mostly male (73%), married/common-law (71%), and median 63 years of age. Disease site consisted of 35% oral cavity, 28% oropharynx, 29% larynx, 6% hypopharynx, and 3% nasopharynx. Most tumors were stage III/IV (67%) and moderately differentiated (65%). Patients were categorized with 7% severe, 17% moderate, 47% mild, and 30% with no comorbidity. 79% of patients were ever-smokers and 84% were ever-drinkers. 481/688 OPC tumors were HPV+. Overall survival (OS) was better in younger patients (HR 1.03 per year, p < 0.0001), and those in married/common-law relationships (HR 1.48, p < 0.001). Greater comorbidity was associated with poorer OS: HR 1.5 (p < 0.0001), 2.0 (p < 0.0001), and 2.4 (p < 0.0001) for mild, moderate, and severe comorbidity, respectively. Univariately, oropharyngeal cancer had superior OS than cancers of the hypopharynx (HR 2.3, p < 0.0001) and oral cavity (HR 1.2, p = 0.006) but not the larynx (HR 0.97, p = 0.59) and worse OS than cancers of the nasopharynx (HR 0.54, p = 0.001). Advanced overall TNM stage was associated with poorer OS (HR 2.42, p < 0.0001), but grade was not significantly associated with survival. In terms of treatment, surgery alone (HR 0.63, p < 0.0001), surgery with post-operative radiation (HR 0.82, p = 0.004), and surgery followed by chemoradiotherapy (HR 0.76, p = 0.02) resulted in better OS than radiation alone. Heavier smoking and alcohol consumption resulted in poorer OS. For oropharyngeal cancer patients alone, HPV positivity improved OS (HR 0.30, p < 0.0001). Conclusion: In addition to stage, other important factors including social, demographic, treatment, histologic, and comorbidity variables also impact survival, and should be considered as prognostic factors in analyses of HNC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 666. doi:1538-7445.AM2012-666