Christopher Angel

Differential mechanisms of CDKN2A (p16) alteration in oral tongue squamous cell carcinomas and correlation with patient outcome.

CDKN2A (p16) disruption is reported as a frequent event in head and neck squamous cell carcinomas that confers poor prognosis. We investigated the frequency of different potential mechanisms of CDKN2A inactivation in oral tongue squamous cell carcinomas (OTSCC) and their impact on patient outcome. From a cohort of 153 OTSCC patients, 131 formalin fixed paraffin embedded blocks of pre‐treatment primary tumours were suitable for further molecular analysis. We assessed CDKN2A (p16) levels by immunohistochemistry (IHC), promoter methylation status by methylation‐sensitive high resolution melting, mutation status by Sanger sequencing, gene copy number variation by fluorescence in situ hybridisation, and correlated these with patient outcome. We found that the majority of OTSCC did not overexpress p16 (110/116, 95%), assessed by IHC. The frequency of CDKN2A mutations was 20% (21/103), homozygous loss was 7% (7/97), hemizygous loss 31% (30/97), and promoter methylation was 18% (20/113). We found no evidence of these mechanisms in 24/106 (23%) p16 IHC negative tumours. No significant correlation was identified between any potential mechanism of CDKN2A inactivation and clinical features, including smoking status and age. There was a non‐significant trend for worse overall survival for p16 IHC negative patients versus positive patients (HR = 1.81, 95% CI = 0.44–7.47, p = 0.40). No relationship was found between mechanisms of CDKN2A disruption and patient outcome. In conclusion, we demonstrate that CDKN2A alteration is a frequent event in OTSCC tumourigenesis. However, no correlation was identified between different potential mechanisms of CDKN2A disruption and clinical characteristics or patient outcome.

Frequency of Fibroblast Growth Factor Receptor 1 gene amplification in oral tongue squamous cell carcinomas and associations with clinical features and patient outcome

OBJECTIVES Novel therapies are required for patients with recurrent or metastatic oral tongue squamous cell carcinoma (OTSCC). Fibroblast Growth Factor Receptor 1 (FGFR1) amplification frequently occurs in squamous cell carcinoma of the lung and represents a novel druggable therapeutic target in this and other malignancies. This study examined the frequency and clinical associations of FGFR1 amplification in OTSCC. MATERIALS AND METHODS The frequency of FGFR1 amplification determined by fluorescence in situ hybridization was evaluated in a cohort of 123 OTSCC patients. Associations of FGFR1 amplification with clinical characteristics and outcome were determined. RESULTS FGFR1 gene amplification was present in 9.3% (10/107) of cases and was significantly associated with smoking status (P = 0.03). FGFR1 amplification was seen more commonly in males (9/10 amplified cases male, P = 0.16) and there were no associations with age, stage, T stage, nodal status, alcohol history or performance status (all P>0.05). Outcome was not significantly different between FGFR1 amplified and non-amplified patients. CONCLUSIONS Copy number variations of the FGFR1 gene occur in a subset of OTSCC with approximately 10% of cases showing amplification of the gene. FGFR1 amplification may represent a therapeutic target in OTSCC.

A community-based model of rapid autopsy in end-stage cancer patients

To the Editor: Systematic genomic studies, including the Cancer Genome Atlas (TCGA)1 and the International Cancer Genome Consortium (ICGC)2, have provided an unprecedented catalog of driver mutations in human cancer. However, these studies use mainly primary, pre-treatment tumor material obtained at surgery with curative intent. There is an urgent need to identify and characterize resistance mechanisms to understand how cancers can evade even the best medical efforts and kill patients; therefore, access to end-stage disease is important. Solid cancers show considerable spatial3, temporal4,5 and genomic heterogeneity at diagnosis. Selective pressure and mutagenic impact of treatment6 drives intra-patient evolution of cancer cell populations4,7. Understanding acquired resistance requires access to paired preand post-treatment samples4,7; however, curative surgery is typically confined to patients with locoregional disease, and opportunities for tumor sampling in advanced disseminated disease are limited. Here, we describe Cancer Tissue Collection After Death (CASCADE), an autopsy program that overcomes logistical challenges to enable collection of samples at end stage for research in melanoma and breast, ovarian and prostate cancers. For the CASCADE study, we aimed to recruit cancer patients close to the end of life, including those outside the minority of patients who die in hospitals. To preserve tissue integrity, autopsies must commence within a few hours of death, requiring access to around-the-clock services. Intervention in the emotionally charged end-of-life environment must be managed in an ethical manner and to a high standard. Finally, we aimed for the study to be highly cost-effective. We believe our approach to meeting these challenges is applicable to researchers in other large urban centers. Here we summarize the main steps in CASCADE’s operating protocol and our experiences from the initial 3 years and 30 autopsies performed (Fig. 1). Information about institutional review board approvals (including a detailed patient informationand-consent form), the autopsy procedure and certain laboratory processes is given in Supplementary Methods and Supplementary Figure 1. Recruitment of participants was led by the clinicians. Such discussions require careful consideration, in timing and in language, and were initiated only if there was a perception that tissue donation would be acceptable to the patients and their families. Factors suggesting acceptability include the emotional stability of the participant and family members and their clarity about and acceptance of the terminal nature of the disease. On occasion, participants prompted discussion by asking about organ or body donation. Consent discussions typically involved oncologists and/or palliative care physicians employed at recruiting hospitals who had established a care relationship with the participant and their family during the patient’s cancer journey. Frequently, the study was introduced at one meeting and discussed over several subsequent clinic visits, allowing patients and their families time to consider participation. We view the involvement of family members in the consent process as essential to support the participant and facilitate decisionmaking. Involvement of family members also ensures that they are fully aware of the autopsy process and helps to clarify funeral arrangements for the study team. After obtaining consent, study investigators collated clinical information, including that related to past and current treatment and diagnostic procedures such as imaging, on an ongoing basis. Between September 2012 and August 2015, 40 patients were approached, and 37 (92.5%) expressed interest in participating. Of those 32 patients (80%) consented; the other 5 had rapid clinical deterioration precluding

Quantitative methodology is critical for assessing DNA methylation and impacts on correlation with patient outcome

BackgroundDNA hypermethylation is reported as a frequent event and prognostic marker in head and neck squamous cell carcinomas (HNSCC). Methylation has been commonly assessed with non-quantitative methodologies, such as methylation-specific PCR (MSP). We investigated previously reported hypermethylated genes with quantitative methodology in oral tongue squamous cell carcinomas (OTSCC).ResultsThe methylation status of 12 genes in 115 OTSCC samples was assessed by one or more of three quantitative analyses: methylation sensitive high resolution melting (MS-HRM), sensitive-melting analysis after real time-methylation specific PCR (SMART-MSP), and bisulfite pyrosequencing.In contrast to much of the literature, either no or infrequent locus-specific methylation was identified by MS-HRM for DAPK1, RASSF1A, MGMT, MLH1, APC, CDH1, CDH13, BRCA1, ERCC1, and ATM. The most frequently methylated loci were RUNX3 (18/108 methylated) and ABO (22/107 methylated). Interrogation of the Cancer Genome Atlas (TCGA) HNSCC cohort confirmed the frequency of significant methylation for the loci investigated.Heterogeneous methylation of RUNX3 (18/108) and ABO (22/107) detected by MS-HRM, conferred significantly worse survival (P = 0.01, and P = 0.03). However, following quantification of methylation levels using pyrosequencing, only four tumors had significant quantities (>15%) of RUNX3 methylation which correlated with a worse patient outcome (P <0.001), while the prognostic significance of ABO hypermethylation was lost. RUNX3 methylation was not prognostic for the TCGA cohort (P = 0.76).ConclusionsWe demonstrated the critical need for quantification of methylation levels and its impact on correlative analyses. In OTSCC, we found little evidence of significant or frequent hypermethylation of many loci reported to be commonly methylated. It is likely that previous reports have overestimated the frequency of significant methylation events as a consequence of the use of non-quantitative methodology.

Prognostic Significance of PD-L1 and CD8 Immune Cells in HPV Oropharyngeal Squamous Cell Carcinoma.

Assessment of CD8+ cell infiltration and PD-L1 expression on intratumoral immune cells identified a subgroup of HPV+ OPSCC patients with excellent clinical outcomes. Immunophenotyping tumors provided prognostic information beyond that provided by existing TNM-based staging systems. Human papilloma virus–positive oropharyngeal squamous cell carcinoma (HPV+ OPSCC) represents a distinct subgroup of head and neck cancers associated with clinical outcomes that are not accurately categorized by existing tumor–node–metastasis-based staging methods. Given the significant impact of immune parameters, such as tumor-infiltrating lymphocytes (TIL) in many cancers, we sought to determine if immunophenotyping tumors can improve categorization of HPV+ OPSCCs for prognostic purposes. In a cohort of 190 patients with HPV+ OPSCC, we quantified and determined the localization of CD8+ TILs, as well as PD-L1–expressing tumor cells (TC) and immune cells (IC). The prognostic significance of these parameters on overall survival (OS) was evaluated, and their contribution to existing prognostic models was determined. High CD8+ TIL abundance (≥30% on stromal or intratumoral ICs) was seen in 61.3% patients and was associated with improved OS [HR, 0.4; 95% confidence interval (CI), 0.2–0.9; P = 0.017]. Although the expression of PD-L1 on TC was not prognostic, high expression of PD-L1 on ≥5% of intratumoral ICs was found in 38.5% patients and was significantly associated with improved OS (HR, 0.37; 95% CI, 0.15–0.93; P = 0. 023). Both high intratumoral IC PD-L1 expression and abundant CD8+ TILs in HPV+ OPSCCs identify subgroups of patients with excellent outcomes and provide additional prognostic information beyond existing staging systems. Cancer Immunol Res; 6(3); 295–304. ©2018 AACR.

Smoking habits and clinical patterns can alter the inflammatory infiltrate in oral lichenoid lesions

OBJECTIVE The present immunohistochemical study aimed to investigate the possible correlation between demographic variables and clinical presentation of oral lichenoid lesions (OLL), in addition to the potential effects of these variables and smoking status on OLL inflammatory infiltrate. STUDY DESIGN A total of 53 patients with OLL were assigned, according to their smoking status at the time of diagnosis, to either a smokers group (n = 27) or a nonsmokers group (n = 26). Demographic and clinical data, including the site and pattern of the OLL, symptoms, and medical history, were analyzed. Immunohistochemical expression of clusters of differentiation, including CD3, CD4, CD8, CD68, and CD1a, was compared between the two groups. RESULTS Gingival involvement in OLL was found to be significantly associated with older age. Buccal mucosa as the sole OLL site showed a significantly higher expression of CD3+ cells compared with other sites (P < .05). OLL presenting as a reticular type alone was significantly associated with less CD3+ expression (P < .05), whereas a significantly higher CD1a+ expression was seen with plaque-like type OLL (P < .05). Smoking was significantly associated with less expression of macrophages (CD68+ cells) and less clinical symptoms (P < .05 and P < .01, respectively). CONCLUSION The inflammatory infiltrate in OLL can be affected by their clinical distribution and presentation. Smoking reduces the expression of macrophages in OLL, and this may alter the immune surveillance and the mechanisms of malignant transformation.

A Double-Clear Variant of Epithelial-Myoepithelial Carcinoma of the Parotid Gland

The hallmark of the histology of epithelial-myoepithelial carcinoma (EMC) is the presence of a regular repetitive mixture of bilayered duct-like structures with an outer layer of myoepithelial cells and inner ductal epithelial cells. Clear cell change in the myoepithelial component is common, but clearing of both cell types, giving an impression of a monocellular neoplasm, is rare. A parotid biopsy was received from an 83-year-old male and subject to routine histologic processing for conventional staining and immunohistochemistry. The encapsulated tumour was composed of sheets of PAS/diastase negative clear cells, separated by fibrous septae. The clear myoepithelial cells were positive for S-100 protein, SMA, and p63 and negative for CK19 and surrounded CK19-positive luminal cells. It is important to utilise immunohistochemistry to differentiate this tumour from others with a similar histologic pattern. Information about the behaviour of the double-clear EMC is limited since there are few cases reported.

Histopathological analysis of oral squamous cell carcinoma in nonsmokers and nondrinkers

A distinct clinical subgroup of nonsmoking (NS) and nondrinking (ND) patients with oral squamous cell carcinoma (OSCC) has been identified. The objective of the study was to assess this cohort for molecular variations in the disease process and if these could be attributed to clinical or epidemiological characteristics. One hundred and twenty-nine consecutive patients (71 males, 58 females) treated for OSCC were assessed at the Royal Melbourne Hospital between January 2007 and July 2010. Formalin-fixed paraffin embedded (FFPE) sections were stained for p53, p16, cyclin D1, and epidermal growth factor receptor (EGFR). Biomarker overexpression was observed in 72 (56%) cases for p53, 23 (18%) for p16, 45 (35%) for cyclin D1, and 72 (56%) for EGFR. Multiple logistic regression analysis revealed that tongue tumors (p = 0.012) and late stage cancers (p = 0.031) were more likely to have cyclin D1 overexpression. Further, older patients significantly more often had cyclin D1 overexpression (p = 0.008) and NSND patients had more p16 expression (p = 0.043). In contrast, smokers were more likely to have EGFR overexpression (p = 0.033). Concurrent overexpression of p53 and cyclin D1 were observed (p = 0.030). Smoking, site, and stage of OSCC can influence biomarker expression, with p16 overexpression specifically observed in NSND, indicating fundamental differences in the mechanisms of oral carcinogenesis among different patient cohorts.

Ameloblastoma, Human Papillomavirus, and p16- is there An Association?

Background: The aetiology of the ameloblastoma is still unclear. Several studies have searched for the presence of human papillomavirus (HPV) in ameloblastomas, however the results have been mixed. Our aim is to clarify this possible association, and to determine if p16 is a reliable surrogate marker for the presence of HPV in these tumours.

Abstract 4693: Characterization of the differential mechanisms of CDKN2A inactivation in oral tongue squamous cell carcinomas and correlation with patient outcome.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background. CDKN2A inactivation has been reported to be a frequent event in head and neck squamous cell carcinomas that correlates with worse patient outcome. The differential mechanisms of CDKN2A inactivation in oral tongue squamous cell carcinomas (OTSCC) have been poorly characterised. We sought to determine the frequency of potential mechanisms of CDKN2A inactivation in OTSCC and explore their impact on patient outcome. Material and methods. We investigated a cohort of 134 OTSCC patients treated between 2002 to 2008 with combinations of surgery, radiotherapy and chemotherapy, who have been comprehensively annotated for patient/tumour characteristics and outcome. 134 formalin fixed paraffin embedded blocks representative of primary tumours prior to treatment were collected. DNA was extracted from 115 patient blocks and a tissue microarray was made representing 123 patient samples. We assessed p16/CDKN2A expression by immunohistochemistry (IHC), promoter methylation status by methylation-sensitive high resolution melting, mutation status by Sanger sequencing, gene copy number variation by fluorescence in-situ hybridisation and correlated each of these with patient outcome. Results. The majority of OTSCC demonstrated loss of p16 expression (107/118, 90.7%), assessed by IHC. The frequency of CDKN2A inactivating mutations was 20.3% (21/103), homozygous CDKN2A loss was 4.1% (7/98), hemizygous CDKN2A loss 30.6% (30/98), and CDKN2A promoter methylation >10% was 17.7% (20/113). 34/107(31.8%) p16 IHC negative tumours had no evidence of CDKN2A mutation, copy number variation or promoter methylation identified. Of eleven p16 IHC positive patients, only two demonstrated evidence of HPV type 16 and 33 on PCR. No correlation was identified between any of the mechanisms of CDKN2A inactivation and clinicopathological features. Smoking status and age did not influence the frequency of any mechanism of CDKN2A inactivation or patient outcome. There was a non-significant trend for worse outcome for p16 IHC negative patients versus IHC positive patients (HR=0.65, 95% CI=0.23-1.79, p=0.40). No significant relationship between mechanisms of p16 inactivation and patient outcome were found. Conclusion. Loss of p16 is a frequent event in oral tongue squamous cell carcinomas that can occur through gene copy number variation, mutation, promoter methylation or through other unidentified mechanisms. We identified no correlation between mechanisms of p16 loss and clinicopathological characteristics or patient outcome. Citation Format: Annette M. Lim, Hongdo Do, Richard J. Young, Stephen Q. Wong, Christopher Angel, Marnie Collins, Elena Takano, June Corry, Stephen B. Fox, David Wiesenfeld, Stephen Kleid, Elizabeth Sigston, Bernard Lyon, Danny Rischin, Alexander Dobrovic, Benjamin Solomon. Characterization of the differential mechanisms of CDKN2A inactivation in oral tongue squamous cell carcinomas and correlation with patient outcome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4693. doi:10.1158/1538-7445.AM2013-4693