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Staging Methods KM curves

Dual stain example

p16 OS KM curves

Scoring Method

Consort diagram

BackgroundMelanoma brain metastases (MBMs) are a challenging clinical problem with high morbidity and mortality. Although first-line dabrafenib–trametinib and ipilimumab–nivolumab have similar intracranial response rates (50%–55%), central nervous system (CNS) resistance to BRAF-MEK inhibitors (BRAF-MEKi) usually occurs around 6 months, and durable responses are only seen with combination immunotherapy. We sought to investigate the utility of ipilimumab–nivolumab after MBM progression on BRAF-MEKi and identify mechanisms of resistance.MethodsPatients who received first-line ipilimumab–nivolumab for MBMs or second/third line ipilimumab–nivolumab for intracranial metastases with BRAFV600 mutations with prior progression on BRAF-MEKi and MRI brain staging from March 1, 2015 to June 30, 2018 were included. Modified intracranial RECIST was used to assess response. Formalin-fixed paraffin-embedded samples of BRAFV600 mutant MBMs that were naïve to systemic treatment (n=18) or excised after progression on BRAF-MEKi (n=14) underwent whole transcriptome sequencing. Comparative analyses of MBMs naïve to systemic treatment versus BRAF-MEKi progression were performed.ResultsTwenty-five and 30 patients who received first and second/third line ipilimumab–nivolumab, were included respectively. Median sum of MBM diameters was 13 and 20.5 mm for the first and second/third line ipilimumab–nivolumab groups, respectively. Intracranial response rate was 75.0% (12/16), and median progression-free survival (PFS) was 41.6 months for first-line ipilimumab–nivolumab. Efficacy of second/third line ipilimumab-nivolumab after BRAF-MEKi progression was poor with an intracranial response rate of 4.8% (1/21) and median PFS of 1.3 months. Given the poor activity of ipilimumab–nivolumab after BRAF-MEKi MBM progression, we performed whole transcriptome sequencing to identify mechanisms of drug resistance. We identified a set of 178 differentially expressed genes (DEGs) between naïve and MBMs with progression on BRAF-MEKi treatment (p value ConclusionsSecond-line ipilimumab–nivolumab for MBMs after BRAF-MEKi progression has poor activity. MBMs that are resistant to BRAF-MEKi that also conferred resistance to second-line ipilimumab–nivolumab showed enrichment of the IPRES gene signature.

"The Philippine’s Commission on Higher Education (CHED) is mandated to take appropriate steps to ensure that education shall be accessible to all. In times where classes were disrupted due to health emergency and community lockdown imposed, the commission handed down Memorandum Order No. 4. Series of 2020, which directed Higher Education Institutions (HEI) to use flexible platform for academic track to better facilitate learning scheme. The sudden paradigm shin lends students to grapple in the new way of teaching and learning process. The underlying circumstance leads to determining the Academic Learning Process between face-to-face and online class of Medical Technology students during the time of contagion. This descriptive-comparative study verified the learning process between online class and face-to face strategic learning. There were fourty six (46) 3rd Year and 4th Year Medical Technology students of World Citi Colleges who assessed the two learning processes based on class preparation, absorbing and capturing new information, and reviewing of course materials. Results revealed that majority of the respondents are in the age bracket of 22-25 and mostly female students. In terms of year level, there is an equal distribution of 3rd year and 4th year Medical Technology students. It is also observed that majority of the respondents experienced a half semester of online class and are highly prepared for face-to-face class but are moderately prepared for online class. The level of preparedness for face-to-face class is greater than the level of preparedness for online class. In terms of academic learning process, the respondents have high ability to absorb new information in face-to-face class but with moderate ability compared to online class. The mean score of the level of capturing new information in face-to-face class is greater than the mean score in online class. However, the respondents have high ability to capture new information in both modes of learning. Relatively, scores on capturing new information in online class are more dispersed than scores for face-to-face class. Additionally, as to reviewing course materials, respondents have moderate ability in reviewing course materials in online class. The mean score for the level of reviewing in face-to-face class is greater than the mean score in online class. Based on the results of independent samples t-test, the researchers concluded that there is no significant difference in the academic learning process between the face-to-face and online class of 3rd and 4th-year Medical Technology students during pandemic."

Molecular alterations in 176 patients with oral squamous cell carcinomas (OSCC) were evaluated to delineate differences in non-smoking non-drinking (NSND) patients. Somatic mutations and DNA copy number variations (CNVs) in a 68-gene panel and human papilloma virus (HPV) status were interrogated using targeted next-generation sequencing. In the entire cohort, TP53 (60%) and CDKN2A (24%) were most frequently mutated, and the most common CNVs were EGFR amplifications (9%) and deletions of BRCA2 (5%) and CDKN2A (4%). Significant associations were found for TP53 mutation and nodal disease, lymphovascular invasion and extracapsular spread, CDKN2A mutation or deletion with advanced tumour stage, and EGFR amplification with perineural invasion and extracapsular spread. PIK3CA mutation, CDKN2A deletion, and EGFR amplification were associated with worse survival in univariate analyses (p &lt, 0.05 for all comparisons). There were 59 NSND patients who tended to be female and older than patients who smoke and/or drink, and showed enrichment of CDKN2A mutations, EGFR amplifications, and BRCA2 deletions (p &lt, 0.05 for all comparisons), with a younger subset showing higher mutation burden. HPV was detected in three OSCC patients and not associated with smoking and drinking habits. NSND OSCC exhibits distinct genomic profiles and further exploration to elucidate the molecular aetiology in these patients is warranted.

Lacrimal gland pleomorphic adenomas (LGPA) are benign mixed tumors. Diagnosis is based on clinical and radiological findings which usually prompts complete excision of the lesion to minimise recurrence and a cumulative risk of malignant transformation. Necrosis in pleomorphic adenoma has been rarely reported in salivary gland PA, either spontaneously or due to iatrogenic interventions. Necrosis is suggestive of a malignant process and makes interpretation of histology specimens difficult. A 23 year old woman, while awaiting biopsy for a mass in the left lacrimal gland, which had been symptomatic for only several months, presented with acute pain and swelling of the left lateral lid. An incisional biopsy showed an inflamed lacrimal gland with focal necrosis and atypia of adjacent cytology and gland architecture. Subsequent excisional biopsy confirmed an LGPA with some inflammation but no necrosis. Necrosis may occur as an atypical presentation in LGPA.

Succinate dehydrogenase (SDH)-deficient gastrointestinal stromal tumor (GIST) is a unique and distinctive subtype of gastric GIST. The literature on this subtype from developing countries is exceedingly sparse. Patients with SDH-deficient GIST often experience a lack or delay in genomic profiling, despite stereotypical clinicopathologic features, potentially resulting in sub-optimal management. SDH-deficient GISTs are highly syndromic, typically have more indolent behavior, a prognosis not predicted by size and mitotic rate, a tendency to lymph node metastases, and are insensitive to standard tyrosine kinase inhibitors (TKIs). We report two women with SDH-deficient GIST. In the first case, SDH deficiency was identified late due to lack of awareness and poor access to diagnostic facilities. The patient progressed through TKI therapy, but responded to temozolomide, which is under investigation in clinical trials. In the second case, SDH deficiency was identified at diagnosis, and the patient responded well to 177Lutetium peptide radionuclide receptor therapy (PRRT) after progressing through two lines of TKIs. We aim to highlight the need for more awareness and access to genomic diagnostic facilities for GIST patients, temozolomide as a novel therapy for SDH-deficient GIST, and the potential value of DOTATATE positron emission tomography (PET) and PRRT as a novel imaging modality and therapy for TKI insensitive GIST patients.

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.

Background The aim of the present study was to identify the presence and frequency of human papillomavirus (HPV) nucleic acid in p16 positive oral squamous cell carcinomas (OSCC), to assess if the virus were transcriptionally active, and to assess the utility of p16 overexpression as a surrogate marker for HPV in OSCC. Methods Forty-six OSCC patients treated between 2007 and 2011 with available formalin-fixed paraffin embedded (FFPE) specimens were included. Twenty-three patients were positive for p16 by immunohistochemistry (IHC) and these were matched with 23 patients with p16 negative tumours. Laser capture micro-dissection of the FFPE OSCC tissues was undertaken to isolate invasive tumour tissue. DNA was extracted and tested for high-risk HPV types using a PCR-ELISA method based on the L1 SPF10 consensus primers, and a real-time PCR method targeting HPV-16 and HPV-18 E6 region. Genotyping of HPV positive cases was performed using a reverse line blot hybridization assay (Inno-LiPA). RNAScope® (a chromogenic RNA in situ hybridization assay), was utilized to detect E6/E7 mRNA of known high-risk HPV types for detection of transcriptionally active virus. Results HPV DNA was found in 3 OSCC cases, all of which were p16 IHC positive. Two cases were genotyped as HPV-16 and one as HPV-33. Only one of the HPV-16 cases was confirmed to harbour transcriptionally active virus via HPV RNA ISH. Conclusion We have shown that the presence of transcriptionally active HPV rarely occurs in OSCC and that p16 is not an appropriate surrogate marker for HPV in OSCC cases. We propose that non-viral mechanisms are responsible for the majority of IHC p16 overexpression in OSCC. This article is protected by copyright. All rights reserved.

Objective The immune system can halt cancer progression by suppressing outgrowth of clinically occult micrometastases in a state of cancer‐immune equilibrium. Cutaneous melanoma provides a unique opportunity to study the immune contexture of such lesions, as miniscule skin metastases are accessible to clinical inspection and diagnostic biopsy. Methods Here, we analysed by multiplex immunofluorescence microscopy samples from a melanoma patient presenting with an overt and an occult in‐transit metastasis (ITM), the latter of which appeared as a small erythematous papule. Results Microarchitecture and immune composition in the two lesions were vastly different. CD4+ and CD8+ T cells accumulated around the margin of the overt SOX10+ Melan A+ ITM but were largely excluded from the tumor centre. By contrast, the occult micrometastasis contained only few SOX10+ Melan A− melanoma cells which were scattered within a dense infiltrate of T cells, including a prominent population of CD103+ CD8+ T cells resembling tissue‐resident memory T (TRM) cells. Notably, almost every single melanoma cell in the micrometastasis was in close proximity to these TRM‐like cells. Conclusion Such results support the emerging concept that CD103+ CD8+ TRM cells are key mediators of cancer surveillance and imply an important function of these cells in controlling clinically occult micrometastases in humans., Analysing an occult in‐transit metastasis in a melanoma patient, we identify CD103+CD8+ T cells with a resident memory phenotype as the dominant T‐cell subset. Such results support the emerging concept that CD103+CD8+ tissue‐resident memory T cells are key mediators of cancer surveillance and imply an important function of these cells in controlling clinically occult micrometastases in humans.

Background Pleomorphic dermal sarcoma (PDS) is a rare, poorly defined skin neoplasm with features similar to atypical fibroxanthoma, but with adverse histopathological characteristics indicating metastatic potential such as tumour necrosis, invasion beyond superficial subcutis or vascular and/or perineural infiltration. Optimal treatment for PDS is uncertain and reported outcomes vary due to the rarity of this diagnosis and uncertainty over histopathological categorization. The aim of this study was to review the clinical and histopathological features of PDS in a single Australian centre. Methods A retrospective review of all patients managed at the Peter MacCallum Cancer Centre with PDS between 2003 and 2017 was performed by a search of electronic records and histories reviewed. Results A total of 27 patients were identified, mostly elderly males (85.2%, mean age 79.8 years). Lesions were seen most commonly on the head and neck region (96.3%), predominantly on the scalp (63%). Mean tumour radial surgical excision margin was 12.8 mm. Eighteen patients (66.7%) underwent radiotherapy; 13 adjuvant, three neoadjuvant and two with palliative intent. After median follow-up of 46.4 months, two patients had recurrence (7.4%); both had inadequate deep margins at first excision. There were three all-cause deaths in the cohort. There was one disease-specific mortality with metastatic PDS disease at the time of initial presentation. Conclusion PDS is a rare cutaneous malignancy most commonly found in the head and neck region in elderly men, which is best managed with adequate surgical excision. The role of radiotherapy is undefined and an area for future investigation.

The aims of this study are to elucidate if molecular markers can be used to differentiate between the two main types of ameloblastoma (unicystic and solid/multicystic), and to determine whether a biologically 'less-aggressive' subtype exists.A retrospective analysis of 33 solid/multicystic ameloblastomas and six unicystic ameloblastomas was completed using immunohistochemistry for five molecular markers: P16, P53, MMP-9, Survivin, and Ki-67. Tumors were graded as either negative or positive (mild, moderate, strong), and the results were related to both ameloblastoma subtypes and outcomes following treatment.Unicystic ameloblastomas were more likely to test strongly positive for P53 than solid/multicystic ameloblastomas (Immunohistochemistry can be valuable in lesions where histological sub-typing of an ameloblastoma is unclear. A biologically 'less-aggressive' subtype may exist, and hence further research into this area is required.

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.

Objective Accurate determination of human papilloma virus (HPV) status is critical when identifying patients with oropharyngeal squamous cell carcinoma (OPSCC) who may be candidates for de-escalation trials. In this study we investigated whether local p16 screening, by immunohistochemistry (IHC), has high positive predictive value (PPV) for HPV status in a good prognosis HPV positive OPSCC (HPVOPSCC) population treated on a clinical trial. Methods and materials Patients enrolled on the TROG 12.01 randomised trial for good prognosis HPVOPSCC were randomised based on local p16 IHC testing but subsequently had central p16 IHC and HPV RNA in situ hybridisation (HPV RNA ISH) testing. Correlations between the local and central p16 and central HPV RNA ISH were studied. The main outcome was the positive predictive value (PPV) of local pathology laboratory testing of p16. Results 176/182 patients had samples available for central testing. 172/176 were evaluable for central testing of p16, and all were confirmed to be p16 positive (172/172, 100%, 95% CI = [97.9%, 100%]). Similarly, 100% of those evaluable for HPV RNA ISH (155/155, 100%, 95% CI = [97.6%, 100%]) were confirmed HPV positive, indicating p16 overexpression driven by transcriptionally active HPV and a PPV of 100% for local p16 testing. Conclusions Our results validate the suitability of local pathology laboratory p16 testing alone, in populations with a high attributable fraction of OPSCC due to HPV, to screen and enrol low risk HPVOPSCC patients onto de-intensification trials. This obviates the need for upfront more complex and expensive HPV assays and/or central laboratory testing.

Background: Human papillomavirus (HPV) infection is a powerful prognostic biomarker in a subset of head and neck squamous cell carcinomas, specifically oropharyngeal cancers. However, the role of HPV in non-oropharyngeal sites, such as the larynx, remains unconfirmed. Methods: We evaluated a cohort of 324 laryngeal squamous cell carcinoma (LSCC) patients for the expression of p16INK4A (p16) protein by immunohistochemistry (IHC) and for high-risk HPV E6 and E7 mRNA transcripts by RNA in situ hybridisation (ISH). p16 expression and HPV status were correlated with clinicopathological features and outcomes. Results: Of 307 patients assessable for p16 IHC, 20 (6.5%) were p16 positive. Females and node-positive patients were more likely to be p16 positive (P

Human papilloma virus-positive oropharyngeal squamous cell carcinoma (HPV

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.

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.

The efficacy of targeted monotherapy for BRAF(V600E)-positive anaplastic thyroid carcinomas (ATC) is not established. We report 2 cases of BRAF(V600E)-positive ATC treated with a BRAF inhibitor. A 49-year-old woman with a T4bN1bM0 ATC manifested symptomatic metastatic disease 8 weeks after radical chemoradiotherapy. Within 1 month of BRAF inhibitor monotherapy, a complete symptomatic response was observed, with FDG-PET scan confirming metabolic and radiologic response. Treatment was terminated after 3 months because of disease progression. The patient died 11 months after primary diagnosis. A 67-year-old man received first-line BRAF inhibitor for a T4aN1bM0 ATC. Within 10 days of treatment his pain had stabilized and his tumor had clinically halved in size. Stable disease was achieved for 11 weeks but the patient died 11 months after diagnosis because of disease progression. BRAF inhibitor monotherapy in ATC may obtain clinical benefit of short duration. Upfront combination therapy should be investigated in this patient subgroup.

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.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.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).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.

Salivary duct carcinoma (SDC) is a rare, aggressive salivary gland malignancy with limited evidence guiding standard treatment. SDC is known to overexpress the androgen receptor, with only a handful of cases reporting responses to androgen blockade. This report presents a case of SDC responding to multiple lines of androgen blockade, including a rapid response to abiraterone, a CYP17 inhibitor effective in prostate cancer. This case represents the first published report of SDC responding to abiraterone and illustrates that androgen receptor expressing SDC may be treated with multiple lines of androgen blockade, including newer agents such as abiraterone. This case suggests that SDC may continue to be androgen-dependent after progression on androgen deprivation, which is analogous to prostate cancer.

Introduction: Oral cavity squamous cell carcinomas (OSCCs) are the most prevalent head and neck malignancy and are a significant cause of mortality and morbidity, especially in the developing world. A better understanding of the molecular pathogenesis of OSCCs is essential for improved management of this disease. Mutations in the NOTCH1 gene have been identified in 9-18% patients with head and neck malignancies. A large proportion of these mutations lie in its extracellular domain and are thought to be inactivating, in contrast to the activating NOTCH1 mutations found in hematological malignancies such as T-ALL. This dichotomy raises interesting questions about the role of the Notch pathway in malignancies. In this study, we aim to characterize NOTCH1 mutations in a cohort of OSCC patients and use an in vitro model to investigate the role of NOTCH1 inactivation in OSCC tumorigenesis. Methods: Targeted next-generation sequencing of a custom 69 gene panel was carried out on fresh frozen and formalin fixed paraffin embedded (FFPE) tissue from prospectively and retrospectively recruited OSCC patients presenting to a tertiary cancer center (n = 94). Detailed clinical annotation was collected for all patients. The BWA/GATK pipeline was used for sequence alignment and mutation calling. Downstream sequencing pipelines were designed in the R statistical environment to optimize identification of somatic mutations in the FFPE samples. Notch pathway mutations were interrogated, and comparisons made against NOTCH1 mutations across all cancer subtypes in The Cancer Genome Atlas (TCGA) database. Five OSCC cell lines (H314, H357, H400, BICR16, BICR56) were sequenced and Notch1 expression characterized by Western blotting. A lentiviral CRISPR/Cas9 system using custom designed guides was used to knock out NOTCH1 in the cell lines expressing wild-type Notch1. Wild-typeNOTCH1-mCherry was expressed in the cell lines with mutated NOTCH1. Non-adherent clonal growth assays and RNAseq were used to assess the impact of these modifications. Results: In our patient cohort, a high frequency of mutations were identified in known tumor suppressor genes TP53 (57%) and CDKN2A (22%). Sixteen patients (17%) were NOTCH1 mutant. The majority of these mutations fell within the extracellular domain and 10 were predicted to be nonsense mutations. Ten patients (11%) had a NOTCH3 mutation. Two of the five OSCC cell lines were NOTCH1 mutant and expressed no detectable Notch1 protein. A wild-type Notch1-mCherry construct was introduced into the mutant cell lines and Notch1 was knocked out in the three cell lines in which functional Notch1 protein was detected. The results of the growth assays and RNA sequencing for all of the pre- and post- modification cell lines will be presented. Conclusions: We have built a bioinformatics pipeline to successfully analyze FFPE tissue and have identified potentially inactivating NOTCH1 mutations in our patients at similar frequencies to international cohorts. We have identified NOTCH3 mutations, which are of uncertain significance. Successful knockout of NOTCH1 in OSCC cell lines was achieved. Citation Format: Kendrick Koo, Christopher Angel, Nadia Kershaw, Dmitri Mouradov, Anderly Chueh, David Wiesenfeld, Tim A. Iseli, Michael McCullough, Oliver Sieber, Antony W. Burgess. Characterization of NOTCH1 mutations in oral cavity squamous cell carcinomas. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A01.

Background DNA 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). Results The 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

Summary Angioleiomyoma in the mouth is described as a rare symptomless, slowly growing mass, most commonly presenting on the lip. Here we present a case of angioleiomyoma on the hard palate with atypical symptoms of pain and fluctuation in size.

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.

Summary 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.

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.

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

The BRAF(T1799A) mutation encodes BRAF(V600E) that leads to activation of the mitogen-activated protein kinase pathway. This study aimed to assess the clinico-pathological features of primary invasive melanomas containing the BRAF(T1799A) mutation. Patients (n=251) with invasive primary melanomas from Australia were interviewed and examined with respect to their melanoma characteristics and risk factors. Independent review of pathology, allele-specific PCR for the BRAF(T1799A) mutation, immunohistochemical staining with Ki67, and phospho-histone-H3 (PH3) were performed. The BRAF(T1799A) mutation was found in 112 (45%) of the primary melanomas. Associations with the BRAF(T1799A) mutation (P0.05) were as follows: low tumor thickness (odds ratio (OR)=3.3); low mitotic rate (OR=2.0); low Ki67 score (OR=5.0); low PH3 score (OR=3.3); superficial spreading melanoma (OR=10.0); pigmented melanoma (OR=3.7); a lack of history of solar keratoses (OR=2.7); a location on the trunk (OR=3.4) or extremity (OR=2.0); a high level of self-reported childhood sun exposure (OR=2.0);or =50 years of age (OR=2.5); and fewer freckles (OR=2.5). We conclude that the BRAF(T1799A) mutation has associations with host phenotype, tumor location, and pigmentation. Although implicated in the control of the cell cycle, the BRAF(T1799A) mutation is associated with a lower rate of tumor proliferation.