The histopathological spectrum and molecular changes associated with KRAS G12C mutation in non-small cell lung carcinoma.

KRAS G12C is the most common KRAS mutation in non-small cell lung carcinoma (NSCLC), for which targeted therapy has recently been developed. From the 732 cases of NSCLC that underwent next-generation sequencing at the Department of Anatomical Pathology, Liverpool Hospital, between July 2021 and May 2023, we retrieved 83 (11%) consecutive cases of KRAS G12C mutated NSCLC, and analysed their clinical, pathological, and molecular features. Of the 83 cases of KRAS G12C mutated NSCLC, there were 46 (55%) men and 37 (45%) women, with mean age of 72 years. Of the 49 cases with known clinical information, 94% were current or ex-smokers, and 49% were stage IV at diagnosis with median survival of 12 months. Sixty-three percent were histology cases and the remainder were cytology cases. Eighty-two percent were non-mucinous adenocarcinomas, with conventional histology including lepidic, acinar, solid, single cells and micropapillary patterns, and 62% were poorly differentiated. There were five (6%) cases of mucinous adenocarcinoma, one case of pleomorphic carcinoma and one case of high-grade fetal adenocarcinoma. TTF1 was positive in the majority (89%) of cases. Nineteen (23%) cases had TP53 co-mutation, and these cases had trends towards higher PD-L1 expression, poor differentiation, and presentation as stage IV disease, but the differences were not statistically significant. KRAS G12C mutated NSCLCs almost exclusively occurred in smokers and were mostly non-mucinous adenocarcinomas with conventional histological patterns which ranged from well to poorly differentiated. Around a quarter had TP53 co-mutation, the histological impacts and immune profile of which need to be assessed in a larger study.

Utility of multigene panel next-generation sequencing in routine clinical practice for identifying genomic alterations in newly diagnosed metastatic nonsmall cell lung cancer.

BACKGROUND: The standard of care in newly diagnosed metastatic non-small cell lung cancer (NSCLC) is to test for aberrations in three genes for driver mutations - ALK, ROS1 and epidermal growth factor receptor (EGFR) - and also for immunohistochemistry to be performed for programmed death-ligand 1 expression level. Next-generation sequencing (NGS), with or without RNA fusion testing, is increasingly used in standard clinical practice to identify patients with potentially actionable mutations. Stratification of NGS mutation tiers is currently based on the European Society of Medical Oncology Scale for Clinical Actionability of Molecular Targets (ESCAT) Tiers I-V and X. AIM: Our aim was to analyse NSCLC tumour samples for the prevalence of Tiers I-V mutations to establish guidance for current and novel treatments in patients with metastatic disease. METHODS: NGS was performed employing the Oncomine Precision Assay (without RNA fusion testing) that interrogates DNA hotspot variants across 45 genes to screen 210 NSCLC tissue samples obtained across six Sydney hospitals between June 2021 and March 2022. RESULTS: In our cohort, 161 of 210 (77%) had at least one gene mutation identified, with 41 of 210 (20%) having two or more concurrent mutations. Tier I mutations included 42 of 210 (20%) EGFR mutations (EIA) and five of 210 (3%) MET exon 14 skipping mutations (EIB). Non-Tier I variants included 22 of 210 (11%) KRAS G12C hotspot mutations (EIIB), with a further 47 of 210 (22%) having non-G12C KRAS (EX) mutations. NGS testing revealed an additional 15% of cases with Tier II ESCAT mutations in NSCLC. Forty-six percent of patients also demonstrated potential Tier III and IV mutations that are currently under investigation in early-phase clinical trials. CONCLUSIONS: In addition to identifying patients with genomic alterations suitable for clinically proven standard-of-care therapeutic options, the 45-gene NGS panel has significant potential in identifying potentially actionable non-Tier 1 mutations that may become future standard clinical practice in NSCLC.

Multigene panel next generation sequencing in metastatic colorectal cancer in an Australian population.

BACKGROUND: Next generation sequencing (NGS) is increasingly used in standard clinical practice to identify patients with potentially actionable mutations. Stratification of NGS mutation tiers is currently based on the European Society of Medical Oncology (ESMO) Scale for Clinical Actionability of Molecular Targets (ESCAT[E]) Tier I-V & X. Allele frequency is also increasingly recognised as an important prognostic tool in advanced cancer. The aim of this study was to determine the genomic mutations in metastatic colorectal cancer (CRC) in an Australian multicultural population and their influence on survival outcomes. METHODS: Next generation sequencing with the 50-gene panel Oncomine Precision Assay™ was used on 180 CRC tissue samples obtained across six Sydney hospitals between June 2021 and March 2022. RESULTS: From 180 samples, 147 (82%) had at least one gene mutation identified with 68 (38%) having two or more concurrent mutations. Tier I variants included RAS wild-type [EI] in 73 (41%) and BRAF V600E [EIA] in 27 (15%). Non-tier I variants include 2 (1%) ERBB2 amplification [EIIB], 26 (15%) PIK3CA hotspot mutations [EIIIA] and 9 (5%) MET focal amplifications [EIIIA]. NGS testing revealed an additional 22% of cases with Tier II & III mutations. 43% of patients also presented with potentially actionable Tier III & IV mutations. Patients with concurrent TP53 and RAS mutations had significantly reduced overall survival (6.1 months versus 21.1 months, p <0.01). High KRAS allele frequency, as defined by those with over 20% variant allele frequency (VAF), also demonstrated reduced overall survival (12.1 months versus 42.9 months, p = 0.04). CONCLUSIONS: In addition to identifying patients with genomic alterations suitable for clinically proven standard of care therapeutic options, the 50 gene NGS panel has significant potential in identifying potentially actionable non-tier 1 mutations and therefore may become future standard clinical practice.

MYB RNA detection by in situ hybridisation has high sensitivity and specificity for the diagnosis of adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is one of the most common primary salivary gland cancers. ACC has several benign and malignant mimics amongst salivary gland neoplasms. An accurate diagnosis of ACC is essential for optimal management of the patients and their follow-up. Upregulation of MYB has been described in 85-90% of ACC, but not in other salivary gland neoplasms. In ACC, MYB upregulation can occur as a result of a genetic rearrangement t(6;9) (q22-23;p23-24), MYB copy number variation (CNV), or enhancer hijacking of MYB. All mechanisms of MYB upregulation result in increased RNA transcription that can be detected using RNA in situ hybridisation (ISH) methods. In this study, utilising 138 primary salivary gland neoplasms including 78 ACC, we evaluate the diagnostic utility of MYB RNA ISH for distinguishing ACC from other primary salivary gland neoplasms with a prominent cribriform architecture including pleomorphic adenoma, basal cell adenoma, basal cell adenocarcinoma, epithelial myoepithelial carcinoma, and polymorphous adenocarcinoma. Fluorescent in situ hybridisation and next generation sequencing were also performed to evaluate the sensitivity and specificity of RNA ISH for detecting increased MYB RNA when MYB gene alterations were present. Detection of MYB RNA has 92.3% sensitivity and 98.2% specificity for a diagnosis of ACC amongst salivary gland neoplasms. The sensitivity of MYB RNA detection by ISH (92.3%) is significantly higher than that of the FISH MYB break-apart probe (42%) for ACC. Next generation sequencing did not demonstrate MYB alterations in cases that lacked MYB RNA overexpression indicating high sensitivity of MYB RNA ISH for detecting MYB gene alterations. The possibility that the sensitivity may be higher in clinical practice with contemporary samples as compared with older retrospective tissue samples with RNA degradation is not entirely excluded. In addition to the high sensitivity and specificity, MYB RNA testing can be performed using standard IHC platforms and protocols and evaluated using brightfield microscopy making it a time and cost-efficient diagnostic tool in routine clinical practice.

Whole genome duplication in oral squamous cell carcinoma in patients younger than 50 years: implications for prognosis and adverse clinicopathological factors.

INTRODUCTION: Oral squamous cell carcinoma (OSCC) in the young (<50 years), without known carcinogenic risk factors, is on the rise globally. Whole genome duplication (WGD) has been shown to occur at higher rates in cancers without an identifiable carcinogenic agent. We aimed to evaluate the prevalence of WGD in a cohort of OSCC patients under the age of 50 years. METHODS: Whole genome sequencing (WGS) was performed on 28 OSCC patients from the Sydney Head and Neck Cancer Institute (SHNCI) biobank. An additional nine cases were obtained from The Cancer Genome Atlas (TCGA). RESULTS: WGD was seen in 27 of 37 (73%) cases. Non-synonymous, somatic TP53 mutations occurred in 25 of 27 (93%) cases of WGD and were predicted to precede WGD in 21 (77%). WGD was significantly associated with larger tumor size (p = 0.01) and was frequent in patients with recurrences (87%, p = 0.36). Overall survival was significantly worse in those with WGD (p = 0.05). CONCLUSIONS: Our data, based on one of the largest WGS datasets of young patients with OSCC, demonstrates a high frequency of WGD and its association with adverse pathologic characteristics and clinical outcomes. TP53 mutations also preceded WGD, as has been described in other tumors without a clear mutagenic driver.

BRAF mutation testing for patients diagnosed with stage III or stage IV melanoma: practical guidance for the Australian setting.

Targeted therapy (BRAF inhibitor plus MEK inhibitor) is now among the possible treatment options for patients with BRAF mutation-positive stage III or stage IV melanoma. This makes prompt BRAF mutation testing an important step in the management of patients diagnosed with stage III or IV melanoma; one that can help better ensure that the optimal choice of systemic treatment is initiated with minimal delay. This article offers guidance about when and how BRAF mutation testing should be conducted when patients are diagnosed with melanoma in Australia. Notably, it recommends that pathologists reflexively order BRAF mutation testing whenever a patient is found to have American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC) stage III or IV melanoma (i.e., any metastatic spread beyond the primary tumour) and that patient's BRAF mutation status is hitherto unknown, even if BRAF mutation testing has not been specifically requested by the treating clinician (in Australia, Medicare-subsidised BRAFV600 mutation testing does not need to be requested by the treating clinician). When performed in centres with appropriate expertise and experience, immunohistochemistry (IHC) using the anti-BRAF V600E monoclonal antibody (VE1) can be a highly sensitive and specific means of detecting BRAFV600E mutations, and may be used as a rapid and relatively inexpensive initial screening test. However, VE1 immunostaining can be technically challenging and difficult to interpret, particularly in heavily pigmented tumours; melanomas with weak, moderate or focal BRAFV600E immunostaining should be regarded as equivocal. It must also be remembered that other activating BRAFV600 mutations (including BRAFV600K), which account for ∼10-20% of BRAFV600 mutations, are not detected with currently available IHC antibodies. For these reasons, if available and practicable, we recommend that DNA-based BRAF mutation testing always be performed, regardless of whether IHC-based testing is also conducted. Advice about tissue/specimen selection for BRAF mutation testing of patients diagnosed with stage III or IV melanoma is also offered in this article; and potential pitfalls when interpreting BRAF mutation tests are highlighted.

Molecular patterns in salivary duct carcinoma identify prognostic subgroups.

Salivary duct carcinoma (SDCa) is a rare cancer with high rate of metastases and poor survival despite aggressive multimodality treatment. This study analyzes the genetic changes in SDCa, their impact on cancer pathways, and evaluates whether molecular patterns can identify subgroups with distinct clinical characteristics and outcome. Clinicopathologic details and tissue samples from 66 patients (48 males, 18 females) treated between 1995 and 2018 were obtained from multiple institutions. Androgen receptor (AR) was assessed by immunohistochemistry, and the Illumina TruSight 170 gene panel was used for DNA sequencing. Male gender, lympho-vascular invasion, lymph node metastasis, and smoking were significant predictors of disease-free survival. AR was present in 79%. Frequently encountered alterations were mutations in TP53 (51%), PIK3CA (32%) and HRAS (22%), as well as amplifications of CDK4/6 (22%), ERBB2 (21%), MYC (16%), and deletions of CDKN2A (13%). TP53 mutation and MYC amplifications were associated with decreased disease-free survival. Analysis of cancer pathways revealed that the PI3K pathway was most commonly affected. Alterations in the cell cycle pathway were associated with impaired disease-free survival (HR 2.6, P = 0.038). Three subgroups based on AR and ERBB2 status were identified, which featured distinct molecular patterns and outcome. Among AR positive SDCa, HRAS mutations were restricted to AR positive tumors without ERBB2 amplification and HRAS mutations featured high co-occurrence with PIK3CA alterations, which seems specific to SDCa. AR negative SDCa were associated with poor disease-free survival in multivariate analysis (HR 4.5, P = 0.010) and none of these tumors exhibited ERBB2 amplification or HRAS mutations. AR and ERBB2 status in SDCa thus classifies tumors with distinct molecular profiles relevant to future targeted therapy. Furthermore, clinical factors such as smoking and molecular features such as MYC amplification may serve as markers of poor prognosis of SDCa.

Breast ductal carcinoma in situ carry mutational driver events representative of invasive breast cancer.

The spectrum of genomic alterations in ductal carcinoma in situ (DCIS) is relatively unexplored, but is likely to provide useful insights into its biology, its progression to invasive carcinoma and the risk of recurrence. DCIS (n=20) with a range of phenotypes was assessed by massively parallel sequencing for mutations and copy number alterations and variants validated by Sanger sequencing. PIK3CA mutations were identified in 11/20 (55%), TP53 mutations in 6/20 (30%), and GATA3 mutations in 9/20 (45%). Screening an additional 91 cases for GATA3 mutations identified a final frequency of 27% (30/111), with a high proportion of missense variants (8/30). TP53 mutations were exclusive to high grade DCIS and more frequent in PR-negative tumors compared with PR-positive tumors (P=0.037). TP53 mutant tumors also had a significantly higher fraction of the genome altered by copy number than wild-type tumors (P=0.005), including a significant positive association with amplification or gain of ERBB2 (P<0.05). The association between TP53 mutation and ERBB2 amplification was confirmed in a wider DCIS cohort using p53 immunohistochemistry as a surrogate marker for TP53 mutations (P=0.03). RUNX1 mutations and MAP2K4 copy number loss were novel findings in DCIS. Frequent copy number alterations included gains on 1q, 8q, 17q, and 20q and losses on 8p, 11q, 16q, and 17p. Patterns of genomic alterations observed in DCIS were similar to those previously reported for invasive breast cancers, with all DCIS having at least one bona fide breast cancer driver event. However, an increase in GATA3 mutations and fewer copy number changes were noted in DCIS compared with invasive carcinomas. The role of such alterations as prognostic and predictive biomarkers in DCIS is an avenue for further investigation.

A rat model of ataxia-telangiectasia: evidence for a neurodegenerative phenotype.

Ataxia-telangiectasia (A-T), an autosomal recessive disease caused by mutations in the ATM gene is characterised by cerebellar atrophy and progressive neurodegeneration which has been poorly recapitulated in Atm mutant mice. Consequently, pathways leading to neurodegeneration in A-T are poorly understood. We describe here the generation of an Atm knockout rat model that does not display cerebellar atrophy but instead paralysis and spinal cord atrophy, reminiscent of that seen in older patients and milder forms of the disorder. Loss of Atm in neurons and glia leads to accumulation of cytosolic DNA, increased cytokine production and constitutive activation of microglia consistent with a neuroinflammatory phenotype. Rats lacking ATM had significant loss of motor neurons and microgliosis in the spinal cord, consistent with onset of paralysis. Since short term treatment with steroids has been shown to improve the neurological signs in A-T patients we determined if that was also the case for Atm-deficient rats. Betamethasone treatment extended the lifespan of Atm knockout rats, prevented microglial activation and significantly decreased neuroinflammatory changes and motor neuron loss. These results point to unrepaired damage to DNA leading to significant levels of cytosolic DNA in Atm-deficient neurons and microglia and as a consequence activation of the cGAS-STING pathway and cytokine production. This in turn would increase the inflammatory microenvironment leading to dysfunction and death of neurons. Thus the rat model represents a suitable one for studying neurodegeneration in A-T and adds support for the use of anti-inflammatory drugs for the treatment of neurodegeneration in A-T patients.

Rats with a missense mutation in Atm display neuroinflammation and neurodegeneration subsequent to accumulation of cytosolic DNA following unrepaired DNA damage.

Mutations in the ataxia-telangiectasia (A-T)-mutated (ATM) gene give rise to the human genetic disorder A-T, characterized by immunodeficiency, cancer predisposition, and neurodegeneration. Whereas a series of animal models recapitulate much of the A-T phenotype, they fail to present with ataxia or neurodegeneration. We describe here the generation of an Atm missense mutant [amino acid change of leucine (L) to proline (P) at position 2262 (L2262P)] rat by intracytoplasmic injection (ICSI) of mutant sperm into oocytes. Atm-mutant rats (AtmL2262P/L2262P ) expressed low levels of ATM protein, suggesting a destabilizing effect of the mutation, and had a significantly reduced lifespan compared with Atm+/+ Whereas these rats did not show cerebellar atrophy, they succumbed to hind-limb paralysis (45%), and the remainder developed tumors. Closer examination revealed the presence of both dsDNA and ssDNA in the cytoplasm of cells in the hippocampus, cerebellum, and spinal cord of AtmL2262P/L2262P rats. Significantly increased levels of IFN-ß and IL-1ß in all 3 tissues were indicative of DNA damage induction of the type 1 IFN response. This was further supported by NF-κB activation, as evidenced by p65 phosphorylation (P65) and translocation to the nucleus in the spinal cord and parahippocampus. Other evidence of neuroinflammation in the brain and spinal cord was the loss of motor neurons and the presence of increased activation of microglia. These data provide support for a proinflammatory phenotype that is manifested in the Atm mutant rat as hind-limb paralysis. This mutant represents a useful model to investigate the importance of neuroinflammation in A-T.

MRE11 and ATM Expression Levels Predict Rectal Cancer Survival and Their Association with Radiotherapy Response.

BACKGROUND: Aberrant expression of DNA repair proteins is associated with poor survival in cancer patients. We investigated the combined expression of MRE11 and ATM as a predictive marker of response to radiotherapy in rectal cancer. METHODS: MRE11 and ATM expression were examined in tumor samples from 262 rectal cancer patients who underwent surgery for rectal cancer, including a sub-cohort of 54 patients who were treated with neoadjuvant radiotherapy. The relationship between expression of the two-protein panel and tumor regression grade (TRG) was assessed by Mann-Whitney U test and receiver operating characteristics area under curve (ROC-AUC) analysis. The association between expression of the two-protein panel and clinicopathologic variables and survival was examined by Kaplan-Meier methods and Cox regression analysis. RESULTS: A high score for two-protein combined expression in the tumor center (TC) was significantly associated with worse disease-free survival (DFS) (P = 0.035) and overall survival (OS) (P = 0.003) in the whole cohort, and with DFS (P = 0.028) and OS (P = 0.024) in the neoadjuvant subgroup (n = 54). In multivariate analysis, the two-protein combination panel (HR = 2.178, 95% CI 1.115-4.256, P = 0.023) and perineural invasion (HR = 2.183, 95% CI 1.222-3.899, P = 0.008) were significantly associated with DFS. Using ROC-AUC analysis of good versus poor histological tumor response among patients treated preoperatively with radiotherapy, the average ROC-AUC was 0.745 for the combined panel, 0.618 for ATM alone, and 0.711 for MRE11 alone. CONCLUSIONS: The MRE11/ATM two-protein panel developed in this study may have clinical value as a predictive marker of tumor response to neoadjuvant radiotherapy, and a prognostic marker for disease-free and overall survival.

PD-L1 expression in tonsillar cancer is associated with human papillomavirus positivity and improved survival: implications for anti-PD1 clinical trials.

In this study, we examined PD-L1 expression by immunohistochemistry in 99 patients with tonsillar cancer and known human papillomavirus (HPV) status to assess its clinical significance. We showed that the pattern of PD-L1 expression is strongly related to HPV status. The PD-L1 positivity rate was 83.3% in HPV-positive cases and 56.9% in HPV-negative cases (p < 0.05). Patients with HPV-positive/PD-L1-positive cancer had significantly better event free survival and overall survival compared with patients with HPV-negative/PD-L1-negative cancer. Relative to those patients with HPV-negative/PD-L1-negative disease who had the highest risk of death, patients with HPV-positive/PD-L1-positive cancers had a 2.85 fold lower risk of developing an event (HR 0.35, 95% CI: 0.16-0.79) and a 4.5 fold lower risk of death (HR =0.22, 95% CI: 0.09-0.53). Our findings will help to guide future clinical trial design in immunotherapy based on PD-L1 expression in tonsillar cancer.

Guttiferone K suppresses cell motility and metastasis of hepatocellular carcinoma by restoring aberrantly reduced profilin 1.

Hepatocellular carcinoma (HCC) is an aggressive malignancy and the 5-year survival rate of advanced HCC is < 10%. Guttiferone K (GUTK) isolated from the Garcinia genus inhibited HCC cells migration and invasion in vitro and metastasis in vivo without apparent toxicity. Proteomic analysis revealed that actin-binding protein profilin 1 (PFN1) was markedly increased in the presence of GUTK. Over-expression of PFN1 mimicked the effect of GUTK on HCC cell motility and metastasis. The effect of GUTK on cell motility was diminished when PFN1 was over-expressed or silenced. Over-expression of PFN1 or incubation with GUTK decreased F-actin levels and the expression of proteins involved in actin nucleation, branching and polymerization. Moreover, a reduction of PFN1 protein levels was common in advanced human HCC and associated with poor survival rate. In conclusion, GUTK effectively suppresses the motility and metastasis of HCC cells mainly by restoration of aberrantly reduced PFN1 protein expression.

Validation of the Singapore nomogram for outcome prediction in breast phyllodes tumours: an Australian cohort.

AIM: To validate the utility of the Singapore nomogram for outcome prediction in breast phyllodes tumours. METHODS: Histological parameters, surgical margin status and clinical follow-up data of 34 women diagnosed with phyllodes tumours were analysed. Biostatistics modelling was performed, and the concordance between predicted and observed survivals was calculated. RESULTS: Women with a high nomogram score had an increased risk of developing relapse, which was predicted using the parameters defined by the Singapore nomogram. CONCLUSIONS: The Singapore nomogram is useful in predicting outcome in breast phyllodes tumours when applied to an Australian cohort of 34 women.

Relationships between p53 mutation, HPV status and outcome in oropharyngeal squamous cell carcinoma.

BACKGROUND AND PURPOSE: This study aimed to examine the rate and type of p53 mutation in oropharyngeal cancer (OSCC). Relationships were sought between human papillomavirus (HPV) status and p53 mutation. The role of p53 mutation as a prognostic factor independent of HPV status and as a modifier of the effect of HPV on outcomes was also examined. METHODS: The HPV status of 202 cases was determined by HPV DNA by RT-PCR and p16 immunohistochemistry. P53 mutation in exon 5-8 was determined by pyrosequencing. Findings were correlated with known clinicopathological factors and outcomes. RESULTS: 48% of the cases were HPV positive and they were significantly less likely to have a p53 mutation than HPV-negative OSCCs (25.8% vs 46.7%, p=0.0021). Mutation was most common in exon 5. Among patients with HPV-positive OSCC, there was no significant difference in p53 mutation by smoking status (22.2% for never smokers and 30.8% for current or ex-smokers). Patients with p53 mutant OSCC had significantly worse overall survival (p=0.01). There was no statistical evidence that p53 mutation modified the effect of HPV status on outcomes. In the multivariate analysis, positive HPV status remained the strongest predictor of outcomes. p53 mutation status was not a significant predictor of outcome after adjusting for age, gender, T stage, N stage and HPV status. CONCLUSIONS: In summary, HPV-positive OSCC are less likely to have mutant p53 than HPV-negative OSCC. Our study did not show any evidence that p53 mutation could modify the effect of HPV status on outcomes.

Redox cycling metals: Pedaling their roles in metabolism and their use in the development of novel therapeutics.

Essential metals, such as iron and copper, play a critical role in a plethora of cellular processes including cell growth and proliferation. However, concomitantly, excess of these metal ions in the body can have deleterious effects due to their ability to generate cytotoxic reactive oxygen species (ROS). Thus, the human body has evolved a very well-orchestrated metabolic system that keeps tight control on the levels of these metal ions. Considering their very high proliferation rate, cancer cells require a high abundance of these metals compared to their normal counterparts. Interestingly, new anti-cancer agents that take advantage of the sensitivity of cancer cells to metal sequestration and their susceptibility to ROS have been developed. These ligands can avidly bind metal ions to form redox active metal complexes, which lead to generation of cytotoxic ROS. Furthermore, these agents also act as potent metastasis suppressors due to their ability to up-regulate the metastasis suppressor gene, N-myc downstream regulated gene 1. This review discusses the importance of iron and copper in the metabolism and progression of cancer, how they can be exploited to target tumors and the clinical translation of novel anti-cancer chemotherapeutics.

Regulation of the tumour suppressor PDCD4 by miR-499 and miR-21 in oropharyngeal cancers.

BACKGROUND: The rates of oropharyngeal cancers such as tonsil cancers are increasing. The tumour suppressor protein Programmed Cell Death Protein 4 (PDCD4) has been implicated in the development of various human cancers and small RNAs such as microRNAs (miRNAs) can regulate its expression. However the exact regulation of PDCD4 by multiple miRNAs in oropharyngeal squamous cell carcinoma (SCC) is not well understood. RESULTS: Using two independent oropharyngeal SCC cohorts with a focus on the tonsillar region, we identified a miRNA profile differentiating SCC tissue from normal. Both miR-21 and miR-499 were highly expressed in tonsil SCC tissues displaying a loss of PDCD4. Interestingly, expression of the miRNA machinery, Dicer1, Drosha, DDX5 (Dead Box Helicase 5) and DGCR8 (DiGeorge Syndrome Critical Region Gene 8) were all elevated by greater than 2 fold in the tonsil SCC tissue. The 3'UTR of PDCD4 contains three binding-sites for miR-499 and one for miR-21. Using a wild-type and truncated 3'UTR of PDCD4, we demonstrated that the initial suppression of PDCD4 was mediated by miR-21 whilst sustained suppression was mediated by miR-499. Moreover the single miR-21 site was able to elicit the same magnitude of suppression as the three miR-499 sites. CONCLUSION: This study describes the regulation of PDCD4 specifically in tonsil SCC by miR-499 and miR-21 and has documented the loss of PDCD4 in tonsil SCCs. These findings highlight the complex interplay between miRNAs and tumour suppressor gene regulation and suggest that PDCD4 loss may be an important step in tonsillar carcinogenesis.

E6 viral protein ratio correlates with outcomes in human papillomavirus related oropharyngeal cancer.

BACKGROUND: The study aimed to identify prognostic markers to improve the management of patients with HPV positive OSCC Methods: We determined the ratio of HPV E6*I and E6*II splice variants by quantitative RT-PCR in 177 HPV positive OSCC and correlated the findings with other clinicopathological data Results: There was no significant difference in locoregional recurrence (HR 1.72 p = 0.24) and death (HR 1.65, p = 0.13) among patients whose tumors had an E6*I/*II ratio ≥1 compared with an E6*I/*II ratio of <1. Univariate analysis showed that patients with E6*I/*II ≥1 OSCC were more likely to have an event. In the multivariable analysis, there was a trend for more events in patients with E6*I/*II ratio ≥1 (HR 1.70, 95% CI 0.95-3.03, p = 0.07) CONCLUSION: Our data suggest that the use of HPV 16 spliced transcripts may help to predict for poorer outcomes in patients with HPV positive OSCC.