Mast cells dysregulate apoptotic and cell cycle genes in mucosal squamous cell carcinoma

BACKGROUND: Mucosal squamous cell carcinoma of the head and neck is a disease of high mortality and morbidity. Interactions between the squamous cell carcinoma and the host's local immunity, and how the latter contributes to the biological behavior of the tumor are unclear. In vivo studies have demonstrated sequential mast cell infiltration and degranulation during squamous cell carcinogenesis. The degree of mast cell activation correlates closely with distinct phases of hyperkeratosis, dysplasia, carcinoma in-situ and invasive carcinoma. However, the role of mast cells in carcinogenesis is unclear. AIM: This study explores the effects of mast cells on the proliferation and gene expression profile of mucosal squamous cell carcinoma using human mast cell line (HMC-1) and human glossal squamous cell carcinoma cell line (SCC25). METHODS: HMC-1 and SCC25 were co-cultured in a two-compartment chamber, separated by a polycarbonate membrane. HMC-1 was stimulated to degranulate with calcium ionophore A23187. The experiments were done in quadruplicate. Negative controls were established where SCC25 were cultured alone without HMC-1. At 12, 24, 48 and 72 hours, proliferation and viability of SCC25 were assessed with MTT colorimetric assay. cDNA microarray was employed to study differential gene expression between co-cultured and control SCC25. RESULTS: HMC-1/SCC25 co-culture resulted in suppression of growth rate for SCC-25 (34% compared with 110% for the control by 72 hours, p < 0.001), and dysregulation of genes TRAIL, BIRC4, CDK6, Cyclin G2 and CDC6 in SCC25. CONCLUSION: We show that mast cells have a direct inhibitory effect on the proliferation of mucosal squamous cell carcinoma in vitro by dysregulating key genes in apoptosis and cell cycle control

Tumor Biology of Head and Neck Cutaneous Squamous Cell Carcinoma

Epidemiologic studies worldwide have consistently shown that the incidence of cutaneous squamous cell carcinoma (cSCC) has steadily increased over the last four decades, possibly due to a combination of depletion of the ozone layer, an aging population, increasing use of immunosuppression, and changing social trends. In fact, the incidence of cutaneous malignancies has reached endemic proportions in New Zealand and Australia, as a result of the additional factors of a predominantly Anglo-Celtic population and a high level of ambient solar irradiation. 75% of cSCC arise in the head and neck, and metastatic disease is not uncommon. Metastatic cSCC to the parotid and cervical lymph nodes carries high morbidity and mortality in both the disease and treatment. Despite its high prevalence, the tumor biology of cSCC is poorly understood. The previous (sixth) American Joint Committee on Cancer (AJCC) TNM staging system for cSCC that existed during much of the duration of this PhD study was rudimentary, and lacking in its power to prognosticate for more advanced disease. A comprehensive staging system with adequate stratification of disease severity was necessary for comparison of treatment outcome in research, and for guiding clinical decision making in whether intensification or de-escalation of treatment is necessary. This provided the impetus for a local study based in Wellington of survival outcome in patients with metastatic head and neck cSCC. This regional study then extended to include the four major head and neck cancer centers in New Zealand. There is an increasing interest and an expanding role in oncology for more-targeted therapies, e.g., immunotherapy and targeted molecular therapy. Among the immune cells (i.e., tumor-associated macrophages, dendritic cells, neutrophils, T cells and mast cells) in the microenvironment, the mast cell has received the least attention despite well-established evidence for its roles in carcinogenesis. In this study, mast cells were shown to deregulate key apoptotic and cell cycle genes, including tumor necrosis factor-related apoptosis-related ligand (TRAIL), baculoviral inhibitor of apoptosis repeat-containing protein 4 (BIRC4), cyclin-dependent kinase 6 (CDK6) and cyclin G2 (CCNG2) in human squamous cell carcinoma cell line SCC25 in vitro, and causing an overall inhibitory effect on the growth of SCC25. Within the tumor microenvironment, angiogenesis is strongly associated with the metastatic phenotype, but the interaction between mast cells and angiogenesis remains to be elucidated. Epidermal growth factor receptor (EGFR) targeted molecular treatment has enjoyed reasonable success in the treatment of colorectal, lung and head and neck mucosal squamous cell carcinoma. This PhD study showed that the role of EGFR is not as straight-forward in cSCC. Whilst clearly over-expressed in primary lesions that subsequently metastasized, EGFR was only weakly detectable in established nodal disease, with a clear implication for EGFRtargeted treatment in metastatic cSCC. The mechanisms of EGFR overexpression, and hence, potential points of intervention remain as yet unclear. This research contributes to better understanding of the tumor biology of head and neck cSCC, which is essential in the future development of novel therapies

Tumor Biology of Head and Neck Cutaneous Squamous Cell Carcinoma

Epidemiologic studies worldwide have consistently shown that the incidence of cutaneous squamous cell carcinoma (cSCC) has steadily increased over the last four decades, possibly due to a combination of depletion of the ozone layer, an aging population, increasing use of immunosuppression, and changing social trends. In fact, the incidence of cutaneous malignancies has reached endemic proportions in New Zealand and Australia, as a result of the additional factors of a predominantly Anglo-Celtic population and a high level of ambient solar irradiation. 75% of cSCC arise in the head and neck, and metastatic disease is not uncommon. Metastatic cSCC to the parotid and cervical lymph nodes carries high morbidity and mortality in both the disease and treatment. Despite its high prevalence, the tumor biology of cSCC is poorly understood. The previous (sixth) American Joint Committee on Cancer (AJCC) TNM staging system for cSCC that existed during much of the duration of this PhD study was rudimentary, and lacking in its power to prognosticate for more advanced disease. A comprehensive staging system with adequate stratification of disease severity was necessary for comparison of treatment outcome in research, and for guiding clinical decision making in whether intensification or de-escalation of treatment is necessary. This provided the impetus for a local study based in Wellington of survival outcome in patients with metastatic head and neck cSCC. This regional study then extended to include the four major head and neck cancer centers in New Zealand. There is an increasing interest and an expanding role in oncology for more-targeted therapies, e.g., immunotherapy and targeted molecular therapy. Among the immune cells (i.e., tumor-associated macrophages, dendritic cells, neutrophils, T cells and mast cells) in the microenvironment, the mast cell has received the least attention despite well-established evidence for its roles in carcinogenesis. In this study, mast cells were shown to deregulate key apoptotic and cell cycle genes, including tumor necrosis factor-related apoptosis-related ligand (TRAIL), baculoviral inhibitor of apoptosis repeat-containing protein 4 (BIRC4), cyclin-dependent kinase 6 (CDK6) and cyclin G2 (CCNG2) in human squamous cell carcinoma cell line SCC25 in vitro, and causing an overall inhibitory effect on the growth of SCC25. Within the tumor microenvironment, angiogenesis is strongly associated with the metastatic phenotype, but the interaction between mast cells and angiogenesis remains to be elucidated. Epidermal growth factor receptor (EGFR) targeted molecular treatment has enjoyed reasonable success in the treatment of colorectal, lung and head and neck mucosal squamous cell carcinoma. This PhD study showed that the role of EGFR is not as straight-forward in cSCC. Whilst clearly over-expressed in primary lesions that subsequently metastasized, EGFR was only weakly detectable in established nodal disease, with a clear implication for EGFRtargeted treatment in metastatic cSCC. The mechanisms of EGFR overexpression, and hence, potential points of intervention remain as yet unclear. This research contributes to better understanding of the tumor biology of head and neck cSCC, which is essential in the future development of novel therapies

Mutational Patterns in Metastatic Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinoma from the head and neck typically metastasize to the lymph nodes of the neck and parotid glands. When a primary is not identified, they are difficult to distinguish from metastases of mucosal origin and primary salivary gland squamous cell carcinoma. UV radiation causes a mutation pattern that predominantly features cytosine to thymine transitions at dipyrimidine sites and has been associated with cutaneous squamous cell carcinoma. In this study, we used whole genome sequencing data from 15 cutaneous squamous cell carcinoma metastases and show that a UV mutation signature is pervasive across the cohort and distinct from mucosal squamous cell carcinoma. The mutational burden was exceptionally high and concentrated in some regions of the genome, especially insulator elements (mean 162 mutations/megabase). We therefore evaluated the likely impact of UV-induced mutations on the dipyrimidine-rich binding site of the main human insulator protein, CCCTC-binding factor, and the possible implications on CCCTC-binding factor function and the spatial organization of the genome. Our findings suggest that mutation signature analysis may be useful in determining the origin of metastases in the neck and the parotid gland. Furthermore, UV-induced DNA damage to insulator binding sites may play a role in the carcinogenesis and progression of cutaneous squamous cell carcinoma

Improved Risk Prediction Calculator for Sentinel Node Positivity in Patients With Melanoma: The Melanoma Institute Australia Nomogram

PURPOSE For patients with primary cutaneous melanoma, the risk of sentinel node (SN) metastasis varies according to several clinicopathologic parameters. Patient selection for SN biopsy can be assisted by National Comprehensive Cancer Network (NCCN) and ASCO/Society of Surgical Oncology (SSO) guidelines and the Memorial Sloan Kettering Cancer Center (MSKCC) online nomogram. We sought to develop an improved online risk calculator using alternative clinicopathologic parameters to more accurately predict SN positivity. PATIENTS AND METHODS Data from 3,477 patients with melanoma who underwent SN biopsy at Melanoma Institute Australia (MIA) were analyzed. A new nomogram was developed by replacing body site and Clark level from the MSKCC model with mitotic rate, melanoma subtype, and lymphovascular invasion. The predictive performance of the new nomogram was externally validated using data from The University of Texas MD Anderson Cancer Center (n = 3,496). RESULTS The MSKCC model receiver operating characteristic curve had a predictive accuracy of 67.7% (95% CI, 65.3% to 70.0%). The MIA model had a predictive accuracy of 73.9% (95% CI, 71.9% to 75.9%), a 9.2% increase in accuracy over the MSKCC model (P < .001). Among the 2,748 SN-negative patients, SN biopsy would not have been offered to 22.1%, 13.4%, and 12.4% based on the MIA model, the MSKCC model, and NCCN or ASCO/SSO criteria, respectively. External validation generated a C-statistic of 75.0% (95% CI, 73.2% to 76.7%). CONCLUSION A robust nomogram was developed that more accurately estimates the risk of SN positivity in patients with melanoma than currently available methods. The model only requires the input of 6 widely available clinicopathologic parameters. Importantly, the number of patients undergoing unnecessary SN biopsy would be significantly reduced compared with use of the MSKCC nomogram or the NCCN or ASCO/SSO guidelines, without losing sensitivity. An online calculator is available at www.melanomarisk.org.au.Supported by an Australian National Health and Medical Research Council Practitioner Fellowship (R.A.S), an Australia NHMRC program grant (to R.A.S., G.J.M., and J.F.T.), the Medical Foundation of the University of Sydney (J.F.T.), Melanoma Institute Australia (J.R.S., R.P.M.S., and S.N.L.), the Friends of the Mater Foundation (A.J.S.), an Australian Medical Research Future Fund Rapid Applied Research Translation Grant via Sydney Health Partners (A.H.R.V.), the Robert and Lynne Grossman Family Foundation (J.E.G.), the Michael and Patricia Booker Melanoma Research Endowment (J.E.G.), the National Institutes of Health Specialized Programs of Research Excellence grant in melanoma at The University of Texas MD Anderson Cancer Center

Analysis of clinically relevant somatic mutations in high-risk head and neck cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma is the second most prevalent malignancy, most frequently occurring in the head and neck (head and neck cutaneous squamous cell carcinoma). Treatment of locally advanced or metastatic disease is associated with functional morbidity and disfigurement. Underlying genetic mechanisms are poorly understood. Targeted sequencing of 48 clinically relevant genes was performed on DNA extracted from formalinfixed and paraffin-embedded high-risk primary head and neck cutaneous squamous cell carcinomas that remained non-metastatic at minimum follow-up of 24 months. Associations of somatic mutations with clinicopathologic characteristics were evaluated and compared with those described in the literature for metastatic disease. Alterations in 44 cancer-associated genes were identified. TP53 was mutated in 100% of cases; APC, ATM, ERBB4, GNAQ, KIT, RB1 and ABL1 were altered in 60% of cases. FGFR2 mutations (40%) were exclusively seen in patients with perineural invasion. MLH1 mutations were exclusively seen in the two younger patients (o45 years). Lower incidences of NOTCH1 mutations were observed compared with that described in metastatic head and neck cutaneous squamous cell carcinoma in the literature. Somatic mutations susceptible to EGFR inhibitors, and other small molecular targeted therapeutics were seen in 60% of cases. This study provides insights into somatic mutations in non-metastatic, high-risk head and neck cutaneous squamous cell carcinoma and identifies potential therapeutic targets. Alterations in FGFR2 and NOTCH1 may have roles in local and distant disease progression. © 2018 USCAP, Inc All rights reserved

Effect of the time interval between melanoma diagnosis and sentinel node biopsy on the size of metastatic tumour deposits in node-positive patients

Introduction: This study sought to assess whether the interval between diagnostic excision-biopsy of a primary melanoma and definitive wide excision with sentinel node biopsy (SNB) influenced the size of SN metastatic deposits, which might have implications for management and prognosis. Methods: Data were collected for (i) a Dutch population-based cohort of patients treated between 2004 and 2014 who underwent SNB within 100 days of complete excision of their primary melanoma and who were SN-positive with known SN metastasis diameter (n = 1027) and (ii) a cohort from a large Australian melanoma treatment centre (n = 541) who presented in the same time period. The effects of SNB timing on the size of SN metastatic deposits were analysed. Results: Dutch patients whose SNB was performed in the second or third months after diagnosis had significantly larger SN metastasis diameters than patients who had their SNB in the first month (median increases of 17% (95%CI -14, 60%, p = 0.211) and 71% (95%CI 15, 119%, p = 0.004), respectively). No significant difference in tumour diameter for early and late SNB was found in the Australian cohort. Conclusions: SN metastasis diameter became progressively greater with SN biopsy in the second and third months after primary melanoma diagnosis in the larger, population-based patient cohort. An increase in metastasis diameter was not observed in the smaller, institutional cohort, possibly due to detection of larger SN metastases by routine pre-operative ultrasound, with fine-needle biopsy confirmation. These patients did not proceed to SNB and were therefore not included in the study

Time interval between diagnostic excision-biopsy of a primary melanoma and sentinel node biopsy: effects on the sentinel node positivity rate and survival outcomes

INTRODUCTION: The optimal time interval between diagnostic excision of a primary cutaneous melanoma and sentinel node (SN) biopsy is unknown. The current study sought to determine whether this interval influenced the SN-positivity rate, recurrence or survival. METHODS: Data collected from 2004 to 2014 for a Dutch population-based cohort of patients with melanoma who underwent SN biopsy (SNB) within 100 days of initial diagnosis (n = 7660) and for a similarly specified cohort from a large Australian melanoma treatment centre (n = 3478) were analysed. Time to SNB was analysed continuously (in weeks) and categorically (per month). The effects of SNB timing on SN-positivity were assessed using multivariable logistic regression, and its effects on recurrence-free survival (RFS) and overall survival (OS) were assessed using Cox proportional hazard regression analyses. Advanced modelling using a multivariable Cox model with penalised splines for modelling the continuous effects of time to SNB on RFS and OS was also performed. RESULTS: In neither the Dutch nor the Australian cohort was there a significant association between time to SNB and SN-positivity in either cohort, nor was there an impact of time to SNB on RFS or OS in either cohort. The spline-based HR curves for RFS and OS confirmed these findings. CONCLUSIONS: The time interval between diagnostic excision of a primary melanoma and SNB did not influence the SN-positivity rate or survival outcomes. This provides reassurance that neither early nor delayed definitive wide excision and SNB will adversely affect prognosis