Advances in long-read single-cell transcriptomics.

Long-read single-cell transcriptomics (scRNA-Seq) is revolutionizing the way we profile heterogeneity in disease. Traditional short-read scRNA-Seq methods are limited in their ability to provide complete transcript coverage, resolve isoforms, and identify novel transcripts. The scRNA-Seq protocols developed for long-read sequencing platforms overcome these limitations by enabling the characterization of full-length transcripts. Long-read scRNA-Seq techniques initially suffered from comparatively poor accuracy compared to short read scRNA-Seq. However, with improvements in accuracy, accessibility, and cost efficiency, long-reads are gaining popularity in the field of scRNA-Seq. This review details the advances in long-read scRNA-Seq, with an emphasis on library preparation protocols and downstream bioinformatics analysis tools.

Ex vivo therapeutic screening of metastatic cSCC: A review of methodological considerations for clinical implementation.

Cutaneous squamous cell carcinoma (cSCC) is the second most common malignancy worldwide, with most deaths caused by locally advanced and metastatic disease. Treatment of resectable metastases is typically limited to invasive surgery with adjuvant radiotherapy; however, many patients fail to respond and there is minimal data to predict response or propose effective alternatives. Precision medicine could improve this, though genomic biomarkers remain elusive in the high mutational background and genomic complexity of cSCC. A phenotypic approach to precision medicine using patient-derived ex vivo tumour models is gaining favour for its capacity to directly assess biological responses to therapeutics as a functional, predictive biomarker. However, the use of ex vivo models for guiding therapeutic selection has yet to be employed for metastatic cSCC. This review will therefore evaluate the existing experimental models of metastatic cSCC and discuss how ex vivo methods could overcome the shortcomings of these existing models. Disease-specific considerations for a prospective methodological pipeline will also be discussed in the context of precision medicine.

Rethinking the tiered trauma team response: A case-series study in a regional trauma centre.

OBJECTIVE: To reduce perceived unnecessary resource use, we modified our tiered trauma response. If a patient was not physiologically compromised, surgical registrar attendance was not mandated. We investigated the effect of this change on missed injury, unplanned representation to ED, diagnostic imaging rates and staff satisfaction. METHODS: A retrospective case series study assessing the 3-month period before and after the intervention was conducted. Logistic regression analyses were used to examine the association between ordering of computerised tomography (CT) and ED length of stay (LOS), injury severity (ISS), age, surgical review and admission. A staff survey was conducted to investigate staff perceptions of the practice change. Free text data were analysed using inductive content analysis. RESULTS: There were 105 patients in the control and 166 in the intervention group and their mean (SD) ISS was the same (ISS [SD] = 4 [±4] [P = 0.608]). A higher proportion of the control group were admitted (56.3% vs 42.2% [P = 0.032]) and they had a shorter ED LOS (274 min [202-456] vs 326 min [225-560], P = 0.044). The rate of missed injury was unchanged. A surgical review resulted in a 26-fold increase in receipt of a whole-body CT scan (odds ratio = 26.89, 95% confidence interval = 3.31-218.17). Just over half of survey respondents felt the change was safe (54.4%), and more surgical (90%) than ED staff (69%) reported the change as positive. CONCLUSION: The removal of the surgical registrar from the initial trauma standby response did not result in any adverse events, reduced admissions, pathology and imaging, but resulted in an increased ED LOS and time to surgical review.

PIK Your Poison: The Effects of Combining PI3K and CDK Inhibitors against Metastatic Cutaneous Squamous Cell Carcinoma In Vitro.

Cutaneous squamous cell carcinoma (cSCC) is a very common skin malignancy with poor prognosis for patients with locally advanced or metastatic cSCC (mcSCC). PI3K/AKT/mTOR and cell cycle signalling pathways are often dysregulated in mcSCC. A combination drug approach has been theorised to overcome the underwhelming clinical performance of targeted inhibitors as single agents. This study investigates the potential of targeted inhibition of the p110α-subunit of PI3K with PIK-75 or BGT226 (P13Ki), and of CDK1/2/5/9 with dinaciclib (CDKi) as single agents and in combination. The patient-derived mcSCC cell lines, UW-CSCC1 and UW-CSCC2, were used to assess cell viability, migration, cell signalling, cell cycle distribution, and apoptosis. PIK-75, BGT226, and dinaciclib exhibited strong cytotoxic potency as single agents. Notably, the non-malignant HaCaT cell line was unaffected. In 2D cultures, PIK-75 synergistically enhanced the cytotoxic effects of dinaciclib in UW-CSCC2, but not UW-CSCC1. Interestingly, this pattern was reversed in 3D spheroid models. Despite the combination of PIK-75 and dinaciclib resulting in an increase in cell cycle arrest and apoptosis, and reduced cell motility, these differences were largely negligible compared to their single-agent counterpart. The differential responses between the cell lines correlated with driver gene mutation profiles. These findings suggest that personalised medicine approaches targeting PI3K and CDK pathways in combination may yield some benefit for mcSCC, and that more complex 3D models should be considered for drug responsiveness studies in this disease.

Sex as a Predictor of Response to Immunotherapy in Advanced Cutaneous Squamous Cell Carcinoma.

Approximately 3-5% of patients with cutaneous squamous cell carcinoma (CSCC) develop advanced disease, accounting for roughly 1% of all cancer deaths in Australia. Immunotherapy has demonstrated significant clinical benefit in advanced CSCC in several key phase II studies; however, there are limited data for patients treated outside of clinical trials. This is particularly relevant in advanced CSCC, which is most often seen in elderly patients with significant comorbidities. Thus, we aim to describe our experience with immunotherapy in a cohort of patients with advanced CSCC in Australia. We retrospectively reviewed all advanced CSCC patients treated with immunotherapy within the Illawarra and Shoalhaven Local Health District. Among the 51 patients treated with immunotherapy, there was an objective response rate (ORR) of 53% and disease control rate (DCR) of 67%. Our most significant predictor of response was sex, with male patients more likely to have better responses compared to female patients (DCR 85% vs. 41%, p < 0.0001), as well as improved progression-free survival (HR 4.6, 95%CI 1.9-10.8, p = 0.0007) and overall survival (HR 3.0, 95%CI 1.3-7.1, p = 0.006). Differential expression analysis of 770 immune-related genes demonstrated an impaired CD8 T-cell response in female patients. Our observed ORR of 53% is similar to that described in current literature with durable responses seen in the majority of patients.

Implementing structured pathology reporting protocol for non-melanocytic skin cancers: practical considerations.

Non-melanocytic skin cancers (NMSCs) account for five times the incidence of all other cancers combined and cost US $6 billion annually. These are the most frequent specimens encountered in community pathology practice in many Western countries. Lack of standardised structured pathology reporting protocols (SPRPs) can result in omission of critical information or miscommunication leading to suboptimal patient management. The lack of standardised data has significant downstream public health implications, including insufficient data for reliable development of prognostic tools and health-economy planning. The Royal College of Pathologists of Australasia has developed an NMSC SPRP. A multidisciplinary expert committee including pathologists, surgeons, dermatologists, and radiation and medical oncologists from high volume cancer centres was convened. A systematic literature review was performed to identify evidence for including elements as mandatory standards or best practice guidelines. The SPRP and accompanying commentary of evidence, definitions and criteria was peer reviewed by external stakeholders. Finally, the protocol was revised following feedback and trialled in multiple centres prior to implementation. Some parameters utilised clinically for determining management and prognosis including tumour depth, lymphovascular invasion or distance to the margins lack high level evidence in NMSC. Dermatologists, surgeons, and radiation oncologists welcomed the SPRP. Pathologists indicated that the variety of NMSC specimens ranging from curettes to radical resections as well as significant differences in the biological behaviour of different tumours covered by the NMSC umbrella made use of a single protocol difficult. The feedback included that using a SPRP for low risk NMSC was neither clinically justified nor compensated adequately by the Australian Medicare Reimbursement Schedule. Following stakeholder feedback, the SPRP implementation was restricted to excision specimens of head and neck NMSC; and low-risk NMSC, such as superficial basal cell carcinoma, were excluded. Implementing NMSC SPRP fulfils an unmet clinical need. Unlike other cancers, NMSCs generate a range of specimen types and are reported in a wide range of pathology practices. Limiting use of SPRP to NMSC at higher risk of progression and providing formatted templates for easy incorporation into laboratory information systems were essential to successful deployment. In the future, further consideration should be given to implementing the SPRP to include all relevant specimens, including non-head and neck and low-risk NMSC specimens.

Protocol for the generation and automated confocal imaging of whole multi-cellular tumor spheroids.

Multi-cellular tumor spheroids (MCTS) have found widespread use in pre-clinical research. However, their complex three-dimensional structure makes immunofluorescent staining and imaging challenging. Here, we present a protocol for whole spheroid staining and automated imaging using laser-scanning confocal microscopy. We describe steps for cell culture, seeding of spheroids and transfer of MCTS, and adhesion to Ibidi chamber slides. We then detail fixation, immunofluorescent staining based on optimized reagent concentrations and incubation times, and confocal imaging facilitated by glycerol-based optical clearing.

Quality of Life Impacts Following Surgery for Advanced Head and Neck Cancer.

Background: Understanding the impact of surgery on patients will enable clinicians to provide evidence-based perioperative management. This study aimed to investigate the quality of life (QoL) impacts following head and neck surgery for advanced stage head and neck cancer. Methods: Head and neck cancer survivors were invited to complete five validated questionnaires to investigate QoL. Associations between QoL and patient variables were analyzed. Variables included age, time since operation, length of surgery, length of stay, Comorbidity Index, estimated 10-year survival, sex, flap type, treatment and cancer type. Outcome measures were also compared to normative outcomes. Results: The majority of participants (N = 27; 55% male; mean (standard deviation) age: 62.6 (13.8) years; mean time since operation: 801 days) had a squamous cell carcinoma (88.9%) and free flap repair (100%). Time since operation was significantly (P < 0.05) associated with higher rates of depression (r = -0.533), psychological needs (r = -0.0415) and physical/daily living needs (r = -0.527). Length of surgery and length of stay were significantly associated with depression (r = 0.442; r = 0.435) and length of stay was significantly associated with speaking difficulties (r = -0.456). There was a significant association between work and education scores with age (r = 0.471), length of surgery (r = 0.424), Comorbidity Index (r = 0.456) and estimated 10-year survival (r = -0.523). Conclusions: Age, time since operation, length of surgery, length of stay, Comorbidity Index and estimated 10-year survival were the outcomes associated with QoL. Patient-reported outcome measures and psychological support could be included in the standard care pathway for head and neck cancer patients to ensure holistic management of their condition.

Comparing Genomic Landscapes of Oral and Cutaneous Squamous Cell Carcinoma of the Head and Neck: Quest for Novel Diagnostic Markers.

Squamous cell carcinoma is the most common head and neck malignancy arising from the oral mucosa and the skin. The histologic and immunohistochemical features of oral squamous cell carcinoma (OSCC) and head and neck cutaneous squamous cell carcinoma (HNcSCC) are similar, making it difficult to identify the primary site in cases of metastases. With the advent of immunotherapy, reliable distinction of OSCC and HNcSCC at metastatic sites has important treatment and prognostic implications. Here, we investigate and compare the genomic landscape of OSCC and HNcSCC to identify diagnostically useful biomarkers. Whole-genome sequencing data from 57 OSCC and 41 HNcSCC patients were obtained for tumor and matched normal samples. Tumor mutation burden (TMB), Catalogue of Somatic Mutations in Cancer (COSMIC) mutational signatures, frequent chromosomal alterations, somatic single nucleotide, and copy number variations were analyzed. The median TMB of 3.75 in primary OSCC was significantly lower (P < .001) than that of 147.51 mutations/Mb in primary HNcSCC. The COSMIC mutation signatures were significantly different (P < .001) between OSCC and HNcSCC. OSCC showed COSMIC single-base substitution (SBS) mutation signature 1 and AID/APOBEC activity-associated signature 2 and/or 13. All except 1 HNcSCC from hair-bearing scalp showed UV damage-associated COSMIC SBS mutation signature 7. Both OSCC and HNcSCC demonstrated a predominance of tumor suppressor gene mutations, predominantly TP53. The most frequently mutated oncogenes were PIK3CA and MUC4 in OSCC and HNcSCC, respectively. The metastases of OSCC and HNcSCC demonstrated TMB and COSMIC SBS mutation signatures similar to their primary counterparts. The combination of high TMB and UV signature in a metastatic keratinizing squamous cell carcinoma suggests HNcSCC as the primary site and may also facilitate decisions regarding immunotherapy. HNcSCC and OSCC show distinct genomic profiles despite histologic and immunohistochemical similarities. Their genomic characteristics may underlie differences in behavior and guide treatment decisions in recurrent and metastatic settings.

How to reduce processing times for site-specific assessments from 29 to 5 days using a common-sense approach: it does not have to be that hard.

Researchers have reported limitations with research governance processes across Australia. This study aimed to streamline research governance processes across a local health district. Four basic principles were applied to remove non-value-adding and non-risk-mitigating processes. Average processing times were reduced from 29 to 5 days and end-user satisfaction was improved, all within the same staffing levels.

Sex Disparity for Patients with Cutaneous Squamous Cell Carcinoma of the Head and Neck: A Systematic Review.

The incidence of head and neck cutaneous squamous cell carcinoma (HNcSCC) is unevenly distributed between men and women. At present, the mechanism behind this disparity remains elusive. This study conducted a systematic review and meta-analysis of proportions to investigate the disparity between sexes for patients with HNcSCC. PubMed, Scopus, EMBASE, MEDLINE, Emcare and CINAHL were searched in November 2021 and June 2022 (N > 50, English, human), and studies which examined the association between sex and HNcSCC were included. Analysis was conducted using RStudio with data and forest plots displaying males as a proportion of total patients with HNcSCC. Two independent researchers performed study selection, data extraction, data analysis and risk of bias. Eighty-two studies (1948 to 2018) comprising approximately 186,000 participants (67% male, 33% female) from 29 countries were included. Significantly more males had HNcSCC overall (71%; CI: 67−74). Males were also significantly more affected by cSCC of the ear (92%; CI: 89−94), lip (74%; CI: 66−81), and eyelid (56%; CI: 51−62). This study found HNcSCC disproportionately affected males overall and across all subtypes. Improving our understanding of sex-specific mechanisms in HNcSCC will better inform our preventive, therapeutic and prognostic practices.

Evolution of Head and Neck Cutaneous Squamous Cell Carcinoma Nodal Staging-An Australian Perspective.

Cutaneous squamous cell carcinoma of the head and neck (HNcSCC) is one of the commonest malignancies. When patients present with regional metastatic disease, treatment escalation results in considerable morbidity and survival is markedly reduced. Owing to the high incidence, Australian institutions have been at the forefront of advocating for reliable, accurate, and clinically useful staging systems that recognise the distinct biological characteristics of HNcSCC. As a result, an extensive body of literature has been produced over the past two decades, which has defined critical prognostic factors, critiqued existing staging systems, and proposed alternative staging models. Notwithstanding, a suitable staging system has proved elusive. The goal of cancer staging is to group patients according to cancer characteristics for which survival differs between groups (distinctiveness), consistently decreases with increasing stage (monotonicity), and is similar within a group (homogeneity). Despite implementing major changes based on published data, the latest edition of the American Joint Committee on Cancer (AJCC) staging manual fails to satisfy these fundamental requirements. This review chronologically explores and summarises the Australian contribution to prognostication and nodal staging of HNcSCC and highlights the ongoing challenges.

Whole genome analysis reveals the genomic complexity in metastatic cutaneous squamous cell carcinoma.

Metastatic cutaneous squamous cell carcinoma (CSCC) is a highly morbid disease requiring radical surgery and adjuvant therapy, which is associated with a poor prognosis. Yet, compared to other advanced malignancies, relatively little is known of the genomic landscape of metastatic CSCC. We have previously reported the mutational signatures and mutational patterns of CCCTC-binding factor (CTCF) regions in metastatic CSCC. However, many other genomic components (indel signatures, non-coding drivers, and structural variants) of metastatic CSCC have not been reported. To this end, we performed whole genome sequencing on lymph node metastases and blood DNA from 25 CSCC patients with regional metastases of the head and neck. We designed a multifaceted computational analysis at the whole genome level to provide a more comprehensive perspective of the genomic landscape of metastatic CSCC. In the non-coding genome, 3' untranslated region (3'UTR) regions of EVC (48% of specimens), PPP1R1A (48% of specimens), and ABCA4 (20% of specimens) along with the tumor-suppressing long non-coding RNA (lncRNA) LINC01003 (64% of specimens) were significantly functionally altered (Q-value < 0.05) and represent potential non-coding biomarkers of CSCC. Recurrent copy number loss in the tumor suppressor gene PTPRD was observed. Gene amplification was much less frequent, and few genes were recurrently amplified. Single nucleotide variants driver analyses from three tools confirmed TP53 and CDKN2A as recurrently mutated genes but also identified C9 as a potential novel driver in this disease. Furthermore, indel signature analysis highlighted the dominance of ID signature 13 (ID13) followed by ID8 and ID9. ID9 has previously been shown to have no association with skin melanoma, unlike ID13 and ID8, suggesting a novel pattern of indel variation in metastatic CSCC. The enrichment analysis of various genetically altered candidates shows enrichment of "TGF-beta regulation of extracellular matrix" and "cell cycle G1 to S check points." These enriched terms are associated with genetic instability, cell proliferation, and migration as mechanisms of genomic drivers of metastatic CSCC.

High-Dimensional and Spatial Analysis Reveals Immune Landscape-Dependent Progression in Cutaneous Squamous Cell Carcinoma.

PURPOSE: The tumor immune microenvironment impacts the biological behavior of the tumor, but its effect on clinical outcomes in head and neck cutaneous squamous cell carcinomas (HNcSCC) is largely unknown. EXPERIMENTAL DESIGN: We compared the immune milieu of high-risk HNcSCC that never progressed to metastasis with those that metastasized using multiparameter imaging mass cytometry. The cohort included both immunosuppressed patients (IS) and patients with an absence of clinical immune-suppression (ACIS). Spatial analyses were used to identify cellular interactions that were associated with tumor behavior. RESULTS: Nonprogressing primary HNcSCC were characterized by higher CD8+ and CD4+ T-cell responses, including numerically increased regulatory T cells. In contrast, primary lesions from HNcSCC patients who progressed were largely devoid of T cells with lower numbers of innate immune cells and increased expression of checkpoint receptors and in the metastatic lesions were characterized by an accumulation of B cells. Spatial analysis reveals multiple cellular interactions associated with nonprogressing primary tumors that were distinct in primary tumors of disease-progressing patients. Cellular regional analysis of the tumor microenvironment also shows squamous cell-enriched tumor regions associated with primary nonprogressing tumors. CONCLUSIONS: Effective responses from both CD8+ and CD4+ T cells in the tumor microenvironment are essential for immune control of primary HNcSCC. Our findings indicate that the early events that shape the immune responses in primary tumors dictate progression and disease outcomes in HNcSCC.

A tEMTing target? Clinical and experimental evidence for epithelial-mesenchymal transition in the progression of cutaneous squamous cell carcinoma (a scoping systematic review).

Cutaneous squamous cell carcinoma (cSCC) is a disease with globally rising incidence and poor prognosis for patients with advanced or metastatic disease. Epithelial-mesenchymal transition (EMT) is a driver of metastasis in many carcinomas, and cSCC is no exception. We aimed to provide a systematic overview of the clinical and experimental evidence for EMT in cSCC, with critical appraisal of type and quality of the methodology used. We then used this information as rationale for potential drug targets against advanced and metastatic cSCC. All primary literature encompassing clinical and cell-based or xenograft experimental studies reporting on the role of EMT markers or related signalling pathways in the progression of cSCC were considered. A screen of 3443 search results yielded 86 eligible studies comprising 44 experimental studies, 22 clinical studies, and 20 studies integrating both. From the clinical studies a timeline illustrating the alteration of EMT markers and related signalling was evident based on clinical progression of the disease. The experimental studies reveal connections of EMT with a multitude of factors such as genetic disorders, cancer-associated fibroblasts, and matrix remodelling via matrix metalloproteinases and urokinase plasminogen activator. Additionally, EMT was found to be closely tied to environmental factors as well as to stemness in cSCC via NFκB and ß-catenin. We conclude that the canonical EGFR, canonical TGF-ßR, PI3K/AKT and NFκB signalling are the four signalling pillars that induce EMT in cSCC and could be valuable therapeutic targets. Despite the complexity, EMT markers and pathways are desirable biomarkers and drug targets for the treatment of advanced or metastatic cSCC.

How to streamline the low and negligible research ethics and governance review process from 80 to 10 days: submission to decision.

Most clinicians find research ethics and governance difficult and time consuming. This study aimed to develop a better local review process for low-risk research. We used real-time processing, leveraged local expertise and streamlined paperwork. As a result, turnaround times decreased from more than 80 days to 10 days, creating an efficient review process for low-risk projects.

Cancer Progression Gene Expression Profiling Identifies the Urokinase Plasminogen Activator Receptor as a Biomarker of Metastasis in Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinoma (cSCC) of the head and neck region is the second most prevalent skin cancer, with metastases to regional lymph nodes occurring in 2%-5% of cases. To further our understanding of the molecular events characterizing cSCC invasion and metastasis, we conducted targeted cancer progression gene expression and pathway analysis in non-metastasizing (PRI-) and metastasizing primary (PRI+) cSCC tumors of the head and neck region, cognate lymph node metastases (MET), and matched sun-exposed skin (SES). The highest differentially expressed genes in metastatic (MET and PRI+) versus non-metastatic tumors (PRI-) and SES included PLAU, PLAUR, MMP1, MMP10, MMP13, ITGA5, VEGFA, and various inflammatory cytokine genes. Pathway enrichment analyses implicated these genes in cellular pathways and functions promoting matrix remodeling, cell survival and migration, and epithelial to mesenchymal transition, which were all significantly activated in metastatic compared to non-metastatic tumors (PRI-) and SES. We validated the overexpression of urokinase plasminogen activator receptor (uPAR, encoded by PLAUR) in an extended patient cohort by demonstrating higher uPAR staining intensity in metastasizing tumors. As pathway analyses identified epidermal growth factor (EGF) as a potential upstream regulator of PLAUR, the effect of EGF on uPAR expression levels and cell motility was functionally validated in human metastatic cSCC cells. In conclusion, we propose that uPAR is an important driver of metastasis in cSCC and represents a potential therapeutic target in this disease.

Defining the incidence of cutaneous squamous cell carcinoma in coastal NSW Australia.

BACKGROUND/OBJECTIVES: To describe the incidence of primary cutaneous squamous cell carcinoma in coastal NSW Australia. METHODS: The design is a case-controlled study of reported cSCC from 2016 to 2019 within a defined region of coastal southern NSW. Participants include all reported pathological diagnoses of cSCC in patients greater than 20 years of age. The main outcome measures the incidence and relative risk of cSCC. RESULTS: The age-adjusted incidence rate of primary cSCC was 856/105 /year. Men over 60 years of age had an age-adjusted incidence rate of 2875/106 /year. Histologically diagnosed invasive SCC samples were included using SNOMED clinical term codes. Keratoacanthomas and SCC in situ SNOWMED codes were not included. SCC in situ results was found within the sample analysis and was offset by including one SCC per annum per person. CONCLUSIONS: The rates of cSCC are far higher than previously reported and demand a reappraisal of our national management of this disease.