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.

Micro RNA Dysregulation in Keratinocyte Carcinomas: Clinical Evidence, Functional Impact, and Future Directions.

The keratinocyte carcinomas, basal cell carcinoma (BCC), and cutaneous squamous cell carcinoma (cSCC), are the most common cancers in humans. Recently, an increasing body of literature has investigated the role of miRNAs in keratinocyte carcinoma pathogenesis, progression and their use as therapeutic agents and targets, or biomarkers. However, there is very little consistency in the literature regarding the identity of and/or role of individual miRNAs in cSCC (and to a lesser extent BCC) biology. miRNA analyses that combine clinical evidence with experimental elucidation of targets and functional impact provide far more compelling evidence than studies purely based on clinical findings or bioinformatic analyses. In this study, we review the clinical evidence associated with miRNA dysregulation in KCs, assessing the quality of validation evidence provided, identify gaps, and provide recommendations for future studies based on relevant studies that investigated miRNA levels in human cSCC and BCC. Furthermore, we demonstrate how miRNAs contribute to the regulation of a diverse network of cellular functions, and that large-scale changes in tumor cell biology can be attributed to miRNA dysregulation. We highlight the need for further studies investigating the role of miRNAs as communicators between different cell types in the tumor microenvironment. Finally, we explore the clinical benefits of miRNAs as biomarkers of keratinocyte carcinoma prognosis and treatment.

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.

Demystifying emerging bulk RNA-Seq applications: the application and utility of bioinformatic methodology.

Significant innovations in next-generation sequencing techniques and bioinformatics tools have impacted our appreciation and understanding of RNA. Practical RNA sequencing (RNA-Seq) applications have evolved in conjunction with sequence technology and bioinformatic tools advances. In most projects, bulk RNA-Seq data is used to measure gene expression patterns, isoform expression, alternative splicing and single-nucleotide polymorphisms. However, RNA-Seq holds far more hidden biological information including details of copy number alteration, microbial contamination, transposable elements, cell type (deconvolution) and the presence of neoantigens. Recent novel and advanced bioinformatic algorithms developed the capacity to retrieve this information from bulk RNA-Seq data, thus broadening its scope. The focus of this review is to comprehend the emerging bulk RNA-Seq-based analyses, emphasizing less familiar and underused applications. In doing so, we highlight the power of bulk RNA-Seq in providing biological insights.

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.

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.

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.

The experience of nutritional care according to patients with head and neck cancer involved with a combined dietitian, specialist nurse and speech pathologist clinic in a regional Australia: a qualitative longitudinal study.

PURPOSE: Malnutrition is a co-morbidity of head and neck cancer (HNC) that has negative consequences for patients. Evidence-based guidelines (EBGs) provide recommendations to prevent and manage malnutrition. A clinic that combines the services of a dietitian, specialist oncology nurse and speech pathologist may promote the implementation of nutritional EBGs in regional Australia. This study aimed to explore the nutritional care experience that patients with HNC had in this setting. METHODS: A qualitative longitudinal study collected data via semi-structured interviews with HNC patients who were treated in one regional cancer care network in Australia. Interviews were conducted at key points in their HNC journey from diagnosis to 4 months post-radiotherapy. Data was analysed using a grounded theory approach. RESULTS: Ten participants completed a total of thirty-six interviews. The findings were grouped into four categories: "preparing for nutritional challenges", "multidisciplinary care directed by patient needs", "the battle to eat", and "incongruence between patient values and nutritional priorities". CONCLUSION: These findings highlight the nutritional burden associated with HNC and barriers to patients accepting nutritional support from healthcare professionals. Information provided by doctors and nurses prior to treatment may help patients prepare for the nutritional challenges ahead and accept support from dietitians. Furthermore, clinics that promote continuity through treatment and allow dietitians to lead aspects of nutritional care, in collaboration with nurses, speech pathologists and doctors, may also enhance the nutritional care experience. More qualitative research within HNC teams would provide further insight on enhancing the implementation of nutritional EBGs to improve outcomes for these patients.

Targeted ordering of investigations reduces costs of treatment for surgical inpatients.

BACKGROUND: Laboratory testing forms an important part of diagnostic investigation in modern medicine; however, the overuse of 'routine blood tests' can result in significant potential harm and financial cost to the patient and the healthcare system. In 2018, a new protocol targeting the ordering of investigations was implemented within the General Surgical Teams of Wollongong Hospital in New South Wales, an Australian tertiary referral hospital, to reduce the number of 'routine blood tests' as a quality improvement initiative. OBJECTIVE: To identify whether there was a reduction in the number of 'routine blood tests' and associated costs following implementation of the new protocol. METHODS: The protocol involved regular review of the laboratory investigations being ordered for the following day with a senior team member. The medical records of all patients admitted under the general surgery service at Wollongong Hospital were retrospectively reviewed over two 10-week periods in 2017 and 2018 (control and study, respectively). The casemix was categorized into Minor, Intermediate, Major or Unscored, depending on case complexity coding. RESULTS: A total of 838 patients were identified during the control period (2017) and 805 patients were identified during the study period (2018). Ten thousand and thirty tests were included in the control period, compared to 8610 over the study period, resulting in a 16% (or greater) reduction in 'routine blood tests' per patient, per day of admission and a 6% reduction in costs in the study group (P < 0.001). CONCLUSION: Targeted ordering of investigations with personalized education and feedback to junior staff during review of clinical status of each patient as a part of normal workflow can reduce inappropriate ordering of 'routine blood tests' and associated costs to the patient and the healthcare system.

Comprehensive Mutational and Phenotypic Characterization of New Metastatic Cutaneous Squamous Cell Carcinoma Cell Lines Reveal Novel Drug Susceptibilities.

Cutaneous squamous cell carcinoma (cSCC) is a common skin cancer. Most patients who develop metastases (2-5%) present with advanced disease that requires a combination of radical surgery and adjuvant radiation therapy. There are few effective therapies for refractory disease. In this study, we describe novel patient-derived cell lines from cSCC metastases of the head and neck (designated UW-CSCC1 and UW-CSCC2). The cell lines genotypically and phenotypically resembled the original patient tumor and were tumorogenic in mice. Differences in cancer-related gene expression between the tumor and cell lines after various culturing conditions could be largely reversed by xenografting and reculturing. The novel drug susceptibilities of UW-CSCC1 and an irradiated subclone UW-CSCC1-R to drugs targeting cell cycle, PI3K/AKT/mTOR, and DNA damage pathways were observed using high-throughput anti-cancer and kinase-inhibitor compound libraries, which correlate with either copy number variations, targetable mutations and/or the upregulation of gene expression. A secondary screen of top hits in all three cell lines including PIK3CA-targeting drugs supports the utility of targeting the PI3K/AKT/mTOR pathway in this disease. UW-CSCC cell lines are thus useful preclinical models for determining targetable pathways and candidate therapeutics.

Sentinel Node Biopsy in 105 High-Risk Cutaneous SCCs of the Head and Neck: Results of a Multicenter Prospective Study.

BACKGROUND: Regional nodal metastases from cutaneous squamous cell carcinoma (cSCC) is strongly associated with a poor prognosis, but these metastases are difficult to predict clinically. Sentinel node biopsy (SNB) has been used for a wide range of malignancies to assess for regional nodal metastasis, but is not widely used for cSCC. METHODS: Patients presenting with high-risk cSCC of the head and neck with clinically N0 necks were offered SNB at the time of primary cSCC excision or secondary wide local excision. Patients with positive sentinel nodes were offered completion lymph node dissection, and all the patients were followed up at regular intervals for up to 5 years. RESULTS: In this study, 105 lesions underwent SNB, and 10 sentinel nodes (9.5%) were positive. In an additional five patients, regional recurrence developed after a negative sentinel node, with a total subclinical nodal metastasis rate of 14.3%. Nodal metastases were significantly associated with reduced disease-specific survival. The significant predictors of metastasis were four or more high-risk features or tumors with a concurrent invasion deeper than 5 mm and PNI. CONCLUSION: For high-risk cSCC, SNB is a safe and feasible staging technique. The total number of high risk features and certain combinations of high-risk features predicted metastasis better than individual high-risk features.

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 formalin-fixed 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 (<45 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.

Analysis and Comparison of the 8th Edition American Joint Committee on Cancer (AJCC) Nodal Staging System in Cutaneous and Oral Squamous Cell Cancer of the Head and Neck.

BACKGROUND: The American Joint Committee on Cancer (AJCC) uses the same nodal staging system for cutaneous and mucosal squamous cell carcinoma of the head and neck in its 8th edition (AJCC 8) despite differences in the etiology, risk factors, and clinical behavior of the two diseases. This study aims to evaluate the performance of the AJCC 8 nodal staging system by direct comparison of cutaneous (cSCC) versus oral squamous cell carcinoma (oSCC) patients. METHODS: Patients with metastatic cSCC (N = 382) and oSCC (N = 325) were identified from a prospective database (years 1987-2016). Multivariable analysis was performed using Cox proportional hazards competing risk model. To assess staging system performance, an explained variation measure (proportion of variation explained, PVE) as well as a discrimination measure (Harrell's concordance index, C-index) were used. RESULTS: Inclusion of extranodal extension (ENE) in AJCC 8 increased the proportion of patients in N3b category (48.7% in cSCC, 40.3% in oSCC). AJCC 8 stratified poorly with regards to risk of death from cSCC and oSCC and showed limited monotonicity of the nodal categories. Estimates of model performance revealed modest predictive capacity for overall survival (OS) and disease-specific survival (DSS) in oSCC (Harrell's C of 0.66 in both) and weak predictive capacity in cSCC (Harrell's C of 0.58 and 0.61, respectively). CONCLUSIONS: The AJCC 8 nodal staging system performs poorly in terms of stratifying survival by N category, especially in cSCC. The data indicate that cSCC merits an independent nodal staging system from that for mucosal SCC.

Predictive value of the 8th edition American Joint Commission Cancer (AJCC) nodal staging system for patients with cutaneous squamous cell carcinoma of the head and neck.

BACKGROUND: The 8th edition American Joint Committee on Cancer (AJCC8) provides the same nodal staging system for mucosal and cutaneous squamous cell carcinoma of the head and neck (HNcSCC) and includes extranodal extension (ENE) as an adverse prognostic criterion. This study evaluates the prognostic efficacy of the AJCC8 pathologic nodal staging system (pN) for HNcSCC. METHODS: Univariate analysis of 382 patients with metastatic HNcSCC staged according to both the 7th (AJCC7) and the 8th edition staging systems. RESULTS: The AJCC7 pN3 category was associated with reduced disease specific survival (DSS HR 5.49; 95% CI: 1.83-16.53; P = 0.002) and overall survival (OS HR 3.42; 95% CI: 1.54-7.58; P = 0.002) as compared with pN1. However, no difference was observed between pN1, pN2, and pN3 categories as defined by the AJCC8. Also, when comparing Stages III and IV as defined by AJCC8, there was no difference in DSS (HR 0.75; 95% CI: 0.34-1.67; P = 0.478) or OS (HR 0.88; 95% CI: 0.51-1.51; P = 0.648). CONCLUSION: The AJCC8 performed poorly as a prognostic indicator for patients with metastatic HNcSCC in this cohort. HNcSCC would benefit from a staging system that accounts for its unique biologic characteristics distinct from mucosal SCC.

Education and support needs in patients with head and neck cancer: A multi-institutional survey.

BACKGROUND: Head and neck cancer (HNC) encompasses a diverse group of tumors, and thus providing appropriate and tailored information to patients before, during, and after treatment is a challenge. The objective of the current study was to characterize the experience and unmet needs of patients with HNC with regard to information and support provision. METHODS: A 28-question, cross-sectional survey was completed by patients treated for HNC at 1 of 4 institutions in New South Wales, Australia (Chris O'Brien Lifehouse and Liverpool, Westmead, and Wollongong hospitals). It consisted of the adapted Kessler Psychological Distress Scale and questions assessing information quality, quantity, and format. RESULTS: A total of 597 patients responded. The mean age of the patients was 58 years (range, 21-94 years) with 284 men and 313 women (1:1.1). The majority of patients reported information concerning the disease process (76%), prognosis (67%), and treatment (77%) was sufficient, and approximately 50% reporting having received little or no information regarding coping with stress and anxiety. A substantial percentage of patients reported receiving minimal information concerning psychosexual health (56%) or the availability of patient support groups (56%). The majority of patients preferred access to multiple modes of information delivery (72%), with the preferred modality being one-on-one meetings with a health educator (37%) followed by internet-based written information (19%). CONCLUSIONS: Patients with HNC are a diverse group, with complex educational and support needs. Patients appear to be given information regarding survivorship topics such as psychological well-being, patient support groups, and psychosexual health less frequently than information concerning disease and treatment. Verbal communication needs to be reinforced by accessible, well-constructed, written and multimedia resources appropriate to the patient's educational level. Cancer 2017;123:1949-1957. © 2017 American Cancer Society.

Bone resection for facial cutaneous malignancies.

BACKGROUND AND OBJECTIVES: The aim of this study is to analyze the clinical outcomes of patients who underwent bone resection for cutaneous malignancy of the face and scalp. METHODS: We retrospectively collected patient data from 62 patients who underwent bone resection for craniofacial cutaneous malignancy of the face and scalp over the last 10 years. We investigated risk factors for disease progression and assessed the utility of pre-operative imaging to predict bone, dura, and brain infiltration. RESULTS: Out of all factors analyzed, brain invasion, surgical margin involvement, and dural margin involvement were found to significantly reduce survival. CT and MRI correctly predicted bone infiltration in 88% and 89% of cases. MRI correctly predicted dura invasion in 89% but grossly underestimated the amount of dural invasion in 23% of reports. CONCLUSIONS: Our data indicate that the resection of bone is a reasonable surgical option in the treatment of patients with advanced cutaneous malignancies of the face and scalp. Brain invasion and positive margins reduced the probability of survival.

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.

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.