Proliferating macrophages in human tumours show characteristics of monocytes responding to myelopoietic growth factors.

Macrophages play essential roles in maintaining tissue homeostasis and immune defence. However, their extensive infiltration into tumours has been linked to adverse outcomes in multiple human cancers. Within the tumour microenvironment (TME), tumour-associated macrophages (TAMs) promote tumour growth and metastasis, making them prime targets for cancer immunotherapy. Recent single-cell analysis suggest that proliferating TAMs accumulate in human cancers, yet their origins and differentiation pathways remain uncertain. Here, we show that a subpopulation of CD163+ TAMs proliferates in situ within the TME of melanoma, lung cancer, and breast cancer. Consistent with their potential role in suppressing anti-tumour activities of T cells, CD163+ TAMs express a range of potent immunosuppressive molecules, including PD-L1, PD-L2, IL-10, and TGF-ß. Other phenotypic markers strongly suggested that these cells originate from CD14+ CCR2+ monocytes, a cell population believed to have minimal capacity for proliferation. However, we demonstrate in vitro that certain myelopoietic cytokines commonly available within the TME induce robust proliferation of human monocytes, especially the combination of interleukin 3 (IL-3) and Macrophage Colony-Stimulating Factor 1 (M-CSF). Monocytic cells cultured with these cytokines efficiently modulate T cell proliferation, and their molecular phenotype recapitulates that of CD163+ TAMs. IL-3-driven proliferation of monocytic cells can be completely blocked by IL-4, associated with the induction of CDKN1A, alongside the upregulation of transcription factors linked to dendritic cell function, such as BATF3 and IRF4. Taken together, our work suggests several novel therapeutic routes to reducing immunosuppressive TAMs in human tumours, from blocking chemokine-mediated recruitment of monocytes to blocking their proliferation.

Seven-colour multiplex immunochemistry/immunofluorescence and whole slide imaging of frozen sections.

Multiplex Immunochemistry/Immunofluorescence (mIHC/IF) aims to visualise multiple biomarkers in a single tissue section and is especially powerful when used on slide scanners coupled with digital analysis tools. mIHC/IF is commonly employed in immuno-oncology to characterise features of the tumour microenvironment (TME) and correlate them with clinical parameters to guide prognostication and therapy. However, mIHC/IF can be applied to a wide range of organisms in any physiological or disease context. Recent innovation has extended the number of markers that can be detected using slide scanners well beyond the 3-4 markers typically reported in traditional fluorescence microscopy. However, these methods often require sequential antibody staining and stripping, and are not compatible with frozen tissue sections. Using fluorophore-conjugated antibodies, we have established a simple mIHC/IF imaging workflow that enables simultaneous staining and detection of seven markers in a single section of frozen tissue. Coupled with automated whole slide imaging and digital quantification, our data efficiently revealed the tumour-immune complexity in metastatic melanoma. Computational image analysis quantified the immune and stromal cell populations present in the TME as well as their spatial interactions. This imaging workflow can also be performed with an indirect labelling panel consisting of primary and secondary antibodies. Our new methods, combined with digital quantification, will provide a valuable tool for high-quality mIHC/IF assays in immuno-oncology research and other translational studies, especially in circumstances where frozen sections are required for detection of particular markers, or for applications where frozen sections may be preferred, such as spatial transcriptomics.

Effect of Microdoses of Incisional Antibiotics on the Rate of Surgical Site Infections in Skin Cancer Surgery: A Randomized Clinical Trial.

Importance: Surgical site infections (SSIs) represent a costly and preventable complication of cutaneous surgery. However, there is a paucity of randomized clinical trials investigating antibiotic prophylaxis for reducing SSIs in skin cancer surgery, and evidence-based guidelines are lacking. Incisional antibiotics have been shown to reduce the rate of SSIs before Mohs micrographic surgery, but this represents a small subset of skin cancer surgery. Objective: To determine whether microdosed incisional antibiotics reduce the rate of SSIs before skin cancer surgery. Design, Setting, and Participants: In this double-blind, controlled, parallel-design randomized clinical trial, adult patients presenting to a high-volume skin cancer treatment center in Auckland, New Zealand, for any form of skin cancer surgery over 6 months from February to July 2019 were included. Patient presentations were randomized to one of 3 treatment arms. Data were analyzed from October 2021 to February 2022. Interventions: Patients received an incision site injection of buffered local anesthetic alone (control), buffered local anesthetic with microdosed flucloxacillin (500 µg/mL), or buffered local anesthetic with microdosed clindamycin (500 µg/mL). Main Outcomes and Measures: The primary end point was the rate of postoperative SSI (calculated as number of lesions with SSI per total number of lesions in the group), defined as a standardized postoperative wound infection score of 5 or more. Results: A total of 681 patients (721 total presentations; 1133 total lesions) returned for postoperative assessments and were analyzed. Of these, 413 (60.6%) were male, and the mean (SD) age was 70.4 (14.8) years. Based on treatment received, the proportion of lesions exhibiting a postoperative wound infection score of 5 or greater was 5.7% (22 of 388) in the control arm, 5.3% (17 of 323) in the flucloxacillin arm, and 2.1% (9 of 422) in the clindamycin arm (P = .01 for clindamycin vs control). Findings were similar after adjusting for baseline differences among arms. Compared with lesions in the control arm (31 of 388 [8.0%]), significantly fewer lesions in the clindamycin arm (9 of 422 [2.1%]; P < .001) and flucloxacillin (13 of 323 [4.0%]; P = .03) arms required postoperative systemic antibiotics. Conclusions and Relevance: This study evaluated the use of incisional antibiotics for SSI prophylaxis in general skin cancer surgery and compared the efficacy of flucloxacillin vs clindamycin relative to control in cutaneous surgery. The significant reduction in SSI with locally applied microdosed incisional clindamycin provides robust evidence to inform treatment guidelines in this area, which are currently lacking. Trial Registration: anzctr.org.au Identifier: ACTRN12616000364471.

Dynamic ctDNA Mutational Complexity in Patients with Melanoma Receiving Immunotherapy.

BACKGROUND: Circulating tumour DNA (ctDNA) analysis promises to improve the clinical care of people with cancer, address health inequities and guide translational research. This observational cohort study used ctDNA to follow 29 patients with advanced-stage cutaneous melanoma through multiple cycles of immunotherapy. METHOD: A melanoma-specific ctDNA next-generation sequencing (NGS) panel, droplet digital polymerase chain reaction (ddPCR) and mass spectrometry analysis were used to identify ctDNA mutations in longitudinal blood plasma samples from Aotearoa New Zealand (NZ) patients receiving immunotherapy for melanoma. These technologies were used in conjunction to identify the breadth and complexity of tumour genomic information that ctDNA analysis can reliably report. RESULTS: During the course of immunotherapy treatment, a high level of dynamic mutational complexity was identified in blood plasma, including multiple BRAF mutations in the same patient, clinically relevant BRAF mutations emerging through therapy and co-occurring sub-clonal BRAF and NRAS mutations. The technical validity of this ctDNA analysis was supported by high sample analysis-reanalysis concordance, as well as concordance between different ctDNA measurement technologies. In addition, we observed > 90% concordance in the detection of ctDNA when using cell-stabilising collection tubes followed by 7-day delayed processing, compared with standard EDTA blood collection protocols with rapid processing. We also found that the undetectability of ctDNA at a proportion of treatment cycles was associated with durable clinical benefit (DCB). CONCLUSION: We found that multiple ctDNA processing and analysis methods consistently identified complex longitudinal patterns of clinically relevant mutations, adding support for expanded clinical trials of this technology in a variety of oncology settings.

Readability, reliability and credibility of online patient information on skin grafts.

BACKGROUND: Skin grafting reflects a common dermatological procedure for closing skin defects. Patient education is important for managing expectation and optimising skin graft take. While health information is increasingly accessed on the internet, there are no existing studies assessing their quality. METHODS: The first 25 results from Google, Microsoft Bing and Yahoo! search engines using the term 'skin graft' were analysed using a variety of standard instruments. Readability was assessed using the Flesch-Kincaid Grade score (FKG), Gunning Fog Index (GFI), Simple Measure of Gobbledygook (SMOG) and the New Dale-Chall Readability Index (NDC). Reliability was assessed using the DISCERN instrument and credibility with the Journal of the American Medical Association Benchmark Criteria (JAMA). Transparency was identified by presence of the Health On the Net Foundation Code certification (HON-code). RESULTS: Seventy-five websites were identified. After exclusion, forty-three remaining websites were analysed with average FKG, GFI and SMOG scores of 7.8, 10.1 and 10.7, respectively. The average NDC was 5.9. The average reliability was fair with a DISCERN score based on the first 15 questions of the instrument of 42.6. The mean JAMA score was 2, and 9 websites displayed the HON-code certificate. CONCLUSIONS: Readability, reliability and credibility of online health information regarding skin grafting can be improved. Health care providers should critically assess existing online patient information or develop alternative material to educate patients undergoing skin graft surgery.

Microbiology of surgical site infections (SSIs) following skin cancer surgery.

BACKGROUNDS: Surgical site infections (SSIs) represent one of the most common and potentially preventable sources of morbidity and healthcare cost escalation associated with skin cancer surgery. There is a lack of data reporting organisms cultured from SSIs in skin surgery, with guidelines for antibiotic prophylaxis based on common skin pathogens rather than actual cultured organisms. In this study, we sought to define the cultured microbiology of SSIs specific to skin cancer surgery and test these against empiric treatment guidelines. METHODS: All consenting patients presenting to the Auckland regional skin cancer treatment centre over a 6-month period were included. Patients receiving any form of antibiotics within a week prior to surgery were excluded. All wounds were assessed postoperatively, with clinically significant infections identified as those with a standardized wound infection score of 4 (range 0-7) and/or prescribed post-operative antibiotics within 3 weeks of surgery. Wound cultures were recorded. RESULTS: About 104 clinically significant SSIs were identified from 333 lesions treated, with cultures available in 27%. Cultured organisms included MSSA (79%), MRSA (14%), coagulase-negative Staphylococci (11%), and 'skin flora' (14%). Empiric guidelines inaccurately predicted effective treatment in 14% of cases, exclusively due to MRSA. CONCLUSION: To our knowledge this is the first comprehensive report of SSI microbiology following skin cancer surgery. The overwhelmingly predominant organisms were Staphylococcus sp. (76%), with the rate of MRSA approaching prevalence warranting empiric first-line treatment. These data help inform effective rationalized empiric antibiotic treatment, when indicated, for optimal outcome following skin surgery.

A survey of surgical management of the sentinel node positive melanoma patient in the post-MSLT2 era.

BACKGROUND: The evidence-based management of melanoma patients with a positive sentinel lymph node biopsy (SLNB) has undergone a dramatic shift following publication of practice-changing surgical trials demonstrating no melanoma-specific survival advantage for completion lymph node dissection (CLND) in this scenario. We aimed to survey how surgeons' clinical practice had shifted in response to new evidence from these trials, and at a time when there was starting to become available systemic adjuvant treatments for AJCC Stage III melanoma patients. METHODS: A web-based survey consisting of practice-based questions and hypothetical clinical scenarios about current melanoma practice with regard to positive sentinel node biopsy was developed and sent to the surgical members of a Melanoma and Skin Cancer (MASC) Trials group in December 2018. Responses were analysed using descriptive statistics. RESULTS: There were 212 invitations sent and 65 respondents (31%). Respondents were from 17 countries, 94% of whom practice in specialist melanoma centres or at referral centres. Of these 97% were familiar with the MSLT2 and DeCOG-SLT clinical trials. At survey, 5% of respondents reported routinely recommending CLND and 55% recommend CLND in selected cases. Respondents were most likely to recommend CLND when multiple SLNs were positive. Important factors for surgical decision-making mentioned included size of SLN deposit, number of positive SLNs and likely compliance with the recommended surveillance regimen. CONCLUSION: In line with rapid adoption of published evidence, surgical management of Stage III melanoma has altered significantly, with few surgeons within the cohort now performing routine CLNDs after positive SLNB.

Impact of Shave Biopsy on Diagnosis and Management of Cutaneous Melanoma: A Systematic Review and Meta-Analysis.

BACKGROUND: Melanoma is the most lethal skin cancer. Excision biopsy is generally recommended for clinically suspicious pigmented lesions; however, a proportion of cutaneous melanomas are diagnosed by shave biopsy. A systematic review was undertaken to investigate the impact of shave biopsy on tumor staging, treatment recommendations, and prognosis. METHODOLOGY: The MEDLINE, Embase, and Cochrane Library databases were searched for relevant articles. Data on deep margin status on shave biopsy, tumor upstaging, and additional treatments on wide local excision (WLE), disease recurrence, and survival effect were analyzed across studies. RESULTS: Fourteen articles from 2010 to 2020 were included. In total, 3713 patients had melanoma diagnosed on shave biopsy. Meta-analysis revealed a positive deep margin in 42.9% of shave biopsies. Following WLE, change in tumor stage was reported in 7.7% of patients. Additional treatment was recommended for 2.3% of patients in the form of either further WLE and/or sentinel lymph node biopsy. There was high heterogeneity across studies in all outcomes. Four studies reported survival, while no studies found any significant difference in disease-free or overall survival between shave biopsy and other biopsy modalities. CONCLUSIONS: Just over 40% of melanomas diagnosed on shave biopsy report a positive deep margin; however, this translated into a change in tumor stage or treatment recommendations in relatively few patients (7.7% and 2.3%, respectively), with no impact on local recurrence or survival among the studies analyzed.

Is Surgical Site Infection (SSI) after skin surgery affected by home municipal versus non-municipal water supply?

PURPOSE: SSI represent one of the most common sources of morbidity and escalated healthcare costs in skin cancer management. It has been shown that exposing wounds to treated water does not increase SSIs, however a large proportion of Australasian patients reside in rural areas dependant on roof or bore collected water for their primary water supply, and no data exist regarding the association between tank water supply and SSI following skin surgery. METHODOLOGY: A nine-month retrospective analysis of patients undergoing skin cancer surgery at the Auckland Regional Plastic Surgery Unit was performed. Wounds assessed using a validated wound infection scoring system. Rates of SSI analysed against various clinical factors (water supply, smoking status, immunocompromise, glucose intolerance) and surgical factors (type of reconstruction, ulceration, lesion site, surface area of lesion). RESULTS: 857 lesions were excised from 357 patients over the period studied. 718 lesions (83.7%) had municipal and 139 lesions (16.3%) had non-municipal water as their primary supply. Overall rate of clinically significant SSI was 15.6%, with no difference between municipal and non-municipal water groups (15.6% vs. 15.8% P = 0.946). Further subgroup analysis did not reveal any difference in rate of SSI based on type of surgical closure (direct closure, skin graft vs. flap). CONCLUSION: Non-municipal water supply was not associated with change in SSI relative to home municipal water supply in patients receiving skin cancer surgery. Our data supplements existing literature that water exposure does not influence SSI following skin surgery irrespective of primary home water supply.

Distinctive Subpopulations of Stromal Cells Are Present in Human Lymph Nodes Infiltrated with Melanoma.

Metastasis of human tumors to lymph nodes (LN) is a universally negative prognostic factor. LN stromal cells (SC) play a crucial role in enabling T-cell responses, and because tumor metastases modulate their structure and function, this interaction may suppress immune responses to tumor antigens. The SC subpopulations that respond to infiltration of malignant cells into human LNs have not been defined. Here, we identify distinctive subpopulations of CD90+ SCs present in melanoma-infiltrated LNs and compare them with their counterparts in normal LNs. The first population (CD90+ podoplanin+ CD105+ CD146+ CD271+ VCAM-1+ ICAM-1+ α-SMA+) corresponds to fibroblastic reticular cells that express various T-cell modulating cytokines, chemokines, and adhesion molecules. The second (CD90+ CD34+ CD105+ CD271+) represents a novel population of CD34+ SCs embedded in collagenous structures, such as the capsule and trabeculae, that predominantly produce extracellular matrix. We also demonstrated that these two SC subpopulations are distinct from two subsets of human LN pericytes, CD90+ CD146+ CD36+ NG2- pericytes in the walls of high endothelial venules and other small vessels, and CD90+ CD146+ NG2+ CD36- pericytes in the walls of larger vessels. Distinguishing between these CD90+ SC subpopulations in human LNs allows for further study of their respective impact on T-cell responses to tumor antigens and clinical outcomes.

Evaluation of a novel database for quality assurance at a head and neck service in New Zealand: an audit of free flap head and neck reconstruction.

BACKGROUND: Clinical audit is a critical quality improvement exercise, yet efficient audit tools are lacking. The main objective of this study was to evaluate a recently deployed database in facilitating the process of clinical audit, and the secondary objective was to evaluate the outcomes of free flap reconstruction of the head and neck at our centre. METHODS: A head and neck cancer-specific database was customized to suit the needs of our head and neck multidisciplinary team. Data has been entered prospectively into this database since March of 2018. An audit of free flap reconstruction of the head and neck over a 12-month period was performed using the database and analysed as a case study to examine its efficacy as a clinical audit tool. Additionally, the outcomes of free flap reconstruction at our centre were compared to those reported in the international literature. RESULTS: The database allows flexible and specific queries, analysis and export of data, and can provide immediate results. However, issues with data quality and completeness were identified. In this audit, the overall 30-day complication rate and 30-day mortality in patients undergoing free flap reconstruction of the head and neck were 58% and 3%, respectively. CONCLUSION: The database is fit for its intended purpose as an audit tool. Outcomes of free flap reconstruction of the head and neck at our centre are comparable to those of institutions overseas.

Both Bone Forearm Infected Nonunion: Report of a One-Bone Free Fibula Flap Salvage and Literature Review.

Background: Both bone forearm infective nonunions represent a rare but functionally limb threatening condition. Method: We report a successful salvage of a severe near total both bone diaphysial osteomyelitis by conversion to a one-bone forearm with free fibula flap. A literature review on forearm salvage addressing both bone defects was performed. Results: Bony union was achieved at 4 months with a highly functional extremity salvage in our case. Conclusion: While very little prior experience has been reported for long segmental both bone forearm infected nonunions, we report of this highly satisfactory salvage using one-bone free tissue transfer strategy. We also provided our literature review with history, indication and evolution of individualized treatment options for this difficult surgical condition.

Evolving Threat of Community Acquired Methicillin Resistant Staphylococcus aureus Upper Extremity Infections in the South Pacific: 2011-2015.

Background: Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) rates have been increasing worldwide and contribute to a growing "global health security threat" as reported by the WHO. Our group previously reported an overall rate of 7% in CA-MRSA upper extremity infections between 2004-2009 at the Auckland Regional Hand Unit. This fell below the Center for Disease Control (CDC) recommendation for empiric antimicrobial cover once local rates exceed 10-15%. We examined prevalence and characteristics of CA-MRSA upper extremity infections in our region over a subsequent 5-year period. Methods: One thousand two hundred and fifty-two patients with upper extremity infections requiring operative management between 2011 and 2015 inclusive were included in this study. Associated clinical characteristics were recorded including ethnicity, cultured organisms, antibiotic sensitivities, infection rate, and treatment practice. Results: One hundred and fifty (12%) of patients had culture positive CA-MRSA upper extremity infections. There was an increasing annual trend. Of note, rates of CA-MRSA in the Maori and Pacific Island ethnic subpopulations exceeded 15% in 2014 and 2015. Susceptibilities, associated factors and patient demographics are reported. Conclusions: Our unit enjoys significantly lower rates of CA-MRSA upper extremity infections than has been reported internationally. However, trends are increasing relative to our prior 6-year report, and the threshold for empiric treatment has been met within the Maori and Pacific Island ethnic subpopulations. This evolving threat is also highlighted by increasing cases of multi-drug resistant CA-MRSA. Evolving regional guidelines for empiric coverage of CA-MRSA among high-risk ethnic subpopulations identified by this study are underway.

Epidemiology and survival of Merkel cell carcinoma in New Zealand: A population-based study between 2000 and 2015 with international comparison.

BACKGROUND/OBJECTIVES: Merkel cell carcinoma is an aggressive neuroendocrine skin cancer. Australian studies report high incidence and poor survival rates compared internationally. While New Zealand has a comparable UV index and racial composition to Australia, survival outcomes are currently unknown. The role of Merkel cell polyoma virus in oncogenesis of Merkel cell carcinoma is an active area of research. We describe the incidence and survival of Merkel cell carcinoma in New Zealand with correlation to demographic and clinical factors including regional polyoma virus prevalence. METHODS: Retrospective study of population-based data from the New Zealand Cancer Registry. Incidence rates were directly standardised to the US standard 2000 population. Survival was investigated using Kaplan-Meier and multivariable Cox regression models. RESULTS: Six hundred and one cases were diagnosed in New Zealand between 2000 and 2015. The overall incidence rate was 0.96/100 000 population. Merkel cell carcinoma is more common in males, elderly and on sun-exposed areas. Eighteen percent of patients were diagnosed with distant metastasis at time of presentation. The overall 5-year survival rate and relative 5-year survival rate were 31% and 45%, respectively. Mortality was 1.9 and 2.5 times higher for stage III and IV disease, respectively, relative to stage I/II disease. Patients over age 80 had twice the mortality compared to those aged 60-69. CONCLUSIONS: New Zealand has a high incidence of Merkel cell carcinoma and poor survival outcomes when compared internationally. We have the highest proportion of distant metastatic disease at time of diagnosis. Further research into the role of nonpolyoma-related Merkel cell carcinoma is warranted to improve Merkel cell carcinoma outcomes in New Zealand and abroad.

Essential Components of Melanoma Histopathological Reporting: The Surgical Oncologist's Perspective.

Histopathological reporting plays a critical role in guiding the surgical oncologist's management plan in treatment of primary cutaneous melanoma. The International Collaboration on Cancer Reporting (ICCR) espouses various components of structured histopathological reporting as "essential" or "recommended." From a surgical oncologist's perspective, we discuss the clinical relevance of each essential component, as well as prognostic and treatment implications with regard to treatment planning.