Whole-genome sequencing of acral melanoma reveals genomic complexity and diversity.

To increase understanding of the genomic landscape of acral melanoma, a rare form of melanoma occurring on palms, soles or nail beds, whole genome sequencing of 87 tumors with matching transcriptome sequencing for 63 tumors was performed. Here we report that mutational signature analysis reveals a subset of tumors, mostly subungual, with an ultraviolet radiation signature. Significantly mutated genes are BRAF, NRAS, NF1, NOTCH2, PTEN and TYRP1. Mutations and amplification of KIT are also common. Structural rearrangement and copy number signatures show that whole genome duplication, aneuploidy and complex rearrangements are common. Complex rearrangements occur recurrently and are associated with amplification of TERT, CDK4, MDM2, CCND1, PAK1 and GAB2, indicating potential therapeutic options.

Whole-genome landscape of mucosal melanoma reveals diverse drivers and therapeutic targets.

Knowledge of key drivers and therapeutic targets in mucosal melanoma is limited due to the paucity of comprehensive mutation data on this rare tumor type. To better understand the genomic landscape of mucosal melanoma, here we describe whole genome sequencing analysis of 67 tumors and validation of driver gene mutations by exome sequencing of 45 tumors. Tumors have a low point mutation burden and high numbers of structural variants, including recurrent structural rearrangements targeting TERT, CDK4 and MDM2. Significantly mutated genes are NRAS, BRAF, NF1, KIT, SF3B1, TP53, SPRED1, ATRX, HLA-A and CHD8. SF3B1 mutations occur more commonly in female genital and anorectal melanomas and CTNNB1 mutations implicate a role for WNT signaling defects in the genesis of some mucosal melanomas. TERT aberrations and ATRX mutations are associated with alterations in telomere length. Mutation profiles of the majority of mucosal melanomas suggest potential susceptibility to CDK4/6 and/or MEK inhibitors.

Significant association of PD-L1 expression with human papillomavirus positivity and its prognostic impact in oropharyngeal cancer.

BACKGROUND: The programmed death pathway plays a role in persistent human papillomavirus (HPV) infection as well as in resistance to immune elimination during malignant progression. In this study, we examined PD-L1 expression by immunohistochemistry and tumour infiltrating lymphocytes (TIL) in 214 patients with oropharyngeal squamous cell cancer (OPSCC) to assess its clinical significance. RESULTS: HPV-positive OPSCC were significantly more likely to express PD-L1 than HPV-negative OPSCC (85.2% vs 57.1%, p < 0.05). PD-L1 staining was more likely to be associated with TILs in HPV-positive OPSCC (67.9% vs 49.6%, p = 0.01). Relative to those patients with HPV-positive/PD-L1-positive OPSCC, patients with HPV negative/PD-L1 negative OPSCC were 6.4 times more likely to develop a local recurrence, 5.8 times more likely to develop an event and 6.5 times more likely to die. Within the HPV positive cases, PD-L1 expression also significantly impacted on the outcomes with PD-L1 negative cases more likely to develop a locoregional recurrence (HR 4.16), to have an event (HR 2.5) and to die (HR 3.16). Evidence of an interaction between HPV status and PD-L1 expression was found for overall survival (p < 0.005). CONCLUSION: Our findings suggested that different immune profiles in oropharyngeal cancer by HPV status and the effect of HPV on the outcomes is modified by PD-L1 expression.

Correlation Between Surgical and Histologic Margins in Melanoma Wide Excision Specimens.

INTRODUCTION: Wide surgical excision is the standard treatment for localized primary cutaneous melanomas, with a narrow histologic margin associated with an increased risk of local recurrence. The correlation between surgical and histologic margins is poorly documented in the literature. METHODS: An audit was performed to (1) document the shrinkage of formalin-fixed specimens, and (2) use a precisely measured surgical margin in vivo to predict the histologic margin. For patients presenting for wide excision of melanomas and other malignant skin tumors, measured surgical margin, in vivo and ex vivo specimen width, and histologic margins after formalin fixation were recorded. The effects of clinicopathologic characteristics, including age, sex, body mass index (BMI), tumor type, anatomic site, and presence of visible tumor in predicting specimen shrinkage and histologic margin were assessed. RESULTS: In total, 252 specimens were evaluated. When compared with measured width in vivo, the formalin-fixed specimens showed a mean shrinkage of 14% (R2 = 0.98), regardless of patient age, sex, BMI, or site of the lesion. The measured surgical margin was not a strong predictor of the histologic margin, with a high degree of variability (R2 = 0.55) not explained by patient factors, tumor subtype, or presence of visible tumor at the time of excision (p > 0.05). CONCLUSIONS: A consistent 14% shrinkage rate of wide excision specimens was found across all patients and excision sites, and we propose a clinically useful 15% correction factor that will account for fixation and shrinkage of cutaneous excision specimens. Excision margins measured by the surgeon were a poor predictor of the histologic margins.

Primary anorectal melanoma: clinical, immunohistology and DNA analysis of 43 cases.

Primary melanoma involving the anorectal region is rare, accounting for <1% of all melanomas in most Western countries. It characteristically presents at an advanced clinical stage and is associated with poor clinical outcomes. Preliminary reports suggest that response rates to immunotherapies in patients with advanced stage mucosal melanoma are much lower than in cutaneous (or acral) melanoma patients but reasons for this are unclear. Comprehensive characterisation of the immune microenvironment in anorectal melanoma has not previously been performed. A single-institution cohort of 43 primary anorectal melanoma patients was examined to describe clinicopathological features and characterise the immune microenvironment to provide insights into the behaviour of this rare melanoma subtype. The tumours displayed multiple adverse prognostic attributes including deep thickness (median 11.5 mm), ulceration (81%) and high mitotic rate (median 12/mm2). The median overall survival was 24 months and the median recurrence-free survival was 9 months. Tumour-infiltrating lymphocytes (TILs) were absent or mild in most tumours (75%); PD-L1 positive staining (>1% of tumour cells) was present in 44% of cases, however in 86% of positive tumours the percentage of positive cells was ≤10%. Four tumours underwent whole genome sequencing; no ultraviolet signature was identified, and there was a lower mutational load but higher structural chromosomal variant load compared with cutaneous melanomas. Poor responses of anorectal melanomas to immunotherapy may be caused by lower immunogenicity of these tumours as characterised by low mutation burden (and therefore low neoantigenicity), low TILs infiltrates and low PD-L1 expression. Further investigation is required to determine whether TILs and PD-L1 expression predict response to immunotherapy in patients with mucosal melanoma.

Melanoma protective antitumor immunity activated by catalytic DNA.

Melanoma incidence is increasing worldwide, and although drugs such as BRAF/MEK small-molecule inhibitors and immune checkpoint antibodies improve patient outcomes, most patients ultimately fail these therapies and alternative treatment strategies are urgently needed. DNAzymes have recently undergone clinical trials with signs of efficacy and no serious adverse events attributable to the DNAzyme. Here we investigated c-Jun expression in human primary and metastatic melanoma. We also explored the role of T cell immunity in DNAzyme inhibition of primary melanoma growth and the prevention of growth in non-treated tumors after the cessation of treatment in a mouse model. c-Jun was expressed in 80% of melanoma cells in human primary melanomas (n = 17) and in 83% of metastatic melanoma cells (n = 38). In contrast, c-Jun was expressed in only 11% of melanocytes in benign nevi (n = 24). Dz13, a DNAzyme targeting c-Jun/AP-1, suppressed both Dz13-injected and untreated B16F10 melanoma growth in the same mice, an abscopal effect relieved in each case by administration of anti-CD4/anti-CD8 antibodies. Dz13 increased levels of cleaved caspase-3 within the tumors. New, untreated melanomas grew poorly in mice previously treated with Dz13. Administration of anti-CD4/anti-CD8 antibodies ablated this inhibitory effect and the tumors grew rapidly. Dz13 inhibited c-Jun expression, reduced intratumoral vascularity (vascular lumina area defined by CD31 staining), and increased CD4+ cells within the tumors. This study provides the first demonstration of an abscopal effect of a DNAzyme on tumor growth and shows that Dz13 treatment prevents growth of subsequent new tumors in the same animal. Dz13 may be useful clinically as a therapeutic antitumor agent by preventing tumor relapse through adaptive immunity.

Punch 'scoring': a technique that facilitates melanoma diagnosis of clinically suspicious pigmented lesions.

AIMS: Early recognition and accurate diagnosis underpins melanoma survival. Identifying early melanomas arising in association with pre-existing lesions is often challenging. Clinically suspicious foci, however small, must be identified and examined histologically. This study assessed the accuracy of punch biopsy 'scoring' of suspicious foci in excised atypical pigmented skin lesions to identify early melanomas. METHODS AND RESULTS: Forty-one excised pigmented skin lesions with a clinically/dermoscopically focal area of concern for melanoma, with the suspicious focus marked prior to excision with a punch biopsy 'score' (a partial incision into the skin surface), were analysed. Melanoma was diagnosed in nine of 41 cases (22%). In eight of nine cases (89%) the melanoma was associated with a naevus, and in seven of nine (88%) cases the melanoma was identified preferentially by the scored focus. In six of nine cases (67%), the melanoma was entirely encompassed by the scored focus. In one case of melanoma in situ, the diagnostic material was identified only on further levelling through the scored focus. In 28 of 32 of non-melanoma cases (88%), the scored focus identified either diagnostic features of a particular lesion or pathological features that correlated with the clinical impression of change/atypia including altered architecture or distribution of pigmentation, features of irritation or regression. CONCLUSIONS: The 'punch scoring technique' allows direct clinicopathological correlation and facilitates early melanoma diagnosis by focusing attention on clinically suspicious areas. Furthermore, it does not require special expertise in ex-vivo clinical techniques for implementation. Nevertheless, in some cases examination of the lesion beyond the scored focus is also necessary to make a diagnosis of melanoma.

Prognostic Significance of Periadnexal Extension in Cutaneous Melanoma and its Implications for Pathologic Reporting and Staging.

Tumor thickness is the strongest predictor of outcome for clinically localized melanoma. Therefore, accurate assessment is critical for appropriate staging, reliable estimation of prognosis, and management. When melanoma extends alongside skin adnexal structures more deeply than the main tumor mass (periadnexal extension), it is currently unknown whether the prognosis is more accurately reflected by the deepest point of periadnexal tumor extension or the main tumor mass. This study sought to address this question. Survival outcomes of 257 primary cutaneous melanoma patients with periadnexal extension diagnosed between 2005 and 2015 and managed at Melanoma Institute Australia were identified and compared with a control cohort of 514 patients who were matched for tumor thickness, sex, age, mitotic rate, ulceration status, and year of diagnosis but lacked periadnexal extension. The incidence of periadnexal extension at Melanoma Institute Australia was 1.5% (257/16,692 cutaneous melanomas diagnosed between 2005 and 2015). The patient characteristics between the 2 groups were otherwise very similar; median Breslow thickness was 0.9 mm for the periadnexal group and 1.0 mm for the control group. The median extension beyond the Breslow thickness in the tumors with periadnexal extension was 0.45 mm (mean, 0.4 mm). Median follow-up was 46 months for the periadnexal group and 44 months for the control group. Measures of clinical outcomes all showed trends for improved survival in the periadnexal extension group; these were melanoma-specific survival (hazard ratio [HR], 0.78; 95% confidence interval [CI], 0.44, 1.38), overall survival (HR, 0.91; 95% CI, 0.59, 1.41), disease-free survival (HR, 0.68; 95% CI, 0.45, 1.03), and distant disease-free survival (HR, 0.69; 95% CI, 0.4, 1.17), although none were statistically significant. There was a higher rate of sentinel lymph node (SLN) metastasis in the periadnexal group versus the control group in patients whose tumors were >1 mm thick (24/100=24% vs. 23/187=12.3%). Periadnexal extension was significantly associated with SLN metastasis on univariate logistic regression analysis (odds ratio [95% CI], 2.25 [1.20, 4.24], P=0.01). If the periadnexal extension had been included in the measurement of tumor thickness, 42.8% of patients would have been upstaged to a higher American Joint Committee on Cancer T category. The findings of this study indicate that periadnexal involvement that extends more deeply than the thickness of the main tumor mass increases the risk of SLN metastasis in tumours >1 mm thick, however, does not worsen clinical outcomes overall, and tumor thickness measurements should not include deeper foci of periadnexal tumor.

Systemic inflammation is an independent predictive marker of clinical outcomes in mucosal squamous cell carcinoma of the head and neck in oropharyngeal and non-oropharyngeal patients.

BACKGROUND: Currently there are very few biomarkers to identify head and neck squamous cell carcinoma (HNSCC) cancer patients at a greater risk of recurrence and shortened survival. This study aimed to investigate whether a marker of systemic inflammation, the neutrophil-to-lymphocyte ratio (NLR), was predictive of clinical outcomes in a heterogeneous cohort of HNSCC cancer patients. METHODS: We performed a retrospective analysis to identify associations between NLR and clinicopathological features to recurrence free survival (RFS) and overall survival (OS). Univariate analysis was used to identify associations and selected variables were included in multivariable Cox regression analysis to determine predictive value. RESULTS: A total of 145 patients with stage I-IV HNSCC that had undergone radiotherapy were analysed. Seventy-six of these patients had oropharyngeal cancer and 69 had non-oropharyngeal HNSCC and these populations were analysed separately. NLR was not associated to any clinicopathological variable. On univariate analysis, NLR showed associations with RFS and OS in both sub-populations. Multivariable analysis showed patients with NLR > 5 had shortened OS in both sub-populations but NLR > 5 only predicted RFS in oropharyngeal patients. Poor performance status predicted OS in both sub-populations and current smokers had shortened OS and RFS in non-oropharyngeal patients. CONCLUSIONS: The results show patients with NLR > 5 predict for shorter overall survival. Further prospective validation studies in larger cohorts are required to determine the clinical applicability of NLR for prognostication in HNSCC patients.

Oxyphil Cell Parathyroid Adenomas Causing Primary Hyperparathyroidism: a Clinico-Pathological Correlation.

Oxyphil cell parathyroid adenomas (OPA) are considered to be an uncommon cause of primary hyperparathyroidism (PHPT), and were historically thought to be clinically silent. It has been our clinical impression that these adenomas present more often than previously thought and may manifest a more severe form of primary hyperparathyroidism than classical adenoma. The aim of this study was to describe the incidence and clinical presentation of OPA. An observational case-control study was undertaken. The study group comprised patients undergoing parathyroidectomy for PHPT where the final pathology confirmed OPA. The controls were made up of an age- and sex-matched group of patients having parathyroidectomy in the same time period where the final pathology confirmed a classical or non-oxyphil adenoma. OPA were defined as parathyroid tumours containing >75% oxyphilic cells. The OPA cases were obtained by reviewing all histopathology slides over an 11-year period (2002-12) where the reports contained the words 'oxyphil' or 'oxyphilic' parathyroid adenomas. These were then reviewed by two independent pathologists to confirm a diagnosis of OPA. The primary outcome measures were preoperative serum calcium and parathyroid hormone (PTH) levels. Secondary outcome measures were symptoms at presentation, accuracy of preoperative localization studies, parathyroid gland weight following surgery, and type of surgery undertaken. In the period 2002-2012, 2739 patients underwent surgery for PHPT. Following pathological review, 91 cases were confirmed as being OPA and formed the study group. A control group (n = 91) from the same period was selected following matching on the basis of age at presentation and sex. OPA were associated with higher preoperative serum calcium (10.84 versus 10.48 mg/dL, p < 0.001) and parathyroid hormone (139 versus 64 ng/L, p < 0.001). At presentation, a lower proportion of OPA cases had asymptomatic disease (15 versus 29%, p = 0.03). There was a trend toward a higher rate of renal calculi at presentation in the OPA group (9 versus 3%, p = 0.07). Preoperative ultrasound was less accurate in localization of OPA when compared with classical adenoma. The rate of minimally invasive surgery was 67% for OPA and 78% for the control group (p = 0.06). All patients were cured of hypercalcaemia at 6-month follow up. There was no significant difference in the weight of removed parathyroid tissue between the groups (868 mg for OPA versus 789 mg for the control group, p = 0.6). OPA are frequently symptomatic and are associated with higher preoperative serum calcium and parathyroid hormone levels than classical types of parathyroid adenomas. OPA are less likely to be localised on preoperative ultrasound examination.

Linezolid versus vancomycin for MRSA skin and soft tissue infections (systematic review and meta-analysis).

BACKGROUND: This review aims to compare the effectiveness of linezolid to vancomycin for the treatment of Methicillin Resistant Staphylococcus Aureus (MRSA) skin and soft tissue infections (SSTIs) in inpatients. METHODS: MEDLINE, EMBASE, the Cochrane Central Register of Controlled Trials and reference lists were searched in March 2007. We included randomized controlled trials that looked at inpatients treated with linezolid versus vancomycin for the treatment of hospital-acquired MRSA SSTIs. Outcome measures were clinical cure (resolution of symptoms and signs) and microbiological cure (eradication of MRSA on wound culture). The validity of the included trials was assessed. The results were combined in meta-analyses, the robustness of which was tested in sensitivity analyses. RESULTS: Four trials were included in this review: three for clinical outcomes (174 participants) and three for microbiological outcomes (439 participants). For clinical outcomes there were non-significant trends in favour of linezolid (RR 0.34; 95% CI 0.04, 2.89; P = 0.32). For microbiological outcomes there was weak evidence of linezolid outperforming vancomycin (RR 0.55; 95% CI 0.30, 1.01; P = 0.05). Sensitivity analyses did not change the conclusions taken from the main analysis. CONCLUSION: With the current available data no difference could be detected between the two treatments, but a trend towards higher effectiveness of linezolid was observed. More data will be required to determine if linezolid is superior to vancomycin for the treatment of MRSA SSTIs. Further systematic reviews are needed to look at other outcomes (length of hospital stay, safety and tolerability, cost-effectiveness) and at MRSA infections at other sites.