Low incidence of BRAF and NRAS mutations in a population with a high incidence of melanoma.

Reported rates of BRAF mutation in Irish cutaneous melanoma cohorts are lower than the reported international data. We aimed to assess the mutational status of a cohort of primary cutaneous melanomas and to correlate it with clinical follow-up data.A total of 92 cases of primary cutaneous melanoma diagnosed at a single institution in 2012 were analyzed. Regions containing common mutations in the BRAF, NRAS, KIT, and KRAS genes were investigated by PCR amplification followed by Sanger sequencing. Demographic details, tumor characteristics, and 10-year outcome data were also obtained.Ten cases with BRAF V600E mutations (11.6%) and five (5.49%) NRAS mutations (4 at Q61R, 1 at Q61K) were detected. No statistically significant differences were noted between groups for age, gender, depth of invasion, nodal status, or recurrence status (p ≥ 0.05).These findings suggest that the Irish population has a markedly lower incidence of BRAF and NRAS mutations in melanoma than those reported in other cohorts.

Refining the application of PRAME-a useful marker in high CSD and acral melanoma subtypes.

Pathologic discordance affecting patient management may approach 20% in melanocytic cases following specialist review. The diagnostic utility of PRAME has been highlighted in several studies but interpretative challenges exist including its use in severely dysplastic compound nevi showing progression to melanoma in situ, nevoid melanoma, and coexisting nevi with melanoma. We examine the PRAME status of a broad spectrum of melanocytic lesions including challenging, dysplastic nevi with severe atypia from a large Irish patient cohort. Retrospective review of the dermatopathology database was conducted to evaluate the PRAME staining characteristics of two hundred and twenty-one melanocytic lesions using a commercially available PRAME antibody (EPR20330). The proportion of nuclear labeling and intensity of staining was recorded. The sensitivity and specificity of PRAME for in situ and malignant melanocytic lesions was 77% and 100%, respectively. Virtually all of our melanoma in situ from high-cumulative sun damaged (CSD) skin (22/23) and all acral lentiginous melanoma (5/5) were PRAME positive while 80% (8/10) of our lentigo maligna melanoma showed diffuse expression. None of our benign subgroup showed diffuse immunoexpression (0/82), including thirty-seven moderate or severely dysplastic nevi. In all cases of melanoma in situ arising in association with a dysplastic compound nevus (0/10), no immunoexpression was observed in the nevic component while in five cases of melanoma in situ with coexistent, intradermal nevus immunostaining was confined to the in situ component. A total of 100% (2/2) of desmoplastic melanomas and 50% (4/8) of nodular melanomas were PRAME positive. PRAME is a sensitive and highly specific immunostain in the diagnosis of in situ and invasive melanoma and we emphasize its application in the evaluation of high CSD and acral melanoma subtypes as well as in challenging threshold cases.

The lupus band test: A review of the sensitivity and specificity in the diagnosis of lupus erythematosus.

Background: The lupus band test (LBT) is a direct immunofluorescence (DIF) technique which shows a band of localised immunoglobulins at the dermo-epidermal junction in lesional, non-sun-exposed skin of patients with both systemic and cutaneous lupus erythematosus (LE), and in perilesional skin of patients with systemic LE. However, low sensitivity and poor concordance between histological and clinical diagnoses warrant a review of the application of the LBT in the diagnosis of LE. Objectives: To assess the sensitivity and specificity of the LBT in diagnosing LE following clinico-pathological correlation (CPC). Methods: All cases sent to our pathology department between 2011 and 2018 for DIF with a clinical query of LE were reviewed. Data collection included demographic details, pathology requests, histology and DIF reports, clinical reports and diagnoses, and serology. Results: Of 256 histology requests, 9% (n = 23) had a positive LBT. This was discordant with the prevalence of LE diagnosis, as 46.3% were diagnosed with LE following CPC. The sensitivity and specificity of the LBT for LE was 17.6% and 98.8% respectively, with a positive predictive value of 92.9% and negative predictive value of 58.2%. Conclusion: The LBT is not a sensitive diagnostic test for LE, but is highly specific, and should be considered as a supportive diagnostic tool for LE. This is the largest reported case series evaluating the efficacy of the LBT in the diagnosis of LE.

Aberrant p16, p53 and Ki-67 immunohistochemistry staining patterns can distinguish solitary keratoacanthoma from cutaneous squamous cell carcinoma.

Keratoacanthoma (KA) is widely considered a benign, usually self-resolving, neoplasm distinct from cutaneous squamous cell carcinoma (cSCC), while some consider KA to be indistinguishable from cSCC. Published studies indicate utility for p16, p53, Ki-67 immunostaining and elastic van Gieson (EVG) in the assessment of KA and cSCC. We compared clinical features and staining patterns for p16, p53, Ki-67 and EVG in fully excised KA, cSCC with KA-like features (cSCC-KAL) and other cSCC (cSCC-OTHER). Significant differences between KA, cSCC-KAL and cSCC-OTHER were found for head and neck location (20%, 86%, 84%), and duration <5 months (95%, 63%, 36%). KA shows both a mosaic pattern for p16 (>25-90% of neoplasm area) and peripheral graded pattern for p53 (up to 50% moderate and strong nuclear staining) in 92% compared with 0% of cSCC-KAL and 0% of cSCC-OTHER. In contrast, a highly aberrant pattern (usually null) for one or both p16 and p53, was present in 0% of KA, 83.8% of cSCC-KAL and 90.9% of cSCC-OTHER. Abnormal distribution of Ki-67 beyond the peripheral 1-3 cells was uncommon in KA (4.2%) and common in cSCC-KAL (67.6%) and cSCC-OTHER (88.4%). Moderate to striking entrapment of elastic and collagen fibres was present in the majority of KA (84%), cSCC-KAL (81%) and cSCC-OTHER (65%). KA are clinically distinct neoplasms typically of short duration occurring preferentially outside the head and neck and generally lacking aberrations of p16, p53 and Ki-67, compared with cSCC that have high rates of aberrant or highly aberrant p16, p53 and Ki-67, but EVG lacked specificity.

A single-centre experience of secondary cutaneous tumours with special reference to precocious metastases.

BACKGROUND: Secondary involvement of skin by tumour comprises 2% of cutaneous neoplasia, in a small proportion of cases serving as the primary manifestation of occult disease. METHODS: Cases of cutaneous metastases (CM) were retrieved from our pathology files between 2013 and 2018 and clinical and histopathological data reviewed. RESULTS: There were 159 cases (median age 70). A majority of clinical presentations comprised isolated, papulonodular lesions. While the anatomic distribution of lesions often bore a proximate relationship to the primary tumour, distant sites of involvement were frequently encountered. Melanoma gave rise to the greatest number of metastases, followed by tumours of the breast, colorectum, and squamous cell carcinoma. In six cases (3.8%), CM served as the presenting feature of occult malignancy. These patients presented at a more advanced age and with distant sites of involvement. The microscopic features of CM include nodules, nests, and cords or single cell infiltrates typically in deeper compartments in the absence of overlying epidermal or adnexal precursor lesions. CONCLUSIONS: CMs are a frequent development in the natural history of melanoma and breast tumours. In practice, a wide spectrum of tumours may give rise to CM and a small proportion more importantly, signal the existence of previously unknown neoplasia.

Therapeutic Response Evaluation in Advanced Melanoma Patients Incorporating Plasma cfDNA, LDH, VEGF, PD-L1, and IFN-γ Measurements.

BACKGROUND/AIM: Current treatment strategies for advanced melanoma require serial assessment of disease status in affected patients. In this study, we sought to examine the relationship between radiographic tumour burden and blood borne biomarkers including plasma cfDNA, serum LDH, plasma VEGF, PD-L1 and IFN-γ in advanced melanoma patients receiving immunotherapy. We hypothesized that a combination of these explanatory variables in a suitable regression analysis model may predict changes in tumour burden during patient treatment. MATERIALS AND METHODS: We extracted and quantified circulating cfDNA, LDH, VEGF, PD-L1, and IFN-γ from thirty patients with stage IV melanoma at baseline and at six months. All participating patients were evaluated with paired blood sample collection and CT scan assessments during treatment. RESULTS: Changes in radiographic tumour burden correlated with changes in levels of cfDNA (p≤0.001), LDH (p≤0.001), VEGF (p≤0.001), and PD-L1 (p<0.05) during treatment. Multiple regression analysis consisting of the follow-up to baseline assessment ratios of cfDNA, LDH, VEGF and PD-L1 explained changes in tumour burden (F (4, 23)=32.05, p<0.001); with an R2 of 0.8479 (Y=ß0+ß1*B+ß2*C+ß3*D+ß4*E). CONCLUSION: A quantitative measure of cfDNA, LDH, VEGF and PD-L1 may complement current methods of assessing tumour burden in advanced melanoma patients.

Cytokeratin 7 and 19 expression in oropharyngeal and oral squamous cell carcinoma.

PURPOSE: The precise etiopathogenesis of human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC), and reasons for predilection for crypt epithelium, remain uncertain. The purpose of this study is to investigate the interaction between HPV and specific cytokeratins 7 (CK7) and 19 (CK19) in crypt epithelium. METHODS: This is a retrospective cohort study of patients presenting between 1999 and 2015 at a tertiary referral center. CK7 and CK19 positivity and H Scores were determined by immunohistochemistry. Disease-specific and overall survival rates were analyzed. RESULTS: There were 253 patients presenting with OPSCC (134), squamous cell carcinoma (SCC) of unknown primary site (22), and oral tongue SCC (97). Primary tumor CK7 and CK19 positivity and H Scores were significantly higher in HPV-positive OPSCC than HPV-negative OPSCC and oral tongue SCC. Higher CK19 Scores, but not CK7 Scores, were also seen in regional metastases from HPV-positive OPSCC than other sites. No impact on disease-specific or overall survival was identified on multivariate analysis. CONCLUSION: The increased expression of CK7 and CK19 in HPV-positive OPSCC compared to HPV-negative disease supports the theory for a role for these cytokeratins in the etiopathogenesis of HPV-related OPSCC.

Pembrolizumab-induced follicular eruption and response to isotretinoin.

Background: Pembrolizumab is a monoclonal antibody targeting PD-1. Folliculitis secondary to pembrolizumab has rarely been reported in the treatment of malignant melanoma. Case: A 49-year-old with a history of mild lower limb folliculitis developed metastatic malignant melanoma, and immunotherapy with pembrolizumab was initiated. Following 19 doses of pembrolizumab, a folliculocentric pustular eruption developed on the lower legs. Biopsy was consistent with folliculitis. Treatment with topical corticosteroids, high-dose prednisolone, lymecycline, clarithromycin, trimethoprim and clindamycin was unsuccessful. Pembrolizumab was stopped after 22 cycles, but the folliculitis persisted. Oral isotretinoin was required for disease control. Discussion: Drug-induced follicular eruptions have rarely been described with anti PD-1 therapy. Isotretinoin may be required to achieve remission.

Lay abstract Pembrolizumab (trade name Keytruda^©) is a type of immune therapy that stimulates the body's immune system to fight cancer cells. This immune therapy can cause a variety of rashes. In this article, the authors describe a patient who developed a rash around hair follicles that is not commonly described with pembrolizumab. A man with a history of mildly infected hair follicles on his legs was diagnosed with advanced melanoma and was treated with pembrolizumab. After 19 treatments, he developed a rash on his legs, centered around hair follicles. Treatment with steroid ointments, steroid tablets and antibiotic tablets was not helpful. Pembrolizumab was stopped, but the rash persisted. A medicine called isotretinoin was required to control the rash. This type of rash has rarely been described with this kind of immune therapy, and isotretinoin might be required to treat it.

The effect of calcium electroporation on viability, phenotype and function of melanoma conditioned macrophages.

Electroporation in combination with chemotherapy is an established treatment used on solid malignancies that results in enhanced chemotherapeutic uptake. Recent advances have begun to transition to the use of non-toxic compounds, such as calcium, in lieu of chemotherapy, which can also induce tumour cell death. While the effect of treatment on tumour cell death has been well characterized and has been shown to induce an immunogenic form of cell death, the effect of treatment on intratumoural immune cells has not been investigated. Here we present data showing the effect of calcium electroporation on immune cells, using melanoma-conditioned bone marrow-derived macrophages. Similar to tumour cells, macrophage cell membranes are susceptible to poration following treatment and subsequently reseal. Macrophages are less susceptible to calcium electroporation induced cell death in comparison to B16F10 melanoma cells. However treatment with electroporation with or without bleomycin or calcium was shown to affect macrophage phenotype and function. Coculture of calcium electroporated macrophages revealed that both the capacity of macrophages to stimulate and direct T cell responses are affected following exposure to treatment. We conclude that calcium electroporation has the potential to boost the immunogenic capacity of exposed tumour associated macrophages, and further research is warranted to determine if calcium electroporation can be optimised to generate systemic anti-cancer immune responses.

Differential association of CD68+ and CD163+ macrophages with macrophage enzymes, whole tumour gene expression and overall survival in advanced melanoma.

BACKGROUND: The density and phenotype of tumour-associated macrophages have been linked with prognosis in a range of solid tumours. While there is strong preclinical evidence that tumour-associated macrophages promote aspects of tumour progression, it can be challenging to infer clinical activity from surface markers and ex vivo behaviour. We investigated the association of macrophage infiltration with prognosis and functional changes in the tumour microenvironment in primary human melanoma. METHODS: Fifty-seven formalin-fixed, paraffin-embedded primary melanomas were analysed by immunohistochemical analysis of CD68, CD163, inducible nitric oxide synthase (iNOS) and arginase expression. RNA sequencing was performed on serial sections of 20 of the stained tumours to determine the influence of macrophage infiltration on gene expression. RESULTS: CD68+ cells are a functionally active subset of macrophages that are associated with increased iNOS and arginase staining and altered gene expression. In comparison, while there is a greater accumulation of CD163+ macrophages in larger tumours, these cells are comparatively inactive, with no association with the level of iNOS or arginase staining, and no effect on gene expression within the tumour. The infiltration of either subset of macrophages did not correlate to overall survival. CONCLUSIONS: Thus, melanomas contain distinct macrophage populations with diverse phenotypes, but with no observable prognostic role.

The role of tumour morphology in assigning HPV status in oropharyngeal squamous cell carcinoma.

OBJECTIVES: There is no consensus on the optimal approach to human papilloma virus (HPV) testing in oropharyngeal squamous cell carcinoma (OPSCC). Our objective was to classify OPSCC as HPV positive or negative based on (1) morphology alone, (2) p16 status alone, (3) combined morphology and p16 status with additional HPV testing in discordant cases in keeping with 2012 College of American Pathologists (CAP) guidelines (combined approach), and to evaluate and compare survival outcomes. MATERIALS AND METHODS: Retrospective review of 168 patients, 146 with OPSCC and 22 with cervical SCC of unknown primary site (SCCUP). Morphology was classified as keratinizing or non-keratinizing, p16 immunohistochemistry (IHC) performed and additional HPV DNA PCR testing undertaken in cases in which morphology and p16 status were discordant. Survival statistics were evaluated and compared for the 3 different approaches to classification. RESULTS: On univariate analysis all 3 classification methods significantly predicted for overall survival (OS). Both p16 status and the combined approach also predicted for disease specific survival (DSS), whereas morphology fell just outside significance (p = 0.06). On multivariate analysis only the combined approach retained significance for both OS and DSS, whilst morphology was also significant for DSS. CONCLUSIONS: Our findings confirm that tumour morphology significantly predicts for survival in OPSCC. However, we found combined tumour morphology and p16 IHC, with additional testing for discordant cases to be superior to either morphology or p16 IHC alone. Further study is required to establish the optimal testing method for HPV in OPSCC particularly in low prevalence populations.

Tumour Cell Anaplasia and Multinucleation as Prognosticators in Oropharyngeal Squamous Cell Carcinoma.

Human papilloma virus (HPV)-positive oropharyngeal squamous cell carcinomas (OPSCC) tend to have good outcomes, however a subset does not share this favourable prognosis. The aim of this paper is to investigate the utility of tumour cell anaplasia and multinucleation as prognostic markers in oropharyngeal squamous cell carcinoma. Retrospective review of 104 patients with OPSCC or squamous cell carcinoma of unknown primary site (SCCUP) who underwent primary resection and/or lymph node dissection. Slides of both primary and nodal metastatic disease were assessed for the presence of anaplasia and multinucleation. 53 patients were HPV-positive. Anaplasia was more frequent in males (p = 0.005), smokers (p = 0.003), and HPV-negative disease (p = 0.04). HPV status and > 10 pack-year smoking history were independent predictors of recurrence-free survival (RFS) and disease-specific survival (DSS). Neither anaplasia, nor multinucleation, at the primary site or in cervical metastases, had any significant impact on RFS or DSS. We did not find either anaplasia or multinucleation to have any significant prognostic impact in OPSCC. However, given the small number of adverse events in the HPV-positive cohort, we may have lacked sufficient power to detect significance in what was the subgroup of primary interest. Our study highlights the challenge of identifying markers of poor prognosis in HPV-positive OPSCC.

Should We Always Biopsy in Clinically Evident Lichen Sclerosus?

The aims of the study were to review cases of clinically diagnosed lichen sclerosus (LS) and to compare the histological features found on biopsy to clinical features seen on examination. METHODS: A retrospective chart review was undertaken of patients attending a specialist vulval service between 2013 and 2015 with a clinical diagnosis of LS. Patients in whom there was clinical diagnostic uncertainty or those with features of lichen planus or lichen planus/LS overlap were excluded. We determined the proportion of these patients who underwent vulval biopsy and reviewed their histology. RESULTS: One hundred fifteen charts were reviewed. Sixty-nine (69/115, 60%) had a firm diagnosis of established LS, and of these, 39 (39/69, 56.5%) had a biopsy performed in the previous 5 years. Thirteen (13/39, 33.3%) of these biopsies fell short of a diagnosis of LS histologically with some suggestive but nondiagnostic and some showing nonspecific features. CONCLUSIONS: Reliance on biopsies solely to establish or exclude the diagnosis of LS is inadvisable. A good level of knowledge of the characteristic clinical features is imperative among gynecologists, dermatologists, and general practitioners.

High concordance of BRAF mutational status in matched primary and metastatic melanoma.

BACKGROUND: Techniques for the accurate identification of activating mutations of BRAF in metastatic melanoma are of great clinical importance, due to the availability of targeted therapies for these tumors. There is uncertainty regarding the frequency with which BRAF status differs between primary and metastatic sites. METHODS: Between 2011 and 2016, 219 melanoma cases underwent BRAF testing in our institution. In 53 of these cases, paired primary and metastatic specimens were available for polymerase chain reaction (PCR) and immunohistochemical evaluation. RESULTS: Fifty-two out of 53 cases (98%) showed concordant BRAF status between primary and metastatic site by immunohistochemistry (IHC). In one case, a metastasis and its matched primary were positive by IHC, but the metastasis was negative on PCR. On further investigation, PCR was positive in the primary, and repeat PCR in the metastasis was positive, following macrodissection. CONCLUSIONS: Our results suggest that discordance of BRAF mutational status between primaries and metastases is a rare occurrence. In one case, IHC provided strong evidence that initial PCR testing had provided a false-negative result due to low tumor volume. Thus, in cases where tissue is difficult to obtain from a metastasis or unavailable, the primary tumor can be used with confidence.

Sampling of wide local excision specimens in cutaneous malignant melanoma.

BACKGROUND: Wide local excisions (WLEs) are frequently undertaken in the management of cutaneous melanoma; however, there is a considerable variability in their macroscopic sampling. The aim of our study was to establish evidence-based guidelines for the macroscopic handling of these specimens with a subsequent review of the impact on our service. METHODS: The study group of 128 cases with initial biopsy and subsequent WLE in our institution in 2010 were identified by a computer-generated search. From analysis of this group, guidelines for macroscopic sampling were derived with a repeat search performed in 2012. RESULTS: Residual melanoma was detected only in those cases in which the original specimen had clear margins of ≤1 mm or with a pigmented lesion. A 32% increase in case numbers was noted over this period with a reduction of 6.2% in block numbers. Average block numbers per case were reduced by 2.3 (30.7%). CONCLUSIONS: We have shown that for WLE specimens with no evidence of a macroscopic lesion and clear margins on original biopsy, little is to be gained from extensive sampling. In these cases we recommend a maximum of 3 blocks per case. Reduction in sampling based on this evidence would result in saving valuable laboratory resources.