Glioblastoma biomarkers in urinary extracellular vesicles reveal the potential for a 'liquid gold' biopsy.

BACKGROUND: Biomarkers that reflect glioblastoma tumour activity and treatment response are urgently needed to help guide clinical management, particularly for recurrent disease. As the urinary system is a major clearance route of circulating extracellular vesicles (EVs; 30-1000 nm nanoparticles) we explored whether sampling urinary-EVs could serve as a simple and non-invasive liquid biopsy approach for measuring glioblastoma-associated biomarkers. METHODS: Fifty urine specimens (15-60 ml) were collected from 24 catheterised glioblastoma patients immediately prior to primary (n = 17) and recurrence (n = 7) surgeries, following gross total resection (n = 9), and from age/gender-matched healthy participants (n = 14). EVs isolated by differential ultracentrifugation were characterised and extracted proteomes were analysed by high-resolution data-independent acquisition liquid chromatography tandem mass spectrometry (DIA-LC-MS/MS). RESULTS: Overall, 6857 proteins were confidently identified in urinary-EVs (q-value ≤ 0.01), including 94 EV marker proteins. Glioblastoma-specific proteomic signatures were determined, and putative urinary-EV biomarkers corresponding to tumour burden and recurrence were identified (FC ≥ | 2 | , adjust p-val≤0.05, AUC > 0.9). CONCLUSION: In-depth DIA-LC-MS/MS characterisation of urinary-EVs substantiates urine as a viable source of glioblastoma biomarkers. The promising 'liquid gold' biomarker panels described here warrant further investigation.

CDKN2A/B Homozygous Deletions in Astrocytomas: A Literature Review.

Genomic alterations of CDKN2A and CDKN2B in astrocytomas have been an evolving area of study for decades. Most recently, there has been considerable interest in the effect of CDKN2A and/or CDKN2B (CDKN2A/B) homozygous deletions (HD) on the prognosis of isocitrate dehydrogenase (IDH)-mutant astrocytomas. This is highlighted by the adoption of CDKN2A/B HD as an essential criterion for astrocytoma and IDH-mutant central nervous system (CNS) WHO grade 4 in the fifth edition of the World Health Organisation (WHO) Classification of Central Nervous System Tumours (2021). The CDKN2A and CDKN2B genes are located on the short arm of chromosome 9. CDKN2A encodes for two proteins, p14 and p16, and CDKN2B encodes for p15. These proteins regulate cell growth and angiogenesis. Interpreting the impact of CDKN2A/B alterations on astrocytoma prognosis is complicated by recent changes in tumour classification and a lack of uniform standards for testing CDKN2A/B. While the prognostic impact of CDKN2A/B HD is established, the role of different CDKN2A/B alterations-heterozygous deletions (HeD), point mutations, and promoter methylation-is less clear. Consequently, how these alternations should be incorporated into patient management remains controversial. To this end, we reviewed the literature on different CDKN2A/B alterations in IDH-mutant astrocytomas and their impact on diagnosis and management. We also provided a historical review of the changing impact of CDKN2A/B alterations as glioma classification has evolved over time. Through this historical context, we demonstrate that CDKN2A/B HD is an important negative prognostic marker in IDH-mutant astrocytomas; however, the historical data is challenging to interpret given changes in tumour classification over time, variation in the quality of evidence, and variations in the techniques used to identify CDKN2A/B deletions. Therefore, future prospective studies using uniform classification and detection techniques are required to improve the clinical interpretation of this molecular marker.

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.

Glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA): a molecularly distinct brain tumor type with recurrent NTRK gene fusions.

Glioneuronal tumors are a heterogenous group of CNS neoplasms that can be challenging to accurately diagnose. Molecular methods are highly useful in classifying these tumors-distinguishing precise classes from their histological mimics and identifying previously unrecognized types of tumors. Using an unsupervised visualization approach of DNA methylation data, we identified a novel group of tumors (n = 20) that formed a cluster separate from all established CNS tumor types. Molecular analyses revealed ATRX alterations (in 16/16 cases by DNA sequencing and/or immunohistochemistry) as well as potentially targetable gene fusions involving receptor tyrosine-kinases (RTK; mostly NTRK1-3) in all of these tumors (16/16; 100%). In addition, copy number profiling showed homozygous deletions of CDKN2A/B in 55% of cases. Histological and immunohistochemical investigations revealed glioneuronal tumors with isomorphic, round and often condensed nuclei, perinuclear clearing, high mitotic activity and microvascular proliferation. Tumors were mainly located supratentorially (84%) and occurred in patients with a median age of 19 years. Survival data were limited (n = 18) but point towards a more aggressive biology as compared to other glioneuronal tumors (median progression-free survival 12.5 months). Given their molecular characteristics in addition to anaplastic features, we suggest the term glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA) to describe these tumors. In summary, our findings highlight a novel type of glioneuronal tumor driven by different RTK fusions accompanied by recurrent alterations in ATRX and homozygous deletions of CDKN2A/B. Targeted approaches such as NTRK inhibition might represent a therapeutic option for patients suffering from these tumors.

Whole genome duplication in oral squamous cell carcinoma in patients younger than 50 years: implications for prognosis and adverse clinicopathological factors.

INTRODUCTION: Oral squamous cell carcinoma (OSCC) in the young (<50 years), without known carcinogenic risk factors, is on the rise globally. Whole genome duplication (WGD) has been shown to occur at higher rates in cancers without an identifiable carcinogenic agent. We aimed to evaluate the prevalence of WGD in a cohort of OSCC patients under the age of 50 years. METHODS: Whole genome sequencing (WGS) was performed on 28 OSCC patients from the Sydney Head and Neck Cancer Institute (SHNCI) biobank. An additional nine cases were obtained from The Cancer Genome Atlas (TCGA). RESULTS: WGD was seen in 27 of 37 (73%) cases. Non-synonymous, somatic TP53 mutations occurred in 25 of 27 (93%) cases of WGD and were predicted to precede WGD in 21 (77%). WGD was significantly associated with larger tumor size (p = 0.01) and was frequent in patients with recurrences (87%, p = 0.36). Overall survival was significantly worse in those with WGD (p = 0.05). CONCLUSIONS: Our data, based on one of the largest WGS datasets of young patients with OSCC, demonstrates a high frequency of WGD and its association with adverse pathologic characteristics and clinical outcomes. TP53 mutations also preceded WGD, as has been described in other tumors without a clear mutagenic driver.

Implications of Concurrent IDH1 and IDH2 Mutations on Survival in Glioma-A Case Report and Systematic Review.

Both IDH1 (isocitrate dehydrogenase 1) and IDH2 (isocitrate dehydrogenase 2) mutations play a vital role in the development of gliomas through disruption of normal cellular metabolic processes. Here we describe a case of a patient with an IDH-mutant astrocytoma, in which both IDH1 and IDH2 mutations were detected within the same tumour. The patient remains disease-free, nine and a half years after her initial diagnosis. Interrogation of cancer genomic databases and a systematic review was undertaken, demonstrating the rarity of the co-occurrence of IDH1 and IDH2 mutations in a variety of cancer types, and in glioma specifically. Due to the favourable outcome observed in this patient, the potential effect of concurrent IDH1 and IDH2 mutations on survival was also investigated.

Standards for the classification of pathogenicity of somatic variants in cancer (oncogenicity): Joint recommendations of Clinical Genome Resource (ClinGen), Cancer Genomics Consortium (CGC), and Variant Interpretation for Cancer Consortium (VICC).

PURPOSE: Several professional societies have published guidelines for the clinical interpretation of somatic variants, which specifically address diagnostic, prognostic, and therapeutic implications. Although these guidelines for the clinical interpretation of variants include data types that may be used to determine the oncogenicity of a variant (eg, population frequency, functional, and in silico data or somatic frequency), they do not provide a direct, systematic, and comprehensive set of standards and rules to classify the oncogenicity of a somatic variant. This insufficient guidance leads to inconsistent classification of rare somatic variants in cancer, generates variability in their clinical interpretation, and, importantly, affects patient care. Therefore, it is essential to address this unmet need. METHODS: Clinical Genome Resource (ClinGen) Somatic Cancer Clinical Domain Working Group and ClinGen Germline/Somatic Variant Subcommittee, the Cancer Genomics Consortium, and the Variant Interpretation for Cancer Consortium used a consensus approach to develop a standard operating procedure (SOP) for the classification of oncogenicity of somatic variants. RESULTS: This comprehensive SOP has been developed to improve consistency in somatic variant classification and has been validated on 94 somatic variants in 10 common cancer-related genes. CONCLUSION: The comprehensive SOP is now available for classification of oncogenicity of somatic variants.

Mismatch repair protein loss in cutaneous head and neck squamous cell carcinoma.

BACKGROUND: The treatment of advanced cutaneous head and neck cutaneous squamous cell carcinomas (HNcSCC) results in significant morbidity. Recently, immune checkpoint inhibitor treatment has been approved for DNA mismatch repair (MMR) deficient patients in a histology-agnostic manner. This study aims to evaluate the incidence of MMR deficiency in advanced HNcSCC and its association with clinicopathologic factors. METHODS: The cohort included 176 consecutive HNcSCC cases treated with curative intent. Immunohistochemistry for MMR proteins (hMLH1, hMSH2, hMSH6, and hPMS2) was performed. Clinicopathological and survival data was collected prospectively. RESULTS: The incidence of MMR protein deficiency was 9.1%. There was no association between age, incidence of metachronous malignancies, clinicopathological factors, or survival outcomes. CONCLUSION: A higher incidence of MMR deficiency was observed in this cohort of advanced HNcSCC. The lack of association with young age at onset or increased incidence of metachronous malignancies suggests that MMR deficiency is likely to be sporadic in HNcSCC.

Atypical Ewing sarcoma breakpoint region 1 fluorescence in-situ hybridization signal patterns in bone and soft tissue tumours: diagnostic experience with 135 cases.

AIMS: Recurrent Ewing sarcoma breakpoint region 1 (EWSR1) gene rearrangements characterize a select group of bone and soft tissue tumours. In our routine diagnostic practice with fluorescence in-situ hybridization (FISH), we have occasionally observed EWSR1 gene rearrangements in tumours not associated classically with EWSR1 translocations. This study aimed to review our institutional experience of this phenomenon and also to highlight the occurrence of unusual EWSR1 FISH signals (i.e. 5' centromeric region or 3' telomeric region signals) that do not fulfil the published diagnostic criteria for rearrangements. METHODS AND RESULTS: Using an EWSR1 break-apart probe, we performed FISH assays on formalin-fixed paraffin-embedded tissue sections from 135 bone and soft tissue specimens as part of their routine diagnostic work-up. EWSR1 gene rearrangements were identified in 51% of cases, 56% of which also showed an abnormal FISH signal pattern (in addition to classically rearranged signals). However, atypical FISH signals were present in 45% of the non-rearranged cases. In addition, we observed tumours unrelated to those described classically as EWSR1-associated that were technically EWSR1-rearranged in 6% of cases. Borderline levels of rearrangement (affecting 10-30% of lesional cells) were present in an additional 17% of these cases. CONCLUSIONS: While our study confirmed that FISH is a sensitive and specific tool in the diagnosis of EWSR1-associated tumours, atypical FISH signals and classical rearrangement in entities other than EWSR1-associated tumours can occur. Therefore, it is essential that the FISH result not be used as an isolated test, but must be evaluated in the context of clinical features, imaging, pathological and immunohistochemical findings.

Rapid Vision Loss Due to Multifocal Glioma: A Diagnostic Challenge.

INTRODUCTION: This is a unique case report in medical literature for its detailing of diagnostics of an uncommon presentation of a rapid unexplained bilateral vision loss of a 73-year-old male diabetic patient. This report highlights the crucial role of advanced molecular diagnostics in difficult neurological cases and also elucidates the difficulties involved in diagnosing optic nerve glioblastoma, an exceptionally rare and aggressive tumour. MAIN CONCERNS AND CLINICAL FINDINGS OF THE PATIENT: Slow and progressive loss of vision over 2 months, ultimately developing almost complete visual impairment in both eyes and a defect of right eye field of vision conclusively highlighted that the likely etiology was neuro-ophthalmic. Initially, the conditions were suspected to be an extended spectrum of diabetic eye disease complications but further deterioration was a hint towards something more substantive. PRIMARY DIAGNOSES, INTERVENTIONS AND OUTCOMES: This entailed in-depth diagnosis processes that included an MRI and the analysis of cerebrospinal fluid. The important discovery was through stereotactic biopsies of the optic nerve revealing a high-grade glial neoplasm. Next generation sequencing confirmed the pathology as IDH-wildtype glioblastoma. Despite management, his vision continued to deteriorate. Hence, an aggressive clinical course was followed. CONCLUSION: This case highlights the important learning need in considering glioblastoma of the optic chiasm as part of the differential diagnosis of rapid vision loss, which may present as multifocal brain lesions, especially in cases of rapid loss of vision where initial workup is negative. Quite a useful lesson that can be drawn from this case relates to the diagnostic process with advanced molecular profiling, more attention given to clinical suspicion and cutting-edge diagnostic tools applied in atypical presentation of neurological conditions.

Prognostic and predictive biomarkers in central nervous system tumours: the molecular state of play.

Central nervous system (CNS) tumours were one of the first cancer types to adopt and integrate molecular profiling into routine clinical diagnosis in 2016. The vast majority of these biomarkers, used to discriminate between tumour types, also offered prognostic information. With the advent of The Cancer Genome Atlas (TCGA) and other large genomic datasets, further prognostic sub-stratification was possible within tumour types, leading to increased precision in CNS tumour grading. This review outlines the evolution of the molecular landscape of adult CNS tumours, through the prism of World Health Organization (WHO) Classifications. We begin our journey in the pre-molecular era, where high-grade gliomas were divided into 'primary' and 'secondary' glioblastomas. Molecular alterations explaining these clinicopathological observations were the first branching points of glioma diagnostics, with the discovery of IDH1/2 mutations and 1p/19q codeletion. Subsequently, the rigorous characterisation of paediatric gliomas led to the unearthing of histone H3 alterations as a key event in gliomagenesis, which also had implications for young adult patients. Simultaneously, studies investigating prognostic biomarkers within tumour types were undertaken. Certain genomic phenotypes were found to portend unfavourable outcomes, for example, MYCN amplification in spinal ependymoma. The arrival of methylation profiling, having revolutionised the diagnosis of CNS tumours, now promises to bring increased prognostic accuracy, as has been shown in meningiomas. While MGMT promoter hypermethylation has remained a reliable biomarker of response to cytotoxic chemotherapy, targeted therapy in CNS tumours has unfortunately not had the success of other cancers. Therefore, predictive biomarkers have lagged behind the identification of prognostic biomarkers in CNS tumours. Emerging research from new clinical trials is cause for guarded optimism and may shift our conceptualisation of predictive biomarker testing in CNS tumours.

'The Reports of My Death Are Greatly Exaggerated'-Evaluating the Effect of Necrosis on MGMT Promoter Methylation Testing in High-Grade Glioma.

(1) Background: MGMT (O-6-methylguanine-DNA methyltransferase) promoter methylation remains an important predictive biomarker in high-grade gliomas (HGGs). The influence of necrosis on the fidelity of MGMT promoter (MGMTp) hypermethylation testing is currently unknown. Therefore, our study aims to evaluate the effect of varying degrees of necrosis on MGMTp status, as determined by pyrosequencing, in a series of primary and recurrent HGGs; (2) Methods: Within each case, the most viable blocks (assigned as 'true' MGMTp status) and the most necrotic block were determined by histopathology review. MGMTp status was determined by pyrosequencing. Comparisons of MGMTp status were made between the most viable and most necrotic blocks. (3) Results: 163 samples from 64 patients with HGGs were analyzed. MGMTp status was maintained in 84.6% of primary and 78.3% of recurrent HGGs between the most viable and necrotic blocks. A threshold of ≥60% tumor cellularity was established at which MGMTp status was unaltered, irrespective of the degree of necrosis. (4) Conclusions: MGMTp methylation status, as determined by pyrosequencing, does not appear to be influenced by necrosis in the majority of cases at a cellularity of at least 60%. Further investigation into the role of intratumoral heterogeneity on MGMTp status will increase our understanding of this predictive marker.

Understanding the Epitranscriptome for Avant-Garde Brain Tumour Diagnostics.

RNA modifications are diverse, dynamic, and reversible transcript alterations rapidly gaining attention due to their newly defined RNA regulatory roles in cellular pathways and pathogenic mechanisms. The exciting emerging field of 'epitranscriptomics' is predominantly centred on studying the most abundant mRNA modification, N6-methyladenine (m6A). The m6A mark, similar to many other RNA modifications, is strictly regulated by so-called 'writer', 'reader', and 'eraser' protein species. The abundance of genes coding for the expression of these regulator proteins and m6A levels shows great potential as diagnostic and predictive tools across several cancer fields. This review explores our current understanding of RNA modifications in glioma biology and the potential of epitranscriptomics to develop new diagnostic and predictive classification tools that can stratify these highly complex and heterogeneous brain tumours.

Correlations between histopathological diagnosis of chemotherapy-induced hepatic injury, clinical features, and perioperative morbidity.

BACKGROUND: Chemotherapy has in some series been linked with increased morbidity after a hepatectomy. Hepatic injuries may result from the treatment with chemotherapy, but can also be secondary to co-morbid diseases. The aim of the present study was to draw correlations between clinical features, treatment with chemotherapy and injury phenotypes and assess the impact of each upon perioperative morbidity. PATIENTS AND METHODS: Retrospective samples (n= 232) were scored grading steatosis, steatohepatitis and sinusoidal injury (SI). Clinical data were retrieved from medical records. Correlations were drawn between injury, clinical features and perioperative morbidity. RESULTS: Injury rates were 18%, 4% and 19% for steatosis, steatohepatitis and SI, respectively. High-grade steatosis was more common in patients with diabetes [odds ratio (OR) = 3.33, P= 0.01] and patients with a higher weight (OR/kg = 1.04, P= 0.02). Steatohepatitis was increased with metabolic syndrome (OR = 5.88, P= 0.02). Chemotherapy overall demonstrated a trend towards an approximately doubled risk of high-grade steatosis and steatohepatitis although not affecting SI. However, pre-operative chemotherapy was associated with an increased SI (OR = 2.18, P= 0.05). Operative morbidity was not increased with chemotherapy, but was increased with steatosis (OR = 2.38, P= 0.02). CONCLUSIONS: Diabetes and higher weight significantly increased the risk of steatosis, whereas metabolic syndrome significantly increased risk of steatohepatitis. The presence of high-grade steatosis increases perioperative morbidity, not administration of chemotherapy per se.

Hemorrhagic Meningitis Due to Varicella-Zoster Virus: A Case Report With Clinical, Cytologic, and Radiologic Correlation.

Although varicella-zoster virus (VZV) is known to affect the central nervous system in a protean manner, hemorrhagic VZV meningitis has not been well documented in the literature. Here, we correlate the clinical, cytologic, and radiologic findings in an immunocompromised patient presenting with subarachnoid hemorrhage associated with VZV meningitis. Clinical findings included multidermatomal zoster, myelitis, and neurapraxia. Magnetic resonance imaging findings included superficial siderosis and diffused linear and nodular leptomeningeal enhancement. Cerebrospinal fluid cytology revealed hemorrhage and lymphocytic pleocytosis. This case report adds hemorrhagic meningitis to the spectrum of complications associated with disseminated VZV infection.

"MYH9 mutation and squamous cell cancer of the tongue in a young adult: a novel case report".

BACKGROUND: The incidence of tongue cancer in young adults is on the rise. This trend is more pronounced in females. Although the aetiology is still unclear, there is mounting evidence that genetic syndromes can play a key role in development of oral cancers in this patient group. We report the first case of oral squamous cell carcinoma (oSCC) in a young adult with an MYH9-related disorder (MYH9-RD). CASE PRESENTATION: A 19-year-old female with a germline MYH9 variant (missense variant in exon 2: c.287C > T, (p.Ser96Leu)) was referred to the head and neck surgery department for investigation of a painful, thick right tongue ulcer. She was diagnosed with Epstein syndrome, an MYH9-RD, at 12 years of age. Her main phenotypic features were profound thrombocytopenia and marked renal impairment. The tongue biopsy confirmed SCC. Preoperative positron emission tomography (PET) revealed avidity in the right tongue and ipsilateral level 2A neck lymph node. With substantial preoperative multidisciplinary input, she underwent cancer ablation and microvascular free flap reconstruction. Her pathology showed a 35 mm diameter, 14 mm thick moderately differentiated SCC with perineural and lymphovascular invasion. Two out of 38 right neck nodes were positive for metastasis with extranodal extension. None of the 34 left neck nodes was involved. She had an uneventful recovery and was discharged home on day 6 postoperative day. On day 15 postoperative day, she had catastrophic bleeding in the neck with a respiratory arrest after a forceful cough. She required an emergency tracheostomy and returned to the theatre for haemostasis. Following a short inpatient stay, she was again discharged home and underwent adjuvant therapy consisting of external beam radiotherapy of 60Gy in 30 fractions. On clinical examination and PET at 6 months after surgery, she had no evidence of disease recurrence. CONCLUSIONS: MYH9-RD can present with advanced locoregional oral cavity malignancy at an early age. The combination of profound thrombocytopenia and marked renal impairment can impact heavily on routine major head and neck cancer surgery and adjuvant treatment. This rare and challenging condition underlines the importance of early detection of cancer and multidisciplinary team input.

Residual melanoma in wide local excision specimens after 'complete' excision of primary cutaneous in situ and invasive melanomas.

Wide local excision (WLE) to achieve adequate clearance margins is the standard initial definitive treatment for patients with biopsy-proven primary cutaneous melanoma. Residual melanoma in WLE specimens after prior complete excision-biopsy (CEB) is reported in 0-6.3% of cases. However, studies evaluating the prevalence, clinicopathological features and relevance of persistent disease in WLE specimens are limited. This study sought to determine the frequency of and clinicopathological characteristics associated with residual melanoma in WLE specimens performed after a CEB of primary cutaneous or acral melanoma (in situ or invasive) with clinically and histologically tumour-free margins, and assess its relevance. A review of the research database and pathology archives of a large Australian tertiary referral melanoma treatment centre was performed. Eligible patients were those for whom a definitive WLE was performed after CEB of a primary melanoma (in situ or invasive) with negative clinical and histological margins, between May 2013 and May 2015. All partial biopsies were excluded. Of 640 eligible patients, 510 (79.7%) had invasive melanoma and 130 (20.3%) had melanoma in situ. Residual disease was identified in 20 cases (20/640, 3.1%), of which three (15%) were melanoma in situ on CEB and 17 (85%) were invasive melanoma. On univariate analysis, the presence of residual disease in WLE specimens was associated with lentigo maligna (LM)/LM melanoma (LMM) subtype [odds ratio (OR) 10.33; 95% confidence interval (CI) 2.84-37.54; p=0.004], nodular melanoma (NM) subtype (OR 4.92; 95% CI 1.53-15.85; p=0.0076) and, for invasive tumours, higher tumour mitotic rate (mean 7.7, SD 7.51 vs 3.4, SD 4.83; OR 1.11; 95% CI 1.04-1.18; p=0.0014). Breslow thickness >4 mm was associated with a higher risk of residual disease (OR 7.30; 95% CI 1.88, 28.26; p=0.004). Cases with residual disease had primary tumours with a significantly larger diameter (median 14 mm, range 4-25) than those without residual disease (median 9 mm, range 2-60), (OR 1.07; 95% CI 1.03-1.11; p≤0.001) and were also more likely to be amelanotic (38% vs 14%), (OR 3.69; 95% CI 1.17, 11.60; p=0.026). Residual disease was associated with assessment of >3 slides of tissue (OR 6.98; 95% CI 1.54-31.62; p=0.0118) and complete blocking of the scar (OR 31.69; 95% CI 3.98-252.21; p=0.0011). Residual melanoma in WLE specimens is an infrequent occurrence. Risk factors for residual disease are LM/LMM and NM melanoma subtypes, higher mitotic rate, larger lesion diameter and amelanosis. Tumours with these features warrant more extensive pathological sampling. WLE after CEB for melanoma remains an important procedure to reduce local recurrence; however, limited pathological sampling of the WLE scar is probably appropriate for cases lacking high risk features.