Expression profiling of single cells and patient cohorts identifies multiple immunosuppressive pathways and an altered NK cell phenotype in glioblastoma.

Glioblastoma (GBM) is an aggressive cancer with a very poor prognosis. Generally viewed as weakly immunogenic, GBM responds poorly to current immunotherapies. To understand this problem more clearly we used a combination of natural killer (NK) cell functional assays together with gene and protein expression profiling to define the NK cell response to GBM and explore immunosuppression in the GBM microenvironment. In addition, we used transcriptome data from patient cohorts to classify GBM according to immunological profiles. We show that glioma stem-like cells, a source of post-treatment tumour recurrence, express multiple immunomodulatory cell surface molecules and are targeted in preference to normal neural progenitor cells by natural killer (NK) cells ex vivo. In contrast, GBM-infiltrating NK cells express reduced levels of activation receptors within the tumour microenvironment, with hallmarks of transforming growth factor (TGF)-ß-mediated inhibition. This NK cell inhibition is accompanied by expression of multiple immune checkpoint molecules on T cells. Single-cell transcriptomics demonstrated that both tumour and haematopoietic-derived cells in GBM express multiple, diverse mediators of immune evasion. Despite this, immunome analysis across a patient cohort identifies a spectrum of immunological activity in GBM, with active immunity marked by co-expression of immune effector molecules and feedback inhibitory mechanisms. Our data show that GBM is recognized by the immune system but that anti-tumour immunity is restrained by multiple immunosuppressive pathways, some of which operate in the healthy brain. The presence of immune activity in a subset of patients suggests that these patients will more probably benefit from combination immunotherapies directed against multiple immunosuppressive pathways.

Development and external validation study of a melanoma risk prediction model incorporating clinically assessed naevi and solar lentigines.

BACKGROUND: Melanoma risk prediction models could be useful for matching preventive interventions to patients' risk. OBJECTIVES: To develop and validate a model for incident first-primary cutaneous melanoma using clinically assessed risk factors. METHODS: We used unconditional logistic regression with backward selection from the Australian Melanoma Family Study (461 cases and 329 controls) in which age, sex and city of recruitment were kept in each step, and we externally validated it using the Leeds Melanoma Case-Control Study (960 cases and 513 controls). Candidate predictors included clinically assessed whole-body naevi and solar lentigines, and self-assessed pigmentation phenotype, sun exposure, family history and history of keratinocyte cancer. We evaluated the predictive strength and discrimination of the model risk factors using odds per age- and sex-adjusted SD (OPERA) and the area under curve (AUC), and calibration using the Hosmer-Lemeshow test. RESULTS: The final model included the number of naevi ≥ 2 mm in diameter on the whole body, solar lentigines on the upper back (a six-level scale), hair colour at age 18 years and personal history of keratinocyte cancer. Naevi was the strongest risk factor; the OPERA was 3·51 [95% confidence interval (CI) 2·71-4·54] in the Australian study and 2·56 (95% CI 2·23-2·95) in the Leeds study. The AUC was 0·79 (95% CI 0·76-0·83) in the Australian study and 0·73 (95% CI 0·70-0·75) in the Leeds study. The Hosmer-Lemeshow test P-value was 0·30 in the Australian study and < 0·001 in the Leeds study. CONCLUSIONS: This model had good discrimination and could be used by clinicians to stratify patients by melanoma risk for the targeting of preventive interventions. What's already known about this topic? Melanoma risk prediction models may be useful in prevention by tailoring interventions to personalized risk levels. For reasons of feasibility, time and cost many melanoma prediction models use self-assessed risk factors. However, individuals tend to underestimate their naevus numbers. What does this study add? We present a melanoma risk prediction model, which includes clinically-assessed whole-body naevi and solar lentigines, and self-assessed risk factors including pigmentation phenotype and history of keratinocyte cancer. This model performs well on discrimination, the model's ability to distinguish between individuals with and without melanoma, and may assist clinicians to stratify patients by melanoma risk for targeted preventive interventions.

Associations of pigmentary and naevus phenotype with melanoma risk in two populations with comparable ancestry but contrasting levels of ambient sun exposure.

BACKGROUND: People at high risk of developing melanoma are usually identified by pigmentary and naevus phenotypes. OBJECTIVE: We examined whether associations of these phenotypes with melanoma risk differed by ambient sun exposure or participant characteristics in two population-based, case-control studies with comparable ancestry but different ambient sun exposure. METHODS: Data were analysed from 616 cases and 496 controls from the Australian Melanoma Family Study and 2012 cases and 504 controls from the Leeds (UK) case-control study. Questionnaire, interview and dermatological skin examination data were collected using the same measurement protocols. Relative risks were estimated as odds ratios using unconditional logistic regression, adjusted for potential confounders. RESULTS: Hair and skin colour were the strongest pigmentary phenotype risk factors. All associations of pigmentary phenotype with melanoma risk were similar across countries. The median number of clinically assessed naevi was approximately three times higher in Australia than Leeds, but the relative risks for melanoma associated with each additional common or dysplastic naevus were higher for Leeds than Australia, especially for naevi on the upper and lower limbs. Higher naevus counts on the head and neck were associated with a stronger relative risk for melanoma for women than men. The two countries had similar relative risks for melanoma based on self-reported naevus density categories, but personal perceptions of naevus number differed by country. There was no consistent evidence of interactions between phenotypes on risk. CONCLUSIONS: Classifying people at high risk of melanoma based on their number of naevi should ideally take into account their country of residence, type of counts (clinical or self-reported), body site on which the naevus counts are measured and sex. The presence of naevi may be a stronger indicator of a genetic predisposition in the UK than in Australia based on less opportunity for sun exposure to influence naevus development.

Vitamin D, vitamin A, the primary melanoma transcriptome and survival.

Survival from melanoma is influenced by several, well-established clinical and histopathological factors, e.g. age, Breslow thickness and microscopic ulceration. We (the Section of Epidemiology and Biostatistics, University of Leeds) have carried out research to better understand the biological basis for these observations. Preliminary results indicated a protective role for vitamin D in melanoma relapse and that higher vitamin D was associated with thinner primary melanomas. Funding from the British Skin Foundation enabled JNB to establish a study of the effects of vitamin A in melanoma. The results suggested that vitamin A could reduce the protective effect of vitamin D in terms of overall survival. Therefore, we propose that vitamin D3 supplementation alone might be preferable to combined multivitamin preparations, where vitamin D supplementation is deemed to be appropriate. Proving a causal link between vitamin D and melanoma-specific survival is challenging. We have shown limited evidence of causation in a Mendelian randomization experiment (described in more detail later). Recent work in Leeds has also shown that higher vitamin D may be protective for microscopic ulceration. Taken together, vitamin D appears to be associated with less aggressive primary melanomas and may itself influence outcome. We continue to explore the role of vitamin D in melanoma survival and the optimum levels that might be crucial.

MC1R gene variants and non-melanoma skin cancer: a pooled-analysis from the M-SKIP project.

BACKGROUND: The melanocortin-1-receptor (MC1R) gene regulates human pigmentation and is highly polymorphic in populations of European origins. The aims of this study were to evaluate the association between MC1R variants and the risk of non-melanoma skin cancer (NMSC), and to investigate whether risk estimates differed by phenotypic characteristics. METHODS: Data on 3527 NMSC cases and 9391 controls were gathered through the M-SKIP Project, an international pooled-analysis on MC1R, skin cancer and phenotypic characteristics. We calculated summary odds ratios (SOR) with random-effect models, and performed stratified analyses. RESULTS: Subjects carrying at least one MC1R variant had an increased risk of NMSC overall, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC): SOR (95%CI) were 1.48 (1.24-1.76), 1.39 (1.15-1.69) and 1.61 (1.35-1.91), respectively. All of the investigated variants showed positive associations with NMSC, with consistent significant results obtained for V60L, D84E, V92M, R151C, R160W, R163Q and D294H: SOR (95%CI) ranged from 1.42 (1.19-1.70) for V60L to 2.66 (1.06-6.65) for D84E variant. In stratified analysis, there was no consistent pattern of association between MC1R and NMSC by skin type, but we consistently observed higher SORs for subjects without red hair. CONCLUSIONS: Our pooled-analysis highlighted a role of MC1R variants in NMSC development and suggested an effect modification by red hair colour phenotype.

Assessing the impact of diagnosis and the related supportive care needs in patients with cutaneous melanoma.

PURPOSE: Despite the large number of people affected by melanoma, little is known about the specific needs of melanoma patients. Understanding the effects of melanoma diagnosis and the specific supportive care needs of this group of patients is a necessary step towards provision of effective psychosocial care. METHODS: Semi-structured interviews were carried out with 15 patients with malignant melanoma of the skin. The sample size, which was purposive, included 8 females and 7 males from 27 to 78 years old. Data were analysed using the NVIVO 8 software and principles of thematic analysis. RESULTS: Four major areas were identified: (a) Emotional effects due to body image, fear of the sun and uncertainty for the future; (b) Effects on Relationships, with some patients in need of more support than others from family and work colleagues; (c) Functional effects due to on-going symptoms such as pain and lymphedema; and (d) Health System and Information Needs, around the clarity, quality and timing of the information received from the health care professionals. CONCLUSIONS: The findings suggest that we often fail to pick up melanoma patients' health and psychosocial needs and fail to refer them appropriately, rather than the services not being available. Interventions should focus on patient and carer education about melanoma and sun protection, psychosocial support and effective information giving. Patient-reported outcome measures should routinely be collected to identify issues of specific concerns to the patients and directing them to the right services based on their individual needs.

Prevalence and correlates of unmet supportive care needs in patients with resected invasive cutaneous melanoma.

BACKGROUND: Knowledge about supportive care needs in patients with cutaneous invasive melanoma is scarce. We examined the unmet needs of melanoma patients treated with surgery and factors associated with these needs to assist health professionals identify areas needing clinical attention. PATIENTS AND METHODS: Cross-sectional multisite survey of UK patients ascertained 3 months to 5 years after complete resection of stage I-III cutaneous melanoma. Participants completed the following validated questionnaires: Supportive Care Needs Survey (SCNS-SF34 with melanoma module), Hospital Anxiety and Depression Scale and 51-item Functional Assessment of Cancer Therapy-Melanoma quality-of-life scale. RESULTS: A total of 472 participants were recruited [319 (67%) clinical stage I-II). Mean age was 60 years (standard deviation = 14) and 255 (54%) were female. One hundred and twenty-three (27%) participants reported at least one unmet need (mostly 'low' level). The most frequently reported unmet needs were fears of cancer returning (n = 138, 29%), uncertainty about the future (n = 119, 25%), lack of information about risk of recurrence (n = 112, 24%) and about possible outcomes if melanoma were to spread (n = 91, 20%). One hundred and thirty-eight (29%) participants reported anxiety and 51 (11%) depression at clinical or subclinical levels. Patients with nodal disease had a significantly higher level of unmet supportive care needs (P < 0.001) as did patients with anxiety or depression (P < 0.001). Key correlates of the total SCNS-SF34 score for unmet supportive care needs were younger age (odds ratio, OR = 2.23, P < 0.001) and leaving school early (OR = 4.85, P < 0.001), while better emotional (OR = 0.89, P < 0.001) and social well-being (OR = 0.91, P < 0.001) were linked with fewer unmet needs. Neither patients' sex nor tumour thickness was associated with unmet needs. CONCLUSIONS: Around a quarter of melanoma patients may have unmet support needs in the mid to long term after primary treatment. In particular, patients who are younger, less educated, distressed or socially isolated could benefit from more support.

An exploration of reported mortality from cutaneous squamous cell carcinoma using death certification and cancer registry data.

BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) is increasing in incidence but mortality rates are low. Identifying high-risk tumours is important when rationalizing clinical review for patients with cSCC. OBJECTIVES: To assess the accuracy of death certification in cases of reported fatal cSCC and to identify risk factors for fatal cSCC. METHODS: A retrospective, observational study of cases of fatal cSCC over 11 years (1993-2004) in Leeds, identified in cancer registry and death certification data. RESULTS: Fifty-eight patients were recorded by the registry as having fatal cSCC in this period. Review of case notes and pathology specimens, where available (34 cases), confirmed that 21/34 patients had died of cSCC. Five were on the ear and none on the lip. Four patients had been treated for leukaemia or lymphoma and one was a renal transplant recipient. On pathology review five patients proved to have had malignant adnexal tumours rather than cSCC, and one a melanoma. In addition, three patients had disease of the ear canal or vulva. CONCLUSIONS: A proportion of deaths were falsely attributed to cSCC as a result of inaccurate histological diagnosis. Some fatalities were related to tumours in sites known to be at higher risk, and a significant proportion was postulated to be related to immunosuppression. In those cases attributed to cSCC in which this could be assessed, the majority were American Joint Committee on Cancer stage 2 and only 24% were in high-risk sites.

Cutaneous adverse events associated with vemurafenib in patients with metastatic melanoma: practical advice on diagnosis, prevention and management of the main treatment-related skin toxicities.

Until recently, no effective treatment was available for patients with metastatic malignant melanoma, and median overall survival was little more than 6 months with the current standard of care, dacarbazine. In 2012, the first specific BRAF mutation inhibitor, vemurafenib, was licensed for the monotherapy of adults with BRAF V600 mutation-positive unresectable or metastatic melanoma. Like other targeted therapies, vemurafenib is associated with a predictable pattern of adverse events, including skin toxicities. We review the most common cutaneous adverse events associated with vemurafenib, based on data from clinical trials, and our own experiences of treating patients in trials and clinical practice. Overall, these toxicities are not preventable, but they rarely necessitate permanent treatment discontinuation and are generally manageable with dose modification and supportive care. We provide a treatment algorithm offering guidance on the most appropriate approach to managing the main skin toxicities to help clinicians unfamiliar with this novel agent to become confident in using vemurafenib effectively in the management of patients with metastatic melanoma.

The determinants of periorbital skin ageing in participants of a melanoma case-control study in the U.K.

BACKGROUND: Skin ageing is said to be caused by multiple factors. The relationship with sun exposure is of particular interest because the detrimental cutaneous effects of the sun may be a strong motivator to sun protection. We report a study of skin ageing in participants of an epidemiological study of melanoma. OBJECTIVES: To determine the predictors of periorbital cutaneous ageing and whether it could be used as an objective marker of sun exposure. METHODS: Photographs of the periorbital skin in 1341 participants were graded for wrinkles, degree of vascularity and blotchy pigmentation and the resultant data assessed in relation to reported sun exposure, sunscreen use, body mass index (BMI), smoking and the melanocortin 1 receptor (MC1R) gene status. Data were analysed using proportional odds regression. RESULTS: Wrinkling was associated with age and heavy smoking. Use of higher sun-protection factor sunscreen was protective (P = 0·01). Age, male sex, MC1R variants ('r', P=0·01; 'R', P=0·02), higher reported daily sun exposure (P=0·02), increased BMI (P=0·01) and smoking (P=0·02) were risk factors for hypervascularity. Blotchy pigmentation was associated with age, male sex, higher education and higher weekday sun exposure (P=0·03). More frequent sunscreen use (P=0·02) and MC1R variants ('r', P=0·03; 'R', P=0·001) were protective. CONCLUSIONS: Periorbital wrinkling is a poor biomarker of reported sun exposure. Vascularity is a better biomarker as is blotchy pigmentation, the latter in darker-skinned individuals. In summary, male sex, sun exposure, smoking, obesity and MC1R variants were associated with measures of cutaneous ageing. Sunscreen use showed some evidence of being protective.

The role of CAF derived exosomal microRNAs in the tumour microenvironment of melanoma.

Exosomes play a crucial role in the crosstalk between cancer associated fibroblasts (CAFs) and cancer cells, contributing to carcinogenesis and the tumour microenvironment. Recent studies have revealed that CAFs, normal fibroblasts and cancer cells all secrete exosomes that contain miRNA, establishing a cell-cell communication network within the tumour microenvironment. For example, miRNA dysregulation in melanoma has been shown to promote CAF activation via induction of epithelial-mesenchymal transition (EMT), which in turn alters the secretory phenotype of CAFs in the stroma. This review assesses the roles of melanoma exosomal miRNAs in CAF formation and how CAF exosome-mediated feedback signalling to melanoma lead to tumour progression and metastasis. Moreover, efforts to exploit exosomal miRNA-mediated network communication between tumour cells and their microenvironment, and their potential as prognostic biomarkers or novel therapeutic targets in melanoma will also be considered.

SFPQ promotes an oncogenic transcriptomic state in melanoma.

The multifunctional protein, splicing factor, proline- and glutamine-rich (SFPQ) has been implicated in numerous cancers often due to interaction with coding and non-coding RNAs, however, its role in melanoma remains unclear. We report that knockdown of SFPQ expression in melanoma cells decelerates several cancer-associated cell phenotypes, including cell growth, migration, epithelial to mesenchymal transition, apoptosis, and glycolysis. RIP-seq analysis revealed that the SFPQ-RNA interactome is reprogrammed in melanoma cells and specifically enriched with key melanoma-associated coding and long non-coding transcripts, including SOX10, AMIGO2 and LINC00511 and in most cases SFPQ is required for the efficient expression of these genes. Functional analysis of two SFPQ-enriched lncRNA, LINC00511 and LINC01234, demonstrated that these genes independently contribute to the melanoma phenotype and a more detailed analysis of LINC00511 indicated that this occurs in part via modulation of the miR-625-5p/PKM2 axis. Importantly, analysis of a large clinical cohort revealed that elevated expression of SFPQ in primary melanoma tumours may have utility as a prognostic biomarker. Together, these data suggest that SFPQ is an important driver of melanoma, likely due to SFPQ-RNA interactions promoting the expression of numerous oncogenic transcripts.

Melanoma sentinel node biopsy and prediction models for relapse and overall survival.

BACKGROUND: To optimise predictive models for sentinal node biopsy (SNB) positivity, relapse and survival, using clinico-pathological characteristics and osteopontin gene expression in primary melanomas. METHODS: A comparison of the clinico-pathological characteristics of SNB positive and negative cases was carried out in 561 melanoma patients. In 199 patients, gene expression in formalin-fixed primary tumours was studied using Illumina's DASL assay. A cross validation approach was used to test prognostic predictive models and receiver operating characteristic curves were produced. RESULTS: Independent predictors of SNB positivity were Breslow thickness, mitotic count and tumour site. Osteopontin expression best predicted SNB positivity (P=2.4 × 10⁻7), remaining significant in multivariable analysis. Osteopontin expression, combined with thickness, mitotic count and site, gave the best area under the curve (AUC) to predict SNB positivity (72.6%). Independent predictors of relapse-free survival were SNB status, thickness, site, ulceration and vessel invasion, whereas only SNB status and thickness predicted overall survival. Using clinico-pathological features (thickness, mitotic count, ulceration, vessel invasion, site, age and sex) gave a better AUC to predict relapse (71.0%) and survival (70.0%) than SNB status alone (57.0, 55.0%). In patients with gene expression data, the SNB status combined with the clinico-pathological features produced the best prediction of relapse (72.7%) and survival (69.0%), which was not increased further with osteopontin expression (72.7, 68.0%). CONCLUSION: Use of these models should be tested in other data sets in order to improve predictive and prognostic data for patients.

Environmental Exposures Such as Smoking and Low Vitamin D Are Predictive of Poor Outcome in Cutaneous Melanoma rather than Other Deprivation Measures.

A lack of basic resources within a society (deprivation) is associated with increased cancer mortality, and this relationship has been described for melanoma. We have previously reported the association of smoking and low vitamin D levels with melanoma death. In this study, we further explored the associations of these with melanoma in addition to deprivation and socio-economic stressors. In this analysis of 2,183 population-ascertained primary cutaneous melanoma patients, clinical, demographic, and socio-economic variables were assessed as predictors of tumor thickness, melanoma death and overall death. Using the Townsend deprivation score, the most deprived group did not have thicker tumors compared to the least deprived. Of the World Health Organization 25x25 risk factors for premature death, smoking and body mass index (BMI) were independently associated with thicker tumors. Low vitamin D was also independently associated with thicker tumors. No socio-economic stressors were independent predictors of thickness. Smoking was confirmed as a key predictor of melanoma death and overall death, as were low vitamin D levels, independent of other measures of deprivation. Neither BMI nor the Townsend deprivation score were predictive in either survival analysis. We report evidence for the role of smoking, vitamin D, and BMI in melanoma progression independent of a postcode-derived measure of deprivation.

Rare variants analysis of cutaneous malignant melanoma genes in Parkinson's disease.

A shared genetic susceptibility between cutaneous malignant melanoma (CMM) and Parkinson's disease (PD) has been suggested. We investigated this by assessing the contribution of rare variants in genes involved in CMM to PD risk. We studied rare variation across 29 CMM risk genes using high-quality genotype data in 6875 PD cases and 6065 controls and sought to replicate findings using whole-exome sequencing data from a second independent cohort totaling 1255 PD cases and 473 controls. No statistically significant enrichment of rare variants across all genes, per gene, or for any individual variant was detected in either cohort. There were nonsignificant trends toward different carrier frequencies between PD cases and controls, under different inheritance models, in the following CMM risk genes: BAP1, DCC, ERBB4, KIT, MAPK2, MITF, PTEN, and TP53. The very rare TYR p.V275F variant, which is a pathogenic allele for recessive albinism, was more common in PD cases than controls in 3 independent cohorts. Tyrosinase, encoded by TYR, is the rate-limiting enzyme for the production of neuromelanin, and has a role in the production of dopamine. These results suggest a possible role for another gene in the dopamine-biosynthetic pathway in susceptibility to neurodegenerative Parkinsonism, but further studies in larger PD cohorts are needed to accurately determine the role of these genes/variants in disease pathogenesis.

Isolation of human tumor-specific antibodies by selection of an antibody phage library on melanoma cells.

A human single-chain Fv (scFv) library as fusion to phage was constructed from donors with a high titer of autoantibodies. The library was subjected to three rounds of positive selection on human melanoma cells and negative selection on human peripheral blood mononuclear cells. Two scFv clones, B3 and B4, were isolated that bound melanoma cells in cell ELISA and fluorescence-activated cell sorting. The scFvs were characterized further by immunohistochemistry on a large number of normal human tissues. No cross-reactivity with normal tissues was observed. On the other hand, the target antigens were expressed in sections from several different melanoma patients and in some breast cancer and basal cell carcinoma sections. The unusually high tumor specificity of the B3 and B4 antigens makes them attractive targets for the specific therapy of melanoma. The selection strategy used should be generally applicable to the identification of novel cell surface antigens by antibody phage display.

Heritability and gene-environment interactions for melanocytic nevus density examined in a U.K. adolescent twin study.

Risk factors for melanoma include environmental (particularly ultraviolet exposure) and genetic factors. In rare families, susceptibility to melanoma is determined by high penetrance mutations in the genes CDKN2A or CDK4, with more common, less penetrant genes also postulated. A further, potent risk factor for melanoma is the presence of large numbers of melanocytic nevi so that genes controlling nevus phenotype could be such melanoma susceptibility genes. A large Australian study involving twins aged 12 y of predominantly U.K. ancestry showed strong evidence for genetic influence on nevus number and density. We carried out essentially the same study in the U.K. to gain insight into gene-environment interactions for nevi. One hundred and three monozygous (MZ) and 118 dizygous (DZ) twin pairs aged 10-18 y were examined in Yorkshire and Surrey, U.K. Nevus counts were, on average, higher in boys (mean = 98.6) than girls (83.8) (p = 0.009) and higher in Australia (110.4) than in the U.K. (79.2, adjusted to age 12 y, p < 0.0001), and nevus densities were higher on sun-exposed sites (92 per m2) than sun-protected sites (58 per m2) (p < 0.0001). Correlations in sex and age adjusted nevus density were higher in MZ pairs (0.94, 95%CI 0.92-0.96) than in DZ pairs (0.61, 95%CI 0.49-0.72), were notably similar to those of the Australian study (MZ = 0.94, DZ = 0.60), and were consistent with high heritability (65% in the U.K., 68% in Australia). We conclude that emergence of nevi in adolescents is under strong genetic control, whereas environmental exposures affect the mean number of nevi.