POT1 and multiple primary melanomas: the dermatological phenotype.

POT1 is the second most frequently reported gene (after CDKN2A) in familial melanoma. Pathogenic variants are associated with earlier onset and/or multiple primary melanomas (MPMs). To date, POT1 phenotypical reports have been largely restricted to associated malignancies, and description of the dermatological landscape has been limited. We identified 10 variants in n=18 of 384 (4.7%) unrelated individuals (n=13 MPMs; n=5 single primary melanomas) of European ancestry. Five variants were rare (minor allele frequency <0.001) or novel (two loss-of-function (LOF), one splice acceptor and two missense) and were predicted to be functionally significant, in five unrelated probands with MPMs (≥3 melanomas). We performed three-dimensional total body photography on both individuals with confirmed pathogenic LOF variants to characterise the dermatological phenotype. Total body naevus counts (≥2 mm diameter) were significantly higher (p=7.72×10-12) in carriers compared with a control population. Majority of naevi were on the probands' back and lower limb regions, where only mild to moderate ultraviolet (UV) damage was observed. Conversely, the head/neck region, where both probands exhibited severe UV damage, had comparably fewer naevi. We hypothesise that carriage of functionally significant POT1 variants is associated with increased naevus counts generally, and naevi >5 mm in diameter specifically and the location of these are independent of UV damage.

The role of artificial intelligence and convolutional neural networks in the management of melanoma: a clinical, pathological, and radiological perspective.

Clinical dermatoscopy and pathological slide assessment are essential in the diagnosis and management of patients with cutaneous melanoma. For those presenting with stage IIC disease and beyond, radiological investigations are often considered. The dermatoscopic, whole slide and radiological images used during clinical care are often stored digitally, enabling artificial intelligence (AI) and convolutional neural networks (CNN) to learn, analyse and contribute to the clinical decision-making. A keyword search of the Medline database was performed to assess the progression, capabilities and limitations of AI and CNN and its use in diagnosis and management of cutaneous melanoma. Full-text articles were reviewed if they related to dermatoscopy, pathological slide assessment or radiology. Through analysis of 95 studies, we demonstrate that diagnostic accuracy of AI/CNN can be superior (or at least equal) to clinicians. However, variability in image acquisition, pre-processing, segmentation, and feature extraction remains challenging. With current technological abilities, AI/CNN and clinicians synergistically working together are better than one another in all subspecialty domains relating to cutaneous melanoma. AI has the potential to enhance the diagnostic capabilities of junior dermatology trainees, primary care skin cancer clinicians and general practitioners. For experienced clinicians, AI provides a cost-efficient second opinion. From a pathological and radiological perspective, CNN has the potential to improve workflow efficiency, allowing clinicians to achieve more in a finite amount of time. Until the challenges of AI/CNN are reliably met, however, they can only remain an adjunct to clinical decision-making.

The oesophageal adenocarcinoma tumour immune microenvironment dictates outcomes with different modalities of neoadjuvant therapy - results from the AGITG DOCTOR trial and the cancer evolution biobank.

A plateau in treatment effect can be seen for the current 'one-size-fits-all' approach to oesophageal adenocarcinoma (OAC) management using neoadjuvant chemoradiotherapy (nCRT) or chemotherapy (nCT). In OAC, the tumour microenvironment (TME) is largely immunosuppressed, however a subgroup of patients with an immune-inflamed TME exist and show improved outcomes. We aimed to understand the overall immune-based mechanisms underlying treatment responses and patient outcomes in OAC, and in relation to neoadjuvant therapy modality. This study included 107 patients; 68 patients were enrolled in the Australian Gastro-Intestinal Trials Group sponsored DOCTOR Trial, and 38 patients were included from the Cancer Evolution Biobank. Matched pre-treatment and post-treatment tumour biopsies were used to perform multi-modality analysis of the OAC TME including NanoString mRNA expression analysis, multiplex and single colour immunohistochemistry (IHC), and peripheral blood mononuclear cell analysis of tumour-antigen specific T cell responses. Patients with the best clinicopathological outcomes and survival had an immune-inflamed TME enriched with anti-tumour immune cells and pathways. Those with the worst survival showed a myeloid T regulatory cell enriched TME, with decreased CD8+ cell infiltration and increased pro-tumour immune cells. Multiplex IHC analysis identified that high intra-tumoural infiltration of CD8+ cells, and low infiltration with CD163+ cells was associated with improved survival. High tumour core CD8+ T cell infiltration, and a low tumour margin infiltration of CD163+ cells was also associated with improved survival. nCRT showed improved survival compared with nCT for patients with low CD8+, or high CD163+ cell infiltration. Poly-functional T cell responses were seen with tumour-antigen specific T cells. Overall, our study supports the development of personalised therapeutic approaches based on the immune microenvironment in OAC. Patients with an immune-inflamed TME show favourable outcomes regardless of treatment modality. However, in those with an immunosuppressed TME with CD163+ cell infiltration, treatment with nCRT can improve outcomes. Our findings support previous studies into the TME of OAC and with more research, immune based biomarker selection of treatment modality may lead in improved outcomes in this deadly disease.

Circulating Tumor DNA: A Promising Biomarker for Predicting Recurrence in Patients with BRAF-Negative Melanoma.

For patients with BRAF wild-type stage III and IV melanoma, there is an urgent clinical need to identify prognostic biomarkers and biomarkers predictive of treatment response. Circulating tumor DNA (ctDNA) is emerging as a blood-based biomarker and has shown promising results for many cancers, including melanoma. The purpose of this study was to identify targetable, tumor-derived mutations in patient blood that may lead to treatment alternatives and improved outcomes for patients with BRAF-negative melanoma. Using a CAncer Personalized Profiling by deep Sequencing (CAPP-seq) pan-cancer gene panel, ctDNA from 150 plasma samples (n = 106 patients) was assessed, including serial blood collections for a subset of patients (n = 16). ctDNA variants were detected in 85% of patients, all in targetable pathways, such as vascular endothelial growth factor receptor, epidermal growth factor receptor, phosphatidylinositol 3-kinase/AKT, Bcl2/mammalian target of rapamycin (mTOR), ALK/MET, and cyclin-dependent kinase 4/6. Patients with stage IV melanoma with low ctDNA concentrations, <10 ng/mL, had significantly better disease-specific survival and progression-free survival. Patients with both a high concentration of ctDNA and any detectable ctDNA variants had the worst prognosis. In addition, these results indicated that longitudinal changes in ctDNA correlated with treatment response and disease progression determined by radiology. This study confirms that ctDNA may be used as a noninvasive liquid biopsy to identify recurrent disease and detect targetable variants in patients with late-stage melanoma.

C-reactive protein is a prognostic biomarker in pancreatic ductal adenocarcinoma patients.

AIM: The 5-year survival rate of pancreatic ductal adenocarcinoma (PDAC) is approximately 11% and has only improved marginally over the last three decades. For operable PDAC, resection and adjuvant FOLFIRINOX chemotherapy is standard of care. There is growing interest in perioperative regimens to improve outcomes. The non-randomized Phase II study "Gemcitabine and Abraxane for resectable Pancreatic cancer" (GAP) demonstrated the feasibility of perioperative gemcitabine/abraxane. Long-term survival in PDAC requires an effective immune response; hence, we undertook this translational study of the GAP trial cohort to identify immune-oncology biomarkers for clinical use. METHODS: We combined Nanostring nCounter technology with immunohistochemistry to investigate the correlation between gene expression and overall patient survival. Findings were investigated in samples from the International Cancer Genome Consortium (ICGC, n = 88) and the Australian Pancreatic Genome Initiative (APGI, n = 227). RESULTS: We confirmed that human equilibrative nucleoside transporter 1 (hENT1) expression was not a prognostic marker in PDAC but patients with high levels of hENT1 were more likely to live longer than 24 months post-surgery. Additionally, CD274 (PD-L1) and two novel biomarkers of survival, cathepsin W (CTSW) and C-reactive protein (CRP), were identified in the GAP cohort (n = 19). CRP expression was confirmed in data from the ICGC. Although PD-L1 and CTSW proteins were not significant across all three cohorts, results show that low CRP mRNA and protein expression are associated with longer overall survival in all three patient groups. CONCLUSION: PDAC patients with long survival have higher hENT1 expression levels. Furthermore, CRP expression is a biomarker of poor prognosis following perioperative chemotherapy and resection in PDAC patients and thus may be useful for identifying patients who could benefit from more aggressive adjuvant strategies.

Multi-omic features of oesophageal adenocarcinoma in patients treated with preoperative neoadjuvant therapy.

Oesophageal adenocarcinoma is a poor prognosis cancer and the molecular features underpinning response to treatment remain unclear. We investigate whole genome, transcriptomic and methylation data from 115 oesophageal adenocarcinoma patients mostly from the DOCTOR phase II clinical trial (Australian New Zealand Clinical Trials Registry-ACTRN12609000665235), with exploratory analysis pre-specified in the study protocol of the trial. We report genomic features associated with poorer overall survival, such as the APOBEC mutational and RS3-like rearrangement signatures. We also show that positron emission tomography non-responders have more sub-clonal genomic copy number alterations. Transcriptomic analysis categorises patients into four immune clusters correlated with survival. The immune suppressed cluster is associated with worse survival, enriched with myeloid-derived cells, and an epithelial-mesenchymal transition signature. The immune hot cluster is associated with better survival, enriched with lymphocytes, myeloid-derived cells, and an immune signature including CCL5, CD8A, and NKG7. The immune clusters highlight patients who may respond to immunotherapy and thus may guide future clinical trials.

Unusual suspects in hereditary melanoma: POT1, POLE, BAP1.

Systematic literature searches on POT1/POLE/BAP1 found that limited skin phenotypic characteristics have been documented in mutation carriers; 248 variants were annotated, and high-cluster variant regions associated with cutaneous melanoma were found in all three genes. Genotype-phenotype correlations can be used to identify patient disease predisposition based on mutation position and cluster regions.

Characterizing the Heterogeneity of Small Extracellular Vesicle Populations in Multiple Cancer Types via an Ultrasensitive Chip.

Identifying small extracellular vesicle (sEV) subpopulations based on their different molecular signatures could potentially reveal the functional roles in physiology and pathology. However, it is a challenge to achieve this aim due to the nano-sized dimensions of sEVs, low quantities of biological cargo each sEV carries, and our incomplete knowledge of identifying features capable of separating heterogeneous sEV subpopulations. Here, a sensitive, multiplexed, and nano-mixing-enhanced sEV subpopulation characterization platform (ESCP) is proposed to precisely determine the sEV phenotypic heterogeneity and understand the role of sEV heterogeneity in cancer progression and metastasis. The ESCP utilizes spatially patterned anti-tetraspanin-functionalized micro-arrays for sEV subpopulation sorting and nanobarcode-based surface-enhanced Raman spectroscopy for multiplexed read-outs. An ESCP has been used for investigating sEV phenotypic heterogeneity in terms of canonical sEV tetraspanin molecules and cancer-associated protein biomarkers in both cancer cell line models and cancer patient samples. Our data explicitly demonstrate the selective enrichment of tetraspanins and cancer-associated protein biomarkers, in particular sEV subpopulations. Therefore, it is believed that the ESCP could enable the evaluation and broader application of sEV subpopulations as potential diagnostic disease biomarkers.

Radiomics Biomarkers Correlate with CD8 Expression and Predict Immune Signatures in Melanoma Patients.

Treatment for metastatic melanoma includes targeted and/or immunotherapy. Although many patients respond, only a subset has complete response. As late-stage patients often have multiple tumors in difficult access sites, non-invasive techniques are necessary for the development of predictive/prognostic biomarkers. PET/CT scans from 52 patients with stage III/IV melanoma were assessed and CT image parameters were evaluated as prognostic biomarkers. Analysis indicated patients with high standard deviation or high mean of positive pixels (MPP) had worse progression-free survival (P = 0.00047 and P = 0.0014, respectively) and worse overall survival (P = 0.0223 and P = 0.0465, respectively). Whole-exome sequencing showed high MPP was associated with BRAF mutation status (P = 0.0389). RNA-sequencing indicated patients with immune "cold" signatures had worse survival, which was associated with CT biomarker, MPP4 (P = 0.0284). Multiplex immunofluorescence confirmed a correlation between CD8 expression and image biomarkers (P = 0.0028). IMPLICATIONS: CT parameters have the potential to be cost-effective biomarkers of survival in melanoma, and reflect the tumor immune-microenvironment. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/6/950/F1.large.jpg.

Pathogenic germline variants are associated with poor survival in stage III/IV melanoma patients.

Patients with late stage resected cutaneous melanoma have poor overall survival (OS) and experience irreversible adverse events from systemic therapy. There is a clinical need to identify biomarkers to predict outcome. Performing germline/tumour whole-exome sequencing of 44 stage III/IV melanoma patients we identified pathogenic germline mutations in CDKN2A, CDK4, ATM, POLH, MRE11A, RECQL4 and XPC, affecting 7/44 patients. These mutations were associated with poor OS (p = 0.0082). We confirmed our findings in The Cancer Genome Atlas (TCGA) human skin cutaneous melanoma cohort where we identified pathogenic variants in 40/455 patients (p = 0.0203). Combining these cohorts (n = 499) further strengthened these findings showing germline carriers had worse OS (p = 0.0009). Additionally, we determined whether tumour mutation burden (TMB) or BRAF status were prognostic markers of survival. Low TMB rate (< 20 Mut/Mb; p = 0.0034) and BRAF p.V600 mutation (p = 0.0355) were associated with worse progression-free survival. Combining these biomarkers indicated that V600 mutant patients had significantly lower TMB (p = 0.0155). This was confirmed in the TCGA (n = 443, p = 0.0007). Integrative analysis showed germline mutation status conferred the highest risk (HR 5.2, 95% CI 1.72-15.7). Stage IV (HR 2.5, 0.74-8.6) and low TMB (HR 2.3, 0.57-9.4) were similar, whereas BRAF V600 status was the weakest prognostic biomarker (HR 1.5, 95% CI 0.44-5.2).

Multiplex melanoma families are enriched for polygenic risk.

Cancers, including cutaneous melanoma, can cluster in families. In addition to environmental etiological factors such as ultraviolet radiation, cutaneous melanoma has a strong genetic component. Genetic risks for cutaneous melanoma range from rare, high-penetrance mutations to common, low-penetrance variants. Known high-penetrance mutations account for only about half of all densely affected cutaneous melanoma families, and the causes of familial clustering in the remainder are unknown. We hypothesize that some clustering is due to the cumulative effect of a large number of variants of individually small effect. Common, low-penetrance genetic risk variants can be combined into polygenic risk scores. We used a polygenic risk score for cutaneous melanoma to compare families without known high-penetrance mutations with unrelated melanoma cases and melanoma-free controls. Family members had significantly higher mean polygenic load for cutaneous melanoma than unrelated cases or melanoma-free healthy controls (Bonferroni-corrected t-test P = 1.5 × 10-5 and 6.3 × 10-45, respectively). Whole genome sequencing of germline DNA from 51 members of 21 families with low polygenic risk for melanoma identified a CDKN2A p.G101W mutation in a single family but no other candidate high-penetrance melanoma susceptibility genes. This work provides further evidence that melanoma, like many other common complex disorders, can arise from the joint action of multiple predisposing factors, including rare high-penetrance mutations, as well as via a combination of large numbers of alleles of small effect.

Complex structural rearrangements are present in high-grade dysplastic Barrett's oesophagus samples.

BACKGROUND: Oesophageal adenocarcinoma (EAC) incidence is increasing and has a poor survival rate. Barrett's oesophagus (BE) is a precursor condition that is associated with EAC and often occurs in conjunction with chronic gastro-oesophageal reflux, however many individuals diagnosed with BE never progress to cancer. An understanding of the genomic features of BE and EAC may help with the early identification of at-risk individuals. METHODS: In this study, we assessed the genomic features of 16 BE samples using whole-genome sequencing. These included non-dysplastic samples collected at two time-points from two BE patients who had not progressed to EAC over several years. Seven other non-dysplastic samples and five dysplastic BE samples with high-grade dysplasia were also examined. We compared the genome profiles of these 16 BE samples with 22 EAC samples. RESULTS: We observed that samples from the two non-progressor individuals had low numbers of somatic single nucleotide variants, indels and structural variation events compared to dysplastic and the remaining non-dysplastic BE. EAC had the highest level of somatic genomic variations. Mutational signature 17, which is common in EAC, was also present in non-dysplastic and dysplastic BE, but was not present in the non-progressors. Many dysplastic samples had mutations in genes previously reported in EAC, whereas only mutations in CDKN2A or in the fragile site genes appeared common in non-dysplastic samples. Rearrangement signatures were used to identify a signature associated with localised complex events such as chromothripsis and breakage fusion-bridge that are characteristic of EACs. Two dysplastic BE samples had a high contribution of this signature and contained evidence of localised rearrangements. Two other dysplastic samples also had regions of localised structural rearrangements. There was no evidence for complex events in non-dysplastic samples. CONCLUSIONS: The presence of complex localised rearrangements in dysplastic samples indicates a need for further investigations into the role such events play in the progression from BE to EAC.

Germline TERT promoter mutations are rare in familial melanoma.

Germline CDKN2A mutations occur in 40 % of 3-or-more case melanoma families while mutations of CDK4, BAP1, and genes involved in telomere function (ACD, TERF2IP, POT1), have also been implicated in melanomagenesis. Mutation of the promoter of the telomerase reverse transcriptase (TERT) gene (c.-57 T>G variant) has been reported in one family. We tested for the TERT promoter variant in 675 multicase families wild-type for the known high penetrance familial melanoma genes, 1863 UK population-based melanoma cases and 529 controls. Germline lymphocyte telomere length was estimated in carriers. The c.-57 T>G TERT promoter variant was identified in one 7-case family with multiple primaries and early age of onset (earliest, 15 years) but not among population cases or controls. One family member had multiple primary melanomas, basal cell carcinomas and a bladder tumour. The blood leukocyte telomere length of a carrier was similar to wild-type cases. We provide evidence confirming that a rare promoter variant of TERT (c.-57 T>G) is associated with high penetrance, early onset melanoma and potentially other cancers, and explains <1 % of UK melanoma multicase families. The identification of POT1 and TERT germline mutations highlights the importance of telomere integrity in melanoma biology.

Deep sequencing of uveal melanoma identifies a recurrent mutation in PLCB4.

Next generation sequencing of uveal melanoma (UM) samples has identified a number of recurrent oncogenic or loss-of-function mutations in key driver genes including: GNAQ, GNA11, EIF1AX, SF3B1 and BAP1. To search for additional driver mutations in this tumor type we carried out whole-genome or whole-exome sequencing of 28 tumors or primary cell lines. These samples have a low mutation burden, with a mean of 10.6 protein changing mutations per sample (range 0 to 53). As expected for these sun-shielded melanomas the mutation spectrum was not consistent with an ultraviolet radiation signature, instead, a BRCA mutation signature predominated. In addition to mutations in the known UM driver genes, we found a recurrent mutation in PLCB4 (c.G1888T, p.D630Y, NM_000933), which was validated using Sanger sequencing. The identical mutation was also found in published UM sequence data (1 of 56 tumors), supporting its role as a novel driver mutation in UM. PLCB4 p.D630Y mutations are mutually exclusive with mutations in GNA11 and GNAQ, consistent with PLCB4 being the canonical downstream target of the former gene products. Taken together these data suggest that the PLCB4 hotspot mutation is similarly a gain-of-function mutation leading to activation of the same signaling pathway, promoting UM tumorigenesis.

POLE mutations in families predisposed to cutaneous melanoma.

Germline mutations in the exonuclease domain of POLE have been shown to predispose to colorectal cancers and adenomas. POLE is an enzyme involved in DNA repair and chromosomal DNA replication. In order to assess whether such mutations might also predispose to cutaneous melanoma, we interrogated whole-genome and exome data from probands of 34 melanoma families lacking pathogenic mutations in known high penetrance melanoma susceptibility genes: CDKN2A, CDK4, BAP1, TERT, POT1, ACD and TERF2IP. We found a novel germline mutation, POLE p.(Trp347Cys), in a 7-case cutaneous melanoma family. Functional assays in S. pombe showed that this mutation led to an increased DNA mutation rate comparable to that seen with a Pol ε mutant with no exonuclease activity. We then performed targeted sequencing of POLE in 1243 cutaneous melanoma cases and found that a further ten probands had novel or rare variants in the exonuclease domain of POLE. Although this frequency is not significantly higher than that in unselected Caucasian controls, we observed multiple cancer types in the melanoma families, suggesting that some germline POLE mutations may predispose to a broad spectrum of cancers, including melanoma. In addition, we found the first mutation outside the exonuclease domain, p.(Gln520Arg), in a family with an extensive history of colorectal cancer.

Molecular characterization of melanoma cases in Denmark suspected of genetic predisposition.

Both environmental and host factors influence risk of cutaneous melanoma (CM), and worldwide, the incidence varies depending on constitutional determinants of skin type and pigmentation, latitude, and patterns of sun exposure. We performed genetic analysis of CDKN2A, CDK4, BAP1, MC1R, and MITFp.E318K in Danish high-risk melanoma cases and found CDKN2A germline mutations in 11.3% of CM families with three or more affected individuals, including four previously undescribed mutations. Rare mutations were also seen in CDK4 and BAP1, while MC1R variants were common, occurring at more than twice the frequency compared to Danish controls. The MITF p.E318K variant similarly occurred at an approximately three-fold higher frequency in melanoma cases than controls. To conclude, we propose that mutation screening of CDKN2A and CDK4 in Denmark should predominantly be performed in families with at least 3 cases of CM. In addition, we recommend that testing of BAP1 should not be conducted routinely in CM families but should be reserved for families with CM and uveal melanoma, or mesothelioma.

Prevalence of Germline BAP1, CDKN2A, and CDK4 Mutations in an Australian Population-Based Sample of Cutaneous Melanoma Cases.

Mutations in Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A) and Cyclin-Dependent Kinase 4 (CDK4) contribute to susceptibility in approximately 40% of high-density cutaneous melanoma (CMM) families and about 2% of unselected CMM cases. BRCA-1 associated protein-1 (BAP1) has been more recently shown to predispose to CMM and uveal melanoma (UMM) in some families; however, its contribution to CMM development in the general population is unreported. We sought to determine the contribution of these genes to CMM susceptibility in a population-based sample of cases from Australia. We genotyped 1,109 probands from Queensland families and found that approximately 1.31% harbored mutations in CDKN2A, including some with novel missense mutations (p.R22W, p.G35R and p.I49F). BAP1 missense variants occurred in 0.63% of cases but no CDK4 variants were observed in the sample. This is the first estimate of the contribution of BAP1 and CDK4 to a population-based sample of CMM and supports the previously reported estimate of CDKN2A germline mutation prevalence.

Germline RAD51B truncating mutation in a family with cutaneous melanoma.

Known melanoma predisposition genes only account for around 40% of high-density melanoma families. Other rare mutations are likely to play a role in melanoma predisposition. RAD51B plays an important role in DNA repair through homologous recombination, and inactivation of RAD51B has been implicated in tumorigenesis. Thus RAD51B is a good candidate melanoma susceptibility gene, and previously, a germline splicing mutation in RAD51B has been identified in a family with early-onset breast cancer. In order to find genetic variants associated with melanoma predisposition, whole-exome sequencing was carried out on blood samples from a three-case cutaneous melanoma family. We identified a novel germline RAD51B nonsense mutation, and we demonstrate reduced expression of RAD51B in melanoma cells indicating inactivation of RAD51B. This is only the second report of a germline truncating RAD51B mutation. While this case report is consistent with melanoma being part of the RAD51B cancer spectrum further population-based screening of large case-control sample series will be needed to definitively establish if this is the case.

Genetics of familial melanoma: 20 years after CDKN2A.

Twenty years ago, the first familial melanoma susceptibility gene, CDKN2A, was identified. Two years later, another high-penetrance gene, CDK4, was found to be responsible for melanoma development in some families. Progress in identifying new familial melanoma genes was subsequently slow; however, with the advent of next-generation sequencing, a small number of new high-penetrance genes have recently been uncovered. This approach has identified the lineage-specific oncogene MITF as a susceptibility gene both in melanoma families and in the general population, as well as the discovery of telomere maintenance as a key pathway underlying melanoma predisposition. Given these rapid recent advances, this approach seems likely to continue to pay dividends. Here, we review the currently known familial melanoma genes, providing evidence that most additionally confer risk to other cancers, indicating that they are likely general tumour suppressor genes or oncogenes, which has significant implications for surveillance and screening.