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Melanoma is a cancer of melanocytes, with multiple subtypes based on body site location. Cutaneous melanoma is associated with skin exposed to ultraviolet radiation; uveal melanoma occurs in the eyes; mucosal melanoma occurs in internal mucous membranes; and acral melanoma occurs on the palms, soles, and nail beds. Here, we present the largest whole-genome sequencing study of melanoma to date, with 570 tumors profiled, as well as methylation and RNA sequencing for subsets of tumors. Uveal melanoma is genomically distinct from other melanoma subtypes, harboring the lowest tumor mutation burden and with significantly mutated genes in the G-protein signaling pathway. Most cutaneous, acral, and mucosal melanomas share alterations in components of the MAPK, PI3K, p53, p16, and telomere pathways. However, the mechanism by which these pathways are activated or inactivated varies between melanoma subtypes. Additionally, we identify potential novel germline predisposition genes for some of the less common melanoma subtypes. Significance: This is the largest whole-genome analysis of melanoma to date, comprehensively comparing the genomics of the four major melanoma subtypes. This study highlights both similarities and differences between the subtypes, providing insights into the etiology and biology of melanoma. This article is highlighted in the In This Issue feature, p. 2711

To examine the vestibulo-ocular reflex (VOR) and compensatory-saccades before and after complete unilateral vestibular deafferentation (UVD).Forty patients were studied before and after surgery for vestibular or facial schwannoma using the video head-impulse test (vHIT) and multivariable regression.Prior to UVD (median(IQR), 14(58.4) days), the average VOR-gain towards the lesioned-ear was lower than in normal for all semicircular canals (lateral, anterior, posterior: 0.69, 0.72, 0.49). One-week after UVD (5(3.0) days) VOR gains were further reduced (0.22, 0.37, 0.27), however, within one-year after UVD (171(125.0) days) the lesioned-ear VOR gains had slightly increased (+0.08, +0.11, +0.03), maximally for the anterior-canal. After UVD, the VOR gain asymmetry (gain towards minus away from intact-ear) was lower for the intact posterior-canal plane (0.56, 0.56, 0.22). For the lesioned canals, the frequency and amplitude of the first compensatory-saccade increased from 61-93% and 1.9-3.6° pre-surgery, to 98-99% and to 3.1-5.9° one-week post-surgery and remained unchanged over one-year; second saccade frequency and amplitude decreased over the same timespan.After UVD the high-acceleration VOR for the intact posterior-canal plane is more symmetrical than the other canals. First compensatory-saccades adapt within one week and subsequently change only marginally.Saccade compensation from surgical UVD is near complete by one-week.

Supplementary Data from Comparative Genomics Provides Etiologic and Biological Insight into Melanoma Subtypes

Melanoma is a cancer of melanocytes, with multiple subtypes based on body site location. Cutaneous melanoma is associated with skin exposed to ultraviolet radiation; uveal melanoma occurs in the eyes; mucosal melanoma occurs in internal mucous membranes; and acral melanoma occurs on the palms, soles, and nail beds. Here, we present the largest whole-genome sequencing study of melanoma to date, with 570 tumors profiled, as well as methylation and RNA sequencing for subsets of tumors. Uveal melanoma is genomically distinct from other melanoma subtypes, harboring the lowest tumor mutation burden and with significantly mutated genes in the G-protein signaling pathway. Most cutaneous, acral, and mucosal melanomas share alterations in components of the MAPK, PI3K, p53, p16, and telomere pathways. However, the mechanism by which these pathways are activated or inactivated varies between melanoma subtypes. Additionally, we identify potential novel germline predisposition genes for some of the less common melanoma subtypes.Significance:This is the largest whole-genome analysis of melanoma to date, comprehensively comparing the genomics of the four major melanoma subtypes. This study highlights both similarities and differences between the subtypes, providing insights into the etiology and biology of melanoma.This article is highlighted in the In This Issue feature, p. 2711

Supplementary Table S1. Representativeness of study participants Supplementary Table S2. Analysis of the intracranial response duration in the COMBI-MB trial. Supplementary Table S3. Analysis of overall survival in the COMBI-MB trial. Supplementary Table S4. Baseline clinical features of the COMBI-BRV trial. Supplementary Table S5. Gene expression score (SingScore) and multiplex immunohistochemical results. Supplementary Table S6. Summary statistics of the linear mixed models utilized to assess the association between protein/pathway expression/score and biopsy sites. Supplementary Table S7. Mutation annotated file of exome sequencing from melanoma biopsies.

Purpose:This study aimed to identify baseline clinical features associated with the outcomes of patients enrolled in the COMBI-MB phase II study of dabrafenib and trametinib treatment in patients with V600 BRAF-mutant metastatic melanoma with melanoma brain metastases (MBM). Exploratory biomarker analysis was also conducted as part of the synergistic COMBI-BRV trial (BRV116521), to identify molecular and immunologic changes associated with dabrafenib in MBMs and extracranial metastases (ECM).Patients and Methods:Post hoc analysis was performed for baseline features of patients (n = 125) enrolled in COMBI-MB. Analyses were performed to identify baseline clinical features associated with intracranial response rate (ICRR), progression-free survival (PFS), and overall survival (OS).Exploratory biomarker analysis was performed on biospecimen collected in the COMBI-BRV trial in which patients with BRAF-mutant, resectable MBM were treated with dabrafenib for 10 to 14 days prior to craniotomy. Accessible ECM were resected or biopsied at the time of craniotomy. Biospecimens underwent molecular and immunologic profiling for comparative analyses.Results:In COMBI-MB baseline treatment with corticosteroids was independently associated with lower ICRR [39% vs. 63%; OR, 0.323; 95 % confidence interval (CI), 0.105–0.996; P = 0.049] and shorter PFS (HR, 1.93; 95% CI, 1.06–3.51; P = 0.031). Additional significant associations identified in the multivariate analysis were improved PFS in patients with a BRAFV600E genotype (HR, 0.565; 95% CI, 0.321–0.996; P = 0.048) and improved OS in patients with Eastern Cooperative Oncology Group 0 (HR, 0.44; 95% CI, 0.25–0.78; P = 0.005).Conclusions:Corticosteroid treatment was associated with reduced ICRR and PFS in COMBI-MB, similar to results with immunotherapy for MBMs. Baseline corticosteroid treatment is a key factor to consider in MBM patient management and clinical trial design/interpretation.

Gene expression of major signalling pathways and multiplex immunohistochemical comparison of markers between timepoints and tumor sites. A) Gene expression of related oncogenic signalling pathways between treatment timepoints and site, B) Estimated mean difference of proteins and pathways between biopsy categories (95% confidence interval for the estimated mean difference is depicted as a segment), C) Multiplex immunohistochemical staining for key intracellular signalling proteins, D) Changes in immune intratumoral immune cell densities between treatment timepoints and site, E and F) Multiplex immunohistochemical staining depicted marked decrease in immune cell densities EDT in PT4 (steroid treated). PRE, collected prior to treatment; EDT, collected early during treatment (i.e., after 10-14 days of dabrafenib). MBM, melanoma brain metastasis; ECM, extracranial metastasis. PT, patient.

Purpose: This study aimed to identify baseline clinical features associated with the outcomes of patients enrolled in the COMBI-MB phase II study of dabrafenib and trametinib treatment in patients with V600 BRAF-mutant metastatic melanoma with melanoma brain metastases (MBM). Exploratory biomarker analysis was also conducted as part of the synergistic COMBI-BRV trial (BRV116521), to identify molecular and immunologic changes associated with dabrafenib in MBMs and extracranial metastases (ECM). Patients and Methods: Post hoc analysis was performed for baseline features of patients (n = 125) enrolled in COMBI-MB. Analyses were performed to identify baseline clinical features associated with intracranial response rate (ICRR), progression-free survival (PFS), and overall survival (OS). Exploratory biomarker analysis was performed on biospecimen collected in the COMBI-BRV trial in which patients with BRAF-mutant, resectable MBM were treated with dabrafenib for 10 to 14 days prior to craniotomy. Accessible ECM were resected or biopsied at the time of craniotomy. Biospecimens underwent molecular and immunologic profiling for comparative analyses. Results: In COMBI-MB baseline treatment with corticosteroids was independently associated with lower ICRR [39% vs. 63%; OR, 0.323; 95 % confidence interval (CI), 0.105–0.996; P = 0.049] and shorter PFS (HR, 1.93; 95% CI, 1.06–3.51; P = 0.031). Additional significant associations identified in the multivariate analysis were improved PFS in patients with a BRAFV600E genotype (HR, 0.565; 95% CI, 0.321–0.996; P = 0.048) and improved OS in patients with Eastern Cooperative Oncology Group 0 (HR, 0.44; 95% CI, 0.25–0.78; P = 0.005). Conclusions: Corticosteroid treatment was associated with reduced ICRR and PFS in COMBI-MB, similar to results with immunotherapy for MBMs. Baseline corticosteroid treatment is a key factor to consider in MBM patient management and clinical trial design/interpretation.

In this retrospective study, we have explored the anatomical factors that lead to the development of radiation necrosis (RN) in the setting of stereotactic radiosurgery (SRS) for melanoma brain metastases (MBM). Between 2014 and 2018, 137 patients underwent SRS for 311 MBM. Lesions were assessed according to anatomical zones: zone 1—peripheral grey-white matter junction and cortical mantle, zone 2—deep white matter, including tumours located at base of sulci, zone 3—tumours adjacent to ependymal lining or in deep locations such as brainstem, basal ganglia and thalamus. Other anatomical factors including lobes, medial-peripheral, supra or infratentorial locations were also recorded. In all, 12.4% (n = 17) of patients and 6.1% (n = 20) of lesions developed RN, actuarial incidence of RN at 12 and 24 months was 10% and 14.2% respectively. Zone 2 lesions recorded the highest rate of development of RN (n = 7/19; 36%), zone 3 (N = 4/24; 16%) and zone 1 (n = 9/268; 3%). Five of 17 patients developed symptomatic RN and 7/17 patients underwent surgery for RN. This study raises awareness of the increased likelihood of deep lesions particularly in white matter structures to develop RN after SRS. Further studies including larger cohorts would be useful in identifying statistical differences in the rate of development of RN in different anatomical zones.

Primary scalp melanomas are associated with a higher rate of brain metastasis than primary cutaneous melanomas occurring at other head and neck and body sites, but the reason is unclear. Spread to brain parenchyma via emissary veins draining from the scalp to dural sinuses has been suggested. We sought to examine the locations of metastases from primary scalp and nonscalp head and neck melanomas to determine whether there was anatomical evidence supporting direct venous spread to the brain. Data from patients who developed distant metastases from cutaneous head and neck melanomas (CHNMs) between 2000 and 2018 were analyzed. Anatomical sites of primary scalp melanomas and their respective intracranial metastases were compared. Times to first brain and nonbrain metastases were investigated for scalp and nonscalp primary CHNMs. Of 693 patients with CHNMs, 244 developed brain metastases: 109 (44.7%) had scalp primaries and 135 (55.3%) had nonscalp primaries. There was no significant association between anatomical sites of scalp primary melanomas and brain metastases (Cramer's V = 0.21; Chi-square P = 0.63). Compared with nonscalp CHNMs, scalp melanomas had no greater propensity for the brain as the first distant metastatic site ( P = 0.52) but had a shorter time to both brain metastasis (76.3 vs. 168.5 months; P0.001) and nonbrain metastasis (22.6 vs. 35.8 months; P0.001). No evidence was found to support a direct vascular pathway for metastatic spread of scalp melanomas to the brain. The increased incidence of brain metastases from scalp melanomas is probably driven by aggressive biological mechanisms.

Purpose Patients with melanoma liver metastases have significantly reduced overall response and survival when treated with immune checkpoint inhibitors (ICI) compared to those without liver metastases. Melanoma liver metastases are less likely to respond to ICI compared to other sites of metastases, and the presence of liver metastases has also been associated with reduced responses to ICI at other metastatic sites. We aimed to profile circulating and tumor immune profiles of melanoma patients with versus without liver metastases to elucidate the factors behind this observation. Methods Pre-treatment PBMCs from 37 advanced melanoma patients (Cohort A) were profiled using mass cytometry (CyTOF) spanning 46 markers. Expression of specific immune cells were compared between those with (n = 8) vs without (n = 29) liver metastases. In a separate independent cohort of 93 untreated metastatic melanoma patients (Cohort B), FFPE tumour samples comprised of subcutaneous, lymph node (LN) and brain metastases were stained for myeloid and T cell markers using multiplex IHC. Immune cell populations in cohort B tissues were compared between patients with (n=40) vs without (n=53) liver metastases. Results PBMCs from patients in Cohort A with liver metastases had an increased proportion of a myeloid population characterised as HLA-DR+CD14+CD16- compared to patients without liver metastases (p = 0.035). However, a difference in CD68+CD14+CD16- myeloid cells was not seen in cohort B melanoma tissues (subcut, LN, brain metastases) between patients with vs without liver metastases. In subcutaneous tissues, there was a significantly reduced density of T cells in patients with liver metastases (median = 59 cells/mm2) compared to those without (median = 217 cells/mm2; p = 0.037). This trend was also observed in LN and brain metastases but did not reach significance. In brain metastases, a higher proportion of FoxP3+ T cells was observed in patients with liver metastases (p = 0.003). An increase in this population was also observed in the LN and subcutaneous metastases in the presence (vs absence) of liver metastases, however this did not reach significance. LN metastases also showed a reduced proportion of both TIM3+ (p = 0.049) and CD103+ (p = 0.048) T cells in patients with liver metastases (vs absence), and a similar trend was observed in subcutaneous metastases. In contrast, brain metastases showed the opposite trend as well as higher proportion of PD1+ T cells in patients with liver metastases vs those without (p = 0.033). Conclusion These data provide insights into the differences in the anti-tumor immune profiles of patients with versus without liver metastases; the presence of liver metastases may have a specific impact at different metastatic sites. This highlights the need for further validation and investigation into the mechanism by which the presence of liver metastases may exert this effect. Citation Format: Jordan W. Conway, Felix Marsh-Wakefield, Kazi J. Nahar, Serigne N. Lo, Ismael A. Vergara, Tuba N. Gide, Grace H. Attrill, Jorja Braden, Matteo S. Carlino, Robyn P. Saw, John F. Thompson, Andrew J. Spillane, Kerwin F. Shannon, Brindha Shivalingam, Alexander M. Menzies, Umaimainthan Palendira, James S. Wilmott, Georgina V. Long, Richard A. Scolyer, Ines Pires Da Silva. The association between melanoma liver metastases (mets) and the systemic anti-tumor immune profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 81.

BACKGROUND Glioblastoma is the most common and aggressive primary brain tumor in adults. Despite maximal surgical resection followed by concomitant chemo-/radiotherapy, the three-year survival remains less than 5%. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) overexpression is associated with poor prognosis in several cancers. ROR1 therapeutics are in Phase I/II clinical trials making it an exciting novel target with translational potential to explore for glioblastoma treatment. The aim of this study was to examine the association between ROR1 mRNA expression and overall survival, by applying the WHO 2021 classification to transcriptomic glioma datasets. METHODS Clinical, histological, and molecular data was extracted from The Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA), Repository for Molecular Brain Neoplasia (REMBRANDT), and GSE16011 (GRAVENDEEL) projects via the GlioVis portal. Using the WHO 2021 classification, the dataset was appropriately re-classified. Only confirmed cases of astrocytoma, oligodendroglioma, or glioblastoma, and ROR1 mRNA expression data were included in the analysis, which included a total of 2,303 cases. 981 cases comprised the low-grade glioma cohort and 1322 cases were included in the high-grade glioma cohort. ROR1 mRNA expression from the four datasets was normalized within each dataset, combined, and divided into high and low expression groups. ROR1 expression and survival correlations were estimated with Kaplan-Meier survival analysis and Mantel-Cox test using GraphPad Prism v9. RESULTS Those with high ROR1 expression had an overall median survival of 4.5 months, as compared to 21.4 months in the low ROR1 expression cohort (p< 0.0001). High-grade gliomas had the highest ROR1 mRNA expression across the consortium when compared to the low-grade glioma cohort (p< 0.0001). CONCLUSION The results of this study indicate an association between overall survival in glioma and ROR1 expression. In addition, targeting ROR1 could hold translational importance for novel putative treatment in glioblastoma patients.

Funder: Wellcome Trust (Wellcome); doi: https://doi.org/10.13039/100004440, Brain metastases are a major cause of melanoma-related mortality and morbidity. We undertook whole-exome sequencing of 50 tumours from patients undergoing surgical resection of brain metastases presenting as the first site of visceral disease spread and validated our findings in an independent dataset of 18 patients. Brain metastases had a similar driver mutational landscape to cutaneous melanomas in TCGA. However, KRAS was the most significantly enriched driver gene, with 4/50 (8%) of brain metastases harbouring non-synonymous mutations. Hotspot KRAS mutations were mutually exclusive from BRAFV600, NRAS and HRAS mutations and were associated with a reduced overall survival from the resection of brain metastases (HR 10.01, p = 0.001). Mutations in KRAS were clonal and concordant with extracranial disease, suggesting that these mutations are likely present within the primary. Our analyses suggest that KRAS mutations could help identify patients with primary melanoma at higher risk of brain metastases who may benefit from more intensive, protracted surveillance.

Introduction The brain is a common site of metastatic disease for patients with advanced melanoma. Brain metastasis portends a poor prognosis, often causing deterioration in neurological function and quality of life, and leading to neurological death. Treatment approaches including surgery, radiotherapy and systemic therapy can lead to better control of this problem. Therefore, appropriate guidelines for the management of melanoma brain metastases need to be established, with regular updating when new treatment options become available. Methods A multidisciplinary working party established by Cancer Council Australia has produced up-to-date, evidence-based clinical practice guidelines for the management of melanoma. After selecting key clinical questions, a comprehensive literature search for relevant studies was conducted, followed by systematic review of those studies. Data were summarised and the evidence was assessed, leading to the development of recommendations. Main recommendations Symptomatic lesions are best treated with surgery, when possible; this provides safe and effective local control. For patients with single or a small number of asymptomatic brain metastases, stereotactic radiotherapy is recommended, but in asymptomatic patients who have not previously received systemic treatment, drug therapy can be considered as a first-line treatment option. Whole brain radiotherapy may provide palliative benefits in patients with multiple brain metastases. Whenever possible, melanoma patients with brain metastases should be managed by a multidisciplinary team of melanoma specialists that considers the optimal combination and sequencing of surgery, radiotherapy and systemic therapy.

Extracellular vesicles (EVs) play key roles in glioblastoma (GBM, astrocytoma grade IV) biology and are novel sources of biomarkers. EVs released from GBM tumors can cross the blood-brain-barrier into the periphery carrying GBM molecules, including small non-coding RNA (sncRNA). Biomarkers cargoed in circulating EVs have shown great promise for assessing the molecular state of brain tumors in situ. Neurosurgical aspirate fluids captured during tumor resections are a rich source of GBM-EVs isolated directly from tumor microenvironments. Using density gradient ultracentrifugation, EVs were purified from cavitron ultrasonic surgical aspirate (CUSA) washings from GBM (n = 12) and astrocytoma II-III (GII-III, n = 5) surgeries. The sncRNA contents of surgically captured EVs were profiled using the Illumina&reg, NextSeqTM 500 NGS System. Differential expression analysis identified 27 miRNA and 10 piRNA species in GBM relative to GII-III CUSA-EVs. Resolved CUSA-EV sncRNAs could discriminate serum-EV sncRNA profiles from GBM and GII-III patients and healthy controls and 14 miRNAs (including miR-486-3p and miR-106b-3p) and cancer-associated piRNAs (piR_016658, _016659, _020829 and _204090) were also significantly expressed in serum-EVs. Circulating EV markers that correlate with histological, neuroradiographic and clinical parameters will provide objective measures of tumor activity and improve the accuracy of GBM tumor surveillance.

Improving outcomes for diffuse glioma patients requires methods that can accurately and sensitively monitor tumour activity and treatment response. Extracellular vesicles (EV) are membranous nanoparticles that can traverse the blood&ndash, brain-barrier, carrying oncogenic molecules into the circulation. Measuring clinically relevant glioma biomarkers cargoed in circulating EVs could revolutionise how glioma patients are managed. Despite their suitability for biomarker discovery, the co-isolation of highly abundant complex blood proteins has hindered comprehensive proteomic studies of circulating-EVs. Plasma-EVs isolated from pre-operative glioma grade II&ndash, IV patients (n = 41) and controls (n = 11) were sequenced by Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) and data extraction was performed by aligning against a custom 8662-protein library. Overall, 4054 proteins were measured in plasma-EVs. Differentially expressed proteins and putative circulating-EV markers were identified (adj. p-value &lt, 0.05), including those reported in previous in-vitro and ex-vivo glioma-EV studies. Principal component analysis showed that plasma-EV protein profiles clustered according to glioma histological-subtype and grade, and plasma-EVs resampled from patients with recurrent tumour progression grouped with more aggressive glioma samples. The extensive plasma-EV proteome profiles achieved here highlight the potential for SWATH-MS to define circulating-EV biomarkers for objective blood-based measurements of glioma activity that could serve as ideal surrogate endpoints to assess tumour progression and allow more dynamic, patient-centred treatment protocols.

IMPORTANCEBrain metastases occur in 60% of patients with advanced melanoma and are a major cause of melanoma-related mortality and morbidity. Although our understanding of the molecular alterations associated with melanoma progression is improving, there are currently no validated biomarkers which might help identify those patients at highest risk of developing brain metastases.OBJECTIVETo examine the somatic mutational and copy-number landscape of brain metastases that develop as the isolated first visceral site of recurrence – “early brain-metastasis” compared to extracranial melanoma metastases.DESIGN, SETTING AND PARTICIPANTSWhole-exome sequencing of 50 tumors from patients undergoing surgical resection of one or more brain metastasis occurring as the first site of visceral relapse were identified from prospectively maintained databases in Sydney, Wellington, New York and Cambridge. Whole exome sequencing analyses allowed mutational profiles to be compared to cutaneous melanomas in The Cancer Genome Atlas (SKCM-TCGA; n=358) and the Memorial Sloan Kettering (SKCM-MSK-IMPACT; n=186) datasets. An external dataset comprising a further 18 patients with surgically resected early brain metastasis from two additional academic centers served as an independent validation cohort.MAIN OUTCOMES AND MEASURESTo assess the frequency of driver mutations in early brain metastasis and their influence on survival.RESULTSIn concordance with the landmark melanoma sequencing studies, we identified mutations in BRAF (21/50, 42%), NRAS (14/50, 28%) and NF1 (11/50, 22%) as the most frequently mutated melanoma driver genes. When compared to the mutational landscape of cutaneous melanomas in TCGA (SKCM-TCGA), KRAS was the most significantly enriched driver gene, with 5/50 (10%) of brain metastases harboring non-synonymous mutations, of which 4/5 (80%) were in the hotspot positions of codons 12 and 61. This was significantly higher than the corresponding frequency ofKRAS-mutations within the entire SKCM-TCGA (2% (7/358), p=0.009, Fisher’s Exact Test) as well as the SKCM-MSK-IMPACT cohort (1.6% (3/186), p=0.016). Variants in KRAS were mutually exclusive fromBRAFV600,NRASandHRASmutations and were associated with a significantly reduced overall survival from resection of brain metastasis (relative toKRAS-wild type brain metastases) in multivariate Cox proportional hazard models (HR 1.80, 95% CI 1.46-24.89, p=0.013). Mutations inKRASwere also clonal and concordant with extracranial disease, which suggests these mutations are present within the primary tumorCONCLUSIONS AND RELEVANCEOur analysis, the largest to date, suggests that early metastases to the brain (presenting as the first site of visceral relapse) are characterized by significant enrichment of hotspotKRASmutations, potentially implicating constitutive RAS-driven cellular programs in neurotropic metastatic behavior in these cases. Based on these data, we suggest that screening forKRASmutations might help identify those patients with primary melanoma at higher risk of brain metastases or poor survival, and could help inform future surveillance strategies.Key PointsQuestionWhat is the frequency of driver mutations in early melanoma brain metastases?FindingsIn this study of 50 patients with melanoma metastasizing first to the brain,KRASmutations were the most significantly enriched driver gene (n=5, 10% of patients) when compared to landmark cutaneous melanoma studies. The highKRASmutation frequency was also observed in an external validation cohort of 18 patients with early brain metastases. Mutations inKRASwere mutually exclusive from mutations in the key RAS signaling genes and conferred a worse overall survival from resection of brain metastasis.MeaningHotspotKRASmutations could help identify those patients with primary melanoma at higher risk of brain metastases that may benefit from more intensive, protracted surveillance as well as earlier use of adjuvant therapy.

Vaginal discharge is a common gynaecological presentation. This is commonly physiological but may be associated with a genital tract infection. An unusual cause of a vaginal discharge is presented ...

Diffuse gliomas (grades II-IV) are amongst the most frequent and devastating primary brain tumours of adults. Currently, patients are monitored by clinical examination and radiographic imaging, which can be challenging to interpret and insensitive to early signs of treatment failure and tumour relapse. While brain biopsy and histologic analysis can evaluate disease progression, serial biopsies are invasive and impractical given the cumulative surgical risk, and may not capture the complete molecular landscape of an evolving tumour. The availability of a minimally invasive 'liquid biopsy' that could assess tumour activity and molecular phenotype in situ has the potential to greatly enhance patient care. Circulating extracellular vesicles (EVs) hold significant promise as robust disease-specific biomarkers accessible in the blood of patients with glioblastoma and other diffuse gliomas. EVs are membrane-bound nanoparticles shed from most if not all cells of the body, and carry DNA, RNA, protein, and lipids that reflect the identity and molecular state of their cell-of-origin. EVs can cross the blood-brain barrier and their release is upregulated in neoplasia. In this review, we describe the current knowledge of EV biology, the role of EVs in glioma biology and the current experience and challenges in profiling glioma-EVs from the circulation.

Simple Summary Immunotherapy leads to durable responses in a proportion of patients with advanced melanoma. Combination immunotherapy is more efficacious than single-agent immunotherapy, yet it is associated with significant toxicity. Currently there are no robust biomarkers to guide first-line immunotherapy selection. We have developed a flow cytometry-based score, to quantify the expression of antigen-presenting molecules MHC-I and MHC-II on melanoma cells, that incorporates both the fraction of tumor cells expressing MHC molecules and the level of expression. We demonstrate that the evaluation of tumor cell surface MHC-I expression aids in treatment selection, with combination immunotherapy providing clinical benefit over single-agent immunotherapy in MHC-I low melanoma with poor immune cell infiltration. Abstract Immunotherapy targeting T-cell inhibitory receptors, namely programmed cell death-1 (PD-1) and/or cytotoxic T-lymphocyte associated protein-4 (CTLA-4), leads to durable responses in a proportion of patients with advanced metastatic melanoma. Combination immunotherapy results in higher rates of response compared to anti-PD-1 monotherapy, at the expense of higher toxicity. Currently, there are no robust molecular biomarkers for the selection of first-line immunotherapy. We used flow cytometry to profile pretreatment tumor biopsies from 36 melanoma patients treated with anti-PD-1 or combination (anti-PD-1 plus anti-CTLA-4) immunotherapy. A novel quantitative score was developed to determine the tumor cell expression of antigen-presenting MHC class I (MHC-I) molecules, and to correlate expression data with treatment response. Melanoma MHC-I expression was intact in all tumors derived from patients who demonstrated durable response to anti-PD-1 monotherapy. In contrast, melanoma MHC-I expression was low in 67% of tumors derived from patients with durable response to combination immunotherapy. Compared to MHC-I high tumors, MHC-I low tumors displayed reduced T-cell infiltration and a myeloid cell-enriched microenvironment. Our data emphasize the importance of robust MHC-I expression for anti-PD-1 monotherapy response and provide a rationale for the selection of combination immunotherapy as the first-line treatment in MHC-I low melanoma.

Exosomes are nano-sized extracellular vesicles released by many cells that contain molecules characteristic of their cell of origin, including microRNA. Exosomes released by glioblastoma cross the blood–brain barrier into the peripheral circulation and carry molecular cargo distinct to that of “free-circulating” miRNA. In this pilot study, serum exosomal microRNAs were isolated from glioblastoma (n = 12) patients and analyzed using unbiased deep sequencing. Results were compared to sera from age- and gender-matched healthy controls and to grade II–III (n = 10) glioma patients. Significant differentially expressed microRNAs were identified, and the predictive power of individual and subsets of microRNAs were tested using univariate and multivariate analyses. Additional sera from glioblastoma patients (n = 4) and independent sets of healthy (n = 9) and non-glioma (n = 10) controls were used to further test the specificity and predictive power of this unique exosomal microRNA signature. Twenty-six microRNAs were differentially expressed in serum exosomes from glioblastoma patients relative to healthy controls. Random forest modeling and data partitioning selected seven miRNAs (miR-182-5p, miR-328-3p, miR-339-5p, miR-340-5p, miR-485-3p, miR-486-5p, and miR-543) as the most stable for classifying glioblastoma. Strikingly, within this model, six iterations of these miRNA classifiers could distinguish glioblastoma patients from controls with perfect accuracy. The seven miRNA panel was able to correctly classify all specimens in validation cohorts (n = 23). Also identified were 23 dysregulated miRNAs in IDHMUT gliomas, a partially overlapping yet distinct signature of lower-grade glioma. Serum exosomal miRNA signatures can accurately diagnose glioblastoma preoperatively. miRNA signatures identified are distinct from previously reported “free-circulating” miRNA studies in GBM patients and appear to be superior., Brain cancer: Vesicle-contained microRNAs offer window into glioblastoma A diagnostic test for short regulatory RNA molecules contained within tiny secreted vesicles in the bloodstream can accurately pick up signs of glioblastoma brain cancer. Researchers in Australia led by Michael Buckland and Kim Kaufman from the Royal Prince Alfred Hospital and the University of Sydney isolated circulating vesicles, called exosomes, from patients with glioblastoma or lower-grade brain cancers known as gliomas as well as healthy controls. Next-generation sequencing revealed a panel of 26 microRNAs contained within the exosomes that were differentially expressed in glioblastoma samples relative to healthy controls. (A different but partially overlapping set of 23 microRNAs also helped distinguish patients with a mutant subtype of glioma.) The researchers narrowed down the list to the seven microRNAs with the most predictive power. Testing for just these microRNAs reliably diagnosed glioblastoma with greater precision than previously reported panels of “free-circulating” microRNAs.

Glioblastoma, WHO-grade IV glioma, carries a dismal prognosis owing to its infiltrative growth and limited treatment options. Glioblastoma-derived extracellular vesicles (EVs; 30-1000 nm membranous particles) influence the microenvironment to mediate tumor aggressiveness and carry oncogenic cargo across the blood-brain barrier into the circulation. As such, EVs are biomarker reservoirs with enormous potential for assessing glioblastoma tumors in situ. Neurosurgical aspirates are rich sources of EVs, isolated directly from glioma microenvironments. EV proteomes enriched from glioblastoma (n = 15) and glioma grade II-III (n = 7) aspirates are compared and 298 differentially-abundant proteins (p-value < 0.00496) are identified using quantitative LC-MS/MS. Along with previously reported glioblastoma-associated biomarkers, levels of all eight subunits of the key molecular chaperone, T-complex protein 1 Ring complex (TRiC), are higher in glioblastoma-EVs, including CCT2, CCT3, CCT5, CCT6A, CCT7, and TCP1 (p < 0.00496). Analogous increases in TRiC transcript levels and DNA copy numbers are detected in silico; CCT6A has the greatest induction of expression and amplification in glioblastoma and shows a negative association with survival (p = 0.006). CCT6A is co-localized with EGFR at 7p11.2, with a strong tendency for co-amplification (p < 0.001). Immunohistochemistry corroborates the CCT6A proteomics measurements and indicated a potential link between EGFR and CCT6A tissue expression. Putative EV-biomarkers described here should be further assessed in peripheral blood.

Exosomes are nano-sized extracellular vesicles released by many cells that contain molecules characteristic of their cell-of-origin, including microRNA. Exosomes released by glioblastoma cross the blood-brain-barrier into the peripheral circulation, and carry molecular cargo distinct to that of ‘free-circulating’ miRNA. In this pilot study, serum exosomal-microRNAs were isolated from glioblastoma (n=12) patients and analyzed using unbiased deep sequencing. Results were compared to sera from age- and gender-matched healthy controls, and to grades II-III (n=10) glioma patients. Significant differentially expressed microRNAs were identified, and the predictive power of individual and subsets of microRNAs were tested using univariate and multivariate analyses. Additional sera from glioblastoma patients (n=4) and independent sets of healthy (n=9) and non-glioma (n=10) controls were used to further test the specificity and predictive power of this unique exosomal-microRNA signature. Twenty-six microRNAs were differentially expressed in serum exosomes from glioblastoma patients’ relative to healthy controls. Random forest modeling and data partitioning selected seven miRNAs (miR-182-5p, miR-328-3p, miR-339-5p, miR-340-5p, miR-485-3p, miR-486-5p and miR-543) as the most stable for classifying glioblastoma. Strikingly, within this model, six iterations of these miRNA classifiers could distinguish glioblastoma patients from controls with perfect accuracy. The seven-miRNA panel was able to correctly classify all specimens in validation cohorts (n=23). Also identified were 23 dysregulated miRNAs in IDHMUTgliomas, a partially overlapping yet distinct signature of lower grade glioma. Serum exosomal-miRNA signatures can accurately diagnose glioblastoma preoperatively. miRNA signatures identified are distinct from previously reported ‘free-circulating’ miRNA studies in GBM patients, and appear to be superior.

Historically, the prognosis of patients with melanoma brain metastases is poor, with median overall survival (OS) of 4-6 months. Little is known of OS in the era of modern systemic therapies and local therapy with stereotactic radiosurgery (SRS) or surgery. Patients diagnosed with melanoma brain metastases at Melanoma Institute Australia from January 2011 to December 2014 were included. OS and prognostic factors were analysed using Cox regression and Kaplan-Meier survival analyses.355 patients were included. The median OS was 7.1 months (95% confidence interval [CI] 6.0-8.1). Median OS differed by treatment modality: systemic therapy and SRS and/or surgery 14.9 months (95% CI 10.7-19.0), SRS and/or surgery with or without whole brain radiotherapy (WBRT) 6.4 months (95% CI 5.4-7.5), systemic therapy 5.4 months (95% CI 3.1-7.7), systemic therapy and WBRT 5.2 months (95% CI 4.1-6.4), WBRT 4.4 months (95% CI 2.4-6.3), and best supportive care 1.8 months (95% CI 1.2-2.3). OS for patients with melanoma brain metastases appears improved in the modern era, particularly for patients who are candidates for systemic therapy with SRS and/or surgery.

Melanoma brain metastasis (MBM) represents a frequent complication of cutaneous melanoma. Despite aggressive multi-modality therapy, patients with MBM often have a survival rate of

Introduction With new systemic therapies demonstrating activity in melanoma brain metastasis, most of the previously reported stereotactic radiosurgery (SRS) data are superseded. In this study, we report the outcomes (overall survival [OS] and brain control [BC]) and identify factors that associate with such outcomes in the era of modern systemic therapy. Method A total of 108 patients treated with SRS from 2010 to 2015 were included. Systemic treatment use within 6 weeks of SRS was noted. OS was defined as time from SRS to death or last follow-up, and BC was defined as absence of any active intracranial disease during follow-up. Univariate and multivariate Cox proportional hazard analyses were performed on clinico-pathological prognostic features associated with OS and BC. Results The median age was 64.3 years, and the median follow-up was 8.6 months. Seventy-nine (73.1%) patients received systemic treatment. The median OS were as follows: anti-CTLA4 – 7.5 months (95% CI: 4.4–15.6), anti-PD1 – 20.4 months (95% CI: 8.8 – N/A) and BRAF inhibitor (BRAFi) ± MEK inhibitor (MEKi) – 17.8 months (95% CI: 11.8 – N/A). Median BC was as follows: anti-CTLA4 – 7.5 months (95% CI: 4.0–15.6), anti-PD1 – 12.7 months (95% CI: 5.5 – N/A) and BRAFi ± MEKi – 12.7 months (95% CI: 8.3–18.5). In multivariate analysis, age and type of systemic therapy were strongly associated with OS. Age, Eastern Cooperative Oncology Group performance status, Graded Prognostic Assessment (GPA) score, and presence of symptoms were associated with BC. Conclusions Favourable outcomes are seen in patients treated with SRS and with the best survival seen in patients treated with anti-PD1. Known independent prognostic factors for survival such as age and performance status and GPA score remain relevant in this setting.

Every year 170,000 patients are diagnosed with brain metastases (BMs) in the United States. Traditionally, adjuvant whole brain radiotherapy (AWBRT) has been offered following local therapy with neurosurgery (NSx) and/or stereotactic radiosurgery (SRS) to BMs. The aim is to increase intracranial control, thereby decreasing symptoms from intracranial progression and a neurological death. There is a rapidly evolving change in the radiation treatment of BMs happening around the world. AWBRT is now being passed over in favour of repeat scanning at regular intervals and more local therapies as more BMs appear radiologically, BMs that may never become symptomatic. This change has happened after the American Society for Radiation Oncology (ASTRO) in Item 5 of its "Choosing Wisely 2014" list recommended: "Don't routinely add adjuvant whole brain radiation therapy to SRS for limited brain metastases". The guidelines are supposed to be based on the highest evidence to hand at the time. This article debates that the randomised controlled trials (RCTs) published prior to this recommendation consistently showed AWBRT significantly increases intracranial control, and avoids a neurological death, what it is meant to do. It also points out that, despite the enormity of the problem, only 774 patients in total had been randomised over more than three decades. These trials were heterogeneous in many respects. This data can, at best, be regarded as preliminary. In particular, there are no single histology AWBRT trials yet completed. A phase two trial investigating hippocampal avoiding AWBRT (HAWBRT) showed significantly less NCF decline compared to historical controls. We now need more randomised data to confirm the benefit of adjuvant HAWBRT. However, the ASTRO Guideline has particularly impacted accrual to trials investigating this, especially the international ANZMTG 01.07 WBRTMel trial. This is an RCT investigating AWBRT following local treatment in patients with one to three BMs from melanoma. WBRTMel has accrued 196 of a required 220 to date but accrual has slowed. HAWBRT may now never be tested in a randomised setting. Encouraging more data in AWBRT is the wiser choice.

biology, clinical trial design, and future treatment deci-sions. OA should not be ignored in the proposed new clas-sification guidelines [2].Sahm and colleagues studied 43 cases diagnosed as OA based on histology. But in 30 of these cases, immunostain-ing for IDH1 (R132H) mutation was restricted to oligo-dendroglial areas only. The astrocytic component of these tumours was re-interpreted to be reactive in nature [4], and they were reclassified as oligodendroglioma. We propose that this finding indicates that ‘true’ OAs (i.e. tumours com-posed of two distinct

Brain metastases are common in patients with metastatic melanoma and the majority of patients who die of the disease do so as a direct result of them. Not only does the diagnosis of brain metastasis signify a very poor prognosis, but it portends a severe impact on the patient’s quality of life. The major clinical problem of melanoma brain metastases has in the past been addressed by treatment with local therapies—surgery and radiation therapy—but the results have been unimpressive, with median survival times of just a few months reported in most series, little better than the survival times of patients offered supportive care only. However, in recent times more effective and less morbid techniques for both the surgical management of brain metastases and the treatment of them using new radiation therapy methodologies have been introduced and, at last, systemic therapies that can treat melanoma brain metastases effectively have been discovered. In this chapter the range of treatments now available to treat melanoma brain metastases will be discussed, outcomes achieved by their use will be presented, and the importance of a multidisciplinary approach to management of the problem will be emphasized.

Background Brain metastases are a common cause of death in patients with melanoma. The role of adjuvant whole brain radiotherapy (WBRT) following local treatment of intracranial melanoma metastases is controversial. The Australian and New Zealand Melanoma Trials Group (ANZMTG) and the Trans-Tasman Radiation Oncology Group (TROG) are leading the first ever single histology randomised trial investigating this question. The primary endpoint is distant intracranial failure on magnetic resonance imaging (MRI) within twelve months of randomisation. The first planned interim analysis was performed twelve months after randomisation of the 100th patient. The analysis was an opportunity to review completeness of the trial data to date. Methods All data received up to the end of twelve months after randomisation of the 100th patient was reviewed. Results Review of pathology reports confirmed that all 100 patients had stage IV melanoma and were appropriately entered into the study. Of the 47 distant intracranial events, 34 occurred in isolation (i.e. only distant failure was identified), whilst 13 were accompanied by local failure. Data review showed compliance with the protocol mandated MRI schedule and accuracy of intracranial failure reporting was very high. The Quality of Life (QoL) component of the study achieved a 91% completion rate. For the neurocognitive function (NCF) assessments, a high completion rate was maintained throughout the 12 month period. Where assessments were not performed at expected time points, valid reasons were noted. Radiotherapy quality was high. Of 50 patients who received WBRT, 32 were reviewed as per protocol design and there was only one major variation out of 308 data points reviewed (0.3%). There were minimal trial related adverse events (AEs) and no serious adverse events (SAEs). Pre-specified protocol stopping rules were not met. Conclusions The Data Safety Monitoring Committee (DSMC) recommended the trial continue recruitment after reviewing the unblinded data. The data provision and quality to date indicates that a reliable outcome will be obtained when the final analysis is performed. Accrual is ongoing with 156 out of 200 patients randomised to date (26th November 2014).

Background Brain metastases (BMs) are common in melanoma patients. Adjuvant whole brain radiotherapy (WBRT) following local treatment of intracranial melanoma metastases with neurosurgery and/or stereotactic radiosurgery is controversial. A randomised trial is needed. However, accrual to WBRT trials has been problematic. A pilot study by Australia and New Zealand Melanoma Trials Group (ANZMTG) was conducted to see if accrual was feasible. Methods Sites canvassed for interest included those who treat melanoma patients, had a proven accrual in previous melanoma trials and who had the relevant infrastructure support. Feasibility forecasts from interested sites were sought. These were compared to the patient numbers documented in the research contracts. A target accrual of 60 patients in 2 years was set. Funding was sought for the pilot study. Basic demographics of the pilot study cohort were collected. Results The first centre opened December 2008; the first patient was randomised in April 2009. The pilot accruing period concluded in September, 2011. In 30 months, 54 patients from 10 of a total of 17 activated sites in Australia (39, 72%) and in Norway (15, 28%) had been accrued. Feasibility forecasts predicted 133 trial eligible patients per year (including 108 Australian + 25 International patients). Site estimates generally overestimated accrual with 4 of 17 active sites estimating within 50% of target numbers. Sites with patient estimates calculated from records were more accurate than those estimated from memory. The overall recruitment target was lower in the research contracts when compared to the feasibility evaluation. Basic demographics of the pilot study revealed 62% of patients were males; 58% had a single metastasis, 28% had two and 14% had three metastases. 12-month overall survival was 50%. Conclusions Despite only 54 patients and not the full 60 patient target being accrued in two years the Trial Management Committee and Data Safely Monitoring Committee approved the continuation of the pilot study to the main trial. On the basis of this successful pilot study, funding was achieved for the full study. 143 patients of a target of 200 have been randomised by June 2014.

Four cases previously treated with ipilimumab with a total of six histologically confirmed symptomatic lesions of RNB without any sign of active tumour following stereotactic irradiation of MBM are reported. These lesions were all originally thought to be disease recurrence. In two cases, ipilimumab was given prior to SRT; in the other two ipilimumab was given after SRT. The average time from first ipilimumab to RNB was 15 months. The average time from SRT to RNB was 11 months. The average time from first diagnosis of MBM to last follow-up was 20 months at which time three patients were still alive, one with no evidence of disease. These cases represent approximately three percent of the total cases of melanoma and ten percent of those cases treated with ipilimumab irradiated in our respective centres collectively. We report this to highlight this new problem so that others may have a high index of suspicion, allowing, if clinically warranted, aggressive surgical salvage, possibly resulting in increased survival. Further studies prospectively collecting data to understand the denominator of this problem are needed to determine whether this problem is just the result of longer survival or whether there is some synergy between these two modalities that are increasingly being used together.

Background Cerebral metastases are a common cause of death in patients with melanoma. Systemic drug treatment of these metastases is rarely effective, and where possible surgical resection and/or stereotactic radiosurgery (SRS) are the preferred treatment options. Treatment with adjuvant whole brain radiotherapy (WBRT) following neurosurgery and/or SRS is controversial. Proponents of WBRT report prolongation of intracranial control with reduced neurological events and better palliation. Opponents state melanoma is radioresistant; that WBRT yields no survival benefit and may impair neurocognitive function. These opinions are based largely on studies in other tumour types in which assessment of neurocognitive function has been incomplete. Methods/Design This trial is an international, prospective multi-centre, open-label, phase III randomised controlled trial comparing WBRT to observation following local treatment of intracranial melanoma metastases with surgery and/or SRS. Patients aged 18 years or older with 1-3 brain metastases excised and/or stereotactically irradiated and an ECOG status of 0-2 are eligible. Patients with leptomeningeal disease, or who have had previous WBRT or localised treatment for brain metastases are ineligible. WBRT prescription is at least 30 Gy in 10 fractions commenced within 8 weeks of surgery and/or SRS. Randomisation is stratified by the number of cerebral metastases, presence or absence of extracranial disease, treatment centre, sex, radiotherapy dose and patient age. The primary endpoint is the proportion of patients with distant intracranial failure as determined by MRI assessment at 12 months. Secondary end points include: survival, quality of life, performance status and neurocognitive function. Discussion Accrual to previous trials for patients with brain metastases has been difficult, mainly due to referral bias for or against WBRT. This trial should provide the evidence that is currently lacking in treatment decision-making for patients with melanoma brain metastases. The trial is conducted by the Australia and New Zealand Melanoma Trials Group (ANZMTG-study 01-07), and the Trans Tasman Radiation Oncology Group (TROG) but international participation is encouraged. Twelve sites are open to date with 43 patients randomised as of the 31st March 2011. The target accrual is 200 patients. Trial registration Australia and New Zealand Clinical Trials Register (ANZCTR): ACTRN12607000512426