Short-term growth assays showing cell growth after 24 hours following treatment with DMSO or dabrafenib (Dab; 1 M) in RPMI or 50% CSF using CellTiter-Glo ****p 0.0001; NS, not significant. protein arrays and immunoblotting on NNT1 patient-derived cell lines using dabrafenib in the presence or absence of cerebrospinal fluid (CSF) in vitro. Patient-derived xenografts were also developed to analyse response to dabrafenib. Results Immune escape following checkpoint blockade was not due to loss of tumour cell recognition by the immune system or low neoantigen burden, but was associated with distinct changes in the microenvironment. Similarly, resistance to targeted therapy was not associated with acquired mutations but upregulation of the AKT/phospho-inositide 3-kinase pathway in the presence of CSF. Conclusion Heterogeneous tumour interactions within the brain microenvironment enable progression on immune and targeted therapies and should be targeted in salvage treatments. mutations more common (15%C20% vs 1%C2%) and mutations less common (15% vs 45%).8 9 Furthermore, acral melanoma is associated with a lower mutational burden and higher copy number variation than common cutaneous melanoma.8 We show here that metastatic acral melanoma responds to both immune and targeted therapies but, as often seen with common cutaneous melanoma, resistance develops to both modalities. Through longitudinal sampling of an exceptional case of a patient with acral melanoma who had isolated progressions in the brain while responding in all other sites, first to immune and then to targeted therapy, we provide insight into the heterogeneity of the tumour microenvironment resulting in resistance to standard melanoma treatments. Improved understanding of tumour-microenvironment relationships will provide hypothesis-driven salvage therapies that target the drivers of tumourigenesis, and the unique tumour microenvironments. Methods RNA/DNA extraction Following microdissection of the tumour to ensure tumour purity, RNA/DNA was extracted using the AllPrep DNA/RNA Micro Kit (Qiagen) relating to manufacturers instructions. Whole-exome sequencing (WES) and RNA-Seq were performed as previously explained.10 11 RNA-Seq analysis The RNA-Seq data were aligned to human GRCh37 assembly by MapSplice (V.2.1.6)12 and further analysis performed in R (V.3.1.0). Gene counts were extracted using featureCounts13 for Ensembl V.73 annotation (V.1.16.1), which were then converted into reads per kilobase million (RPKM). The RPKM ideals are used for all expression-level quantification of the genes. Genes overexpressed in the brain lesion (log2FC2) as compared with the baseline lesion were used to perform over-representation analysis using the gProfileR package (V.0.5.3).14 From your over-representation analysis on gene ontology, we represented biological processes inside a network using Cytoscape.15 WES analysis WES data were aligned to human GRCh37 with bwa-mem (V.0.7.7).16 Deduplication, realignment and recalibration was performed within the aligned data as suggested in the Genome Analysis Toolkit (GATK) framework.17 Somatic mutation calling was then done using MuTect (V.1.1.7).18 Neoantigen determination Non-synonymous mutations were scanned and candidate nonamer peptides identified. The human being leukocyte antigen (HLA) type of the patient was determined by Central Manchester University or college Hospital (laboratory research no 03 GB-009.991) to be HLA A 02:01. Candidate sequences were input into NetCTLpan V.1.1 previously validated by additional organizations. 19 Droplet digital PCR Each bad control or cell collection sample was run over three replicate wells. Per well, 10 ng input DNA was added to 11 l GSK3145095 ddPCR Supermix for probes (no Uridine-5′-triphosphate (UTP)) (Bio-Rad) and 0.55 l of custom designed probe (Lifetech) for phospho-inositide 3-kinase (PI3K) L25S made up to a total volume of 22 l with water. Droplets were generated using a QX200 automated droplet generator (Bio-Rad) and a PCR reaction was performed using the following cycling conditions: 95C for 10 min; 40 cycles of 94C for 30 s and 55C for 1 min; GSK3145095 followed by 98C for 10 min (all at a ramp rate of 2C/s), and a final hold at 4C (ramp rate 1C/s). Droplets were read using a QX200 droplet reader (Bio-Rad) and the data analysed using QuantaSoft software V.1.4.0.99 (Bio-Rad). CIBERSORT analysis The leucocyte signature matrix LM22 (547 genes) that differentiates 22 types of tumour-infiltrating immune cells was utilized for the analysis. Normalised gene manifestation data from RNA-Seq analysis of each sample (subcutaneous and mind lesions) were processed with the Cell type Recognition By Estimating Relative Subsets Of known RNA Transcripts (CIBERSORT) web tool (http://cibersort.stanford.edu/) using default guidelines at 100 permutations. Cell tradition Cell lines from patient samples were established from new tumour biopsies, cultured under standard conditions and were regularly monitored for mycoplasma contamination by PCR. Cells were cultured in Roswell Park Memorial Institute (cell tradition press) (RPMI) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Short-term growth inhibition assays Cultured cells from your subcutaneous and mind metastases were seeded into 96-well plates (3000 cells per well). Twenty-four hours later on, serial dilutions of dabrafenib (Selleckchem) were added. Cells were incubated for a further 72 hours.**p 0.01; ***p 0.001; NS, not significant; PDGFR, platelet-derived growth-factor receptor; PI3K, phospho-inositide 3-kinase. Intriguingly, despite rescuing proliferation and survival, the presence of CSF did not result in reactivation of ERK in the cells from your resistant lesion (number 3C), so we used RPPA to identify which pathways were triggered. in the microenvironment. Similarly, resistance to targeted therapy was not associated with acquired mutations but upregulation of the AKT/phospho-inositide 3-kinase pathway in the presence of CSF. Summary Heterogeneous tumour relationships within the brain microenvironment enable progression on immune and targeted therapies and should become targeted in salvage treatments. mutations more common (15%C20% vs 1%C2%) and mutations less common (15% vs 45%).8 9 Furthermore, acral melanoma is associated with a lower mutational burden and higher copy quantity variation than common cutaneous melanoma.8 We show here that metastatic acral melanoma responds to both immune and targeted therapies but, as often seen with common cutaneous melanoma, resistance develops to both modalities. Through longitudinal sampling of an exceptional case of a patient with acral melanoma who experienced isolated progressions in the brain while responding in all other sites, 1st to immune and then to targeted therapy, we provide insight into the heterogeneity of the tumour microenvironment resulting in resistance to standard melanoma treatments. Improved understanding of tumour-microenvironment relationships will provide hypothesis-driven salvage therapies that target the drivers of tumourigenesis, and the unique tumour microenvironments. Methods RNA/DNA extraction Following microdissection of the tumour to ensure tumour purity, RNA/DNA was extracted using the AllPrep DNA/RNA Micro Kit (Qiagen) relating to manufacturers instructions. Whole-exome sequencing (WES) and RNA-Seq were performed as previously explained.10 11 RNA-Seq analysis The RNA-Seq data were aligned to human GRCh37 assembly by MapSplice (V.2.1.6)12 and further analysis performed in R (V.3.1.0). Gene counts were extracted using featureCounts13 for Ensembl V.73 annotation (V.1.16.1), which were then converted into reads per kilobase million (RPKM). The RPKM ideals are used for all expression-level quantification of the genes. Genes overexpressed in the brain lesion (log2FC2) as compared with the baseline lesion were used to perform over-representation analysis using the gProfileR package (V.0.5.3).14 From your over-representation analysis on gene ontology, we represented biological processes inside a network using Cytoscape.15 WES analysis WES data were aligned to human GRCh37 with bwa-mem (V.0.7.7).16 Deduplication, realignment and recalibration was performed within the aligned data as suggested in the Genome Analysis Toolkit (GATK) framework.17 Somatic mutation calling was then done using MuTect (V.1.1.7).18 Neoantigen determination Non-synonymous mutations were scanned and candidate nonamer peptides identified. The human being leukocyte antigen (HLA) type of the patient was determined by Central Manchester University or college Hospital (laboratory research no 03 GB-009.991) to be HLA A 02:01. Candidate sequences were input into NetCTLpan V.1.1 previously validated by additional organizations.19 Droplet digital PCR Each negative control or cell line sample was run over three replicate wells. Per well, 10 ng input DNA was added to 11 l ddPCR Supermix for probes (no Uridine-5′-triphosphate (UTP)) (Bio-Rad) and 0.55 l of custom designed probe (Lifetech) for phospho-inositide 3-kinase (PI3K) L25S made up to a total volume of 22 l with water. Droplets were generated using a QX200 automated droplet generator (Bio-Rad) and a PCR reaction was performed using the following cycling conditions: 95C for 10 min; 40 cycles of 94C for 30 s and 55C for 1 min; followed by 98C for 10 min (all at a ramp rate of 2C/s), and a final hold at 4C (ramp rate 1C/s). Droplets were read using a QX200 droplet reader (Bio-Rad) and the data analysed using QuantaSoft software V.1.4.0.99 (Bio-Rad). CIBERSORT analysis The leucocyte signature matrix LM22 (547 genes) that differentiates 22 types of tumour-infiltrating immune cells was used for the analysis. Normalised gene expression data from RNA-Seq analysis of each sample (subcutaneous and brain lesions) were processed with the Cell type Identification By Estimating Relative Subsets Of known RNA Transcripts (CIBERSORT) web tool (http://cibersort.stanford.edu/) using default parameters at 100 permutations. Cell culture Cell lines from patient samples were established from fresh tumour biopsies, cultured under standard conditions and were routinely monitored for mycoplasma contamination by PCR. Cells were cultured in Roswell Park Memorial Institute (cell culture media) (RPMI) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Short-term growth inhibition assays GSK3145095 Cultured cells from the subcutaneous and brain metastases were seeded into 96-well plates (3000 cells per well). Twenty-four hours later, serial dilutions of dabrafenib (Selleckchem) were added. Cells were incubated for a further 72 hours and viability measured by CellTiter-Glo assays (Promega). Relative survival.