Triple Wild Type Research Articles

Targeted DNA Sequencing of Cutaneous Melanoma Identifies Prognostic and Predictive Alterations.

Cutaneous melanoma (CM) can be molecularly classified into four groups: BRAF mutant, NRAS mutant, NF1 mutant and triple wild-type (TWT) tumors lacking any of these three alterations. In the era of immune checkpoint inhibition (ICI) and targeted molecular therapy, the clinical significance of these groups remains unclear. Here, we integrate targeted DNA sequencing with comprehensive clinical follow-up in CM patients. This was a retrospective cohort study that assessed clinical and molecular features from patients with localized or metastatic CM who underwent targeted next-generation sequencing as part of routine clinical care. A total of 254 patients with CM who had a CLIA-certified targeted sequencing assay performed on their tumor tissue were included. Of the 254 patients with cutaneous melanoma, 77 were BRAF mutant (30.3%), 77 were NRAS mutant (30.3%), 47 were NF1 mutant (18.5%), 33 were TWT (13.0%) and the remaining 20 (7.9%) carried mutations in multiple driver genes (BRAF/NRAS/NF1 co-mutated). The majority of this co-mutation group carried mutations in NF1 (n = 19 or 90%) with co-occurring mutations in BRAF or NRAS, often with a weaker oncogenic variant. Consistently, NF1 mutant tumors harbored numerous significantly co-altered genes compared to BRAF or NRAS mutant tumors. The majority of TWT tumors (n = 29, 87.9%) harbor a pathogenic mutation within a known Ras/MAPK signaling pathway component. Of the 154 cases with available TMB data, the median TMB was 20 (range 0.7-266 mutations/Mb). A total of 14 cases (9.1%) were classified as having a low TMB (≤5 mutations/Mb), 64 of 154 (41.6%) had an intermediate TMB (>5 and ≤20 mutations/Mb), 40 of 154 (26.0%) had a high TMB (>20 and ≤50 mutations/Mb) and 36 of 154 (23.4%) were classified as having a very high TMB (>50 mutations/Mb). NRAS mutant melanoma demonstrated significantly decreased overall survival on multivariable analysis (HR for death 2.95, 95% CI 1.13-7.69, p = 0.027, log-rank test) compared with other TCGA molecular subgroups. Of the 116 patients in our cohort with available treatment data, 36 received a combination of dual ICI with anti-CTLA4 and anti-PD1 inhibition as first-line therapy. Elevated TMB was associated with significantly longer progression-free survival following dual-agent ICI (HR 0.26, 95% CI 0.07-0.90, p = 0.033, log-rank test). NRAS mutation in CMs correlated with significantly worse overall survival. Elevated TMB was associated with increased progression-free survival for patients treated with a combination of dual ICI, supporting the potential utility of TMB as a predictive biomarker for ICI response in melanoma.

Assessing melanoma prognosis: the interplay between patient profiles, survival, and BRAF, NRAS, KIT, and TWT mutations in a retrospective multi-study analysis.

The incidence and prevalence of melanoma are increasing globally, presenting a significant public health concern. The main genetic drivers of melanoma include BRAF, NRAS, KIT and triple wild-type (TWT) mutations. Little is known about the effects of these mutations on outcomes in terms of demographics and patient characteristics. We examined differences in melanoma mortality risk and mutation count across mutation type and patient disease profile. We extrapolated primary melanoma patient data from 14 studies via the cBioportal database. Patients were divided into demographic groups and classified according to BRAF, NRAS, KIT and TWT mutation status. Analyses included two-sample Student t -test and two-way analysis of variance tests analysis with Tukey's post hoc test. Survival outcomes were compared via Kaplan-Meier curve and Cox regression. NRAS-mutated patients exhibited decreased overall survival compared to BRAF-mutated patients. Male patients had higher mutation counts across all gene groups than females, with the fewest TWT mutations in comparison to BRAF, NRAS and KIT mutations. Males also exhibited increased mortality risk for NRAS, KIT and TWT mutations compared to BRAF mutations. An unknown primary melanoma was associated with increased mortality risk across all gene groups. NRAS-mutated acral melanoma patients had an increased mortality risk compared to NRAS-mutated cutaneous melanoma patients. Older patients had a higher mortality risk than younger patients. Patients with heavier versus lower weights had lower mortality risk, which was more pronounced for BRAF-mutated patients. These relationships highlight the importance of demographic and pathologic relationships to aid in risk assessment and personalize treatment plans.

ERK mediates interferon gamma-induced melanoma cell death

BackgroundInterferon-gamma (IFNγ) exerts potent growth inhibitory effects on a wide range of cancer cells through unknown signaling pathways. We pursued complementary screening approaches to characterize the growth inhibition pathway.MethodsWe performed chemical genomics and whole genome targeting CRISPR/Cas9 screens using patient-derived melanoma lines to uncover essential nodes in the IFNγ-mediated growth inhibition pathway. We used transcriptomic profiling to identify cell death pathways activated upon IFNγ exposure. Live imaging experiments coupled with apoptosis assays confirmed the involvement of these pathways in IFNγ-mediated cell death.ResultsWe show that IFNγ signaling activated ERK. Blocking ERK activation rescued IFNγ-mediated apoptosis in 17 of 23 (~ 74%) cell lines representing BRAF, NRAS, NF1 mutant, and triple wild type subtypes of cutaneous melanoma. ERK signaling induced a stress response, ultimately leading to apoptosis through the activity of DR5 and NOXA proteins.ConclusionsOur results provide a new understanding of the IFNγ growth inhibition pathway, which will be crucial in defining mechanisms of immunotherapy response and resistance.

Molecular Features of Resected Melanoma Brain Metastases, Clinical Outcomes, and Responses to Immunotherapy

Central nervous system (CNS)-penetrant systemic therapies have significantly advanced care for patients with melanoma brain metastases. However, improved understanding of the molecular landscape and microenvironment of these lesions is needed to both optimize patient selection and advance treatment approaches. To evaluate how bulk and single-cell genomic features of melanoma brain metastases are associated with clinical outcome and treatment response. This cohort study analyzed bulk DNA sequencing and single nuclear RNA-sequencing data from resected melanoma brain metastases and included 94 consecutive patients with a histopathologically confirmed diagnosis of melanoma brain metastasis who underwent surgical resection at a single National Comprehensive Cancer Network cancer center in San Francisco, California, from January 1, 2009, to December 31, 2022. A Clinical Laboratory Improvement Amendments-certified targeted sequencing assay was used to analyze tumor resection specimens, with a focus on BRAF V600E alteration. For frozen pathologic specimens from CNS treatment-naive patients undergoing surgical resection, commercial single nuclear RNA sequencing approaches were used. The primary outcome was overall survival (OS). Secondary outcomes included CNS progression-free survival (PFS), microenvironmental composition with decreased T-cell and macrophage populations, and responses to immunotherapy. To correlate molecular status with clinical outcome, Kaplan-Meier survival analysis of 94 consecutive patients (median age, 64 years [range, 24-82 years]; 70 men [74%]) with targeted BRAF alteration testing showed worse median intracranial PFS (BRAF variant: 3.6 months [IQR, 0.1-30.6 months]; BRAF wildtype: 11.0 months [IQR, 0.8-81.5 months]; P < .001) and OS (BRAF variant: 9.8 months [IQR, 2.5-69.4 months]; BRAF wildtype: 23.2 months [IQR, 1.1-102.5 months]; P = .005; log-rank test) in BRAF V600E variant tumors. Multivariable Cox proportional hazards regression analysis revealed that BRAF V600E status was an independent variable significantly associated with both PFS (hazard ratio [HR], 2.65; 95% CI, 1.54-4.57; P < .001) and OS (HR, 1.96; 95% CI, 1.08-3.55; P = .03). For the 45 patients with resected melanoma brain metastases undergoing targeted DNA sequencing, molecular classification recapitulated The Cancer Genome Atlas groups (NRAS variant, BRAF variant, NF1 variant, and triple wildtype) with no subtype enrichment within the brain metastasis cohort. On a molecular level, BRAF V600E variant lesions were found to have a significantly decreased tumor mutation burden. Moreover, single nuclear RNA sequencing of treatment-naive BRAF V600E variant (n = 3) brain metastases compared with BRAF wildtype (n = 3) brain metastases revealed increased immune cell populations in BRAF wildtype tumors (mean [SD], 11% [4.1%] vs 3% [1.6%] CD45-positive cells; P = .04). Survival analysis of postoperative immunotherapy responses by BRAF status revealed that BRAF wildtype lesions were associated with a response to checkpoint inhibition (median OS: with immunotherapy, undefined; without immunotherapy, 13.0 months [range, 1.1-61.7 months]; P = .001; log-rank test) while BRAF variant lesions (median OS: with immunotherapy, 9.8 months [range, 2.9-39.8 months]; without immunotherapy, 9.5 months [range, 2.5-67.2 months]; P = .81; log-rank test) were not. This molecular analysis of patients with resected melanoma brain metastases found that BRAF V600E alteration is an important translational biomarker associated with worse clinical outcomes, differential microenvironmental composition, and benefit from immunotherapy. Patients with BRAF V600E variant melanoma brain metastases may thus benefit from alternative CNS-penetrant systemic regimens.

Response and Resistance to Trametinib in MAP2K1-Mutant Triple-Negative Melanoma

The development of targeted therapies for non-BRAF p.Val600-mutant melanomas remains a challenge. Triple wildtype (TWT) melanomas that lack mutations in BRAF, NRAS, or NF1 form 10% of human melanomas and are heterogeneous in their genomic drivers. MAP2K1 mutations are enriched in BRAF-mutant melanoma and function as an innate or adaptive resistance mechanism to BRAF inhibition. Here we report the case of a patient with TWT melanoma with a bona fide MAP2K1 mutation without any BRAF mutations. We performed a structural analysis to validate that the MEK inhibitor trametinib could block this mutation. Although the patient initially responded to trametinib, he eventually progressed. The presence of a CDKN2A deletion prompted us to combine a CDK4/6 inhibitor, palbociclib, with trametinib but without clinical benefit. Genomic analysis at progression showed multiple novel copy number alterations. Our case illustrates the challenges of combining MEK1 and CDK4/6 inhibitors in case of resistance to MEK inhibitor monotherapy.

BIOM-02. MUTATIONAL ANALYSIS AND SINGLE CELL SEQUENCING OF MELANOMA BRAIN METASTASES REVEALS BRAF STATUS CORRELATES WITH CLINICAL OUTCOME AND DIFFERENTIAL IMMUNE POPULATIONS

Abstract Understanding the molecular landscape and microenvironment of melanoma brain metastases is critical to devise improved treatments. Here, we perform bulk and single cell genomic analysis of melanoma brain metastases to identify molecular correlates of clinical outcome. 84 consecutive patients who underwent surgical resection at a single institution with a histo-pathologically confirmed diagnosis of melanoma brain metastasis were retrospectively identified. In 60 patients (71%) with sufficient brain metastasis tissue for targeted next generation sequencing, DNA mutations were assessed with a CLIA certified sequencing assay. Single nuclear RNA sequencing using the 10x platform was performed on n=6 samples from treatment naïve patients. Overall survival (OS) and CNS progression free survival (CNS PFS) from time of brain metastasis diagnosis were estimated using the Kaplan-Meier method. The median patient age was 62 years old (range: 25-78 years), and the median clinical follow up was 17 months. A total of 33 patients (39%) had BRAFV600E melanoma brain metastases. Multivariate analysis incorporating age, performance status, and extracranial disease revealed BRAF status was an independent prognostic factor for OS (p&amp;lt; 0.05). In patients undergoing targeted next generation sequencing, the most common pathogenic variant was TERT promoter mutation (n=44; 73%). With regard to TCGA molecular melanoma subgroups, NRAS mutant (n=22; 37%) brain metastases were most common followed by BRAF mutant (n=20; 33%), NF1 mutant (n=11; 18%), and triple wildtype (n=7; 12%). Evaluation of clinical outcomes in the context of next generation sequencing results revealed no differences by TERT status but demonstrated worse overall survival in the BRAF mutant molecular group (p&amp;lt; 0.01, log-rank test). Single nuclear sequencing of 36,115 nuclei across 6 samples revealed BRAF wildtype tumors exhibit greater infiltrating immune cell populations including microglia and T cell subtypes. Future work will require integration of these findings with different systemic therapy regimens and across larger, prospective, multi-institutional cohorts.

Mutational Analysis and Single Cell Sequencing of Melanoma Brain Metastases

<h3>Purpose/Objective(s)</h3> Melanoma brain metastases are a significant source of morbidity and mortality. While radiosurgery and (CNS) penetrant therapies have improved clinical outcomes, understanding the molecular landscape and microenvironment of brain metastases is critical to devise improved treatments for patients. Here, we perform bulk and single cell genomic analysis of melanoma brain metastases to identify molecular mechanisms underlying therapeutic response. <h3>Materials/Methods</h3> 50 consecutive patients who underwent surgical resection with histo-pathologically confirmed melanoma brain metastases and known BRAF^V600E status treated at a single institution were retrospectively identified. In 25 patients (50%) with sufficient brain metastasis tissue for targeted next generation sequencing, DNA mutations were assessed with a CLIA certified sequencing assay spanning over 500 cancer genes. Single cell RNA sequencing was performed on n=5 samples with sufficient frozen tissue. Overall survival (OS) and CNS progression free survival (CNS PFS) were estimated using the Kaplan-Meier method from time of brain metastasis diagnosis. Single cell analysis was performed using the 10x platform and Seurat R package. <h3>Results</h3> The median patient age was 64 years old (27 – 78 years), and the median clinical follow up was 17 months. 21 patients (42%) had BRAF^V600E melanoma brain metastases. BRAF^V600E mutation was associated with worse CNS PFS (p=0.04, log-rank test) and OS (p=0.03, log-rank test). Multivariate analysis incorporating age, Karnofsky performance score, and presence of extracranial disease supports <i>BRAF</i> status as an independent prognostic factor for OS (p<0.05). In patients undergoing targeted next generation sequencing, the most common pathogenic variant was <i>TERT</i> promoter mutation (n=19; 76%) followed by <i>TP53</i> mutation (n=9; 36%). The four TCGA molecular melanoma subgroups were equally represented: <i>NRAS</i> mutant (n=8; 32%), <i>NF1</i> mutant (n=6; 24%), <i>BRAF</i> mutant (n=6; 24%) or triple wildtype (n=5; 20%) Evaluation of clinical outcomes in the context of next generation sequencing results revealed no differences by <i>TERT</i> status but demonstrated worse overall survival in <i>BRAF</i> mutant tumors (median OS: 11 months; p=0.01, log-rank test). Single cell sequencing of 31,219 nuclei across 5 samples revealed distinct subpopulations between <i>BRAF</i> mutant and <i>BRAF</i> wildtype tumors in both the tumor and the brain microenvironment, with increased immune infiltration in <i>BRAF</i> wildtype tumors and greater neuronal subpopulations in <i>BRAF</i> mutant lesions. <h3>Conclusion</h3> Our data suggest <i>BRAF</i> may be useful as a prognostic biomarker and <i>TERT</i> promoter mutations as a recurrent intracranial molecular alteration in melanoma. Single cell analysis shows brain metastases exhibit distinct neuronal and immune populations. Future work will require integration of molecular status with treatment to help guide therapy and interrogation of larger, prospective, multi-institutional cohorts to validate these observations.

Landscape of mutations in early stage primary cutaneous melanoma: An InterMEL study.

It is unclear why some melanomas aggressively metastasize while others remain indolent. Available studies employing multi-omic profiling of melanomas are based on large primary or metastatic tumors. We examine the genomic landscape of early-stage melanomas diagnosed prior to the modern era of immunological treatments. Untreated cases with Stage II/III cutaneous melanoma were identified from institutions throughout the United States, Australia and Spain. FFPE tumor sections were profiled for mutation, methylation and microRNAs. Preliminary results from mutation profiling and clinical pathologic correlates show the distribution of four driver mutation sub-types: 31% BRAF; 18% NRAS; 21% NF1; 26% Triple Wild Type. BRAF mutant tumors had younger age at diagnosis, more associated nevi, more tumor infiltrating lymphocytes, and fewer thick tumors although at generally more advanced stage. NF1 mutant tumors were frequent on the head/neck in older patients with severe solar elastosis, thicker tumors but in earlier stages. Triple Wild Type tumors were predominantly male, frequently on the leg, with more perineural invasion. Mutations in TERT, TP53, CDKN2A and ARID2 were observed often, with TP53 mutations occurring particularly frequently in the NF1 sub-type. The InterMEL study will provide the most extensive multi-omic profiling of early-stage melanoma to date. Initial results demonstrate a nuanced understanding of the mutational and clinicopathological landscape of these early-stage tumors.

Candidate therapeutic agents in a newly established triple wild-type mucosal melanoma cell line.

BackgroundMucosal melanoma has characteristically distinct genetic features and typically poor prognosis. The lack of representative mucosal melanoma models, especially cell lines, has hindered translational research on this melanoma subtype. In this study, we aimed to establish and provide the biological properties, genomic features and the pharmacological profiles of a mucosal melanoma cell line that would contribute to the understanding and treatment optimization of molecularly‐defined mucosal melanoma subtype.MethodsThe sample was collected from a 67‐year‐old mucosal melanoma patient and processed into pieces for the establishment of cell line and patient‐derived xenograft (PDX) model. The proliferation and tumorigenic property of cancer cells from different passages were evaluated, and whole‐genome sequencing (WGS) was performed on the original tumor, PDX, established cell line, and the matched blood to confirm the establishment and define the genomic features of this cell line. AmpliconArchitect was conducted to depict the architecture of amplified regions detected by WGS. High‐throughput drug screening (HTDS) assay including a total of 103 therapeutic agents was implemented on the established cell line, and selected candidate agents were validated in the corresponding PDX model.ResultsA mucosal melanoma cell line, MM9H‐1, was established which exhibited robust proliferation and tumorigenicity after more than 100 serial passages. Genomic analysis of MM9H‐1, corresponding PDX, and the original tumor showed genetic fidelity across genomes, and MM9H‐1 was defined as a triple wild‐type (TWT) melanoma subtype lacking well‐characterized “driver mutations”. Instead, the amplification of several oncogenes, telomerase reverse transcriptase (TERT), v‐Raf murine sarcoma viral oncogene homolog B1 (BRAF), melanocyte Inducing transcription factor (MITF) and INO80 complex ATPase subunit (INO80), via large‐scale genomic rearrangement potentially contributed to oncogenesis of MM9H‐1. Moreover, HTDS identified proteasome inhibitors, especially bortezomib, as promising therapeutic candidates for MM9H‐1, which was verified in the corresponding PDX model in vivo.ConclusionsWe established and characterized a new mucosal melanoma cell line, MM9H‐1, and defined this cell line as a TWT melanoma subtype lacking well‐characterized “driver mutations”. The MM9H‐1 cell line could be adopted as a unique model for the preclinical investigation of mucosal melanoma.

Abstract P2-01-09: Clinical impact of ESR1 mutation ctDNA on survival outcome is dependent on PI3KCA/TP53 ctDNA mutation status

Abstract Background: With more endocrine therapies- (ET) based treatment (tx) available, genomic markers that could assist in the prediction of tx outcome is critical. The role of ctDNA mutations in ER+/HER2- metastatic breast cancer (MBC) after prior ET is based on retrospective study results. Methods: ER+/HER2- MBC patients (pts) starting ET-based salvage tx were eligible (NCT04212702). Cell-free DNA (cfDNA) was extracted from plasma before tx, and prepared for next-generation sequencing (NGS) analysis. The targeted NGS for ctDNA included regions of the ESR1 ligand-binding domain, PIK3CA hotspot mutations, and TP53 DNA-bonding domain mutations. 96% of the samples were sequenced at an average depths &amp;gt;10000x using the Ion Torrent platform. Progression-free survival (PFS) was defined from the start of the salvage tx to the date of progression. Results: From 2015/08 to 2020/05, a total of 163 pts treated with ET-based tx were prospectively enrolled. The median age was 60 (32-92). 13%, 15%, 48%, and 17% of pts received ET only, ET + CDK4/6 inhibitor, ET + everolimus, and ET + metronomic chemotherapy, respectively. Only 14 patients received fulvestrant as ET. The median level of recovered cfDNA was 38.5 ng (range 4.4-1935) and the level of cfDNA was significantly and inversely correlated with PFS (p = 0.0032). With mutation ctDNA ≥ 0.5% as a threshold for positive calling, 100 (61.3%), 41 (25.1%), and 25 (15.3) pts have at least one ESR1, PIK3CA, and TP53 mutation, respectively and 61 (37.4%) pts had &amp;gt;1 ESR1 mutation genotypes. The median PFS of the cohort (n=163) was 8.3 mos (95% CI 5.7 – 11.1 mos). PIK3CA mutation (MT) in ctDNA was associated with a worse outcome in all patients (HR 1.91, 95% CI 1.20 to 3.04, p = 0.0064) and the subgroups of ET + everolimus (HR 2.20, 95% CI 1.10 – 4.39, p = 0.025) and ET + metronomic chemotherapy (HR 5.34, 95% CI 1.63- 17.54, p = 0.006). The presence of TP53 MT ctDNA was also associated with worse PFS (HR 1.81, p = 0.043, n = 163) but also exerted a poor prognostic impact in pts with wild type (WT) PIK3CA (HR 3.28, 95% CI 1.44 – 7.48, p = 0.0048). However, the variant allelic frequency (VAF) of PIK3CA MT (p = 0.0421), but not TP53 MT (p = 0.7723), had a inverse linear correlation with PFS. Surprisingly, pts with ESR1 MT had a better PFS as compared to ESR1 WT pts (HR 0.68 95% CI 0.46 – 0.99, p = 0.049). However, if the threshold for. variant calling was raised to 2%, then ESR1 MT (n= 52, 31.9%) vs WT pts had similar PFS (median PFS 8.6 vs 7.8 mos, HR 0.92, 95% CI 0.62-1.37, p = 0.69), suggesting that defining different VAF threshold of MT ESR1 may have divergent PFS impact. How ERS1 MT ctDNA affected PFS was dependent on PIK3CA/TP53 status. When either PIK3CA or TP53 MT ctDNA was present, the ESR1 MT ctDNA did not have any impact on PFS, regardless of VAF. In pts with WT PIK3CA/TP53, pts with ESR1 MT ctDNA VAF 0.5 – 2.0% had a significant better PFS as compared with triple WT pts (HR 1.9, p = 0.0035). Conclusion: Using a 3-gene panel for ctDNA testing with MT ctDNA ≥ 0.5% as a threshold for positive calling in ER+/HER2- MBC pts treated with ET-based tx, the presence of PIK3CA and TP53 mut in ctDNA conferred a worse prognosis. The positive prognostic impact of ESR1 was only noticeable in pts with PIK3CA and TP53 WT ctDNA, and the presence of a low VAF ESR1 MT ctDNA, which may suggest an ER denpendency, was significantly correlated with a better outcome. Table 1.Median PFS of pts with and without PIK3CA, TP53 and ESR in ctDNAPopulation (n)Genotype(s)Median PFS (mos)Hazard Ratiop-valueAll (163)PIK3CA MT (41) vs. WT (122)(VAF ≥ 0.5%)5.4 vs. 10.31.910.0064TP53 MT (25) vs. WT (143)(VAF ≥ 0.5%)4.1 vs. 8.91.810.0439ESR1 MT vs. WT(VAF ≥ 0.5%)9.8 vs. 5.80.680.0493ET + everolimus (82)PIK3CA MT vs. WT(VAF ≥ 0.5%)2.8 vs. 5.92.200.0254PIK3CA and TP53 WT (106)WT vs ESR1 MT (VAF ≥ 0.5% - &amp;lt; 2%)6 vs 15.61.910.0035WT vs ESR1 MT (VAF ≥ 2%)6 vs 121.360.355 Citation Format: Tom Wei-Wu Chen, Wen Hsiao, Ming-Shen Dai, Ching-Hung Lin, Dwang-Ying Chang, I-Chun Chen, Ming-Yang Wang, Ling-Yi Huang, Shu-Han Chang, Shu-Min Huang, Ann-Lii Cheng, Kien Thiam Tan, Yen-Shen Lu. Clinical impact of ESR1 mutation ctDNA on survival outcome is dependent on PI3KCA/TP53 ctDNA mutation status [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-09.

1043P NF1 mutations and immune checkpoint inhibitor outcomes in patients with BRAF wildtype melanoma

Three genomic subtypes of BRAF wildtype (WT) cutaneous melanoma (CM) include NRAS mutant (mut), NF1 mut, triple wildtype (tWT). NF1 mut melanoma have a high tumor mutation burden and have been associated with increased responsiveness to immune checkpoint inhibitors (ICI) in some retrospective studies. We evaluated whether the NF1 subtype was associated with improved ICI outcomes in our population.

Genetic markers for characterization and prediction of prognosis of melanoma subtypes: a 2021 update.

In this article we examined the most important genetic markers involved in melanoma susceptibility, initiation and progression, and their impact on the prognosis of the disease. Current knowledge in melanoma genetics identifies distinct pathways to the development of different melanoma subtypes characterized by specific clinico-pathological features and partially known genetic markers, modulated by high, low or absence of cumulative sun damage. The most prevalent somatic mutations are related to the activation of the MAPK pathway, which are classified into four major subtypes: BRAF mutant, NRAS mutant, NF1 mutant and triple wild type. Moreover, germinal mutations are also involved in the characterization and predictions of prognosis in melanoma. Currently, CDKN2A is seen as the main high-risk gene involved in melanoma susceptibility being mutated in around 20% of melanoma-prone families. Other high-risk susceptibility genes described include CDK4, POT1, BAP1, TERT promoter, ACD, and TERF2IP. Melanoma is one of the most genetically predisposed among all cancers in humans, and ultraviolet light from the sun is the main environmental factor. This genetic predisposition is starting to be understood, impacting not only on the risk of developing melanoma but also on the risk of developing other types of cancer, as well as on the prognosis of the disease.

Integrated molecular drivers coordinate biological and clinical states in melanoma.

We performed harmonized molecular and clinical analysis on 1,048 melanomas and discovered markedly different global genomic properties among subtypes (BRAF, (N)RAS, NF1, Triple Wild-Type), subtype-specific preferences for secondary driver genes, and active mutational processes previously unreported in melanoma. Secondary driver genes significantly enriched in specific subtypes reflected preferential dysregulation of additional pathways, such as induction of TGF-β signaling in BRAF melanomas and inactivation of the SWI/SNF complex in (N)RAS melanomas, and select co-mutation patterns coordinated selective response to immune checkpoint blockade. We also defined the mutational landscape of Triple Wild-Type melanomas and identified enrichment of DNA repair defect signatures in this subtype, which were associated with transcriptional downregulation of key DNA repair genes and may revive previously discarded or currently unconsidered therapeutic modalities for genomically stratified melanoma patient subsets. Broadly, harmonized meta-analysis of melanoma whole-exomes identified distinct molecular drivers that may point to multiple opportunities for biological and therapeutic investigation.

The tumor genetics of acral melanoma: What should a dermatologist know?

Dermatologists stand at the gateway of individualization of classification, treatment, and outcomes of acral melanoma patients. The acral melanoma genetic landscape differs in vital ways from that of other cutaneous melanomas. These differences have important implications in understanding pathogenesis, treatment, and prognosis. The selection of molecularly targeted therapy must be adapted for acral melanoma. It is also critical to recognize that tumor development is far more complex than an isolated event, reliably treated by a medication acting on a single target. Tumors exhibit intratumor genetic heterogeneity, metastasis may have different genetic or epigenetic features than primary tumors, and tumor resistance may develop because of the activation of alternative genetic pathways. Microenvironmental, immune, and epigenetic events contribute and sustain tumors in complex ways. Treatment strategies with multiple targets are required to effectively disrupt the tumor ecosystem. This review attempts to translate the current molecular understanding of acral melanoma into digestible concepts relevant to the practice of dermatology. The focus is tumor genetics defining potentially treatable cancer pathways, contextualized within the relevant pathologic and molecular features.

Melanomas with activating RAF1 fusions: clinical, histopathologic, and molecular profiles

A subset of melanomas is characterized by fusions involving genes that encode kinases. Melanomas with RAF1 fusions have been rarely reported, mostly in clinical literature. To investigate this distinctive group of melanomas, we searched for melanomas with activating structural variants in RAF1, utilizing our case archive of clinical samples with comprehensive genomic profiling (CGP) by a hybrid capture-based DNA sequencing platform. Clinical data, pathology reports, and histopathology were reviewed for each case. RAF1 breakpoints, fusion partners, and co-occurring genetic alterations were characterized. From a cohort of 7119 melanomas, 40 cases (0.6%) featured fusions that created activating structural variants in RAF1. Cases with activating RAF1 fusions had median age of 62 years, were 58% male, and consisted of 9 primary tumors and 31 metastases. Thirty-nine cases were cutaneous primary, while one case was mucosal (anal) primary. Primary cutaneous melanomas showed variable architectures, including wedge-shaped and nodular growth patterns. Cytomorphology was predominantly epithelioid, with only one case, a desmoplastic melanoma, consisting predominantly of spindle cells. RAF1 5′ rearrangement partners were predominantly intrachromosomal (n = 18), and recurrent partners included MAP4 (n = 3), CTNNA1 (n = 2), LRCH3 (n = 2), GOLGA4 (n = 2), CTDSPL (n = 2), and PRKAR2A (n = 2), all 5′ of the region encoding the kinase domain. RAF1 breakpoints occurred in intron 7 (n = 32), intron 9 (n = 4), intron 5 (n = 2), and intron 6 (n = 2). Ninety-eight percent (n = 39) were wild type for BRAF, NRAS, and NF1 genomic alterations (triple wild type). Activating RAF1 fusions were present in 2.1% of triple wild-type melanomas overall (39/1882). In melanomas with activating RAF1 fusions, frequently mutated genes included TERTp (62%), CDKN2A (60%), TP53 (13%), ARID2 (10%), and PTEN (10%). Activating RAF1 fusions characterize a significant subset of triple wild-type melanoma (2.1%) with frequent accompanying mutations in TERTp and CDKN2A. CGP of melanomas may improve tumor classification and inform potential therapeutic options, such as consideration of specific kinase inhibitors.

Comprehensive genomic profiling and outcomes among metastatic melanoma patients (pts) treated with first-line cancer immunotherapy (CIT) in a real-world setting

Abstract Background Distinctive genomic subtypes (BRAF-mutated [BRAFmut], NRASmut, NF1mut, and triple wild type [wt]) have been identified in melanoma, but little is known about their distribution and association with outcomes outside of clinical trials. By linking longitudinal electronic health records (EHR) and comprehensive genomic profiling we aim to describe characteristics and outcomes among CIT-treated pts in a real-world setting. Methods Metastatic melanoma pts in the de-identified Flatiron Health (FH)-Foundation Medicine (FMI) Clinico-Genomic Database (CGDB), in which EHR-derived data from FH are linked to genomic data from FMI, who received first-line pembrolizumab (pembro), nivolumab (nivo), ipilimumab (ipi), or ipi + nivo between Jan 1, 2011, and Nov 30, 2018, were analyzed. Median OS was calculated from the start of first-line therapy and was evaluated according to BRAF status and genomic subtypes. Results Of 656 melanoma pts in CGDB, 236 received first-line CIT. The majority of pts (69%, n = 165) were BRAFwt and the rest (31%, n = 71) were BRAFmut. Median age was 62.9 yrs (57.3 yrs for BRAFmut pts and 65.6 yrs for BRAFwt pts), 89% were white, and 97% were treated in community clinics. Among BRAFwt pts, 40% (n = 66) were NRASmut, 32% (n = 53) were NF1mut, and 28% (n = 46) were triple wt. Pembro was used in 36% of pts, followed by ipi + nivo (25%), nivo (20%), and ipi (18%). Almost half (47%) of the pts received subsequent therapy; of these pts, 53% (n = 59) received CIT, 23% (n = 26) received targeted therapy, and 24% (n = 27) received other therapies. Median OS for BRAFwt and BRAFmut pts after first-line CIT was 38.6 mo (95% CI 20.2–not estimable [NE]) and 28.9 mo (95% CI 23.3–NE), respectively. Among BRAFwt pts, median OS was 44.9 mo (95% CI 28.9–NE) for NRASmut pts, 27.1 mo (95% CI 19.4–NE) for NF1mut pts, and 19.8 mo (95% CI 11.8-NE) for triple wt pts. Conclusions Our study demonstrated that outcomes among melanoma pts receiving first-line CIT in real-world setting vary based on genomic subtypes and revealed, for the first time, differences in outcomes of major genomic subtypes in BRAFwt pts. Continued investigation of the association between specific genomic subtypes and survival in a real-world setting is needed. Editorial acknowledgement Melanie Sweetlove, MSc (ApotheCom, Yardley, PA, USA). Legal entity responsible for the study Genentech, a member of the Roche Group. Funding Genentech, a member of the Roche Group. Disclosure N. Sadetsky: Shareholder / Stockholder / Stock options, Full / Part-time employment: Genentech, a member of the Roche Group. P. Lambert: Shareholder / Stockholder / Stock options, Full / Part-time employment: Genentech, a member of the Roche Group. C. Julian: Shareholder / Stockholder / Stock options, Full / Part-time employment: Genentech, a member of the Roche Group. J. Chen: Shareholder / Stockholder / Stock options, Full / Part-time employment: Genentech, a member of the Roche Group. Y. Yan: Shareholder / Stockholder / Stock options, Full / Part-time employment: Genentech, a member of the Roche Group.

Abstract 3376: The secondary genomic architecture of melanoma

Abstract Introduction: While targeted- and immuno-therapies have revolutionized the treatment of melanoma, a significant proportion of patients develop resistance to therapy and lack targetable tumor drivers. Thus, there remains a critical need for the discovery of novel tumor drivers to identify additional therapeutic targets. Melanoma has been divided into 4 genomic subtypes based on the presence of mutations in the three most frequently mutated, mutually exclusive, driver genes: BRAF, NRAS, NF1, and triple wild-type (TWT), and, in particular, no significantly mutated genes (SMGs) have been identified in TWT tumors. We hypothesized that uniform genomic analysis of expanded cohorts would provide power to identify driver genes altered at low frequencies, identify novel biological targets and therapeutic vulnerabilities unique to genomic subclasses, and specifically enable discovery of additional molecular properties unique to TWT melanoma. Methods: We aggregated 1,013 melanoma tumor and matched germline samples that passed joint quality control metrics and performed harmonized genomic analysis. Mutational significance analysis was performed using MutSigCV2 and OncodriveFML, and candidate genes filtered using TCGA expression data. Mutational signature analysis was performed using deconstructSigs, and validated in a separate cohort of melanomas. DESeq2 and edgeR were used to perform differential expression analysis. Results: We identified a set of 38 high-confidence drivers in melanoma (MutSigCV2, q &amp;lt; 0.1; OncodriveFML, q &amp;lt; 0.1), 21 of which were in either the COSMIC Cancer Gene Census or OncoKB. Several of these genes (e.g. ARID1A, CDK4) have not been considered significantly mutated or previously reported in melanoma. Further, mutational significance analysis within each subtype identified 63 BRAF, 73 NRAS, 26 NF1 and 11 TWT SMGs (MutSigCV2, q &amp;lt; 0.1), and revealed that secondary driver genes are rarely shared between the subtypes. Mutational signature analysis determined that a subset of melanomas (7%) have homologous recombination (HR) deficiency (signature 3), and this signature is significantly enriched in TWT melanomas (21.6%, p = 1.21 x 10-10, Fisher’s). ATM, APLF and SMARCD1 expression were significantly correlated with signature 3 contribution (p &amp;lt; 0.05), and significantly differentially expressed between signature 3 and non-signature 3 tumors in TWT melanoma (q &amp;lt; 0.05). Conclusion: Through uniform analysis of the largest melanoma whole-exome-sequenced cohort to date, we identified a set of high confidence driver genes, many of which were not previously associated with melanoma, as well as secondary driver genes unique to each genomic subtype. Mutational signature analysis identified the enrichment of HR deficiency in TWT melanomas, which appears to be facilitated through downregulation of ATM and may have immediate translational implications for patient-stratification of therapies that target HR deficient melanomas. Citation Format: Jake Conway, Amaro Taylor-Weiner, Saud AlDubayan, Brendan Reardon, Felix Dietlein, David Liu, Eliezer M. Van Allen. The secondary genomic architecture of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3376.

Therapeutic implications of a novel driver classification system for cutaneous and unknown primary melanomas.

9539 Background: Cutaneous and “unknown primary” melanomas frequently harbor alterations that activate the Mitogen Activated Protein Kinase (MAPK) pathway, and are often classified as BRAF, NRAS, NF1 mutant, or “triple wild type.” Multigene sequencing may identify additional oncogenic drivers, but the clinical impact of this information is unknown. Methods: Patients with BRAF inhibitor naïve melanoma underwent prospective tumor molecular profiling using a targeted capture-based assay (MSK-IMPACT), and demographic and treatment data were collected. Time to treatment failure was assessed for patients who received frontline PD-1 monotherapy or nivolumab plus ipilimumab. Results: 576 patients were successfully sequenced. 533 samples (96%) harbored a known or presumed oncogenic mutation in 1 of 28 genes in the RTK-RAS-MAPK pathway. 187 tumors (32%) had two or more drivers in this pathway, and the rates of driver co-alterations varied widely by specific driver. A hierarchical classification of 9 driver groups (BRAF V600E; V600K/R/M; BRAF non-V600; NRAS Q61; Other RAS; NF1; KIT; Other driver; Unknown driver) was significantly associated with tumor mutational burden, primary melanoma site, and patient age. Time to treatment failure varied by driver class and site of primary melanoma for PD-1 monotherapy but not nivolumab plus ipilimumab. 150 patients with BRAF V600 wild-type melanoma required systemic therapy after progression on checkpoint blockade. 21 were given genomically matched therapy, and 5/21 had clinical benefit for ≥6 months. Complete and durable responses were observed with TRK and ROS1 inhibitors in patients with NTRK1/2/3 and ROS1 fusion positive tumors. Conclusions: Oncogenic alterations in the RTK-RAS-MAPK pathway can be detected using targeted capture NGS in the vast majority of cutaneous and unknown primary melanomas. A hierarchical classification of 9 driver groups revealed clinically relevant melanoma subsets with varying clinical outcomes to PD-1 monotherapy. Select patients with oncogenic kinase fusions can achieve durable therapeutic benefit with targeted inhibitors of these rare drivers.

Targeting MAP2K1 mutation with trametinib in a triple wild-type melanoma patient.

e21027 Background: MAP2K1 inhibitors combined with BRAF inhibitors provide a prolonged progression-free survival for BRAF mutant patients. However, the impact of MEK inhibition of MAP2K1 mutant and BRAF/NRAS/NF1 wild type melanoma patients remains elusive. Methods: We performed next-generation sequencing using an in-house developed 400 full exon panel for one of our triple wild type (BRAF/NRAS/NF1) melanoma patient. The patient received off-label, trametinib at a dose of 2 mg o.d. We performed additional sequencing at progression. After progression, the patient was treated off-label with combined trametinib and palbociclib therapy. Furthermore, we screened the TCGA melanoma and MSK-IMPAKT databases for patients with similar mutation profiles. Results: Our patient presented with dual somatic MAP2K1 mutations at Cys121Ser and Pro124Arg with a 68% allele frequency. The tumor also harbored a minor EGFR mutant clone (11% allele frequency) and a complete loss of the CDKN2A locus. The patient received trametinib and after two months presented a partial response as per RECIST1.1. However, after two additional months, the patient progressed. A new biopsy was performed, and the treatment was modified to combine trametinib and palbociclib to co-target MAP2K1 and the loss of the CKDN2A locus. The patient progressed under dual trametinib/palbociclib therapy without any objective benefit. The on-progression biopsy showed the absence of a gatekeeper mutation of MAP2K1 and the persistence of the loss of the CDKN2A locus. We detected a large number of novel copy number variations, most notable amplification of MITF (9x), FGFR1 (10X) and of MDM2 (16x). We also detected a loss of the minor, EGFR mutant clone. The analysis of large-scale state transitions (LSTs) for deficiency in homolog recombination (HRDness) was negative in pre and post-treatment biopsies. Conclusions: Our patient provides unique evidence to target solitary MAP2K1 mutations in triple wild melanoma by MEK1 inhibitors. Resistance to MEK1 inhibition developed rapidly and likely involved FGFR1 and MDM2, which have not been previously associated with resistance to MEK inhibitor monotherapy. The addition of palbociclib could not rescue these potential resistance mechanisms.

Synergy from gene expression and network mining (SynGeNet) method predicts synergistic drug combinations for diverse melanoma genomic subtypes

Systems biology perspectives are crucial for understanding the pathophysiology of complex diseases, and therefore hold great promise for the discovery of novel treatment strategies. Drug combinations have been shown to improve durability and reduce resistance to available first-line therapies in a variety of cancers; however, traditional drug discovery approaches are prohibitively cost and labor-intensive to evaluate large-scale matrices of potential drug combinations. Computational methods are needed to efficiently model complex interactions of drug target pathways and identify mechanisms underlying drug combination synergy. In this study, we employ a computational approach, SynGeNet (Synergy from Gene expression and Network mining), which integrates transcriptomics-based connectivity mapping and network centrality analysis to analyze disease networks and predict drug combinations. As an exemplar of a disease in which combination therapies demonstrate efficacy in genomic-specific contexts, we investigate malignant melanoma. We employed SynGeNet to generate drug combination predictions for each of the four major genomic subtypes of melanoma (BRAF, NRAS, NF1, and triple wild type) using publicly available gene expression and mutation data. We validated synergistic drug combinations predicted by our method across all genomic subtypes using results from a high-throughput drug screening study across. Finally, we prospectively validated the drug combination for BRAF-mutant melanoma that was top ranked by our approach, vemurafenib (BRAF inhibitor) + tretinoin (retinoic acid receptor agonist), using both in vitro and in vivo models of BRAF-mutant melanoma and RNA-sequencing analysis of drug-treated melanoma cells to validate the predicted mechanisms. Our approach is applicable to a wide range of disease domains, and, importantly, can model disease-relevant protein subnetworks in precision medicine contexts.