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Supplementary Data from Loss-of-Function Fibroblast Growth Factor Receptor-2 Mutations in Melanoma

Supplementary Data from Elevated Cutaneous Smad Activation Associates with Enhanced Skin Tumor Susceptibility in Organ Transplant Recipients

MET tyrosine kinase domain modeling location of the F1200 residue

Demographics of the METex14-mutated and EGFR-mutated cohorts.

Purpose:Although patients with advanced-stage non–small cell lung cancers (NSCLC) harboring MET exon 14 skipping mutations (METex14) often benefit from MET tyrosine kinase inhibitor (TKI) treatment, clinical benefit is limited by primary and acquired drug resistance. The molecular basis for this resistance remains incompletely understood.Experimental Design:Targeted sequencing analysis was performed on cell-free circulating tumor DNA obtained from 289 patients with advanced-stage METex14-mutated NSCLC.Results:Prominent co-occurring RAS–MAPK pathway gene alterations (e.g., in KRAS, NF1) were detected in NSCLCs with METex14 skipping alterations as compared with EGFR-mutated NSCLCs. There was an association between decreased MET TKI treatment response and RAS–MAPK pathway co-occurring alterations. In a preclinical model expressing a canonical METex14 mutation, KRAS overexpression or NF1 downregulation hyperactivated MAPK signaling to promote MET TKI resistance. This resistance was overcome by cotreatment with crizotinib and the MEK inhibitor trametinib.Conclusions:Our study provides a genomic landscape of co-occurring alterations in advanced-stage METex14-mutated NSCLC and suggests a potential combination therapy strategy targeting MAPK pathway signaling to enhance clinical outcomes.

The MET F1200 residue is conserved across multiple tyrosine kinases

Purpose: Nonmelanoma skin cancer incidence is enhanced >50-fold in patients taking antirejection drugs (ARD) following organ transplantation. Preclinical studies suggest that ARD treatment increases transforming growth factor-β1 (TGF-β1) levels, which contribute to enhanced tumor susceptibility independent of the immunosuppressive effects of ARDs. This study investigates whether TGF-β signaling is elevated in transplant patients.Experimental Design: Immunohistochemical tissue microarray analysis was used to determine the levels of TGF-β1, TGF-β2, TGF-β3, TβRII, and activated P-Smad2/3 and P-Smad1/5/8, which are phosphorylated directly by distinct TGF-β/BMP receptor complexes. We analyzed >200 cutaneous lesions and adjacent nonlesional skin samples from 87 organ transplant recipients, and 184 cutaneous lesions and adjacent skin samples from 184 individuals who had never received ARDs.Results: We found significantly higher levels of P-Smad2 in both nonlesional and lesional tissue from transplant recipients compared with those not exposed to ARDs (P ≤ 0.001). In contrast, P-Smad1/5/8, a marker of activation of the bone morphogenetic protein signaling pathway, was generally not expressed at higher levels in patients taking ARDs, including analysis of nonlesional skin, actinic keratoses, carcinoma in situ, or squamous cell carcinoma but was differentially expressed between keratoacanthoma from transplant recipients compared with those from non–transplant recipients (P ≤ 0.005).Conclusions: Observation of elevated P-Smad2 levels in transplant recipients is consistent with the notion that elevated TGF-β signaling may contribute to malignancy in organ transplant recipients. Disparate P-Smad1/5/8 expression levels between keratoacanthoma from the two patient groups might reflect the distinct BMP-responsive cell of origin for this hair follicle–derived lesion. (Clin Cancer Res 2009;15(16):5101–7)

Genes included in NGS Panels

Supplemental Table 1. Adverse events observed and adjudicated as possibly, probably or definitely related to therapy (n = 19).

Purpose: Although durable responses can be achieved with tyrosine kinase inhibitors such as imatinib in melanomas harboring KIT mutations, the efficacy of alternative inhibitors after progression to imatinib and the activity of these agents on brain metastases are unknown.Experimental Design: We conducted a phase II study of nilotinib 400 mg twice a day in two cohorts of patients with melanomas harboring KIT mutations or amplification: (A) those refractory or intolerant to a prior KIT inhibitor; and (B) those with brain metastases. The primary endpoint was 4-month disease control rate. Secondary endpoints included response rate, time-to-progression (TTP), and overall survival (OS). A Simon two-stage and a single-stage design was planned to assess for the primary endpoint in cohorts A and B, respectively.Results: Twenty patients were enrolled and 19 treated (11 in cohort A; 8 in cohort B). Three patients on cohort A [27%; 95% confidence interval (CI), 8%–56%] and 1 on cohort B (12.5%; 90% CI, 0.6%–47%) achieved the primary endpoint. Two partial responses were observed in cohort A (18.2%; 90% CI, 3%–47%); none were observed in cohort B. The median TTP and OS was 3.3 (90% CI, 2.1–3.9 months) and 9.1 months (90% CI, 4.3–14.2 months), respectively, in all treated patients.Conclusions: Nilotinib may achieve disease control in patients with melanoma harboring KIT alterations and whose disease progressed after imatinib therapy. The efficacy of this agent in KIT-altered melanoma with brain metastasis is limited. Clin Cancer Res; 21(10); 2289–96. ©2015 AACR.

PDF file, 173K, Transient transfection of SOX10-specific siRNAs in 0380-MMU and UACC 1022 cells.

PDF file, 122K, Senescence associated heterochromatin foci (SAHF) are more pronounced in melanoma cells when SOX10shRNA is expressed compared to non-silencing (NS) control.

Supplementary Figures 1-2 from In Melanoma, RAS Mutations Are Accompanied by Switching Signaling from BRAF to CRAF and Disrupted Cyclic AMP Signaling

Melanocytes require the RAS/RAF/MEK/ERK and the cyclic AMP (cAMP) signaling pathways to maintain the fine balance between proliferation and differentiation. We have investigated how cross-talk between these pathways affects melanoma progression. We show that cAMP suppresses CRAF activity in melanocytes and that this is essential to suppress the oncogenic potential of CRAF in these cells. As a consequence, BRAF alone is responsible for signaling to MEK. However, when RAS is mutated in melanoma, the cells switch their signaling from BRAF to CRAF. This switch is accompanied by dysregulated cAMP signaling, a step that is necessary to allow CRAF to signal to MEK. Thus, a fundamental switch in RAF isoform usage occurs when RAS is mutated in melanoma, and this occurs in the context of disrupted cAMP signaling. These data have important implications for the development of therapeutic strategies to treat this life-threatening disease. (Cancer Res 2006; 66(19): 9483-91)

Supplementary Figure Legends 1-2 from In Melanoma, RAS Mutations Are Accompanied by Switching Signaling from BRAF to CRAF and Disrupted Cyclic AMP Signaling

PDF file, 164K, KitlacZ and Mitfvga are not sufficient genetic alterations to suppress melanoma in Grm1Tg mice.

PDF file, 152K, Loss of SOX10 expression does not consistently result in changes in protein levels of Cyclin/CDK proteins responsible for RB phosphorylation in early G1 phase.

Background Acral and mucosal melanomas are aggressive subtypes of melanoma, which have a significantly lower burden of somatic mutations than cutaneous melanomas, but more frequent copy number variations, focused gene amplifications, and structural alterations. The landscapes of their genomic alterations remain to be fully characterized. Methods We compiled sequencing data of 240 human acral and mucosal melanoma samples from 11 previously published studies and applied a uniform pipeline to call tumor cell content, ploidy, somatic and germline mutations, as well as CNVs, LOH, and SVs. We identified genes that are significantly mutated or recurrently affected by CNVs and implicated in oncogenesis. We further examined the difference in the frequency of recurrent pathogenic alterations between the two melanoma subtypes, correlation between pathogenic alterations, and their association with clinical features. Results We nominated PTPRJ, mutated and homozygously deleted in 3.8% (9/240) and 0.8% (2/240) of samples, respectively, as a probable tumor suppressor gene, and FER and SKP2, amplified in 3.8% and 11.7% of samples, respectively, as probable oncogenes. We further identified a long tail of infrequent pathogenic alterations, involving genes such as CIC and LZTR1. Pathogenic germline mutations were observed on MITF, PTEN, ATM, and PRKN. We found BRAF V600E mutations in acral melanomas with fewer structural variations, suggesting that they are distinct and related to cutaneous melanomas. Amplifications of PAK1 and GAB2 were more commonly observed in acral melanomas, whereas SF3B1 R625 codon mutations were unique to mucosal melanomas (12.9%). Amplifications at 11q13-14 were frequently accompanied by fusion to a region on chromosome 6q12, revealing a recurrent novel structural rearrangement whose role remains to be elucidated. Conclusions Our meta-analysis expands the catalog of driver mutations in acral and mucosal melanomas, sheds new light on their pathogenesis and broadens the catalog of therapeutic targets for these difficult-to-treat cancers.

Tools to visualize genetic alterations within tissues remain underdeveloped despite the growth of spatial transcriptomic technologies, which measure gene expression in different regions of tissues. Since genetic alterations can be detected in RNA-sequencing data, we explored the feasibility of observing somatic alterations in spatial transcriptomics data. Extracting genetic information from spatial transcriptomic data would illuminate the spatial distribution of clones and allow for correlations with regional changes in gene expression to support genotype-phenotype studies. Recent work demonstrates that copy number alterations can be inferred from spatial transcriptomics data1. Here, we describe new software to further enhance the inference of copy number from spatial transcriptomics data. Moreover, we demonstrate that single nucleotide variants are also detectable in spatial transcriptomic data. We applied these approaches to map the location of point mutations, copy number alterations, and allelic imbalances in spatial transcriptomic data of two cutaneous squamous cell carcinomas. We show that both tumors are dominated by a single clone of cells, suggesting that their regional variations in gene expression2are likely driven by non-genetic factors. Furthermore, we observe mutant cells in histologically normal tissue surrounding one tumor, which were not discernible upon histopathologic evaluation. Finally, we detected mono-allelic expression of immunoglobulin heavy chains in B-cells, revealing clonal populations of plasma cells surrounding one tumor. In summary, we put forward solutions to add the genetic dimension to spatial transcriptomic datasets, augmenting the potential of this new technology.

Cancer is caused by the accumulation of pathogenic alterations of the genome and epigenome that result in permanent changes that disrupt cellular homeostasis. The genes that become corrupted in this process vary among different tumour types, reflecting specific vulnerabilities and dependencies of the cell from which the cancer originated. This also applies to 'melanoma', a cancer that constitutes not one, but multiple diseases that can be separated based on their cell of origin, aetiology, clinical appearance and course, and response to treatment. In this article, we review the current classification of melanoma within distinct evolutionary pathways and the associated genetic alterations. In addition, we review the application of molecular diagnostics to the diagnosis of melanocytic tumours in the context of histopathological assessment.

Uveal melanoma (UM) is a rare subtype of melanoma that originates from melanocytes of the choroidal plexus, ciliary body and iris. Despite successful treatment of the primary by radiation or surgery, 50% of UM patients develop metastases, mostly in the liver, as an invariably lethal complication. Over 90% of UM harbor activating mutations in the closely related GNAQ or GNA11 genes, mainly at codons Q209, compromising the GTPase activity. Mutant GNAQ and GNA11 can be successfully targeted by the cyclic depsipeptide YM-254890. To anticipate mechanisms of resistance that might arise under treatment with this novel class of Gαq inhibitors, we generated UM cell lines resistant to YM-254890 using an in-vitro cell culture system. Upon chronic growth suppression of a GNA11Q209L mutant UM cell line, we established 12 YM-254890 resistance clones. We identified a secondary GNA11F75Y mutation in one clone and GNA11Y192H mutations in the other 11 clones. The secondary mutations were present in cis with the original GNA11 Q209L mutations. To validate the functional role of these secondary mutations in conveying resistance, we engineered two double mutants, GNA11Q209L/F75Y and GNA11Q209L/Y192H, and introduced them into 293FT cells respectively and confirmed that both F75Y and Y192H convey resistance to YM-254890 treatment. The proliferation of YM-254890-resistant cell lines was still dependent on GNA11 and downstream PKC/MAPK signaling. Combined inhibition of PKC and MEK synergistically reduced cell viability in YM-254890 resistant UM cells. Analysis of the crystal structure of YM-2548890 in complex with GNAQ predicts both F75Y and Y192H mutations directly affect the binding of YM-254890 to Gαq. Our data suggest that direct targeting mutant GNAQ/11 is promising but will select for secondary mutations within GNAQ/11 that will result in resistance. Combinatorial targeting of other components in the Gαq signaling pathway will increase clinical efficacy. Citation Format: Jiafang Ma, Meng Wang, Aashish Manglik, Boris C. Bastian, Xu Chen. A secondary Gαq mutation confers resistance to Gαq inhibitors in uveal melanoma. [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 3885.

ObjectiveTo analyze the genetic features of melanocytomas and melanomas of the anterior uvea and assess the value of molecular testing for diagnosis and prognostication.DesignRetrospective case-control study.SubjectsPatients with melanocytoma (n= 16) and melanoma (n= 19) of the anterior uvea.MethodsTargeted next-generation sequencing was performed on formalin-fixed, paraffin-embedded tumor tissue from anterior uveal melanocytic tumors and correlated with clinicopathologic features.Main outcome measuresPresence or absence of accompanying oncogenic alterations beyond GNAQ/GNA11 and their association with histologic features and local recurrence.ResultsHotspot missense mutations in GNAQ/GNA11 were identified in 91% (32/35) of all cases. None of the melanocytomas with or without atypia demonstrated chromosomal imbalances or additional oncogenic variants beyond GNAQ mutation, and none recurred over a median follow-up of 36 months. Additional alterations identified in a subset of melanomas include mutations in BAP1 (n= 3), EIF1AX (n= 4), SRSF2 (n= 1), PTEN (n=1), and EP300 (n= 1); monosomy 3p (n= 6); trisomy 6p (n= 3); trisomy 8q (n= 2); and an ultraviolet mutational signature (n= 5). Local recurrences were limited to melanomas, all of which demonstrated oncogenic alterations in addition to GNAQ/GNA11 (n= 5). A single melanoma harboring GNAQ and BAP1 mutations and monosomy 3 was the only tumor that metastasized.ConclusionsIn this study, anterior segment uveal melanocytomas did not display oncogenic alterations beyond GNAQ/GNA11. Therefore, they are genetically similar to uveal nevi rather than uveal melanoma based on their molecular features known from the literature. Molecular testing can be performed on borderline cases to aidrisk stratification and clinical management decisions.

Evidence shows that adolescents need later wake times for sufficient sleep and that starting school later improves sleep outcomes. In recent years, there has been increased interest in the effect of school start times on educational outcomes. We aim to summarize recent studies, evaluate key findings, and identify limitations in the literature. Recent studies examined the relationship of school start times to attendance, discipline, grades, test scores, and other outcomes. Many studies found that later start times improved attendance and grades. The results for test scores and other outcomes were more mixed. Nascent evidence suggests the relationship between start times and educational outcomes exists for younger students as well. While findings suggest that later school start times were associated with better educational outcomes, the methodological approaches employed have limitations. Few studies used rigorous methods to examine within school changes in start times.

Every cell in the human body has a unique set of somatic mutations, yet it remains difficult to comprehensively genotype an individual cell1. Here, we developed solutions to overcome this obstacle in the context of normal human skin, thus offering the first glimpse into the genomic landscapes of individual melanocytes from human skin. As expected, sun-shielded melanocytes had fewer mutations than sun-exposed melanocytes. However, within sun-exposed sites, melanocytes on chronically sun-exposed skin (e.g. the face) displayed a lower mutation burden than melanocytes on intermittently sun-exposed skin (e.g. the back). Melanocytes located adjacent to a skin cancer had higher mutation burdens than melanocytes from donors without skin cancer, implying that the mutation burden of normal skin can be harnessed to measure cumulative sun damage and skin cancer risk. Moreover, melanocytes from healthy skin commonly harbor pathogenic mutations, though these mutations tended to be weakly oncogenic, likely explaining why they did not give rise to discernible lesions. Phylogenetic analyses identified groups of related melanocytes, suggesting that melanocytes spread throughout skin as fields of clonally related cells, invisible to the naked eye. Overall, our study offers an unprecedented view into the genomic landscapes of individual melanocytes, revealing key insights into the causes and origins of melanoma.

In the present study we consider whether certain pre-service teachers (PSTs) particularly benefit from high-quality student teaching experiences. To conduct these analyses, we connect student teaching and K-12 workforce data for six educator preparation programs (EPPs) and assess whether placement school and cooperating teacher characteristics predict the effectiveness of early-career teachers. Results show that high-quality student teaching placements especially benefit PSTs with lower GPAs and narrow effectiveness gaps between teachers with lower versus higher GPAs. These findings call for closer partnerships between EPPs and school districts and suggest that EPPs may wish to prioritize placements for PSTs with lower GPAs.

Melanomas that have histopathologic features that overlap with those of Spitz nevus are referred to as spitzoid melanomas. However, the diagnostic concept is used inconsistently and genomic analyses suggest it is a heterogeneous category. Spitz tumors, the spectrum of melanocytic neoplasms extending from Spitz nevi to their malignant counterpart Spitz melanoma, are defined in the 2018 WHO classification of skin tumors by the presence of specific genetic alterations, such as kinase fusions or HRAS mutations. It is unclear what fraction of “spitzoid melanomas” defined solely by their histopathologic features belong to the category of Spitz melanoma or to other melanoma subtypes. We assembled a cohort of 25 spitzoid melanomas diagnosed at a single institution over an 8-year period and performed high-coverage DNA sequencing of 480 cancer related genes. Transcriptome wide RNA sequencing was performed for select cases. Only nine cases (36%) had genetic alterations characteristic of Spitz melanoma, including HRAS mutation or fusion involving BRAF, ALK, NTRK1, or MAP3K8. The remaining cases were divided into those with an MAPK activating mutation and those without an MAPK activating mutation. Both Spitz melanoma and spitzoid melanomas in which an MAPK-activating mutation could not be identified tended to occur in younger patients on skin with little solar elastosis, infrequently harbored TERT promoter mutations, and had a lower burden of pathogenic mutations than spitzoid melanomas with non-Spitz MAPK-activating mutations. The MAPK-activating mutations identified affected non- V600 residues of BRAF as well as NRAS, MAP2K1/2, NF1, and KIT, while BRAF V600 mutations, the most common mutations in melanomas of the WHO low-CSD category, were entirely absent. While the “spitzoid melanomas” comprising our cohort were enriched for bona fide Spitz melanomas, the majority of melanomas fell outside of the genetically defined category of Spitz melanomas, indicating that histomorphology is an unreliable predictor of Spitz lineage.

The deubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with a high risk for mesothelioma and melanocytic tumors. Here, we show that pancreatic intraepithelial neoplasia driven by oncogenic mutant KrasG12D progressed to pancreatic adenocarcinoma in the absence of BAP1. The Hippo pathway was deregulated in BAP1-deficient pancreatic tumors, with the tumor suppressor LATS exhibiting enhanced ubiquitin-dependent proteasomal degradation. Therefore, BAP1 may limit tumor progression by stabilizing LATS and thereby promoting activity of the Hippo tumor suppressor pathway. Significance: BAP1 is mutated in a broad spectrum of tumors. Pancreatic Bap1 deficiency causes acinar atrophy but combines with oncogenic Ras to produce pancreatic tumors. BAP1-deficient tumors exhibit deregulation of the Hippo pathway. See related commentary by Brekken, p. 1624

Whereas subject-area specialization is common practice in secondary grades, little is known about its incidence and impact in elementary schools. In this study we use data from North Carolina elementary schools to assess which teachers specialize and estimate whether specialization is associated with teacher effectiveness and school achievement. We find that specialization is prevalent in upper-elementary grades—approximately 25 percent of fourth-grade teachers and 37 percent of fifth-grade teachers specialize—and schools assign relatively more effective teachers to specialize. Analyses indicate that specialization is not leading to its theorized benefits in mathematics and reading. Teachers are less effective than they were before specializing and school-level achievement is not associated with more specialization. However, science results suggest benefits to subject-area specialization. These findings question the use of specialization in elementary grades but invite continued research to more fully assess its impact.