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Point-of-care hepatitis C testing from needle and syringe programs: An Australian feasibility study
Pigmentation and Melanoma | ABSTRACTS 815
816
Genetic alterations in primary melanoma in Taiwan C Chu1, Y Sheen1, K Tan2 and K Tse2 1 Department of Dermatology, National Taiwan University Hospital, Taipei, Taiwan and 2 ACT Genomics, Taipei, Taiwan Acral melanoma is the most common subtype of melanoma in Asians. However, differences in genetic alterations between acral melanoma and cutaneous melanoma in Asians are not well studied. To augment the understanding of the prevalence, patterns, and associations of mutations between different subtypes of melanoma, we performed next-generation sequencing of coding regions in 409 cancer-associated genes of 66 primary melanomas comprised of 45 acral melanomas and 21 cutaneous melanomas. The most common mutation in cutaneous melanoma was BRAF (66.7%), while in acral melanoma, KIT (24.4%), KRAS (15.6%), and NRAS (13.3%) were the most common mutations. Compared with acral melanoma, the mutation frequency of BRAF in cutaneous melanoma is significantly higher. Meanwhile, frequencies of NRAS/KRAS mutations, altered cell cycle regulation, BIRC2/3/5 gain, and amplification of receptor tyrosine kinase genes were significantly enriched in acral melanomas. Of note, cell cycle dysregulation and BIRC2/3/5 gain were significantly associated with poor melanoma-specific survival in all melanoma patients and in acral melanomas. The multivariate analysis showed that lymph node metastasis and cell cycle-related alterations are independent prognostic factor for melanoma-specific survival. This study strengthens our understanding of the patterns and clinical associations of oncogenic mutations in melanomas in Taiwan.
Autophagy inhibition sensitizes targeted therapy-resistant melanoma to MEK1/2 inhibitors A Truong1,2, C Kinsey2, M Foth2, M Scherzer1,2, J Sanchez3 and M McMahon2,4 1 Department of Oncological Sciences, University of Utah, Salt Lake City, Utah, United States, 2 Huntsman Cancer Institute, Salt Lake City, Utah, United States, 3 Department of Pathology, University of Utah, Salt Lake City, Utah, United States and 4 Department of Dermatology, University of Utah, Salt Lake City, Utah, United States Targeted therapy has dramatically improved the treatment landscape for metastatic melanoma, however is limited to those with BRAF(V600) mutations. GNAQ/GNA11 mutations are found in approximately 2% of melanoma, including 80% of uveal melanoma. Mutations in these G-alpha proteins lead to constitutive activation of multiple oncogenic pathways, including MAPK (RAF–>MEK1/2–>ERK1/2) signaling. Unfortunately, metastatic uveal melanoma is refractory to pharmacologic targeted therapies inhibiting MEK1/2, including trametinib and binimetinib. We showed that MEK1/2 inhibition increased autophagic flux in GNAQ/GNA11 mutated melanoma cell lines. The addition of autophagy inhibitors, such as chloroquine, resulted in synergistic cytotoxicity (Loewe model), demonstrating the cytoprotective role of autophagy as a result of MEK1/2 inhibitor treatment. Furthermore, combining trametinib with chloroquine or hydroxychloroquine resulted in a rapid and profound decrease in tumor burden in vivo compared to either treatment as monotherapy. This was also demonstrated in patient derived xenografts of NRAS-mutated cutaneous melanomas, a more common genetic subset of melanoma in which targeted therapy is also ineffective. Additionally, we showed that BRAF-mutated melanoma cell lines with genetically engineered or drug selected resistance to BRAF and MEK1/2 inhibitors displayed increased baseline autophagic flux, regardless of the presence of drug. The addition of autophagy inhibitors sensitized these resistant cells to BRAF and MEK1/2 inhibition. Our findings suggest a novel and potentially effective treatment strategy for melanomas with innate or acquired resistance to MEK1/2 inhibition.
817
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Ethanol induces skin hyperpigmentation in mice with aldehyde dehydrogenase 2 deficiency T Yamauchi1, A Matsumoto2, S Ito3, K Wakamatsu3, T Suzuki4 and M Fujita1 1 Dermatology, University of Colorado Denver, Aurora, Colorado, United States, 2 Saga University School of Medicine, Saga, Japan, 3 Fujita Health University School of Health Sciences, Aichi, Japan and 4 Yamagata University, Yamagata, Japan Excessive alcohol consumption leads to alcohol use disorder. Alcohol is metabolized to acetaldehyde, which is then oxidized to acetic acid by aldehyde dehydrogenases (ALDH), a class of enzymes that facilitate the conversion of aldehydes to their corresponding acids. Among ALDHs, mitochondrial ALDH2 is the primary enzyme involved in the metabolism of acetaldehyde. In addition to its well-known role in ethanol metabolism, recent studies have suggested that ALDH2 dysfunction is associated with a variety of human diseases including cardiovascular diseases, diabetes, neurodegenerative diseases, stroke, cancer, anemia, pain, osteoporosis and aging. However, the role of ALDH2 has not been well investigated in the skin. In this study, we studied the effect of the functional defect of Aldh2 using Aldh2 KO mice. Ten-week-old Aldh2 KO mice and control C57BL/6 (WT) mice were fed with a standard hard diet and bottled water with ethanol for 10 weeks. A dose-dependent skin pigmentation was observed in the ears, feet, tail and genital area in Aldh2 KO mice but not WT mice (p ¼ 0.08e0.95; p interaction <0.001). The pigmentation was mainly localized in the epidermis and partially in the dermis, and found to be melanin. Analysis of degradation products of melanin demonstrated ethanol dose-dependent production of eumelanin in the mouse skin. Immunohistochemical analysis showed ethanol-induced melanocyte proliferation in Aldh2 KO mice. Acetaldehyde exposure occurs not only by ethanol consumption but also by a volatile organic compound, smoking and food products. ALDH2 enzyme is inactivated by post-translational modification under increased oxidative/nitrosative stress, suggesting the role of ALDH2 in acetaldehyde-induced skin even in individuals without a defective polymorphism of ALDH2.
Oriented analysis for transposon mutagenesis screening to study drug resistance J Hong1,2, T Lee2, W Wee2 and Y Chen1 1 Graduate Institute of Medical Genomics and Proteomics, Taipei, Taiwan and 2 Department of Dermatology, National Taiwan University Hospital, Taipei, Taiwan Several high-throughput screenings have been performed to study drug resistance. In comparison to the reverse genetic screening, the forward screening by transposon insertional mutagenesis does not need a priori target panel. We have performed piggyBac transposon mutagenesis screening by hyperactive transposase and pGG134 transposon carrying MSCV promoter in SK-MEL-28 melanoma cell line for resistant factor of the BRAF inhibitor, encorafenib. This study aimed to develop an appropriate computational analysis to identify the candidate genes. After sequencing the splinkerette PCR products, the original baseline mutagenesis library showed insertions distributed across all chromosomes. The first analysis for common insertion sites was done by Kernel Convolved Rule-Based Mapping. The results of the baseline library and the encorafenib selection group both predominantly marked by large genes, including DMD, PTPRD, and MACROD2. There were still over 2000 transposon insertion sites identified in the encorafenib group, which suggested no pure single mutagenic clone after drug selection. Considering the orientation-specific insertion by pGG134, the trapped genes were then filtered by sense/anti-sense insertion ratio. The filtered results in the encorafenib group showed that MITF had the highest insertion number, followed by USP47, SYCP2L, TNRC6C and MAP3K4. By comparing the baseline library, MITF, USP47 and MAP3K4 had obviously increased sense insertions after encorafenib selection. Overexpression of MAP3K4 was proved to offer resistance to BRAF inhibitor in the sensitive melanoma cells. Overall, the results of our transposon screening revealed that common insertion sites alone may not be sufficient to identify the candidate genes for drug resistance. Orientated analysis by filtering the sense insertional mutation and comparing the baseline mutagenesis library should be performed.
819
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Extragenital lichen sclerosus is an immune-related adverse event with nivolumab E Williams1, B Mackool2 and R Nazarian1 1 Pathology, Massachusetts General Hospital, Boston, Massachusetts, United States and 2 Dermatology, Massachusetts General Hospital, Boston, Massachusetts, United States Nivolumab is a programmed cell death (PD)-1 ligand inhibitor used in treatment of melanoma and other malignancies. Immune-related adverse events (IRAEs) affect nearly all organ systems, most commonly the skin, the gastrointestinal tract (enterocolitis), the liver (hepatitis), and the endocrine system. Skin reactions related to immunotherapy occur in 4-27% of patients, including lichenoid mucocutaneous eruptions, vitiligo, psoriasis, lichen planus, and bullous pemphigoid. Most recently, genital lichen sclerosus (LS) and relapse of morphea have been reported. We seek to better describe this category of IRAE, and describe a patient with metastatic melanoma treated with nivolumab, who developed exacerbation of an existing plaque of extragenital LS. A 78-year-old white woman was diagnosed with metastatic melanoma of unknown primary following excisional biopsy of one of two deep masses in her right lower extremity. Mutational analysis revealed that the patient was NRAS mutant and BRAF wildtype. A year prior, an oval white plaque on her right abdomen was histologically diagnosed as mild LS. The patient was started on nivolumab, and the right abdomen site developed into a large purpuric plaque with numerous roughened papules. Histologic examination again confirmed features of LS. Improvement was achieved with pulse oral steroids. This case demonstrates a flare of extragenital LS in a patient commenced on nivolumab. The etiology of LS is not fully known, but increasing evidence suggests that it is a T cellmediated disorder. It is associated with other autoimmune conditions, such as viligo and thyroid disease, which are also IRAEs. This case highlights a possible major role of PD-1 in the inhibition of self-reactive T cells. In reporting this association, we expand the spectrum of PD1 inhibitor therapy-related dermatoses. LS should be considered on the differential for IRAEs of the skin as it responds well to standard treatment.
m6A mRNA demethylase FTO regulates tumorigenicity and response to anti-PD-1 immunotherapy in melanoma S Yang1, J Wei2, Y Cui1, P Shah1, Y Deng1, A Aplin3, Z Lu2, C He2 and Y He1 1 University of Chicago, Chicago, Illinois, United States, 2 University of Chicago, Chicago, Illinois, United States and 3 Thomas Jefferson University, Philadelphia, Pennsylvania, United States Analogous to epigenetic regulation of gene expression through the reversible DNA and histone modifications, post-transcriptional N6-methyladenosine (m6A) methylation of adenosine residues in mRNA as well as lncRNA provides an additional layer of regulation that may alter mRNA metabolism and gene expression. Recently, m6A RNA methylation research was revived by the discovery of the fat mass- and obesity-associated protein (FTO) as the first RNA demethylase, implying that m6A RNA methylation is a reversible and dynamic modification and may have critical biological functions. However the role of FTO in melanoma tumorigenicity remains unknown. Here we show that N6-methyladenosine (m6A) mRNA demethylation by FTO promotes melanoma growth and inhibits response to anti-PD-1 blockade therapy and cell killing induced by interferon gamma (IFNg). FTO is up-regulated in human melanoma, suggesting an oncogenic role of FTO in melanoma. FTO knockdown inhibited melanoma cell proliferation, migration, and invasion in vitro, which is reversed by knockdown of the m6A methyltransferases METTL3 and METTL14. FTO knockdown inhibited melanoma tumor growth in vivoin both xenograft and syngeneic melanoma mouse models. FTO is up-regulated by metabolic stress through the autophagy/NF-kB pathway. m6A immunoprecipitation sequencing (m6A-seq) and RNA-seq analyses showed that FTO knockdown increases m6A modifications in the critical melanoma-promoting genes including PDCD1, CXCR4 and SOX10, leading to increased RNA decay through the m6A reader YTHDF2. Our findings demonstrate a key role of FTO as a RNA demethylase in promoting melanoma growth, and suggest that the combination of FTO inhibition with targeted therapy or immunotherapies may improve therapeutic response.
www.jidonline.org S141
816
Genetic alterations in primary melanoma in Taiwan C Chu1, Y Sheen1, K Tan2 and K Tse2 1 Department of Dermatology, National Taiwan University Hospital, Taipei, Taiwan and 2 ACT Genomics, Taipei, Taiwan Acral melanoma is the most common subtype of melanoma in Asians. However, differences in genetic alterations between acral melanoma and cutaneous melanoma in Asians are not well studied. To augment the understanding of the prevalence, patterns, and associations of mutations between different subtypes of melanoma, we performed next-generation sequencing of coding regions in 409 cancer-associated genes of 66 primary melanomas comprised of 45 acral melanomas and 21 cutaneous melanomas. The most common mutation in cutaneous melanoma was BRAF (66.7%), while in acral melanoma, KIT (24.4%), KRAS (15.6%), and NRAS (13.3%) were the most common mutations. Compared with acral melanoma, the mutation frequency of BRAF in cutaneous melanoma is significantly higher. Meanwhile, frequencies of NRAS/KRAS mutations, altered cell cycle regulation, BIRC2/3/5 gain, and amplification of receptor tyrosine kinase genes were significantly enriched in acral melanomas. Of note, cell cycle dysregulation and BIRC2/3/5 gain were significantly associated with poor melanoma-specific survival in all melanoma patients and in acral melanomas. The multivariate analysis showed that lymph node metastasis and cell cycle-related alterations are independent prognostic factor for melanoma-specific survival. This study strengthens our understanding of the patterns and clinical associations of oncogenic mutations in melanomas in Taiwan.
Autophagy inhibition sensitizes targeted therapy-resistant melanoma to MEK1/2 inhibitors A Truong1,2, C Kinsey2, M Foth2, M Scherzer1,2, J Sanchez3 and M McMahon2,4 1 Department of Oncological Sciences, University of Utah, Salt Lake City, Utah, United States, 2 Huntsman Cancer Institute, Salt Lake City, Utah, United States, 3 Department of Pathology, University of Utah, Salt Lake City, Utah, United States and 4 Department of Dermatology, University of Utah, Salt Lake City, Utah, United States Targeted therapy has dramatically improved the treatment landscape for metastatic melanoma, however is limited to those with BRAF(V600) mutations. GNAQ/GNA11 mutations are found in approximately 2% of melanoma, including 80% of uveal melanoma. Mutations in these G-alpha proteins lead to constitutive activation of multiple oncogenic pathways, including MAPK (RAF–>MEK1/2–>ERK1/2) signaling. Unfortunately, metastatic uveal melanoma is refractory to pharmacologic targeted therapies inhibiting MEK1/2, including trametinib and binimetinib. We showed that MEK1/2 inhibition increased autophagic flux in GNAQ/GNA11 mutated melanoma cell lines. The addition of autophagy inhibitors, such as chloroquine, resulted in synergistic cytotoxicity (Loewe model), demonstrating the cytoprotective role of autophagy as a result of MEK1/2 inhibitor treatment. Furthermore, combining trametinib with chloroquine or hydroxychloroquine resulted in a rapid and profound decrease in tumor burden in vivo compared to either treatment as monotherapy. This was also demonstrated in patient derived xenografts of NRAS-mutated cutaneous melanomas, a more common genetic subset of melanoma in which targeted therapy is also ineffective. Additionally, we showed that BRAF-mutated melanoma cell lines with genetically engineered or drug selected resistance to BRAF and MEK1/2 inhibitors displayed increased baseline autophagic flux, regardless of the presence of drug. The addition of autophagy inhibitors sensitized these resistant cells to BRAF and MEK1/2 inhibition. Our findings suggest a novel and potentially effective treatment strategy for melanomas with innate or acquired resistance to MEK1/2 inhibition.
817
818
Ethanol induces skin hyperpigmentation in mice with aldehyde dehydrogenase 2 deficiency T Yamauchi1, A Matsumoto2, S Ito3, K Wakamatsu3, T Suzuki4 and M Fujita1 1 Dermatology, University of Colorado Denver, Aurora, Colorado, United States, 2 Saga University School of Medicine, Saga, Japan, 3 Fujita Health University School of Health Sciences, Aichi, Japan and 4 Yamagata University, Yamagata, Japan Excessive alcohol consumption leads to alcohol use disorder. Alcohol is metabolized to acetaldehyde, which is then oxidized to acetic acid by aldehyde dehydrogenases (ALDH), a class of enzymes that facilitate the conversion of aldehydes to their corresponding acids. Among ALDHs, mitochondrial ALDH2 is the primary enzyme involved in the metabolism of acetaldehyde. In addition to its well-known role in ethanol metabolism, recent studies have suggested that ALDH2 dysfunction is associated with a variety of human diseases including cardiovascular diseases, diabetes, neurodegenerative diseases, stroke, cancer, anemia, pain, osteoporosis and aging. However, the role of ALDH2 has not been well investigated in the skin. In this study, we studied the effect of the functional defect of Aldh2 using Aldh2 KO mice. Ten-week-old Aldh2 KO mice and control C57BL/6 (WT) mice were fed with a standard hard diet and bottled water with ethanol for 10 weeks. A dose-dependent skin pigmentation was observed in the ears, feet, tail and genital area in Aldh2 KO mice but not WT mice (p ¼ 0.08e0.95; p interaction <0.001). The pigmentation was mainly localized in the epidermis and partially in the dermis, and found to be melanin. Analysis of degradation products of melanin demonstrated ethanol dose-dependent production of eumelanin in the mouse skin. Immunohistochemical analysis showed ethanol-induced melanocyte proliferation in Aldh2 KO mice. Acetaldehyde exposure occurs not only by ethanol consumption but also by a volatile organic compound, smoking and food products. ALDH2 enzyme is inactivated by post-translational modification under increased oxidative/nitrosative stress, suggesting the role of ALDH2 in acetaldehyde-induced skin even in individuals without a defective polymorphism of ALDH2.
Oriented analysis for transposon mutagenesis screening to study drug resistance J Hong1,2, T Lee2, W Wee2 and Y Chen1 1 Graduate Institute of Medical Genomics and Proteomics, Taipei, Taiwan and 2 Department of Dermatology, National Taiwan University Hospital, Taipei, Taiwan Several high-throughput screenings have been performed to study drug resistance. In comparison to the reverse genetic screening, the forward screening by transposon insertional mutagenesis does not need a priori target panel. We have performed piggyBac transposon mutagenesis screening by hyperactive transposase and pGG134 transposon carrying MSCV promoter in SK-MEL-28 melanoma cell line for resistant factor of the BRAF inhibitor, encorafenib. This study aimed to develop an appropriate computational analysis to identify the candidate genes. After sequencing the splinkerette PCR products, the original baseline mutagenesis library showed insertions distributed across all chromosomes. The first analysis for common insertion sites was done by Kernel Convolved Rule-Based Mapping. The results of the baseline library and the encorafenib selection group both predominantly marked by large genes, including DMD, PTPRD, and MACROD2. There were still over 2000 transposon insertion sites identified in the encorafenib group, which suggested no pure single mutagenic clone after drug selection. Considering the orientation-specific insertion by pGG134, the trapped genes were then filtered by sense/anti-sense insertion ratio. The filtered results in the encorafenib group showed that MITF had the highest insertion number, followed by USP47, SYCP2L, TNRC6C and MAP3K4. By comparing the baseline library, MITF, USP47 and MAP3K4 had obviously increased sense insertions after encorafenib selection. Overexpression of MAP3K4 was proved to offer resistance to BRAF inhibitor in the sensitive melanoma cells. Overall, the results of our transposon screening revealed that common insertion sites alone may not be sufficient to identify the candidate genes for drug resistance. Orientated analysis by filtering the sense insertional mutation and comparing the baseline mutagenesis library should be performed.
819
820
Extragenital lichen sclerosus is an immune-related adverse event with nivolumab E Williams1, B Mackool2 and R Nazarian1 1 Pathology, Massachusetts General Hospital, Boston, Massachusetts, United States and 2 Dermatology, Massachusetts General Hospital, Boston, Massachusetts, United States Nivolumab is a programmed cell death (PD)-1 ligand inhibitor used in treatment of melanoma and other malignancies. Immune-related adverse events (IRAEs) affect nearly all organ systems, most commonly the skin, the gastrointestinal tract (enterocolitis), the liver (hepatitis), and the endocrine system. Skin reactions related to immunotherapy occur in 4-27% of patients, including lichenoid mucocutaneous eruptions, vitiligo, psoriasis, lichen planus, and bullous pemphigoid. Most recently, genital lichen sclerosus (LS) and relapse of morphea have been reported. We seek to better describe this category of IRAE, and describe a patient with metastatic melanoma treated with nivolumab, who developed exacerbation of an existing plaque of extragenital LS. A 78-year-old white woman was diagnosed with metastatic melanoma of unknown primary following excisional biopsy of one of two deep masses in her right lower extremity. Mutational analysis revealed that the patient was NRAS mutant and BRAF wildtype. A year prior, an oval white plaque on her right abdomen was histologically diagnosed as mild LS. The patient was started on nivolumab, and the right abdomen site developed into a large purpuric plaque with numerous roughened papules. Histologic examination again confirmed features of LS. Improvement was achieved with pulse oral steroids. This case demonstrates a flare of extragenital LS in a patient commenced on nivolumab. The etiology of LS is not fully known, but increasing evidence suggests that it is a T cellmediated disorder. It is associated with other autoimmune conditions, such as viligo and thyroid disease, which are also IRAEs. This case highlights a possible major role of PD-1 in the inhibition of self-reactive T cells. In reporting this association, we expand the spectrum of PD1 inhibitor therapy-related dermatoses. LS should be considered on the differential for IRAEs of the skin as it responds well to standard treatment.
m6A mRNA demethylase FTO regulates tumorigenicity and response to anti-PD-1 immunotherapy in melanoma S Yang1, J Wei2, Y Cui1, P Shah1, Y Deng1, A Aplin3, Z Lu2, C He2 and Y He1 1 University of Chicago, Chicago, Illinois, United States, 2 University of Chicago, Chicago, Illinois, United States and 3 Thomas Jefferson University, Philadelphia, Pennsylvania, United States Analogous to epigenetic regulation of gene expression through the reversible DNA and histone modifications, post-transcriptional N6-methyladenosine (m6A) methylation of adenosine residues in mRNA as well as lncRNA provides an additional layer of regulation that may alter mRNA metabolism and gene expression. Recently, m6A RNA methylation research was revived by the discovery of the fat mass- and obesity-associated protein (FTO) as the first RNA demethylase, implying that m6A RNA methylation is a reversible and dynamic modification and may have critical biological functions. However the role of FTO in melanoma tumorigenicity remains unknown. Here we show that N6-methyladenosine (m6A) mRNA demethylation by FTO promotes melanoma growth and inhibits response to anti-PD-1 blockade therapy and cell killing induced by interferon gamma (IFNg). FTO is up-regulated in human melanoma, suggesting an oncogenic role of FTO in melanoma. FTO knockdown inhibited melanoma cell proliferation, migration, and invasion in vitro, which is reversed by knockdown of the m6A methyltransferases METTL3 and METTL14. FTO knockdown inhibited melanoma tumor growth in vivoin both xenograft and syngeneic melanoma mouse models. FTO is up-regulated by metabolic stress through the autophagy/NF-kB pathway. m6A immunoprecipitation sequencing (m6A-seq) and RNA-seq analyses showed that FTO knockdown increases m6A modifications in the critical melanoma-promoting genes including PDCD1, CXCR4 and SOX10, leading to increased RNA decay through the m6A reader YTHDF2. Our findings demonstrate a key role of FTO as a RNA demethylase in promoting melanoma growth, and suggest that the combination of FTO inhibition with targeted therapy or immunotherapies may improve therapeutic response.
www.jidonline.org S141