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Targeted sequencing identifies many potentially actionable mutations in melanomas
1270
1323
b-arrestins as key regulators of response to p53 reactivation drugs in melanoma Ada Girnita, MD, PhD, Dermatology Department and Cancer Centrum Karolinska, Stockholm, Sweden; Claire Worall, PhD, Cancer Centrum Karolinska, Stockholm, Sweden; Naida Suleymanova, MD, Cancer Centrum Karolinska, Stockholm, Sweden; Caitrin Crudden, MMSc, Cancer Centrum Karolinska, Stockholm, Sweden; Leonard Girnita, MD, PhD, Cancer Centrum Karolinska, Stockholm, Sweden Background: Malignant melanoma represents a major clinical challenge as shown by the steadily rising incidence in Sweden in the past 20 years. This is due to the modern intense sun exposure and new holiday habits with trips when the unprotected skin is exposed to high amounts of UV radiation from sun or tanning beds. Although the UV exposure is accepted as etiologic factor, its molecular signature, p53 mutation, is rarely found in suggesting there is another mechanism of p53 pathway inactivation. In addition, several tyrosine kinase receptors, including IGF-1R pathway, are shown to play an essential role in melanoma progression and metastasis. The IGF-1R and p53 pathways are cross-regulated and share the same ubiquitin ligase, Mdm2. Downregulation of p53 redirects Mdm2 towards IGF-1R. IGF-1R ubiquitination is controlled by b-arrestin which act as an adaptor protein for Mdm2. In both p53 mutant and p53 wild type melanoma cells disruption of p53Mdm2 complex increased cell death. Purpose: In this study we investigated the effect of p53 reactivation on MM cell proliferation as related to IGF-1R expression and signaling.
Targeted sequencing identifies many potentially actionable mutations in melanomas Nathaniel Slater, University of North Carolina School of Medicine, Chapel Hill, NC, United States; Edith Bowers, MD, PhD, University of North Carolina School of Medicine, Chapel Hill, NC, United States; Joel Parker, PhD, University of North Carolina School of Medicine, Chapel Hill, NC, United States; Nirali Patel, MD, University of North Carolina School of Medicine, Chapel Hill, NC, United States; David Eberhard, MD, PhD, University of North Carolina School of Medicine, Chapel Hill, NC, United States; Shelton Earp, MD, University of North Carolina School of Medicine, Chapel Hill, NC, United States; David Ollila, MD, University of North Carolina School of Medicine, Chapel Hill, NC, United States; Stergios Moschos, MD, University of North Carolina School of Medicine, Chapel Hill, NC, United States; Neil Hayes, MD, MPH, University of North Carolina School of Medicine, Chapel Hill, NC, United States; Nancy Thomas, MD, PhD, University of North Carolina School of Medicine, Chapel Hill, NC, United States
Methods: We analyzed the functional effects of p53 reactivation on IGF-1R expression and function in different melanoma cell lines. By modulating levels of b-arrestin we investigated the mechanisms controlling the Mdm2/IGF-1R interaction following disruption of the p53/Mdm2 loop. Results: p53 reactivation by either downregulation of its natural inhibitor Mdm2 or Nutlin-3 treatment causes a decrease in MM cell viability. Yet, the decreased cell viability is higher following p53 reactivation through Nutlin-3 treatment thus suggesting p53-independent cell death. Nutlin-3 treatment decreases expression of the IGF-1R in a ligand dependent manner, indicating this pathway is accountable for MM cell death. Moreover, b-arrestin overexpression enhances the effects of Nutlin3 on IGF-1R downregulation decreasing cell viability while b-arrestin inhibition reverses these effects. Taken together these results confirm the mechanism of Mdm2-b-arrestin 1 dependent IGF-1R degradation following disruption of the p53/Mdm2 interaction. Conclusion: This study highlights the complex bidirectional interplay between the IGF and p53 signaling pathways: a control feedback mechanism in normal cells is converted into a feed-forward mechanism in cancer cells. Interplay between p53 and IGF-1R directed by b-arrestins has important implications for therapy targeting either pathway. Commercial support: None identified.
Melanoma, a genetically heterogeneous malignancy, has among the highest rates of somatic mutation. Next generation sequencing (NGS) holds the potential to assist in tailoring therapy for advanced melanomas based on clinically actionable protein alterations. This study retrospectively reviewed the mutational profiles and clinicopathologic characteristics of 24 patients with late stage cutaneous melanomas whose tumors were sequenced between March 2012 and November 2013. Genetic data were acquired using a high-throughput platform developed at the University of North Carolina, comprising a panel of 247 genes with clinical relevance to cancer. These include genes for which targeted drugs are approved or in trials, tumor suppressors with the potential to impact treatment decisions, and genes with prognostic significance. Samples harbored an average of 20.2 proteinaltering mutations (range 1e74), 88% of which were nonsynonymous coding variants. Variants included 98 alterations in 27 discrete genes for which targeted drug therapy is commercially available; of these, 23 are involved in a transmembrane receptor protein tyrosine kinase signaling pathway. Sixty-seven percent of patients had potentially actionable alterations other than BRAF, NRAS, or KIT. In addition to BRAF and NRAS, alterations in ROS1, NF1, and FGFR2 were most commonly found (present in 11, 8, and 7 patients, respectively). BRAF mutations outside of codon 600, undetected by conventional testing, were also found in 3 patients. As of August 2014, patients had received a mean of 1.9 systemic therapies, including combination BRAF and MEK inhibition, immunotherapies, and other chemotherapies. While BRAF and MEK inhibitors are the only small molecule pharmaceuticals approved in melanoma, at least 20 genes inform eligibility for clinical trials investigating drugs approved for other cancer types or experimental therapies. In the future, novel systemic therapies may benefit melanoma patients who are BRAF wildtype, BRAF inhibitor-resistant, unable to tolerate immunotherapy, or not surgical candidates. Next generation sequencing techniques are already being translated to the clinic in some centers, including our own. As the number of actionable targets continues to expand, rational combination therapy offers the hope of long term management of unresectable or metastatic melanoma. Targeted, individualized therapy will likely become increasingly important in the treatment of patients with advanced melanomas. Commercial support: None identified.
1518 Study of mutations of CDKN2A - MITF in patients with familial melanoma in Chilean population Luis Mondaca, MD, Pontificia Universidad Cat olica de Chile, Santiago, Chile; Montserrat Molg o, MD, Pontificia Universidad Cat olica de Chile, Santiago, Chile; Pablo Uribe, MD, PhD, Pontificia Universidad Cat olica de Chile, Santiago, Chile; Sergio Gonzalez, MD, Pontificia Universidad Cat olica de Chile, Santiago, Chile Purpose: The objective of this series of cases is to determine the clinical features and mutations of CDKN2A and MITF in Chilean patients who are bearers of familial melanoma (FM). Mutations in the CDKN2A gene, personal (sporadic melanoma, multiple primary melanoma) and family history of melanoma, are potent risk factors to develop cutaneous malignant melanoma (CMM). We studied two high susceptibility genes to develop MMC: CDKN2A tumor suppressor gene associated with FM, encodes two proteins: p16 (exons 1a, 2 and 3) and p14 (exons 1b, 2, 3) and MITF, whose mutations are associated with FM (in the absence of mutations in CDKN2A/CDK4 genes). Patients and methods: We studied 26 patients (21 families) with FM, we carry out evaluation clinicoepidemiologic and genetic study by PCR and sequencing DNA from leukocytes of peripheral blood of selected patients, we analyzed mutations in exon 1a, 2, 3 and allele specific - 34 G [ T 1a of CDKN2A and exon 9 of MITF. Results: Total patients studied, a 42.3 % had mutations in CDKN2A (11 patients), a 23% in those with family history of CMM and more than one melanoma. In exon 1a (34G [ T) 7 patients (4 families); in exon 2, three patients (2 families), in 1 patient mutations were found in intron 2 of CDKN2A, IVS2 +19A [ G; polymorphisms 540 C [ T and 500 C [ G of exon 3 in 12 patients (8 families); mutations in MITF or patients with more than one mutation was not found. In relation to skin types of the patients in the study, predominated skin types 2 and 3 (76% of the total), history of atypical nevi removed in 17 of 26 patients in the series (65%), count of greater than 50 nevi in 69% of patients, In addition, 10 patients of the total (38%) did not use sunscreen never before his first melanoma diagnosis, data that confirm clasical melanoma risk factors.
321 The correlation of skin color with dermoscopic nevus patterns in a Queensland high risk melanoma cohort Natalie Ong, MBBS, MPH, Dermatology Research Center, University of Queensland, School of Medicine, TRI, Brisbane, Australia; David Duffy, MBBS, PhDQIMR Berghofer Medical Research Institute, Brisbane, Australia; Katie Lee, Dermatology Research Center, University of Queensland, School of Medicine, TRI, Brisbane, Australia; Phil McClenahan, Dermatology Research Center, University of Queensland, School of Medicine, TRI, Brisbane, Australia; Kasturee Jagirdar, Dermatology Research Center, The University of Queensland, School of Medicine, TRI, Brisbane, Australia; Richard Sturm, PhD, Dermatology Research Center, The University of Queensland, School of Medicine, TRI, Brisbane, Australia; H. Peter Soyer, MD, Dermatology Research Center, The University of Queensland, School of Medicine, TRI, Brisbane, Australia
Conclusion: This study identified mutations in CDKN2A on Chilean population with familial melanoma, epidemiologic and phenotypic characteristics in FM, it can develop individual, family, preventive public health measures and increase knowledge about the genetic basis of CMM in Chile.
In the risk stratification process, the intrinsic link between the rising incidence of cutaneous melanoma and constitutive skin color is considered. There are established pigmentation and nevogenic gene polymorphisms responsible for the production of pheomelanin/eumelanin pigment, the tanning response, and increased risk of melanocytic nevi formation. The aim of this study was to examine the relationship between counts of nevi of the three major dermatoscopic nevi patterns (globular, reticular, and nonspecific) and the individual’s signature nevus pattern with constitutive and facultative skin color measured by skin reflectance on nonexposed and sun-exposed body sites. In this study, skin reflectance was determined using a handheld spectrophotometer, and a device for standard and dermatoscopic sequential imaging was utilised for total body photography and dermatoscopic image capture of nevi. Skin color and freckling were scored from total body images. Our results established significant differences between constitutive skin reflectance with signature nevus patterns (Kruskal Wallis test, P \.001). There were higher counts of nonspecific nevi as constitutional skin reflectance increased correlating with lighter skin color. This relationship was observed in accordance with MC1R genotype determining constitutional skin reflectance. There was also considerable variation between nevus pattern counts and freckling score (P \ .001). This study may aid in melanoma risk stratification, whilst exemplifying the influence of genetics on phenotype.
Commercial support: None identified.
Commercial support: None identified.
AB176
J AM ACAD DERMATOL
MAY 2015
1323
b-arrestins as key regulators of response to p53 reactivation drugs in melanoma Ada Girnita, MD, PhD, Dermatology Department and Cancer Centrum Karolinska, Stockholm, Sweden; Claire Worall, PhD, Cancer Centrum Karolinska, Stockholm, Sweden; Naida Suleymanova, MD, Cancer Centrum Karolinska, Stockholm, Sweden; Caitrin Crudden, MMSc, Cancer Centrum Karolinska, Stockholm, Sweden; Leonard Girnita, MD, PhD, Cancer Centrum Karolinska, Stockholm, Sweden Background: Malignant melanoma represents a major clinical challenge as shown by the steadily rising incidence in Sweden in the past 20 years. This is due to the modern intense sun exposure and new holiday habits with trips when the unprotected skin is exposed to high amounts of UV radiation from sun or tanning beds. Although the UV exposure is accepted as etiologic factor, its molecular signature, p53 mutation, is rarely found in suggesting there is another mechanism of p53 pathway inactivation. In addition, several tyrosine kinase receptors, including IGF-1R pathway, are shown to play an essential role in melanoma progression and metastasis. The IGF-1R and p53 pathways are cross-regulated and share the same ubiquitin ligase, Mdm2. Downregulation of p53 redirects Mdm2 towards IGF-1R. IGF-1R ubiquitination is controlled by b-arrestin which act as an adaptor protein for Mdm2. In both p53 mutant and p53 wild type melanoma cells disruption of p53Mdm2 complex increased cell death. Purpose: In this study we investigated the effect of p53 reactivation on MM cell proliferation as related to IGF-1R expression and signaling.
Targeted sequencing identifies many potentially actionable mutations in melanomas Nathaniel Slater, University of North Carolina School of Medicine, Chapel Hill, NC, United States; Edith Bowers, MD, PhD, University of North Carolina School of Medicine, Chapel Hill, NC, United States; Joel Parker, PhD, University of North Carolina School of Medicine, Chapel Hill, NC, United States; Nirali Patel, MD, University of North Carolina School of Medicine, Chapel Hill, NC, United States; David Eberhard, MD, PhD, University of North Carolina School of Medicine, Chapel Hill, NC, United States; Shelton Earp, MD, University of North Carolina School of Medicine, Chapel Hill, NC, United States; David Ollila, MD, University of North Carolina School of Medicine, Chapel Hill, NC, United States; Stergios Moschos, MD, University of North Carolina School of Medicine, Chapel Hill, NC, United States; Neil Hayes, MD, MPH, University of North Carolina School of Medicine, Chapel Hill, NC, United States; Nancy Thomas, MD, PhD, University of North Carolina School of Medicine, Chapel Hill, NC, United States
Methods: We analyzed the functional effects of p53 reactivation on IGF-1R expression and function in different melanoma cell lines. By modulating levels of b-arrestin we investigated the mechanisms controlling the Mdm2/IGF-1R interaction following disruption of the p53/Mdm2 loop. Results: p53 reactivation by either downregulation of its natural inhibitor Mdm2 or Nutlin-3 treatment causes a decrease in MM cell viability. Yet, the decreased cell viability is higher following p53 reactivation through Nutlin-3 treatment thus suggesting p53-independent cell death. Nutlin-3 treatment decreases expression of the IGF-1R in a ligand dependent manner, indicating this pathway is accountable for MM cell death. Moreover, b-arrestin overexpression enhances the effects of Nutlin3 on IGF-1R downregulation decreasing cell viability while b-arrestin inhibition reverses these effects. Taken together these results confirm the mechanism of Mdm2-b-arrestin 1 dependent IGF-1R degradation following disruption of the p53/Mdm2 interaction. Conclusion: This study highlights the complex bidirectional interplay between the IGF and p53 signaling pathways: a control feedback mechanism in normal cells is converted into a feed-forward mechanism in cancer cells. Interplay between p53 and IGF-1R directed by b-arrestins has important implications for therapy targeting either pathway. Commercial support: None identified.
Melanoma, a genetically heterogeneous malignancy, has among the highest rates of somatic mutation. Next generation sequencing (NGS) holds the potential to assist in tailoring therapy for advanced melanomas based on clinically actionable protein alterations. This study retrospectively reviewed the mutational profiles and clinicopathologic characteristics of 24 patients with late stage cutaneous melanomas whose tumors were sequenced between March 2012 and November 2013. Genetic data were acquired using a high-throughput platform developed at the University of North Carolina, comprising a panel of 247 genes with clinical relevance to cancer. These include genes for which targeted drugs are approved or in trials, tumor suppressors with the potential to impact treatment decisions, and genes with prognostic significance. Samples harbored an average of 20.2 proteinaltering mutations (range 1e74), 88% of which were nonsynonymous coding variants. Variants included 98 alterations in 27 discrete genes for which targeted drug therapy is commercially available; of these, 23 are involved in a transmembrane receptor protein tyrosine kinase signaling pathway. Sixty-seven percent of patients had potentially actionable alterations other than BRAF, NRAS, or KIT. In addition to BRAF and NRAS, alterations in ROS1, NF1, and FGFR2 were most commonly found (present in 11, 8, and 7 patients, respectively). BRAF mutations outside of codon 600, undetected by conventional testing, were also found in 3 patients. As of August 2014, patients had received a mean of 1.9 systemic therapies, including combination BRAF and MEK inhibition, immunotherapies, and other chemotherapies. While BRAF and MEK inhibitors are the only small molecule pharmaceuticals approved in melanoma, at least 20 genes inform eligibility for clinical trials investigating drugs approved for other cancer types or experimental therapies. In the future, novel systemic therapies may benefit melanoma patients who are BRAF wildtype, BRAF inhibitor-resistant, unable to tolerate immunotherapy, or not surgical candidates. Next generation sequencing techniques are already being translated to the clinic in some centers, including our own. As the number of actionable targets continues to expand, rational combination therapy offers the hope of long term management of unresectable or metastatic melanoma. Targeted, individualized therapy will likely become increasingly important in the treatment of patients with advanced melanomas. Commercial support: None identified.
1518 Study of mutations of CDKN2A - MITF in patients with familial melanoma in Chilean population Luis Mondaca, MD, Pontificia Universidad Cat olica de Chile, Santiago, Chile; Montserrat Molg o, MD, Pontificia Universidad Cat olica de Chile, Santiago, Chile; Pablo Uribe, MD, PhD, Pontificia Universidad Cat olica de Chile, Santiago, Chile; Sergio Gonzalez, MD, Pontificia Universidad Cat olica de Chile, Santiago, Chile Purpose: The objective of this series of cases is to determine the clinical features and mutations of CDKN2A and MITF in Chilean patients who are bearers of familial melanoma (FM). Mutations in the CDKN2A gene, personal (sporadic melanoma, multiple primary melanoma) and family history of melanoma, are potent risk factors to develop cutaneous malignant melanoma (CMM). We studied two high susceptibility genes to develop MMC: CDKN2A tumor suppressor gene associated with FM, encodes two proteins: p16 (exons 1a, 2 and 3) and p14 (exons 1b, 2, 3) and MITF, whose mutations are associated with FM (in the absence of mutations in CDKN2A/CDK4 genes). Patients and methods: We studied 26 patients (21 families) with FM, we carry out evaluation clinicoepidemiologic and genetic study by PCR and sequencing DNA from leukocytes of peripheral blood of selected patients, we analyzed mutations in exon 1a, 2, 3 and allele specific - 34 G [ T 1a of CDKN2A and exon 9 of MITF. Results: Total patients studied, a 42.3 % had mutations in CDKN2A (11 patients), a 23% in those with family history of CMM and more than one melanoma. In exon 1a (34G [ T) 7 patients (4 families); in exon 2, three patients (2 families), in 1 patient mutations were found in intron 2 of CDKN2A, IVS2 +19A [ G; polymorphisms 540 C [ T and 500 C [ G of exon 3 in 12 patients (8 families); mutations in MITF or patients with more than one mutation was not found. In relation to skin types of the patients in the study, predominated skin types 2 and 3 (76% of the total), history of atypical nevi removed in 17 of 26 patients in the series (65%), count of greater than 50 nevi in 69% of patients, In addition, 10 patients of the total (38%) did not use sunscreen never before his first melanoma diagnosis, data that confirm clasical melanoma risk factors.
321 The correlation of skin color with dermoscopic nevus patterns in a Queensland high risk melanoma cohort Natalie Ong, MBBS, MPH, Dermatology Research Center, University of Queensland, School of Medicine, TRI, Brisbane, Australia; David Duffy, MBBS, PhDQIMR Berghofer Medical Research Institute, Brisbane, Australia; Katie Lee, Dermatology Research Center, University of Queensland, School of Medicine, TRI, Brisbane, Australia; Phil McClenahan, Dermatology Research Center, University of Queensland, School of Medicine, TRI, Brisbane, Australia; Kasturee Jagirdar, Dermatology Research Center, The University of Queensland, School of Medicine, TRI, Brisbane, Australia; Richard Sturm, PhD, Dermatology Research Center, The University of Queensland, School of Medicine, TRI, Brisbane, Australia; H. Peter Soyer, MD, Dermatology Research Center, The University of Queensland, School of Medicine, TRI, Brisbane, Australia
Conclusion: This study identified mutations in CDKN2A on Chilean population with familial melanoma, epidemiologic and phenotypic characteristics in FM, it can develop individual, family, preventive public health measures and increase knowledge about the genetic basis of CMM in Chile.
In the risk stratification process, the intrinsic link between the rising incidence of cutaneous melanoma and constitutive skin color is considered. There are established pigmentation and nevogenic gene polymorphisms responsible for the production of pheomelanin/eumelanin pigment, the tanning response, and increased risk of melanocytic nevi formation. The aim of this study was to examine the relationship between counts of nevi of the three major dermatoscopic nevi patterns (globular, reticular, and nonspecific) and the individual’s signature nevus pattern with constitutive and facultative skin color measured by skin reflectance on nonexposed and sun-exposed body sites. In this study, skin reflectance was determined using a handheld spectrophotometer, and a device for standard and dermatoscopic sequential imaging was utilised for total body photography and dermatoscopic image capture of nevi. Skin color and freckling were scored from total body images. Our results established significant differences between constitutive skin reflectance with signature nevus patterns (Kruskal Wallis test, P \.001). There were higher counts of nonspecific nevi as constitutional skin reflectance increased correlating with lighter skin color. This relationship was observed in accordance with MC1R genotype determining constitutional skin reflectance. There was also considerable variation between nevus pattern counts and freckling score (P \ .001). This study may aid in melanoma risk stratification, whilst exemplifying the influence of genetics on phenotype.
Commercial support: None identified.
Commercial support: None identified.
AB176
J AM ACAD DERMATOL
MAY 2015