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Detection of Driver Mutations in FFPE Samples from Patients with Verified Malignant Melanoma

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Detection of Driver Mutations in FFPE Samples from Patients with Verified Malignant Melanoma Neoplasma 2019; 66(1): 33–38 33 doi:10.4149/neo_2018_180115N31 Detection of driver mutations in FFPE samples from patients with verified malignant melanoma B. MALICHEROVA1,2,#,*, T. BURJANIVOVA1,2,#, E. MINARIKOVA3, I. KASUBOVA1,2, T. PECOVA3, M. BOBROVSKA4, I. HOMOLA5, Z. LASABOVA1,2,6, L. PLANK1,2,4 1Biomedical Center Martin, Jessenius Faculty of Medicine in Martin, Commenius University in Bratislava (JFM CU), Slovakia; 2Division of Oncology JFM CU, Martin, Slovakia; 3Clinic of Dermatovenerology, Jessenius Faculty of Medicine and University Hospital in Martin, Martin, Slovakia; 4Department of Pathological Anatomy, Jessenius Faculty of Medicine and University Hospital in Martin, Martin, Slovakia; 5Depart- ment of Plastic Surgery, Jessenius Faculty of Medicine and University Hospital in Martin, Martin, Slovakia; 6Department of Molecular Biology, JFM CU, Martin Slovakia *Correspondence: [email protected] #Contributed equally to this work. Received January 15, 2018 / Accepted April 24, 2018 Malignant melanoma is an oncological disease characterized by etiologic heterogeneity and it has increasing incidence and mortality in the Slovak Republic. While it is treated surgically in combination with chemotherapy, targeted therapy, and immunotherapy, malignant melanomas can ulcerate and are susceptible to infections. These are highly aggressive cancers with metastasis, and recent studies have shown the presence of mutations in RAC1, PPP6C and STK19 genes in melanoma patients. Mutations in these genes are driver mutations; important in oncogenesis and providing selective advantage to tumor cells. The aim of our study is to establish a method to detect driver mutations in formalin-fixed, paraffin embedded (FFPE) tissue DNA. We applied Sanger sequencing to detect driver somatic mutations in RAC1, PPP6C, STK19 and BRAF genes in patients with malignant melanoma. Confirmation of BRAF V600E mutation was obtained by allele-specific PCR. The BRAF V600E mutation was present in 15 of 113 patients (13.2%) and the driver mutation in 7 of 113 patients (6.2 %). Our results demonstrate that Sanger sequencing analysis detects mutations in FFPE clinical samples. The identification of these somatic driver mutations in samples with verified malignant melanomas enabled development of a molecular classi- fication of melanomas, and our study provides evidence of diversity of novel driver mutations implicated in malignant melanoma pathogenesis. These findings could have very important implications for targeted therapy. Key words: malignant melanoma, genetic analysis, driver mutations, diagnostic biomarker The incidence of malignant melanomas has been melanoma is associated with constitutive activation of the increasing around the world, mainly in Caucasians. People mitogen-activated protein kinase pathway (MAPK). This is with red hair and fair skin have a higher chance of devel- a complex signaling pathway with RAS/RAF proteins and oping melanoma. In the Slovak Republic, although malig- other protein kinases including MEK and ERK. This pathway nant melanoma incidence is not high compared to other appears to be critical for the growth-phase of melanoma [2] malignant tumors, it has relatively high mortality, especially and it is estimated that almost half malignant melanomas are when detected in advanced stages of the disease. The key associated with a MAPK activation mutation [3, 4]. risk factor for its appearance is exposure to UV radiation, so A typical initiating oncogene involved in the MAPK tumor may be prevented in predisposed people by avoiding pathway is a BRAF gene mutation (B-Raf Proto-Oncogene, direct sunlight and use of protective agents Serine/Threonine Kinase). Over 60% of melanomas carry a Newer techniques of molecular biology improve the BRAF gene mutation, and 80% of these carry the BRAF V600E prognosis of the disease because these allow us to find genetic mutation [5, 6]. For patients with metastatic melanoma and changes in nucleic acids, increase the specificity of diagnostic BRAF V600E driver mutation, therapy targeting the mitogen- criteria and reduce time for patient diagnosis [1]. The exact activated protein kinase pathway – a combination of BRAF mechanism for initiation of tumorigenesis of malignant inhibitors and MEK inhibitors – is an important treatment 34 B. MALICHEROVA, T. BURJANIVOVA, E. MINARIKOVA, I. KASUBOVA, T. PECOVA, M. BOBROVSKA, I. HOMOLA, Z. LASABOVA, L. PLANK option [7]. The incidence of mutations in the BRAF and assembly of actin filaments at the leading edge of cells [14], NRAS genes is significantly increased in skin melanomas not and it has recently been described as a common mutation in chronically exposed to UV radiation [8–12]. cutaneous melanomas together with BRAF and NRAS; and it Other genes also have features which suggest a role in is the third most frequent hotspot gain-of-function mutation melanoma oncogenesis; three of these are protein phospha- in sun-exposed melanomas. RAC1 mutation is associ- tase 6 catalytic subunit (PPP6C), Ras-related C3 botulinum ated with UV damage [15] but a further newly discovered toxin substrate 1 (RAC1) and serine/threonine kinase 19 melanoma-associated gene is the PPP6C potential tumor (STK19). Mutations in STK19 (a predicted kinase with suppressor gene [12]. PPP6C encodes the catalytic subunit an unknown function) generally cluster around hot-spot of the PP6 serine/threonine phosphatase complex. This gene regions, and subsequent somatic hotspot point mutations has been reported to play an important role in several cancer- provide strong evidence that STK19 is a novel cancer gene related pathways, including de-phosphorylation of Aurora in melanoma [12]. Activating mutations in RAC1 gene have kinase A which regulates mitosis [16]. Results from both been identified in 4% of all melanomas [12] and in 9% of groups included R264C mutations in PPP6C, thus indicating sun-exposed melanomas [13]. a tumor suppression function for this protein in melanoma RAC1 is a member of Rho family of small GTPases which [13]. PPP6C mutations may result in the loss-of-function have an important role in the control of cell proliferation typical for a tumor suppressor, and while PPP6C mutations and cell migration. The RAC1 gene is associated with the occur in melanoma patient primary tumors and metastases, the cells with PPP6C mutations are sensitive to Aurora kinase inhibitors. This has therapeutic implications [16] because identification of mutations in cancer cells that drive malig- Table 1. Clinical-pathological data of the cohort of 113 malignant mela- nant transformation may provide targeted therapy that nomas, from the Department of Dermatovenerology, University Hospital improves overall survival [13]. in Martin, 62 men and 51 women, diagnosed in years 2015–2017. No. of patients Patients n=113(%) Patients and methods Men 62 (54.8%) Women 51 (45.1%) A total of 113 clinical samples of formalin-fixed, paraffin Average age embedded tissue (FFPE) with malignant melanomas were Men 63.9 (yr) included in this study; 85 were primary tumors and 28 were Women 64 (yr) metastatic infiltrated lymph nodes. Melanoma samples were Types of melanoma collected from 62 men and 51 women diagnosed in 2015–2017 Superficial spreading melanoma 48 (42.4%) (Table 1). Clinical-pathological information obtained from Nodular melanoma 34 (30.0%) the patients with malignant melanoma are presented in Acral lentiginous melanoma 6 (5.3%) Table 1 and Supplementary Table S1. All studied formalin- Lentigo malignant melanoma 4 (3.5%) fixed and paraffin embedded (FFPE) samples were collected Unspecified type of melanoma 9 (7.9%) from the archives of the Departments of Dermatovenerology Melanoma in situ 9 (7.9%) and Pathology, University Hospital in Martin. The clinical- Desmoplastic malignant melanoma 1 (0.88%) pathological data was extracted from the patient history. The Medium and late regression malignant melanoma 1 (0.88%) lesion skin surface was examined in detail by dermatoscope Stage (pT) status, Thickness of malignant melanoma and histological testing included basic immuno-histological Tx unidentified 3 (2.6%) examination (MelanA, HBM45, S100, VIM) [17]. Immuno- T1 0-1mm 17 (15.0%) histopathologic parameters including the extent of the tumor T2 1,1-2,0 mm 26 (23.0%) (pT) and lymph node (pN) and distant metastasis status (pM) T3 2,1-4,0 mm 45 (39.8%) were recorded (Table 1). Histological staging and grading T4 more than 4,1 mm 22 (19.4%) was performed according to the revised pTNM classification Lymph node metastases status (pN) of the American Joint Committee on Cancer (AJCC) and Nx 91(80.5%) the Union for International Cancer Control (UICC) [18]. N1 1lymph node 8 (7.1%) All participants enrolled in our study gave written informed N2 2-3lymph nodes 9 (7.9%) consent and the study was approved by the Ethics Committee N3 4 lymph nodesmetastases 5 (4.4%) of University Hospital in Martin. Distant metastases status (pM) DNA extraction. DNA from paraffin embedded tissue M1a Skin, subcutaneous tissue or 6 (5.3%) was isolated by the black PREP FFPE DNA kit following the lymph nodes beyond the regional LU manufacturer’s protocol. The optimized procedure included M1b Lung metastases the extra step of de-paraffinisation and DNA was measured M1c Other localization on NanoDrop™ 2000/2000C (Thermo Fisher Scientific, Mx 107 (94.6%) Germany). DRIVER MUTATIONS IN PATIENTS WITH MALIGNANT MELANOMA 35 Table 2. Primer sequence, annealing temperature and length of the products used in this study. Gene Primer Sequence Annealing temperature
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