Available online at www.annclinlabsci.org Annals of Clinical & Laboratory Science, vol. 47, no. 5, 2017 523 Role of CDKN2C Fluorescence In Situ Hybridization in the Management of Medullary Thyroid Carcinoma Maha El Naofal1, Adriel Kim1, Hui Yi Yon1, Mohamed Baity1, Zhao Ming2, Jacquelin Bui-Griffith5, Zhenya Tang4, Melissa Robinson4, Elizabeth G. Grubbs5, Gilbert J. Cote3, and Peter Hu1 1School of Health Professions Program in Diagnostic Genetics, 2Program in Cytogenetics, 3Department of Endocrine Neoplasia and Hormonal Disorders, 4Department of Clinical Cytogenetics, and 5Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Abstract. Medullary thyroid carcinoma (MTC), an aggressive form of thyroid cancer, occurs sporadically in approximately 75% of MTCs. RET and RAS mutations play a role in about 40% and 15%, respectively, of sporadic MTCs and are predominant drivers in MTC pathways. These mutations are some of the most comprehensively described and screened for in MTC patients; however, in recent studies, other mutations in the CDKN2C gene (p18) have been implicated in the tumorigenesis of MTC. Comparative genomic hybridization analysis revealed that approximately 40% of sporadic MTC samples have loss of CDKN2C at chromosome 1p32 in addition to frequent losses of CDKN2D (p19) at chromosome 19p13. However, no feasible routine method had been established to detect loss of heterozygosity (LOH) of CDKN2C and CD- KN2D. The aim of this study is to assess the feasibility of using Fluorescence in situ Hybridization (FISH) to screen MTC patients for CDKN2C and CDKN2D deletions. We subjected 5 formalin-fixed, paraffin- embedded (FFPE) MTC samples with defined RET/RAS mutations to dual-color FISH assays to detect loss of CDKN2C and/or CDKN2D. We prepared spectrum orange probes using the bacterial artificial chro- mosomes RP11-779F9 for CDKN2C (p18) and RP11-177J4 for CDKN2D (p19) and prepared spectrum green control probes to the 1q25.2 and 19q11 regions (RP11-1146A3 and RP11-942P7, respectively). Nine FFPE normal thyroid tissue samples were used to establish the cutoff values for the FISH signal pat- terns. Of the five FFPE MTC samples, four and one yielded a positive significant result for CDKNN2C loss and CDKN2D loss, respectively. The results of a Clinical Laboratory Improvement Amendments validation with a CDKN2C/CKS1B probe set for CDKN2C (p18) loss of heterozygosity were 100% concordant with the FISH results obtained in this study. Thus, FISH is a fast and reliable diagnostic or prognostic indicator of gene loss in MTC. Key words: MTC, RET, RAS, FISH. Introduction of RET, causing constitutive activation of the pro- to-oncogene, were found to be responsible for the Medullary thyroid carcinoma (MTC), which arises disease in 1993 [4-6]. Underscoring the importance from the calcitonin-producing parafollicular C cells of RET in MTC, the U.S. Food and Drug of the thyroid [1], accounts for 5–10% of all thy- Administration recently approved the use of the roid cancers. Hereditary, MEN2-associated MTC molecular targeted therapies Vandetanib and accounts for approximately 25% of MTC cases, Cabozantinib, both of which inhibit the action of whereas sporadic MTC accounts for 75% of cases RET [7], for the treatment of MTC. Although [2,3]. The predisposition to developing hereditary RET mutations are the major drivers of sporadic MTC was mapped to chromosome 10 by genetic MTC, they occur in only about 40% of sporadic linkage analysis in 1987; and germline mutations MTCs [8]. In addition, mutations in RAS had been implicated in approximately 10-15% of sporadic Address correspondence to Peter Hu, PhD., FACSc; Diagnostic cases, labeling it as a second driver of MTC [9]. Genetics Program, Unit 2, The University of Texas MD Anderson These mutations, even though they are predomi- Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA: phone: 713 563 3095; fax: 713 745 3337; e mail: pchu@mdanderson. nant drivers of MTCs, still do not account for all org cases of sporadic MTC and its tumorigenesis. 0091-7370/17/0500-523. © 2017 by the Association of Clinical Scientists, Inc. 524 Annals of Clinical & Laboratory Science, vol. 47, no. 5, 2017 Figure 1. We searched the University of California Santa Cruz Genome Browser to identify BAC clones. The BAC clone RP11-779F9 spans CDKN2C on chromosome 1p; the control BAC clone RP11-1146A3 is on the q-arm. The BAC clone RP11-177J4 spans CDKN2D on chromosome 19p; the control BAC clone RP11-932P7 is on the q-arm. To date, no other major drivers of C-cell oncogen- in MTC implicates the p19 gene, CDKN2D, esis have been identified. An array-based compara- though the exact nature of that role remains unclear tive genomic hybridization study first performed [10,17]. Those genes involved in the CDKN path- by Ye et al. revealed many copy number alterations way, such as CDKN2C and CDKN2D, loss of het- in MTC, indicating that C-cell oncogenesis may be erozygosity (LOH) suggests that MTC patients driven by copy number alterations rather than gene with RET/RAS mutations have additional molecu- mutations [10]. Other studies found increased lar abnormalities. This is supported by a more de- MTC risk to be associated with cell cycle genes, tailed analysis of available array comparative ge- including those in the cyclin D–Cdk4-6/INK4/ nomic hybridization studies [10,17]. In the MD Rb/E2F pathway, which integrates mitogenic and Anderson cancer center data set, which included 20 antimitogenic stimuli to control cell growth sporadic MTCs, 14 (70%) had LOH in regions [11,12]. Cyclin-dependent kinase inhibitor containing either CDKN2C or CDKN2D. (CDKN) family members, which include p15, Furthermore, an independent analysis of 41 spo- p16, p18, and p19, play a crucial role in preventing radic MTC tumors revealed loss of heterozygosity inappropriate cell division by binding to CDK4 or (LOH) in CDKN2C, at chromosome 1p32 and CDK6 and inhibiting the action of cyclin D, lead- CDKN2D, at chromosome 19p13 for 39% and ing to G1 cycle arrest. Frequently mutated or de- 24% of cases, respectively [17]. These findings sug- leted in a wide variety of tumors, CDKN genes gest that CDKN2C and CDKN2D loss need to be increase RB phosphorylation, which in turn dis- routinely screened for in patients with sporadic rupts the inhibitory RB-E2F interaction. The free MTCs and eventually correlated with clinical out- E2F transcribes genes and then drives the cell comes to provide a more rational approach to se- through S phase, suggesting that the CDKN family lecting individualized therapies targeting CDK in- members have important functions as tumor sup- hibition in MTC of which current research on pressors [13]. CDK4 inhibitors for various cancer therapies is ongoing [18-20]. Loss of the p18 gene, CDKN2C, has been associ- ated with RET-mediated MTC [14-17]. Even though CDKN2C and CDKN2D LOH had Additionally, the frequent loss of the 19p13 region been shown to be highly implicated in sporadic CDKN2C/CKS1B FISH for Medullary Thyroid Carcinoma 525 Figure 2. Validation of the homebrew probes for the CDKN2C and CDKN2D genes and their respec- tive control probes. (A) The probe for CDKN2C, lo- cated at 1p32, is shown in the 1p region (red), and its control probe is shown in the 1q region (green). (B) The probe for CDKN2D, lo- cated at 19p13, is shown in the 19p region (red), and the control probe is shown in the 19q region (green). MTC cases, no feasible routine method had been RP11-177J4 (for CDKN2D), along with their respec- established to assess for those losses. The aim of the tive controls RP11-1146A3 (located on 1q25.2) and present study is to assess the feasibility of using RP11-942P7 (located on 19q11) from http://bacpac. fluorescence in situ hybridization (FISH) to screen chori.org/. Bacterial stocks were plated and cultured MTC patients for CDKN2C and CDKN2D overnight at 37°C and a single colony was then inocu- lated in 25 ml of Luria-Bertani broth with 25 µg/ml deletions. chloramphenicol. The culture was placed in a controlled environment incubator shaker set to 260 rpm at 37°C Materials and Methods for 8-12 hours. DNA extraction was then performed us- ing the Purelink HiPure Plasmid Miniprep kit Probe Design and Preparation. In FISH, the probes (Invitrogen) according to the manufacturer’s instruc- are fluorescently labeled single stranded DNA that are tions. A Vysis Nick Translation kit (Abbott Laboratories, complementary to the targeted genomic sequence. In Abbott Park, IL) was used according to the manufactur- order to attain those DNA fragments complementary to er’s instructions to directly label BAC DNA with our regions of interest, we searched the University of SpectrumOrangefortargetedregionsandSpectrumGreen California Santa Cruz Genome Browser (http://genome. for control regions. Working solutions for each of the ucsc.edu/cgi-bin/hgGateway) to identify bacterial artifi- BAC probes were hybridized on blood slides for valida- cial chromosome (BAC) clones (Figure 1). We ordered tion. The validation included hybridizing the probes on the BAC clones RP11-779F9 (for CDKN2C) and blood slide metaphases followed by reverse DAPI 526 Annals of Clinical & Laboratory Science, vol. 47, no. 5, 2017 Figure 3. Normal thyroid tissue showed an overall normal FISH 2R2G signal pattern. MTC tissue showed an abnormal FISH 1R2G signal pattern, indicating a deletion in the 1p32 region. through Cytovision AI imaging system to confirm the The probes were initially denatured at 75°C for 10 proper position of the probes on chromosome 1 and 19 minutes to improve their hybridization. The CDKN2C with the absence of non-specific or specific hybridization and CDKN2D probes with their respective controls on other chromosomes’ regions. were added to the pretreated FFPE slides. The slides were coverslipped, allowing the probes to disperse, and Pretreatment of Formalin-Fixed, Paraffin-Embedded then sealed with rubber cement.