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 (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 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 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 genes, tailed analysis of available array comparative ge- including those in the D–Cdk4-6/INK4/ nomic hybridization studies [10,17]. In the MD Rb/ 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 inhibitor containing either CDKN2C or CDKN2D. (CDKN) family members, which include p15, Furthermore, an independent analysis of 41 spo- , p18, and p19, play a crucial role in preventing radic MTC tumors revealed loss of heterozygosity inappropriate by binding to CDK4 or (LOH) in CDKN2C, at chromosome 1p32 and CDK6 and inhibiting the action of , 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 , 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 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 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 ’ 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. Probe and slide are Tissues. Nine slides of formalin-fixed, paraffin-embedded co-denatured at 85°C for 10 minutes and hybridiza- (FFPE) normal thyroid tissue and five slides of FFPE MTC tion at 37°C for 24–48 hours in a Thermobrite ma- with defined RET/RAS mutations derived from patients chine. The slides were washed in 2xSSC for 2 minutes who were treated at The University of Texas MD Anderson each followed by washes in 2x SSC/0.025% Tween at Cancer Center were randomly selected. All cases were de- 65°C for 5-10 minutes and 2SSC/0.025% Tween at rived from patients who were treated at The University of room temperature for 2 minutes. The slides were sub- Texas MD Anderson Cancer Center. Pathologically con- jected to washes of 2xSSC and dehydrated via serial firmed normal and medullary thyroid carcinoma tumor up-graded washes of ethanol (70%, 95%, and 100%). tissue was obtained with the approval of The University of The slides were air-dried subjected to serial CitriSolv Texas MD Anderson Cancer Center’s Institutional Review washes and then subjected to two 100% ethanol wash- Board. The slides were initially deparaffinized by placing es for 5 minutes each step. Approximately 20 µl of them in the oven at 65°C overnight and then washing room-temperature DAPI was applied to the hybridiza- them in a series of CitriSolv. The slides were then pretreat- tion area. The slides were cover slipped and stored in ed with the Spot-LightTissue Pretreatment Kit (Invitrogen). darkness for 20 minutes at 4°C and then examined Briefly, the slides were dehydrated by serial washes of 100% under a fluorescence microscope. ethanol and then placed in pretreatment solution for 33 minutes at 95–98°C. The slides were then washed with FISH in CLIA Laboratory. For the cytogenetic buffer (2x saline sodium citrate [SSC]/0.025% Tween 20) Clinical Laboratory Improvement Amendments several times and dehydrated with serial up-graded washes (CLIA) test, the commercial probe set developed by of ethanol (50%, 70%, 90%, and 100%). Tumor borders Vysis for detecting CDKN2C loss in multiple myelo- were delineated on each slide by using a DAKO cytoma- ma was utilized to expand the FISH analysis in clinical tion pen. The slides were then incubated with the settings [21]. We used the CDKN2C/CKS1B probe reagent for 30 minutes at 37°C, washed several times with set, which consists of a red-labeled 180-kbp probe that buffer, and finally subjected to serial up-graded washes of spans the entire CKS1B gene and a green-labeled 168- ethanol. kbp probe that spans the entire CDKN2C gene. The initial validation determines its clinical sensitivity and Fluorescence in Situ Hybridization. CDKN2C and/or specificity as well as gathering the normal reference CDKN2D loss were detected by a dual-color FISH assay. range and cut off for its future applicability in the clinical setting. CDKN2C/CKS1B FISH for Medullary Thyroid Carcinoma 527

Results Table 1. Cutoff values for the CDKN2C and CDKN2D genes for the 1R2G and 1R1G fluorescence in situ hybrid- ization signal patterns. In a validation test with metaphases from human blood, developed homebrew FISH probes for CDKN2C and CDKN2D was hybridized at chro- Gene 1R2G (%) 1R1G (%) mosome 1p32 and chromosome 19p13, respec- CDKN2C 10.0 12.0 tively (Figure 2). Among nine selected normal CDKN2D 9.5 10.5 thyroid FFPE samples, all had a normal 2R2G FISH signal pattern (Figure 3). The cutoff values Table 2. Percentages of 1R2G and 1R1G fluorescence in obtained with the BETAINV statistical tool for situ hybridization signal patterns observed for genes and its different signal expression patterns are given in significance in relation to the cutoff shown in Table 1. Table 1. The CDKN2C gene had a cut off of 10% and 12 % for 1R2G and 1R1G signal pattern, re- Sample Gene 1R2G 1R1G Relation spectively. The CDKN2D gene, on the other hand, (%) (%) to cutoff value had a cut off of 9.5% and 10.5% for 1R2G and 1R1G signal pattern, respectively. Of the five 1 CDKN2C 48.5 13.5 + FFPE MTC samples with defined RET/RAS muta- CDKN2D 10.0 14.0 + tions, four and one yielded a positive significant 2 CDKN2C 6.0 9.5 - result for CDKN2C loss and CDKN2D loss, re- CDKN2D 8.0 10.0 - spectively (Table 2). The results of the cytogenetic 3 CDKN2C 13.5 10.5 + CLIA test were 100% concordant with our FISH CDKN2D 3.0 1.0 - results (Table 3). 4 CDKN2C 11.5 12.0 + CDKN2D 1.0 3.0 - Discussion 5 CDKN2C 55.0 10.0 + CDKN2D 9.0 7.0 - Previous studies have shown that CDKN2C loss is Table 3. Percentages of CDKN2C loss in 4 medullary thy- associated with RET-mediated MTC [14-16]. roid carcinoma samples according to research fluorescence Furthermore, the implication of CDKN2C and in situ hybridization (FISH) and Clinical Laboratory CDKN2D LOH in sporadic MTC was document- Improvement Amendments (CLIA) FISH. ed as well. Initial comparative genomic hybridiza- tion data indicated that 40% of MTC tumors have Sample CDKN2C loss by CDKN2C CDKN2C loss. Screening for CDKN2C loss with Research FISH (%) loss by CLIA other methodologies is not feasible; therefore, we FISH (%) surmised that FISH would be the best way to screen for LOH and that this screening could be 1 62 67 3 24 40 implemented into the clinical care of patients. In 4 24 20 this study, we used a dual-color FISH assay to ana- 5 65 78 lyze chromosome 1p32 for CDKN2C loss and 19p13 for CDKN2D loss.

Given the nature of the FFPE tissue sections used Statistical Analysis. Dual-color FISH data were deter- mined to be significant using a cutoff value established in the present study, we expected to encounter with the BETAINV statistical tool through Microsoft some challenges when performing FISH. The Excel. The cutoff for FISH analysis is usually conducted MTC sections were heavily fibrous and had pro- from the analysis of 200 cells, 100 cells by two different tein cross-linking due to formalin fixation, and technologists. This is performed by identifying the nor- these characteristics can mask DNA sites, which mal case with the highest number of false positive nuclei can affect the probes’ affinity to their binding sites. for any given signal pattern and inserted into the To account for these factors, the samples were rig- BETAINV function with a 95% confidence interval orously pretreated at 95–98°C and then subjected [22]. 528 Annals of Clinical & Laboratory Science, vol. 47, no. 5, 2017

to enzymatic digestion at 37°C, which maintained proto-oncogene in multiple endocrine neoplasia type 2A. Nature, 363(6428), 458–60. https://doi.org/10.1038/363458a0 the integrity of the cells. In addition, we evaluated 7. Wells, S. A., Robinson, B. G., Gagel, R. F., Dralle, H., Fagin, J. each batch of probes with high stringency. The vali- A., Santoro, M., Schlumberger, M. J. (2012). Vandetanib in pa- dation eliminated any chance of false specific hybrid- tients with locally advanced or metastatic medullary thyroid can- cer: A randomized, double-blind phase III trial. J Clin Oncol, ization due to the probe design in addition to con- 30(2), 134–141. https://doi.org/10.1200/JCO.2011.35.5040 firming that the probes bound to chromosome 1 and 8. Hu, M. I.,& Cote, G. J. (2012). Medullary thyroid carcinoma: 19 proper regions with adequate fluorescence intensi- who’s on first? Thyroid, 22(5), 451–453. https://doi.org/10.1089/ thy.2012.2205.ed ties. The protocol we used also proved to be highly 9. Ciampi, R., Mian, C., Fugazzola, L., Cosci, B., Romei, C., efficient for hybridization with direct fluorochrome- Barollo, S., Elisei, R. (2013). Evidence of a low prevalence of RAS mutations in a large medullary thyroid cancer series. Thyroid, labeled probes on FFPE tissue sections. 23(1), 50–7. https://doi.org/10.1089/thy.2012.0207 10. Ye, L., Santarpia, L., Cote, G. J., El-naggar, A. K.,& Gagel, R. F. In conclusion, although various techniques are avail- (2008). High resolution array-comparative genomic hybridiza- tion profiling reveals deoxyribonucleic acid copy number altera- able to assess CDKN2C loss, only FISH fulfills the tions associated with medullary thyroid carcinoma. J Clin criteria as a fast and reliable method for doing so, es- Endocrinol Metab, 93, 4367–4372. https://doi.org/10.1210/ pecially for FFPE MTC samples. Of the 5 MTC jc.2008-0912 11. Barbieri, R. B., Bufalo, N. E., Secolin, R., Assumpcao, L. V. M., samples we tested, 4 had CDKN2C loss; however, Maciel, R. M. B., Cerutti, J. M.,& Ward, L. S. (2014). only 1 had loss of both CDKN2C and CDKN2D. We Polymorphisms of cell cycle control genes influence the develop- ment of sporadic medullary thyroid carcinoma. Eur J Endocinol, thus found frequent loss of CDKN2C in 80% of the 171(6), 761–767. https://doi.org/10.1530/EJE-14-0461 MTC samples. Furthermore, the results of a cytoge- 12. Cánepa, E. T., Scassa, M. E., Ceruti, J. M., Marazita, M. C., netic CLIA test for CDKN2C were 100% concor- Carcagno, A. L., Sirkin, P. F.,& Ogara, M. F. (2007). INK4 , a family of mammalian CDK inhibitors with novel bio- dant with our research FISH data. This study sug- logical functions. IUBMB Life, 59(7), 419–426. https://doi. gests that FISH offers a great advantage as a routine org/10.1080/15216540701488358 diagnostic tool for screening MTC patients. 13. Knudsen, E. S.,& Wang, J. Y. J. (2010). Targeting the RB- pathway in cancer therapy. Clin Cancer Res, 16(4), 1094–1099. Acknowledgement https://doi.org/10.1158/1078-0432.CCR-09-0787 14. Van Veelen, W., Van Gasteren, C. J. R., Acton, D. S., Franklin, D. S., Berger, R., Lips, C. J. M., & Höppener, J. W. M. (2008). We thank Dean Shirley Richmond for supporting the Synergistic effect of oncogenic RET and loss of p18 on medullary Project-Based Integrated Curriculum Development thyroid carcinoma development. Cancer Res, 68(5), 1329–1337. Initiative (PICDIn); laboratory exercises implanted in https://doi.org/10.1158/0008-5472.CAN-07-5754 PICDIn formed the basis for this manuscript. We would 15. Van Veelen, W., Klompmaker, R., Gloerich, M., van Gasteren, C. also like to acknowledge the Department of Scientific J. R., Kalkhoven, E., Berger, R., Acton, D. S. (2009). P18 is a Publications at MD Anderson Cancer Center for their tumor suppressor gene involved in human medullary thyroid car- careful review and edits of this manuscript. This work was cinoma and pheochromocytoma development. Int J Cancer, supported in part by National Cancer Institute Grant P50 124(2), 339–45. https://doi.org/10.1002/ijc.23977 CA168505 (GJC and EGG). 16. Grubbs, E. G., Williams, M. D., Scheet, P., Vattathil, S., Perrier, N. D., Lee, J. E.,… Cote, G. J. (2016). Role of CDKN2C copy References number in sporadic medullary thyroid carcinoma. Thyroid, 26(11). https://doi.org/10.1089/thy.2016.0224 1. Hazard, JB, Hawk, WA,& Crile, G. (1959). Medullary (solid) 17. Flicker, K., Ulz, P., Höger, H., Zeitlhofer, P., Haas, O. A., carcinoma of the thyroid; a clinicopathologic entity.J Clin Behmel, A., Speicher, M. R. (2012). High-resolution analysis of Endocrinol Metab, 19, 152–161. https://doi.org/10.1210/ alterations in medullary thyroid carcinoma genomes. Int J jcem-19-1-152 Cancer, 131(2), 66–73. https://doi.org/10.1002/ijc.26494 2. Raue, F., Kotzerke, J., Reinwein, D., Schröder, S., Röher, H. D., 18. Dickson, M. A. (2014). Molecular pathways: CDK4 inhibitors Deckart, H.,… Vogt, H. (1993). Prognostic factors in medullary for cancer therapy. Clinical Cancer Research, 20(13), 3379–3383. thyroid carcinoma: evaluation of 741 patients from the German https://doi.org/10.1158/1078-0432.CCR-13-1551 Medullary Thyroid Carcinoma Register. Clin Investig, 71(1), 19. Mayer, E. L. (2015). Targeting Breast Cancer with CDK 7–12. https://doi.org/10.1007/BF00210956 Inhibitors. Current Oncology Reports. https://doi.org/10.1007/ 3. Saad, M., Ordonez, N., Rashid, R., Guido, J., Hill, C. J., Hickey, s11912-015-0443-3 R.,& Samaan, N. (1984). Medullary carcinoma of the thyroid. A 20. Sheppard, K. E., & McArthur, G. A. (2013). The cell-cycle regu- study of the clinical features and prognostic factors in 161 pa- lator CDK4: An emerging therapeutic target in melanoma. tients. Medicine (Baltimore), 63, 319–342. Clinical Cancer Research, 19(19), 5320–5328. https://doi. 4. Mathew, C., Chin, K., Easton, D., Thorpe, K., Carter, C., Liou, org/10.1158/1078-0432.CCR-13-0259 G., Kryseman, A. (1987).A linked genetic marker for multiple 21. Leone, P. E., Walker, B. A., Jenner, M. W., Chiecchio, L., endocrine neoplasia type 2A on chromosome 10. Nature, 328, Protheroe, R. K. M., Johnson, D. C., Morgan, G. J. (2009). 527–528. https://doi.org/doi:10.1038/328527a0 Deletions of CDKN2C in multiple myeloma: biological and 5. Donis-Keller, H., Dou, S., Chi, D., Carlson, K., Toshima, K., clinical implications. Clin Cancer Res, 14(19), 6033–6041. Lairmore, T., Wells, S. J. (1993). Mutations in the RET proto- https://doi.org/10.1158/1078-0432.CCR-08-0347.Deletions oncogene are associated with MEN 2A and FMTC. Hum Mol 22. Wolff, D. J., Bagg, A., Cooley, L. D., Dewald, G. W., Hirsch, B. Genet., 2, 851–856. A., Jacky, P. B., Rao, P. N. (2007). Guidance for fluorescence in 6. Mulligan, L. M., Kwok, J. B., Healey, C. S., Elsdon, M. J., Eng, situ hybridization testing in hematologic disorders. The Journal C., Gardner, E., Papi, L. (1993). Germ-line mutations of the RET of Molecular Diagnostics, 9(2), 134–143. https://doi.org/10.2353/ jmoldx.2007.060128