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CDKN2A (P14arf, P16ink4a), and MTAP Genes in Head and Neck Squamous Cell Carcinoma

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CDKN2A (P14arf, P16ink4a), and MTAP Genes in Head and Neck Squamous Cell Carcinoma ORIGINAL ARTICLE Fine-Mapping Loss of Gene Architecture at the CDKN2B (p15INK4b), CDKN2A (p14ARF, p16INK4a), and MTAP Genes in Head and Neck Squamous Cell Carcinoma Maria J. Worsham, PhD; Kang Mei Chen, MD; Nivedita Tiwari, MS; Gerard Pals, PhD; Jan P. Schouten, PhD; Seema Sethi, MD; Michael S. Benninger, MD Objective: To identify the extent and the smallest region Results: Cell line UMSCC-11B retained all 9p loci of loss for CDKN2BINK4b, CDKN2AARF,INK4a, and MTAP. tested in the region. Cell lines UMSCC-17A/B indi- Homozygous deletions of human chromosome 9p21 occur cated homozygous deletion of CDKN2AARF, INK4a start- frequently in malignant cell lines and are common in squa- ing at p16INK4 exon 1␣ to include exons 2 and 3. mous cell carcinoma of the head and neck (HNSCC). This Homozygous loss was indicated for CDKN2BINK4b and complex region encodes the tumor suppressor genes CDKN2AARF,INK4a in UMSCC-11A, and UMSCC-81A. Cell cyclin-dependent kinase 2B (CDKN2B)(p15INK4b) and line UMSCC-81B indicated retention of all 9p loci ex- CDKN2A (p14ARF,p16INK4a) and the housekeeping gene me- cept for exon 1␣ (p16INK4a). Selective loss of the 3Ј end thylthioadenosine phosphorylase (MTAP). of MTAP was observed in UMSCC-11A. Genomic alter- ations by fine-mapping MLPA were validated at the DNA Design: A targeted probe panel designed to finely map level for CDKN2A. the region of 9p21 loss comprised 3 probes for CDKN2BINK4b, 7 for CDKN2AARF, INK4a, and 3 for MTAP and Conclusions: We identified exon 1␣ (p16INK4a) as the small- was interrogated using the multiplex ligation- est region of loss in the CDKN2AARF, INK4a gene. The fre- dependent probe amplification assay (MLPA). The MLPA quency and precise loss of CDKN2BINK4b, CDKN2AARF, INK4a, genomic copy number alterations for CDKN2A were vali- and MTAP in the prognosis of 9p21-deleted HNSCC may dated using real-time polymerase chain reaction. provide impetus for use of these targets as therapeutic bio- markers in head and neck cancer. Subjects: Six HNSCC primary (A) and recurrent or meta- static (B) cell lines were examined: UMSCC-11A/11B, UMSCC-17A/17B, and UMSCC-81A/81B. Arch Otolaryngol Head Neck Surg. 2006;132:409-415 HE MOST COMMON CHRO- tory function is achieved through the p16 mosome rearrangements protein product, which functions up- associated with squamous stream of Rb, and the p14 protein, which cell carcinomas of the head blocks MDM2 inhibition of p53 activity, and neck (HNSCCs) are preventing p53 degradation, and thus per- unbalanced translocations leading to chro- mitting p53-induced apoptosis or growth T 1 8 mosomal deletions. Chromosome 9p21 arrest. The CDKN2D/p14 gene has a has been reported to be a critical region unique first exon 1␤ that, under the con- of loss in various cancers. Genetic alter- trol of its own promoter, splices onto exon ations at the 9p21 locus have been linked 2ofCDKN2A/p16 in an alternative read- to malignant progression in HNSCC.2,3 The ing frame, allowing production of the 2 cyclin-dependent kinase 2A (CDKN2A) totally unrelated proteins, p14 and p16, Author Affiliations: and CDKN2B genes map to 9p21 and are respectively (Figure 1).8 Department of in tandem, spanning a region of approxi- Whereas CDKN2A/p16 mutations se- Otolaryngology–Head and Neck mately 80 kilobases (kb), with CDKN2B lectively inactivate the Rb pathway, dele- Surgery and Research Division, located 25 kb centromeric to CDKN2A4,5 tion of the CDKN2A locus impairs the Rb Henry Ford Hospital, Detroit, (Figure 1). The CDKN2A locus con- and p53 pathways.6-8 Deletion of the Mich (Drs Worsham, Chen, trols the Rb pathway (which regulates G1/ CDKN2A locus also frequently affects the Sethi, and Benninger and Ms Tiwari); and Department of S-phase transition) and the p53 pathway CDKN2B locus, which encodes p15, an im- Clinical Genetics, VU Medical (which induces growth arrest or apopto- portant mediator of the antiproliferative 9 Center (Dr Pals), and sis in response to either DNA damage or effect of transforming growth factor ␤. In- MRC-Holland (Dr Schouten), inappropriate mitogenic stimuli by gen- activation of the CDKN2B (p15), CDKN2A Amsterdam, the Netherlands. erating 2 gene products).6,7 This regula- (p14), and CDKN2A (p16) genes is a fre- (REPRINTED) ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 132, APR 2006 WWW.ARCHOTO.COM 409 ©2006 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 fected by deletion, offering additional therapeutic tar- A gets of consideration in HNSCC. Centromere Chromosome 9p21 Telomere CDKN2B CDKN2A MTAP METHODS HNSCC CELL LINES AND DNA EXTRACTION 121β 1α 2 3 1 2 3 4 5 6 7 8 9 10 11 132 1 Tumor sample acquisition, tissue culture, karyotype analysis, and genomewide mapping for altered individual gene loci meth- B ods have been detailed elsewhere.19-21 Cell lines UMSCC-11A UMSCC-11A and UMSCC-11B were derived from tumor tissue obtained from UMSCC-11B the primary tumor site (larynx) before and after chemo- therapy, respectively. Cell lines UMSCC-17A (supraglottis) and UMSCC-17A /B UMSCC-17B (neck soft tissue) were derived from tumor tis- UMSCC-81A sue obtained simultaneously from primary (A) and metastatic UMSCC-81B (B) sites. Cell lines UMSCC-81A (larynx) and UMSCC-81B (ton- sillar pillar) were derived from tumor tissue obtained from pri- Figure 1. Fine mapping of loss of gene architecture at the cyclin-dependent mary (A) and second primary (B) sites. kinase 2A (CDKN2A)(p14 ARF, p16 INK4) and methylthioadenosine DNA from the 6 cell lines was extracted using the QIAamp phosphorylase (MTAP ) loci in squamous cell carcinomas of the head and Kit (Qiagen Inc, Chatsworth, Calif) at passages 83 and 90 for neck. A, Genomic organization of the CDKN2B, CDKN2A, and MTAP genes at UMSCC-11A and UMSCC-11B, respectively; 138 and 184 for 9p21 illustrating 3 multiplex ligation-dependent probe amplification assay UMSCC-17A and UMSCC-17B, respectively; and 24 and 129 probes for CDKN2B, 7 for CDKN2A, and 3 for MTAP. B, Loss of gene segments is illustrated by dashed lines and retention by solid lines. The for UMSCC-81A and UMSCC-81B, respectively. UMSCC-11B cell line retained all 9p21 loci tested. Note the homozygous loss of CDKN2B, CDKN2A, and the 3Ј end of MTAP in UMSCC-11A. The FINE-MAPPING LOSS AT THE 9p21 LOCUS UMSCC-81A cell line had homozygous loss of CDKN2B and CDKN2A but retained MTAP. The UMSCC-81B cell line indicated loss in only exon 1␣ of p16 INK4a. The multiplex ligation-dependent probe amplification assay (MLPA)21,22 provides relative quantification of loss and gain of gene loci using the candidate gene approach. Our group21 quent event in human oral SCCs.10 The main modes of recently showed that for HNSCC cell lines UMSCC-11A/B, p16INK4a inactivation in HNSCC are known to include UMSCC-17A/B, and UMSCC-81A/B, loss and gain of genetic homozygous deletions (40%-60%), mutations (15%- loci using a genomewide probe panel concurred with tumor 20%), and gene hypermethylation events (5%).10,11 karyotypes. The 112–gene probe panel with single probes for the CDKN2A and CDKN2B genes indicated loss Somatic alterations in the CDKN2A gene occur in of CDKN2A in all except UMSCC-11B, with concomitant many cancer types, and germline mutation carriers are loss of CDKN2B in UMSCC-11A, UMSCC-17B, and predisposed to a high risk of pancreatic and breast UMSCC-81A. cancers.12 To examine the extent of deletion at the CDKN2B/ Although inactivation of p16 has been reported as CDKN2A/MTAP locus, a targeted MLPA probe panel was the most common genetic alteration in HNSCC, mak- designed to dissect out the region of 9p21 loss. This panel of ing it an ideal target for gene replacement,13 the extent 38 probes comprises 17 control non–chromosome 9p probes to which p14 is independently altered, or the fre- and 21 chromosome 9p21 probes (Table). The latter start quency with which p14 and p16 are affected, has not 3349 kb from the centromeric region and extend up to been examined thoroughly. Because these proteins are 600 kb to the 9p telomere to include 3 probes for CDKN2BINK4b, 7 for CDKN2AARF, INK4a, and 3 for MTAP (Table completely unrelated, bearing no amino acid homol- and Figure 1A). ogy to one another, each of these targets has the potential to independently serve as therapeutic inter- ventions in HNSCC. INTERPRETATION The human methylthioadenosine phosphorylase Normal tissue from each cancer patient serves as an internal (MTAP) gene at 9p21, approximately 100-kb telomeric reference when available. For cell lines, when normal DNA to CKDN2A, is an essential enzyme for the salvage of ad- samples are not available, control (normal) male and female enine and methionine (Figure 1). Cells that lack this en- DNA samples are run with each probe set. Quantification— zyme become sensitive to purine synthesis inhibitors or loss or gain of gene loci—is determined through a process of methionine starvation and can be therapeutically ex- normalization. This process addresses variations in the sur- ploited for selective therapy.14-16 Deletion of MTAP was face area of a peak (intensity) encountered due to fluctuations the most frequent alteration in genomewide profiling stud- in the assay run, such as amount of DNA, ploidy variations, ies of oral squamous cell carcinoma.17 Because deletion and polymerase chain reaction (PCR) conditions. Briefly, the INK4a ARF peak area for each probe is expressed as a percentage of the total of CDKN2A (p16) and CDKN2A (p14) causes dys- 21 regulation of the 2 pathways, Rb and p53, important in surface area of all peaks of a sample in an assay run (Figure 2). Relative copy number for each probe is obtained as a ratio of most cancers, loss of MTAP activity is thought to be in- 18 the normalized value for each locus (peak) of the sample to cidental and not of pathogenic consequence.
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