Oncogene (1997) 14, 369 ± 373 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 Deletion mapping of chromosome 4 in head and neck squamous cell carcinoma Mark A Pershouse1,3, Adel K El-Naggar2, Kenneth Hurr2, Huai Lin1,3, WK Alfred Yung1,3 and Peter A Steck1,3 Departments of 1Neuro-Oncology and 2Pathology and 3The Brain Tumor Center, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA Genomic deletions involving chromosome 4 have recently Cytogenetic studies have identi®ed recurring, but been implicated in several human cancers. To identify widely varied alterations of chromosomes 1, 3, 4, 5, 7, and characterize genetic events associated with the 8, 9, 11, 14, 15 and 17 (Jin et al., 1993; Sreekantaiah et development of head and neck squamous cell carinoma al., 1994). Although several molecular studies have (HNSCC), a ®ne mapping of allelic losses associated shown that mutation of p53 and ampli®cation of with chromosome 4 was performed on DNA isolated epidermal growth factor receptor are relatively from 27 matched primary tumor specimens and normal common events. However, the exact genes that are tissues. Loss of heterozygosity (LOH) of at least one targeted in the majority of the observed chromosomal chromosome 4 polymorphic allele was seen in the alterations are unknown (Brachman et al., 1992; majority of tumors (92%). Allelic deletions were con®ned Grandis et al., 1993; Shin et al., 1994). Recently, to short arm loci in four tumors and to the long arm loci several groups have performed allelotyping studies on in 12 tumors, suggesting the presence of two regions of HNSCC specimens to further de®ne regions of common deletion. One region of frequent deletion was consistent loss. Frequent loss of heterozygosity centered at D4S405 on 4p and included the loci D4S1546 (LOH) was observed on chromosome arms 3p, 5q, to D4S428 in *41% of the tumors. The common region 9p, 11q and 17p, with less frequent losses on 4, 6p, 8, of deletion on 4q was more complex and extended from 14q, 18 and 19q (Nawroz et al., 1994; Ah-See et al., D4S1571 to D4S1573. Frequent genetic alterations were 1994; El-Naggar et al., 1995). The combination of these observed within this region (4q25) and one marker, observations suggest that multiple molecular altera- D4S407, exhibited a high frequency of LOH (475%). tions are responsible for the oncogenesis of HNSCC. These results indicate that alterations of chromosome 4 Our interest in chromosome 4 results from an regions are associated with HNSCC tumorigenesis and observation that the microcell-mediated chromosomal further localizes the regions that may harbor tumor transfer of chromosome 4 in human gliomas resulted in suppressor genes. the suppression of the tumorigenicity of glioma cells (Pershouse et al., submitted). The putative tumor Keywords: chromosome 4; loss of heterozygosity; suppressor locus associated with this observed pheno- carcinoma typic suppression was further localized to a region (47 cM) near the epidermal growth factor gene on 4q. This study was undertaken to further examine the occurrence and location of regions of common deletion Introduction involving chromosome 4, particularly associated with the long arm in HNSCC. Approximately 50 000 North Americans will be diagnosed with head and neck squamous cell carcinoma (HNSCC) this year and over 14 000 will Results succumb to the disease (DeVesa et al., 1995). Etiological factors for HNSCC include tobacco and Tumor specimens from 27 patients with various grades alcohol use, along with occupational exposure to of HNSCC were screened for LOH with a panel of 27 carcinogens, implying that damage to DNA plays a microsatellite markers speci®c for chromosome 4 loci. major role in the oncogenesis of this cancer (Vokes et Typical autoradiographs of the markers D4S408 and al., 1993). This is further supported by studies D4S1546 illustrating LOH and retention of alleles are demonstrating mutagen-induced chromosomal fragi- shown in Figure 1. The overall frequency of deletion at lity as an independent risk factor (Spitz et al., 1993). each locus and their ordering is depicted in Figure 2. However, the molecular events in HNSCC are still Two tumors showed no loss of chromosome 4 loci poorly understood (Hong et al., 1995; Sidransky, (tumors #36 and 48). Alternatively, two other tumors 1995). By better de®ning the molecular genetic basis (#4 and 8) displayed extensive losses, suggesting the of tumor initiation and progression in HNSCC, new deletion of all or most of chromosome 4 in these insights into the biological behavior, diagnosis, and neoplasms. Allelic alterations (alleles not present in the possible new therapeutic approaches may be forth- matched normal specimens) were observed in six of 27 coming. specimens (22%), with three tumors (#41, 44, and 46) showing alterations at several markers. Correspondence: PA Steck The majority of tumors (22 of 27, 81%) had Received 28 June 1996; revised 17 September 1996; accepted 17 deletions and/or alterations associated with the long September 1996 arm of chromosome 4. Twelve tumor specimens Chromosome 4 loss in HNSCC MA Pershouse et al 370 6 7 8 N T N T N T D4S1546 41 42 46 N T N T N T D4S408 Figure 1 Illustration of microsatellite polymorphism analysis in head and neck squamous cell carcinoma specimens. Paired normal lymphocyte (N) and tumor (T) DNA samples for patients 6, 7 and 8 using microsatellite markers D4S1546 demonstrating non-informative, retention of heterozygosity, and loss of the upper allele, respectively. For patients 41, 42 and 43 D4S408 marker shows retention of heterozygosity, non-informa- tive, and loss of the lower allele, respectively revealed deletions con®ned only to 4q (Table 1; Figure 3, groups A and B). All of the tumors with deletions on 4q involved at least an area at 4q25 from the markers D4S1571 to D4S1573 which represents a region of about 7 cM (Gyapay et al., 1994). Two patterns of allelic losses within this region were de®ned. One series of tumor specimens exhibited a continuous region of possible deletions within this critical area at 4q25 (boxed markers Figure 3a). A common region of loss was centered at D4S1616 and involved the Figure 2 Illustration of the frequency and approximate location of LOH at loci on chromosome 4 in HNSCC and several other adjacent markers D4S1553 to D4S407 (*1.5 mb). human cancers. Genetic markers are listed in their most probable Breakpoints involving LOH and retention of alleles in order and the approximate physical map of several markers are tumors #6 and 41 would indicate a consistent region of shown deletion surrounding D4S1616. A similar pattern of continuous deletions was de®ned for tumors with allelic losses involving both the long and short arm of the retention. A similar pattern was seen for tumors with chromosome (Figure 3, group C1). However, for this and without allelic losses on the p arm (Figure 3, series of tumors, only the region surrounding D4S407 groups C2 and B, respectively). The most centromeric was consistently involved. The combination of these region of consistent deletion was centered at D4S1553, two series of tumors would suggest a minimal region of while the more telomeric region was centered at involvement between D4S1616 and D4S407, as D4S1616 and extended from D4S1651 to D4S1611. illustrated by breakpoints in tumors (#6 and 43). Four tumors (#5, #43, #44, and #45) displayed losses Two tumors (#11 and 53) revealed continuous deletion associated only with the short arm of the chromosome of the area D4S1616-D4S407, but had an additional (Table 1). Overall, LOH on 4p was shown in 11 of the 27 area of deletion telomeric to 4q25. (41%) of the tumors examined. Of these, four (14%) had A second more complex pattern of allelic losses for potentially lost the entire arm (Figure 4). A minimum 4q25 was also observed involving the same region. area of deletion involved the markers D4S405 and However, this series of tumors displayed two areas of D4S428. Three tumors (#44, 45, and 52) have single p- deletions accompanied by two markers within this arm, deletions at D4S405. One tumor (#53) displayed a region, EGF and D4S193, that showed frequent deletion towards the centromeric side suggesting a Chromosome 4 loss in HNSCC MA Pershouse et al 371 Table 1 Diagnosis, grading and site of HNSCC tissue samples mas including hepatocellular, bladder, ovarian and Tumor Allelic cervical (Buetow et al., 1989; Sato et al., 1991; Mitra et no. Gradea Stageb Site deletionc Diagnosisd al., 1994; Fujimoto et al., 1994; Polascik et al., 1995). 2 2 IV Tongue 4q MDSqCa For HNSCC the allelic deletions associated with 3 2 III Tongue 4q PDSqCa chromosome 4 were considered relatively infrequent 4 2 IV Tongue 4p+qe InvSqCa compared to other chromosomal alterations and 5 2 II H & Neck 4p MDMetSqCa `unexpected' as previous reports failed to implicate 6 2 IV Larynx 4q MDSqCa 7 3 IV Tongue 4q PDSqCa this chromosome (Narwoz et al., 1994). Although only 8 2 IV Maxilla 4p+qe InvSqCa one marker per arm was used in the latter study. The 9 2 III Tongue 4q MDSqCa results from our study illustrate a higher frequency of 10 3 IV Tongue 4q PDSqCa loss associated with chromosome 4 with 92% of the 11 2 IV Tongue 4q MDSqCa 13 1 IV Tongue 4p+q WDSqCa specimens showing allelic deletion involving chromo- 36 2 IV Larynx ±f MDSqCa some 4. Furthermore, two regions of common deletion 37 3 IV Neck 4q PDSqCa were observed: (1) a large area involving the 41 2 IV Tongue 4q MDSqCa centromeric region of the short arm centered at 42 2 III Tongue 4q MDSqCa D4S405 and (2) a smaller region showing complex 43 2 II Larynx 4p+q MDSqCa 44 1 I Tongue 4p+q InvSqCa alterations that involved at least 4q25.