COMPARATIVE GENOMIC HYBRIDIZATION ANALYSIS OF TONSILLAR CANCER REVEALS A DIFFERENT PATTERN OF GENOMIC IMBALANCES IN HUMAN PAPILLOMAVIRUS-POSITIVE AND -NEGATIVE TUMORS Liselotte DAHLGREN 1 * , Hanna MELLIN 1 , Danny WANGSA 2 , Kerstin HESELMEYER-HADDAD 2 , Linda BJ ¨ ORNESTÅL 3 , Johan LINDHOLM 1 , Eva MUNCK-WIKLAND 3 , Gert AUER 1 , Thomas RIED 2 and Tina DALIANIS 1 1 Department of Oncology-Pathology, Karolinska Institute, Karolinska Hospital, Stockholm, Sweden 2 Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 3 Department of Otorhinolaryngology, Head and Neck Surgery, Karolinska Institute, Karolinska Hospital, Stockholm, Sweden Our aim was to map and compare genomic imbalances in human papillomavirus (HPV)-positive and -negative squa- mous cell carcinomas of the tonsil. Twenty-five primary car- cinomas were analyzed by comparative genomic hybridiza- tion. Fifteen (60%) were found to be HPV-positive by PCR, and the majority were HPV-16. There were statistically sig- nificant differences in the distribution of DNA gains and losses between the HPV-positive and -negative samples. Eleven of 15 HPV-positive samples (73%) showed gain on chromosome 3q24-qter, while only 4/10 (40%) HPV-negative samples had the same gain (p  0.049). Furthermore, 4/10 (40%) HPV-negative samples but no HPV-positive samples had gain on chromosome 7q11.2-q22 (p  0.017). As ex- pected, and similar to previous studies, patients with an HPV-positive tumor had a statistically significantly better disease-specific survival than patients with an HPV-negative tumor (p  0.002). The most common changes, e.g., gain on 3q or 8q, loss on 11q or 13 and loss on chromosome 7q in HPV-negative tumors, did not have any influence on progno- sis. However the number of cases in each subgroup was limited. © 2003 Wiley-Liss, Inc. Key words: human papillomavirus; tonsillar cancer; comparative genomic hybridization; chromosome 3q Head-and-neck cancer constitutes 3.4% of all cancer cases each year in Europe 1 and is the fifth most common cancer type in the United States. 2 In approximately 50 – 60% of patients, the tumor has spread to regional lymph nodes by the time of diagnosis, and it is known that formation of metastases reduces the chance of survival by about 50%. 2 Treatment of head-and-neck cancer has not improved greatly over the last years, and the 5-year survival rate remains low. 3 The main reasons for the low survival rate are advanced tumor stage at detection, high prevalence of recurrence and multiple primary tumors. 3 The major risk factors of head-and-neck squamous cell carci- noma (HNSCC) in the Western world are smoking and alcohol consumption. However, during the past 2 decades the role of high-risk human papillomavirus (HPV) has been studied, and data supporting HPV as a causative agent in the development and progression of a subset of these cancers have accumulated. 4–6 The overall frequency of HPV in HNSCC is around 25–30%, with considerable variability depending on the tumor location. 7 The highest frequency is reported from studies on tonsillar cancer, 7–9 where 35–70% of tumors are HPV-positive, most commonly with HPV-16 and/or HPV-33. Furthermore, we and others have shown that patients with HPV-positive tonsillar cancer have a statistically significant reduction in risk of death from cancer compared to patients with HPV-negative tumors and that this is independent of tumor stage. 4,8 In addition, in a relatively limited study, patients with high viral load tumors had a significantly better prognosis compared to patients with low viral load tumors. 10 The fact that HPV is a favorable predictive/prognostic factor in tonsillar cancer prompted us to analyze whether differences in the pattern of chromosomal gains and losses were correlated with the presence of HPV. We hypothesized that such differences could explain the variable clinical course of HPV-positive cancer. In high-risk HPV-positive cancer of the tonsil, the early proteins E6 and E7 are generally expressed, 11 and it is known that the presence of these proteins is sufficient to immortalize and trans- form keratinocytes. 12 In cervical cancer, constant expression of E6 and E7 is required for malignancy. 13 E6 and E7 interfere with p53 and pRb, respectively, leading to cell-cycle progression and accu- mulation of genetic damage. 13–15 E6 binds and initiates degrada- tion of p53 and activates c-myc, leading to activated telomerases, 16 while the E7 protein forms complexes with proteins in the Rb gene family, liberating the E2F transcription factor and promoting cell division. 17 Furthermore, it is known that multiple genetic changes are involved in the development of HNSCC and that the karyotypes observed in HNSCC are among the most complex so far described in solid tumors. 18 Losses involving 9p, 3p and 17q have been identified in preneoplastic lesions; and 9p and 3p are also the most frequently reported losses in established HNSCC. 19 The target genes for some chromosomal losses are known. For instance, the p16 gene maps to chromosome band 9p21, and p53 localizes to 17p13. 20 Also, inactivation of p16, by mutation, loss of heterozy- gosity or hypermethylation, is an early event, which occurs in 59% of HNSCC cases. 21 Gain of 3q is an early sign of metastatic disease, and gain of 1q and 2q usually results in a clinically poor outcome. 22 Other examples of genetic changes are somatic muta- tions of p53 (17p13), which are detected in 25–50% of HNSCCs, notably in HPV-negative tumors, and expression of cyclin D1, which has been described to be inversely proportional to HPV infection. 23 In addition, DNA gains that map to 3q26-qter, 5p14- pter and 8q are frequent in HNSCC. 24,25 Although several studies have been performed regarding the genetic imbalances in HNSCC, 18,25 to our knowledge none has correlated genomic imbalances with the presence or absence of HPV. We compared genetic imbalances in 25 fresh-frozen HPV-pos- itive and -negative tonsillar carcinomas. HPV status was investi- gated by PCR using the consensus primers GP5+/6+ 26 and CPI/ CPIIG. 27 Sequencing and/or type-specific HPV PCR was Grant sponsor: UICC; Grant sponsor: Swedish Cancer Foundation; Grant sponsor: Stockholm Cancer Society; Grant sponsor: Stockholm City Council; Grant sponsor: Karolinska Institute. *Correspondence to: Department of Oncology-Pathology, Cancer Cen- ter Karolinska, R8:01, Karolinska Institute, Karolinska Hospital, SE-171 76, Stockholm, Sweden. Fax: +46-8-309195. E-mail: Liselotte.Dahlgren@cck.ki.se Received 3 January 2003; Revised 4 April, 8 May 2003; Accepted 16 May 2003 DOI 10.1002/ijc.11371 Int. J. Cancer: 107, 244 –249 (2003) © 2003 Wiley-Liss, Inc. Publication of the International Union Against Cancer