DNA의추출 Table 1. Sequences of oligonucleotide primers Primers L1 consensus primer Sequence CGT CCM ARR GGA WAC TGA TC Size of amplified products

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1 KISEP Rhinology Korean J Otolaryngol 2000;43: 비 부비동반전성유두종에서인유두종바이러스및 Epstein-Barr 바이러스의검출과 p53 단백및 Proliferating Cell Nuclear Antigen 의발현 조진희 서병도 전범조 장한성 양명재 윤희로 노우영 Detection of Human Papillomavirus and Epstein-Barr Virus in Sinonasal Inverted Papillomas and Its Correlation with p53 and Proliferating Cell Nuclear Antigen Expression Jin Hee Cho, MD, Byung Do Suh, MD, Beom Cho Jun, MD, Han Sung Jang, MD, Myung Jae Yang, MD, He Ro Yoon, MD and Woo Young Ro, MD Department of Otolaryngology-HNS, College of Medicine, The Catholic University of Korea, Seoul, Korea ABSTRACT Background and ObjectivesSinonasal inverted papillomas are benign but topographically aggressive neoplasms that have a high recurrence rate and seem to be associated with malignancy. The etiology of inverted papilloma remains unknown, but some hypotheses suggest that nasal polyps proliferation and chronic inflammation are due to allergy or various infectious lesions. This study was to elucidate the biological characteristics and the role of human papillomavirus HPV and Ebstein - Barr virus EBV and the expression of p53 protein and proliferating cell nuclear antigen PCNA in sinonasal inverted papillomas. Materials and MethodsWe examined 26 specimens from 26 individuals with normal nasal mucosae n10 and inverted papillomas n16 to determine the occurance of HPV and EBV infection and the expression of p53 protein and PCNA. ResultsOf the 16 Inverted papillomas, HPV DNA was detected in eight cases, HPV 18 was detected in two cases 18, HPV 16 and HPV 33 were both found in every case 6, HPV 6 and HPV 16 were coinfected in one case 6, and other types were found in 3 cases. HPV DNA was not detected in the normal nasal mucosae. EBV DNA was detected in 10 cases 62 out of 16 inverted papillomas and in two cases 20 of 10 normal nasal mocosae. The altered p53 protein expression was observed in four cases 25, and positive PCNA staining was detected in four cases 25 out of 16 inverted papillomas. One positive PCNA staining was detected among 10 normal mucosae. The mean PC10 index was 16.0 in the inverted papillomas group and 4.1 in normal nasal mucosae group. ConclusionAn inverse correlation may exist between oncogenic HPV infection and p53 alteration in sinonasal inverted papillomas. Korean J Otolaryngol 2000;43: KEY WORDSInverted papillomas p53 Proliferating cell nuclear antigen PCNA. 836

2 DNA의추출 Table 1. Sequences of oligonucleotide primers Primers L1 consensus primer Sequence CGT CCM ARR GGA WAC TGA TC Size of amplified productsbase pair 5 -GCM CAG GGW CAT AAY AAT GG 450 Type specific E6 primer HPV AGA CCA GTT GTG CAA GAC GTT TAA GCA CGT CTA AGA TGT CTT GTT TAG HPV AGA CCA GTT GTG CAA GAC FTT TAA AAG GGA AAG TTG TCT CFC CAC ACA HPV AAG GGC GTA ACC GAA ATC GG CAT ATA CCT CAC GTC GCA G HPV AAG GGC ATA ACC GAA ATA GG TTC TGC TGG ATT CAA CGG T HPV TAA GCT CGG CAT TGG AAA TAC CCT CCT TCC TCC TAT GTT GTG GAA TCG HPV GCA GTA AGG TAC TGC ACG ACT ATG CGA CCC CAA ATA TTA TGA AAT CGT Primer for Epstein-Barr virus detection TC60 5 -CCA CAG GTA AGT GGA CTT TC61 5 -GAC CGG TGC CTT CTT AGG-3 837

3 인유두종바이러스의중합효소연쇄반응 Table 2. Clinicopathologic findings of the detection of HPV DNA, EBV DNA, p53 and PCNA in patients with sinonasal inverted papillomas Case No Sex Age Involved structure 1 F 58 Lt. nasal cavity maxillary sinus 2 M 37 Rt. nasal cavity 3 M 46 Lt. nasal cavity ant. 4 M 56 Lt. nasal cavity ant. Recurrence Operation procudure Lt. medial 5 M 56 Rt. nasal cavity 6 F 57 Lt. nasal cavity sphenoid, 7 M 55 Rt. nasal cavity 8 M 30 Lt. nasal cavity maxillary sinus, 9 F 46 Lt. nasal cavity 10 M 42 Rt. nasal cavity, sphenoid 11 M 36 Lt. nasal cavity maxillary sinus, 12 F 68 Lt. nasal cavity, maxillary sinus, frontal 13 M 51 Rt. maxillary sinus 14 M 66 Lt. maxillary sinus 15 M 49 Rt. nasal cavity, maxillary sinus 16 M 75 Rt. maxillary sinus, frontal *HPV DNA was detected, but didn t confirmed subtypes exophytic exophytic Growth pattern HPV type EBV p53 PCNA inverted * inverted inverted 16 inverted 18 inverted inverted * inverted inverted 18 inverted 6, 16 inverted inverted inverted 33 inverted inverted inverted * inverted 838 Korean J Otolaryngol 2000;43:836-43

Lane DHexamples of amplified products of patients with sinonasal inverted papillomas. D,G,H demonstrste the positive signal for the presence of HPV. Fig. 2.

4 EBV의중합효소연쇄반응 p53과 PCNA의조직내면역조직화학적검색 탈파라핀 자료분석과통계처리 Fig. 1. Photography of agarose gel electrophoresis of amplified 450 base pairs DNA arrow with L1 consensus primers HPV Lane AMarker 100 base pairs ladder, Lane Bpositive control, Lane Cnegative control, Lane DHexamples of amplified products of patients with sinonasal inverted papillomas. D,G,H demonstrste the positive signal for the presence of HPV. Fig. 2. Photography of agarose gel electrophoresis showing amplified products of various HPV subtypes. lane AMarker 100 base pairs ladder, Lane Bamplified products of 263 base pairs with HPV 6 specific E6 primer, Lane C206 base pairs with HPV 16 specific E6 primer, Lane D419 base pairs with HPV 18 specific E6 primer, Lane E413 base pairs with HPV 33 specific E6 primer. 839

5 Table 3. HPV and EBV identified in sinonasal inverted papillomas and normal nasal mucosae Total No. HPV* HPV type EBV* of cases Others Inverted papillomas % % 6 Normal nasal mucosae 10 00% % 14 *Positive sign indicates the detection of HPV DNA and EBV DNA, except negative sign. One case was coinfected with HPV 6 and HPV 16 Comparison between sinonasal inverted papillomas and normal nasal mucosae p0.01 Comparison between sinonasal inverted papillomas and normal nasal mucosae p0.05 Fig. 3. Photography of agarose gel electrophoresis showing positive control amplified with TC 60 and TC 61 primer EBV and amplified products of 125 base pairs arrow with TC 60 and TC 61 primer on lane DK. Lane AMarker 50 base pairs ladder, Lane Bpositive control. Lane Cnegative control, Lane DKA examples of amplified products of patients with sinonasal inverted papillomas. D,F,H,I,J demonstrste the positive signal for the presence of EBV. Table 4. Comparision of the detection of HPV DNA, EBV DNA, p53 and PCNA between recurrent and non-recurrent group of sinonasal inverted papillomas No. of cases HPV DNA EBV DNA p53 PCNA Recurrent group % 3 50% 2 33% 1 17% Non-recurrent group % 7 70% 2 20% 3 30% Total No Korean J Otolaryngol 2000;43:836-43

조진희 외 Fig. 4. Immunohistochemical staining of p53 protein with APAAP (alkaline phosphatase anti-alkaline phosphatase) method in sinonasal inverted papilloma tissue.

Immunohistochemical staining of proliferating cell nuclear antigen with APAAP (alkaline phosphatase anti-alkaline phosphatase) method in sinonasal inverted papilloma tissue.

6 조진희 외 Fig. 4. Immunohistochemical staining of p53 protein with APAAP (alkaline phosphatase anti-alkaline phosphatase) method in sinonasal inverted papilloma tissue. The positive immunoreactivity is localized in the nucleus (arrow), 400. Fig. 5. Immunohistochemical staining of proliferating cell nuclear antigen with APAAP (alkaline phosphatase anti-alkaline phosphatase) method in sinonasal inverted papilloma tissue. The positive immunoreactivity is localized in the nucleus (arrow), 400. Table 5. The results of p53 and PCNA immunostaining and PC10 index in sinonasal inverted papillomas and normal nasal mucosae No. of cases Inverted papilllomas 16 p53 PCNA - 4 (25%) (25%) Normal nasal mucosae 10 0 (0%) 10 1 (10%) *PC10 index was calculated the fomular, PC10 index=sum of PCNA (+) cell/sum of tumor cell 100 Positive sign indicates mutant p53 protein expressiom, except negative sign Positive sign indicates the PCNA positivity, except negative sign PC10 indx* - (%) 반전성 유두종 환자의 연령 및 성별분포는 40 60세에 호 차이가 없었다(Fig. 4, Table 4). 발하여 평균연령은 50세, 남성에서 많다고 알려져 있으며, 저 자의 연구에서도 50대에 많았고 평균연령는 51.7세였으며, PCNA의 면역조직학적 소견 반전성 유두종 16례중 4례(25%), 대조군에서는 10례중 1 남녀비는 3 1이었다. 또한 반전성 유두종에서 악성 변화를 례에서 양성반응을 나타냈으며(Fig. 5), PC10 지표는 반전성 동반하는 빈도는 2 50%로 보고되었지만 대부분 5 15% 유두종군에서는 평균 16.0%, 대조군에서는 평균 4.1%로 관 정도가 악성변화를 동반한다고 하며 편평상피암이 대부분이 찰되어 반전성 유두종군에서 높게 관찰되는 양상을 보였으 지만 본 연구에서 조사한 16례에서는 이 형성이나 악성 변화 나, 자료수가 적어 통계학적 의의는 밝힐 수 없었고(Table 5), 를 동반한 예가 없었다. 또한 재발군과 비재발군에서 PCNA의 반응률에는 차이가 없 인유두종 바이러스는 호흡기와 소화기 점막조직에서 양성 및 악성 종양을 일으키는 원인 인자로 알려져 있으며 80여 었다(Table 4). 종의 아형이 있고 약 8000 bp 크기의 이중쇄 DNA 바이러 고 찰 스로, 상기도에 잠복감염상태로 존재하다가 여러 가지 요인 에 의해 활성화되어 종양발생에 보조적인 역할을 하는 것으 비 부비동 반전성 유두종은 종양의 상피세포가 기질내로 16) 로 알려져 있다. 병소의 위치와 병변의 진행과정에 따라 인유 반전하는 양성상피종양으로 Ward 가 처음 비유두종을 발 두종 바이러스의 다른 아형이 검출되는데, HPV 6, 11은 주 표하였고, 종양의 상피가 기질내로 반전한다는 조직학적 특 로 비현성 감염이나 후두 유두종 같은 양성질환을 유발하며, 성을 기술한 이래 여러 가지 명칭으로 불려져왔으나 근래에 HPV 16, 18, 31, 35, 51은 자궁경부 및 비뇨기계의 악성종 는 반전성 유두종으로 주로 불리우고 있다. 반전성 유두종은 양과 연관이 있는 것으로 알려져 있다. HPV와 비 부비동 반 비강내 원발성종양의 0.5 4%로 비교적 드물게 발생하나 수 전성 유두종의 연관성은 HPV 16, 11, 16, 18, 57 등 여러 아 술적 치료후에도 재발율이 높고, 주위조직의 파괴를 야기하며, 형에 대하여 연구되어져 왔는데 본 연구에서는 Manos 등14) 악성종양과의 연관 등으로 지속적인 추적관찰이 필요한 질환 이 고안한 L1 consensus primer를 이용하는 PCR 방법으 이다. 로 반전성 유두종 16례중 8례(50%)에서 HPV DNA를 검 841

7 842 Korean J Otolaryngol 2000;43:836-43

8 REFERENCES 1) Skolnik EM, Loewy A, Friedman JE. Inverted papilloma of the nasal cavity. Arch Otolaryngol 1996;64,84: ) Segal K, Atar E, Mor C, Har-El G, Sidi J. Inverting papilloma of the nose and paranasal sinusesl. Laryngoscope 1986;96: ) Chang P, Wang L, Syrjanen S, Syrjanan K. Human papilloma virus infection in the respiratory tract. Am J Otolaryngol 1992;13: ) Calcaterra TC, Thompson JW, Paglia DE. Inverting papillomas of the nose nd paranasal sinuses. Laryngoscope 1980;90: ) Lawson W, Biller HF, Jacobson A, Som P. The role of conservative surgery in the managment of inverted papilloma. Laryngoscope 1983;93: ) Weissler MC, Montgomery WW, Turner PA, Montgomery SK, Joseph MP. Inverted papilloma. Ann Otol Rhinol Laryngol 1986;95: ) Kashima HK, Kessis T, Hruban RH, W TC, Zinreich SJ, Shah KV. Human papilloma virus in sinonasal papillomas and squamous cell carcinoma. Laryngoscope 1992;102: ) Macdonald MR, Le KT, Freeman J, Hui MF, Cheung RK, Dosch HM. Amajority of inverted sinonasal papillomas carries Epstein- Barr virus genomes. Cancer 1995;75: ) Gaffey MJ, Frierson HF, Weiss LM, Barber CM, Baber GB, Stoler MH. Human papillo, avirus and Epstein-Barr virus in sinonasal schneiderian papillomas. An in sity hybridization and polymerase chain reaction study. Am J Clin Pathol 1996;106: ) Lesperance MM, Esclamado RM. Squamous cell carcinoma arising in inverted papilloma. Laryngoscope 1995;105: ) Caruana SM, Zwiebel N, Cocker R, McCormick SA, Eberle RC, Lazarus P. p53 alteration and human papillomavirus infection in paranasal sinus cancer. Cancer 1997;79: ) Garcia RL, Coltrera MD, Gown AM. Analysis of proliferative grade using anti-pcna/cyclin monoclonal antivodies in fixed, embedded tissues. Am J Pathol 1991;34: ) Garcia RL, Coltrera MD, Gown AM. Analysis of proliferative grade using anti-pcna/cyclin monoclonal antibodies in fixed, embedded tissues. AM J Pathol 1991;34: ) Manos MM, Ting Y, Wright DK. Use of polymerase chain reaction amplification for the detection of genital human papillomaviruses in molecular dignostics of human cancer. Furth M, Graves M, editors cancer cell. New York: Cold Spring Harbor ; ) Wood AL, Hall PA, Shepard NAm Hanby AM, Waseem NH, Lane DP, Levison DA. The assessment of proliferating cell nuclear antigen PCNA immunostaining in primary gastrointestinal lymphomas and its relationship to histological grade, S G 2 M phase fraction flow cytometric analysis and prognosis. Histopathology 1991;19: ) Ward N. A miror of the pratice of medicine and surgery in the hospital of London. Hospital Lancet 1854;2: ) Byun JY, Cho JS, Hong IH, Kim WS, et al. A study on the expression of p53 and the detection of Human papilloma virus in the sinonasal inverted papilloma associated with carcinoma. Korean J Otolaryngol 1998;41: ) Park IY, Hong WP, Baik SS, Lim HS, Jo HR. The significance of E-B virus in nasopharyngeal cancer patients. Korean J Ootlaryngol 1986;29: ) Suh JS, Lee JH, Kim YD, Lee TY. Detection of Ebstien-Barr virus in nasopharyngeal tissue of healthy subjects by polymerase cahin reaction. Korean J Ootlaryngol 1996;39:

중합효소연쇄반응 (polymerase chain reaction:pcr) 에의한 HPV와 EBV의검출 Table 1. Oligonucleotide primers of PCR for HPV and EBV Nucleotide sequence Product size Huma

중합효소연쇄반응 (polymerase chain reaction:pcr) 에의한 HPV와 EBV의검출 Table 1. Oligonucleotide primers of PCR for HPV and EBV Nucleotide sequence Product size Huma KISEP Head and Neck Korean J Otolaryngol 2001;44:0611 상악암에서중합효소연쇄반응을이용한인형유두종바이러스와 EpsteinBarr Virus 의검출 조재식 임상철 정형수 임회정 김정현 The Detection of Human Papillomavirus and EpsteinBarr Virus DNA in Maxillary