교신저자 : 김민식, 137-040 서울 서초구 반포동 505 가톨릭대학교 의과대학 이비인후과학교실 전화 : (02) 2258-6211 · 전송 : (02) 595-1354 E-mail : [email protected]
대한 두경부 종양 학회지 제 27 권 제 1 호 2011
Relationship between High Risk Human Papillomavirus and Young Patients and Nonsmokers in Squamous Cell
Carcinoma of the Tonsil in Korea
Young-Hoon Joo, MD
1, Chan-Kwon Jung, MD
2, Kwang-Jae Cho, MD
1, Dong-Il Sun, MD
1, Jun-Ook Park, MD
1, Min-Sik Kim, MD
1Department of Otolaryngology-Head and Neck Surgery
1and Pathology,
2College of Medicine,
The Catholic University of Korea, Seoul, Korea
한국인 편도 편평상피암종에서 고위험 인간 유두종 바이러스와 젊은 연령 및 비흡연자와의 관계
가톨릭대학교 의과대학 이비인후과학교실,1 병리학교실2
주영훈1·정찬권2·조광재1·선동일1·박준욱1·김민식1
= 국 문 초 록 =
연구 및 목적
이번 연구의 목적은 한국인에서 편도 편평세포암종의 발생에 고위험 인간유두종 바이러스(high risk human pap- illomavirus)의 역할 및 관여 인자에 대해 밝히고자 한다.
재료 및 방법
연구는 편도 편평상피세포암종으로 진단받은 54명을 대상으로 하였다. 고위험 인간유두종 바이러스의 감염을 알 기위해 in situ hybridization 방법을 이용하였다. 고위험 인간유두종 바이러스의 감염과 나이, 성별, 흡연, 음주, 병기, 병리학적 특징 등과의 관계를 분석하였다.
결 과
고위험 인간유두종 바이러스의 양성율은 31.5%(17/54)였다. 고위험 인간유두종 바이러스 감염은 젊은 연령(50세 미만), 비흡연자, 림프절 전이와 유의한 상관관계가 있었다(각각 p=0.008, p=0.042, p=0.027). 하지만 성별, 음주, 원발부위 병기, 종양분화도, 피막외침범, 혈관 및 신경 침범과 고위험 인간유두종 바이러스와의 관계는 통계적으 로 유의하지 않았다. 6개월 이상 추적 관찰이 가능한 환자를 대상으로 조사한 5년 전체 생존율 및 질병 특이 생존율 은 각각 60%와 62%였다. 고위험 인간유두종 바이러스의 양성율은 질병 특이 생존율과 유의한 상관관계가 있었다 (p=0.019).
결 론
한국인의 고위험 인간유두종 바이러스 감염은 젊은 연령 및 비흡연자의 편도 편평세포암종의 발생에 관련이 있고 예후 인자로 중요한 역할을 한다.
중심 단어：편도종양ㆍ인간유두종 바이러스 16ㆍ인간유두종 바이러스 18ㆍ나이인자ㆍ흡연.
online©MLComm
Introduction
Squamous cell carcinoma(SCC) of the tonsil represents 15% to 20% of all oropharyngeal malignancies and is the most common type of oropharyngeal carcinoma.1) Recent data shows that the incidence of tonsillar cancer has in- creased between 2 and 3% per year from 1973 to 1995 in Caucasian and African-American men, respectively.2) Annu- ally, 3400 cases of head and neck cancer develop, account- ing for 2.6% of all new Korean cases.3) Oropharyngeal SCC is the third most frequent head and neck cancer following laryngeal and oral cavity cancers, and tonsillar SCC ac- counts for 6.5% of all head and neck cancers.
Tobacco smoking and the heavy use of alcohol are proba- bly the best known risk factors for SCC of the head and neck.
Around 4-10% of patients with this disease are not exposed to these risk factors, and only a small proportion of tobacco- exposed individuals develop SCC of the head and neck.4) Re- cently, human papillomavirus(HPV) has been implicated in the literature as an etiologic factor in tonsillar SCC. Some studies suggest that HPV, especially type 16 and 18, may be responsible for a small subgroup of oral squamous cell carci- noma and up to 50% of oropharyngeal squamous cell carci- nomas, especially the tonsillar squamous cell carcinoma.5,6) A strong association between the presence of HPV and ex- pression of key cell cycle proteins has been reported in a se- ries of tonsillar squamous cell carcinoma.5,6) Because of dif- ferences in risk factors and outcomes, the development and clinical behavior of HPV-positive tumors can be considered a discrete entity.5-7)
The aim of the present study was to examine the presence of HPV in samples of the tonsillar cancer, collected from Korea. Furthermore, clinicopathological characteristics and disease outcome were correlated with HPV status.
Patients and Methods
1. Patients and tumor samples
The clinical and pathological data of patients who were diagnosed with tonsillar carcinoma and underwent surgery at the Department of Otolaryngology-HNS, The Catholic University of Korea, Seoul, Korea, from 1994 to 2009, were reviewed. The study group consisted of 54 patients with previously untreated SCC of the tonsil whose original path- ological specimens were available for evaluation and for whose follow-up was possible. The follow-up duration ranged from 2 to 128 months, with a mean of 38.7 months. All pa-
tients were treated with primary tumor resection and neck dissection and 31 patients(57.4%) patients received postop- erative radio(chemo) therapy. The patients were staged ac- cording to the 2002 edition of the TNM classification of the American Joint Committee on Cancer(AJCC). The Institution- al Review Board of Seoul St Mary’s Hospital(Seoul, Korea) approved the retrospective review of the medical records and the use of archived tumor specimens.
All archival tissue samples were routinely fixed in forma- lin and embedded in paraffin. Representative tissue areas were marked on standard hematoxylin and eosin sections, punched out of the paraffin block using a 2.0-mm punch, and inserted in a recipient paraffin block, to produce a 5×6 array of 30 cases. Duplicate tissue cores per specimen were arrayed on a recipient paraffin block in order to decrease the error introduced by sampling and to minimize the im- pact of tissue loss during processing. Thirty tissue cores were arrayed on one slide. Sections(5µm) were cut from the completed array blocks and transferred to silanized glass slides.
2. Fluorescence in situ hybridization
Tumor slides were used to perform chromogenic in situ hybridization(ISH), according to standard protocols. The probe sets used for the assay included are well validated to identify low- or high-risk HPV(Ventana Medical Systems).
The high-risk family 16 probe cocktail has an affinity to high- risk HPV genotypes 16, 18, 31, 33, 35, 39, 51, 52, 56, 58, and 66. The low-risk family 6 probe cocktail has an affinity to HPV genotypes 6 and 11. The slides were examined for the presence of integrated HPV using light microscopy. Positive signal is indicated by dark blue dotlike staining within the cytoplasm of tumor cells. Copy number can be estimated by comparison to the controls, which are xenograft controls that have known copy numbers. Internal negative controls were ex- amined for each tissue section.
3. Semiquantitative analyses of FISH result
Evaluation of nuclear hybridization signal was assessed by a pathologist specialized in head and neck pathology(CK Jung). Signal morphology was categorized as follows : 1) When nuclei were completely and homogeneously stained, the signal was classified as diffuse. Often, the other nuclei exhibited multiple fluorescent signals with variation in spot number between individual cells within the lesion ; 2) When nuclear signals varied significantly in size and intensity this pattern was termed granular and 3) Discrete nuclear signals (1-3 per nucleus) in a clean background were classified as punctuate.
4. Statistical analysis
To determine the significance between high risk HPV and clinicopathological factors(such as age, gender, smoking status, alcohol consumption, T stage, cervical metastasis, tumor cell differentiation, extracapsular spread, perineural invasion, perivascular invasion), the chi-square test, Fisher’s exact test, multiple logistic regression analysis, multiple lin- ear regression analysis, and correlation analysis were used, as appropriate. The overall survival was determined using the Kaplan-Meier method. p＜0.05 was considered statisti- cally significant. All calculations were performed using SPSS software ver. 13.0(SPSS, Chicago, IL, USA).
Results
1. Patient characteristics
The mean age of the subjects was 56(range from 27 to 77) years, and the male-to-female ratio was 48 : 6. Smoking habit was recorded in all patients: non-smoker(24 never smoked, 10 stopped at least 1year ago at the time of surgery) or smok- er(20 patients continued to smoke). Alcohol consumption was recorded : 15 non-drinker, 23 light drinker, 16 heavy drinker. Regarding their pathological stages, there were 9 (17%), 35(65%), 9(17%), and 1(2%) patients with stage T1 to T4 cancers, respectively. Concerning the disease stage of the cervical lymph nodes, there were 20(37%), 8(15%), and 26(48%) cases with N0 to N2, respectively. Regarding tu- mor cell differentiation, there were 10(19%), 38(70%), and 6 (11%) well-(G1), moderate-(G2), and poorly- differentiated (G3) cases, respectively.
2. Relationship between high risk HPV and 2. clinico-pathological factors
High risk HPV in situ hybridization was observed in 17 (31.5%) of 54 tonsillar SCC patients(Fig. 1). We analyzed the correlation between high risk HPV and several clinical fac- tors in all 54 patients with tonsillar SCC(Table 1). There was a significant correlation in high risk HPV and younger age (younger than 50 years of age), non-smoking status and cer- vical metastasis(p=0.008, p=0.042 and p=0.027, respective- ly), while there was no significant relationship between gen- der, alcohol consumption, T stage, tumor cell differentiation, extracapsular spread, perineural invasion, perivascular in- vasion and high risk HPV. In the multivariate analysis, there was no relationship between high risk HPV and clinicopath- ological factors.
3. Relationship between high risk HPV and survival rate
Patients who were followed for more than 6 months had a5-year overall survival and disease-specific survival rate of 60% and 62%. The 5-year disease-specific survival rates of the 17 patients with high risk HPV positivity was 86%, where-
Fig. 1. Positive in situ hybridization staining in squamous cell car- cinoma of the tonsil. The blue dotlike pattern is indicative of positive staining(human papillomavirus high-risk probe, original magnification ×400).
Table 1. Demographic Profiles and association with incidence of high risk human papillomavirus in patients with tonsillar squa- mous cell carcinoma
Parameter No of
patients(%) High risk HPV positivity(%) p-value Age(years)
≤50
＞ 50 Gender
Male Female
Tobacco consumption Yes
No
Alcohol consumption Yes
No
T classification T1 and T2 T3 and T4 Cervical metastasis
Yes No
Histologic differentiation Well/Moderately Poorly
Extracapsular spread Yes
No
Perineural invasion Yes
No
Perivascular invasion Yes
No
15(27.8) 39(72.2)
48(88.9)
06(11.1)20(37.0) 34(63.0)
16(29.6) 38(70.4)
44(81.5) 10(18.5)
34(63.0) 20(37.0)
48(88.9)
06(11.1)23(42.6) 31(57.4)
07(13.0)
47(87.0)
08(14.8)
46(85.2)
60.0 20.5
29.2 50.0
15.0 41.2
25.0 34.2
31.8 30.0
20.6 50.0
31.2 33.3
34.8 29.0
42.9 29.8
37.5 30.4
0.008*
0.275
0.042*
0.371
0.615
0.027*
0.623
0.437
0.384
0.491
as those of the 37 high risk HPV negative patients was 50%
(Fig. 2). High risk HPV status was statistically significant prognostic factor for disease-specific survival(p=0.019).
The 5-year disease-specific survival rates with cervical lymph node metastasis was 52%, whereas those of the metastasis- free patients was 78%. However, this result did not reach sta- tistical significance(p=0.097). Age, gender, smoking and alcohol consumption, T stage, tumor cell differentiation, extra- capsular spread, perineural invasion, perivascular invasion, and postoperative radiotherapy were not associated with dis- ease-specific survival. In a multivariate survival analysis, there was no adverse factor affecting disease-specific survival.
Discussion
Tonsillar carcinoma is not uncommon, representing 15- 20% of all oropharyngeal malignancies.1) A well documented association has been established between tobacco and alco- hol use and the development of tonsillar carcinoma, howev- er, more commonly individuals without a history of such
traditional risk factors have been witnessed to develop ton- sillar carcinoma.2) This phenomenon has led to intense inves- tigation regarding the role of non-traditional risk factors for the development of tonsillar carcinoma, such as viral patho- gens. HPV are small epitheliotropic DNA viruses that have been demonstrated as the etiologic agent in such benign pro- cesses as hyperplastic, papillomatous, and verrucoid squa- mous epithelial lesions.8,9) Although over a 100 HPV geno- types have been described, HPV-6 and HPV-11 are considered benign while HPV-16 and HPV-18 are considered malignant because of their oncogenic genotypes.
Over the past several decades, South Korea has experi- enced rapid socioeconomic growth; indeed, one of the high- est growth rates in the world. Korea also went through dra- matic changes in its demography and lifestyle. The number and proportion of elderly people is growing, and Korea has one of the fastest growing populations among the world’s major regions. As South Korea went through rapid econom- ic development, there were significant changes in diet and nutrition in the population. Recently, incidence of oropha- ryngeal cancer in the younger patient group is increasing in Korea.3) Young people are probably less exposed to carcino- gens that lead to a malignant transformation. Other causes or factors of oropharyngeal cancer in young people have not yet been disclosed. In this series, nine of 15 patients(60.0%) younger than 50 years had high risk HPV positivity, while 8 out of 39 patients older than 50 years had high risk HPV negativity. Therefore, our findings suggest that younger pa- tients with high risk HPV positivity are at increased risk for SCC of the tonsil.
Adult smoking rate in South Korea was 30.4% in 2001 and it was one of the highest prevalence among Organization for Economic Co-operation and Development(OECD) member countries based on Health statistics by the OECD reports.10) Recently, Korean government rapidly increased the spend- ing on public antismoking campaign and passed the laws prohibiting smoking in public places as well as requiring private workplaces to be non smoking area.
In this study, smoking rate was 30.7%. Thirty-four non- smokers in our patient group had a 41.2% high risk HPV pos- itive rate, while 20 current smokers had the 15.0% positive rate by the univariate analysis.
Conclusion
High risk HPV infection is significantly associated with the SCC of the tonsil among young patients and nonsmokers in Korean population.
Fig. 2. Kaplan-Meier disease-specific survival curve(A) and dis- ease-specific survival curve according to the high risk HPV status(B).
1.0 0.8 0.6 0.4 0.2
0.0
0 25 50 75 100 125 F/U month
Cum survival
A
1.0 0.8 0.6 0.4 0.2
0.0
0 25 50 75 100 125 F/U month
Cum survival
B
p=0.019
High risk HPV positive, 86%
High risk HPV negative, 50%
