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Clinical Effect of Human Papillomavirus Genotypes in Patients With Cervical Cancer Undergoing Primary Radiotherapy
Int. J. Radiation Oncology Biol. Phys., Vol. 78, No. 4, pp. 1111–1120, 2010 Copyright Ó 2010 Elsevier Inc. Printed in the USA. All rights reserved 0360-3016/$–see front matter
doi:10.1016/j.ijrobp.2009.09.021
CLINICAL INVESTIGATION
Cervix
CLINICAL EFFECT OF HUMAN PAPILLOMAVIRUS GENOTYPES IN PATIENTS WITH CERVICAL CANCER UNDERGOING PRIMARY RADIOTHERAPY CHUN-CHIEH WANG, M.D., PH.D.,*y CHYONG-HUEY LAI, M.D.,z HUEI-JEAN HUANG, M.D.,z ANGEL CHAO, M.D., PH.D.,z CHEE-JEN CHANG, PH.D.,x TING-CHANG CHANG, M.D.,z HUNG-HSUEH CHOU, M.D.,z AND JI-HONG HONG, M.D., PH.D.*y Departments of *Radiation Oncology and zObstetrics and Gynecology, Chang Gung Memorial Hospital, Taoyuan, Taiwan; xGraduate Institutes of Clinical Medical Sciences, and yDepartment of Medical Imaging and Radiological Science, Chang Gung University School of Medicine, Taoyuan, Taiwan Purpose: To study the prognostic value of the human papillomavirus (HPV) genotypes in cervical cancer patients undergoing radiotherapy. Patients and Methods: A total of 1,010 patients with cervical cancer after radiotherapy between 1993 and 2000 were eligible for this study. The HPV genotypes were determined by a genechip, which detects 38 types of HPV. The patient characteristics and treatment outcomes were analyzed using the Cox regression hazard model and classification and regression tree decision tree method. Results: A total of 25 genotypes of HPV were detected in 992 specimens (98.2%). The leading 8 types were HPV16, 58, 18, 33, 52, 39, 31, and 45. These types belong to two high-risk HPV species: alpha-7 (HPV18, 39, 45) and alpha-9 (HPV16, 31, 33, 52, 58). Three HPV-based risk groups, which were independent of established prognostic factors, such as International Federation of Gynecology and Obstetrics stage, age, pathologic features, squamous cell carcinoma antigen, and lymph node metastasis, were associated with the survival outcomes. The high-risk group consisted of the patients without HPV infection or the ones infected with the alpha-7 species only. Patients co-infected with the alpha-7 and alpha-9 species belonged to the medium-risk group, and the others were included in the lowrisk group. Conclusion: The results of the present study have confirmed the prognostic value of HPV genotypes in cervical cancer treated with radiotherapy. The different effect of the alpha-7 and alpha-9 species on the radiation response deserves additional exploration. Ó 2010 Elsevier Inc. Human papillomavirus, Cervical cancer, Radiotherapy, Prognosis.
Cervical cancer is the second most common cancer among women worldwide. Infection with the human papillomavirus (HPV) is suggested as the initiating event of cervical cancer, and the prevalence of HPV in women with cervical cancer is 95–100% using current techniques (1–3). More than 100 HPV genotypes have been identified and classified by their L1 open reading frame (4). When HPVs share 60–70% nucleotide identity, they are clustered into the same species. Two HPV species, alpha-7 (HPV18, 39, 45, 59, 68, and 70) and alpha-9 (HPV16, 31, 33, 35, 52, 58, and 67), are responsible for >80% of all cervical cancer cases (5). The HPV genotypes could pose differing prognoses for cervical cancer patients, in addition to a number of prognostic factors, such as the International Federation of Gynecology
and Obstetrics stage, lymph node status, patient age, histologic features, or squamous cell carcinoma antigen (SCCAg) level. Accumulating research, including our recent study of patients who underwent primary surgery (6), has suggested that HPV18 infection is associated with a more aggressive form of cervical cancer than other HPV infections (7–9). In addition, some studies have shown that the presence of an HPV33-related tumor has a favorable outcome (10, 11). However, other reports have not been able to identify the link between HPV genotypes and clinical outcome (12–14). These inconsistent results have highlighted the problem of determining the prognostic role of HPV genotypes in cervical cancer. The small numbers of patients studied, insufficient follow-up, lack of accounting for known prognostic factors, and variable laboratory techniques have made the
Reprint requests to: Ji-Hong Hong, M.D., Ph.D., Department of Radiation Oncology, Chung Gung Memorial Hospital, No. 5, Fu-Shin St., KweiShan, Taoyuan, Taiwan. Tel: (886) 3-328-2177; Fax: (886) 3-328-0797; E-mail: [email protected] Supported by the National Science Council-Taiwan (Grant
NSC94-2314-B-182-011) and Chang Gung Memorial Hospital (Grant CMRPG340693). Conflict of interest: none. Received April 10, 2009, and in revised form Sept 4, 2009. Accepted for publication Sept 4, 2009.
INTRODUCTION
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interpretation of published results difficult. The heterogeneity of treatment modalities within and among studies has also made this issue perplexing. Because radiotherapy (RT) is one of the mainstay treatments of cervical cancer, we report the prognosis of cervical cancer patients in relation to the HPV genotypes analyzed using a highly sensitive DNA chip in a large series of patients primarily treated with RT. PATIENTS AND METHODS Patient population Between August 1993 and May 2000, a list of consecutive patients with invasive cervical cancer at Chang Gung Memorial Hospital was retrieved with the approval of the institutional review board. The data of the 1,067 patients who had undergone primary surgery have been previously published (6). Samples from the other 1,036 patients who had undergone RT as their primary treatment were genotyped for HPV infection. Of the 1,036 patients, 26 were excluded because of having undergone incomplete RT (dose <50 Gy); thus, 1,010 patients were eligible for the present analysis.
HPV genotyping The detailed procedure for the HPV genotyping has been previously reported (6, 15). In brief, DNA was extracted from paraffin-embedded specimens and used as a template for the amplification of the L1 open reading frame with the biotinylated GP6+ and SPF1 consensus primers. The resultant amplifiers were then hybridized with the Easychip HPV Blot Membrane (King Car, I-Lan, Taiwan), which detects 38 types of HPV (6, 11, 16, 18, 26, 31, 32, 33, 35, 37, 39, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72, 74, 81, 82, 83, 84, and L1AE5). After hybridization, the membranes were incubated with Avidex-AP (Applied Biosystems Inc. Foster City, CA), and then with NBT/BCIP tablet (Sigma-Aldrich, St. Louis). The results were determined according to the HPV type format on the chip. Samples with HPV-negative results were reconfirmed by polymerase chain reaction of glyceraldehydes-3-phosphate dehydrogenase to validate the DNA quality and the leading 8 types of HPV. The samples with multiple types on HPV blotting were verified by E6 type-specific polymerase chain reaction.
Surveillance and treatment The pretreatment workup and radiation technique were identical to those reported in our previous study (16). Pelvic and abdominal computed tomography or magnetic resonance imaging, chest Xray, and serum marker analysis (SCC-Ag and carcinoembryonic antigen) was performed before treatment. Patients usually underwent a combination of external beam RT and intracavitary brachytherapy. External beam RT was delivered using a daily fraction of 1.8–2 Gy in five fractions weekly. The initial radiation dose to the whole pelvis was 40–45 Gy. The irradiation fields were extended to the abdominal para-aortic region when involvement of the para-aortic nodes was suspected from the imaging findings. For the patients who underwent brachytherapy, a parametrial boost was given to a total dose of #54–58 Gy. Brachytherapy was administrated in six fractions with 4.3 Gy/fraction to Point A. For the patients who did not undergo brachytherapy, a total dose of 68–72 Gy was given to the primary tumor. Cisplatin-based chemotherapy was administered to 270 patients. Of the 270 patients, 90% were treated with concurrent chemoradiotherapy (CCRT), which was adopted as the standard treatment of cervical cancer after August 1999 (115 patients) at our hospital. Patients were followed regularly to May 2007, with a min-
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imal follow-up period of for 7 years. The post-therapy surveillance protocol was the same as that used in our recent study (6).
Statistical analysis The data were analyzed using the Statistical Package for Social Sciences, version 11 (SPSS, Chicago, IL). Pearson’s chi-square test was used to evaluate the association between the variables. Survival curves were generated using the Kaplan-Meier method, and comparisons were made using the log–rank test. Multivariate analyses of the factors associated with local and distant failure were done using Cox regression analysis. Classification and regression tree (CART) analysis was performed using Answer Tree software, version 2.01 (SPSS). Patients with small cell carcinoma (n = 11) were excluded from the present analysis because of its rarity and distinct clinical course. The remaining 999 patients were considered as the primary node. The minimal node size was set to 5, and the group size (parental node) was $30 cases. The missing data for SCC-Ag and carcinoembryonic antigen were imputed using their medium values (4.5 and 3.1, respectively). The survival differences between the CART nodes were further verified using the log–rank test.
RESULTS Clinical characteristics and HPV genotypes The clinicopathologic characteristics of the 1,010 patients are summarized in Table 1. The patient age range was 21–91 years (median, 61). Squamous cell carcinoma was the most common cell type (92%), followed by adenocarcinoma/adenosquamous carcinoma (6.9%) and small cell carcinoma (1.1%). Regarding HPV status, only 18 patients (1.8%) had HPV-negative tumors. In the 992 HPV-positive specimens, 25 genotypes of HPV were detected. The leading eight genotypes were HPV16 (36%), HPV58 (26.2%), HPV18 (18.9%), HPV33 (13.4%), HPV52 (8.2%), HPV39 (5.4%), HPV31 (3.4%), and HPV45 (2.6%). These types belong to two high-risk HPV species: alpha-7 (HPV18, 39, and 45) and alpha-9 (HPV16, 31, 33, 52, and 58). The other HPV genotypes had a prevalence rate of <2%. Of the HPV-positive specimens, 204 (20.6%) contained two types and 58 (5.8%) were infected by more than two types of HPV. All samples with multiple infections had at least one HPV type that was a member of the alpha-7 or alpha-9 species. To reduce the interference among the different HPV genotypes, the 730 patients whose specimens contained only one HPV type were analyzed first. The association between the HPV genotypes and various clinicopathologic characteristics is summarized in Table 2. HPV33, 52, and 58 were common (>50%) in patients >65 years old. The two leading types in those with squamous cell carcinoma were HPV16 (38.1%) and HPV58 (23.1%). In contrast, the predominant type in those with adenocarcinoma/adenosquamous carcinoma and small cell carcinoma was HPV18. HPV18 infection was associated with a probability of having nodal metastasis detectable on computed tomography/magnetic resonance imaging as great as 58.3% in patients with late-stage disease (Stage IIIa-IVa). In patients with early-stage disease, the presence of HPV52 and 58 was associated with a relatively low incidence of nodal metastasis, 7.1% and 8%, respectively. Tumors with the alpha-9 species tended to have greater
Clinical effect of HPV in cervical cancer d C.-C. WANG et al.
Table 1. Population characteristics (n = 1,010) (Continued )
Table 1. Population characteristics (n = 1,010) Characteristic Age (y) <45 45–65 >65 Stage Ib IIa IIb III IVa Pathologic type SCC AC/ASC Small cell carcinoma Differentiation Well to moderate Poor Unclassified SCC-Ag level <1.5 1.5–4 >4 Missing CEA level #5 >5 Missing LN metastasis No Yes Undetermined HPV infection Negative Positive Single Multiple
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n (%) 120 (11.9) 482 (47.8) 408 (40.3) 202 (20) 191 (18.9) 341 (33.8) 255 (25.2) 21 (2.1) 930 (92) 69 (6.9) 11 (1.1) 484 (47.9) 346 (34.3) 180 (17.9) 258 (25.5) 183 (18.1) 472 (46.7) 97 (9.6) 600 (59.4) 223 (22.1) 187 (18.5) 778 (77) 204 (20.2) 28 (2.8) 18 (1.8) 992 (98.2) 730 (73.6) 262 (26.4) (Continued )
expression of SCC-Ag: >50% had an SCC-Ag level >4 ng/ mL compared with approximate 30% of those with the alpha-7 species (p < .0001). For the 262 specimens with multiple HPV infections, HPV18 (44.7%) and HPV58 (43.9%) were the two predominant types, with HPV16 (37%) third. The incidence of adenocarcinoma/adenosquamous in the multiple HPV group (10.7%) was greater than that in the single HPV group (5.2%; p = .004). In the whole cohort, 17.9% of HPV18-positive tumors were adenocarcinoma/adenosquamous carcinoma, and similar patterns were also observed for the other two members of the alpha-7 species: HPV39 (14.5%) and HPV45 (15.4%). In contrast, the prevalence of adenocarcinoma/adenosquamous carcinoma in the alpha-9 species was relatively low, ranging from 0% for HPV52 to 7.5% for HPV33 (HPV16, 2.8%; HPV31, 2.9%; HPV52, 0%; and HPV58, 6.4%; p < .0001). Follow-up data and survival analysis After a median follow-up of 78 months, 471 patients (46.6%) were still alive, and 390 (38.6%) had experienced
Characteristic
n (%)
Genotype 16 Single Multiple 18 Single Multiple 31 Single Multiple 33 Single Multiple 39 Single Multiple 45 Single Multiple 52 Single Multiple 58 Single Multiple 26, 35, 42, 44, 51, 53, 54, 56, 59, 61, 66, 67, 68, 69, 70, 74, 82, and 84
364 (36.7) 267 (36.6) 97 (37) 191 (19.2) 74 (10.1) 117 (44.7) 34 (3.4) 22 (3) 12 (4.6) 135 (13.6) 68 (9.3) 67 (25.6) 55 (5.5) 19 (2.6) 36 (13.7) 26 (2.6) 15 (2) 11 (4.2) 83 (8.4) 53 (7.3) 30 (11.5) 265 (26.7) 150 (20.5) 115 (43.9) <2)
Abbreviations: SCC = squamous cell carcinoma; AC/ASC = adenocarcinoma/adenosquamous carcinoma; SCC-Ag = SCC antigen; CEA = carcinoembryonic antigen; LN = lymph node; HPV = human papillomavirus. Data presented as numbers of patients, with percentages in parentheses.
treatment failure, including 212 local failures and 253 distant relapses (75 patients had both). Of the 539 deaths, 175 patients (32.5%) had died of intercurrent disease. These patients were censored at the date of death for the calculation of disease-specific survival (DSS). The 5- and 10-year overall survival rate for the entire cohort was 58.6% and 44.1%, respectively. The corresponding DSS rates were 66.9% and 60.9%. On univariate and multivariate analyses (Table 3), patient age, stage, pathologic features, SCC-Ag level, and lymph node metastasis status were confirmed as prognostic indicators for overall survival and DSS. Although previous studies have suggested that the number of HPV infections is associated with the tumor response after RT (17–19), this factor did not achieve statistical significance on multivariate analysis (p = .19 and p = .201 for overall survival and DSS, respectively). A trend toward a poor prognosis for the HPVnegative group in DSS compared to the HPV-positive group was noted on Kaplan-Meier analysis (p = .085). CART modeling To further explore the effect of the individual HPV genotypes on the results of RT for cervical cancer, CART modeling was performed. The variables listed in Table 2, the predominant eight HPV genotypes, and the alpha-7 and
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HPV genotype Single Variable
18
31*
33*
39
45
52*
39 (14.6) 147 (55.1) 81 (30.3)
13 (17.6) 41 (55.4) 20 (27)
4 (18.2) 9 (40.9) 9 (40.9)
3 (4.5) 24 (35.8) 40 (59.7)
0 11 (57.9) 8 (42.1)
2 (13.3) 7 (46.7) 6 (40)
1 (1.9) 20 (37.7) 32 (60.4)
198 (74.2) 69 (25.8)
61 (82.4) 13 (17.6)
15 (68.2) 7 (32.8)
48 (70.6) 20 (29.4)
14 (73.7) 5 (26.3)
11 (73.3) 4 (26.7)
41 (77.4) 12 (22.6)
260 (38.1)y 7 (18.4)y 0
52 (7.6) 20 (52.6) 5 (62.5)y
23 (3.4) 1 (2.6) 0
55 (8.0) 1 (2.6) 1 (12.5)
19 (2.8) 1 (2.6) 0
13 (1.9) 2 (5.2) 0
55 (23.1) 53 (22.3) 130 (54.6)
30 (44.8) 15 (22.4) 22 (32.8)
5 (25) 3 (15) 12 (60)
19 (30.2) 12 (19) 32 (50.8)
158 (72.8) 59 (27.2)
38 (62.3) 23 (37.7)
14 (77.8) 4 (22.2)
40 (74.1) 14 (25.9)
40/192 (20.8) 15/68 (22.1)
17/64 (26.6) 7/12 (58.3)
3/14 (21.4) 1/8 (12.5)
8/41 (19.5) 5/16 (31.3)
7 (41.2) 5 (29.4) 5 (29.4) 12 (75) 4 (25) 4/14 (28.6) 1/5 (20)
Abbreviations as in Table 1. Data presented as numbers of patients, with percentages in parentheses, unless otherwise specified. * Alpha-9 species. y Prevalence in each pathologic type.
58* 11 (7.3) 59 (39.3) 80 (53.3)
All
Multiple
79 (10.8) 341 (46.6) 311 (42.5)
38 (14.6) 130 (49.8) 93 (35.6)
108 (72) 42 (28)
538 (73.6) 193 (26.4)
183 (70.1) 78 (29.9)
54 (7.8) 0 1 (12.5)
158 (23.1) 5 (13.2) 0
684 (93.7) 38 (5.2) 8 (1.1)
229 (88.1) 28 (10.7) 3 (1.2)
5 (35.7) 5 (35.7) 4 (28.6)
12 (23.1) 10 (19.2) 30 (57.7)
31 (24.2) 20 (15.6) 77 (60.2)
185 (28.2) 131 (20) 339 (51.8)
69 (28.4) 50 (20.6) 124 (51)
5 (55.6) 4 (44.4)
37 (77.1) 11 (22.9)
83 (73.5) 30 (26.5)
430 (73.1) 158 (26.9)
161 (71.9) 63 (28.1)
2/11 (18.2) 0/4 (0)
3/42 (7.1) 2/12 (16.7)
9/104 (8) 15/46 (32.6)
88/524 (16.8) 54/187 (28.9)
30/176 (17) 28/77 (36.4)
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Age (y) <45 45–65 >65 Stage Early (Ia-IIb) Late (IIIa-IVa) Pathologic type SCC AC/ASC Small cell carcinoma SCC-Ag level <1.5 1.5–4 >4 CEA level <5 >5 LN metastasis Early stage Late stage
16*
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Table 2. Correlations between clinicopathologic factors and HPV genotypes
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Table 3. Univariate and multivariate analysis of clinicopathologic covariates on overall and disease-specific survival Overall survival Variable Stage Early (Ia–IIb) Late (IIIa–IVa) Age (y) <45 45–65 >65 Pathologic type SCC AC/ASC Small cell carcinoma SCC-Ag level <1.5 1.5–4 >4 CEA level #5 >5 LN metastasis No Yes Differentiation Well to moderate Poor Unclassified HPV infection Negative Single Multiple Chemotherapy No Yes
Disease-specific survival
Deaths/total patients (n) 5-y (%) 10-y (%)
p
Deaths/total patients (n) 5-y (%) 10-y (%)
<.001/<.001 345/734 194/276
66.1 38.7
50.8 26.4
67/120 225/482 247/407
50.0 62.2 56.6
44.4 51.5 34.9
479/928 47/69 9/11
60.4 39.1 27.3
45.4 32.3 18.2
109/258 89/182 280/472
68.2 63.4 54.2
54.7 48.8 38.3
297/600 138/223
63.3 49.3
47.9 35.8
390/778 135/204
62.8 42.2
47.4 32.6
261/484 191/346 87/180
58.5 54.6 66.7
43.6 42.8 48.3
11/18 383/730 154/262
44.4 60 55.7
38.1 45.4 40.9
392/740 147/270
60.7 52.6
45.4 41.8
p <.001/<.001
215/734 147/276
74.1 48.3
68.2 42.2
63/120 168/482 131/407
50.4 68.1 71.2
45.9 63.2 63.2
312/928 42/69 6/11
69.3 44.2 44.4
63.4 36.8 29.6
60/258 59/182 198/472
78.6 70.8 62.4
74.7 65.1 54.4
191/600 102/223
71.4 57.7
65.9 49.2
246/778 107/204
71.5 49.9
65.6 44.2
176/484 136/346 50/180
67.6 63.5 73.8
60.2 57.7 71.0
9/18 251/730 105/262
48.5 68.7 63.2
48.5 62.6 57.4
242/740 120/270
70.5 57.7
64.5 52.5
<.001/.041
<.001/.049
<.001/<.001
<.001/<.001
<.001/.016
<.001/.002
<.001/.200
<.001/.149
<.001/.005
<.001/.004
.228/.416
.033/.108
.182/.190
.036/.201
.017/.486
<.001/.667
Abbreviations as in Table 1. p Values presented as univariate/multivariate values.
alpha-9 HPV species were used to identify subsets of patients with distinctly different survival distributions. A summary of this analysis is shown in Fig. 1. The International Federation of Gynecology and Obstetrics stage was the principle discriminator for both DSS and local control. The lymph node status was the first node for the risk of distant metastasis. Similar to the results of the previous multivariate analysis, the pathologic features, patient age, and SCC-Ag level were the other determinants of these classification trees. Regarding the importance of the HPV genotype in the prognosis, the presence of HPV33 predicted for better DSS in patients with advanced squamous cell cancer (p = .029; Fig. 1a). In the same subgroup with negative HPV33 and a high SCC-Ag levels, patients with HPV39 infection had a worse outcome, although it did not achieve statistical significance using the log–rank test (p = .223). In the CART analysis of local control (Fig. 1b), the presence of two members of the alpha-7 species, HPV39 and HPV45, were associated with a greater incidence of local failure in the subgroup with early-stage squamous cell carcinoma (both p < .001). In patients with early-stage adenocarcinoma, HPV16 positivity predicted a trend toward better local control after RT
(p = .094). Similarly, patients in the advanced squamous cell carcinoma group with significantly better local control were those with alpha-9 species infection (p = .005). Chemotherapy was shown to improve local control in those with advanced squamous cell carcinoma without HPV alpha-9 infection; however, it failed to reach statistical significance (5-year control rate, 59.7% vs. 34.5%, respectively; p = .226). In addition, the presence of the alpha-7 species indicated a greater incidence of metastasis in patients with nodal disease and a greater SCC-Ag level (p = .184; Fig. 1c). HPV risk group The results of the CART analysis suggested that various HPV genotypes have a different effect on the prognosis of cervical cancer after RT. In general, these results imply that cervical cancer associated with HPV alpha-7 species has a worse prognosis. To further examine the effect of HPV genotypes on the patients with multiple HPV infections, we divided patients into three subgroups: patients with the alpha-7 types only, those with the alpha-9 types only, and those with both alpha-7 and alpha-9 types. As shown in Fig. 2, patients with the alpha-9 types only had the best outcome for DSS (p = .075) and
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Fig 1. Classification and regression tree (CART) survival analysis to identify which variables were most strongly associated with survival in cervical cancer patients undergoing primary radiotherapy. Number within each square indicates number of events and patients in that subset. (a) Disease-specific survival. (b) Local control. (c) Distant metastasis. FIGO = International Federation of Gynecology and Obstetrics; Im = improvement; SCC = squamous cell carcinoma; Ad/AS = adenocarcinoma/adenosquamous carcinoma; HPV = human papillomavirus; SCC-Ag = SCC antigen; UC = unclassified; W,M/D = well, moderately differentiated; P/D = poorly differentiated; CEA = carcinoembryonic antigen.
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DISCUSSION
Fig 2. Kaplan-Meier estimates of (a) disease-specific survival and (b) local control by human papillomavirus (HPV) genotypes in patients with multiple human papillomavirus infections.
local control (p = .013), followed by the patients with both alpha-7 and alpha-9 types. Patients infected with multiple alpha7 types only had the greatest risk of treatment failure. From that analysis, patients were categorized into low-, medium-, and high-risk groups by their HPV genotypes. The low-risk group consisted of patients who had HPV infections, but excluded those with alpha-7 species positivity. Patients co-infected with the alpha-7 and alpha-9 species were in the medium-risk group. The high-risk group consisted of the patients without HPV infection or the ones infected with the alpha-7 species only. The risk factors associated with DSS, local control, and distant metastasis examined by multivariate analysis are summarized in Table 4. These results were consistent with those from our previous study in which the International Federation of Gynecology and Obstetrics stage, pathologic features, SCC-Ag level, and lymph node status were the significant prognostic factors for DSS (16). The present study has further demonstrated that the HPV risk group is an independent risk factor for DSS. The effect of the HPV group on DSS mainly resulted from the effect of local control, in contrast to the SCC-Ag level and lymph node status, which were strongly associated with distant metastasis.
The presence of HPV DNA has been shown to be essential for the development of invasive cervical cancer (1, 2). However, contradictory results have been presented concerning the influence of HPV genotypes on the clinical outcome of cervical cancer patients (6–9, 11–14, 20). The present study with a large population (1,010 patients) and long-term follow-up (median, 78 months) has illustrated the significant clinical effect of HPV genotypes in cervical cancer patients undergoing primary RT. The presence of HPV alpha-7 species or HPV negativity (high-risk group) was significantly associated with a poorer outcome. Thus, the HPV genotypes should be considered as a biomolecular marker for cervical cancer. In accordance with the results from other groups, patients with HPV-negative tumors tended to have a worse survival rate (9, 18, 21–23), although a contradictory result was suggested by Pilch et al. (20). Recently, HPV infection has also been associated with improved outcomes in patients with squamous cell carcinoma of the head and neck, especially of the oropharynx (24). The better outcome in association with HPV positivity in patients undergoing RT could be attributed to the method of p53 dysfunction. In HPV-positive cancers, most of the loss of p53 function results from the HPV E6-related degradation, which is different from the mutation of p53 in HPV-negative cancers (25). These two types of p53 dysfunction might not be equivalent in terms of their response to RT (26, 27). In addition, cell cycle-related and other genes that are upregulated in HPV-positive tumors could affect the radiation response (28). The high sensitivity and broad spectrum of the chip-based technique enabled us to detect multiple infections and uncommon HPV genotypes, which were seldom discussed in previous studies. For example, HPV18 is the well-known primary HPV type associated with cervical adenocarcinoma (5). Our data have further demonstrated that HPV39 and HPV45 have a similar rate of association with adenocarcinoma/adenosquamous carcinoma. This observation is consistent with the report by Clifford and Franceschi (29). Of the 992 patients with HPV infection, HPV16 was the most prevalent type (36.7%), followed by HPV58 (26.7%) and HPV18 (19.2%). The prevalence of HPV58 in our cohort was relatively greater than the average 3.4% reported in the other studies (30). In our previous study of patients treated with primary surgery, the prevalence of HPV16, 18, and 58 was 63.8%, 16.5%, and 6.8%, respectively (6). This discrepancy could be attributed to the age distribution: the surgery group had a median age of 50 years compared with that of 61 years in the RT group. It has been shown that older patients have a high prevalence of HPV 52 and 58 infection by several epidemiologic studies (9, 31, 32) and better DSS after RT (16). However, older patients have tended to have short follow-up periods for overall survival owing to the high risk of death from intercurrent disease. This could explain the observation that patients with HPV52 or 58 infections had the worst overall survival in some studies (10).
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Table 4. Multivariate analysis of potential predictors of disease-specific survival, local control and distant metastasis Disease-specific survival Variable Stage Ib IIa-IIb III IVa Age (y) <45 45–65 >65 Pathologic type SCC AC/ASC Small cell carcinoma SCC-Ag level <1.5 1.5–4 >4 CEA level #5 >5 LN metastasis No Yes Differentiation Well Moderate Poor HPV (risk group) Low High Medium Chemotherapy No Yes
RR (95% CI)
Local control p
p
RR (95% CI)
<.001 1 1.383 (1.043–1.833) 2.624 (1.974–3.488) 2.795 (1.489–5.245)
Distant metastasis
<.001 1 1.670 (1.133–2.462) 4.213 (2.862–6.202) 5.563 (2.619–11.814)
<.001
<.001
.240 1 1.256 (0.829–1.903) 1.393 (0.940–2.064) .028 1 0.684 (0.484–0.967) .825 1 0.995 (0.710–1.392) .144 1 0.779 (0.567–1.071) 0.720 (0.479–1.083)
.020
.113 1 1.142 (0.869–1.501) 0.743 (0.499–1.108)
<.001 1 2.028 (1.429–2.878) 1.437 (0.975–2.117)
.536 1 1.085 (0.838–1.404)
<.001 1 2.124 (1.601–2.816)
.062
1 1.485 (1.121–1.968) 1.131 (0.834–1.535)
.116 1 1.253 (0.939–1.671)
.002
1 0.970 (0.769–1.223) 0.676 (0.485–0.941)
.002 1 1.814 (1.214–2.710) 1.976 (1.356–2.881)
.588
1 1.452 (1.135–1.858)
<.001 1 2.032 (1.283–3.219) 4.579 (1.896–11.059)
.001
1 1.066 (0.834–1.362)
.097 1 0.706 (0.491–1.015) 0.694 (0.470–1.023)
1 3.749 (2.444–5.750) 3.032 (1.057–8.693)
1 1.729 (1.239–2.413) 1.795 (1.310–2.461)
.003
.001 1 0.545 (0.376–0.790) 0.467 (0.313–0.699)
1 2.326 (1.617–3.346) 2.547 (1.081–5.997)
p
1 1.050 (0.760–1.451) 1.684 (1.209–2.348) 0.916 (0.356–2.360)
.051 1 0.692 (0.511–0.938) 0.728 (0.527–1.005)
RR (95% CI)
.603 1 1.095 (0.763–1.571) 1.158 (0.814–1.648)
.393 1 0.869 (0.629–1.2)
.243 1 1.206 (0.881–1.652)
Abbreviations: RR = relative risk; CI = confidence interval; other abbreviations as in Table 1.
In terms of the prognostic effect, several previous studies have tried to group some HPV genotypes for analysis. For example, HPV18 was grouped with HPV45 (9), and HPV33, 52, and 58 were considered related types (11). In the present study, HPV grouping according to their DNA similarity was supported by the results of the CART analysis. In the CART analysis, the presence of the alpha-7 species, HPV39 or HPV45, was associated with a worse prognosis. In contrast, women whose tumors harbored the alpha-9 species, HPV16 or HPV33, had a better outcome. It was not surprising that the results from the univariate analysis (Table 3) showed that the use of chemotherapy conferred a worse prognosis because the selection for chemotherapy was usually determined by high-risk clinical features during the study period. However, that HPV genotypes could affect the efficacy of CCRT in patients with advanced squamous cell carcinoma of the cervix was suggested by the CART analysis results. With regard to the incidence of multiple HPV infections, 26.4% of the patients enrolled in the present study presented with two or more genotypes. These data are slightly greater
than those reported in some previous studies, in which multiple infections were documented in 4–18% of patients (19, 32–34). Bachtiary et al. (17) reported a high incidence (43.7%, n = 106) of patients infected with multiple HPV types. Also, this group of patients had a significantly greater risk of disease progression than patients with a single HPV infection after RT. A similar conclusion was suggested by Munagala et al. (19), who demonstrated nearly fivefold greater treatment failure in cases of multiple infections compared to cases of single infection (57% vs. 12%) in a cohort of 43 patients. These observations are consistent with the results of our univariate analysis; however, the presence of multiple HPV infections was not a statistically significant prognostic factor on multivariate analysis (Table 3). Our data also showed that cervical cancer with multiple HPV infections is a heterogeneous group, in which patients with both alpha-7 and alpha-9 infections had an outcome better than the patients with alpha-7 infection only and worse than those with alpha-9 infection only (Fig. 2). Therefore, a risk grouping according to the HPV genotype was derived for our cervical cancer patients undergoing primary RT. These results
Clinical effect of HPV in cervical cancer d C.-C. WANG et al.
are consistent with those from several studies that HPV18-related cervical cancer, particularly if diagnosed at an early stage, is associated with an increase in the risk of cervical cancer-specific death (6, 9, 34). The results of our various approaches point to the same conclusion: patients with HPV18 and its related types (HPV39 and 45; alpha-7 species) have shorter DSS and greater local recurrent rates than those with HPV16 infection and its related types (HPV31, 33, 52, and 58; alpha-9 species). However, the underlying mechanisms that result in the tumors with the alpha-7 species being more resistant to RT than those with the alpha-9 species are still undetermined. In vitro studies have revealed several features of HPV18 infection that differ from those of HPV16 infection, including enhanced E7 phosphorylation (35) and increased transformation (36). Furthermore, HPV18 is associated with significant less apoptosis than HPV16, affording one possible explanation for greater radioresistant cervical cancer with HPV18 infection (37). Another possible mechanism is the difference in the E6 oncoprotein activities. Overexpression of the E6 protein confers a radiation-resistant phenotype (38). E6 protein plays a pivotal role in carcinogenesis by targeting the host proteins, such as p53 and the proteins with the PDZ domain, for proteasome-mediated degradation (see Liu and Baleja for review [39]). The proteins with the PDZ domain, such as hDlg, hScrib, MUPP-1, and MAGIs, are degraded by HPV16 and HPV18 E6 with distinct relative efficiency because a critical difference exists in the amino acid residue
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at the extreme C termini of the two E6 proteins (40). This difference exists not only between HPV16 and HPV18, but also between the alpha-9 and alpha-7 species. Whether the variation in the PDZ domain-binding capacities determines the RT response is worth additional exploration.
CONCLUSION Our results have demonstrated that HPV genotypes, independent of the established prognostic factors, are associated with the survival outcomes of cervical cancer patients undergoing RT. Although our study had many strengths compared with most previous studies, it was limited in a few respects. First, the high incidence of intercurrent disease in elderly patients made the comparisons of overall survival among the various prognostic factors less meaningful. Therefore, we used DSS as the endpoint for most analyses. Second, the treatment protocols did not follow the current consensus to treat most patients with CCRT. The influence of HPV genotypes on CCRT could not be fully determined from the present study. In addition, the mechanisms of the different radiation responses between the alpha-7 and alpha-9 species remain unclear. Identifying the mechanisms by which cervical carcinoma harboring the alpha-7 species is associated with a poorer prognosis could be capitalized on to improve the outcome of cervical cancer patients undergoing primary RT.
REFERENCES 1. de Villiers EM, Wagner D, Schneider A, et al. Human papillomavirus infections in women with and without abnormal cervical cytology. Lancet 1987;2:703–706. 2. Walboomers JM, Jacobs MV, Manos MM, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999;189:12–19. 3. Parkin DM, Bray F. Chapter 2: The burden of HPV-related cancers. Vaccine 2006;24(Suppl. 3):S11–S25. 4. de Villiers EM, Fauquet C, Broker TR, et al. Classification of papillomaviruses. Virology 2004;324:17–27. 5. Doorbar J. Molecular biology of human papillomavirus infection and cervical cancer. Clin Sci (Lond) 2006;110:525–541. 6. Lai CH, Chang CJ, Huang HJ, et al. Role of human papillomavirus genotype in prognosis of early-stage cervical cancer undergoing primary surgery. J Clin Oncol 2007;25:3628–3634. 7. Rose BR, Thompson CH, Simpson JM, et al. Human papillomavirus deoxyribonucleic acid as a prognostic indicator in earlystage cervical cancer: A possible role for type 18. Am J Obstet Gynecol 1995;173:1461–1468. 8. Burger RA, Monk BJ, Kurosaki T, et al. Human papillomavirus type 18: Association with poor prognosis in early stage cervical cancer. J Natl Cancer Inst 1996;88:1361–1368. 9. Lombard I, Vincent-Salomon A, Validire P, et al. Human papillomavirus genotype as a major determinant of the course of cervical cancer. J Clin Oncol 1998;16:2613–2619. 10. Huang LW, Chao SL, Hwang JL. Human papillomavirus-31-related types predict better survival in cervical carcinoma. Cancer 2004;100:327–334. 11. Lai HC, Sun CA, Yu MH, et al. Favorable clinical outcome of cervical cancers infected with human papilloma virus type 58 and related types. Int J Cancer 1999;84:553–557.
12. Kristensen GB, Karlsen F, Jenkins A, et al. Human papilloma virus has no prognostic significance in cervical carcinoma. Eur J Cancer 1996;32A:1349–1353. 13. Viladiu P, Bosch FX, Castellsague X, et al. Human papillomavirus DNA and antibodies to human papillomaviruses 16 E2, L2, and E7 peptides as predictors of survival in patients with squamous cell cervical cancer. J Clin Oncol 1997;15:610–619. 14. van Muyden RC, ter Harmsel BW, Smedts FM, et al. Detection and typing of human papillomavirus in cervical carcinomas in Russian women: a prognostic study. Cancer 1999;85:2011– 2016. 15. Huang HJ, Huang SL, Lin CY, et al. Human papillomavirus genotyping by a polymerase chain reaction-based genechip method in cervical carcinoma treated with neoadjuvant chemotherapy plus radical surgery. Int J Gynecol Cancer 2004;14: 639–649. 16. Hong JH, Tsai CS, Lai CH, et al. Risk stratification of patients with advanced squamous cell carcinoma of cervix treated by radiotherapy alone. Int J Radiat Oncol Biol Phys 2005;63:492– 499. 17. Bachtiary B, Obermair A, Dreier B, et al. Impact of multiple HPV infection on response to treatment and survival in patients receiving radical radiotherapy for cervical cancer. Int J Cancer 2002;102:237–243. 18. Lindel K, Burri P, Studer HU, et al. Human papillomavirus status in advanced cervical cancer: Predictive and prognostic significance for curative radiation treatment. Int J Gynecol Cancer 2005;15:278–284. 19. Munagala R, Dona MG, Rai SN, et al. Significance of multiple HPV infection in cervical cancer patients and its impact on treatment response. Int J Oncol 2009;34:263–271.
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20. Pilch H, Gunzel S, Schaffer U, et al. Human papillomavirus (HPV) DNA in primary cervical cancer and in cancer free pelvic lymph nodes—Correlation with clinico-pathological parameters and prognostic significance. Zentralbl Gynakol 2001;123: 91–101. 21. Higgins GD, Davy M, Roder D, et al. Increased age and mortality associated with cervical carcinomas negative for human papillomavirus RNA. Lancet 1991;338:910–913. 22. Harima Y, Sawada S, Nagata K, et al. Human papilloma virus (HPV) DNA associated with prognosis of cervical cancer after radiotherapy. Int J Radiat Oncol Biol Phys 2002;52: 1345–1351. 23. Graflund M, Sorbe B, Sigurdardottir S, et al. HPV-DNA, vascular space invasion, and their impact on the clinical outcome in early-stage cervical carcinomas. Int J Gynecol Cancer 2004; 14:896–902. 24. Fakhry C, Gillison ML. Clinical implications of human papillomavirus in head and neck cancers. J Clin Oncol 2006;24:2606– 2611. 25. Crook T, Wrede D, Vousden KH. p53 Point mutation in HPV negative human cervical carcinoma cell lines. Oncogene 1991;6:873–875. 26. DeWeese TL, Walsh JC, Dillehay LE, et al. Human papillomavirus E6 and E7 oncoproteins alter cell cycle progression but not radiosensitivity of carcinoma cells treated with low-dose-rate radiation. Int J Radiat Oncol Biol Phys 1997;37:145–154. 27. Franken NA, Van Bree C, Haveman J. Differential response to radiation of TP53-inactivated cells by overexpression of dominant-negative mutant TP53 or HPVE6. Radiat Res 2004;161: 504–510. 28. Pyeon D, Newton MA, Lambert PF, et al. Fundamental differences in cell cycle deregulation in human papillomavirus-positive and human papillomavirus-negative head/neck and cervical cancers. Cancer Res 2007;67:4605–4619. 29. Clifford G, Franceschi S. Members of the human papillomavirus type 18 family (alpha-7 species) share a common association
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30. 31. 32. 33.
34. 35. 36. 37. 38. 39. 40.
with adenocarcinoma of the cervix. Int J Cancer 2008;122: 1684–1685. Smith JS, Lindsay L, Hoots B, et al. Human papillomavirus type distribution in invasive cervical cancer and high-grade cervical lesions: A meta-analysis update. Int J Cancer 2007;121:621–632. Munoz N, Bosch FX, de Sanjose S, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003;348:518–527. Lai CH, Huang HJ, Hsueh S, et al. Human papillomavirus genotype in cervical cancer: A population-based study. Int J Cancer 2007;120:1999–2006. Bosch FX, Manos MM, Munoz N, et al. for the A worldwide perspective. International Biological Study on Cervical Cancer (IBSCC) Study Group. Prevalence of human papillomavirus in cervical cancer. J Natl Cancer Inst 1995;87:796–802. Schwartz SM, Daling JR, Shera KA, et al. Human papillomavirus and prognosis of invasive cervical cancer: A populationbased study. J Clin Oncol 2001;19:1906–1915. Werness BA, Levine AJ, Howley PM. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 1990;248:76–79. Villa LL, Schlegel R. Differences in transformation activity between HPV-18 and HPV-16 map to the viral LCR-E6-E7 region. Virology 1991;181:374–377. Arends MJ, Wyllie AH, Bird CC. Human papillomavirus type 18 is associated with less apoptosis in fibroblast tumours than human papillomavirus type 16. Br J Cancer 1995;72:646–649. Hampson L, El Hady ES, Moore JV, et al. The HPV16 E6 and E7 proteins and the radiation resistance of cervical carcinoma. FASEB J 2001;15:1445–1447. Liu Y, Baleja JD. Structure and function of the papillomavirus E6 protein and its interacting proteins. Front Biosci 2008;13: 121–134. Thomas M, Massimi P, Navarro C, et al. The hScrib/Dlg apicobasal control complex is differentially targeted by HPV-16 and HPV-18 E6 proteins. Oncogene 2005;24:6222–6230.
doi:10.1016/j.ijrobp.2009.09.021
CLINICAL INVESTIGATION
Cervix
CLINICAL EFFECT OF HUMAN PAPILLOMAVIRUS GENOTYPES IN PATIENTS WITH CERVICAL CANCER UNDERGOING PRIMARY RADIOTHERAPY CHUN-CHIEH WANG, M.D., PH.D.,*y CHYONG-HUEY LAI, M.D.,z HUEI-JEAN HUANG, M.D.,z ANGEL CHAO, M.D., PH.D.,z CHEE-JEN CHANG, PH.D.,x TING-CHANG CHANG, M.D.,z HUNG-HSUEH CHOU, M.D.,z AND JI-HONG HONG, M.D., PH.D.*y Departments of *Radiation Oncology and zObstetrics and Gynecology, Chang Gung Memorial Hospital, Taoyuan, Taiwan; xGraduate Institutes of Clinical Medical Sciences, and yDepartment of Medical Imaging and Radiological Science, Chang Gung University School of Medicine, Taoyuan, Taiwan Purpose: To study the prognostic value of the human papillomavirus (HPV) genotypes in cervical cancer patients undergoing radiotherapy. Patients and Methods: A total of 1,010 patients with cervical cancer after radiotherapy between 1993 and 2000 were eligible for this study. The HPV genotypes were determined by a genechip, which detects 38 types of HPV. The patient characteristics and treatment outcomes were analyzed using the Cox regression hazard model and classification and regression tree decision tree method. Results: A total of 25 genotypes of HPV were detected in 992 specimens (98.2%). The leading 8 types were HPV16, 58, 18, 33, 52, 39, 31, and 45. These types belong to two high-risk HPV species: alpha-7 (HPV18, 39, 45) and alpha-9 (HPV16, 31, 33, 52, 58). Three HPV-based risk groups, which were independent of established prognostic factors, such as International Federation of Gynecology and Obstetrics stage, age, pathologic features, squamous cell carcinoma antigen, and lymph node metastasis, were associated with the survival outcomes. The high-risk group consisted of the patients without HPV infection or the ones infected with the alpha-7 species only. Patients co-infected with the alpha-7 and alpha-9 species belonged to the medium-risk group, and the others were included in the lowrisk group. Conclusion: The results of the present study have confirmed the prognostic value of HPV genotypes in cervical cancer treated with radiotherapy. The different effect of the alpha-7 and alpha-9 species on the radiation response deserves additional exploration. Ó 2010 Elsevier Inc. Human papillomavirus, Cervical cancer, Radiotherapy, Prognosis.
Cervical cancer is the second most common cancer among women worldwide. Infection with the human papillomavirus (HPV) is suggested as the initiating event of cervical cancer, and the prevalence of HPV in women with cervical cancer is 95–100% using current techniques (1–3). More than 100 HPV genotypes have been identified and classified by their L1 open reading frame (4). When HPVs share 60–70% nucleotide identity, they are clustered into the same species. Two HPV species, alpha-7 (HPV18, 39, 45, 59, 68, and 70) and alpha-9 (HPV16, 31, 33, 35, 52, 58, and 67), are responsible for >80% of all cervical cancer cases (5). The HPV genotypes could pose differing prognoses for cervical cancer patients, in addition to a number of prognostic factors, such as the International Federation of Gynecology
and Obstetrics stage, lymph node status, patient age, histologic features, or squamous cell carcinoma antigen (SCCAg) level. Accumulating research, including our recent study of patients who underwent primary surgery (6), has suggested that HPV18 infection is associated with a more aggressive form of cervical cancer than other HPV infections (7–9). In addition, some studies have shown that the presence of an HPV33-related tumor has a favorable outcome (10, 11). However, other reports have not been able to identify the link between HPV genotypes and clinical outcome (12–14). These inconsistent results have highlighted the problem of determining the prognostic role of HPV genotypes in cervical cancer. The small numbers of patients studied, insufficient follow-up, lack of accounting for known prognostic factors, and variable laboratory techniques have made the
Reprint requests to: Ji-Hong Hong, M.D., Ph.D., Department of Radiation Oncology, Chung Gung Memorial Hospital, No. 5, Fu-Shin St., KweiShan, Taoyuan, Taiwan. Tel: (886) 3-328-2177; Fax: (886) 3-328-0797; E-mail: [email protected] Supported by the National Science Council-Taiwan (Grant
NSC94-2314-B-182-011) and Chang Gung Memorial Hospital (Grant CMRPG340693). Conflict of interest: none. Received April 10, 2009, and in revised form Sept 4, 2009. Accepted for publication Sept 4, 2009.
INTRODUCTION
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interpretation of published results difficult. The heterogeneity of treatment modalities within and among studies has also made this issue perplexing. Because radiotherapy (RT) is one of the mainstay treatments of cervical cancer, we report the prognosis of cervical cancer patients in relation to the HPV genotypes analyzed using a highly sensitive DNA chip in a large series of patients primarily treated with RT. PATIENTS AND METHODS Patient population Between August 1993 and May 2000, a list of consecutive patients with invasive cervical cancer at Chang Gung Memorial Hospital was retrieved with the approval of the institutional review board. The data of the 1,067 patients who had undergone primary surgery have been previously published (6). Samples from the other 1,036 patients who had undergone RT as their primary treatment were genotyped for HPV infection. Of the 1,036 patients, 26 were excluded because of having undergone incomplete RT (dose <50 Gy); thus, 1,010 patients were eligible for the present analysis.
HPV genotyping The detailed procedure for the HPV genotyping has been previously reported (6, 15). In brief, DNA was extracted from paraffin-embedded specimens and used as a template for the amplification of the L1 open reading frame with the biotinylated GP6+ and SPF1 consensus primers. The resultant amplifiers were then hybridized with the Easychip HPV Blot Membrane (King Car, I-Lan, Taiwan), which detects 38 types of HPV (6, 11, 16, 18, 26, 31, 32, 33, 35, 37, 39, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72, 74, 81, 82, 83, 84, and L1AE5). After hybridization, the membranes were incubated with Avidex-AP (Applied Biosystems Inc. Foster City, CA), and then with NBT/BCIP tablet (Sigma-Aldrich, St. Louis). The results were determined according to the HPV type format on the chip. Samples with HPV-negative results were reconfirmed by polymerase chain reaction of glyceraldehydes-3-phosphate dehydrogenase to validate the DNA quality and the leading 8 types of HPV. The samples with multiple types on HPV blotting were verified by E6 type-specific polymerase chain reaction.
Surveillance and treatment The pretreatment workup and radiation technique were identical to those reported in our previous study (16). Pelvic and abdominal computed tomography or magnetic resonance imaging, chest Xray, and serum marker analysis (SCC-Ag and carcinoembryonic antigen) was performed before treatment. Patients usually underwent a combination of external beam RT and intracavitary brachytherapy. External beam RT was delivered using a daily fraction of 1.8–2 Gy in five fractions weekly. The initial radiation dose to the whole pelvis was 40–45 Gy. The irradiation fields were extended to the abdominal para-aortic region when involvement of the para-aortic nodes was suspected from the imaging findings. For the patients who underwent brachytherapy, a parametrial boost was given to a total dose of #54–58 Gy. Brachytherapy was administrated in six fractions with 4.3 Gy/fraction to Point A. For the patients who did not undergo brachytherapy, a total dose of 68–72 Gy was given to the primary tumor. Cisplatin-based chemotherapy was administered to 270 patients. Of the 270 patients, 90% were treated with concurrent chemoradiotherapy (CCRT), which was adopted as the standard treatment of cervical cancer after August 1999 (115 patients) at our hospital. Patients were followed regularly to May 2007, with a min-
Volume 78, Number 4, 2010
imal follow-up period of for 7 years. The post-therapy surveillance protocol was the same as that used in our recent study (6).
Statistical analysis The data were analyzed using the Statistical Package for Social Sciences, version 11 (SPSS, Chicago, IL). Pearson’s chi-square test was used to evaluate the association between the variables. Survival curves were generated using the Kaplan-Meier method, and comparisons were made using the log–rank test. Multivariate analyses of the factors associated with local and distant failure were done using Cox regression analysis. Classification and regression tree (CART) analysis was performed using Answer Tree software, version 2.01 (SPSS). Patients with small cell carcinoma (n = 11) were excluded from the present analysis because of its rarity and distinct clinical course. The remaining 999 patients were considered as the primary node. The minimal node size was set to 5, and the group size (parental node) was $30 cases. The missing data for SCC-Ag and carcinoembryonic antigen were imputed using their medium values (4.5 and 3.1, respectively). The survival differences between the CART nodes were further verified using the log–rank test.
RESULTS Clinical characteristics and HPV genotypes The clinicopathologic characteristics of the 1,010 patients are summarized in Table 1. The patient age range was 21–91 years (median, 61). Squamous cell carcinoma was the most common cell type (92%), followed by adenocarcinoma/adenosquamous carcinoma (6.9%) and small cell carcinoma (1.1%). Regarding HPV status, only 18 patients (1.8%) had HPV-negative tumors. In the 992 HPV-positive specimens, 25 genotypes of HPV were detected. The leading eight genotypes were HPV16 (36%), HPV58 (26.2%), HPV18 (18.9%), HPV33 (13.4%), HPV52 (8.2%), HPV39 (5.4%), HPV31 (3.4%), and HPV45 (2.6%). These types belong to two high-risk HPV species: alpha-7 (HPV18, 39, and 45) and alpha-9 (HPV16, 31, 33, 52, and 58). The other HPV genotypes had a prevalence rate of <2%. Of the HPV-positive specimens, 204 (20.6%) contained two types and 58 (5.8%) were infected by more than two types of HPV. All samples with multiple infections had at least one HPV type that was a member of the alpha-7 or alpha-9 species. To reduce the interference among the different HPV genotypes, the 730 patients whose specimens contained only one HPV type were analyzed first. The association between the HPV genotypes and various clinicopathologic characteristics is summarized in Table 2. HPV33, 52, and 58 were common (>50%) in patients >65 years old. The two leading types in those with squamous cell carcinoma were HPV16 (38.1%) and HPV58 (23.1%). In contrast, the predominant type in those with adenocarcinoma/adenosquamous carcinoma and small cell carcinoma was HPV18. HPV18 infection was associated with a probability of having nodal metastasis detectable on computed tomography/magnetic resonance imaging as great as 58.3% in patients with late-stage disease (Stage IIIa-IVa). In patients with early-stage disease, the presence of HPV52 and 58 was associated with a relatively low incidence of nodal metastasis, 7.1% and 8%, respectively. Tumors with the alpha-9 species tended to have greater
Clinical effect of HPV in cervical cancer d C.-C. WANG et al.
Table 1. Population characteristics (n = 1,010) (Continued )
Table 1. Population characteristics (n = 1,010) Characteristic Age (y) <45 45–65 >65 Stage Ib IIa IIb III IVa Pathologic type SCC AC/ASC Small cell carcinoma Differentiation Well to moderate Poor Unclassified SCC-Ag level <1.5 1.5–4 >4 Missing CEA level #5 >5 Missing LN metastasis No Yes Undetermined HPV infection Negative Positive Single Multiple
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n (%) 120 (11.9) 482 (47.8) 408 (40.3) 202 (20) 191 (18.9) 341 (33.8) 255 (25.2) 21 (2.1) 930 (92) 69 (6.9) 11 (1.1) 484 (47.9) 346 (34.3) 180 (17.9) 258 (25.5) 183 (18.1) 472 (46.7) 97 (9.6) 600 (59.4) 223 (22.1) 187 (18.5) 778 (77) 204 (20.2) 28 (2.8) 18 (1.8) 992 (98.2) 730 (73.6) 262 (26.4) (Continued )
expression of SCC-Ag: >50% had an SCC-Ag level >4 ng/ mL compared with approximate 30% of those with the alpha-7 species (p < .0001). For the 262 specimens with multiple HPV infections, HPV18 (44.7%) and HPV58 (43.9%) were the two predominant types, with HPV16 (37%) third. The incidence of adenocarcinoma/adenosquamous in the multiple HPV group (10.7%) was greater than that in the single HPV group (5.2%; p = .004). In the whole cohort, 17.9% of HPV18-positive tumors were adenocarcinoma/adenosquamous carcinoma, and similar patterns were also observed for the other two members of the alpha-7 species: HPV39 (14.5%) and HPV45 (15.4%). In contrast, the prevalence of adenocarcinoma/adenosquamous carcinoma in the alpha-9 species was relatively low, ranging from 0% for HPV52 to 7.5% for HPV33 (HPV16, 2.8%; HPV31, 2.9%; HPV52, 0%; and HPV58, 6.4%; p < .0001). Follow-up data and survival analysis After a median follow-up of 78 months, 471 patients (46.6%) were still alive, and 390 (38.6%) had experienced
Characteristic
n (%)
Genotype 16 Single Multiple 18 Single Multiple 31 Single Multiple 33 Single Multiple 39 Single Multiple 45 Single Multiple 52 Single Multiple 58 Single Multiple 26, 35, 42, 44, 51, 53, 54, 56, 59, 61, 66, 67, 68, 69, 70, 74, 82, and 84
364 (36.7) 267 (36.6) 97 (37) 191 (19.2) 74 (10.1) 117 (44.7) 34 (3.4) 22 (3) 12 (4.6) 135 (13.6) 68 (9.3) 67 (25.6) 55 (5.5) 19 (2.6) 36 (13.7) 26 (2.6) 15 (2) 11 (4.2) 83 (8.4) 53 (7.3) 30 (11.5) 265 (26.7) 150 (20.5) 115 (43.9) <2)
Abbreviations: SCC = squamous cell carcinoma; AC/ASC = adenocarcinoma/adenosquamous carcinoma; SCC-Ag = SCC antigen; CEA = carcinoembryonic antigen; LN = lymph node; HPV = human papillomavirus. Data presented as numbers of patients, with percentages in parentheses.
treatment failure, including 212 local failures and 253 distant relapses (75 patients had both). Of the 539 deaths, 175 patients (32.5%) had died of intercurrent disease. These patients were censored at the date of death for the calculation of disease-specific survival (DSS). The 5- and 10-year overall survival rate for the entire cohort was 58.6% and 44.1%, respectively. The corresponding DSS rates were 66.9% and 60.9%. On univariate and multivariate analyses (Table 3), patient age, stage, pathologic features, SCC-Ag level, and lymph node metastasis status were confirmed as prognostic indicators for overall survival and DSS. Although previous studies have suggested that the number of HPV infections is associated with the tumor response after RT (17–19), this factor did not achieve statistical significance on multivariate analysis (p = .19 and p = .201 for overall survival and DSS, respectively). A trend toward a poor prognosis for the HPVnegative group in DSS compared to the HPV-positive group was noted on Kaplan-Meier analysis (p = .085). CART modeling To further explore the effect of the individual HPV genotypes on the results of RT for cervical cancer, CART modeling was performed. The variables listed in Table 2, the predominant eight HPV genotypes, and the alpha-7 and
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HPV genotype Single Variable
18
31*
33*
39
45
52*
39 (14.6) 147 (55.1) 81 (30.3)
13 (17.6) 41 (55.4) 20 (27)
4 (18.2) 9 (40.9) 9 (40.9)
3 (4.5) 24 (35.8) 40 (59.7)
0 11 (57.9) 8 (42.1)
2 (13.3) 7 (46.7) 6 (40)
1 (1.9) 20 (37.7) 32 (60.4)
198 (74.2) 69 (25.8)
61 (82.4) 13 (17.6)
15 (68.2) 7 (32.8)
48 (70.6) 20 (29.4)
14 (73.7) 5 (26.3)
11 (73.3) 4 (26.7)
41 (77.4) 12 (22.6)
260 (38.1)y 7 (18.4)y 0
52 (7.6) 20 (52.6) 5 (62.5)y
23 (3.4) 1 (2.6) 0
55 (8.0) 1 (2.6) 1 (12.5)
19 (2.8) 1 (2.6) 0
13 (1.9) 2 (5.2) 0
55 (23.1) 53 (22.3) 130 (54.6)
30 (44.8) 15 (22.4) 22 (32.8)
5 (25) 3 (15) 12 (60)
19 (30.2) 12 (19) 32 (50.8)
158 (72.8) 59 (27.2)
38 (62.3) 23 (37.7)
14 (77.8) 4 (22.2)
40 (74.1) 14 (25.9)
40/192 (20.8) 15/68 (22.1)
17/64 (26.6) 7/12 (58.3)
3/14 (21.4) 1/8 (12.5)
8/41 (19.5) 5/16 (31.3)
7 (41.2) 5 (29.4) 5 (29.4) 12 (75) 4 (25) 4/14 (28.6) 1/5 (20)
Abbreviations as in Table 1. Data presented as numbers of patients, with percentages in parentheses, unless otherwise specified. * Alpha-9 species. y Prevalence in each pathologic type.
58* 11 (7.3) 59 (39.3) 80 (53.3)
All
Multiple
79 (10.8) 341 (46.6) 311 (42.5)
38 (14.6) 130 (49.8) 93 (35.6)
108 (72) 42 (28)
538 (73.6) 193 (26.4)
183 (70.1) 78 (29.9)
54 (7.8) 0 1 (12.5)
158 (23.1) 5 (13.2) 0
684 (93.7) 38 (5.2) 8 (1.1)
229 (88.1) 28 (10.7) 3 (1.2)
5 (35.7) 5 (35.7) 4 (28.6)
12 (23.1) 10 (19.2) 30 (57.7)
31 (24.2) 20 (15.6) 77 (60.2)
185 (28.2) 131 (20) 339 (51.8)
69 (28.4) 50 (20.6) 124 (51)
5 (55.6) 4 (44.4)
37 (77.1) 11 (22.9)
83 (73.5) 30 (26.5)
430 (73.1) 158 (26.9)
161 (71.9) 63 (28.1)
2/11 (18.2) 0/4 (0)
3/42 (7.1) 2/12 (16.7)
9/104 (8) 15/46 (32.6)
88/524 (16.8) 54/187 (28.9)
30/176 (17) 28/77 (36.4)
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Age (y) <45 45–65 >65 Stage Early (Ia-IIb) Late (IIIa-IVa) Pathologic type SCC AC/ASC Small cell carcinoma SCC-Ag level <1.5 1.5–4 >4 CEA level <5 >5 LN metastasis Early stage Late stage
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Table 2. Correlations between clinicopathologic factors and HPV genotypes
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Table 3. Univariate and multivariate analysis of clinicopathologic covariates on overall and disease-specific survival Overall survival Variable Stage Early (Ia–IIb) Late (IIIa–IVa) Age (y) <45 45–65 >65 Pathologic type SCC AC/ASC Small cell carcinoma SCC-Ag level <1.5 1.5–4 >4 CEA level #5 >5 LN metastasis No Yes Differentiation Well to moderate Poor Unclassified HPV infection Negative Single Multiple Chemotherapy No Yes
Disease-specific survival
Deaths/total patients (n) 5-y (%) 10-y (%)
p
Deaths/total patients (n) 5-y (%) 10-y (%)
<.001/<.001 345/734 194/276
66.1 38.7
50.8 26.4
67/120 225/482 247/407
50.0 62.2 56.6
44.4 51.5 34.9
479/928 47/69 9/11
60.4 39.1 27.3
45.4 32.3 18.2
109/258 89/182 280/472
68.2 63.4 54.2
54.7 48.8 38.3
297/600 138/223
63.3 49.3
47.9 35.8
390/778 135/204
62.8 42.2
47.4 32.6
261/484 191/346 87/180
58.5 54.6 66.7
43.6 42.8 48.3
11/18 383/730 154/262
44.4 60 55.7
38.1 45.4 40.9
392/740 147/270
60.7 52.6
45.4 41.8
p <.001/<.001
215/734 147/276
74.1 48.3
68.2 42.2
63/120 168/482 131/407
50.4 68.1 71.2
45.9 63.2 63.2
312/928 42/69 6/11
69.3 44.2 44.4
63.4 36.8 29.6
60/258 59/182 198/472
78.6 70.8 62.4
74.7 65.1 54.4
191/600 102/223
71.4 57.7
65.9 49.2
246/778 107/204
71.5 49.9
65.6 44.2
176/484 136/346 50/180
67.6 63.5 73.8
60.2 57.7 71.0
9/18 251/730 105/262
48.5 68.7 63.2
48.5 62.6 57.4
242/740 120/270
70.5 57.7
64.5 52.5
<.001/.041
<.001/.049
<.001/<.001
<.001/<.001
<.001/.016
<.001/.002
<.001/.200
<.001/.149
<.001/.005
<.001/.004
.228/.416
.033/.108
.182/.190
.036/.201
.017/.486
<.001/.667
Abbreviations as in Table 1. p Values presented as univariate/multivariate values.
alpha-9 HPV species were used to identify subsets of patients with distinctly different survival distributions. A summary of this analysis is shown in Fig. 1. The International Federation of Gynecology and Obstetrics stage was the principle discriminator for both DSS and local control. The lymph node status was the first node for the risk of distant metastasis. Similar to the results of the previous multivariate analysis, the pathologic features, patient age, and SCC-Ag level were the other determinants of these classification trees. Regarding the importance of the HPV genotype in the prognosis, the presence of HPV33 predicted for better DSS in patients with advanced squamous cell cancer (p = .029; Fig. 1a). In the same subgroup with negative HPV33 and a high SCC-Ag levels, patients with HPV39 infection had a worse outcome, although it did not achieve statistical significance using the log–rank test (p = .223). In the CART analysis of local control (Fig. 1b), the presence of two members of the alpha-7 species, HPV39 and HPV45, were associated with a greater incidence of local failure in the subgroup with early-stage squamous cell carcinoma (both p < .001). In patients with early-stage adenocarcinoma, HPV16 positivity predicted a trend toward better local control after RT
(p = .094). Similarly, patients in the advanced squamous cell carcinoma group with significantly better local control were those with alpha-9 species infection (p = .005). Chemotherapy was shown to improve local control in those with advanced squamous cell carcinoma without HPV alpha-9 infection; however, it failed to reach statistical significance (5-year control rate, 59.7% vs. 34.5%, respectively; p = .226). In addition, the presence of the alpha-7 species indicated a greater incidence of metastasis in patients with nodal disease and a greater SCC-Ag level (p = .184; Fig. 1c). HPV risk group The results of the CART analysis suggested that various HPV genotypes have a different effect on the prognosis of cervical cancer after RT. In general, these results imply that cervical cancer associated with HPV alpha-7 species has a worse prognosis. To further examine the effect of HPV genotypes on the patients with multiple HPV infections, we divided patients into three subgroups: patients with the alpha-7 types only, those with the alpha-9 types only, and those with both alpha-7 and alpha-9 types. As shown in Fig. 2, patients with the alpha-9 types only had the best outcome for DSS (p = .075) and
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Fig 1. Classification and regression tree (CART) survival analysis to identify which variables were most strongly associated with survival in cervical cancer patients undergoing primary radiotherapy. Number within each square indicates number of events and patients in that subset. (a) Disease-specific survival. (b) Local control. (c) Distant metastasis. FIGO = International Federation of Gynecology and Obstetrics; Im = improvement; SCC = squamous cell carcinoma; Ad/AS = adenocarcinoma/adenosquamous carcinoma; HPV = human papillomavirus; SCC-Ag = SCC antigen; UC = unclassified; W,M/D = well, moderately differentiated; P/D = poorly differentiated; CEA = carcinoembryonic antigen.
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DISCUSSION
Fig 2. Kaplan-Meier estimates of (a) disease-specific survival and (b) local control by human papillomavirus (HPV) genotypes in patients with multiple human papillomavirus infections.
local control (p = .013), followed by the patients with both alpha-7 and alpha-9 types. Patients infected with multiple alpha7 types only had the greatest risk of treatment failure. From that analysis, patients were categorized into low-, medium-, and high-risk groups by their HPV genotypes. The low-risk group consisted of patients who had HPV infections, but excluded those with alpha-7 species positivity. Patients co-infected with the alpha-7 and alpha-9 species were in the medium-risk group. The high-risk group consisted of the patients without HPV infection or the ones infected with the alpha-7 species only. The risk factors associated with DSS, local control, and distant metastasis examined by multivariate analysis are summarized in Table 4. These results were consistent with those from our previous study in which the International Federation of Gynecology and Obstetrics stage, pathologic features, SCC-Ag level, and lymph node status were the significant prognostic factors for DSS (16). The present study has further demonstrated that the HPV risk group is an independent risk factor for DSS. The effect of the HPV group on DSS mainly resulted from the effect of local control, in contrast to the SCC-Ag level and lymph node status, which were strongly associated with distant metastasis.
The presence of HPV DNA has been shown to be essential for the development of invasive cervical cancer (1, 2). However, contradictory results have been presented concerning the influence of HPV genotypes on the clinical outcome of cervical cancer patients (6–9, 11–14, 20). The present study with a large population (1,010 patients) and long-term follow-up (median, 78 months) has illustrated the significant clinical effect of HPV genotypes in cervical cancer patients undergoing primary RT. The presence of HPV alpha-7 species or HPV negativity (high-risk group) was significantly associated with a poorer outcome. Thus, the HPV genotypes should be considered as a biomolecular marker for cervical cancer. In accordance with the results from other groups, patients with HPV-negative tumors tended to have a worse survival rate (9, 18, 21–23), although a contradictory result was suggested by Pilch et al. (20). Recently, HPV infection has also been associated with improved outcomes in patients with squamous cell carcinoma of the head and neck, especially of the oropharynx (24). The better outcome in association with HPV positivity in patients undergoing RT could be attributed to the method of p53 dysfunction. In HPV-positive cancers, most of the loss of p53 function results from the HPV E6-related degradation, which is different from the mutation of p53 in HPV-negative cancers (25). These two types of p53 dysfunction might not be equivalent in terms of their response to RT (26, 27). In addition, cell cycle-related and other genes that are upregulated in HPV-positive tumors could affect the radiation response (28). The high sensitivity and broad spectrum of the chip-based technique enabled us to detect multiple infections and uncommon HPV genotypes, which were seldom discussed in previous studies. For example, HPV18 is the well-known primary HPV type associated with cervical adenocarcinoma (5). Our data have further demonstrated that HPV39 and HPV45 have a similar rate of association with adenocarcinoma/adenosquamous carcinoma. This observation is consistent with the report by Clifford and Franceschi (29). Of the 992 patients with HPV infection, HPV16 was the most prevalent type (36.7%), followed by HPV58 (26.7%) and HPV18 (19.2%). The prevalence of HPV58 in our cohort was relatively greater than the average 3.4% reported in the other studies (30). In our previous study of patients treated with primary surgery, the prevalence of HPV16, 18, and 58 was 63.8%, 16.5%, and 6.8%, respectively (6). This discrepancy could be attributed to the age distribution: the surgery group had a median age of 50 years compared with that of 61 years in the RT group. It has been shown that older patients have a high prevalence of HPV 52 and 58 infection by several epidemiologic studies (9, 31, 32) and better DSS after RT (16). However, older patients have tended to have short follow-up periods for overall survival owing to the high risk of death from intercurrent disease. This could explain the observation that patients with HPV52 or 58 infections had the worst overall survival in some studies (10).
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Table 4. Multivariate analysis of potential predictors of disease-specific survival, local control and distant metastasis Disease-specific survival Variable Stage Ib IIa-IIb III IVa Age (y) <45 45–65 >65 Pathologic type SCC AC/ASC Small cell carcinoma SCC-Ag level <1.5 1.5–4 >4 CEA level #5 >5 LN metastasis No Yes Differentiation Well Moderate Poor HPV (risk group) Low High Medium Chemotherapy No Yes
RR (95% CI)
Local control p
p
RR (95% CI)
<.001 1 1.383 (1.043–1.833) 2.624 (1.974–3.488) 2.795 (1.489–5.245)
Distant metastasis
<.001 1 1.670 (1.133–2.462) 4.213 (2.862–6.202) 5.563 (2.619–11.814)
<.001
<.001
.240 1 1.256 (0.829–1.903) 1.393 (0.940–2.064) .028 1 0.684 (0.484–0.967) .825 1 0.995 (0.710–1.392) .144 1 0.779 (0.567–1.071) 0.720 (0.479–1.083)
.020
.113 1 1.142 (0.869–1.501) 0.743 (0.499–1.108)
<.001 1 2.028 (1.429–2.878) 1.437 (0.975–2.117)
.536 1 1.085 (0.838–1.404)
<.001 1 2.124 (1.601–2.816)
.062
1 1.485 (1.121–1.968) 1.131 (0.834–1.535)
.116 1 1.253 (0.939–1.671)
.002
1 0.970 (0.769–1.223) 0.676 (0.485–0.941)
.002 1 1.814 (1.214–2.710) 1.976 (1.356–2.881)
.588
1 1.452 (1.135–1.858)
<.001 1 2.032 (1.283–3.219) 4.579 (1.896–11.059)
.001
1 1.066 (0.834–1.362)
.097 1 0.706 (0.491–1.015) 0.694 (0.470–1.023)
1 3.749 (2.444–5.750) 3.032 (1.057–8.693)
1 1.729 (1.239–2.413) 1.795 (1.310–2.461)
.003
.001 1 0.545 (0.376–0.790) 0.467 (0.313–0.699)
1 2.326 (1.617–3.346) 2.547 (1.081–5.997)
p
1 1.050 (0.760–1.451) 1.684 (1.209–2.348) 0.916 (0.356–2.360)
.051 1 0.692 (0.511–0.938) 0.728 (0.527–1.005)
RR (95% CI)
.603 1 1.095 (0.763–1.571) 1.158 (0.814–1.648)
.393 1 0.869 (0.629–1.2)
.243 1 1.206 (0.881–1.652)
Abbreviations: RR = relative risk; CI = confidence interval; other abbreviations as in Table 1.
In terms of the prognostic effect, several previous studies have tried to group some HPV genotypes for analysis. For example, HPV18 was grouped with HPV45 (9), and HPV33, 52, and 58 were considered related types (11). In the present study, HPV grouping according to their DNA similarity was supported by the results of the CART analysis. In the CART analysis, the presence of the alpha-7 species, HPV39 or HPV45, was associated with a worse prognosis. In contrast, women whose tumors harbored the alpha-9 species, HPV16 or HPV33, had a better outcome. It was not surprising that the results from the univariate analysis (Table 3) showed that the use of chemotherapy conferred a worse prognosis because the selection for chemotherapy was usually determined by high-risk clinical features during the study period. However, that HPV genotypes could affect the efficacy of CCRT in patients with advanced squamous cell carcinoma of the cervix was suggested by the CART analysis results. With regard to the incidence of multiple HPV infections, 26.4% of the patients enrolled in the present study presented with two or more genotypes. These data are slightly greater
than those reported in some previous studies, in which multiple infections were documented in 4–18% of patients (19, 32–34). Bachtiary et al. (17) reported a high incidence (43.7%, n = 106) of patients infected with multiple HPV types. Also, this group of patients had a significantly greater risk of disease progression than patients with a single HPV infection after RT. A similar conclusion was suggested by Munagala et al. (19), who demonstrated nearly fivefold greater treatment failure in cases of multiple infections compared to cases of single infection (57% vs. 12%) in a cohort of 43 patients. These observations are consistent with the results of our univariate analysis; however, the presence of multiple HPV infections was not a statistically significant prognostic factor on multivariate analysis (Table 3). Our data also showed that cervical cancer with multiple HPV infections is a heterogeneous group, in which patients with both alpha-7 and alpha-9 infections had an outcome better than the patients with alpha-7 infection only and worse than those with alpha-9 infection only (Fig. 2). Therefore, a risk grouping according to the HPV genotype was derived for our cervical cancer patients undergoing primary RT. These results
Clinical effect of HPV in cervical cancer d C.-C. WANG et al.
are consistent with those from several studies that HPV18-related cervical cancer, particularly if diagnosed at an early stage, is associated with an increase in the risk of cervical cancer-specific death (6, 9, 34). The results of our various approaches point to the same conclusion: patients with HPV18 and its related types (HPV39 and 45; alpha-7 species) have shorter DSS and greater local recurrent rates than those with HPV16 infection and its related types (HPV31, 33, 52, and 58; alpha-9 species). However, the underlying mechanisms that result in the tumors with the alpha-7 species being more resistant to RT than those with the alpha-9 species are still undetermined. In vitro studies have revealed several features of HPV18 infection that differ from those of HPV16 infection, including enhanced E7 phosphorylation (35) and increased transformation (36). Furthermore, HPV18 is associated with significant less apoptosis than HPV16, affording one possible explanation for greater radioresistant cervical cancer with HPV18 infection (37). Another possible mechanism is the difference in the E6 oncoprotein activities. Overexpression of the E6 protein confers a radiation-resistant phenotype (38). E6 protein plays a pivotal role in carcinogenesis by targeting the host proteins, such as p53 and the proteins with the PDZ domain, for proteasome-mediated degradation (see Liu and Baleja for review [39]). The proteins with the PDZ domain, such as hDlg, hScrib, MUPP-1, and MAGIs, are degraded by HPV16 and HPV18 E6 with distinct relative efficiency because a critical difference exists in the amino acid residue
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at the extreme C termini of the two E6 proteins (40). This difference exists not only between HPV16 and HPV18, but also between the alpha-9 and alpha-7 species. Whether the variation in the PDZ domain-binding capacities determines the RT response is worth additional exploration.
CONCLUSION Our results have demonstrated that HPV genotypes, independent of the established prognostic factors, are associated with the survival outcomes of cervical cancer patients undergoing RT. Although our study had many strengths compared with most previous studies, it was limited in a few respects. First, the high incidence of intercurrent disease in elderly patients made the comparisons of overall survival among the various prognostic factors less meaningful. Therefore, we used DSS as the endpoint for most analyses. Second, the treatment protocols did not follow the current consensus to treat most patients with CCRT. The influence of HPV genotypes on CCRT could not be fully determined from the present study. In addition, the mechanisms of the different radiation responses between the alpha-7 and alpha-9 species remain unclear. Identifying the mechanisms by which cervical carcinoma harboring the alpha-7 species is associated with a poorer prognosis could be capitalized on to improve the outcome of cervical cancer patients undergoing primary RT.
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