Prior High-Risk HPV Testing and Pap Test Results for 427 Invasive Cervical Cancers in China s Largest CAP-Certified Laboratory

Prior High-Risk HPV Testing and Pap Test Results for 427 Invasive Cervical Cancers in China s Largest CAP-Certified Laboratory Baowen Zheng, MD 1 ; Zaibo Li, MD, PhD 2 ; Christopher C. Griffith, MD, PhD 3 ; Shanshan Yan, MD 1 ; Congde Chen, MD 1 ; Xiangdong Ding, MD 1 ; Xiaoman Liang, MD 1 ; Huaitao Yang, MD, PhD 4 ; and Chengquan Zhao, MD 3 BACKGROUND: Cervical cancer and its precursor lesions are caused by a persistent high-risk human papillomavirus (hrhpv) infection. hrhpv testing has been reported to have higher sensitivity than Papanicolaou (Pap) testing for the detection of cervical precursor lesions. However, limited data are available for prior human papillomavirus (HPV) testing results for patients later diagnosed with invasive cervical cancer, especially in countries lacking a national cervical cancer screening program such as China. This study investigated prior hrhpv testing results for patients with invasive cervical cancer in China. METHODS: Cases with a histologic diagnosis of invasive cervical carcinoma were retrieved from Guangzhou KingMed Diagnostics (the largest independent pathology laboratory in China); prior hrhpv and Pap test results obtained within the year before the cancer diagnosis were recorded. RESULTS: HPV testing was negative in 7.5% of 427 cases of invasive cervical carcinoma, including squamous cell carcinoma (5%) and adenocarcinoma (25%). In 155 cervical cancer cases with prior hrhpv and Pap testing, the negative rate for Pap testing was 1.9%, and the negative rate for HPV was 9.7%. Furthermore, when only cases of adenocarcinoma (n 5 18) were examined, both the hrhpv-negative rate and the Pap-negative rate were higher at 33% and 5.6%, respectively. CONCLUSIONS: These data demonstrate a considerable prior hrhpv-negative rate and a lower prior Papnegative rate in patients with invasive cervical carcinoma (especially adenocarcinoma) from a population of women without access to an established screening program. Cancer (Cancer Cytopathol) 2015;123:428-34. VC 2015 American Cancer Society. KEY WORDS: cervical cancer; China; histology; human papillomavirus (HPV); human papillomavirus test; Papanicolaou (Pap) test. INTRODUCTION Cervical cancer is the second most common type of cancer among women in countries that lack a cervical cancer screening program. Cervicovaginal cytology screening with the Papanicolaou (Pap) test has successfully lowered the incidence and mortality rate of cervical cancer worldwide. Most cervical cancers and their precursors are caused by a persistent high-risk human papillomavirus (hrhpv) infection, and hrhpv testing has been suggested to have a higher sensitivity than Pap testing in the detection of precancerous cervical lesions. 1 A large study from Kaiser Permanente Northern California (Oakland) also reported that the risk of cervical cancer is significantly lower for women who are hrhpv-negative (3.8 per 100,000 women per year) versus women who Corresponding author: Chengquan Zhao, MD, Department of Pathology, Magee-Womens Hospital, University of Pittsburgh Medical Center, 300 Halket Street, Pittsburgh, PA 15213; Fax: (412) 641-1675; zhaoc@upmc.edu 1 Guangzhou KingMed Diagnostics, Guangzhou, People s Republic of China; 2 Department of Pathology, Wexner Medical Center, Ohio State University, Columbus, Ohio; 3 Department of Pathology, Magee-Womens Hospital, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania; 4 Department of Pathology, University of Cincinnati Medical Center, Cincinnati, Ohio See related article on pages 421-7, this issue. The first 2 authors contributed equally to this article. Received: March 11, 2015; Revised: April 9, 2015; Accepted: April 9, 2015 Published online May 8, 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cncy.21557, wileyonlinelibrary.com 428 Cancer Cytopathology July 2015

Prior High-Risk HPV Testing/Zheng et al are Pap test negative (7.5 per 100,000 women per year). 2 On the basis of these and similar studies, hrhpv testing has been widely used for the triaging of patients with atypical squamous cells of undetermined significance. On the basis of results from the Addressing the Need for Advanced HPV Diagnostics trial, the US Food and Drug Administration recently approved the Cobas human papillomavirus (HPV) test (Roche Molecular Diagnostics) for use as a first-line primary screening test for cervical cancer in women who are 25 years old or older. 3 5 Cotesting with both Pap and hrhpv testing has been shown to not only detect significantly more cervical intraepithelial neoplasia 3 but also result in significantly lower rates of cervical intraepithelial neoplasia 3 or invasive cancer in subsequent rounds of screening. 6 8 The aforementioned study by Kaiser Permanente Northern California also reported a significantly lower cancer risk for women who were double-negative for Pap and HPV testing versus women who were HPV-negative only (3.2 vs 3.8 per 100,000 women per year). 2 As a result, cotesting is now recommended as a primary screening modality for women who are 30 to 65 years old. 9,10 Despite the low risk of precancerous cervical lesions in many of these studies, several studies, including the Kaiser Permanente study, found a broad range of hrhpvnegative rates (10%-30%) in cervical cytology specimens from patients with cervical cancer. 2,11 17 Our recent multicenter study in the United States indicated HPVnegative rates of 9%, 23%, and 25% less than 1 year, 1 to 3 years, and 3 to 5 years, respectively, before histologic diagnoses of cervical cancer. 18 Because some HPV infections are persistent for only a few years before invasive squamous cell carcinomas (SCCs) develop, these hrhpvnegative rates in patients developing invasive cervical cancer cause concern for the potential of an unacceptable false-negative rate and poor performance of screening programs if hrhpv testing is used as a single-modality cervical cancer screening tool, especially with extended screening intervals. China accounts for 14% of the world s annual incidence of cervical cancer; however, screening is still not widely accepted in China, especially in rural areas, where cervical cancer rates are highest (up to 81 per 100,000 women). A large pooled analysis in China suggests that the cervical cancer incidence in China may be higher than the currently estimated incidence of 9.6 per 100,000 women. 19,20 This underestimation of the incidence of cervical cancer in Chinese women has several possible explanations: 1) there is no well-established nationwide cancer registry, 2) there is no national cervical cancer screening program, and 3) there is no national standard for cytology quality control. Worldwide, the data for prior hrhpv testing results for patients with invasive cervical cancer are very limited. In the aforementioned multicenter study in the United States, only 70 invasive cervical carcinoma cases diagnosed in 2012 had prior HPV testing. 18 Therefore, we teamed up with Guangzhou KingMed Diagnostics, the largest independent pathology laboratory in China, to obtain a large study cohort to better understand the advantages and limitations of hrhpv testing in detecting invasive cervical carcinoma. This retrospective study examines prior hrhpv and Pap testing in women with histologically diagnosed invasive cervical cancer in China. MATERIALS AND METHODS Patient Selection Guangzhou KingMed Diagnostics is the largest independent pathology laboratory in China that is fully certified by the College of American Pathologists (CAP). Patients with a histologic diagnosis of invasive cervical cancer were retrospectively identified in the pathology database of Guangzhou KingMed Diagnostics over a 46- month period from January 2011 to October 2014. The study protocol received a priori approval by the appropriate institutional review committee. Prior hrhpv and Pap test results at Guangzhou KingMed Diagnostics were recorded, and only cases with prior hrhpv testing results within the year before the cancer diagnosis were included. All surgical pathology slides were retrieved and reviewed by 2 surgical pathologists (C.Z. and X.D.) to confirm the diagnosis and histologic classification. More than 90% of the samples were from Guangdong Province. Surgical specimens were collected from approximately 1000 hospitals throughout Guangdong Province and were referred to Guangzhou KingMed Diagnostics. Most cases originated from local community hospitals serving populations primarily from suburban and rural areas. Pap test specimens and HPV test specimens were collected by clinicians from more than 800 local community hospitals, women s health centers, clinics, and physical examination centers. Because there Cancer Cytopathology July 2015 429

TABLE 1. High-Risk HPV Testing Results Within the Year Before the Histological Diagnosis in 427 Invasive Cervical Cancer Cases Histologic Type No. Age, Average (Range), y HPV-Positive, No. HPV-Positive, % HPV-Negative, % SCC 364 45.9 (23-81) 346 95.0 5.0 ADC 56 43.4 (26-88) 42 75.0 25.0 ADSQ 5 43.2 (34-49) 5 100 0 Small cell carcinoma 2 58.5 (57-60) 2 100 0 Total 427 45.6 (23-81) 395 92.5 7.5 Abbreviations: ADC, adenocarcinoma; ADSQ, adenosquamous cell carcinoma; HPV, human papillomavirus; SCC, squamous cell carcinoma. is no well-established national cancer registry or national cervical cancer screening program, clinicians request Pap and/or HPV testing for a variety of reasons, mostly on the basis of clinical judgment and patient willingness. Pap and HPV testing specimens are received at Guangzhou KingMed Diagnostics separately and analyzed by different departments (the cytology department and the molecular microbiology department, respectively). hrhpv testing was performed at Guangzhou KingMed Diagnostics for all studied cases, but some patients may have had Pap cytology test results reported by another hospital laboratory and were, therefore, unavailable for this study. Pap Testing Pap tests were performed at the Guangzhou KingMed Diagnostics cytology laboratory with a variety of methods, including conventional Pap smears and 4 liquid-based cytology preparations: ThinPrep (Hologic, Bedford, Mass), SurePath (BD Diagnostics, Franklin Lakes, NJ), Liqui-PREP (LGM International, Melbourne, Fla), and Lituo (Lituo Biotechnology Co, Ltd, Hunan, China). Detailed methods, the Bethesda System (TBS) report rates, and additional details of the Guangzhou KingMed Diagnostics cytology laboratory are included in our recent publications. 21,22 hrhpv Testing hrhpv testing was performed at the Guangzhou KingMed Diagnostics molecular laboratory by 1 of 2 HPV testing methods. The majority of HPV tests were performed with the Hybrid Capture 2 (HC2) assay (Qiagen, Hinden, Germany), which tests for high- and intermediate-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). These samples were collected and stored in Digene s Standard Transport Medium. Less frequently, HPV testing was performed with a Tellgenplex 26 HPV genotyping panel nuclear acid detection kit for hrhpv (flow cytometry fluorescence hybridization method; Tellgen, Shanghai, China); this method was used to detect 26 HPV types. 23 Statistical Analysis Statistical analysis with the Pearson chi-square test was conducted with SAS 9.1 software (SAS Institute, Gary, NC). A P value less than.05 was considered statically significant. RESULTS Four hundred twenty-seven patients with invasive cervical cancer had hrhpv testing results within the year before the histological diagnosis; they included 364 patients with SCC, 56 patients with adenocarcinoma (ADC), and 7 patients with other malignancies. The average age of the patients was 45.6 years (range, 23-81 years). HPV Testing Results The average time between HPV testing and the histological diagnosis of cervical cancers was 1.3 months (range, 0.2-9 months). Four hundred fourteen of the 427 patients (97.0%) had hrhpv testing in the 3 months before the histological diagnosis. HPV testing within the 1-year time period before the histologic diagnosis was positive in 92.5% of all invasive cervical carcinoma cases and in 95% of SCC cases (Table 1). HPV testing was performed with HC2 in all but 5 SCC cases, and these 5 cases were all positive for hrhpv according to the Tellgenplex 26 HPV genotyping panel (HPV-16, 2; HPV-18, 1; HPV-33, 1; and HPV-59, 1). Fourteen of 56 cases (25.0%) of cervical ADC had negative HPV testing results within the year before the histologic diagnosis of invasive carcinoma; this was significantly higher than the rate for SCC (25.0% vs 5%, P <.001). Only 4 cases of ADC were of an unusual type, and they included 2 clear cell carcinomas, 1 villoglandular 430 Cancer Cytopathology July 2015

Prior High-Risk HPV Testing/Zheng et al TABLE 2. Papanicolaou Testing Results Within the Year Before the Histological Diagnosis in 155 Invasive Cervical Cancer Cases Papanicolaou Test Result SCC, No. (%) ADC, No. (%) ADSQ, No. (%) Small Cell, No. (%) Total, No. (%) Malignant cells 37 (27.8) 2 (11.1) 0 0 39 (25.2) HSIL 45 (33.8) 2 (11.1) 0 0 47 (30.3) ASC-H 28 (21.1) 1 (5.6) 2 (66.7) 1 (100) 32 (20.6) AGC 4 (3.0) 11 (61.1) 0 0 15 (9.7) LSIL 2 (1.5) 0 1 (33.3) 0 3 (1.9) ASCUS 15 (11.3) 1 (5.6) 0 0 16 (10.3) NILM 2 (1.5) 1 (5.6) 0 0 3 (1.9) Total 133 18 3 1 155 Abbreviations: ADC, adenocarcinoma; ADSQ, adenosquamous cell carcinoma; AGC, atypical glandular cell; ASC-H, atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion; ASCUS, atypical squamous cells of undetermined significance; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion; NILM, negative for intraepithelial lesion or malignancy; SCC, squamous cell carcinoma. carcinoma, and 1 minimal deviation carcinoma. HPV testing was negative in 2 of these 4 cases (50%; 1 clear cell carcinomas and 1 minimal deviation carcinoma). The HPV-negative rate was not statistically significantly different between the usual type of ADC and unusual types of ADC, but this was most likely due to the small sample size. All 5 cases of adenosquamous carcinoma and the 2 cases of small cell carcinoma were HPV-positive. Pap Test Results One hundred fifty-five of the 427 invasive cervical carcinoma cases (36.3%) with HPV testing results also had Pap cytology test results within the year before the histological diagnosis of cancer. Five types of Pap tests were used for the 155 cases: ThinPrep (132 cases or 85.2%), SurePath (5 cases or 3.2%), Liqui-PREP (10 cases or 6.5%), Lituo (6 cases or 3.9%), and conventional Pap smears (2 cases or 1.3%). One hundred fifty-two of the 155 patients (98.1%) had an abnormal Pap test within the 1-year time period before the diagnoses of cervical cancer. The average time period between Pap testing and histological diagnosis was 1.3 months (range, 0.2-8 months). Pap results interpreted as high-grade squamous intraepithelial lesions, positive for malignant cells, and atypical squamous cells (cannot exclude high-grade squamous intraepithelial lesion) were the most common abnormal Pap findings and were reported for 30.3%, 25.2%, and 20.6% of cases, respectively (Table 2). Pap results interpreted as atypical squamous cells of undetermined significance accounted for 10.3% of the abnormal Pap findings (Table 2). Pap results interpreted as atypical glandular cells were the most common abnormal Pap findings before cases of ADC, and they preceded the diagnosis in 61.1% of cases (Table 2). The Pap-negative rate TABLE 3. Comparison of Papanicolaou Results and HPV Results Within the Year Before the Histological Diagnosis in 155 Invasive Cervical Cancer Cases Papanicolaou Test Result HPV-Positive HPV-Negative No. % No. % Malignant 36 25.7 3 20.0 HSIL 45 32.1 2 13.3 ASC-H 29 20.7 3 20.0 AGC 10 7.1 5 33.3 LSIL 3 2.1 0 0 ASCUS 15 10.7 1 6.7 NILM 2 1.4 1 6.7 Total 140 100 15 100 Abbreviations: AGC, atypical glandular cell; ASC-H, atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion; ASCUS, atypical squamous cells of undetermined significance; HPV, human papillomavirus; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion; NILM, negative for intraepithelial lesion or malignancy. showed no statistically significant difference among different histologic types of cervical carcinoma; this was likely due to the small sample sizes. The Pap-negative rate also showed no statistically significant difference among the different Pap preparations, and again, this was most likely due to the fact that most were ThinPrep preparations, with few other preparations. Comparison of Pap and HPV Testing Results All cases with both Pap and HPV testing results (n 5 155) were divided into 2 groups: HPV-positive (n 5 140) and HPV-negative (n 5 15). One hundred thirty-eight of the 140 patients (98.6%) with positive HPV testing had abnormal Pap test results, and 14 of the 15 patients (93.3%) with negative HPV testing had abnormal Pap test results within the year before the histological diagnosis of cervical cancer (Table 3). The abnormal Pap testing rates were not statistically different between these 2 groups (P 5.162). The Cancer Cytopathology July 2015 431

TABLE 4. Comparison of Papanicolaou Results and HPV Results Within the Year Before the Histological Diagnosis in 18 Cervical Adenocarcinoma Cases Papanicolaou Test Result HPV-Positive HPV-Negative No. % No. % Malignant 1 7.7 1 16.7 HSIL 2 15.4 0 0 ASC-H 1 7.7 0 0 AGC 6 46.2 5 8.3 ASCUS 1 7.7 0 0 NILM 1 8.3 0 0 Total 12 100 6 100 Abbreviations: AGC, atypical glandular cell; ASC-H, atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion; ASCUS, atypical squamous cells of undetermined significance; HPV, human papillomavirus; HSIL, high-grade squamous intraepithelial lesion; NILM, negative for intraepithelial lesion or malignancy. overall Pap test negative rate was 1.9% (3 of 155); this was significantly lower than the HPV-negative rate of 9.7% (15 of 155, P 5.0036). Only 1 of the 15 cases with negative HPV test results also had a negative Pap test. This 1 case was the only double-negative screened case of cervical carcinoma in this series (1 or 155 or 0.65%). Eighteen cases of ADC had both Pap and HPV testing results within the year before the histologic diagnosis; they included 12 HPV-positive cases and 6 HPV-negative cases (Table 4). All 6 HPV-negative cases had abnormal Pap test results. One of the 12 HPV-positive cases (8.3%) had a negative Pap test. The Pap test negative rate was significantly lower than the HPV-negative rate in these cases of ADC (5.6% vs 33.3%, P 5.0352). All 6 HPVnegative cases had the usual type of ADC. DISCUSSION In this large, retrospective study of 427 cases of invasive cervical carcinoma, we found an HPV-negative rate of 7.5% within the year before the histological diagnosis (97% of the cases were tested within 3 months). Five percent of SCC cases and 25% of ADC cases had negative HPV test results within the year before the histologic diagnosis, and this was consistent with other studies demonstrating a higher HPV-negative rate for cervical ADC. Although the HPV-negative rates for Pap test specimens from patients with cervical cancer vary widely, these findings are in keeping with previous studies. 2,11 18 A study from a large US academic women s hospital reported a 9.7% HPV-negative rate within the year before the diagnosis of invasive cervical SCC. 11 In another recent multicenter study in the United States, the HPV-negative rate was reported to be 9% for patients with invasive cervical cancer during the 1-year period before the histologic diagnosis. 18 Another study indicated that 14% of patients with cervical glandular neoplasia had prior HPV-negative test results. 14 In addition, the study from Kaiser Permanente showed that 37% of cervical SCC cases and 22% of cervical ADC cases had a baseline negative HPV testing result 5 years before the histologic diagnosis, and this higher rate of HPV-negative cases is not unexpected because of the longer prediagnosis period. 2 The reason for a higher HPV-negative rate in patients with cervical ADC is not certain, but possible explanations include the fact that ADC is less frequently etiologically related to HPV infection (especially unusual types of ADC such as minimal deviation ADC). A recent worldwide analysis of 760 paraffin-embedded samples of cervical ADC using an HPV genotyping method demonstrated HPV positivity of 71.8% for the usual type of ADC and considerably lower rates for unusual types (endometrioid, 27.3%; serous, 25%; clear cell, 20%; not otherwise specified, 13.9%; minimal deviation, 8.3%). 24 A second possible explanation is that ADC is more often focal and/or has a high location in the cervical canal, and this makes sampling more difficult. Like previous studies, the current study once again demonstrates that a certain percentage of invasive cervical carcinomas (especially ADC) can be expected to have negative HPV test results. The proposed causes of negative HPV test results include low-titer or low copy number HPV, rare HPV types not included in routine HPV testing, limited analytic sensitivity, inadequate specimen cellularity, and rapidly developing cervical carcinoma with HPV testing performed before infection. 25 27 Although HPV testing is a highly sensitive method and has been approved as a primary screening modality, we have demonstrated that the Pap-negative rate is significantly lower than the HPV-negative rate in patients with both HPV and Pap test results within the 1-year period before the histological diagnosis. Furthermore, the difference between the Pap-negative rate and the HPV-negative rate is more pronounced in patients with invasive cervical ADC. However, on the basis of these data from a highly selected population of women with cervical carcinoma, we cannot conclude the Pap testing is more sensitive than HPV testing in the detection of cervical cancers in a routine screening population. 432 Cancer Cytopathology July 2015

Prior High-Risk HPV Testing/Zheng et al The Pap-negative rate in this series is somewhat lower than those previously reported for other laboratories in China, and this might be due to the population included in this study and also due to the high-level cytology services at Guangzhou KingMed Diagnostics. Guangzhou KingMed Diagnostics is the first large pathology laboratory fully certified (both for anatomic pathology and clinical pathology) by CAP in China. Guangzhou KingMed Diagnostics has also been certified by the International Organization for Standardization (15189). Laboratory workload standards, quality-control practices, and cytology-histology correlation reviews were all performed in accordance with current CAP Laboratory Accreditation Program checklists. 21,22 Although these data suggest a possible weakness in hrhpv testing, several caveats must be kept in mind. Most samples were taken in close temporal proximity to the histologic diagnosis of cervical cancer, and some, if not many, of these women were likely symptomatic and clinically suspected of having cervical cancer. This situation is different from that of a routine screening population, which is composed of mostly asymptomatic women, with a minority having precancerous lesions. The data presented are, therefore, more relevant to the detection of prevalent cancers than screening for precancerous lesions. Despite this, we feel that the detection of prevalent cancers is still an important aspect of routine screening programs and that an inability to detect such lesions suggests a potential weakness in the detection of even precancerous lesions. The majority of HPV testing performed in this study used HC2 and, therefore, reflects the specific limitations of this method for hrhpv detection. The Cobas HPV test was recently approved for first-line primary screening by the US Food and Drug Administration and has been shown to have improved detection of HPV in comparison with HC2 in women undergoing treatment for cervical dysplasia. 28 Additional studies will be needed to investigate the sensitivity of the Cobas HPV test in a similar population. The HPV status was also not determined in surgical specimens; therefore, there might be some true HPV-negative cases included in the series (especially cases of ADC). Despite this, because the vast majority of cervical carcinomas are now known to be etiologically related to HPV infection, we feel that it is reasonable to conclude that most of the HPV-negative results represent true false-negative results. The greatest limitation of this study is perhaps the fact that a significant number of the patients (272 of 427) had only HPV testing results available and no Pap test results. This was at least partly due to the fact that clinicians in China request Pap testing and/or HPV testing without the benefit of standardized guidelines. In addition, Guangzhou KingMed Diagnostics is a large, independent laboratory in China, and some patients have HPV testing performed by Guangzhou KingMed Diagnostics, whereas Pap cytology testing is performed in a separate hospital system and, therefore, was not available for this study. Because of the absence of well-established screening programs in China and because most cases included in this study were from rural or suburban areas, it is reasonable to conclude that at least some of the cervical cancers developed in patients without prior screening. Given this, we believe that on average the cancers included in this study were larger and at a higher stage than tumors seen in patients with routine screening. However, detailed information on the tumor sizes and stages of the study cohort is unavailable. Another limitation of this study may be a verification bias; for example, women with abnormal test results are more likely to undergo diagnostic testing. This bias may make both screening tests (Pap and HPV tests) appear more sensitive in this setting than either is in reality. In conclusion, this large, retrospective study examines recent HPV testing results for women with invasive cervical cancer from China, where there is no wellestablished national cervical cancer screening program. Our data demonstrate that the HPV testing methods used in this study (mostly HC2) had a higher negative rate for patients with invasive cervical carcinoma and especially for patients with cervical ADC in comparison with Pap cytology. This suggests significant concerns about the potential use of HPV testing alone in the detection of cervical cancer. However, if cost-effectiveness or more sensitive HPV testing methods are considered, it is still uncertain what the best screening strategy might be. This is especially true in areas and nations without established screening programs such as rural China. FUNDING SUPPORT No specific funding was disclosed. CONFLICT OF INTEREST DISCLOSURES The authors made no disclosures. Cancer Cytopathology July 2015 433

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