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© 1999 American Society for Clinical Oncology Detection of Human Papillomavirus Types 16 and 18 mRNA in Peripheral Blood of Advanced Cervical Cancer Patients and Its Association With PrognosisFrom the Departments of Obstetrics and Gynecology, Radiation Oncology, and Pathology, Chang Gung Memorial Hospital, and Department of Biochemistry, Chang Gung University College of Medicine, Taipei, Taiwan. Address reprint requests to Chih-Jen Tseng, MD, Department of Gynecology and Obstetrics, Chang Gung Memorial Hospital, No. 5, Fu-Shing St, Kweishan 333, Taoyuan, Taiwan; email jantseng@ ms1.hinet.net.
PURPOSE: To evaluate the feasibility of detecting human papillomavirus E6 (HPVE6) gene mRNA in the peripheral blood of patients with locally advanced cervical cancer, and the relationship of the circulating HPV viral–specific mRNA with clinicopathologic factors and prognosis of locally advanced cervical cancer. PATIENTS AND METHODS: The presence of types 16 and 18 HPVE6 gene mRNA was determined by reverse transcription followed by nested polymerase chain reaction. Thirty-five patients with locally advanced cervical cancer who were positive for HPV type 16 or 18 DNA were included in the study. All patients received external-beam radiation therapy followed by intracavitary brachytherapy. RESULTS: Eighteen (51.4%) of 35 HPV DNA–positive cervical cancer patients had HPV-specific mRNA in their peripheral blood cells, compared with none of 17 HPV DNA–negative cervical cancer patients and none of 12 control volunteers. The presence of HPVE6 gene mRNA in peripheral blood was associated with bulky tumor volume (> 4 cm) and pelvic lymph node metastasis (tumor volume, P = .03; lymph node status, P = .03). After a median follow-up of 22 months, patients who were positive for peripheral-blood HPVE6 gene mRNA had a significantly higher risk of recurrence than those who were negative (10 of 18 v three of 17, P = .02; mean recurrent time, 20.7 months v 12.6 months, P = .02). There was also a statistically significant association of peripheral-blood HPVE6 gene mRNA positivity with distant metastasis (eight of 18 v one of 17; P = .01). CONCLUSION: Results of this study seem to suggest that the presence of HPVE6 gene mRNA in peripheral blood may provide an early marker that identifies patients who are at risk for metastasis.
OVER THE PAST decades, the prognosis for patients with locally advanced carcinoma of the cervix has been poor, particularly for those with bulky local tumor and extensive parametrial extension.1,2 Radiation therapy has been the primary treatment modality for these patients, and analysis of treatment failure patterns after radiotherapy showed that approximately 60% of patients who experience treatment failure have pelvic recurrence, and approximately half of them develop distant metastasis.1,3-5 Recurrence and metastasis at distant sites continue to pose major problems in the clinical management of patients with locally advanced cervical carcinoma. Therefore, one of the most important aspects of successful treatment of patients with locally advanced cervical cancer is the identification of patients at risk for distant metastasis and the early detection of distant metastasis so that appropriate salvage therapy after radiotherapy or surgery can be provided. Evidence from animal studies has shown that malignant cells are released into the blood stream from primary tumors, leading to an increased incidence of distant metastases.6-8 It has also become clear that the appearance of circulating cancer cells might be a key step in tumor metastasis. The detection of circulating tumor cells using reverse transcription followed by polymerase chain reaction (RT-PCR) assay has recently been applied to a variety of human tumors.9-17 Using this method, investigators have detected circulating cells of malignant colorectal cancer,9 hepatocellular carcinoma,10 prostate cancer,11 melanoma,12,13 neuroblastoma,14 breast cancer,15 and pancreatic cancer16 by targeting the expression of a number of tumor-specific genes, such as alpha-fetoprotein and tyrosinase, or the presence of K-ras codon 12 mutations. We recently reported that malignant cervical cells in peripheral blood may be determined by the expression of the human papillomavirus E6 (HPVE6) transforming gene in cervical cancer patients with clinical evidence of distant metastasis.17 These results seemed to indicate that cervical cancer cells could be released into the circulatory system, and that such cells could be identified by the RT-PCR method. However, the biologic and clinical significance of these observations remains to be proved. Additionally, it remains to be determined whether the detection of circulating HPVE6 gene mRNA can be used to predict the development of future metastasis in these patients. In this article, we report our results from a prospective study on the presence of circulating HPVE6 gene mRNA in patients with locally advanced cervical cancer.
Previously untreated patients with stage IIB to stage IVA advanced cervical carcinoma with positive HPV type 16 or 18 DNA in their cervical cancer tissues were the subjects of the study. Patients with stage IIB tumors had to have bulky tumors larger than 4 cm and extensive parametrial invasion18 to be included in the study. All patients underwent complete physical and gynecologic examinations (while under anesthesia), and the cervical cancer was staged according to the guidelines of the International Federation of Gynecology and Obstetrics (FIGO). Initially, fresh cancer tissue samples obtained from patients with locally advanced cervical carcinoma were analyzed for the presence of HPV types 16 and 18 DNA. All patients had no history of other malignancy and had not undergone chemotherapy or radiotherapy. Patients with documented disease beyond the pelvis were excluded from the study, as were those with positive para-aortic lymph nodes. Cervical cancer tissues and peripheral-blood samples were obtained from all patients before therapy. Fifty female laboratory staff members and hospital nurses voluntarily donated peripheral blood to serve as controls. Human cervical cancer cell lines CaSki, HeLa, SiHa, MT-3, C4-I, and C33-A obtained from the American Type Culture Collection (Rockville, MD) were used as HPV-positive controls. The presence of HPV types 16 and 18 DNA in cervical cancer tissues was analyzed by nested PCR.19,20 Transcriptional products of types 16 and 18 HPVE6 transforming gene in the mononuclear cells recovered from peripheral blood were analyzed by RT-PCR.17 Written informed consent was obtained from all patients, and the study protocols were approved by the Protocol Committee of Chang Gung Memorial Hospital. Eligible patients received external-beam radiation therapy followed by intracavitary brachytherapy. Pretreatment investigation included a complete medical history and physical examination, complete blood count, serum electrolyte evaluation, chemistry profile, tumor marker of squamous cell carcinoma (SCC) antigen, chest radiograph, ECG, and a contrast-enhanced computed tomography (CT) scan of the abdomen and pelvis. All patients underwent a protoscopy, cystoscopy, bone scan, and biopsy of any and all suspected lesions. Patients with radiographically suspicious para-aortic or pelvic lymph nodes underwent a CT-guided percutaneous needle aspiration of the node. Extraperitoneal staging laparotomy was performed if the aspiration cytology was normal. Any enlarged or suspicious nodes were excised and examined for histopathologic interpretation. All patients with stage IIB to IVA carcinoma returned for a follow-up visit every 4 months for the first 2 years after completion of radiotherapy and every 6 months thereafter. The follow-up investigations included a physical examination, cervical cytology, chemistry profile, and tumor marker of SCC. Further work-ups, such as CT scans of the pelvis, bone scan, chest radiograph, or biopsy of any suspected lesions, were performed when clinically indicated. Pelvic failure was defined as disease that recurred in the pelvis, including central and parametrial failure. Distant failure was defined as disease that occurred outside the pelvis. Survival was defined as the observed length of life from protocol entry to death or until May 20, 1997, when the data analysis was performed if the patient was still alive.
Specimen Sampling and Preparation
Determination of the Presence of HPV DNA by PCR
Determination of the Presence of Circulating HPVE6 Gene mRNA
Statistical Considerations
From January 1994 to November 1995, a total of 50 consecutive patients with stage IIB bulky to IVA advanced carcinoma of the uterine cervix were tested for the presence of HPV types 16 and 18 DNA. Among these patients, 35 (70%) with cervical cancer had either HPV type 16 or 18 DNA in their primary cancer tissues and were enrolled onto the study. The mean age of the patients was 56.2 years (range, 33 to 68 years). Data on the characteristics of patients, including age, FIGO stage, tumor size, histologic grade, and lymph node status, are listed in Table 1. Curative radiation therapy was delivered completely for all eligible patients.
Peripheral-blood specimens from 18 (51.4%) of 35 cervical HPV DNA–positive patients were found to contain HPV-specific mRNA, in contrast to none of the 17 patients with HPV types 16 and 18 DNA–negative cervical cancer. Twelve (24%) of 50 healthy volunteers had HPV type 16 or 18 DNA detected by cervical smears, but none was found to have HPV viral-specific mRNA in their peripheral-blood cells. Six (42.9%) of 14 patients with stage IIB carcinoma, 10 (55.6%) of 18 with stage III carcinoma, and two (66.7%) of three with stage IV carcinoma had HPV viral-specific mRNA in their peripheral-blood cells. No statistical significant correlation was found between HPV viral-specific mRNA in the peripheral-blood cells and clinicopathologic parameters, including age, elevated SCC antigen, histology, or cell differentiation. However, the presence of HPV viral-specific mRNA in peripheral-blood cells was found to be associated with bulky tumor volume (> 4 cm) and pelvic lymph node metastasis (tumor volume and lymph node status, P = .03). Relapse patterns of patients are summarized in Table 2. Univariate analysis showed a high cervical cancer recurrence rate of 55.6% for patients with HPVE6 gene mRNA in their peripheral circulation, compared with 17.6% for patients who did not have circulating HPV mRNA (10 of 18 v three of 17, P = .02). Among the 18 patients with positive circulating RT-PCR results, eight had relapses at distant sites and six had central recurrences; in contrast, among 17 patients with negative circulating RT-PCR results, two had central recurrences and one had recurrence at a distant site. The median time to recurrence was 20.7 months (range, 13 to 28 months) for the RT-PCR–negative group and only 12.6 months (range, 8 to 19 months) for the RT-PCR–positive group (P = .02). Statistical analysis also showed a significant correlation between a positive RT-PCR assay and distal metastasis (eight of 18 v one of 17; P = .01). After a median follow-up of 22 months (range, 12 to 35 months), a total of 10 patients died of recurrent cervical cancer. Among these 10 patients, eight were positive and two negative for the circulating HPV viral mRNA. Kaplan-Meier analysis showed that the actuarial disease-free survival rate at 2 years was significantly lower for patients positive for circulating HPV viral mRNA compared with those who were negative (mean survival time, 21 v 33 months; P = .01; Fig 1).
Metastasis of cancer cells is one of the critical factors that determines the prognosis of the disease, and it is also the main reason for the high fatality rate of cancer. Therefore, detecting metastasis at its earliest stage and predicting prognosis are important in the treatment of patients with cancer. Cervical cancer cells can spread through the retroperitoneal route and lymphatic and blood circulation. The presence of circulating tumor cells is an indicator of the systemic spreading of cancer cells, which may lead to metastasis at distant sites. Because the number of tumor cells in peripheral blood is expected to be small at its early stage, techniques for their detection must be both highly sensitive and specific. The PCR method, an extremely sensitive technique, has the ability to amplify a particular sequence of either DNA or RNA and can potentially detect one copy of targeted nucleotide sequences among 106 cells.21,22 In the case of cervical cancer, the mRNA of type 16 or 18 HPVE6 transforming gene is almost invariably expressed in HPV type 16– or HPV type 18–positive cervical cancer cells,23-25 which makes it an ideal target to detect small numbers of cervical cancer cells. We recognize that a positive peripheral-blood RT-PCR test does not necessarily prove the presence of circulating tumor cells. Positive RT-PCR results may be caused by many factors, including bloodborne HPV virus particles, a small amount of circulating naked HPV RNA, or even HPV present in nontumor cervical cells or even noncervical cells. Without proof that the circulating cervical cancer cells have actually been isolated and characterized, the aforementioned possibilities cannot be ruled out. However, the likelihood that the RT-PCR results we observed in this study were caused by these factors is remote. Circulating bloodborne HPV virions have not been shown to exist after extensive research by many investigators, and even if they exist in small amounts, no HPV-specific transcripts would be expected. Naked HPV-specific RNA would not have survived in human blood (unpublished data), probably because of the ribonuclease in the blood. Furthermore, we have previously shown that HPV can be found in peripheral-blood mononuclear cells, but our data have also shown that no HPV-specific RNA could be detected in these cells.26 It is therefore possible that a positive peripheral-blood RT-PCR test may indicate the presence of circulating tumor cells. Studies by other investigators on the metastasis of murine lung carcinoma have suggested that at least 1,000 lung cancer cells are needed to establish metastasis.27,28 If similar kinetics is applicable to human cervical cancer, our RT-PCR analysis would have provided enough sensitivity to detect micrometastatic cervical cancer cells. This assumption also is in accord with our observed positive rate of circulating HPVE6 gene mRNA of 92% in patients with cervical cancer who had clinical evidence of distant metastasis,17 and of 56% in patients with locally advanced cervical cancer who lacked the false-positive results observed among 70 healthy controls or 17 patients with HPV-negative cervical cancer. It is therefore possible that our method is feasible to detect circulating micrometastasis before other clinical evidence becomes evident. Since the first published report on circulating cancer cells in peripheral blood in 1955,29 the existence of circulating cancer cells has been all but confirmed in a number of hematologic malignancies and solid tumors.9-17 The question has now become, "What is the clinical significance of these circulating cancer cells?" According to the rationale that the presence of circulating tumor cells may lead to the establishment of metastatic lesions outside the radiation field, patients with cancer cells in their peripheral blood may have a higher risk of metastasis and a poor prognosis than those without. Therefore, if the presence of circulating HPVE6 gene mRNA is found to correlate with relapse patterns of the disease and the presence of circulating micrometastasis, it might influence and revise our understanding of disease progression, as well as the actuarial meaning of circulating tumor cells. Our results suggest that patients with cervical cancer who have RT-PCR–positive test results for circulating HPVE6 gene mRNA have a significantly higher relapse rate than those without. Our observations also show that a statistically significant correlation seems to exist between the presence of circulating HPVE6 gene mRNA and the incidence of distal metastases. These results suggest that identification of circulating HPVE6 gene mRNA may assist clinicians in assessing tumor progression, as has been suggested by other investigators for circulating colorectal cancer and melanoma cells.9,13 It is also possible that the detection of circulating HPVE6 gene mRNA may become an additional parameter through which clinicians can make a better prognosis and identify patients who may need more intensive therapeutic interventions. However, there are certainly clinicopathologic factors other than circulating HPVE6 gene mRNA that have been used successfully in the prediction of higher risk for recurrence or poor prognosis. These factors include large tumor size, parametrial extension of the disease, metastatic tumors to the pelvic lymph nodes, elevated SCC, and FIGO stage.30-32 Our findings indicate that the presence of circulating HPVE6 gene mRNA is also associated with the size of tumors and lymph node metastasis. Because the two factors could affect survival and the study was based on a small sample size, the clinical and biologic significance of a positive RT-PCR test result is difficult to assess using univariate analysis without a larger cohort study. Nevertheless, these results may indicate that tumor cells were released from the tumor foci into circulation when the tumor size increased, which is in accordance with the observations in hepatocellular carcinoma.10 Although circulating HPVE6 gene mRNA seems to be a sensitive marker for identifying patients at risk for recurrence or distant metastasis, there are several limitations associated with the method used here that may potentially affect its clinical applications. First, although an overwhelming majority of cervical cancer cells contain HPV DNA,33 a small percentage of cervical cancers do not. The association between poor prognosis in cervical cancer and nondetection of HPV DNA has been reported34,35; however, the current method would not be applicable to these patients with cervical cancer. Second, the detection of HPVE6 gene mRNA in the cells recovered from peripheral blood may reflect the presence of tumor cells in circulation, which are spontaneously released from the primary tumor foci into the circulatory system. It remains possible that cells shed from the tumors may no longer be viable or tumorigenic. Multiple variables, such as the immune status of the host, invasive capacity of the tumors, survival of these cells in blood circulation, growth potential of the cells, and colonization potential of the cells once they arrive at the distant sites, all play a role in the establishment of a successful metastasis.36 Solid tumor animal experiments have suggested that relatively small tumors can shed millions of cells into the systemic circulation, with only a small proportion of these cells developing into clinically detectable metastatic disease.6-8 Therefore, the presence of tumor cells in the bloodstream does not necessarily indicate that metastases will develop, which may explain our observations that certain patients with a positive peripheral-blood RT-PCR test result showed no clinical evidence of metastatic disease even after years of follow-up. In conclusion, results of this study suggest that the presence of circulating HPVE6 gene mRNA using RT-PCR is significantly associated with bulky tumor volume and lymph node metastasis, and can be a useful tool for identifying patients who may be at higher risk of developing distant metastasis before clinical signs become evident. This method may also be useful in selecting patients who may need either more or less intensive therapeutic interventions. It is desirable for future studies to ascertain whether the presence of circulating HPVE6 gene mRNA can predict the development of future metastasis for patients with stage IB2 disease. If affirmative, it may be beneficial in re-assessing neoadjuvant chemotherapy for patients with positive RT-PCR results. More studies are needed and should include serial blood analysis and follow-up evaluation of patients with circulating HPVE6 gene mRNA for even longer periods of time to monitor the effect of therapy on the presence and concentrations of circulating tumor cells. Randomized studies are currently underway to evaluate the efficacy of treatment in patients who test positive for circulating HPVE6 gene mRNA. Results from these studies may help to determine whether the identification of HPVE6 gene mRNA may be useful in designing therapeutic protocols to match the specific needs of individual patients.
Supported by medical research grants no. NSC87-2314-B-182A-092 and NSC87-2314-B182-010 from the National Science Council of Taiwan.
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Copyright © 1999 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 test. Significant difference was considered at P < .05. 
