Int J Gynecol Cancer 2006, 16, 10481054

Human papillomavirus prevalence in postradiotherapy uterine cervical carcinoma patients: correlation with recurrence of the disease
R.K. SINGH*, S. MAULIKy, S. MITRA*, R.K. MONDALy, P.S. BASUy, S. ROYCHOWDHURYz & C.K. PANDA*
Departments of *Oncogene Regulation and yGynaecology Oncology, Chittaranjan National Cancer Institute, Kolkata, India; and zHuman Genetics and Genomic Division, Indian Institute of Chemical Biology, Kolkata, India

Abstract. Singh RK, Maulik S, Mitra S, Mondal RK, Basu PS, Roychowdhury S, Panda CK. Human papillomavirus prevalence in postradiotherapy uterine cervical carcinoma patients: correlation with recurrence of the disease. Int J Gynecol Cancer 2006;16:10481054.
To understand the role of human papillomavirus (HPV) in recurrence of uterine cervical cancer (CA-CX) after radiotherapy, we have analyzed the HPV prevalence in the exfoliated cells of 56 patients and their corresponding plasma. HPV DNA was detected in exfoliated cells of 78% (44/56) patients (HPV-16, 68%; HPV-18, 14%; HPV-X [other than 16, 18], 11%; and mixed infection of HPV-16 and HPV-18 in three cases). HPV DNA in plasma was present in only 25% (11/44) of the HPV-positive exfoliated cells (positive predictive value, 100%; negative predictive value, 27%) with concordance in HPV types. The recurrence of the disease was signicantly associated with the presence of HPV in the exfoliated cell (P 0.01) and plasma (P 0.007) as well as high viral load in the exfoliated cell (P 0.0002). KaplanMeier disease-free estimates have also shown the signicant association between HPV prevalence in plasma and recurrence of the disease (P 0.045). Thus, it indicates that in postradiotherapy CA-CX patients, the high viral load in the exfoliated cell as well as HPV presence in the plasma samples could be used in early detection of the patients at increased risk for disease recurrence and progression.
KEYWORDS:

cervical cancer, human papillomavirus, plasma DNA, postradiotherapy, recurrence.

Uterine cervical cancer (CA-CX) is considered to be one of the most common cancers among women worldwide(1). In eastern India, CA-CX constitutes about 18% of the total cancers, occupying second highest position among females(2). Several etiologic factors have been associated with its development(2), most important being infection with human papillomavirus (HPV), with 90100% prevalence(3,4). Although many types of HPV have been associated with CA-CX, HPV-16 and HPV-18 are the two most common high-risk types, accounting for 75100% of the cases(4).
Address correspondence and reprint requests to: Chinmay Kumar Panda, PhD, Department of Oncogene Regulation, Chittaranjan National Cancer Institute, 37, S.P. Mukherjee Road, Kolkata 700026, India. Email: ckpanda@vsnl.net The rst and second authors contributed equally to this work.

Radiation therapy along with surgery is the most effective mode of treatment for cervical cancer. However, treatment failures have been encountered even after radiotherapy, and postirradiation adjuvant hysterectomy or chemotherapy may be recommended for such cases(5). Cervical cancer treatment by radiotherapy is often encountered with local recurrence (38% for stage I to 45% for stage III)(6). Conicting reports have been found in the timing of recurrence. Most recurrence in cervical carcinoma occurs within 5 years after the initial radiation therapy, more than 5 years have been reported in few cases(7). Since HPV is considered to be a necessary factor for CA-CX development and disease severity, its persistence was seen in 57% of the patients after postradiotherapy and was signicantly associated with recurrence(5). Similarly, the HPV persistence and association with recurrence have been detected in 19.6% of
# 2006, Copyright the Authors Journal compilation # 2006, IGCS and ESGO

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the patients with HPV DNApositive cervical intraepithelial neoplasia (CIN) lesions after therapeutic conization,(8) and 50% of the CIN patients with HPV positivity after treatment with diathermic large-loop excision(9). It has been suggested that HPV DNA persistence after successful treatment might be due to the presence of HPV DNA and/or HPV DNA sequence fragments in the degraded tumor cells or cell debris(5). Previous studies have found circulating tumor DNA in the plasma and serum of the cancer patients, reecting the tumor load or metastasis of the patients(10). Though the mechanism of this phenomenon is not clear, the presence of tumor DNA in plasma/serum may have diagnostic and prognostic value(11). HPV DNA has been seen in the plasma of 1250% CA-CX patients before the radiotherapy(12,13). However, during follow-up studies, Yang et al.(13) have found 23% (5/21) HPV prevalence in plasma of postradiotherapy CA-CX patients, but no prognostic signicance has been determined. Moreover, the HPV prevalence was high in plasma of the CA-CX patients having recurrence. It seems that HPV detection after therapy in the exfoliated cells as well as plasma of the patients may be used as a tool for monitoring the efcacy of the treatment and recurrence of the disease. Thus, to nd out the role of HPV in recurrence of CA-CX after radiotherapy, attempt has been made in this study to analyze the prevalence of HPV in the exfoliated cells and plasma of the corresponding postradiotherapy patients. The prevalence of HPV has been correlated with different clinicopathologic parameters and prognosis of the disease.

External-beam irradiation was delivered with telecobalt and linear accelerator 6-MV and 18-MV X-ray, with central shielding through anteroposterior-opposed portals, to the whole pelvic region and, if needed, to the para-aortic region depending on the status of disease. Total dose of external-beam irradiation was to the parametrium, 50 Gy. External-beam irradiation was followed by highdose rate intracavitary brachytherapy that used a remotely controlled afterloading system with an Ir-192 source. Two to three fractions were administered once weekly with a fraction dose of 7 Gy at point A, ranging from 14 to 21 Gy. DNA isolation DNA from the exfoliated cervical cells and plasma was isolated according to the procedure described by Jen et al.(14). Briey, the cervical swab (or brush) was immersed in 1 mL of phosphate-buffered saline and swirled to release the cells. The suspension was centrifuged at 12,000 rpm for 15 min at room temperature, and the pellet was then treated with lysis buffer (10 mM TrisHCl, pH 7.4; 5 mM ethylenediamine-tetra-acetic acid (EDTA), pH 8; and 1% sodium dodecyl sulphate (SDS)). The lysate was further treated with proteinase K at 37C for overnight followed by phenolchloroform extraction and ethanol precipitation of DNA. The DNA precipitate was dissolved in 40 lL Tris-EDTA (10 mM TrisHCl, pH 7.4 + 1 mM EDTA), and the concentration of DNA was measured by spectrophotometry (UV-160A; Shimadzu, Tokyo, Japan). The mean DNA concentration of the samples was 2.44 0.76 lg/lL. Similarly, DNA from the plasma of the corresponding patients was extracted by SDS/proteinase K digestion followed by phenolchloroform extraction and ethanol precipitation. HPV analysis and quantitation The presence of HPV in the cervical lesions was detected by polymerase chain reaction (PCR) using primers (MY09 and MY11) from the consensus L1 region(15). Typing of HPV-16/18 in the L1-positive samples was done by PCR using specic primers from the E6 region of HPV-16(16) and the LCR region of HPV-18(17). The PCR products were electrophoresed in 2% agarose gel, stained with ethidium bromide, visualized under ultraviolet light, and photographed. For nal conrmation of the HPV types, after gel electrophoresis, the PCR products were transferred to Gene Screen nylon membrane for Southern hybridization with [32P]-labeled HPV typespecic probes(18). DNA from the SiHa (for HPV-16) and HeLa (for HPV-18)
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Materials and methods

Patients Cervical exfoliated cells and respective blood were collected at once from the randomly selected CA-CX patients who received last radiation between 1988 and 2004 at hospital section of Chittaranjan National Cancer Institute, Kolkata, India. Informed consent was obtained from all the patients and institutional review board. The median age of the cervical cancer patients was 49, with a range of 3367 years. Clinically, the tumors were staged according to the FIGO classication by the staff of the Department of Gynecology and the Department of Radiology and Radiation Oncology (Table 1). Radiotherapy treatment techniques All the CA-CX patients underwent radical radiotherapy as primary treatment with intent to cure.

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Table 1.

Demographic and clinicopathologic data of postradiotherapy CA-CX patients

HPV status in exfoliated cell HPV status in plasma Typec 16 16 16 16 16 16 18 18 16 16 18 16 16 16 16 16 16 18 16 16 X 16 16 16 X 16 18 16 16 16, 18 16 X 16 18 16 X 16, 18 X 16 16 16 16, 18 16 16 HPV 2 1 2 1 2 2 2 2 2 2 2 1 2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 1 2 2 1 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 1 1 2 1 Concentrationb (pg/mL) 97.6 55.6 88.0 49.6 40 36 314 78.4 218.2 45.6 361.8 Typec 16 16 18 16 16 16 16 16 16 18 16

Tumor samples 98/3068 00/1283 01/262 88/2216 94/939 00/4775 02/3533 02/6260 03/5573 00/5828 00/1648 85/258 94/2431 94/939 97/2023 96/4538 03/939 03/4038 02/1621 02/2828 02/4401 00/549 98/259 02/5887 98/4454 03/3108 00/1648 03/1395 93/2760 91/5551 92/4433 95/4703 01/5190 00/5213 99/4914 02/3803 00/2591 03/1543 03/6143 03/3360 02/3087 98/4929 97/4570 03/5204 03/2941 03/870 02/588 93/1624 00/1997 94/1011 96/2642 94/204 90/2201 02/5678 99/2076 03/3183

Stage IB IB IB IB IB IB IIA IIB IIB IIB IIB IIB IIB IIB IIB IIB IIB IIB IIB IIB IIB IIB IIB IIB IIB IIB IIB IIIB IIIB IIIB IIIB IIIB IIIB IIIB IIIB IIIB IIIB IIIB IIIB IIIB IIIB IIIB IIIB IIIB IIIB IIIB IIIB IIIB IIIB IIIB IIIB IIIB IIIB IVB IVB IVB

Age (years) 38 50 44 62 44 40 53 60 44 50 38 42 40 42 45 55 60 42 55 62 50 45 70 40 45 48 38 44 38 35 64 45 45 52 45 46 50 53 34 52 45 56 63 67 58 62 45 65 61 33 50 62 49 53 41 45

Intervala (months) 86 58 48 199 133 55 34 25 5 224 128 55 49 110 93 91 22 20 35 32 27 62 81 24 62 142 56 24 35 159 151 112 38 50 53 29 55 26 13 17 30 67 87 16 16 23 36 147 55 133 113 135 156 27 75 33

Recurrence 2 1 2 1 2 2 1 2 2 2 1 1 1 2 2 2 2 2 2 2 2 2 1 2 2 2 2 1 1 2 1 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 2 2 1 2 2 2 1 2 2 1

HPV 2 1 1 1 2 1 1 2 2 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 2 2 2 2 2 1 2 1 1 1 1 1 1 1 1 1 1

Concentrationb (pg/mL) 185.7 94.8 611.5 568.5 649.8 44 415.4 707.2 645 63.1 37.4 357.9 85.2 133 171.3 48.8 44 195.3 314.9 649.8 219.2 34.4 582.8 554.1 563.7 468 113.9 90 463.2 721.6 659.4 154 133.1 133.1 123.5 233.5 602 75.6 659.4 99.6 759.9 611.5 712.0 623.5

1, present; 2, absent. a Months from last radiotherapy. b Amount of HPV present in 1 mL phosphate-buffered saline suspension of exfoliated cells. c 16 denotes HPV type 16; 18 denotes HPV type 18; X denotes other than HPV types 16 and 18.

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cell lines and HPV typespecic plasmids were used as positive controls. For quantitation of HPV in the samples, the intensity of the L1-amplied bands in the agarose gel was determined by Shimadzu CS-9000 densitometry scanner. For standard graph, 2 lL of the HPV-16 plasmid after serial dilution was PCR amplied using the L1-specic primers, electrophoresed in 2% agarose gel, and photographed under ultraviolet transilluminator. The intensity of the band was quantitated in the densitometric scanner. Then, the concentration of HPV in the samples was determined from standard graph of the intensity of diluted HPV-16 plasmid. Statistics Correlation between postradiotherapy HPV prevalence and recurrence of the disease was evaluated by the Fisher exact test. Sensitivity, specicity, and predictive values (positive predictive value [PPV] and negative predictive value [NPV]) were calculated using 2 3 2 table and their 95% condence intervals (CI) were calculated by ClopperPearson method using quantitative parasitology (statistical software package at http://bio.univet.hu/QP/QP.htm)(19). Disease-free survival curve was calculated according to KaplanMeier method. Postradiation overall survival was measured from the date of last radiation to the date of last follow-up or recurrence of the disease. The log-rank test was used to assess the differences in patient survival (disease free) between cases with plasma HPV and cases without plasma HPV. Probability value (P value) 0.05 was considered statistically signicant. All the calculations were performed using statistical program SPSS (SPSS Inc., Chicago, IL).
Figure 1. Representative autoradiograph showing HPV-16specic PCR products in A) exfoliated cells and B) plasma, hybridized by HPV-16 probe. M, marker (pUC18 plasmid digested with Msp I); 16 p, HPV-16 plasmid DNA. The numbers in the other lanes indicate patients registration number.

HPV prevalence was more or less comparable among the patients after 2 years of postradiotherapy, whereas average viral load gradually increased up to 5 years of postradiotherapy followed by decrease in the subsequent years (Fig. 2). Moreover, recurrence of the disease was quiet high (64%, 9/14) in the patients treated within 5 years of initial radiotherapy than those more than 5 years of initial radiotherapy (36%, 5/14). Prevalence of HPV in plasma samples HPV DNA was detected in 11 of the 56 (20%, CI 10 32%) cases of the plasma samples. Among the HPVpositive samples, HPV-16 was present in majority (9/ 11) of the cases (Fig. 1B), while rest of the samples were HPV-18 positive. There was concordance between the HPV types present in the plasma and in

Results
Prevalence of HPV in exfoliated cervical cell Using the L1 primer, HPV DNA was detected in 44 of the 56 patients (78%, CI 6688%) (Table 1). Among the HPV-positive cases, only HPV-16 and HPV-18 were identied in 30 and 6 (68%, CI 5281%; 14%, CI 52 28%) cases, respectively (Fig. 1A), whereas HPV-X (other than 16/18) was seen in ve (11%, CI 3825%) cases. Three samples (3803, 1997, 5678) were both HPV-16 and HPV-18 positive. There was wide range (34.4759.9 pg/mL; Table 1) of HPV DNA concentration in the exfoliated cells irrespective of its DNA concentration (data not shown). About 75% (33/44) of the HPV-positive samples showed !100 pg/mL HPV DNA in the exfoliated cells. Interestingly, average
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Figure 2. Average HPV frequency and viral load in the exfoliated cells of postradiotherapy CA-CX patients. pg, picogram. 2006 IGCS, International Journal of Gynecological Cancer 16, 10481054

1052 R.K. Singh et al.

the corresponding exfoliated cells. The PPV of HPV presence in plasma and their corresponding exfoliated cell was 100%, CI 72100%, whereas NPV was 27%, CI 1542%. About 27% (3/11) of the HPV-positive plasma samples showed !100 pg/mL HPV DNA in the blood. Clinical correlation Of the 56 patients, disease recurrence was seen in 25% (14/56) cases irrespective of the clinical stages before radiotherapy (Table 1). The presence of HPV in the exfoliated cells as well as the viral load after radiotherapy are independent of the clinical stage of the disease before therapy (data not shown). However, signicant association of the recurrence was seen in cases with HPV-positive exfoliated cells (P 0.01) as well as high viral load (!100 pg/mL) (P 0.007) (Table 2). The presence of HPV in plasma of the corresponding patients was seen to be signicantly associated with viral load (!100 pg/mL) in the exfoliated cells (P 0.025) and recurrence of the disease (P 0.0002) (Table 2). The sensitivity, specicity, PPV and NPV, and their CI for the HPV tests in detecting recurrence of disease are shown in Table 3. The HPV prevalence in exfoliated cell and their high viral load had higher sensitivity (100%) in detecting recurrence, but their specicities were low (29%, 37%). However, plasma samples have higher specicity (93%) than sensitivity (57%). The PPV were 32%, 42%, and 73%, respectively, for HPV in exfoliated cell, viral load, and plasma; the corresponding NPVs were 100%, 100%, and 87%, respectively. The log-rank test has also uncovered a statistical signicant association between poor clinical outcome of the patient and HPV positivity in the plasma samples (P 0.04) (Fig. 3).
Table 2. Correlation between HPV DNA prevalence and recurrence of disease Recurrence HPV prevalence Positive Negative 30 12 19 11 3 39 Total 44 12 33 11 11 45 P value

Table 3. Diagnostic performance of HPV testing in detecting recurrence of disease HPV prevalence Sensitivity, Specicity, PPV, NPV, % (95% CI) % (95% CI) % (95% CI) % (95% CI)

Exfoliated 100 (77100) 29 (1645) 32 (1948) 100 (73100) cell 100 (77100) 37 (2056) 42 (2661) 100 (72100) Viral load in exfoliated cell Plasma 57 (2982) 93 (8098) 73 (3994) 87 (7395)

Discussion
It is evident from our study that the presence of HPV in the exfoliated cervical cells and corresponding plasma of CA-CX patients after radiotherapy has signicant role in recurrence of the disease. To our knowledge, no one has quantitated the HPV viral load in the exfoliated cells as well as in the plasma of CACX patients after radiotherapy, though HPV DNA is regarded as a diagnostic marker of cervical carcinoma due to its high prevalence (95%) in tumor samples before treatment(3). The signicant association of the HPV prevalence and high viral load in the exfoliated cells after radiotherapy with recurrence of the disease seen in our study suggests the presence of latent tumor cells with HPV infection that are resistant to therapy. The gradual increase of HPV viral load after postradiotherapy treatment up to 5 years is an indicator of the growth of latent tumor cells for clinical manifestation. Similar to our study, Sakurai et al.(7) have seen high frequency (90.5%) of recurrence in CA-CX patients within 5 years after the initial radiotherapy

Exfoliated cell Positive 14 Negative 0 Viral load in exfoliated cell !100 pg/mL 14 100 pg/mL 0 Plasma Positive 8 Negative 6
a

0.01

0.007

0.0002

Fisher exact test.

2006 IGCS, International Journal of Gynecological Cancer 16, 10481054

Figure 3. KaplanMeier estimates of disease-free patients with HPV prevalence in plasma, recurrence of the disease, and interval from last radiotherapy treatment. Strata are shown according to the presence of HPV in plasma. P values are determined by log-rank test.

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compared to more than 5 years after treatment, but they have not analyzed the HPV prevalence in their samples, whereas Nagai et al.(8) have only found HPV prevalence (57%) among postradiotherapy patients and its association with recurrence of the disease. In contrast, contradictory reports were found on HPV status after different mode of surgical treatments of CIN patients(8,9,20). It has been seen that in CIN patients, there is clearance of HPV infection within 6 months after surgical treatment, but patients with persistent HPV infection after therapy are at increased risk of disease recurrence. Many studies have shown the occurrence of tumor DNA in the plasma/serum of cancer patients(11,21), with the aim to use it as a diagnostic marker in management of the disease. In this study, we have found 20% HPV DNA in the plasma sample of patients and is strongly associated with the poor clinical outcome and disease recurrence. The plasma DNA exhibited the HPV type identical to that present in the exfoliated cells of the CA-CX patients. Thus, the possibility of HPV DNA in circulation from another latent infection was remote. Similar to our study, Pornthanakesam et al.(22) have reported that the presence of plasma HPV DNA after complete treatment was an indicator to develop recurrence or to have distant metastasis. Duenas et al.(23) have also observed the clearance of plasma HPV DNA in those achieving complete response after treatment but not in those with persistent disease. Similar phenomenon has also been seen in nasopharyngeal carcinoma patients where Epstein Barr virus DNA was detected in plasma of the patients after radiotherapy(24). Thus, the presence of HPV DNA in plasma might be due to the shredding of the exfoliated tumor cells after radiotherapy in the circulating system for distant metastasis in some patients. The transformation potentiality of circulating DNA in postradiotherapy CA-CX patients has not been determined, though the circulating DNA in the pretherapeutic patients showed high transforming activity in both in vitro and in vivo studies(25,26). Thus, in this study, we report the detection of HPV DNA in the exfoliated cell as well as plasma of postradiotherapy patients. The high HPV prevalence and high viral load detected in the exfoliated cells due to its high sensitivity suggest its usage in early detection of the patients at increased risk for disease recurrence and progression. In contrast, high NPV in the HPV prevalence in exfoliated cells and corresponding plasma as well as high viral load with recurrence of the disease also suggest the same phenomenon.
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Acknowledgments
We are thankful to the Director, Chittaranjan National Cancer Institute, Kolkata 700026, India, for encouragement and help during this work. We are also grateful to Prof. H.Z. Hausen and Dr E.M. de Villiers for their generous gift of HPV-16/18 plasmids. Financial support for this work was provided by grant (27 (0111)/00/EMRII) from the Council of Scientic Industrial Research, Government of India, to C.K.P. and CSIR-JRF/NET Fellowship grant (9/30 (026)/2002-EMR-I) to R.K.S.

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Accepted for publication September 19, 2005

2006 IGCS, International Journal of Gynecological Cancer 16, 10481054