Cryotherapy for Human Papillomavirus Clearance in Biopsy-confirmed Cervical Low-grade Squamous Intraepithelial Lesions
Cryotherapy for Human Papillomavirus Clearance in Biopsy-confirmed Cervical Low-grade Squamous Intraepithelial Lesions
Persistent human papillomavirus (HPV) infections is the single necessary cause of cervical
 cancer. Cervical cancer is still the major health problem in the developing countries. It has
 been the first rank women's cancer in Thailand for many decades. Approximately 10-20% of Thai
 women have the high-risk HPV (HR-HPV) infections in their cervices. This will frequently lead
 to low-grade squamous intraepithelial lesions (LSILs) (10%), high-grade squamous
 intraepithelial lesions (HSILs) (0.8%), and finally, cervical cancers (0.16%) within 10-20
 years. The treatment options for LSILs are either observation or ablative surgery. However,
 in our institute, cryotherapy, which is the one of ablative surgery, is more frequently used
 to comfort our women. It is not only effective but safe with only minimal side effects;
 watery leukorrhea for 2-4 weeks, and local cervical infection not more than 1%.
 Contraindication to this procedure are active cervical infection, lesion of 2 mm-larger than
 probe, lesion inside cervical os and suspected cervical cancer. Additionally, in developing
 countries such as Thailand, this treatment is safe, acceptable, feasible and effective.
 Patients with LSILs could also have this treatment in some rural area from the 10-days
 trained nurses. Fortunately, recent reports showed that cryotherapy has abilities not only in
 clearing LSILs but also clearing the HPV infections, its necessary cause. However, there are
 no randomized controlled trial compared its clearing ability between observation and
 cryotherapy. Therefore, a randomized controlled trial is needed to demonstrate that. Findings
 from this trial will contribute enormously to older women who already get infected by HPV.
 Aside from preventing cervical cancer in treated woman, cryotherapy could also give her
 relief from worrying about having a time-bomb like HPV infection in her cervix.
This will be a hospital-based, parallel, assessor-blinded, randomized controlled trial. Study
 subjects refer to LSIL patients, more than 30 years-old, with positive HPV testing who do not
 have any contraindication of performing cryotherapy. Therefore, after recruitment LSIL
 patients whose age more than 30 years-old, we will exclude only those who have negative HPV
 testing or any contraindications of performing cryotherapy. The primary outcome is HPV
 clearance (negative HPV testing for the baseline HPV type) at 12 months after the treatment.
 Subjects will be randomly allocated to be treated by cryotherapy or observation only by using
 block randomization with varied block size of 2, 4, and 6. The sample size of at least 25 for
 each arm was planned to have the power of 80% to detect 20% difference rates of HPV clearance
 after 12 months of treatment between the two arms, with significance level of 0.05, two-sided
 test.

 Study population:

 Biopsy-confirmed LSIL patients, age more than 30 years-old, referred to either Srinagarind
 Hospital's, Khon Kaen Hospital's, or Roi Et Hospital's colposcopic clinics.

 Inclusion criteria:

 -  Baseline HPV testing is positive.

 -  Be able to give their informed consent to this study.

 Exclusion criteria:

 -  Having active cervical infection

 -  Having lesion of 2 mm-larger than probe

 -  Having lesion inside cervical os

 -  Having suspected cervical cancer lesion

 Sample and sample size:

 From previous studies, HPV clearance rate after cryotherapy at 12 months is 83.9%. (Elfgren,
 2002) Spontaneous HPV clearance rate in young women at 24 months is 70%. (Moscicki, 1998)
 However, this HPV clearance rate in women older than 34 years-old at 12 months is only 53.6%.
 (Clavel, 2005) Using these numbers respectively as PE and PC putting in the formula below, we
 can calculate our sample size in each group. The sample size will be calculated to detect the
 20-40% difference between the HPV clearance rates after 12 months, at significant level of
 95% and power of 80% (two-sided test), using the approach of Lachin that was reviewed in the
 paper of Donner.(Donner, 1984)

 Required sample size at different rate of clearance

 P-control P-experimental Alpha level Power N Difference 95%CI 0.50 0.70 5% 80% 103 20
 6.9-33.1 0.50 0.75 5% 80% 66 25 9-41 0.50 0.80 5% 80% 45 30 11.3-48.7 0.50 0.85 5% 80% 33 35
 14-56 0.50 0.90 5% 80% 25 40 17.1-62.9

 The sample size of at least 25 patients in each group will be chosen because of its precision
 and feasibility. Such sample size can detect 40% difference of HPV clearance rate between the
 two groups, at 95% significant level, with the power of 80%, 95%CI of 17.1-62.9,
 respectively.

 Instruments:

 After signing on the consent form, all eligible patients will be examined by the baseline
 standard procedures and PCR-HPV testing for 37 HPV types. Only positive HPV testing ones will
 be enrolled in the trial and then randomized to receive the cryotherapy or observation. The
 method of block randomization, with block size varies from 2 to 6, will be used to allocate
 treatments.

 The allocation ratio is 1:1. The allocation sequence will be generated by computer, using
 STATA software version 10.0.

 Based on the obtained schedule, the number of sealed envelopes, which are equal to the
 required blocks, will be prepared and labeled. Each envelope contains the corresponding
 sealed allocation cards that will allocate treatments to patients.

 Colposcopy and cryotherapy instruments are available in our colposcopic clinic. However, we
 will reimburse for all CO2-gases tanks and materials used for Pap smears and HPV testing.

 The people, who prepare the randomized scheme, will not involve in the trial. The allocation
 schedule will be concealed to the assessor (who measure the HPV testing) until the end of
 trial.

 Data collection and measurement:

 Because of the difference between two treatments, only the assessor (who measure the HPV
 testing) will be blinded from the treatments.

 The specimens will be sent to the assessor without patient's name or HN that can be linked to
 the treatment received. They will be provided only the study ID number in which only the
 principle investigator can link it to the treatment received.

 The main outcome is HPV clearance. It is measured by the gold-standard HPV testing. This will
 be performed at baseline and 12 months later.

 Data analysis:

 There were two main parts of analysis - describing selected characteristics of the study
 patients and analysis for answering the research questions. The first part, selected baseline
 characteristics of the patients in each treatment were compared i.e. age, sex, sexual
 experiences, underlying diseases, etc. Comparability of the distribution of those selected
 characteristics based on judgment rather than statistical significant tests. In this part,
 proportion was use for summarizing categorical data. For continuous data, the statistics
 include mean, standard deviation, minimum, maximum, and median.

 For the second part, the analysis was based on the group in which the study patients were
 randomized (intention-to-treat basis). The different rate of clearance between the two arms
 of the trial and its 95% confidence interval will be calculated. This will be done at 12
 months after the treatment. Z-test will be used for testing if such differences are different
 from zero. Multiple logistic regression will be used to control effects of any important
 confounding factors. Absolute risk reduction (ARR), relative risk reduction (RRR) and number
 needed to treat (NNT) will also be calculated.

 The statistical analysis of this clinical trial will be performed based on intention-to-treat
 principle. All hypothesis tests are two-tailed at significant level of 0.05. Statistical
 package for all analysis is STATA version 10.0.
Inclusion Criteria:

 -  Biopsy-confirmed LSIL patients

 -  Age more than 30 years-old

 -  Baseline HPV testing is positive.

 -  Be able to give their informed consent to this study.

 Exclusion Criteria:

 -  Having active cervical infection

 -  Having lesion of 2 mm-larger than probe

 -  Having lesion inside cervical os

 -  Having suspected cervical cancer lesion