HPV E6/E7 mRNA Versus HPV DNA as Triage for Cervical Cancer Screening
HPV E6/E7 mRNA Versus HPV DNA as Triage for Cervical Cancer Screening
AIM: To develop and standardize a cost effective methodology or algorithm for mRNA E6/E7 for
 HPV genotypes 16, 18, 31, 33 and 45 as compared to commercially available assays which can be
 incorporated to triage excess false positives from primary screening for cervical cancers

 Objectives:

 1. Development and standardization of methodology /algorithm for mRNA E6/E7 testing for HPV
 genotypes 16, 18, 31, 33 and 45 using Real-time RT-PCR, in cervical samples.

 2. To compare the test performance of this HPV E6/E7 mRNA assay to HPV DNA by HC 2 as
 secondary screening test, with the reference standard of colposcopy with biopsy, to
 triage women found positive in primary screening by VIA , in a population based
 screening for cancer of cervix.

 3. To determine number of false positives in the primary screening test after testing VIA
 positives with a known high specificity secondary screening test (HPV-DNA HC II )
 compared to HPV E6/E7 mRNA testing.

 Study Population: Women in the age group of 30-65 years, who test positive on primary
 cervical screening test VIA will be enrolled for the proposed diagnostic tests along with
 reference standard of colposcopy with guided biopsy.

 Methodology:

 Women in the age group of 30-65 years undergoing routine cervical cancer screening through
 hospital ( Preventive Oncology screening clinic) and community based screening programs with
 abnormal test result using the primary cervical cancer screening test VIA will be recruited
 in the study. The primary screening test VIA will be administered by application of 5% Acetic
 Acid to the cervix and visualizing the cervix with the help of a halogen focus lamp. VIA will
 be considered to be positive if definite acetowhite lesions are visualized close to the
 squamocolumnar junction.
AIM: To develop and standardize a cost effective methodology or algorithm for mRNA E6/E7 for
 HPV genotypes 16, 18, 31, 33 and 45 as compared to commercially available assays which can be
 incorporated to triage excess false positives from primary screening for cervical cancers.

 Objectives:

 1. Development and standardization of methodology /algorithm for mRNA E6/E7 testing for HPV
 genotypes 16, 18, 31, 33 and 45 using Real-time RT-PCR, in cervical samples.

 2. To compare the test performance of this HPV E6/E7 mRNA assay to HPV DNA by HC 2 as
 secondary screening test, with the reference standard of colposcopy with biopsy, to
 triage women found positive in primary screening by VIA , in a population based
 screening for cancer of cervix.

 3. To determine number of false positives in the primary screening test after testing VIA
 positives with a known high specificity secondary screening test (HPV-DNA HC II )
 compared to HPV E6/E7 mRNA testing.

 Study Population: Women in the age group of 30-65 years, who test positive on primary
 cervical screening test VIA will be enrolled for the proposed diagnostic tests along with
 reference standard of colposcopy with guided biopsy.

 Study Site: Women tested positive on primary screening test VIA with referrals for further
 diagnostic testing at TMH will be enrolled under the study for proposed diagnostic tests
 along with reference test at tertiary care facility , Dept. of Preventive Oncology, Tata
 Memorial Hospital in Mumbai.

 Sample Size To detect statistically significant difference in the test characteristics with
 95% confidence limits, 80 % statistical power and alpha =0.05, we require to have around 180
 cases of cervical intraepithelial neolplasia in the study.

 With a demonstrated prevalence of around 2% for detecting high grade CIN lesions from our
 urban and rural community based cervical screening programmes, we need to enroll around 9,000
 women from population based cervical cancer screening, to get 180 cases of cervical
 intraepithelial neolplasia

 Methodology:

 Women in the age group of 30-65 years undergoing routine cervical cancer screening through
 hospital ( Preventive Oncology screening clinic) and community based screening programs with
 abnormal test result using the primary cervical cancer screening test VIA will be recruited
 in the study. The primary screening test VIA will be administered by application of 5% Acetic
 Acid to the cervix and visualizing the cervix with the help of a halogen focus lamp. VIA will
 be considered to be positive if definite acetowhite lesions are visualized close to the
 squamocolumnar junction.

 Criteria for a positive VIA test are presented below:

 Positive VIA test:

 1. Opaque, dense, dull, definite, well defined aceto white lesions, touching the
 squamocolumnar junction or close to the external os.

 2. Strikingly dense acetowhite areas in the columnar epithelium.

 3. Condyloma and Leukoplakia occurring closer to the squamocolumnar junction turning
 intensely white after application of acetic acid.

 4. Growth on the cervix turning acetowhite.

 The screened women who are tested Screen Positive by the primary screening test VIA will
 become eligible for recruitment in the study . Screen positive women from the outreach
 community screening programs will be referred to TMH ( Preventive Oncology screening clinic)
 for further diagnostic verification.

 Participant Recruitment

 The study participants will be recruited from Hospital ( Preventive Oncology screening
 clinic) and community based screening programmes in Mumbai. Medical Social Workers will
 explain the details and the purpose of the study to participants attending the screening
 clinic. Apparently healthy women in the age group of 30-65 yrs with an intact uterus and no
 past history of cervical neoplasia will become eligible to participate in the programme. If
 they are fulfilling the inclusion criteria given above, a written informed consent in the
 vernacular language (either Hindi or Marathi) will be obtained from the participant and a
 unique participant identification number will be assigned to the eligible women. Information
 on socio demographic and reproductive variables will then be collected using a detailed
 questionnaire.

 Demographic, reproductive and sexual history data will be recorded at screening following
 informed consent..

 Screen positive women will be tested for the presence of oncogenic HPV types by Hybrid
 Capture-II (HC-II) and HPV E6/E7 mRNA testing as diagnostic test. In addition to the above
 two tests all the screen positive women will also further receive Colposcopy with guided
 cervical biopsy, the reference gold standard test.

 Colposcopy will be performed by trained doctors and the colposcopy impression will be noted
 down along with a punch biopsy from the aceto white area on the cervix detected by the
 primary screening test VIA. Biopsy specimens will be fixed in 10% formalin and will be
 processed and reported by the pathology laboratory at the Tata Memorial Centre.

 The reference standard for final disease status will be histology.

 Post Test Counseling :

 At the end of all testing procedures post test counseling will be done by the Doctor and
 Medical Social Worker to explain the significance and the results of the testing procedures
 performed. The importance of follow up visit to understand the results of HPV/ mRNA E6/E7 and
 cervical biopsy will be explained to the women.

 Follow up and Treatment:

 Treatment for opportunistic and reproductive tract infections (RTI) will be provided. Women
 with High grade lesions (cervical precancers ) HSIL will be recommended to seek treatment
 with cryotherapy or LEEP (Loop Electrosurgical Excision Procedure) or by cold knife
 conization at the Tata Memorial Centre Mumbai.

 Total Project Period: Three years:

 Proposal submission, training, participant recruitment, data collection and analysis,
 including follow-up of cases at the end of enrollment period
Inclusion Criteria:

 1. Female between 30-65years of age

 2. Not pregnant by menstrual history

 3. No history of prior treatment for cancer of the cervix

 4. No history of hysterectomy

 5. Healthy enough to undergo a pelvic examination, i.e., not seriously ill with a
 debilitating condition

 Exclusion Criteria:

 1. Women below and above this age group

 2. Women already screened and detected for cervical precencers and cancers

 3. Women with debilitating condition

 4. Women with Hysterectomy

 5. Women whom are pregnant