1 ASCO 2005 Gynecologic Oncology MarkPap® Test for Cervical Cancer Screening N.S. Markovic, O. Markovic, E.S. Henderson, BioSciCon’s Cervical Cancer Study Group*

2 *BioSciCon’s Cervical Cancer Study Group Markovic Nenad, BioSciCon, Rockville, MD Markovic Olivera, BioSciCon Shelley Parr, UMD Health Center, College Park, MD Alan Ross, Women’s Health Care Center, Bethesda, MD Rufus Rosser, Ob/Gy Off., Laurel, MD, Washington, DC Lewis Townsend, Contemporary Women Health Care Associates, Bethesda, MD Jed Gould, Ob/Gy Offices, Laurel, Bethesda, Gaithersburg, MD James Sundeen, Diagnostic Pathology Services, Clarksburg, MD William Smith, Jr. & Aruna Kumar, Pathology, Suburban Hospital, Bethesda, MD. Shiniwas Katti, consultant-statistician.

3 Background At ASCO 2001, we introduced a new biomarker- driven technology for improving the visualization of epithelial cells with abnormalities on Pap smears. The biomarker was cervical acid phosphatase labeling dysplastic cells. At ASCO 2004, we presented data of two clinical trials. This time, we are presenting results of comparing the new test with the conventional Pap smear in general population (GP).

4 MarkPap® Test MarkPap® Test (BioSciCon, Rockville, MD) is a double-staining, single slide cytological method for demonstration of cervical acid phosphatase inside Papanicolaou stained abnormal cervical cells on smears or monolayers prepared from specimens collected in solution. The test is available from the manufacturer, the Ricca Chemical Company, Arlington, TX.

5 Cervical acid phosphatase (CAP) Cervical acid phosphatase is visualized as red, granular deposit inside cells counterstained with a modified Papanicolaou technique (Fig. 1-4) The red biomarker is clearly visible on the bluish background of cervical cells. This visual characteristics reduce the probability for omission of labeled cells.

6 Fig. 1: ASC-H (x20) CAP labels 3 small abnormal cells x20 Normal CAP Negative cells

7 Fig. 2: ASC-H (x100) x100 CAP is the red marker Normal CAP negative cell

8 Fig. 3: LSIL (x100) HPV cells Red biomarker Bi-nucleus Micro- vacuolalization Dyskaruosis Koilocyte- like cells

9 Biomarker: Advantage for cervical cancer screening Cervical acid phosphatase (CAP) in cervical squamous cells is a biomarker for cervical dysplasia and HPV infection. CAP is positive in more than 99.98% cervical abnormal cells. CAP is negative in more than 99.99% normal cervical squamous cells.

10 MarkPap Utilization: Hypotheses tested If this biomarker enhances the detection of abnormal cervical cells, how this can be utilized for cervical cancer screening? If MarkPap (CAP+PAP) is superior to PAP, is the difference meaningful to replace PAP? Is the difference [MarkPap-PAP] higher than 30% in General Population (GP) of women coming for regular Pap test Ho: π e < π s + δ (0.3 π s ) H A :π e ≥ π s + δ (0.3 π s )

11 Study Design Multicenter, split-sample (case-control) study, intended: To assess the efficacy of cytoscreeners using MarkPap to improve the detection of abnormal cervical specimens, and To reduce the false negative readings, in comparison with those using the control Pap smear. Measuring endpoints: proportion, sensitivity, specificity, predictive values, false negative rates.

12 Clinical Trial: GP - 1 Population N = 1,067 women age 18 and above who came to doctor’s offices for regular Pap test. Prerequisite: Confirmed eligibility Signed Informed Consent Specimens Collected 2,150 at 8 clinical sites Analyzed for control Pap smear (PAP) and new test (MarkPap) in a single contract research laboratory (DPS, Clarksburg, MD)

13 Study Management Central Research Site Design Organization Financing Monitoring for compliance Monitoring for adverse events Data acquisition Data collection Data base(s) Data management Data evaluation Adjudication Statistical analyses Study evaluation

14 Specimen processing - Laboratory Control smear Receive, register, code, label, assign to Pap test procedure. Stain with Papanicolaou stains using an automatic slide stainer MarkPap smear Receive, register, code, label, assign to MarkPap test procedure. Process for marker visualization. Stain background with Papanicolaou stains

15 Fig. 4 : Quality Control & Quality Assurance COMBO control slide containing HeLa and buccal cells HeLa CAP+ Buccal CAP[-]

16 PAP Slide screening Control slide Primary screening: Screen for epithelial cell abnormalities and report using the Bethesda System terminology. All positive/abnormal slides rescreen for QC Ten percent negative/normal slides and slides from high-risk patients send for second opinion (QC). Send reports back to clinical sites and store into the study dbase.

17 MarkPap slide screening MarkPap slide Primary screening: Screen for biomarker and for epithelial cell abnormalities. All positive/abnormal slides send for QC to pathologist All negative/normal slides send for QC to cytotechnologist Report results in the Laboratory dbase

18 Post-study handling Decoding at the end of study Using dbase information, compare results of screening test and control slides. If results are associated, record this result as “true” value. If results are discrepant, send Pap slides for adjudication. Use adjudicated results as “true” values.

19 Evaluation Validate results of the test and the control screening using “true” values. Calculate sensitivity and specificity of test and control results using “true” values as standard. Use 95%CI for improving the probability of assessment. Test hypothesis for equivalency Pe-Ps < δ (5%-50%) Test for significance of the difference using the McNemar test for comparison of proportions in paired groups

20 Results Pap smear N= 947 TP = 21 TN = 898 Se: 0.46 Sp: 0.99 MarkPap test N= 947 TP = 38 TN = 858 Se: 0.84 Sp: 0.95

21 McNemar Test* (CPT+)(PAP+) = 15(a) (CPT+)(PAP-) = 139(b) (CPT-)(PAP+) = 51(c) (CPT-)(PAP-) = 855(d) ([b-c]-1) 2 /(b+c) = (1df) 2-tailed P value= Odds ratio = %CI: LL = 2.660, UL= *QuickCalcs (

22 Predictive Values* * QuickCalcs ( PAP control Se: 46 Sp: 99 Prevalence: 7 PPV: 77 NPV: 96 FN: 3.94 MarkPap test Se: 84 Sp: 95 Prevalence: 7 PPV: 56 NPV: 99 FN: 1.25

23 Conclusion MarkPap test is superior to the PAP smear for: Detection of abnormal specimens Sensitivity of screening for cervical cancer Allows for less false negative readings

24 Acknowledgment This work was supported in part by NIH- NCI SBIR Grant No. 1 R43 CA , and No. 2 R44 CA NCI, DCP, Biomarker Research Group MdBio and Dr. Charlie Grudzinskas University of Maryland IRB

25 Correspondence Nenad Markovic, M.D., Ph.D. BioSciCon, Inc Forest Landing Circle Rockville, MD Tel Fax