1 ASCO 2004 Tumor Biology & Human Genetics Pap test and a new biomarker- based technology for enhancing the visibility of abnormal cells N.S. Markovic, O. Markovic, J. Sundeen, W. Smith, S.N. Markovic, 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 the Cervical Acid Phosphatase-Papanicolaou (CAP-PAP) test as a new, biomarker–driven technology for detection of abnormal cervical cells on Pap smears. Since this report, we conducted three clinical trials to asses the value of this test for cervical cancer screening in general (N=1,500) and in a high- risk population (N=500). This technology is commercially available as the MarkPap® Research Kit (BioSciCon, Rockville, MD ).

4 Biomarker characteristics 1 Biomarker is red, granular pigment precipitated inside cells at sites of acid phosphatase activity. Cervical acid phosphatase is absent in normal squamous cells.

5 Biomarker characteristics 2 Biomarker is present in: Abnormal squamous cells Internal control: monocytes, endocervical cells Malignant cells

6 MarkPap and ThinPrep Pap

7 Methods Multicenter, random assignment, assessors blinded, split sample, concurrent control 3-group (test and two controls [Pap smear and Thin Prep thin layers]) clinical trial was conducted on 1,500 specimens obtained from eligible subjects coming for regular Pap test. Specimens collected in PreservCyt® and transferred onto microscopic slides using the ThinPrep Processor 2000 were split in two samples before processing. Specimens obtained with cervical epithelium abrading devices, were split by the physician in two samples (smears). Primary efficiency endpoints were the portion of detected abnormal samples and the portion of false-negative readings, Accuracy was measured against cytology standard (association or adjudication).

8 Clinical Site Procedures Recruitment Eligibility IRB approved Informed Consent Clinical Case Report Form Sampling cervical specimen H&S specimen collected in solution Follow-up: recording clinical and adverse events. Reporting Diagnostic procedures (colposcopy, biopsy, histology) when required

9 Laboratory Site Procedures Specimen receiving and sampling (split- sample) Laboratory Case Report Form Coding – assessors blinding Specimen transfer to microscopic slides Specimen processing (test or control) Screening for cervical cancer Interpretation, recording and reporting

10 Safety and Data Management Team Monthly visits to research sites Collection of data from CCRF and LCRF Data input into databases Data management Statistical analyses Reporting

11 Efficacy evaluation Efficacy endpoints: Proportion of detected true abnormal specimens Proportion of detected false negative slides Test accuracy (sensitivity, specificity) measured against adjudicated cytology as standard control.

12 Success Ingredients Clinical Laboratory Trials Results PS RS RW DN + FP DP (TP) FN FP BCC 2x 1/3

13 Results 1 MPT/PAP Association: 0.83 Discrepancy: 0.17 Abnormal: 0.157/0.049 False negative: 0.04/0.09 Sensitivity: 0.83/0.51 Specificity: 0.94/0.99 MPT/ThP* Association: 0.86 Discrepancy: 0.14 Abnormal: 0.097/0.103 False negative: 0.03/0.09 Sensitivity: 0.45/0.60 Specificity: 0.95/0.98

14 Results 2 MPT/PAP Hypothesis testing Pe > Ps + 0.5Ps Superiority for more than 50% of control MPT/ThinPrep Hypothesis testing Pe – Ps < 0.3Ps Equivalence within 30% of control

15 Conclusion These data suggest that the new technology is more sensitive (with equivalent specificity) than either conventional Pap smear or ThinPrep Pap test. However, when specimens were collected in PreservCyt®, due to alcohol inhibition of the enzyme, this sensitivity was reduced. Using a new, enzyme protective, solution for MarkPap specimens was recommended.

16 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 University of Maryland IRB

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