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ASCO 2005Gynecologic Oncology

MarkPap® Test for Cervical Cancer Screening

N.S. Markovic, O. Markovic, E.S. Henderson,

BioSciCon’s Cervical Cancer Study Group*

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*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.

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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).

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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.

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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.

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Fig. 1: ASC-H (x20)

CAP labels 3 small abnormal cells

x20

NormalCAP

Negative cells

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Fig. 2: ASC-H (x100)

x100

CAP is the red marker

NormalCAP

negativecell

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Fig. 3: LSIL (x100)

HPV cells

Red biomarker

Bi-nucleus

Micro-vacuolalization

Dyskaruosis

Koilocyte-like cells

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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.

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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: πe < πs + δ (0.3 πs )

• HA :πe ≥ πs + δ (0.3 πs )

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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.

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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)

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Study ManagementCentral 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

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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

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Fig. 4 : Quality Control & Quality Assurance

COMBO control slide containing HeLa and buccal cells

HeLaCAP+

BuccalCAP[-]

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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.

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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

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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.

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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

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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

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McNemar Test*

• (CPT+)(PAP+) = 15 (a)• (CPT+)(PAP-) = 139 (b)• (CPT-)(PAP+) = 51 (c)• (CPT-)(PAP-) = 855 (d)• ([b-c]-1)2 /(b+c) = 59.45 (1df)• 2-tailed P value = 0.0001• Odds ratio = 3.861• 95%CI: LL = 2.660, UL= 5.737 *QuickCalcs (www.graphpad.com)

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Predictive Values* *QuickCalcs (www.graphpad.com)

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

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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

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Acknowledgment

• This work was supported in part by NIH-NCI SBIR Grant No. 1 R43 CA086767-01, and No. 2 R44 CA086767-02

• NCI, DCP, Biomarker Research Group

• MdBio and Dr. Charlie Grudzinskas

• University of Maryland IRB

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Correspondence

• Nenad Markovic, M.D., Ph.D.• BioSciCon, Inc.• 14905 Forest Landing Circle• Rockville, MD 20850• Tel 301-610-9130• Fax 301-610-7662• E-mail: nsmarkovic@comcast.net