COBAS HPV TEST

P100020 · Roche Molecular Systems, Inc. · MAQ · Apr 19, 2011 · Microbiology

Device Facts

• Record ID: P100020
• Device Name: COBAS HPV TEST
• Applicant: Roche Molecular Systems, Inc.
• Product Code: MAQ · Microbiology
• Decision Date: Apr 19, 2011
• Decision: APPR
• Device Class: Class 3

Intended Use

The cobas HPV Test is a qualitative in vitro test for the detection of Human Papillomavirus (HPV) in patient specimens. The test utilizes amplification of target DNA by the Polymerase Chain Reaction (PCR) and nucleic acid hybridization for the detection of 14 high-risk (HR) HPV types in a single analysis. The test specifically identifies types HPV 16 and HPV 18 while concurrently detecting the rest of the high risk types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68). The cobas HPV Test is indicated: (a) To screen patients 21 years and older with ASC-US (atypical squamous cells of undetermined significance) cervical cytology test results to determine the need for referral to colposcopy. (b) To be used in patients 21 years and older with ASC-US cervical cytology results, to assess the presence or absence of high-risk HPV genotypes 16 and 18. This information, together with the physician’s assessment of cytology history, other risk factors, and professional guidelines, may be used to guide patient management. The results of this test are not intended to prevent women from proceeding to colposcopy. (c) In women 30 years and older, the cobas HPV Test can be used with cervical cytology to adjunctively screen to assess the presence or absence of high risk HPV types. This information, together with the physician’s assessment of cytology history, other risk factors, and professional guidelines, may be used to guide patient management. (d) In women 30 years and older, the cobas HPV Test can be used to assess the presence or absence of HPV genotypes 16 and 18. This information, together with the physician’s assessment of cytology history, other risk factors, and professional guidelines, may be used to guide patient management.

Device Story

The cobas HPV Test is an in vitro diagnostic assay for detecting 14 high-risk HPV genotypes in cervical specimens collected in PreservCyt solution. The device uses automated total nucleic acid isolation, PCR amplification, and real-time detection of fluorescent-labeled probes. It is performed on the cobas 4800 system by laboratory technicians. The system outputs qualitative results (Positive/Negative/Invalid) for HR HPV types collectively and specifically identifies HPV 16 and 18. Healthcare providers use these results alongside cytology and clinical history to manage patient risk for cervical disease (>=CIN2). The test aids in triaging ASC-US patients and adjunctive screening for women >=30, potentially reducing unnecessary colposcopies or identifying high-risk patients for timely intervention.

Clinical Evidence

Clinical performance evaluated in the prospective ATHENA study (n=47,208). In ASC-US patients (>=21 years), sensitivity for >=CIN2 was 90.0% and specificity 70.5%. In NILM patients (>=30 years), unadjusted sensitivity for >=CIN2 was 83.2% and specificity 60.4%. Verification bias-adjusted sensitivity for >=CIN2 was 34.5% and specificity 93.6%.

Technological Characteristics

Qualitative in vitro nucleic acid amplification test (PCR). Detects 14 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) and beta-globin internal control. Uses four-channel fluorescent detection. Run on cobas 4800 system (automated isolation, PCR setup, thermal cycling).

Indications for Use

Indicated for women >=21 years with ASC-US cervical cytology to determine colposcopy referral; women >=30 years for adjunctive screening with cytology; and women >=30 years to assess presence of HPV genotypes 16 and 18. Contraindications: None.

Submission Summary (Full Text)

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SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED)

I. GENERAL INFORMATION

Device Generic Name: Human Papillomavirus DNA detection kit

Device Trade Name: cobas HPV Test

Applicant’s Name and Address:

Roche Molecular Systems, Inc. (RMS)
4300 Hacienda Drive
PO Box 9002
Pleasanton, CA 94588-0900

Date(s) of Panel Recommendation: None

Premarket Approval Application (PMA) Number: P100020

Date of FDA Notice of Approval: April 19, 2011

Expedited: Not applicable

II. INDICATIONS FOR USE

The cobas HPV Test is indicated for use:

The cobas HPV Test is a qualitative *in vitro* test for the detection of Human Papillomavirus (HPV) in patient specimens. The test utilizes amplification of target DNA by the Polymerase Chain Reaction (PCR) and nucleic acid hybridization for the detection of 14 high-risk (HR) HPV types in a single analysis. The test specifically identifies types HPV 16 and HPV 18 while concurrently detecting the rest of the high risk types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68).

The cobas HPV Test is indicated:

(a) To screen patients 21 years and older with ASC-US (atypical squamous cells of undetermined significance) cervical cytology test results to determine the need for referral to colposcopy.

(b) To be used in patients 21 years and older with ASC-US cervical cytology results, to assess the presence or absence of high-risk HPV genotypes 16 and 18. This information, together with the physician’s assessment of cytology history, other risk factors, and professional guidelines, may be used to guide patient management. The results of this test are not intended to prevent women from proceeding to colposcopy.

(c) In women 30 years and older, the cobas HPV Test can be used with cervical cytology to adjunctively screen to assess the presence or absence of high risk HPV types. This

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information, together with the physician’s assessment of cytology history, other risk factors, and professional guidelines, may be used to guide patient management.

(d) In women 30 years and older, the cobas HPV Test can be used to assess the presence or absence of HPV genotypes 16 and 18. This information, together with the physician’s assessment of cytology history, other risk factors, and professional guidelines, may be used to guide patient management.

Cervical specimens that may be tested with the cobas HPV Test include the following liquid based collection media and collection device:

- ThinPrep Pap Test PreservCyt Solution
- Endocervical Brush/Spatula

### III. CONTRAINDICATIONS

None.

### IV. WARNINGS AND PRECAUTIONS

The warnings and precautions can be found in the cobas HPV Test labeling.

### V. DEVICE DESCRIPTION

The cobas HPV Test is a qualitative in vitro test for the detection of Human Papillomavirus (HPV) in patient specimens. The test utilizes amplification of target DNA by the Polymerase Chain Reaction (PCR) and nucleic acid hybridization for the detection of 14 high-risk (HR) HPV types in a single analysis. The test specifically identifies (types) HPV16 and HPV18 while concurrently detecting the rest of the high risk types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). Specimens are limited to cervical cells collected with an endocervical brush/spatula in PreservCyt solution (Hologic Corp.). The cobas HPV Test is based on two major processes: (1) automated specimen preparation to simultaneously extract HPV and cellular DNA; (2) PCR amplification of target DNA sequences using both HPV and beta-globin specific complementary primer pairs and real-time detection of cleaved fluorescent-labeled HPV and beta-globin specific oligonucleotide detection probes. The concurrent extraction, amplification and detection of beta-globin in the cobas HPV Test monitors the entire test process.

The Master Mix reagent for the cobas HPV Test contains primer pairs and probes specific for the 14 high-risk HPV types and beta-globin DNA. The detection of amplified DNA (amplicon) is performed during thermal cycling using oligonucleotide probes labeled with four different fluorescent dyes which are read on 4 different channels. The amplified signal from twelve high-risk HPV types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68), is detected using the same fluorescent dye, while HPV16, HPV18 and beta-globin signals are each detected with their own dedicated fluorescent dye.

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The Test is run on the cobas 4800 system. The cobas 4800 system is a multi-instrument platform that will perform qualitative in vitro nucleic acid amplification tests from human specimens. The cobas 4800 system integrates automated total nucleic acid isolation directly from secondary sample tubes, PCR setup, and real-time PCR.

The cobas 4800 system software includes a validated two stage data analysis algorithm to determine the cycle threshold value (Ct) — the cycle number where the signal of the accumulating PCR product starts to grow exponentially in each channel as well as to check for the integrity of the signal. “Positive,” “Negative,” or “Invalid” result are determined for each sample in each channel based on predefined parameters and Ct cut offs for each channel. The ultimate result reported for each specimen or control is determined as a combination of results from all four detection channels according to predefined data analysis algorithm.

The result reporting architecture includes two options: (1) HR HPV only, and (2) HR HPV plus genotyping of 16 and 18. For the HR HPV only option, any positive signal from Channel 1 and/or channel 2 and/or channel 3 is reported as “HR HPV positive” and channel 4 (beta-globin signal) must be valid. For HR HPV plus genotyping option, positivity is determined for each individual channel, thereby allowing the specific identification of HPV16 or HPV18 with the other HR HPV types detected collectively.

Additional details can be found in the operator’s manual for the device.

## Interpretation of Results

Note: All assay and run validation is performed by the cobas 4800 software.

Note: A valid run may include both valid and invalid specimen results.

For a valid run, specimen results are interpreted as shown below:

*Result Interpretation of the cobas HPV Test for Presence of HPV DNA*

| cobas HPV Test | Result Report and Interpretation |
| --- | --- |
| SubTest “HPV High Risk Panel”: | |
| HR HPV POS | High Risk HPV Positive
Specimen is positive for the DNA of any one of, or combination of, the following high risk HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68. |
| HR HPV NEG | High Risk HPV Negative*
HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 DNA were undetectable or below the pre-set threshold. |
| Invalid | High Risk HPV Invalid
Results are invalid. Original specimen should be re-tested to obtain valid result. |
| Failed | No Result for Specimen
Consult the cobas 4800 system Operator’s Manual for instructions to review run flags and recommended actions. Original specimen should be re-tested to obtain valid result. |
| SubTest “HPV High Risk Panel Plus Genotyping” | |
| Other HR HPV POS, HPV16 POS, HPV18 POS | Other High Risk HPV Positive, HPV16 Positive, HPV18 Positive.
Specimen is positive for HPV types 16 and 18 DNA and the DNA of any one of, or combination of, the following high risk HPV types: 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68. |
| Other HR HPV POS, HPV16 POS, HPV18 NEG | Other High Risk HPV Positive, HPV16 Positive, HPV18 Negative*.
Specimen is positive for HPV type 16 DNA and the DNA of any one of, or combination of, the following high risk HPV types: 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68. |

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| cobas HPV Test | Result Report and Interpretation |
| --- | --- |
| | HPV type 18 DNA was undetectable or below the pre-set threshold. |
| Other HR HPV POS, HPV16 NEG, HPV18 POS | Other High Risk HPV Positive, HPV16 Negative*, HPV18 Positive.
Specimen is positive for HPV type 18 DNA and the DNA of any one of, or combination of, the following high risk HPV types: 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68.
HPV type 16 DNA was undetectable or below the pre-set threshold. |
| Other HR HPV POS, HPV16 NEG, HPV18 NEG | Other High Risk HPV Positive, HPV16 Negative*, HPV18 Negative*.
Specimen is positive for the DNA of any one of, or combination of, the following high risk HPV types: 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68.
HPV types 16 and 18 DNA were undetectable or below the pre-set threshold. |
| Other HR HPV NEG, HPV16 POS, HPV18 POS | Other High Risk HPV Negative*, HPV16 Positive, HPV18 Positive.
HPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 DNA were undetectable or below the pre-set threshold.
Specimen is positive for HPV types 16 and 18 DNA. |
| Other HR HPV NEG, HPV16 NEG, HPV18 POS | Other High Risk HPV Negative*, HPV16 Negative*, HPV18 Positive.
HPV types 16, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 DNA were undetectable or below the pre-set threshold.
Specimen is positive for HPV type 18 DNA. |
| Other HR HPV NEG, HPV16 POS, HPV18 NEG | Other High Risk HPV Negative*, HPV16 Positive, HPV18 Negative*.
HPV types 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 DNA were undetectable or below the pre-set threshold.
Specimen is positive for HPV type 16 DNA. |
| Other HR HPV NEG, HPV16 NEG, HPV18 NEG | Other High Risk HPV Negative*, HPV16 Negative*, HPV18 Negative*.
HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 DNA were undetectable or below the pre-set threshold. |
| Invalid | Invalid.
The results are Invalid. Original specimen should be re-tested no more than two times to obtain valid results. If the results are still invalid a new specimen should be obtained. |
| Failed | No Result for Specimen
Consult the cobas 4800 system Operator's Manual for instructions to review run flags and recommended actions.
Original specimen should be re-tested to obtain valid results. |

*A negative result does not preclude the presence of HPV infection because results depend on adequate specimen collection, absence of inhibitors and sufficient DNA to be detected.

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# Result Interpretation of the cobas HPV Test*

| Results | Interpretation for Patients with ASC-US cytology who are ≥21 years old | Interpretation for Patients with NILM cytology who are ≥ 30 years old |
| --- | --- | --- |
| Other HR HPV** NEG, HPV16 NEG, HPV18 NEG | Very low likelihood of underlying ≥ CIN2; | Lowest likelihood of underlying ≥ CIN2. |
| Other HR HPV** POS, HPV16 NEG, HPV18 NEG | Increased likelihood that underlying ≥ CIN2 will be detected at colposcopy. | Low likelihood of underlying ≥ CIN2. |
| HPV16 POS and/or HPV18 POS | Highest likelihood that underlying ≥ CIN2 will be detected at colposcopy^{1,2}. | Increased likelihood of underlying ≥ CIN2. |

**Other HR HPV DNA includes the following types: 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68.
*According to the 2006 consensus guidelines³, HPV testing should not be performed on women younger than 21 years of age. Also, women 21 years and older with greater than ASC-US cytology (including ASC-H, LSIL or above) should proceed to colposcopy regardless of their HPV test results.

NOTE: HPV negative results are not intended to prevent women from proceeding to colposcopy.

NOTE: In addition to the results tabulated above, invalid results for one or more combinations are also possible. If such a result is obtained, for example:
Other HR HPV NEG, HPV16 POS, HPV18 Invalid
The positive and negative results should be interpreted as shown in Table 1. In this example, HPV 18 results are invalid. The specimen should be re-tested to obtain valid results.

NOTE: Negative results indicate HPV DNA concentrations are undetectable or below the pre-set threshold.

NOTE: Positive test results indicates the presence of any one or more of the high risk types, but since patients are often co-infected with low-risk types it does not rule out the presence of low-risk types in patients with mixed infections.

NOTE: Results of this test should only be interpreted in conjunction with information available from clinical evaluation of the patient and patient history.

# VI. ALTERNATIVE PRACTICES AND PROCEDURES

The patient’s age, medical history and thorough physical examination, including cytology, will provide further information on a patient’s risk of cervical disease, as well as the need for referral to colposcopy. The cobas HPV Test should only be used in conjunction with this clinical information in accordance with appropriate patient management procedures.

Two alternatives for the detection of high-risk HPV DNA and one alternative for the detection of HPV 16 and 18 DNA are currently approved in the United States. At the time of this approval there are no alternative FDA approved devices that detect other HPV targets (such as HPV RNA or protein). Each DNA detection method has its own advantages and disadvantages.

A patient should fully discuss these alternatives with his/her physician to select the screening method(s) that best meets expectations and lifestyle.

# VII. MARKETING HISTORY

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This product is currently being marketed in Australia since the end of August, 2009. The product has also been available for sale in the European Union since December 2009. The product was licensed in Canada in 2010. It has not been withdrawn from these markets for any reason.

## VIII. POTENTIAL ADVERSE EFFECTS OF THE DEVICE ON HEALTH

Below is a list of the potential adverse effects (e.g., complications) associated with the use of the device. As with any *in vitro* diagnostic test, the potential adverse effects are associated with incorrect test results or result interpretations. Failure of this device to perform as expected or failure to correctly interpret results may lead to incorrect HPV test results and subsequently, improper patient management decisions in cervical cancer screening and treatment. False negative results may lead to delays in the timely diagnosis of cervical cancer and treatment, allowing an undetected condition to worsen and potentially increasing morbidity and mortality. False positive results could lead many women to unnecessarily undergo more frequent screening and potentially invasive procedures such as colposcopy and biopsy.

## IX. SUMMARY OF PRECLINICAL STUDIES

### A. Laboratory Studies

#### 1. Clinical Cutoff Determination of the cobas HPV Test

The clinical cutoff for detecting high-grade cervical disease (≥CIN2) for the cobas HPV test was selected based on approximately 29,000 subjects enrolled in Phase 1 of the ATHENA study. The method for selection of cutoff was based on Kondratovich⁴ and was chosen to achieve a pre-defined level of sensitivity of 93% for ≥ CIN2 in the ASC-US population. Based on these criteria, the cutoff values of (40.0, 40.5, 40.0) in the 3 channels (12 Other HR HPV, HPV 16 and HPV 18, respectively) were selected for the cobas HPV test.

#### 2. Limit of Detection at the Clinical Cutoff

The Limit of Detection (LOD) at the clinical cutoff of high risk HPV genotypes HPV16, HPV18 and HPV31 was determined for the cobas HPV Test. The LODs were assessed using 1) plasmids of HPV31, HPV16 and HPV18 in the background of pooled HPV negative patient specimens collected in PreservCyt solution, and 2) HPV positive cell lines SiHa (HPV16) and HeLa (HPV18) in PreservCyt solution containing an HPV negative cell line (HCT-15) background. Plasmid and cell lines were diluted to concentrations below, above and at the expected LOD levels. A minimum of 60 replicates were tested for each plasmid or cell line level for each of 3 reagent lots. A total of 30 runs were performed in a period of 5 days using 4 instrument systems. The LOD at the clinical cutoff is the level of HPV DNA in the sample that has positive test results (above the clinical cutoff) at least 95% of the time. The table below contains results from the reagent lot producing the most conservative (highest) LOD in the analysis.

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Limit of Detection Levels for HPV Types 31, 16, 18 and Cell Lines SiHa (HPV16) and HeLa (HPV18)

| HPV Type | Concentration (copies or cells/mL) | Number of Positive/Tested | Mean CT | % Positives | 95% Confidence Interval | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | | | Lower | Upper |
| 31 | 600 | 60/60 | 36.6 | 100.0% | 94.0% | 100.0% |
| | 300 | 59/61 | 37.9 | 96.7% | 88.7% | 99.6% |
| | 150 | 49/60 | 38.7 | 81.7% | 69.6% | 90.5% |
| 16 | 1500 | 60/60 | 36.5 | 100.0% | 94.0% | 100.0% |
| | 600 | 60/60 | 37.7 | 100.0% | 94.0% | 100.0% |
| | 300 | 55/61 | 39.1 | 90.2% | 79.8% | 96.3% |
| 18 | 1,500 | 60/60 | 36.9 | 100.0% | 94.0% | 100.0% |
| | 600 | 60/60 | 38.0 | 100.0% | 94.0% | 100.0% |
| | 300 | 42/61 | 39.6 | 68.9% | 55.7% | 80.1% |
| SiHa (HPV16) | 200 | 60/60 | 36.9 | 100.0% | 94.6% | 100.0% |
| | 100 | 60/60 | 38.0 | 100.0% | 94.6% | 100.0% |
| | 50 | 53/60 | 39.3 | 88.3% | 77.4% | 95.2% |
| HeLa (HPV18) | 80 | 60/60 | 35.7 | 100.0% | 94.0% | 100.0% |
| | 40 | 60/60 | 36.8 | 100.0% | 94.0% | 100.0% |
| | 20 | 56/60 | 38.2 | 93.3% | 83.8% | 98.1% |

## 3. Inclusivity Verification

To verify that the cobas HPV Test is capable of accurately detecting *all HPV high risk genotypes, the Limit of Detection (LOD) at the clinical cutoff was determined for genotypes 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. Quantified plasmid stocks of each HPV genotype were diluted into a background of pooled HPV negative patient specimens collected in PreservCyt solution to concentrations below, above and at the expected LOD levels. Two lots of reagents were used to produce a minimum of 24 replicates for each positive level with each lot of reagents. For each HPV type, the reported LOD was defined as the lowest testing concentration having a > 95% positive hit rate. Table below contains results from the reagent lot producing the most conservative (higher) LOD in the analysis.

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Summary of High Risk Genotype Limit Of Detection For cobas® HPV Genotype Inclusivity Study

| HPV DNA
*Type | LOD
(copies/mL) | Number of
Positive/Tested | Mean
CT | Hit
Rate | 95% Confidence
Interval | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | | | Lower | Upper |
| 33 | 300 | 24/24 | 38.2 | 100.0% | 85.7% | 100.0% |
| 35 | 600 | 23/24 | 38.4 | 95.8% | 78.8% | 99.8% |
| 39 | 300 | 24/24 | 37.9 | 100.0% | 85.7% | 100.0% |
| 45 | 150 | 23/24 | 38.0 | 95.8% | 78.8% | 99.8% |
| 51 | 300 | 24/24 | 38.4 | 100.0% | 85.7% | 100.0% |
| 52 | 2400 | 24/24 | 39.1 | 100.0% | 85.7% | 100.0% |
| 56 | 1200 | 23/24 | 38.4 | 95.8% | 78.8% | 99.8% |
| 58 | 600 | 24/24 | 38.6 | 100.0% | 85.7% | 100.0% |
| 59 | 300 | 23/24 | 39.0 | 95.8% | 78.8% | 99.8% |
| 66 | 1200 | 24/24 | 37.7 | 100.0% | 85.7% | 100.0% |
| 68 | 1200 | 24/24 | 38.0 | 100.0% | 85.7% | 100.0% |

*The LOD of the cobas HPV Test for HPV genotypes 16, 18 and 31 was determined as described above in this Package Insert.

## 4. Reproducibility

An 18-member panel composed of pools made from clinical samples collected into PreservCyt solution, and from samples derived from SiHa and HeLa cell lines was tested for Reproducibility. Each panel member was tested for 18 days (6 days per kit lot), 2 replicates per run, at 3 testing sites. Two operators at each of 3 sites performed 2 runs per day for 3 days each on each of 3 reagent lots. A run was defined as 36 panel-member aliquots and 1 positive and 1 negative control.

Overall, 111 runs were performed to obtain 108 valid runs. The 3 invalid runs were due to instrument errors (percent of invalid runs was 2.7% (3/111) with 95% CI: 0.6%, 7.7%). A total of 3,888 tests were performed on the 18 panel members in the valid runs; 5 of those tests were invalid due to instrument errors.

All valid test results were included in the analyses that reported the percentage of correct results. There were no false positive results in 216 tests performed on the negative panel members (background negative cell and the pooled negative clinical sample; see Table below).

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Results by Sample Type and Negative Panel Member for Lot and Site/Instrument

| | | | | Number Negative / Total Number Valid Results | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample Type | Panel Member | Ct SD | Ct CV % | Lot | | | Site/ Instrument | | |
| | | | | Lot ID | Negative/ Valid | % | Site ID | Negative/ Valid | % |
| Background cell line | Negative cell line | n/a | n/a | 1 | 72/72 | 100.0 | 1 | 72/72 | 100.0 |
| | | | | 2 | 72/72 | 100.0 | 2 | 72/72 | 100.0 |
| | | | | 3 | 72/72 | 100.0 | 3 | 72/72 | 100.0 |
| Pooled negative clinical sample | Negative | n/a | n/a | 1 | 72/72 | 100.0 | 1 | 72/72 | 100.0 |
| | | | | 2 | 72/72 | 100.0 | 2 | 72/72 | 100.0 |
| | | | | 3 | 72/72 | 100.0 | 3 | 72/72 | 100.0 |

The percents of positive results for the positive panel members are presented below. With respect to sites, site 1 tended to have a lower percent positive for some weak-positive and moderate-positive panel members. This trend can be attributed to operator 1, who tended to have lower percent positive values in the weak positive and moderate positive panel members.

Analysis of variance of the Ct values from valid tests performed on positive panel members yielded overall CV (%) ranges of 1.1% to 2.5% for the SiHa cell lines, 1.5% to 2.5% for the HeLa cell lines, and 3.5% to 10.3% for the pooled clinical samples.

Results by Sample Type and Positive Panel Member for Lot and Site/Instrument

| | | | | Number Positive / Total Number Valid Results | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample Type | Panel Member | Ct SD | Ct CV % | Lot | | | Site/Instrument | | |
| | | | | Lot ID | Positive/ Valid | % | Site ID | Positive/ Valid | % |
| SiHa cell line | HPV16 - weak positive A (25 cells/mL) | 0.45 | 1.1 | 1 | 41/72 | 56.9 | 1 | 22/72 | 30.6 |
| | | | | 2 | 25/72 | 34.7 | 2 | 38/72 | 52.8 |
| | | | | 3 | 23/72 | 31.9 | 3 | 29/72 | 40.3 |
| SiHa cell line | HPV16 - weak positive B (60 cells/mL) | 0.68 | 1.7 | 1 | 66/72 | 91.7 | 1 | 56/72 | 77.8 |
| | | | | 2 | 64/72 | 88.9 | 2 | 71/72 | 98.6 |
| | | | | 3 | 63/72 | 87.5 | 3 | 66/72 | 91.7 |
| SiHa cell line | HPV16 - weak positive C (80 cells/mL) | 0.68 | 1.8 | 1 | 68/72 | 94.4 | 1 | 61/72 | 84.7 |
| | | | | 2 | 67/72 | 93.1 | 2 | 72/72 | 100.0 |
| | | | | 3 | 69/72 | 95.8 | 3 | 71/72 | 98.6 |
| SiHa cell line | HPV16 - positive (150 cells/mL) | 0.94 | 2.5 | 1 | 71/72 | 98.6 | 1 | 71/72 | 98.6 |
| | | | | 2 | 71/72 | 98.6 | 2 | 72/72 | 100.0 |
| | | | | 3 | 72/72 | 100.0 | 3 | 71/72 | 98.6 |
| HeLa cell line | HPV18 - weak positive A (8 cells/mL) | 0.60 | 1.5 | 1 | 43/72 | 59.7 | 1 | 34/72 | 47.2 |
| | | | | 2 | 35/72 | 48.6 | 2 | 46/72 | 63.9 |
| | | | | 3 | 42/72 | 58.3 | 3 | 40/72 | 55.6 |
| HeLa cell line | HPV18 - weak positive B (22 cells/mL) | 0.90 | 2.4 | 1 | 67/72 | 93.1 | 1 | 59/72 | 81.9 |
| | | | | 2 | 63/72 | 87.5 | 2 | 72/72 | 100.0 |
| | | | | 3 | 67/72 | 93.1 | 3 | 66/72 | 91.7 |
| HeLa cell line | HPV18 - weak positive C (27 cells/mL) | 0.90 | 2.4 | 1 | 69/72 | 95.8 | 1 | 65/72 | 90.3 |
| | | | | 2 | 67/72 | 93.1 | 2 | 71/72 | 98.6 |
| | | | | 3 | 72/72 | 100.0 | 3 | 72/72 | 100.0 |
| HeLa cell line | HPV18 - positive (50 cells/mL) | 0.91 | 2.5 | 1 | 70/72 | 97.2 | 1 | 69/72 | 95.8 |
| | | | | 2 | 71/72 | 98.6 | 2 | 72/72 | 100.0 |
| | | | | 3 | 72/72 | 100.0 | 3 | 72/72 | 100.0 |
| Pooled HPV 16 clinical sample | HPV16 - moderate positive | 1.59 | 4.3 | 1 | 66/71 | 93.0 | 1 | 64/72 | 88.9 |
| | | | | 2 | 66/71 | 93.0 | 2 | 68/70 | 97.1 |
| | | | | 3 | 69/72 | 95.8 | 3 | 69/72 | 95.8 |
| Pooled HPV | HPV16 - positive | 1.21 | 3.5 | 1 | 72/72 | 100.0 | 1 | 72/72 | 100.0 |

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| | | | | | Number Positive / Total Number Valid Results | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample Type | Panel Member | Ct SD | Ct CV % | Lot | | | Site/Instrument | | | |
| | | | | | Lot ID | Positive/ Valid | % | Site ID | Positive/ Valid | % |
| 16 clinical sample | | | | 2 | 71/71 | 100.0 | 2 | 72/72 | 100.0 | |
| | | | | 3 | 72/72 | 100.0 | 3 | 71/71 | 100.0 | |
| Pooled HPV 18 clinical sample | HPV18 - moderate positive | 2.30 | 6.1 | 1 | 62/71 | 87.3 | 1 | 56/71 | 78.9 | |
| | | | | 2 | 63/72 | 87.5 | 2 | 71/72 | 98.6 | |
| | | | | 3 | 67/72 | 93.1 | 3 | 65/72 | 90.3 | |
| Pooled HPV 18 clinical sample | HPV18 - positive | 3.51 | 10.3 | 1 | 72/72 | 100.0 | 1 | 71/71 | 100.0 | |
| | | | | 2 | 72/72 | 100.0 | 2 | 72/72 | 100.0 | |
| | | | | 3 | 71/71 | 100.0 | 3 | 72/72 | 100.0 | |
| Pooled HPV 31 clinical sample | HPV31 - moderate positive | 2.95 | 8.0 | 1 | 67/72 | 93.1 | 1 | 61/72 | 84.7 | |
| | | | | 2 | 62/72 | 86.1 | 2 | 68/72 | 94.4 | |
| | | | | 3 | 63/72 | 87.5 | 3 | 63/72 | 87.5 | |
| Pooled HPV 31 clinical sample | HPV31 - positive | 3.01 | 8.3 | 1 | 72/72 | 100.0 | 1 | 70/72 | 97.2 | |
| | | | | 2 | 68/72 | 94.4 | 2 | 72/72 | 100.0 | |
| | | | | 3 | 72/72 | 100.0 | 3 | 70/72 | 97.2 | |
| Pooled HPV 45 clinical sample | HPV45 - moderate positive | 1.88 | 5.0 | 1 | 70/72 | 97.2 | 1 | 66/72 | 91.7 | |
| | | | | 2 | 66/72 | 91.7 | 2 | 70/72 | 97.2 | |
| | | | | 3 | 64/72 | 88.9 | 3 | 64/72 | 88.9 | |
| Pooled HPV 45 clinical sample | HPV45 - positive | 1.80 | 5.0 | 1 | 72/72 | 100.0 | 1 | 72/72 | 100.0 | |
| | | | | 2 | 72/72 | 100.0 | 2 | 72/72 | 100.0 | |
| | | | | 3 | 72/72 | 100.0 | 3 | 72/72 | 100.0 | |

Overall Mean, Standard Deviations, and Coefficients of Variation (%) for Cycle Threshold, Estimated from Valid Samples of Positive Sample Type Panel Members

| | | Standard Deviation [SD] and Percent Coefficient of Variation [CV(%)] | | | | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample Type1/ Conc.2(cells/mL) | | | Within-Run | | Between-Run | | Between-Day | | Between-Operator | | Between-Lot | | Between-Site/Instrument | | Total | |
| | N1/N | MeanCT | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| SiHa GT 16 weak positive A (25/mL) | 89/216 | 39.80 | 0.38 | 0.96% | 0.20 | 0.50% | 0.08 | 0.21% | 0.00 | 0.00% | 0.09 | 0.23% | 0.00 | 0.00% | 0.45 | 1.13% |
| SiHa GT 16 weak positive B (60/mL) | 193/216 | 39.14 | 0.53 | 1.36% | 0.17 | 0.43% | 0.19 | 0.48% | 0.03 | 0.08% | 0.25 | 0.64% | 0.23 | 0.59% | 0.68 | 1.74% |
| SiHa GT 16 weak positive C (80/mL) | 204/216 | 38.73 | 0.58 | 1.50% | 0.00 | 0.00% | 0.18 | 0.47% | 0.08 | 0.21% | 0.21 | 0.55% | 0.21 | 0.54% | 0.68 | 1.76% |
| SiHa GT 16 positive (150/mL) | 214/216 | 37.89 | 0.45 | 1.19% | 0.22 | 0.57% | 0.35 | 0.91% | 0.35 | 0.91% | 0.21 | 0.57% | 0.58 | 1.53% | 0.94 | 2.47% |
| HeLa GT 18 weak positive A (8/mL) | 120/216 | 39.02 | 0.57 | 1.45% | 0.00 | 0.00% | 0.00 | 0.00% | 0.00 | 0.00% | 0.12 | 0.32% | 0.16 | 0.41% | 0.60 | 1.54% |
| HeLa GT 18 weak | 197 | 38.10 | 0.72 | 1.89% | 0.38 | 1.00% | 0.11 | 0.29% | 0.13 | 0.33% | 0.17 | 0.44% | 0.30 | 0.78% | 0.90 | 2.36% |

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| positive B (22/mL) | 216 | | | | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| HeLa GT 18 weak positive C (27/mL) | 208
216 | 37.77 | 0.73 | 1.93% | 0.13 | 0.35% | 0.17 | 0.44% | 0.31 | 0.83% | 0.25 | 0.67% | 0.26 | 0.69% | 0.90 |
| HeLa GT 18 positive (50/mL) | 213
216 | 36.76 | 0.64 | 1.74% | 0.07 | 0.20% | 0.29 | 0.79% | 0.38 | 1.05% | 0.32 | 0.87% | 0.29 | 0.80% | 0.91 |
| Clinical GT 16 weak positive | 201
214 | 37.33 | 1.46 | 3.92% | 0.44 | 1.18% | 0.44 | 1.17% | 0.00 | 0.00% | 0.00 | 0.00% | 0.00 | 0.00% | 1.59 |
| Clinical GT 16 positive | 215
215 | 34.95 | 1.05 | 3.02% | 0.50 | 1.44% | 0.00 | 0.00% | 0.00 | 0.00% | 0.18 | 0.51% | 0.27 | 0.76% | 1.21 |
| Clinical GT 18 weak positive | 192
215 | 37.63 | 2.27 | 6.02% | 0.00 | 0.00% | 0.00 | 0.00% | 0.00 | 0.00% | 0.00 | 0.00% | 0.39 | 1.05% | 2.30 |
| Clinical GT 18 positive | 215
215 | 34.17 | 3.16 | 9.25% | 1.26 | 3.68% | 0.00 | 0.00% | 0.42 | 1.23% | 0.00 | 0.00% | 0.73 | 2.13% | 3.51 |
| Clinical GT 31 weak positive | 192
216 | 36.91 | 2.95 | 7.98 | 0.00 | 0.00% | 0.00 | 0.00% | 0.22 | 0.60% | 0.00 | 0.00% | 0.00 | 0.00% | 2.95 |
| Clinical GT 31 positive | 212
216 | 36.49 | 2.81 | 7.69% | 0.00 | 0.00% | 0.67 | 1.84% | 0.00 | 0.00% | 0.00 | 0.00% | 0.86 | 2.35% | 3.01 |
| Clinical GT 45 weak positive | 200
216 | 37.37 | 1.88 | 5.03% | 0.00 | 0.00% | 0.00 | 0.00% | 0.00 | 0.00% | 0.00 | 0.00% | 0.00 | 0.00% | 1.88 |
| Clinical GT 45 positive | 216
216 | 35.66 | 1.74 | 4.87% | 0.21 | 0.58% | 0.00 | 0.00% | 0.00 | 0.00% | 0.00 | 0.00% | 0.41 | 1.14% | 1.80 |

1. Moderate is abbreviated as mod.
2. Analyte concentrations are given for the SiHa and HeLa cell lines.
3. n is the number of positive tests, which contribute CT values to the analysis. N is the total number of valid tests for the panel member. Because only positive test results were included, estimates of SD (and %CV) may be underestimated.

## 5. Precision

In-house Precision was examined using a panel composed of HPV positive and negative cell lines diluted into PreservCyt solution and pooled HPV positive and negative cervical specimens collected in PreservCyt solution. The precision panel was designed to include members below (< 70% positivity rate), at (90% to 99% positivity rate) and above (> 99% positivity rate) the Limit of Detection of the cobas HPV Test. Panel members 1-9 and 19-22 were prepared with HPV positive and negative cell lines (SiHa, HPV16; HeLa, HPV18; HCT-15, HPV negative) diluted at different levels into PreservCyt solution (panel level 1 was prepared with HPV negative cell line only). Panel members 10-18 were prepared with high risk HPV positive specimen in PreservCyt solution pools (HPV16, HPV18, HPV31 and HPV45) diluted at different levels into pooled HPV negative specimens in PreservCyt solution (panel level 10 was prepared with HPV negative specimen pool only).

A description of the precision panel, anticipated performance in % positivity rate and the actual study performance in % positivity rate are shown in the table below. All panel levels at and above the limit of detection yielded the anticipated positivity rates.

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Summary of the Precision Panel and Hit Rates For cobas HPV Precision Study

| Panel Number | HPV Target | Description | Anticipated Positivity Rate | N Tested | N Pos | Positivity Rate | 95% CI | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | | | | Lower | Upper |
| 1 | N/A | HCT15 cell line (HPV negative) | 0% | 144 | 0 | 0.0% | 0% | 3% |
| 2 | HPV16 | SiHa cell line | < 70% | 143 | 80 | 55.9% | 47% | 64% |
| 3 | HPV16 | SiHa cell line | 90% — 95% | 144 | 138 | 95.8% | 91% | 98% |
| 4 | HPV16 | SiHa cell line | 95% — 99% | 144 | 144 | 100.0% | 97% | 100% |
| 5 | HPV16 | SiHa cell line | > 99% | 143 | 142 | 99.3% | 96% | 100% |
| 6 | HPV18 | HeLa cell line | < 70% | 144 | 96 | 66.7% | 58% | 74% |
| 7 | HPV18 | HeLa cell line | 90% — 95% | 144 | 143 | 93.3% | 96% | 100% |
| 8 | HPV18 | HeLa cell line | 95% — 99% | 144 | 142 | 98.6% | 95% | 100% |
| 9 | HPV18 | HeLa cell line | > 99% | 144 | 144 | 100.0% | 97% | 100% |
| 10 | N/A | Pooled HPV neg specimen | 0% | 141 | 1 | 0.7% | 0% | 4% |
| 11 | HPV16 | High Risk HPV positive specimen | 90% — 99% | 144 | 140 | 97.2% | 93% | 99% |
| 12 | HPV16 | High Risk HPV positive specimen | > 99% | 143 | 143 | 100.0% | 97% | 100% |
| 13 | HPV18 | High Risk HPV positive specimen | 90% — 99% | 144 | 140 | 97.2% | 93% | 99% |
| 14 | HPV18 | High Risk HPV positive specimen | > 99% | 144 | 144 | 100.0% | 97% | 100% |
| 15 | HPV31 | High Risk HPV positive specimen | 90% — 99% | 143 | 142 | 99.3% | 96% | 100% |
| 16 | HPV31 | High Risk HPV positive specimen | > 99% | 144 | 144 | 100.0% | 97% | 100% |
| 17 | HPV45 | High Risk HPV positive specimen | 90% — 99% | 144 | 133 | 92.4% | 87% | 96% |
| 18 | HPV45 | High Risk HPV positive specimen | > 99% | 144 | 144 | 100.0% | 97% | 100% |
| *19 | HPV16 & HPV18 | SiHa & HeLa cell lines | < 70% | 143 | 88 | 61.5% | 53% | 70% |
| *20 | HPV16 & HPV18 | SiHa & HeLa cell lines | 90% — 95% | 144 | 144 | 100.0% | 97% | 100% |
| *21 | HPV16 & HPV18 | SiHa & HeLa cell lines | 95% — 99% | 144 | 144 | 100.0% | 97% | 100% |
| *22 | HPV16 & HPV18 | SiHa & HeLa cell lines | > 99% | 144 | 144 | 100.0% | 97% | 100% |
| **19 | HPV16 & HPV18 | SiHa & HeLa cell lines | < 70% | 143 | 103 | 72.0% | 64% | 79% |
| **20 | HPV16 & HPV18 | SiHa & HeLa cell lines | 90% — 95% | 144 | 143 | 93.3% | 96% | 100% |
| **21 | HPV16 & HPV18 | SiHa & HeLa cell lines | 95% — 99% | 144 | 142 | 98.6% | 95% | 100% |
| **22 | HPV16 & HPV18 | SiHa & HeLa cell lines | > 99% | 144 | 144 | 100.0% | 97% | 100% |

Analysis of variance of the Ct values from valid tests performed on positive panel members (see table below) yielded overall CV (%) ranges of 1.1% to 1.7% for the SiHa cell lines, 1.5% to 2.2% for the HeLa cell lines, and 3.7% to 8.5% for the pooled clinical samples.

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Overall Mean, Standard Deviations, and Coefficients of Variation (%) for Cycle Threshold, Estimated from Valid Samples of Positive Sample Type Precision Panel Members

| | | | | Standard Deviation [SD] and Percent Coefficient of Variation [CV(%)] | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| # | Sample Type/Conc.1(cells/mL) | | | Between-Lot | | Between-Run/System | | Between-Operator | | Between-Day | | Within-Run | | Total | |
| | | N2N | Mean CT | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | SiHa HPV16(25/mL) | 80143 | 39.8 | 0.000 | 0.000% | 0.000 | 0.000% | 0.065 | 0.20% | 0.168 | 0.40% | 0.410 | 1.00% | 0.448 | 1.10% |
| 2 | SiHa HPV16(60/mL) | 138144 | 38.8 | 0.172 | 0.40% | 0.000 | 0.00% | 0.000 | 0.00% | 0.000 | 0.00% | 0.640 | 1.70% | 0.663 | 1.70% |
| 3 | SiHa HPV16(80/mL) | 144144 | 38.4 | 0.055 | 0.10% | 0.000 | 0.00% | 0.116 | 0.30% | 0.142 | 0.40% | 0.569 | 1.50% | 0.601 | 1.60% |
| 4 | SiHa HPV16(150/mL) | 142143 | 37.3 | 0.067 | 0.20% | 0.092 | 0.20% | 0.000 | 0.00% | 0.284 | 0.80% | 0.405 | 1.10% | 0.508 | 1.40% |
| 5 | HeLa HPV18(8/mL) | 96144 | 38.9 | 0.116 | 0.30% | 0.073 | 0.20% | 0.000 | 0.00% | 0.000 | 0.00% | 0.665 | 1.70% | 0.680 | 1.70% |
| 6 | HeLa HPV18(22/mL) | 143144 | 37.7 | 0.000 | 0.00% | 0.000 | 0.00% | 0.076 | 0.20% | 0.074 | 0.20% | 0.811 | 2.20% | 0.818 | 2.20% |
| 7 | HeLa HPV18(27/mL) | 142144 | 37.5 | 0.000 | 0.00% | 0.000 | 0.00% | 0.000 | 0.00% | 0.229 | 0.60% | 0.675 | 1.80% | 0.712 | 1.90% |
| 8 | HeLa HPV18(50/mL) | 144144 | 36.5 | 0.000 | 0.00% | 0.000 | 0.00% | 0.000 | 0.00% | 0.157 | 0.40% | 0.578 | 1.60% | 0.599 | 1.60% |
| 9 | Clinical HPV16 | 140144 | 37.2 | 0.000 | 0.00% | 0.258 | 0.70% | 0.000 | 0.00% | 0.000 | 0.00% | 1.650 | 4.40% | 1.670 | 4.50% |
| 10 | Clinical HPV16 | 143143 | 34.5 | 0.220 | 0.60% | 0.135 | 0.40% | 0.000 | 0.00% | 0.441 | 1.30% | 1.183 | 3.40% | 1.288 | 3.70% |
| 11 | Clinical HPV18 | 140144 | 36.7 | 0.378 | 1.00% | 0.000 | 0.00% | 0.000 | 0.00% | 0.000 | 0.00% | 3.081 | 8.40% | 3.104 | 8.50% |
| 12 | Clinical HPV18 | 144144 | 34.9 | 0.000 | 0.00% | 0.692 | 2.00% | 0.000 | 0.00% | 1.291 | 3.70% | 2.180 | 6.20% | 2.626 | 7.50% |
| 13 | Clinical HPV31 | 142143 | 37.1 | 0.000 | 0.00% | 0.255 | 0.70% | 0.323 | 0.90% | 0.000 | 0.00% | 2.351 | 6.30% | 2.387 | 6.40% |
| 14 | Clinical HPV31 | 144144 | 35.8 | 0.190 | 0.50% | 0.000 | 0.00% | 0.000 | 0.00% | 0.746 | 2.10% | 2.825 | 7.90% | 2.928 | 8.20% |
| 15 | Clinical HPV45 | 133144 | 37.3 | 0.000 | 0.00% | 0.186 | 0.50% | 0.101 | 0.30% | 0.000 | 0.00% | 1.915 | 5.10% | 1.926 | 5.20% |
| 16 | Clinical HPV45 | 144144 | 35.0 | 0.393 | 1.10% | 0.246 | 0.70% | 0.000 | 0.00% | 0.000 | 0.00% | 1.780 | 5.10% | 1.839 | 5.30% |
| *17 | SiHa HPV16(25/mL)HeLa HPV18(8/mL) | 88143 | 39.8 | 0.000 | 0.00% | 0.000 | 0.00% | 0.014 | 0.00% | 0.000 | 0.00% | 0.461 | 1.20% | 0.461 | 1.20% |

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| | | | | Standard Deviation [SD] and Percent Coefficient of Variation [CV(%)] | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| # | Sample Type /Conc.1(cells/mL) | | | Between-Lot | | Between-Run/System | | Between-Operator | | Between-Day | | Within-Run | | Total | |
| | | N/N | Mean CT | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| *18 | SiHa HPV16(60/mL)HeLa HPV18(22/mL) | 144
144 | 38.4 | 0.106 | 0.30% | 0.000 | 0.00% | 0.034 | 0.10% | 0.000 | 0.00% | 0.591 | 1.50% | 0.601 | 1.60% |
| *19 | SiHa HPV16(80/mL)HeLa HPV18(27/mL) | 144
144 | 38.3 | 0.134 | 0.30% | 0.060 | 0.20% | 0.000 | 0.00% | 0.238 | 0.60% | 0.405 | 1.10% | 0.479 | 1.30% |
| *20 | SiHa HPV16(150/mL)HeLa HPV18(50/mL) | 144
144 | 37.2 | 0.088 | 0.20% | 0.039 | 0.10% | 0.000 | 0.00% | 0.238 | 0.60% | 0.405 | 1.10% | 0.479 | 1.30% |
| **17 | SiHa HPV16(25/mL)HeLa HPV18(8/mL) | 103
143 | 38.8 | 0.000 | 0.00% | 0.127 | 0.30% | 0.065 | 0.20% | 0.274 | 0.70% | 0.579 | 1.50% | 0.656 | 1.70% |
| **18 | SiHa HPV16(60/mL)HeLa HPV18(22/mL) | 143
144 | 37.6 | 0.182 | 0.50% | 0.000 | 0.00% | 0.000 | 0.00% | 0.145 | 0.40% | 0.710 | 1.90% | 0.747 | 2.00% |
| **19 | SiHa HPV16(80/mL)HeLa HPV18(27/mL) | 142
144 | 37.3 | 0.000 | 0.00% | 0.062 | 0.20% | 0.000 | 0.00% | 0.131 | 0.40% | 0.626 | 1.70% | 0.643 | 1.70% |
| **20 | SiHa HPV16(150/mL)HeLa HPV18(50/mL) | 144
144 | 36.4 | 0.000 | 0.00% | 0.000 | 0.00% | 0.000 | 0.00% | 0.244 | 0.70% | 0.481 | 1.30% | 0.540 | 1.50% |

1 Analyte concentrations are given for the SiHa and HeLa cell lines.
2 n is the number of positive tests, which contribute CT values to the analysis. N is the total number of valid tests for the panel member. Because only positive test results were included, estimates of SD (and %CV) may be underestimated.
*Results shown from detection channel 2 (HPV16)
** Results shown from detection channel 3 (HPV18)
N/A = Not applicable

## 6. Analytical Specificity

A panel of bacteria, fungi and viruses, including those commonly found in the female urogenital tract, as well as several Human papillomavirus types classified as low or undetermined risk were tested with the cobas HPV Test to assess analytical specificity. The organisms listed in the table below were spiked at high concentrations (≥ 1 × 10⁶ *units/reaction with the exception of Treponema pallidum and Adenovirus-5, which were both tested at ≥ 1 × 10⁵ *units/reaction) into HPV negative specimen in PreservCyt solution and into HPV negative specimen in PreservCyt solution spiked with HPV31,

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HPV16 and HPV18 plasmid DNA at 3 times the limit of detection. Results indicated that none of these organisms interfered with detection of HPV 31, HPV16 and HPV18 or produced false positive results in the HPV negative specimen.

*All bacteria were quantified as Colony Forming Units (CFU) except Chlamydia trachomatis which was quantified as Elementary Bodies (EBs). Treponema pallidum and all HPV genotypes were quantified as DNA copies. Adenovirus was quantified as Plaque Forming Units (PFU). CMV, EBV, HSV-1 and HSV-2 were quantified as Viral Particles (VP). HBV and HIV-1 were quantified in International Units (IU) and SV40 was quantified in Infection Units (IU).

Microorganisms Tested for Analytical Specificity

| Achromobacter xerosis | Erysipelothrix rhusiopathiae | Mycoplasma hominis | Weissella paramesenteroides |
| --- | --- | --- | --- |
| Acinetobacter calcaceticus | Escherichia coli | Neisseria gonorrhea | Yersinia enterocolitica |
| Acinetobacter lwoffii | Ewingella americana | Neisseria meningitidis Serogroup A | HPV 6 |
| Acinetobacter sp. Genospecies 3 | Fusobacterium nucleatum | Pasteurella multocida | HPV 11 |
| Actinomyces isrealii | Gemella morbillorum | Pediococcus acidilactica | HPV 26 |
| Adenovirus 5 | Gardnerella vaginalis | Peptostreptococcus anaerobius | HPV 30 |
| Aerococcus viridans | Haemophilus ducreyi | Propionibacterium acnes | HPV 34 |
| Alcaligenes faecalis | Hepatitis B virus (HBV) | Proteus mirabilis | HPV 40 |
| Bacillus thuringiensis | Herpes simplex virus 1 (HSV-1) | Proteus vulgaris | HPV 42 |
| Bacteroides fragilis | Herpes simplex virus 2 (HSV-2) | Providencia stuartii | HPV 53 |
| Bacteroides ureolyticus | Human immunodeficiency virus (HIV-1) | Pseudomonas aeruginosa | HPV 54 |
| Bifidobacterium longum | Kingella kingae | Ruminococcus productus | HPV 55B |
| Bifidobacterium adolescentis | Klebsiella pneumoniae ss osuenae | Salmonella minnesota | HPV 61 |
| Bifidobacterium brevi | Lactobacillus acidophilus | Serratia marcescens | HPV 62 |
| Campylobacter jejuni | Lactobacillus crispatus | Staphylococcus aureus | HPV 64 |
| Candida albicans | Lactobacillus delbrueckii s. lactis | Staphylococcus epidermidis | HPV 67 |
| Chlamydia trachomatis | Lactobacillus jensenii | Staphylococcus saprophyticus | HPV 69 |
| Chromobacter violaceum | Lactobacillus vaginalis | Streptococcus agalactiae | HPV 70 |
| Citrobacter braakii | Lactococcus lactis cremoris | Streptococcus anginosus | HPV 71 |
| Clostridium perfringens | Legionella pneumophila | Streptococcus pyogenes | HPV 72 |
| Corynebacterium genitalium | Micrococcus luteus | Streptococcus sanguis | HPV 73 |
| Cytomegalovirus (CMV) | Mobiluncus curtsii s. curtsii | Simian Virus 40 (SV40) | HPV 81 |
| Eikenella corrodens | Moraxella osloensis | Treponema Pallidum | HPV 82 |
| Enterobacter cloacae | Morganella morganii | Trichomonas vaginalis | HPV 83 |
| Enterococcus faecalis | Mycobacterium avium | Ureaplasma urealyticum | HPV 84 |
| Enterococcus faecium | Mycobacterium smegmatis | Veillonela parvula | HPV 85 |
| Epstein Barr Virus (EBV) | Mycoplasma genitalium | Vibrio parahaemolyticus | HPV 89 (CP6108) |

## 7. Interfering Substances

HPV positive and HPV negative cervical specimens as well as contrived specimens were used to assess the effects of endogenous and exogenous interfering substances that could potentially be present in cervical specimens. Testing materials used in these studies are described in the table below. The concentrations of endogenous and exogenous substances tested represent conditions that could occur during specimen collection. Whole blood, Peripheral Blood Mononuclear Cells (PBMC) and cervical mucus were tested as potential endogenous interfering substances found in cervical specimens. Levels of each potential interfering substance tested and performance observations are described

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below. No interference was seen for PBMC or cervical mucus at all levels tested. Whole blood showed no interference when present in visually detectable amounts of up to 1.5%.

## Interference Testing Sample Descriptions

| Sample type | Description | Study |
| --- | --- | --- |
| HPV-Positive Cervical Specimens | 10 individual HPV positive cervical specimens in PreservCyt solution were aliquoted for testing with and without endogenous interfering substances | Endogenous Interference |
| HPV Negative Cervical Specimens | 10 individual HPV negative cervical specimens in PreservCyt solution were aliquoted for testing with and without endogenous interfering substances | Endogenous Interference |
| Contrived HPV Positive Cervical Specimen | Cervical specimens in PreservCyt solution positive for one of the high risk HPV types other than HPV16 and/or HPV18 were diluted with HPV negative specimen to generate signal consistent with approximately 3 fold LOD. HPV types 16 and 18 plasmids were then added at concentrations of approximately 3 fold LOD. | Endogenous Interference |
| 3 x LOD PreservCyt Specimen Pools | HPV types 31, 16, 18 plasmids were each diluted to 3 fold LOD into pools of negative cervical specimen in PreservCyt solution. | Exogenous Interference |

## Interference Testing Results with Endogenous Interferents

| Interferent Tested | Concentrations Tested | Interference Observed |
| --- | --- | --- |
| Whole Blood | 1%, 1.5%, 2%, 3% v/v | Above 1.5% |
| PBMC | 10^{4}, 10^{5}, 10^{6} cells/mL | None |
| Cervical Mucus | Mucus obtained from standard cervical cleaning procedure | None |

A total of 18 over-the-counter (OTC) feminine hygiene and contraceptive products were tested as potential interfering substances. Types of potential interferents tested and performance observations in 3 x LOD pools prepared from HPV negative cervical specimens in PreservCyt solution are described below.

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Interference Testing Results with Exogenous Interferents

| Product Name | Active Ingredients | Interference Observed |
| --- | --- | --- |
| Prodium | Phenazopyridine Hydrochloride | None |
| Vaginal Contraceptive Foam | Nonoxynol-9 | None |
| Clotrimazole 7 | Clotrimazole | None |
| Gyne-Lotrimin 7 | Clotrimazole | None |
| Gynecort | Hydrocortisone | None |
| Vagisil Satin | Hydrocortisone | None |
| Vagi-Gard (Douche) | Povidone-iodine | None |
| Miconazole | Miconazole nitrate | None |
| Monistat 3 Cream | Miconazole nitrate | None |
| Equate tioconazole 1 | Tiocanazole | None |
| Vagi-Gard Medicated Cream | Benzocaine | None |
| Vagicaine Anti-Itch Cream | Benzocaine | None |
| Yeast Gard | Pulsatilla, Candida Parapsilosis, Candida Albicans | None |
| Norforms | PEG-32,PEG-18, Peg-20 stearate | None |
| KY Jelly | Hydroxyethylcellulose, Chlorhexidine Gluconate | None |
| Vagisil Moisturizer | DMDM Hydantoin, Diazolidinyl urea | None |
| Replens | Polycarbophil, | None |
| Vagi-Gard (Lube Gel) | Glucano Delta Lactone, Chlorhexidine Gluconate | None |

## 8. Reagent Stability

The cobas HPV Test consists of 5 kits. The following table lists each of these kits, along with their corresponding shelf life and storage temperature, as supported by the results of real-time stability studies:

| Kits for the cobas HPV Test | Shelf Life (months) | Storage Temperature |
| --- | --- | --- |
| cobas 4800 System Sample Preparation Kit | 18 | 2-8°C |
| cobas 4800 System Liquid Cytology Preparation Kit | 18 | 2-8°C |
| cobas 4800 System Wash Buffer Kit | 18 | 15-25°C |
| cobas 4800 HPV Amplification/Detection Kit | 18 | 2-8°C |
| cobas 4800 HPV Controls Kit | 14 | 2-8°C |

Each kit consists of different components required for sample preparation, performance monitoring (assay controls) and amplification/detection. The expiration date for each test kit is defined by the shortest dated component in that kit.

## 9. Sample Handling and Collection

Cervical specimens should be collected in PreservCyt Solution, the ThinPrep Pap Test preservation system, using an Endocervical Brush/Spatula.

Specimen stability studies demonstrated that for the cobas HPV Test cervical specimens can be stored at 2-30°C in PreservCyt Solution for up to 6 months prior to performing the cobas HPV Test. See PreservCyt solution labeling for storage requirements prior to cytology processing. PreservCyt specimens should not be frozen.

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# 10. Cross-Contamination Study

Cross-contamination was examined using 3 cobas 4800 systems. HPV positive and negative samples were processed in a checkerboard configuration. HPV positive samples [PC(+)] were prepared by adding cultured CaSki cells [HPV16 positive, manufactured and quantified by Roche Culture Collection (RMSCC)] to PreservCyt Solution to generate a signal which covers 95% or more of the results found in specimens of diseased patients in the intended use population. PreservCyt Solution without spiked HPV cultures was used as negative samples [PC(-)].

Four runs were done with each cobas 4800 system for a total of 12 runs. The initial runs (run 1 for all systems) contained PreservCyt Solution only. These runs were done to verify that the system was free of contamination prior to the checkerboard runs. The subsequent two runs per system (runs 2 and 3 for all systems) contained both negative HPV samples and positive HPV samples in a checkerboard configuration. The last run (run 4 for all systems) contained negative HPV PreservCyt Solution only. This last negative run was used to determine the rate of run to run carry over. Of the six runs that examined the HPV high positive and negative checkerboard configuration, 2 out of 282 HPV negative samples showed positive results in channel 2, leading to a cross-contamination rate of 0.71%. There were no invalid runs or test results observed.

All negative runs, including prior to and after the checkerboard runs, yielded negative results, indicating no run-to-run contamination had occurred. There were no invalid runs or test results observed. The sample to sample cross-contamination rate was found to be 0.71% and no run to run carry over rate was observed on the cobas 4800 system.

## B. Animal Studies

Not applicable

## C. Additional Studies

Not applicable

# X. SUMMARY OF PRIMARY CLINICAL STUDY

## A. Study Design

Patients were enrolled between May 2008 and August 2009. The database for this PMA/PMA supplement included 47,208 patients. There were 61 investigational sites.

### 1. Clinical Inclusion and Exclusion Criteria

**Inclusion Criteria:**

a. Females age ≥21 years
b. Presenting for routine cervical cancer screening (see Glossary for definition)
c. Intact cervix
d. Willing and able to undergo colposcopy and biopsy and ECC ≤12 weeks (≤84 days) from Study Visit 1

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e. Written informed consent
f. Willing and able to participate in the 3-year Follow-Up Phase

## Exclusion Criteria:

Subjects were excluded from enrollment if ANY of the following criteria were met:

a) Incomplete informed consent (lacking signature of study subject OR signature of appropriate consenting study personnel, ie, either the principal investigator or someone to whom the principal investigator has appropriately delegated consenting authority)
b) Known pregnancy at Baseline Study Visit 1
c) Presenting for colposcopy at Study Visit 1
d) Any medical condition that, in the opinion of the investigator, would result in increased risk of bleeding at biopsy
e) Known history of ablative or excisional therapy (eg, LEEP, cryotherapy, cone biopsy) to the cervix in the 12 months before Baseline Study Visit 1
f) Hysterectomy (including supracervical)
g) Current or planned participation in any clinical trial for HPV treatment (for the 3-year duration of this study)

## 2. Follow-up Schedule:

Patients were scheduled to return for follow-up examinations as described under “follow-up phase” below.

## Baseline phase

A multicenter, prospective study (ATHENA Study) was conducted to evaluate the performance of the cobas HPV Test as a triage test to stratify women with ASC-US cytology results for colposcopy, and also as an adjunctive test to cervical cytology to guide management decisions. The study consisted of a Baseline Phase, as well as a 3 year Follow-up Phase. In the Baseline Phase, Subjects ≥ 21 years old undergoing routine cervical cancer screening were invited to participate in the study. In total, 47,208 subjects were enrolled from May 2008 to August 2009 at 61 clinical sites in the Baseline Phase. Following written informed consent, demographic information and gynecologic histories were obtained. Two cervical samples were collected for HPV testing and ThinPrep liquid based cytology (LBC). HPV testing was performed at five different laboratories and LBC testing at four. Cytology samples were classified according to the criteria of the 2001 Bethesda System. The first cervical sample collected from each study participant was tested with the cobas HPV Test as well as an investigational use only (IUO) HR HPV test and an IUO HPV genotyping test. For testing with the cobas HPV Test, the first ~29,000 samples collected were stored and were within the window for sample stability at the time of testing. The remaining ~18,000 samples collected were tested prospectively, i.e., in “real time” by the testing sites at the time of cervical sample collection. The second sample collected from all subjects with ASC-US Pap test results was tested with an FDA-approved test according to the manufacturer’s instructions.

Those subjects ≥ 21 years old with ASC-US cytology were invited to undergo colposcopy. In addition, all subjects ≥30 years old with NILM (negative for intraepithelial lesions or malignancy) cytology and a positive test result for HR HPV

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DNA (positive by the IUO HR HPV test and/or the IUO HPV genotyping test), as well as a randomly selected subset of subjects (approximately 1:35) with NILM cytology/negative HR HPV DNA (by both the IUO HR HPV and the IUO HPV genotyping test), were invited to proceed to colposcopy. In order to avoid bias, both study participants and colposcopists were blinded to all HPV tests and cytology results until after the colposcopy was completed. Colposcopy was conducted according to a standardized protocol in which biopsies were obtained on all visible lesions; endocervical curettage was performed in all patients in whom the squamocolumnar junction was not visualized and a single random cervical biopsy was obtained if no lesions were visible. All biopsies were examined by a Central Pathology Review Panel (CPR) consisting of three expert pathologists, and discordant results adjudicated according to a pre-defined protocol. For all analyses, the clinical performance of cobas HPV Test was measured against CPR histology results. The analyses were performed for those subjects with histology ≥ CIN2 and ≥ CIN3 by CPR. Subjects with a CPR diagnosis of ≥ CIN2 by CPR exited the study. All subjects who had undergone colposcopy and biopsy, without a diagnosis of ≥ CIN2 by CPR were invited to proceed to the Follow-up Phase of the study.

## Follow-up phase

All subjects who did not have histology ≥ CIN2 by CPR were invited to participate in a 3 year longitudinal study. Approximately 8,000 eligible subjects have entered the follow-up study. Subjects undergo annual visits for cervical sampling for cytology and HPV DNA testing (by the cobas HPV test). All subjects with ≥ ASC-US are invited to proceed to colposcopy. Colposcopy and biopsies are performed in a standardized manner as described above. All cervical biopsies are examined by the Central Pathology Review Panel. All subjects with ≥ CIN2 by CPR exit the study and those with &lt; CIN2 by CPR are invited to proceed to the follow-up year visit. In order to maximize disease ascertainment, an exit colposcopy and endocervical curettage (ECC) will be offered to all subjects in Year 3.

## Study design to demonstrate clinical sensitivity and specificity for screening patients with ASC-US cytology results to determine the need for referral for colposcopy

Those subjects ≥ 21 years old with ASC-US cytology, regardless of HPV results, were invited to undergo colposcopy. Both study participants and colposcopists were blinded to all HPV tests and cytology results until after the colposcopy was completed. Colposcopy was conducted according to a standardized protocol and all biopsies were read by the CPR, as described above. The clinical performance of cobas HPV Test was measured against histology results of ≥ CIN2 and ≥ CIN3 by CPR.

## Study design to demonstrate clinical performance of the cobas HPV Test as an adjunct to cervical cytology in women ≥30 years old

All subjects ≥30 years old with NILM (negative for intraepithelial lesions or malignancy) cytology and a positive test result for HR HPV DNA (positive by the IUO HR HPV test and/or the IUO HPV genotyping test), as well as a randomly selected subset of subjects (approximately 1:35) with NILM cytology/negative HR HPV DNA (by both the IUO HR HPV and the IUO HPV genotyping test), were invited to proceed to colposcopy. The analyses were performed for histology results ≥ CIN2 and ≥ CIN3 by CPR. All subjects ≥

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30 years who were invited to colposcopy and did not have histology ≥ CIN2 by CPR were eligible to participate in a 3 year longitudinal study for the cobas HPV Test. All subjects with follow-up cytology ≥ ASC-US are invited to proceed to colposcopy; colposcopy and biopsies are performed in a standardized manner as described above. All cervical biopsies are examined by the CPR and all subjects with ≥ CIN2 exit the study. Exit colposcopy and ECC are offered to all subjects. The objectives of the follow-up phase of the study are to determine the 3-year risk (cumulative incidence rates, CIRs) of developing &gt; CIN2 and &gt; CIN3 in subjects ≥ 30 years with NILM cytology. Risk will be measured according to the baseline HPV status (as determined by the cobas HPV Test) for: positive and negative for HR HPV DNA and positive for genotype 16 and/or 18, as well as 12 other HR types. As with the baseline study, the histology of &gt; CIN2 and &gt; CIN3 will be determined by CPR.

## B. Accountability of PMA Cohort

### Accountability in ASC-US (≥ 21 years) Population

A total of 1,923 (4.1%) subjects out of 46,887 eligible subjects had an ASC-US cytology result. Of these, 1,918 (99.7%) subjects were evaluable and 5 subjects were not evaluable due to a missing/invalid IUO HR HPV test and/or IUO HPV genotyping test or cobas HPV Test results. Evaluable subjects were those who had ASC-US cytology results and had valid test results from the IUO HR HPV test, the IUO HPV genotyping test, and the cobas HPV Test. Of these, 1,620 (84.5%) proceeded to Study Visit 2. A total of 1,918 subjects were evaluable for the primary objectives of the study. Two additional subjects with valid biopsy results but invalid results for the cobas HPV test are also included as shown below.

No sample was taken at Study Visit 2 for 10 of 1,622 subjects who completed Study Visit 2, and 32 subjects that had undetermined biopsy results [i.e., inadequate tissue for diagnosis or sample(s) taken outside visit window]. A total of 1,612 subjects had valid biopsy results out of which a total of 1,578 evaluable ASC-US subjects had biopsy results. Detailed accountability of ASC-US(≥ 21 years) subjects is shown below:

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# Accountability in NILM (≥ 30 years) population

A total of 32,260 subjects was evaluable in the NILM (≥ 30 years) population. Evaluable subjects were defined as eligible subjects ≥ 30 years old with a NILM cytology result and who had valid results from the IUO HR HPV test, the IUO HPV genotyping test, and the cobas HPV Test. A total of 5,105 subjects were selected/randomized to Study Visit 2, but 683 of these subjects exited the study prior to Study Visit 2. Of the remaining subjects, 4,371 subjects had biopsy results and 4,258 subjects had valid biopsy results from central pathology review (CPR) panel. Detailed accountability of NILM (≥ 30 years) subjects is shown below:

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## C. Study Population Demographics and Baseline Parameters

The demographics of the study population are typical for a prospective study performed in the US.

## ASC-US Population Demographics

The demographics of the ASC-US population are described below. The median age of the ASC-US subjects was 36 years, with 32.6% subjects age 21-29 and 26.5% age 30-39; the remaining 40.9% subjects were ≥40 years. Approximately 80% of the ASC-US subjects were White, 17% were African American, and the remaining 3% were from other races. Most eligible subjects (97.4%) had high school or above education.

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Summary of Demographic Characteristics for Evaluable ASC-US Subjects

| Characteristics | Evaluable Subjects
n = 1,918 |
| --- | --- |
| Age (Years) | |
| Mean | 37.2 |
| Standard Deviation | 11.5 |
| Median | 36 |
| (Min, Max) | (21,80) |
| | |
| | n (%) |
| Age Group (Years) | |
| 21-29 | 626 (32.6) |
| 30-39 | 508 (26.5) |
| ≥40 | 784 (40.9) |
| Race | |
| White | 1,534 (80.0) |
| American Indian or Alaskan Native | 10 (0.5) |
| Black or African American | 322 (16.8) |
| Asian | 32 (1.7) |
| Native Hawaiian or Other Pacific Islander | 2 (0.1) |
| Any Combination* | 18 (0.9) |
| Ethnicity | |
| Hispanic or Latino | 368 (19.2) |
| Not Hispanic or Latino | 1,550 (80.8) |
| Education | |
| Elementary | 50 (2.6) |
| High School (or GED) | 432 (22.5) |
| Vocational/Some College | 522 (27.2) |
| College Degree | 666 (34.7) |
| Some Graduate Work | 54 (2.8) |
| Graduate Degree (Masters or Higher) | 194 (10.1) |

Note: This table summarizes all evaluable ASC-US subjects (evaluable for primary objective). To be considered evaluable, the subject must have been eligible, have had an ASC-US cytology result, and have had valid results from the IUO HR HPV Test, IUO HPV genotyping Test, and the cobas HPV Test.
* Any Combination refers to subjects who selected more than one race.

NILM Population Demographics

The demographics of the NILM population (≥30 years) are presented below. The median age was 44 years with ~35% subjects age 30-39 years and ~65% age ≥40 years. Approximately 84% of the NILM subjects were White, 13% were Black or African American, and the remaining 3% were from other races.

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Summary of Demographic Characteristics for Evaluable NILM Subjects

| Characteristics | Evaluable Subjects
n = 32,260 |
| --- | --- |
| Age (Years) | |
| Mean | 44.9 |
| Standard Deviation | 10.1 |
| Median | 44 |
| (Min, Max) | (30,93) |
| | n (%) |
| Age Group (Years) | |
| 30-39 | 11,398 (35.3) |
| ≥40 | 20,862 (64.7) |
| Race | |
| White | 27,197 (84.3) |
| American Indian or Alaskan Native | 173 (0.5) |
| Black or African American | 4,199 (13.0) |
| Asian | 466 (1.4) |
| Native Hawaiian or Other Pacific Islander | 72 (0.2) |
| Any Combination* | 150 (0.5) |
| Missing | 3 (<0.1) |
| Ethnicity | |
| Hispanic or Latino | 5,736 (17.8) |
| Not Hispanic or Latino | 26,522 (82.2) |
| Missing | 2 (<0.1) |
| Education | |
| Elementary | 648 (2.0) |
| High School (or GED) | 7,656 (23.7) |
| Vocational/Some College | 8,290 (25.7) |
| College Degree | 10,780 (33.4) |
| Some Graduate Work | 847 (2.6) |
| Graduate Degree (Masters or Higher) | 4,030 (12.5) |
| Missing | 9 (<0.1) |

Note: This table summarizes all evaluable NILM subjects. To be considered evaluable, the subject must have been eligible, must have been ≥30 years old, have had a NILM cytology result, and have had valid results from the IUO HR HPV Test, IUO HPV genotyping Test, and the cobas HPV Test.
* Any Combination refers to subjects who selected more than one race.

D. Safety and Effectiveness Results

1. Safety Results
Not applicable, this was an IDE-exempt study

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# 2. Effectiveness Results

The analysis of effectiveness was based on the following data.

## Performance Characteristics in the ASC-US Population (≥ 21 Years)

A total of 1,612 subjects with ASC-US cytology completed Study Visit 2 procedures. The results of the cobas HPV Test reported as (HR HPV) Positive or (HR HPV) Negative together with the CPR diagnosis are presented below. In a total of 1,578 ASC-US subjects with valid CPR panel diagnoses, 80 subjects had a ≥ CIN2 result (prevalence of ~5.1%), and 46 subjects had a ≥ CIN3 result (prevalence of ~2.9%).

## Results of the cobas HPV Test and Central Pathology Review Panel Diagnosis in the ASC-US Population (≥ 21 Years)

| cobas HPV Test Result | Central Pathology Review Panel Diagnosis | | | | | Total |
| --- | --- | --- | --- | --- | --- | --- |
| | Undetermined | Normal | CIN1 | CIN2 | ≥CIN3 | |
| Positive | 13 | 351 | 91 | 29 | 43 | 527 |
| Negative | 19 | 989 | 67 | 5 | 3 | 1,083 |
| Invalid | 0 | 2 | 0 | 0 | 0 | 2 |
| Total | 32 | 1,340 | 158 | 34 | 46 | 1,612 |

Note: The 32 Undetermined CPR results were due to biopsy sample(s) collected out of study visit window or biopsy sample(s) found to be inadequate for diagnosis. These were excluded from the analysis, resulting in 1578 valid biopsy results.
Percent of Invalid cobas HPV Test results was 0.12% (2/1612) with 95% CI: 0.03% to 0.45%

Performance of the cobas HPV Test in detecting high-grade cervical disease (≥ CIN2 and ≥ CIN3) is presented in the table below. The sensitivity and the specificity of the test for detecting ≥ CIN2 histology were 90.0% ((72/80) with 95% CI: 81.5% to 94.8%) and 70.5% ((1,056/1,498) with 95% CI: 68.1% to 72.7%), respectively. The positive likelihood ratio (PLR) was estimated as 3.1, which implies a positive cobas HPV Test result is 3.1 times more likely in subjects with ≥ CIN2 than in subjects with &lt; CIN2. The negative likelihood ratio (NLR) was estimated as 0.1, which implies that a negative cobas HPV Test result is 10 (1/0.1) times more likely in subjects with &lt; CIN2 than in subjects with ≥ CIN2.

The sensitivity and specificity of the cobas HPV Test for detecting ≥ CIN3 histology were 93.5% ((43/46) with 95% CI: 82.5% to 97.8%) and 69.3% ((1,061/1,532) with 95% CI: 66.9% to 71.5%), respectively.

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Performance of the cobas HPV Test in Detecting ≥ CIN2 and ≥ CIN3 in the ASC-US Population (≥ 21 Years)

| Performance | CPR Panel Diagnosis ≥ CIN2 | | CPR Panel Diagnosis ≥ CIN3 | |
| --- | --- | --- | --- | --- |
| | Point Estimate | 95% CI | Point Estimate | 95% CI |
| Sensitivity (%) | 90.0
(72/80) | (81.5, 94.8) | 93.5
(43/46) | (82.5, 97.8) |
| Specificity (%) | 70.5
(1,056/1,498) | (68.1, 72.7) | 69.3
(1,061/1,532) | (66.9, 71.5) |
| PLR | 3.1
(72/80)(442/1,498) | (2.7, 3.4) | 3.0
(43/46)/(471/1,532) | (2.7, 3.4) |
| NLR | 0.1
(8/80)/(1,056/1,498) | (0.1, 0.3) | 0.1
(3 46)/(1,061/1,532) | (0.0, 0.3) |
| PPV (%) | 14.0
(72/514) | (12.8, 15.3) | 8.4
(43/514) | (7.6, 9.2) |
| NPV (%) | 99.2
(1,056/1,064) | (98.6, 99.6) | 99.7
(1,061/1,064) | (99.2, 99.9) |
| Prevalence (%) | 5.1
(80/1,578) | (4.1, 6.3) | 2.9
(46/1,578) | (2.2, 3.9) |

Note: PPV = Positive Predictive Value; NPV = Negative Predictive Value.
PLR = Positive Likelihood Ratio; NLR = Negative Likelihood Ratio.

The performance of the cobas HPV Test in detecting high-grade cervical disease (≥ CIN2 and ≥ CIN3) and the performance of the FDA approved HPV Test are presented in the table below. The sensitivity for detecting ≥ CIN2 histology was 90.0% ((72/80) with 95% CI: 81.5% to 94.8%) for the cobas HPV Test and 87.2% ((68/78) with 95% CI: 78.0% to 92.9%) for the FDA approved HPV Test. The specificity for detecting ≥ CIN2 histology was 70.5% (1,056/1,498) with 95% CI: 68.1% to 72.7%) for the cobas HPV Test and 71.1% ((1,056/1,495) with 95% CI: 68.8% to 73.4%) for the FDA approved HPV Test.

The sensitivity for detecting ≥ CIN3 histology was 93.5% ((43/46) with 95% CI: 82.5% to 97.8%) for the cobas HPV Test and 91.3% ((942/46) with 95% CI: 79.7% to 96.6%) for the FDA approved HPV Test. The specificity for detecting ≥ CIN3 histology was 69.3% ((1,053/1,517) with 95% CI: 66.9% to 71.5%) for the cobas HPV Test and 70.0% ((1,062/1,517) with 95% CI: 67.7% to 72.3%) for the FDA approved HPV Test.

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Comparison of the Performance of the cobas HPV Test and an FDA approved HPV test in Detecting ≥ CIN2 and ≥ CIN3 in the ASC-US Population

| | cobas HPV Test | | FDA approved HPV Test | |
| --- | --- | --- | --- | --- |
| | Point Estimate | 95% CI | Point Estimate | 95% CI |
| ≥ CIN2 | | | | |
| Sensitivity (%) | 90.0 (72/80) | (81.5, 94.8) | 87.2 (68/78)¹ | (78.0, 92.9) |
| Specificity (%) | 70.5 (1,056/1,498) | (68.1, 72.7) | 71.1 (1,056/1,485)² | (68.8, 73.4) |
| PPV (%) | 14.0 (72/514) | (12.8, 15.3) | 13.7 (68/497) | (12.4, 15.1) |
| NPV (%) | 99.2 (1,056/1,064) | (98.6, 99.6) | 99.1 (1,056/1,066) | (98.3, 99.5) |
| Prevalence (%) | 5.1 (80/1578) | (4.1, 6.3) | 5.0 (78/1563) | (4.0, 6.2) |
| ≥ CIN3 | | | | |
| Sensitivity (%) | 93.5 (43/46) | (82.5, 97.8) | 91.3 (42/46) | (79.7, 96.6) |
| Specificity (%) | 69.3 (1,053/1,517) | (66.9, 71.5) | 70.0 (1,062/1,517) | (67.7, 72.3) |
| PPV (%) | 8.4 (43/514) | (7.6, 9.2) | 8.5 (42/497) | (7.6, 9.4) |
| NPV (%) | 99.7 (1,061/1,064) | (99.2, 99.9) | 99.6 (1,062/1,066) | (99.0, 99.9) |
| Prevalence (%) | 2.9 (43/1578) | (2.2, 3.9) | 3.0 (46/1563) | (2.2, 3.9) |

¹ Results for two subjects with a ≥ CIN2 diagnosis could not be determined by the FDA approved HPV Test due to insufficient volume resulting from repeated testing
² Results for thirteen subjects with a &lt; CIN2 diagnosis could not be determined by the FDA approved HPV Test due to insufficient volume resulting from repeated testing.

Performance of the cobas HPV Test in detecting ≥ CIN2 and ≥ CIN3 evaluated by age group is presented in the table below. The sensitivity of the cobas HPV Test for detecting ≥ CIN2 histology was 93.3% ((42/45) with 95% CI: 82.1% to 97.7%) in the 21-29 year age group, 100% ((20/20) with 95% CI: 83.9% to 100%) in the 30-39 year age group, and 66.7% ((10/15) with 95% CI: 41.7% to 84.8%) in the ≥ 40 years age group. The specificity of the test was highest in ≥ 40 years, with an estimate of 85.0% (95% CI: 82.0% to 87.6%).

The sensitivity in detecting ≥ CIN3 was 100% ((24/24) with 95% CI: 74.1% to 100%) in the 21-29 year age group, 100% ((11/11) with 95% CI: 86.2% to 100%) in the 30-39 year age group, and 72.7% ((8/11) with 95% CI: 43.4% to 90.3%) in the ≥ 40 years age group. The specificity of the test was highest in ≥ 40 years, with an estimate of 84.8% ((535/631) with 95% CI: 81.8% to 87.4%).

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Performance of the cobas HPV Test in Detecting ≥ CIN2 and ≥ CIN3 in the ASC-US Population by Age Group

| Performance | 21-29 Years | 30-39 Years | ≥ 40 Years |
| --- | --- | --- | --- |
| N | 514 | 422 | 642 |
| ≥ CIN2 | | | |
| Sensitivity (%) | 93.3 (42/45) | 100.0 (20/20) | 66.7 (10/15) |
| 95% CI (%) | (82.1, 97.7) | (83.9, 100.0) | (41.7, 84.8) |
| Specificity (%) | 49.7 (233/469) | 72.1 (290/402) | 85.0 (533/627) |
| 95% CI (%) | (45.2, 54.2) | (67.6, 76.3) | (82.0, 87.6) |
| PPV (%) | 15.1 (42/278) | 15.2 (20/132) | 9.6 (10/104) |
| 95% CI (%) | (13.6, 16.7) | (13.1, 17.5) | (6.6, 13.7) |
| NPV (%) | 98.7 (233/236) | 100.0 (290/290) | 99.1 (533/538) |
| 95% CI (%) | (96.3, 99.6) | (97.4, 100.0) | (98.1, 99.5) |
| ≥ CIN2 prevalence | 8.8% (45/514) | 4.7% (20/422) | 2.3% (15/642) |
| 95% CI (%) | (6.6, 11.5) | (3.1, 7.2) | (1.4, 3.8) |
| ≥ CIN3 | | | |
| Sensitivity (%) | 100.0 (24/24) | 100.0 (11/11) | 72.7 (8/11) |
| 95% CI (%) | (86.2, 100.0) | (74.1, 100.0) | (43.4, 90.3) |
| Specificity (%) | 48.2 (236/490) | 70.6 (290/411) | 84.8 (535/ 631) |
| 95% CI (%) | (43.8, 52.6) | (66.0, 74.8) | (81.8, 87.4) |
| PPV (%) | 8.6 (24/278) | 8.3 (11/132) | 7.7 (8/104) |
| 95% CI (%) | (7.9, 9.5) | (7.0, 9.9) | (5.3, 11.1) |
| NPV (%) | 100.0 (236/236) | 100.0 (290/290) | 99.4 (535/538) |
| 95% CI (%) | (96.8, 100.0) | (97.5, 100.0) | (98.5, 99.8) |
| ≥ CIN3 prevalence | 4.7% (24/514) | 2.6% (11/422) | 1.7% (11/642) |

Performance of the FDA approved HPV test in detecting ≥ CIN2 and ≥ CIN3 by age group is presented in the table below.

Performance of an FDA approved HPV test in Detecting ≥ CIN2 and ≥ CIN3 in the ASC-US Population by Age Group

| Performance | 21-29 Years | 30-39 Years | ≥ 40 Years |
| --- | --- | --- | --- |
| N | 506 | 417 | 640 |
| ≥ CIN2 | | | |
| Sensitivity (%) | 88.4 (38 / 43) | 100.0 (20 / 20) | 66.7 (10 / 15) |
| 95% CI (%) | (75.5, 94.9) | (83.9, 100.0) | (41.7, 84.8) |
| Specificity (%) | 50.1 (232 / 463) | 73.6 (292 / 397) | 85.1 (532 / 625) |
| 95% CI (%) | (45.6, 54.6) | (69.0, 77.6) | (82.1, 87.7) |
| PPV (%) | 14.1 (38 / 269) | 16.0 (20 / 125) | 9.7 (10 / 103) |
| 95% CI (%) | (12.5, 15.9) | (13.8, 18.5) | (6.7, 13.9) |
| NPV (%) | 97.9 (232 / 237) | 100.0 (292 / 292) | 99.1 (532 / 537) |
| 95% CI (%) | (95.3, 99.1) | (97.4, 100.0) | (98.1, 99.5) |
| ≥ CIN2 prevalence | 8.5 (43/506) | 4.8 (20/417) | 2.3 (15/640) |
| 95% CI (%) | (6.4, 11.3) | (3.1, 7.3) | (1.4, 3.8) |
| ≥ CIN3 | | | |
| Sensitivity (%) | 95.8 (23 / 24) | 100.0 (11 / 11) | 72.7 (8 / 11) |
| 95% CI (%) | (79.8, 99.3) | (74.1, 100.0) | (43.4, 90.3) |
| Specificity (%) | 49.0 (236 / 482) | 71.9 (292 / 406) | 84.9 (534 / 629) |
| 95% CI (%) | (44.5, 53.4) | (67.4, 76.1) | (81.9, 87.5) |
| PPV (%) | 8.6 (23 / 269) | 8.8 (11 / 125) | 7.8 (8 / 103) |
| 95% CI (%) | (7.7, 9.5) | (7.3, 10.5) | (5.3, 11.2) |
| NPV (%) | 99.6 (236 / 237) | 100.0 (292 / 292) | 99.4 (534 / 537) |
| 95% CI (%) | (97.2, 99.9) | (97.5, 100.0) | (98.5, 99.8) |
| ≥ CIN3 prevalence | 4.7 (24/506) | 2.6 (11/417) | 1.7 (11/640) |
| 95% CI (%) | (3.2, 7.0) | (1.5, 4.7) | (1.0, 3.1) |

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# ASC-US (≥ 21 Years) Population – Likelihood Ratios and Risk Estimates

Likelihood ratios (LRs) and the risks of disease (≥ CIN2 and ≥ CIN3) along with 95% CIs for cobas HPV Test results (HR HPV 16 positive/18 positive, 12 Other HR, and HR HPV negative are presented below for the ASC-US (≥21 years) population.

For the ≥ CIN2 histology, the estimate of the LR of HPV16 positive/18 positive was 6.1, indicating that an HPV16 positive/18 positive result is 6.1 times more likely to come from a subject with disease (≥ CIN2) than from a subject without disease (&lt; CIN2). The risk of a ≥ CIN2 outcome for an ASC-US subject with an HPV16 positive/18 positive result was 24.4%. The LRs of 12 Other HR HPV positive was 1.8. Both LRs were significantly greater than 1.

The estimate of the LR of a negative cobas HPV Test result was 0.1, indicating that a negative result was 10 times more likely to come from a subject without disease (&lt; CIN2) than from a subject with disease (≥ CIN2).

The risk of disease (≥ CIN2) is the chance/probability of having the disease given an HPV test outcome. The risk of disease (≥ CIN2) was 5.1% in the ASC-US population regardless of the HPV test result (prevalence =5.1%). The risk of disease was significantly increased for the test results of HPV16 positive/18 positive and 12 Other HR HPV positive and significantly decreased for an HR HPV negative result.

For ≥ CIN3 histology, both LRs of HPV16 positive/18 positive and 12 Other HR HPV positive were statistically significantly greater than 1, and the LR of an HPV negative result was statistically significantly less than 1. The risk of the disease (≥ CIN3) was 2.9% in the ASC-US population. The risk of ≥ CIN3 was significantly increased for the HPV16 positive/18 positive and 12 Other HR HPV positive, and significantly decreased for an HPV negative result.

## Likelihood Ratios and Risk of Disease by cobas HPV Test Result in Detecting ≥CIN2 and ≥CIN3 in the ASC-US Population

| Diagnosis by CPR | cobas HPV Test Result | Likelihood Ratio (95% CI) | Risk of Disease (%)
Given the Test Result (95% CI) |
| --- | --- | --- | --- |
| ≥CIN2 | HPV 16 positive/18 positive | 6.1 (4.7, 7.9) | 24.4 (20.1, 29.7) |
| | 12 Other HR HPV positive | 1.8 (1.3, 2.4) | 8.6 (6.6, 11.6) |
| | HPV Negative | 0.1 (0.1, 0.2) | 0.8 (0.3, 1.0) |
| | Prevalence | 5.1% | |
| ≥CIN3 | HPV 16 positive/18 positive | 6.3 (4.8, 8.3) | 15.9 (12.5, 20.0) |
| | 12 Other HR HPV positive | 1.5 (1.0, 2.3) | 4.4 (2.9, 6.5) |
| | HPV Negative | 0.1 (0.0, 0.3) | 0.3 (0.1, 0.9) |
| | Prevalence | 2.9% | |

# ASC-US (≥ 21 Years) Population – Absolute and Relative Risk Estimates

The table below presents the CPR diagnosis by all possible cobas HPV Test result in ASCUS population.

## Summary of cobas HPV Test Result and Central Pathology Review Panel Diagnosis in the ASC-US Population (&gt;=21 years)

| | Central Pathology Review Diagnosis | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| cobas HPV Test Result | Undetermined | Negative | CIN1 | CIN2 | >=CIN3 | Total |
| Other HR HPV NEG, HPV16 NEG, HPV18 NEG | 19 | 989 | 67 | 5 | 3 | 1,083 |

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| | Central Pathology Review Diagnosis | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| cobas HPV Test Result | Undetermined | Negative | CIN1 | CIN2 | >=CIN3 | Total |
| Other HR HPV NEG, HPV16 NEG, HPV18 POS | 1 | 21 | 3 | 0 | 1 | 26 |
| Other HR HPV NEG, HPV16 POS, HPV18 NEG | 0 | 40 | 8 | 13 | 12 | 73 |
| Other HR HPV NEG, HPV16 POS, HPV18 POS | 0 | 5 | 0 | 0 | 1 | 6 |
| Other HR HPV POS, HPV16 NEG, HPV18 NEG | 9 | 246 | 63 | 14 | 15 | 347 |
| Other HR HPV POS, HPV16 NEG, HPV18 POS | 2 | 12 | 8 | 0 | 1 | 23 |
| Other HR HPV POS, HPV16 POS, HPV18 NEG | 1 | 25 | 9 | 2 | 12 | 49 |
| Other HR HPV POS, HPV16 POS, HPV18 POS | 0 | 2 | 0 | 0 | 1 | 3 |
| Invalid | 0 | 2 | 0 | 0 | 0 | 0 |
| Overall | 32 | 1,342 | 158 | 34 | 46 | 1,612 |

Note1: Undetermined results include inadequate biopsy sample for diagnosis and sample collected outside the Study Visit window.
Note2: None of the subjects in the ASC-US population had a CPR diagnosis &gt;CIN3

The table below presents the CPR diagnosis and the absolute risk of disease (≥ CIN2 and ≥ CIN3) by cobas HPV Test result. HPV16 positive/18 positive had the highest absolute risk for both ≥ CIN2 and ≥ CIN3. In general, the absolute risks for both ≥ CIN2 and ≥ CIN3 were higher in subjects with results of HPV positive, HPV16 positive/18 positive, or 12 Other HR positive than in subjects with an HPV negative result.

Central Pathology Review Diagnosis and Absolute Risk of ≥ CIN2 and ≥ CIN3 for Different cobas HPV Test Results in the A…