BD ONCLARITY HPV ASSAY

P160037 · Becton, Dickinson and Company · MAQ · Feb 12, 2018 · Microbiology

Device Facts

• Record ID: P160037
• Device Name: BD ONCLARITY HPV ASSAY
• Applicant: Becton, Dickinson and Company
• Product Code: MAQ · Microbiology
• Decision Date: Feb 12, 2018
• Decision: APPR
• Device Class: Class 3

Intended Use

The BD Onclarity HPV Assay is a qualitative in vitro test for the detection of Human Papillomavirus in cervical specimens collected by a clinician using an endocervical brush/spatula combination or broom and placed in a BD SurePath vial. The test utilizes amplification of target DNA by 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 16, 18 and 45 while concurrently detecting the other HR HPV types that include 31, 33, 35, 39, 51, 52, 56, 58, 59, 66 and 68. The BD Onclarity HPV Assay is indicated: (a) In women 21 years and older with ASC-US (atypical squamous cells of undetermined significance) cervical cytology test results, the BD Onclarity HPV Assay can be used to determine the need for referral to colposcopy. (b) In women 21 years and older with ASC-US cervical cytology test results, the BD Onclarity HPV assay can be used to detect high-risk HPV genotypes 16, 18 and 45. This information together with physician's assessment of screening 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 BD Onclarity HPV Assay can be used together with cervical cytology to adjunctively screen to detect high risk HPV types. This information, together with the physician's assessment of screening history, other factors, and professional guidelines, may be used to guide patient management. (d) In women 30 years and older, the BD Onclarity HPV Assay can be used to detect high-risk HPV genotypes 16, 18 and 45. This information, together with the physician's assessment of screening history, other factors, and professional guidelines, may be used to guide patient management. (e) In women 25 years and older, the BD Onclarity HPV Assay can be used as a first-line primary cervical cancer screening test to detect high risk HPV, including 16 and 18. Women who test negative for the high risk HPV types by the BD Onclarity HPV Assay should be followed up in accordance with the physician's assessment of screening and medical history, other risk factors, and professional guidelines. Women who test positive for HPV genotypes 16 and/or 18 by the BD Onclarity HPV Assay should be referred to colposcopy. Women who test high risk HPV positive and 16 and 18 negative by the BD Onclarity HPV Assay (12 other HR HPV Positive) should be evaluated by cervical cytology to determine the need for referral to colposcopy.

Device Story

Device detects 14 high-risk HPV DNA types in clinician-collected cervical specimens (SurePath vial). Process: automated specimen homogenization/lysis (BD Pre-Warm Heater/Viper LT System); PCR amplification of E6/E7 oncogene regions; real-time detection via TaqMan probes. Output: qualitative HPV status (positive/negative) and specific genotype identification (16, 18, 45) or groups (31, 51, 52, 33/58, 59/56/66, 35/39/68). Used in clinical laboratories; results interpreted by software. Clinicians use output to guide colposcopy referral or management decisions, potentially enabling earlier detection of cervical cancer precursors.

Clinical Evidence

Prospective multicenter study (n=33,858). Evaluated performance for ASC-US triage (≥21y), adjunctive screening (≥30y), and primary screening (≥25y) against CPR panel histology (≥CIN2/≥CIN3 endpoints). Sensitivity for ≥CIN2 in ASC-US triage was 85.7% (95% CI 77.8-91.1). Primary screening algorithm demonstrated superior sensitivity and specificity compared to cytology-alone and current practice algorithms.

Technological Characteristics

Real-time PCR assay; targets E6/E7 oncogenes. Uses TaqMan probes with fluorescent dyes and Black Hole Quenchers. Automated processing via BD Viper LT System. Internal control: human beta globin gene. Reagents: dried in PCR tubes. Connectivity: automated software-based result interpretation.

Indications for Use

Indicated for women ≥21 years with ASC-US cytology for colposcopy triage; women ≥30 years as adjunctive screening with cytology; and women ≥25 years as first-line primary cervical cancer screening. No known contraindications.

Submission Summary (Full Text)

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SUMMARY OF SAFETY AND EFFECTIVENESS DATA (SSED)

I. GENERAL INFORMATION

Device Generic Name: Human Papillomavirus (HPV) DNA detection kit

Device Trade Name: BD Onclarity HPV Assay

Device Procode: MAQ

Applicant's Name and Address: Becton Dickinson and Company
7 Loveton Circle
Sparks, MD 21152

Date(s) of Panel Recommendation: Not applicable

Premarket Approval Application (PMA) Number: P160037

Date of FDA Notice of Approval: February 12, 2018

Priority Review: Not applicable

II. INDICATIONS FOR USE

The BD Onclarity HPV Assay is a qualitative *in vitro* test for the detection of Human Papillomavirus in cervical specimens collected by a clinician using an endocervical brush/spatula combination or broom and placed in a BD SurePath vial. The test utilizes amplification of target DNA by 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 16, 18 and 45 while concurrently detecting the other HR HPV types that include 31, 33, 35, 39, 51, 52, 56, 58, 59, 66 and 68.

The BD Onclarity HPV Assay is indicated:

(a) In women 21 years and older with ASC-US (atypical squamous cells of undetermined significance) cervical cytology test results, the BD Onclarity HPV Assay can be used to determine the need for referral to colposcopy.

(b) In women 21 years and older with ASC-US cervical cytology test results, the BD Onclarity HPV assay can be used to detect high-risk HPV genotypes 16, 18 and 45. This information together with physician's assessment of screening 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.

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(c) In women 30 years and older, the BD Onclarity HPV Assay can be used together with cervical cytology to adjunctively screen to detect high risk HPV types. This information, together with the physician's assessment of screening history, other factors, and professional guidelines, may be used to guide patient management.

(d) In women 30 years and older, the BD Onclarity HPV Assay can be used to detect high-risk HPV genotypes 16, 18 and 45. This information, together with the physician's assessment of screening history, other factors, and professional guidelines, may be used to guide patient management.

(e) In women 25 years and older, the BD Onclarity HPV Assay can be used as a first-line primary cervical cancer screening test to detect high risk HPV, including 16 and 18. Women who test negative for the high risk HPV types by the BD Onclarity HPV Assay should be followed up in accordance with the physician's assessment of screening and medical history, other risk factors, and professional guidelines. Women who test positive for HPV genotypes 16 and/or 18 by the BD Onclarity HPV Assay should be referred to colposcopy. Women who test high risk HPV positive and 16 and 18 negative by the BD Onclarity HPV Assay (12 other HR HPV Positive) should be evaluated by cervical cytology to determine the need for referral to colposcopy.

## III. CONTRAINDICATIONS

There are no known contraindications.

## IV. WARNINGS AND PRECAUTIONS

The warnings and precautions can be found in the BD Onclarity HPV Assay labeling.

## V. DEVICE DESCRIPTION

The BD Onclarity HPV Assay is based on two major processing steps: 1) automated specimen preparation to homogenize the matrix, lyse cells, and release cellular DNA; and 2) PCR amplification of target DNA sequences using primers and fluorescently-labeled detector probes for both HPV and human beta globin. The human beta globin gene serves as an internal control for the entire test by concurrently assessing specimen processing, extraction, and amplification. The primers and probes of the BD Onclarity HPV Assay target regions in the E6/E7 oncogenes.

The automated specimen preparation for the BD Onclarity HPV Assay is completed by the BD Pre-Warm Heater and the BD Viper LT System. Cervical specimens collected in BD SurePath collection vials are extracted using BD FOX Extraction to release cellular DNA. The purified cellular DNA solution from each extracted specimen is transferred into PCR tubes containing reagents which are then sealed to prevent contamination.

The BD Onclarity HPV Assay reagents are dried in three individual PCR tubes. Each of the three PCR tubes contains specific oligonucleotide sets to detect HPV genotype DNA and an oligonucleotide set to detect a region of the human beta globin gene. These HR HPV genotypes

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are reported either individually (16, 18, 45) or as a genotype group (31, 51, 52, 33/58, 59/56/66, and 35/39/68).

The BD Onclarity HPV Assay uses real-time PCR technology. The detection of the target DNA is accomplished using TaqMan DNA probes, which include a fluorescent dye at the $5^{\prime}$ end and a quenching molecule at the $3^{\prime}$ end of the oligonucleotide. The BD Onclarity HPV Assay utilizes fifteen probes labeled with one of four fluorescent dyes. Each dye is paired with one of two Black Hole Quencher molecules (BHQ Dye). Fluorescent detection of amplification occurs in four separate optical channels on the BD Viper LT System.

# Interpretation of Results:

All calculations are performed automatically by the BD Viper LT software. The presence or absence of clinically relevant HPV DNA is determined by the PCR cycle (Ct) at which the signal crosses a pre-established threshold. The assay will extract, amplify, and detect a fragment of the human beta globin gene as an internal control to assess specimen processing, extraction, amplification, and to indicate the presence of PCR inhibitors. If the HPV-specific signal is greater than a cycle threshold, the internal control is utilized by the algorithm in the interpretation of the result. If the HPV-specific signal is less than or equal to a cycle threshold, the internal control is ignored by the algorithm.

Interpretation of High Risk HPV Genotype HPV Test Results for the BD Onclarity HPV Assay:

| High Risk HPV Result | Interpretation | Result | Report |
| --- | --- | --- | --- |
| HR↑ | Positive for High Risk HPV types | HPV HR Positive | HPV DNA detected by PCR. |
| HR↓ | Negative for High Risk HPV types | HPV HR Negative | HPV DNA not detected by PCR. |
| × | HPV DNA, if present, is not detectable | Internal Control Failure | Internal Control Failure. Repeat test from initial specimen tube or obtain another specimen for testing. |
| × | HPV DNA, if present, is not detectable | Extraction Transfer Failure | Extraction Transfer Failure. Repeat test from initial specimen tube or obtain another specimen for testing. |
| × | HPV DNA, if present, is not detectable. | Liquid Level Failure | Liquid Level Failure. Repeat test from initial specimen tube or obtain another specimen for testing. |
| + | HPV DNA, if present, is not detectable. | Error | Error. Repeat test from initial specimen tube or obtain another specimen for testing. |

Interpretation of Specific HPV Genotype Test Results for the BD Onclarity HPV Assay:

| HPV Genotype Result | Interpretation | Result |
| --- | --- | --- |
| 16↑ | Positive for HPV type 16 | HPV type 16 Positive |
| 16↓ | Negative for HPV type 16 | HPV type 16 Negative |
| 18↑ | Positive for HPV type 18 | HPV type 18 Positive |
| 18↓ | Negative for HPV type 18 | HPV type 18 Negative |
| 45↑ | Positive for HPV type 45 | HPV type 45 Positive |
| 45↓ | Negative for HPV type 45 | HPV type 45 Negative |

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| HPV Genotype Result | Interpretation | Result |
| --- | --- | --- |
| GT↑↑ | Positive HPV Genotype result(s) for HPV 31, 33, 35, 39, 51, 52, 56, 58, 59, 66, and/or 68 | Positive HPV Genotype result(s) other than HPV 16, 18, or 45 |
| GT✧ | Negative HPV Genotype result(s) for HPV 31, 33, 35, 39, 51, 52, 56, 58, 59, 66, and/or 68 | Negative HPV Genotype result(s) masked |
| ■ | HPV genotype result is available for unmasking | Genotype result is masked |
| - - | HPV genotype result is not available for unmasking | HPV Negative result, Internal Control failure, Liquid Level failure or Extraction Transfer failure |

VI. ALTERNATIVE PRACTICES AND PROCEDURES

There are several alternatives for the detection of cervical cancer precursors, including testing by cytology alone, co-testing with HPV alongside or as a follow-up to cytology, or HPV testing as a first line screening test for cervical cancer. Each alternative has its own advantages and disadvantages. A woman should fully discuss these alternatives with her physician to select the method that best meets expectations and lifestyle.

The woman's age, medical history and thorough physical examination will provide further information on the risk of cervical disease, as well as the need for referral to colposcopy. The BD Onclarity HPV Assay should only be used in conjunction with this clinical information in accordance with appropriate clinical patient management guidelines.

VII. MARKETING HISTORY

The BD Onclarity HPV Assay is marketed as an in vitro diagnostic assay in the following countries: Australia, New Zealand, India, Korea, Turkey, Saudi Arabia, Brazil, Mexico, Guatemala, and countries within the European Union, including Austria, Belgium, Luxemburg, Denmark, France, Germany, Italy, Poland, Romania, and Spain. It has not been withdrawn from these markets for any reason related to the device's safety or effectiveness.

VIII. PROBABLE ADVERSE EFFECTS OF THE DEVICE ON HEALTH

The following section outlines the potential adverse effects (e.g., complications) associated with the use of the BD Onclarity HPV Assay. 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. False negative results may lead to delays in the timely diagnosis of cervical cancer, allowing an undetected condition to worsen and potentially increasing morbidity and mortality. False positive

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results could lead many women to unnecessarily undergo more frequent screening and potentially invasive procedures such as colposcopy and biopsy.

## IX. SUMMARY OF NONCLINICAL STUDIES

## A. Laboratory Studies

### 1. Clinical Cutoff Determination

The preliminary clinical cutoff determination for the BD Onclarity HPV Assay was established based on a series of studies with remnant clinical specimens from a screening population of patients with normal cytology. Further evaluation of the preliminary cutoff was evaluated with clinical specimens and in independent, published studies designed to compare performance of multiple HPV assays. ROC curves were generated to determine a cutoff allowing for maximal sensitivity and specificity. The methods used for cutoff selection were prioritized to achieve the maximum sensitivity for detecting ≥CIN2 disease while maintaining a clinically acceptable level of specificity. Based on these studies, the clinical cutoff was set at a Ct of 38.3 for HPV16 and 34.2 for all other genotypes.

### 2. C95 Determination at the Clinical Cutoff

The C95 determination at the clinical cutoff was determined for the BD Onclarity HPV Assay using the following HPV positive cell lines: SiHa (HPV 16), HeLa (HPV 18) and MS751 (HPV 45). The C95 is the level of HPV DNA in the specimen that yields a positive result at least 95% of the time using the clinical cutoff. In addition, cloned plasmid DNA containing the sequences for the following HPV genotypes was also used: HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. These cells and plasmids were diluted in BD SurePath media containing an HPV-negative cell line (C33A) for the preparation of at least five dilution levels. Plasmids and cell lines were diluted to concentrations below, above and at the expected C95 levels. A matrix equivalency study was performed between this contrived background and negative clinical matrix to demonstrate equivalent C95s for HPV types 16, 18 and 45 using either background. A minimum of forty-five replicates were tested for each of the HPV positive cell lines and each of the HPV16, HPV18, and HPV45 plasmids. Twenty replicates were tested for each of the remaining 11 other high risk HPV plasmids. Three lots of reagents were utilized across three BD Viper LT Systems. Probit analysis was performed for the determination of C95 for each genotype and each lot. The C95 for each sample was the maximum C95 value which was produced among the three reagent lots. Table 1 lists the C95s determined for the HPV plasmids and cell lines tested.

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Table 1: C95 Determination at Clinical Cutoff

| Target | C95(Cells or Copies/mL) | 95% Confidence Interval | |
| --- | --- | --- | --- |
| | | Lower | Upper |
| SiHa cells (HPV 16) | 50 (cells/mL) | 37 | 67 |
| HeLa cells (HPV 18) | 199 (cells/mL) | 154 | 256 |
| MS751 cells (HPV 45) | 862 (cells/mL) | 669 | 1111 |
| HPV 16 | 251 | 193 | 326 |
| HPV 18 | 1083 | 1000 | 1267 |
| HPV 45 | 1261 | 1154 | 1358 |
| HPV 31 | 830 | 718 | 879 |
| HPV 33 | 1665 | 1495 | 2030 |
| HPV 35 | 1550 | 1472 | 1655 |
| HPV 39 | 1794 | 1617 | 1862 |
| HPV 51 | 1522 | 1315 | 1613 |
| HPV 52 | 814 | 776 | 951 |
| HPV 56 | 1090 | 937 | 1185 |
| HPV 58 | 2369 | 2231 | 6631 |
| HPV 59 | 1000 | 942 | 1152 |
| HPV 66 | 862 | 823 | 916 |
| HPV 68 | 2392 | 2227 | 2646 |

To verify that the BD Onclarity HPV Assay is capable of accurately detecting all target HPV high risk genotypes, the C95 at the HPV clinical cutoff was confirmed using SiHa (HPV 16), HeLa (HPV 18) and MS751 (HPV 45) cell lines as well as cloned plasmid DNA containing the sequences for the following HPV genotypes: HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 in BD SurePath media containing an HPV-negative cell line (C33A). Twenty replicates of each cell line/plasmid diluted to the C95 concentrations were tested with one reagent lot on one BD Viper LT System. Table 2 lists the confirmed C95 for each cell line and plasmid.

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Table 2: C95 Confirmation

| Target | C95 (Cells or Copies/mL) | Ratio POS to Total | Positivity Rate (%) |
| --- | --- | --- | --- |
| SiHa cells (HPV 16) | 50 (cells/mL) | 20/20 | 100 |
| HeLa cells (HPV 18) | 208 (cells/mL) | 19/20 | 95 |
| MS751 cells (HPV 45) | 862 (cells/mL) | 20/20 | 100 |
| 16 | 251 | 19/20 | 95 |
| 18 | 1083 | 19/20 | 95 |
| 45 | 1261 | 19/20 | 95 |
| 31 | 830 | 20/20 | 100 |
| 33 | 1665 | 20/20 | 100 |
| 35 | 1550 | 20/20 | 100 |
| 39 | 1794 | 20/20 | 100 |
| 51 | 1522 | 20/20 | 100 |
| 52 | 814 | 20/20 | 100 |
| 56 | 1131 | 19/20 | 95 |
| 58 | 2294 | 19/20 | 95 |
| 59 | 1000 | 20/20 | 100 |
| 66 | 862 | 20/20 | 100 |
| 68 | 2367 | 20/20 | 100 |

# 3. Analytical Specificity

A panel of bacteria, yeast, and cultured viruses, including those found in female urogenital specimens, as well as cloned plasmid DNA containing high-risk and low-risk HPV target sequences, was used to evaluate the analytical specificity of the BD Onclarity HPV Assay on the BD Viper LT System. Each potential cross-reactant was tested in BD SurePath media containing an HPV-negative cell line (C33A). The microorganisms are described in Table 3. Bacteria and yeast were tested at $\geq 1\mathrm{x}10^{6}$ CFU/mL, HPV plasmid DNA was tested at $\geq 1\mathrm{x}10^{6}$ copies/mL and non-HPV viruses were tested at $\geq 1\mathrm{x}10^{6}$ vp/mL. The BD Onclarity HPV Assay did not exhibit cross-reactivity with any of the microorganisms tested or any non-targetted HPV types.

Table 3: Panel of Potential Cross Reactants for Analytical Specificity Study

| Actinomyces israelii | Proteus vulgaris | HPV 56 |
| --- | --- | --- |
| Atopobium vaginae | Providencia stuartii | HPV 58 |
| Bacteroides fragilis | Pseudomonas aeruginosa | HPV 59 |
| Bacteroides ureolyticus | Staphylococcus aureus | HPV 66 |
| Bifidobacterium adolescentis | Staphylococcus epidermidis | HPV 68 |
| Bifidobacterium breve | Streptococcus agalactiae | HPV 16 |
| Bifidobacterium longum ssp. | Streptococcus pyogenes | HPV 6 |
| Chlamydia trachomatis | Ureaplasma urealyticum | HPV 11 |
| Clostridium perfringens | Candida albicans | HPV 26 |
| Corynebacterium genitalium | Trichomonas vaginalis | HPV 30 |
| Enterobacter cloacae ssp. | Adenovirus, type 5 | HPV 34 |
| Enterococcus faecalis | HCMV, AD169 Strain | HPV 53 |
| Enterococcus faecium | HSV1 | HPV 67 |
| Escherichia coli | HSV2 | HPV 69 |
| Fusobacterium nucleatum ssp. | EBV-1, B95-8 Strain | HPV 70 |

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| Gardnerella vaginalis | HIV-1 | HPV 73 |
| --- | --- | --- |
| Klebsiella pneumoniae ssp. | HPV 16 | HPV 82 |
| Lactobacillus acidophilus | HPV 18 | HPV 97 |
| Mycobacterium smegmatis | HPV 31 | |
| Mycoplasma genitalium | HPV 33 | |
| Neisseria gonorrhoeae | HPV 35 | |
| Peptostreptococcus anaerobius | HPV 39 | |
| Prevotella bivia | HPV 45 | |
| Prevotella disiens | HPV 51 | |
| Proteus mirabilis | HPV 52 | |

## 4. Interfering Substances

### Endogenous and Exogenous

Contrived HPV negative and positive specimens were tested in the presence or absence of each potential interferent that may be present in clinical cervical specimens. The concentrations of exogenous and endogenous substances tested in this study represent concentrations that could potentially occur during specimen collection. All contrived specimens were prepared from a BD SurePath matrix containing a cellular background of C33A cells. HPV negative contrived specimens contained only the C33A cellular background. HPV positive specimens for this study contained cell lines positive for HPV 16 (SiHa), HPV 18 (HeLa) or HPV 45 (MS751) at 3 x C95. Interfering substances reported in % w/v were weighted and dissolved in the appropriate amount of HPV positive or negative contrived matrix. Vortex and/or incubation in a warm bath were applied as necessary. The potential interfering substances were added to HPV positive or negative contrived matrix and the mixtures were vortexed as necessary. For final sample preparation, a 0.5 mL aliquot from each sample was added to an HPV LBC Diluent tube. Twelve replicates were tested for each interferent.

The tested substances are described in Table 4. The concentrations represent the highest level of substance that did not show any interference in the BD Onclarity HPV Assay:

Table 4: Interference Testing Results with Endogenous and Exogenous Substances

| Potential Interfering Substance | Concentration Tested | Interference Observed |
| --- | --- | --- |
| KY Vaginal Lubricant | 6% (w/v) | None |
| VCF Vaginal Contraceptive Film | 10% (w/v) | None |
| VCF Vaginal Contraceptive Foam | 10% (w/v) | None |
| Conceptrol Contraceptive Gel | 10% (w/v) | None |
| Monistat 3 | 2% (w/v) | None |
| Clotrimazole 7 | 10% (w/v) | None |
| Vagistat-1 Tioconazole | 2% (w/v) | None |
| Clindamycin Vaginal Cream | 8% (w/v) | None |
| Summer's Eve Douche | 10% (v/v) | None |
| Zovirax (Acyclovir) Cream | 7% (w/v) | None |

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| Vandazole Gel (Metronidazole) Vaginal Gel, 0.75%) | 10% (w/v) | None |
| --- | --- | --- |
| Summer's Eve Deodorant | 3% (w/v) | None |
| Bovine Mucin | 8% (v/v) | None |
| Progesterone | 20 ng/mL | None |
| Estradiol | 1.2 ng/mL | None |
| Whole Blood | 4% (v/v) | None |
| Leukocytes | 1x10^{6} cells/mL | None |
| Semen | 10% (v/v) | None |
| Replens | 10%(w/v) | None |

There were four substances that yielded false negative results when tested at concentrations higher than described in Table 4: Whole Blood, Mucin, Zovirax Cream (Acyclovir) and Clindamycin Vaginal Cream.

## Competing Targets:

A study was performed to test whether high concentrations of HR HPV DNA could interfere with the genotyping capability of HPV types 16, 18 and 45. For the competing target conditions, the appropriate plasmids representing the competing HR genotypes were spiked into positive and negative contrived matrix at a concentration of $1 \times 10^{6}$ copies/mL in the SurePath specimen. A $0.5\mathrm{mL}$ aliquot from each competing target condition was added to $1.7\mathrm{mL}$ of HPV Diluent. No interference was observed from the competing targets. As a control, a portion of the HPV positive and negative contrived matrix, devoid of any interfering substances or competing target plasmid, was assayed in the same manner as the test conditions. This matrix control was run once per matrix preparation. External negative and positive run controls were included with each run. No competitive interference among any of the targets was identified.

## 5. Reproducibility

Studies were conducted with panels consisting of contrived samples in negative clinical matrix and pooled clinical specimens. For the contrived panel members, HPV positive cell lines for genotypes 16, 18, and 45 (SiHa, HeLa and MS7541, respectively) were spiked into negative clinical matrix at three different concentrations representing a high negative (i.e., the C5 concentration), low positive (i.e., the C95 concentration), and moderate positive (i.e., $3 \times C95$). Additionally, six different clinical pools that were positive for either HPV 16, 18, 45, 31, 33/58 or 52 were also tested, as well as a sample containing pooled clinical matrix that was negative for all genotypes. These panels were tested at three clinical sites over the course of nine days for each site. Three reagent lots were utilized (each lot was tested for 3 days). Two runs were performed at each site with 3 replicates per run. Two operators per site participated in the study. The total number of replicates per panel member was 162 (54 replicates per site). Invalid results due operator error, a negative control failure, and an extraction error on the BD Viper LT system were excluded from calculation of negativity and positivity rates and from the analysis of the numeric Ct values, resulting in a total of 156-159 replicates per panel member.

All valid test results were included for calculation of negativity or positivity rates. There were no false positive results in 158 tests performed on the negative panel members. Percent positive

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results along with $95\%$ confidence intervals for the negative and positive panel members are shown in Tables 5 and 6, respectively.

Table 5: Results by Sample Type for High Negative and Negative Panel Members for Lot and Site/Instrument:

| | | | | Number Negative/Total Number of Results | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | Lot | | Site/Instrument | | |
| Sample Type | Panel Member | Ct SD | Ct %CV | ID | Percent Negative | ID | Percent Negative | 95% CI |
| SiHa cell line | HPV 16 High Negative (2 cells/mL) | 0.47 | 1.2 | 1 | 96.1 (49/51) | 1 | 92.6 (50/54) | |
| | | | | 2 | 83.3 (45/54) | 2 | 98.1 (53/54) | |
| | | | | 3 | 92.6 (50/54) | 3 | 80.4 (41/51) | |
| | | | | All | 90.6 (144/159) | All | 90.6 (144/159) | 85.0-94.2 |
| HeLa cell line | HPV 18 High Negative (23 cells/mL) | 0.95 | 2.6 | 1 | 100.0 (54/54) | 1 | 100.0 (54/54) | |
| | | | | 2 | 100.0 (51/51) | 2 | 100.0 (51/51) | |
| | | | | 3 | 100.0 (51/51) | 3 | 100.0 (51/51) | |
| | | | | All | 100.0 (156/156) | All | 100.0 (156/156) | 97.6 - 100.0 |
| MS751 cell line | HPV 45 High Negative (90 cells/mL) | 0.72 | 2.0 | 1 | 100.0 (54/54) | 1 | 100.0 (54/54) | |
| | | | | 2 | 100.0 (51/51) | 2 | 100.0 (51/51) | |
| | | | | 3 | 100.0 (51/51) | 3 | 100.0 (51/51) | |
| | | | | All | 100.0 (156/156) | All | 100.0 (156/156) | 97.6 - 100.0 |
| Pooled negative clinical sample | Negative | NA | NA | 1 | 100.0 (51/51) | 1 | 100.0 (53/53) | |
| | | | | 2 | 100.0 (53/53) | 2 | 100.0 (54/54) | |
| | | | | 3 | 100.0 (54/54) | 3 | 100.0 (51/51) | |
| | | | | All | 100.0 (158/158) | All | 100.0 (158/158) | 97.6 - 100.0 |

Table 6: Results by Sample Type for Positive Panel Member for Lot and Site/Instrument

| | Number Positive/Total Number of Results | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | Lot | | Site/Instrument | | |
| Sample Type | Panel Member | ID | Percent Positive | ID | Percent Positive | 95% CI |
| SiHa cell line | HPV 16 Low Positive (50 cells/mL) | 1 | 100.0 (51/51) | 1 | 100.0 (54/54) | |
| | | 2 | 98.1 (53/54) | 2 | 98.1 (53/54) | |
| | | 3 | 100.0 (54/54) | 3 | 100.0 (51/51) | |
| | | All | 99.4 (158/159) | All | 99.4 (158/159) | 96.5 - 99.9 |
| SiHa cell line | HPV 16 Moderate Positive (150 cells/mL) | 1 | 100.0 (50/50) | 1 | 100.0 (53/53) | |
| | | 2 | 100.0 (54/54) | 2 | 100.0 (54/54) | |
| | | 3 | 100.0 (54/54) | 3 | 100.0 (51/51) | |

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| | Number Positive/Total Number of Results | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | Lot | | Site/Instrument | | |
| Sample Type | Panel Member | ID | Percent Positive | ID | Percent Positive | 95% CI |
| | | All | 100.0 (158/158) | All | 100.0 (158/158) | 97.6 - 100.0 |
| HeLa cell line | HPV 18
Low Positive
(208 cells/mL) | 1 | 96.3 (52/54) | 1 | 100.0 (54/54) | |
| | | 2 | 100.0 (51/51) | 2 | 96.1 (49/51) | |
| | | 3 | 98.0 (50/51) | 3 | 98.0 (50/51) | |
| | | All | 98.1 (153/156) | All | 98.1 (153/156) | 94.5 - 99.3 |
| HeLa cell line | HPV 18
Moderate Positive
(623 cells/mL) | 1 | 100.0 (54/54) | 1 | 100.0 (54/54) | |
| | | 2 | 100.0 (51/51) | 2 | 100.0 (51/51) | |
| | | 3 | 100.0 (51/51) | 3 | 100.0 (51/51) | |
| | | All | 100.0 (156/156) | All | 100.0 (156/156) | 97.6 - 100.0 |
| MS751 cell line | HPV 45
Low Positive
(862 cells/mL) | 1 | 96.3 (52/54) | 1 | 94.4 (51/54) | |
| | | 2 | 96.1 (49/51) | 2 | 94.1 (48/51) | |
| | | 3 | 94.1 (48/51) | 3 | 98.0 (50/51) | |
| | | All | 95.5 (149/156) | All | 95.5 (149/156) | 91.0 - 97.8 |
| MS751 cell line | HPV 45
Moderate Positive
(2586 cells/mL) | 1 | 98.1 (53/54) | 1 | 100.0 (54/54) | |
| | | 2 | 100.0 (51/51) | 2 | 98.0 (50/51) | |
| | | 3 | 100.0 (51/51) | 3 | 100.0 (51/51) | |
| | | All | 99.4 (155/156) | All | 99.4 (155/156) | 96.5 - 99.9 |
| Pooled clinical sample | HPV 16 | 1 | 100.0 (51/51) | 1 | 98.1 (53/54) | |
| | | 2 | 100.0 (54/54) | 2 | 96.3 (52/54) | |
| | | 3 | 94.4 (51/54) | 3 | 100 (51/51) | |
| | | All | 98.1 (156/159) | All | 98.1 (156/159) | 94.6 - 99.4 |
| Pooled clinical sample | HPV 18 | 1 | 100.0 (54/54) | 1 | 100.0 (54/54) | |
| | | 2 | 100.0 (51/51) | 2 | 100.0 (51/51) | |
| | | 3 | 100.0 (51/51) | 3 | 100.0 (51/51) | |
| | | All | 100.0 (156/156) | All | 100.0 (156/156) | 97.6 - 100.0 |
| Pooled clinical sample | HPV 45 | 1 | 57.4 (31/54) | 1 | 55.6 (30/54) | |
| | | 2 | 66.7 (34/51) | 2 | 68.6 (35/51) | |
| | | 3 | 60.8 (31/51) | 3 | 60.8 (31/51) | |
| | | All | 61.5 (96/156) | All | 61.5 (96/156) | 53.7 - 68.8 |
| Pooled clinical sample | HPV 31 | 1 | 64.8 (35/54) | 1 | 74.1 (40/54) | |
| | | 2 | 74.5 (38/51) | 2 | 76.5 (39/51) | |
| | | 3 | 76.5 (39/51) | 3 | 64.7 (33/51) | |
| | | All | 71.8 (112/156) | All | 71.8 (112/156) | 64.3 - 78.3 |

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| | Number Positive/Total Number of Results | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | Lot | | Site/Instrument | | |
| Sample Type | Panel Member | ID | Percent Positive | ID | Percent Positive | 95% CI |
| Pooled clinical sample | HPV 33/58 | 1 | 96.3 (52/54) | 1 | 96.3 (52/54) | |
| | | 2 | 100.0 (51/51) | 2 | 96.1 (49/51) | |
| | | 3 | 96.1 (49/51) | 3 | 100.0 (51/51) | |
| | | All | 97.4 (152/156) | All | 97.4 (152/156) | 93.6 - 99.0 |
| Pooled clinical sample | HPV 52 | 1 | 100.0 (54/54) | 1 | 100.0 (54/54) | |
| | | 2 | 100.0 (51/51) | 2 | 100.0 (51/51) | |
| | | 3 | 100.0 (51/51) | 3 | 100.0 (51/51) | |
| | | All | 100.0 (156/156) | All | 100.0 (156/156) | 97.6 - 100.0 |

The overall mean, standard deviation and coefficients of variation for cycle threshold (Ct) for positive panel members is summarized in Table 7. Analysis of variance of the Ct score from the valid tests performed on positive panel members yielded $\%$ CV ranges of $1.7\%$ to $1.8\%$ for SiHa cells, $1.3\%$ to $1.9\%$ for HeLa cells, and $1.7\%$ to $1.8\%$ for MS751 cells. The overall $\%$ CV for pooled HPV positive clinical specimens ranged from $2.9\%$ to $5.6\%$ .

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Table 7: Overall Mean, Standard Deviation, and Coefficients of Variation (%) for Cycle Threshold

| | Stand Deviation (SD) and Percent Coefficient of Variation (%CV) | | | | | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | Within Run | | Between Run | | Between Operator | | Between Site | | Between Lot | | Between Day | | Total |
| Sample Type | Panel Member | N | Mean | SD | CV | SD | CV | SD | CV | SD | CV | SD | CV | SD | CV | SD |
| SiHa cell line | HPV 16 Low Positive (50 cells/mL) | 159 | 36.36 | 0.60 | 1.7 | 0.00 | 0.0 | 0.13 | 0.4 | 0.23 | 0.6 | 0.07 | 0.2 | 0.00 | 0.0 | 0.64 |
| | HPV 16 Moderate Positive (150 cells/mL) | 158 | 35.03 | 0.56 | 1.6 | 0.00 | 0.0 | 0.00 | 0.0 | 0.12 | 0.3 | 0.13 | 0.4 | 0.00 | 0.0 | 0.58 |
| | HPV 16 High Negative (2 cells/mL) | 35 | 38.24 | 0.36 | 0.9 | 0.25 | 0.7 | 0.00 | 0.0 | 0.13 | 0.3 | 0.00 | 0.0 | 0.11 | 0.3 | 0.47 |
| HeLa cell line | HPV 18 Low Positive (208 cells/mL) | 156 | 33.16 | 0.60 | 1.8 | 0.00 | 0.0 | 0.13 | 0.4 | 0.11 | 0.3 | 0.01 | 0.0 | 0.00 | 0.0 | 0.61 |
| | HPV 18 Moderate Positive (623 cells/mL) | 156 | 31.71 | 0.34 | 1.1 | 0.15 | 0.5 | 0.17 | 0.5 | 0.13 | 0.4 | 0.07 | 0.2 | 0.00 | 0.0 | 0.42 |
| | HPV 18 High Negative (23 cells/mL) | 153 | 36.40 | 0.92 | 2.5 | 0.00 | 0.0 | 0.00 | 0.0 | 0.26 | 0.7 | 0.03 | 0.1 | 0.00 | 0.0 | 0.95 |
| MS751 cell line | HPV 45 Low Positive (862 cells/mL) | 156 | 32.90 | 0.54 | 1.6 | 0.08 | 0.2 | 0.00 | 0.0 | 0.26 | 0.8 | 0.12 | 0.4 | 0.00 | 0.0 | 0.60 |
| | HPV 45 Moderate Positive (2586 cells/mL) | 156 | 31.41 | 0.51 | 1.6 | 0.00 | 0.0 | 0.21 | 0.7 | 0.05 | 0.1 | 0.00 | 0.0 | 0.00 | 0.0 | 0.55 |
| | HPV 45 High Negative (90 cells/mL) | 155 | 36.47 | 0.64 | 1.7 | 0.00 | 0.0 | 0.25 | 0.7 | 0.29 | 0.8 | 0.04 | 0.1 | 0.00 | 0.0 | 0.72 |
| Clinical Specimen Pools | HPV 16 | 159 | 35.22 | 1.52 | 4.3 | 0.00 | 0.0 | 0.00 | 0.0 | 0.00 | 0.0 | 0.29 | 0.8 | 0.32 | 0.9 | 1.57 |
| | HPV 18 | 156 | 30.47 | 1.08 | 3.5 | 0.00 | 0.0 | 0.08 | 0.3 | 0.30 | 1.0 | 0.00 | 0.0 | 0.00 | 0.0 | 1.11 |
| | HPV 45 | 156 | 33.35 | 1.78 | 5.3 | 0.34 | 1.0 | 0.00 | 0.0 | 0.25 | 0.8 | 0.00 | 0.0 | 0.00 | 0.0 | 1.83 |
| | HPV 31 | 156 | 33.21 | 1.81 | 5.4 | 0.00 | 0.0 | 0.00 | 0.0 | 0.00 | 0.0 | 0.51 | 1.5 | 0.00 | 0.0 | 1.86 |
| | HPV 33/58 | 156 | 30.73 | 1.38 | 4.5 | 0.20 | 0.7 | 0.00 | 0.0 | 0.12 | 0.4 | 0.19 | 0.6 | 0.00 | 0.0 | 1.41 |
| | HPV 52 | 156 | 30.08 | 0.79 | 2.6 | 0.24 | 0.8 | 0.00 | 0.0 | 0.33 | 1.1 | 0.00 | 0.0 | 0.00 | 0.0 | 0.87 |

Note: Only replicates with detected viral load (Ct score <40.0) were included in the variance components analysis.

## 6. Precision

An in-house precision study was performed using the same panel members as described in the Reproducibility study. Testing was performed over the course of 12 days with 4 instruments, 3 operators and 3 lots. Each operator tested the panel with two runs per day per lot. Three replicates per panel member were performed for each run. Tables 8 and 9 show the agreement results for the contrived and clinical panel members, respectively, with the BD Onclarity Assay on the BD Viper LT system. The total number of measurments per panel member was 214-216. Tables 10 and 11 summarize the variance component analysis results for the contrived and pooled clinical panel members, respectively. The overall %CV ranged from 1.4% to 1.8% for SiHa cells, 0.9% to 2.1% for HeLa cells, and 0.9% to 1.5% for MS751 cells. The overall %CV for pooled HPV positive clinical specimens ranged from 2.0% to 5.6%.

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Table 8: Summary of Precision Panel Contrived Specimens and Agreement Rates

| HPV Genotype | HPV Target Source | HPV Panel Level (cells/mL) | Total Tested | Total Correct | Percent Agreement | 95% CI | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | | | Lower | Upper |
| HPV16 | SiHa cells | 2 | 214 | 205 | 95.79% Negative | 92.20% | 97.77% |
| HPV16 | SiHa cells | 50 | 216 | 207 | 95.83% Positive | 92.27% | 97.79% |
| HPV16 | SiHa cells | 150 | 215 | 213 | 99.07% Positive | 96.67% | 99.74% |
| HPV18 | HeLa cells | 23 | 216 | 214 | 99.07% Negative | 96.69% | 99.75% |
| HPV18 | HeLa cells | 208 | 216 | 216 | 100.00% (Positive) | 98.25% | 100.00% |
| HPV18 | HeLa cells | 623 | 214 | 214 | 100.00% (Positive) | 98.24% | 100.00% |
| HPV45 | MS751 cells | 90 | 214 | 212 | 99.07% (Negative) | 96.66% | 99.74% |
| HPV45 | MS751 cells | 862 | 216 | 214 | 99.07% (Positive) | 96.69% | 99.75% |
| HPV45 | MS751 cells | 2586 | 216 | 216 | 100.00% (Positive) | 98.25% | 100.00% |

Table 9: Summary of Precision Panel Pooled Clinical Specimens and Agreement Rates

| HPV Genotype | HPV Target Source | HPV Panel Level | Mean HPV Ct Score | Total Tested | Total Correct | Percent Agreement | 95% CI | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | | | | Lower | Upper |
| HPV16 | Pooled Clinical | Negative | N/A | 216 | 216 | 100.00% | 98.25% | 100.00% |
| HPV18 | Pooled Clinical | Negative | N/A | 216 | 216 | 100.00% | 98.25% | 100.00% |
| HPV31 | Pooled Clinical | Negative | N/A | 216 | 216 | 100.00% | 98.25% | 100.00% |
| HPV33/58 | Pooled Clinical | Negative | N/A | 216 | 216 | 100.00% | 98.25% | 100.00% |
| HPV39/68/35 | Pooled Clinical | Negative | N/A | 216 | 216 | 100.00% | 98.25% | 100.00% |
| HPV45 | Pooled Clinical | Negative | N/A | 216 | 216 | 100.00% | 98.25% | 100.00% |
| HPV51 | Pooled Clinical | Negative | N/A | 216 | 216 | 100.00% | 98.25% | 100.00% |
| HPV52 | Pooled Clinical | Negative | N/A | 216 | 216 | 100.00% | 98.25% | 100.00% |
| HPV59/56/66 | Pooled Clinical | Negative | N/A | 216 | 216 | 100.00% | 98.25% | 100.00% |
| HPV16 | Pooled Clinical 16 | Positive | 34.75 | 216 | 216 | 100.00 | 98.25% | 100.00 |
| HPV18 | Pooled Clinical 18 | Positive | 30.60 | 216 | 216 | 100.00% | 98.25% | 100.00% |
| HPV31 | Pooled Clinical 31 | Positive | 32.92 | 216 | 182 | 84.26% | 78.81% | 88.51% |
| HPV33/58 | Pooled Clinical 33/58 | Positive | 30.34 | 216 | 216 | 100.00% | 98.25% | 100.00% |
| HPV45 | Pooled Clinical 45 | Positive | 32.48 | 216 | 173 | 80.09% | 74.26% | 84.87% |
| HPV52 | Pooled Clinical 52 | Positive | 31.58 | 216 | 216 | 100.00% | 98.25% | 100.00% |

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Table 10: Variance Component Analysis Results for Contrived Specimens

| HPV Cell Lines (cells/mL) | Description | Mean Ct Score | Between-Operator | | Between-Instrument | | Between-Reagent | | Between-Day | | Between-Run | | Within-run | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SD | % CV | SD | % CV | SD | % CV | SD | % CV | SD | % CV | SD | % CV | SD | % CV |
| SiHa | 2 cells/mL ≥94% Negative | 38.42 | 0.00 | 0.00 | 0.29 | 0.75 | 0.00 | 0.00 | 0.00 | 0.00 | 0.23 | 0.59 | 0.40 | 1.04 | 0.54 | 1.41 |
| | 50 cells/mL ≥94% Positive | 36.59 | 0.10 | 0.28 | 0.11 | 0.30 | 0.08 | 0.22 | 0.00 | 0.00 | 0.00 | 0.00 | 0.65 | 1.78 | 0.67 | 1.84 |
| | 150 cells/mL ≥98% Positive | 34.98 | 0.00 | 0.00 | 0.12 | 0.33 | 0.03 | 0.09 | 0.00 | 0.00 | 0.17 | 0.50 | 0.55 | 1.56 | 0.59 | 1.68 |
| HeLa | 23 cells/mL ≥94% Negative | 35.48 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.09 | 0.27 | 0.30 | 0.85 | 0.67 | 1.88 | 0.74 | 2.08 |
| | 208 cells/mL ≥94% Positive | 32.29 | 0.00 | 0.00 | 0.08 | 0.26 | 0.00 | 0.01 | 0.00 | 0.00 | 0.08 | 0.26 | 0.26 | 0.81 | 0.29 | 0.89 |
| | 623 cells/mL ≥98% Positive | 30.87 | 0.00 | 0.00 | 0.00 | 0.00 | 0.03 | 0.09 | 0.00 | 0.00 | 0.19 | 0.62 | 0.20 | 0.65 | 0.28 | 0.90 |
| MS751 | 90 cells/mL ≥94% Negative | 35.53 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.01 | 0.00 | 0.00 | 0.19 | 0.52 | 0.51 | 1.44 | 0.54 | 1.53 |
| | 862 cells/mL ≥94% Positive | 32.02 | 0.00 | 0.00 | 0.09 | 0.27 | 0.03 | 0.09 | 0.00 | 0.00 | 0.16 | 0.51 | 0.27 | 0.84 | 0.33 | 1.02 |
| | 2586 cells/mL ≥98% Positive | 30.54 | 0.00 | 0.00 | 0.08 | 0.27 | 0.00 | 0.00 | 0.00 | 0.00 | 0.12 | 0.38 | 0.23 | 0.77 | 0.27 | 0.90 |

Table 11: Variance Component Analysis Results for Pooled Clinical Specimens

| HPV Panel Member | Mean Ct. Score | Between-Operator | | Between-Instrument | | Between-Reagent Lot | | Between-Day | | Between-Run | | Within-Run | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD | % CV | SD | % CV | SD | % CV | SD | % CV | SD | % CV | SD | % CV | SD | % CV |
| Clinical Positive 16 | 34.75 | 0.0 | 00 | 0.25 | 0.72 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 1.56 | 4.49 | 58 | 4.54 |
| Clinical Positive 18 | 30.60 | 0.0 | 0.0 | 0.0 | 0.0 | 0.12 | 0.38 | 0.13 | 0.42 | 0.0 | 0.0 | 1.16 | 3.78 | 1.17 | 3.83 |
| Clinical Positive 31 | 32.92 | 0.06 | 0.18 | 0.12 | 0.36 | 0.18 | 0.54 | 0.0 | 0.0 | 0.0 | 0.0 | 1.68 | 5.09 | 1.69 | 5.13 |
| Clinical Positive 33/58 | 30.34 | 0.0 | 0.0 | 0.04 | 0.12 | 0.0 | 0.0 | 0.12 | 0.38 | 0.0 | 0.0 | 0.60 | 1.97 | 0.61 | 2.01 |
| Clinical Positive 45 | 32.48 | 0.23 | 0.69 | 0.35 | 1.07 | 0.18 | 0.56 | 0.0 | 0.0 | 0.0 | 0.0 | 1.77 | 5.46 | 1.83 | 5.63 |
| Clinical Positive 52 | 31.58 | 0.24 | 0.76 | 0.09 | 0.28 | 0.0 | 0.0 | 0.19 | 0.61 | 0.0 | 0.0 | 1.39 | 4.40 | 1.43 | 4.51 |

# 7. Carry-over/Cross Contamination

A study was performed to evaluate the risk of producing a false positive result in either the same run (within run carry-over) or in a subsequent run (between run carry-over carry-over) on the BD Viper LT System. One run was performed per day over five days on each of three instruments comprising a total of 675 test replicates. Each run, arranged in an alternating checkerboard

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pattern, consisted of specimens containing an HPV negative cell line (C33A) with and without SiHa cells (i.e., an HPV 16 positive cell line) spiked at $1.0 \times 10^{5}$ cells/mL in BD SurePath media. There were zero false positive results among 675 results for the HPV negative samples and the contamination rate with BD SurePath media was $0\%$ (0/675) with $95\%$ CI: $0.0\%$ to $0.6\%$ .

Because the BD Onclarity HPV Assay can be run on a post-cytology specimen after processing on the PrepMate Cytology Processor, a carry-over/cross-contamination study was also performed on the PrepMate to ensure no upstream specimen processing events could cause false positive results. An HPV16 positive cell line was spiked in SurePath media containing a background of HPV negative C33A cells. The HPV16 positive cell line was spiked at a concentration that represented the amount of target necessary to achieve an HPV16 Ct in the $5^{\text{th}}$ percentile from the clinical study. For each run, six of these HPV16 high positive samples were run with six HPV16 negative samples in a checkerboard pattern on the PrepMate. Twelve consecutive runs on the PrepMate were performed on one PrepMate system, for a total of 72 replicates. No false positive results among 72 results for the HPV negative samples were found in this study and the contamination rate was $0\%$ (0/72) with $95\%$ CI: $0.0\%$ to $5.1\%$ .

# 8. Reagent Stability

The BD Onclarity Assay consists of five reagents. Table 12 lists each of the reagents along with their corresponding shelf life and storage temperature, as supported by the results of real time stability studies:

Table 12: Reagent Stability

| BD Onclarity HPV Assay Reagents | Shelf Life Dating in days (approximate months) | Storage Temperature |
| --- | --- | --- |
| HPV Reagent Pack | 249 (~8 months) | 2-8°C |
| HPV Positive/Negative Control | 528 (~18 months) | 2-33°C |
| HPV LBC Diluent Tube | 249 (~8 months) | 2-25°C |
| HPV PCR Reagent Trough | 249 (~8 months) | 2-33°C |
| HPV Extraction Tube | 249 (~8 months) | 2-33°C |

The change in Ct value between baseline and the maximum storage duration was $\leq 1$ for all temperatures.

# 9. Specimen Stability

Specimen stability studies demonstrated that for the BD Onclarity HPV Assay, cervical specimens can be stored in SurePath media at $2 - 30^{\circ}\mathrm{C}$ for up to 30 days, $2 - 8^{\circ}\mathrm{C}$ for 180 days, or at $-20^{\circ}\mathrm{C}$ for 180 days from the date of collection to the time of transfer to the HPV LBC Diluent tube. After transfer to a BD Onclarity HPV LBC Diluent tube, the diluted specimen can be stored at $2 - 30^{\circ}\mathrm{C}$ for up to 15 days, or up to 90 days when stored at $-20^{\circ}\mathrm{C}$ . After pre-warming, the specimens may be stored for up to 7 days at $2 - 30^{\circ}\mathrm{C}$ . The change in Ct value between baseline and the maximum storage duration was $\leq 1$ for all temperatures.

# B. Animal Studies

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Not Applicable

## C. Additional Studies

Not applicable

## X. SUMMARY OF PRIMARY CLINICAL STUDY

The applicant performed a clinical study to establish a reasonable assurance of safety and effectiveness of high risk HPV type nucleic acid detection with the BD Onclarity HPV Assay for routine cervical cancer screening in the US. Data from this clinical study were the basis for the PMA approval decision. A summary of the clinical study is presented below.

## A. Study Design

Subjects were enrolled between August 2013 and June 2015. The database for this PMA reflected data collected through June 2015 and included 33,858 women. There were 31 investigational sites. Specific entrance criteria were structured to qualify subjects, specimens, and test results.

The multicenter, prospective clinical study was conducted to evaluate the performance of the BD Onclarity HPV Assay for the following three indications: 1. As a triage test to stratify women aged 21 with ASC-US cytology results for colposcopy; 2. As an adjunctive test to cervical cytology to guide management decisions in women aged ≥30; and 3. As a first-line primary test for cervical cancer screening in women aged ≥25. Women aged ≥21 undergoing routine cervical cancer screening were enrolled from 31 collection sites between August 2013 and June 2015. Following written informed consent, demographic and gynecologic histories were obtained. Two liquid based cytology (LBC) specimens were collected from each woman, the first being collected in SurePath collection media, and the second in PreservCyt collection media. The cytology results from the first collected SurePath specimen were evaluated at one of three central laboratories and classified according to the 2001 Bethesda System. BD Onclarity HPV Assay testing was performed on LBC specimen aliquots removed prior to cytology testing (i.e., "prequots") at one of four testing laboratories. A post-cytology aliquot (i.e., "postquot") was also removed from the first collected SurePath specimen for a subset of randomly selected women and tested with the BD Onclarity Assay. The second collected PreservCyt specimen was tested with the BD Onclarity Assay and an FDA-approved HPV assay. Clinical performance of the BD Onclarity assay was evaluated using the pre-cytology aliquot from the first-collected SurePath specimen.

Women ≥21 years old with ≥ASC-US cytology and women ≥25 years old with unsatisfactory cytology were invited to undergo colposcopy. In addition, all women ≥25 years old with a positive test result for high risk HPV DNA by the BD Onclarity HPV Assay in either SurePath or PreservCyt and/or the FDA-approved HPV test, as well as a randomly selected subset of women (approximately 10%) with NILM cytology and negative HR HPV DNA results (by both the BD Onclarity HPV assay and the FDA-approved HPV DNA test) were invited to

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proceed to colposcopy. In order to avoid bias in the evaluation of HPV and cytology testing, both study participants and colposcopists were blinded to all HPV test and cytology results until after the colposcopy procedure was completed. Colposcopy was conducted according to a standardized protocol in which biopsies were obtained on all visible lesions or acetowhite areas. Endocervical curettage was performed in all patients, and a single random cervical biopsy was obtained if no lesions or acetowhite areas were visible. All biopsies were examined by a Central Pathology Review (CPR) Panel consisting of three expert pathologists. Discordant histology results were adjudicated according to a pre-defined protocol.

The slides that were prepared from the biopsies and reviewed by the CPR panel were stained with hematoxylin and eosin (H&amp;E). In addition, recommendations outlined in the 2012 Lower Anogenital Squamous Terminology (LAST) guidelines were adapted in selecting certain H&amp;E stained slides to also be evaluated using p16 immunohistochemistry assistance. The criteria in selecting slides to be read with p16 assistance were established based on the LAST committee recommendations, where p16 assistance should be used as a supplement to H&amp;E only for slides where the H&amp;E architecture is equivocal in nature. Based on these recommendations, the following criteria were established for using p16 assistance after the reading of H&amp;E stained slides: 1. at least of one the expert CPR adjudicators reported a CIN2 diagnosis for the histologic specimen, and 2. there was disagreement among the diagnoses of the reviewers, whereby at least one of the reviewers reported a diagnosis of ≥CIN2 and at least one of the reviewers reported a diagnosis of <cin2. (i.e.="" 1.="" 2.="" <="" a="" alone”),="" alone”),="" and="" approach="" as="" based="" based on="" by="" cases="" clings="" clings.="" clings.="" different="" diag-="" diagnosed="" diagnoses="" diagnoses:="" each="" endpoint="" evaluation="" f="" for="" in="" is="" it="" known="" les="" les,="" method="" meeting="" misl-adapted="" of="" on="" other="" p16="" performed="" phv="" q.="" quality="" random="" re-exclusion="" reviewers="" reviewings="" should="" slides="" slides="" si="" siams”,="" siams”)="" siams”).="" siams”)="" siams”)="" siams”)="" siams”)="" siams”)="" siams”)="" siams”)="" single="" standard="" study="" study.="" the="" this="" to="" two="" used="" was="" were="" where="" who="" with=""> 65 years of age may be included following USPSTF recommendations.

- Female subjects who provide informed consent.
- Female subjects who have received the HPV vaccine (approximately 10% of total enrollment)
- Subjects who have the following minimum specimen results:
- Cytology result from the BD SurePath Collection Vial
- At least one LBC aliquot for BD Viper LT testing

Patients were not permitted to enroll in the BD-USHPV study if they met any of the following exclusion criteria:

- Subjects known to be pregnant.
- Subjects who had a cervical cytology specimen collected within the last 4 months.

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- Subjects with prior complete or partial hysterectomy involving removal of cervix.
- Subjects with an application of chemical compounds to the cervical area 24 hour prior to study entry—acetic acid, iodine, spermicide, douche, or anti-fungal meds.
- Subjects on whom conization, LEEP, cervical laser surgery or cryosurgery on the cervix has been performed in the last twelve months.
- Subjects who have been previously enrolled in a cervical disease diagnostic trial since 2007.

## 2. Follow-up Schedule

All women with abnormal cytology results (i.e., ≥ASC-US), unsatisfactory cytology results, women who were HPV positive by either the BD Onclarity or FDA-approved HPV assay, as well as a subset of women with normal cytology who were HPV negative by both HPV assays were invited to undergo the colposcopy procedure within approximately three months.

For the primary screening indication, all women aged ≥25 years who had undergone colposcopy and biopsy in the baseline phase but did not receive treatment as well as approximately 10% of NILM women (≥25 years) with HR HPV negative results and no baseline biopsy or treatment were invited to proceed to the three year Follow-up Phase of the study. Approximately 8,900 women from the baseline phase are eligible for the three-year follow-up study, where they will undergo annual cytology and HPV DNA testing with the BD Onclarity HPV Assay. Those women with abnormal cytology will be invited to proceed to colposcopy. Colposcopy and biopsies will be performed in a standardized manner as described above. All cervical tissue will be examined by the Central Pathology Review Panel. An exit colposcopy with biopsy and endocervical curettage (ECC) will be offered to all women in Year 3. All women, regardless of cytology or histology result, will be followed through the duration of the study. Women who receive treatment procedures will exit the study. The three-year follow-up study is currently underway and will be submitted as a post-approval study as a condition of approval for the primary screening claim.

Adverse events and complications were recorded at all visits.

## 3. Clinical Endpoints

With regards to safety, as an in vitro diagnostic test, the BD Onclarity HPV Assay involves sampling cells from the cervix using an endocervical brush/spatula or broom. The test, therefore, presents no more safety hazard to an individual being tested than other tests where cervical cells are sampled in this manner (i.e., cervical cytology). Safety issues regarding false positive and negative test results are discussed in section VIII.

With regards to effectiveness, the following are the clinical endpoints for each indication:

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ASC-US Triage (≥21 years) indication: The clinical performance of the BD Onclarity HPV Assay was evaluated against CPR panel histology diagnoses, with ≥CIN2 and ≥CIN3 as the disease endpoints for all women aged ≥21 with ASC-US cytology.

Adjunct (NILM ≥30 years) indication: Clinical performance of the BD Onclarity HPV Assay was evaluated against CPR panel histology diagnoses, with ≥CIN2 and ≥CIN3 as the disease endpoints for all women aged ≥30 years with NILM cytology. Baseline risks for both ≥CIN2 and ≥CIN3 were evaluated for each BD Onclarity HPV result.

Primary Screening (≥25 years) indication: The clinical performance of the BD Onclarity HPV Assay in the baseline phase was evaluated against CPR panel histology diagnoses, with ≥CIN2 and ≥CIN3 as the disease endpoints for all women aged ≥25 years. The performance of the primary screening algorithm was compared to two different screening algorithms, one based on current practice and the other based on cytology alone. Risks for ≥CIN2 and ≥CIN3 were evaluated according to the baseline HPV status (as determined by the BD Onclarity HPV assay) for each BD Onclarity HPV assay readout as well as different HPV genotype and cytology combinations.

## B. Accountability of PMA Cohort

A total of 33,858 subjects were enrolled in this study. Of the enrolled subjects, 33,634 subjects had evaluable cytology. Subject age and cytology result were used to group the enrolled subjects into the following intended use populations:

## ASC-US Population (≥21 years)

Women ≥21 years with ASC-US cytology were evaluated for the ASC-US triage indication. A total of 1,960 (5.8%) subjects out of 33,634 subjects with evaluable cytology had an ASC-US cytology result and were invited to proceed to the colposcopy/biopsy procedure, of which, 1,618 attended. Out of the 1,618 subjects, two had specimen collection errors/issues, leaving 1,616 subjects with biopsy results. Three of these subjects had unsatisfactory adjudicated results, and six had missing BD Onclarity results, leaving a total of 1,607 evaluable subjects.

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# Adjunct Population (≥30 years)

Women ≥30 years with NILM cytology were evaluated for the Adjunct indication. Out of the 33,634 subjects women with evaluable cytology, 30,489 had NILM cytology. Of these, 22,383 subjects were 30 years or older. Of these, 1,991 women with an HPV positive result and 1,228 randomly selected women who were HPV negative by both the Onclarity and FDA approved tests were invited to proceed to the colposcopy/biopsy procedure, for a total of 3,219 subjects. Of these 3,219 women, 2,610 attended the colposcopy visit and 2,603 had biopsy results. Six subjects had adjudicated results of unsatisfactory and were not used in the data analysis, and six had either missing or invalid HPV results. Evaluable histology and HPV results were obtained for 2,591 subjects.

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# Primary Screening Population (≥25 years)

All women ≥25 years with valid cytology results were evaluated for the primary screening indication. Out of the 33,634 subjects with evaluable cytology, 29,690 were over the age of 25. Fifty-seven of these subjects had unsatisfactory cytology results and were excluded, for a total of 29,633 subjects in the primary screening population. This represented about $88.1\%$ of the

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enrolled subjects. Of these, 27,152 subjects had NILM, 1,625 subjects had ASC-US, and 856 subjects had &gt;ASC-US cytology. A total of 6,803 subjects were invited to return for the colposcopy/biopsy procedure because they either had an abnormal cytology result (2,481), were HPV positive NILM (2,861), or were a random selection of HPV negative NILM (1,461). Of these, 5,568 attended the colposcopy visit and 5,557 had biopsy results. Ten subjects had unsatisfactory results and were not included in the data analysis. Of the 5,547 women with evaluable adjudicated results, 13 had missing HPV Onclarity test results, leaving a total of 5,534 women in the primary screening population with evaluable histology and HPV results.


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# C. Study Population Demographics and Baseline Parameters

The demographics of the study population shown in Table 13 are typical for a cervical cancer screening study performed in the US.

Table 13: Study Demographics for Each Intended Use Population

| Demographics | ASC-US | Adjunct | Primary Screening |
| --- | --- | --- | --- |
| Age at Consent (years) | Mean | 36.2 | 43.9 | 40.7 |
| Standard Deviation | 11.5 | 9.6 | 10.9 |
| Median | 34.0 | 43.0 | 39.0 |
| (Min, Max) | 21, 82 | 30, 83 | 25, 83 |
| Total (n) | 1960 | 22383 | 29633 |
| Ethnicity (%) | Hispanic or Latino | 15.5 303/1960 | 20.5 4592/22383 | 19.9 5887/29633 |
| Not Hispanic or Latino | 84.5 1657/1960 | 79.5 17789/22383 | 80.1 23744/29633 |
| Ethnicity data missing | 0 0/1960 | 0.0 2/22383 | 0.0 2/29633 |
| Race (%) | American Indian or Alaskan Native | 0.4 7/1960 | 0.4 97/22383 | 0.5 149/29633 |
| Asian | 1.0 20/1960 | 1.5 336/22383 | 1.4 416/29633 |
| Black or African American | 23.4 459/1960 | 16.6 3722/22383 | 18.0 5340/29633 |
| Native Hawaiian or Other Pacific Islander | 0.2 4/1960 | 0.3 60/22383 | 0.2 73/29633 |
| White | 73.8 1446/1960 | 80.4 17988/22383 | 79.0 23400/29633 |
| Other | 1.2 24/1960 | 0.8 180/22383 | 0.9 255/29633 |

The median age of the eligible women was 37, with $28.0\%$ of women in the 21-29 years age group, $28.3\%$ in the 30-39 age group, and $43.7\%$ of women in the $\geq 40$ years age group. A total of $90.6\%$ of women had NILM cytology, $5.8\%$ had ASC-US cytology, $3.3\%$ had &gt;ASC-US cytology, and $0.2\%$ had unsatisfactory cytology. The percent of final non-reportable BD Onclarity assay results was $0.24\%$ (79/33,570). Not included in this calculation are specimens that did not yield a result (64/33,634) due to specimen labeling, processing, and volume issues.

Table 14 shows HPV positivity of the BD Onclarity HPV Assay by testing site and study population. HPV prevalence was $39.1\%$ in the ASC-US $(\geq 21$ years) population, $7.9\%$ in the NILM $(\geq 30$ years) population and $12.7\%$ in the Primary Screening $(\geq 25$ years) population.

Table 14: Summary of HPV Positivity of the BD Onclarity HPV Assay by Testing Sites and Study Population

| BD Onclarity HPV Assay HR Positivity Rate | | | |
| --- | --- | --- | --- |
| Testing Site | ASC-US (≥ 21 years) | NILM (≥ 30 years) | Primary Screening (≥ 25 years) |

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Table 15 shows HPV positivity rates by the BD Onclarity HPV Assay by age and study population. HPV positivity decreased with age in each study population.

Table 15: Summary of HPV Positivity Rates of the BD Onclarity HPV Assay by Age and Study Population

| BD Onclarity HPV Assay HR Positivity Rate | | | |
| --- | --- | --- | --- |
| Age Group | ASC-US (≥ 21 years) | NILM (≥ 30 years) | Screening (≥ 25 years) |
| 21 - 29 | 54.6% (398/729) | N/A | 22.4% (1216/5432) |
| 30 - 39 | 39.2% (204/521) | 10.3% (889/8663) | 13.8% (1310/9477) |
| ≥ 40 | 22.9% (161/703) | 6.4% (872/13621) | 8.4% (1222/14604) |
| Total | 39.1% (763/1953) | 7.4% (1761/22284) | 12.7% (3748/29513) |

The BD Onclarity HPV Assay results stratified by age are outlined in Table 16 for the ASC-US population (≥ 21 years), in Table 17 for the NILM population (≥ 30 years), and in Table 18 for the primary screening population (≥ 25 years). In all populations, the 11 other HPV HR positive results were more frequent than HPV16, HPV18, and HPV45 positive results in general and within age groups. HPV positivity rates for each category decreases with age in all three populations.

Table 16: BD Onclarity HPV Assay Result by Age Group for ASC-US (≥ 21 years) Population

| BD Onclarity HPV Assay Result | | | | | |
| --- | --- | --- | --- | --- | --- |
| Age Group | HPV16+ | HPV18+ | HPV45+ | 11 Other HPV HR + | HPV - |
| 21 - 29 | 10.6% (77/729) | 3.4% (25/729) | 3.2% (23/729) | 45.8% (334/729) | 45.4% (331/729) |
| 30 - 39 | 7.7% (40/521) | 2.5% (13/521) | 2.1% (11/521) | 31.1% (162/521) | 60.8% (317/521) |
| ≥ 40 | 3.8% (27/703) | 1.4% (10/703) | 1.8% (13/703) | 18.9% (133/703) | 77.1% (542/703) |
| Total | 7.4% (144/1953) | 2.5% (48/1953) | 2.4% (47/1953) | 32.2% (629/1953) | 60.9% (1190/1953) |

Note: For this table, women with mixed infections were counted for each HPV type they were infected with (i.e., if a woman was infected with both HPV16 and 18, then she was included in both the HPV16+ and HPV18+ analysis).

Table 17: BD Onclarity HPV Assay Result by Age Group for NILM (≥ 30 years) Population

| BD Onclarity HPV Assay Result | | | | | |
| --- | --- | --- | --- | --- | --- |
| Age Group | HPV16+ | HPV18+ | HPV45+ | 11 Other HPV HR | HPV - |
| 30 - 39 | 2.0% (173/8663) | 0.5% (45/8663) | 0.8% (68/8663) | 7.7% (671/8663) | 89.7% (7774/8663) |
| ≥ 40 | 1.2% (157/13621) | 0.4% (53/13621) | 0.4% (56/13621) | 4.9% (671/13621) | 93.6% (12749/13621) |
| Total | 1.5% (330/22284) | 0.4% (98/22284) | 0.6% (124/22284) | 6.0% (1342/22284) | 92.1% (20523/22284) |

Note: For this table, women with mixed infections were counted for each HPV type they were infected with (i.e., if a woman was infected with both HPV16 and 18, then she was included in both the HPV16+ and HPV18+ analysis).

Table 18: BD Onclarity HPV Assay Result by Age Group for Screening (≥ 25 years) Population

| Age Group | HPV16+ | HPV18+ | 12 Other HPV HR + | HPV - |
| --- | --- | --- | --- | --- |
| 25 - 29 | 4.5% (246/5432) | 1.2% (63/5432) | 18.9% (1025/5432) | 77.6% (4216/5432) |
| 30 - 39 | 2.9% (274/9477) | 0.8% (78/9477) | 11.2% (1063/9477) | 86.2% (8167/9477) |

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| ≥40 | 1.5% (223/14604) | 0.6% (84/14604) | 6.9% (1010/14604) | 91.6% (13382/14604) |
| --- | --- | --- | --- | --- |
| Total | 2.5% (743/29513) | 0.8% (225/29513) | 10.5% (3098/29513) | 87.3% (25765/29513) |

Note: For this table, women with mixed infections were counted for each HPV type they were infected with (i.e., if a woman was infected with both HPV16 and 18, then she was included in both the HPV16+ and HPV18+ analysis).

## Safety and Effectiveness Results

### 1. Safety Results

As an in vitro diagnostic test, the BD Onclarity HPV Assay involves sampling cells from the cervix using an endocervical brush/spatula combination or broom. Collection of the sample, therefore, presents no more safety hazard to an individual being tested than other tests where cervical cells are sampled in this manner (such as cervical cytology).

### Adverse effects that occurred in the PMA clinical study:

All serious adverse events (AE) reported during the study were captured in the subjects' chart and in the electronic database. The adverse events collected during this study were only those events related to the study-required colposcopy and biopsy procedure. Each AE was assigned a severity criterion (mild, moderate, marked) by the principal investigator or designee. Each AE was also evaluated for determination as a Serious Adverse Event by the principal investigator or designee. During the clinical study, there were 37 events reported, 28 of which were adverse events and 9 were serious adverse events.

Twenty-two subjects out of the 33,858 enrolled in the study (0.06%) reported a total of 28 adverse events. Of the 28 adverse events, 8 were assigned an unlikely relationship to the procedure, 8 were assigned a possible relationship to the procedure and 12 were assigned a probable relationship to the procedure. The adverse events with a possible relationship to the procedure were comprised of 1 bacterial vaginosis, 3 abdominal cramps or lower abdominal pain, 1 low grade fever, 2 vaginal bleeding and 1 urinary tract infection. The 12 adverse events with a probable relationship to the procedure were comprised of 3 cervical or vaginal bleeding, 3 uterine contractions or cramping, 2 vaginal or cervical pain, 1 abdominal bloating, 2 syncopal episodes and 1 fall with a laceration due to the syncopal episode. All the adverse events with a possible or probable relationship to the procedure resolved within 33 days of event onset.

Of the 9 serious adverse events, all were unlikely to have been a result of the BD HPV Assay or the investigational procedure. The serious adverse events were comprised of 6 cancers, 1 myomectomy, 1 reported death due to a prescription overdose, and 1 diverticulitis and sepsis resulting in death. Five of the 9 serious adverse events were resolved within 58 days of onset. Four serious adverse events remained unresolved at the study closure.

### 2. Effectiveness Results

The analysis of effectiveness was based on the data in the following sections for each claimed indication. The clinical performance data in this section are based on histology diagnoses using H&amp;E+p16 assistance for slides meeting the LAST-adapted criteria and H&amp;E alone for all other slides. For clinical performance estimates using H&amp;E alone as the histologic endpoint for all slides, please see section XI: Summary of Supplemental Clinical Information. It should be noted

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that no significant differences in performance were apparent when using either histologic endpoint for any of the three indications.

# PERFORMANCE CHARACTERISTICS IN THE ASC-US POPULATION (≥21 YEARS)

Study enrollment was completed in June 2015 with a total of 33,858 subjects. Of the total enrollment, 1,960 subjects ≥21 years of age had ASC-US cytology results. All subjects were referred to the colposcopy/biopsy visit, of which 1,619 returned. Satisfactory histological diagnoses were determined for 1,613 subjects. Six subjects (all <cin2) (all="" 1,607="" 19="" <table="" [bbox]0.545,0.063,0.927,0.284[="" [bbox]0.545,0.063,0.892,0.284[="" [bbox]0.745,0.063,0.892,0.284[="" [bbox]0.285,0.063,0.565[="" [bbox]0.285,0.063,0.465[="" asa="" ASC-us="" asc-us.="" bdp="" baseline="" by="" cpr="" claim.="" cln2="" clinic="" data="" de="" de:="" de:="" dept="" dept-="" diagnosis="" diagnosis.="" each="" effectives="" endpoint="" evaluable="" evaluable'="" every="" evaluate="" evaluation.="" evacuated="" evacuated.="" field="" from="" g="" g.="" g0="" had="" history="" how="" in="" include="" including="" inc="" inc="" inc]="" inc]="" is="" it="" known="" l607="" l607).="" l607).="" l607).="" l607).="" l607).="" l607).="" l607).<="" l607).<sup="" adjacent="" adjusted="" age="" age*="" adjusted:="" adjudicated="" adjudicated="" adrs.="" adrs..="" adrs.0.36%="" adrs.105="" adjusted="" adjusted.="" after="" after»="" after»<sup="" adjudicated="" adjudicated.="" adrs.="" adrs.0.36%="" adrs.105="" adjusted.="" after»="" after»<sup.="" adjusted.="" and="" are="" asa="" associated="" association="" association.="" at="" bdp="" baseline="" blood="" bloods="" by="" cpr="" cpr.="" case="" ce="" ce.="" ce.2015]="" ce.2016]="" ce.2016].="" ce.2017]="" ce.2017].="" cpr.="" data="" de="" de.="" de.="" de.2.39="" de.2.39,="" de.2.39.="" detected="" detected.="" different="" did="" diagnosis="" diagnosis.="" d="" d)="" d)20.="" d)20.2,="" each="" eligible="" effect="" effects="" each.="" end="" endpoint="" evaluation="" evaluation.="" evacuated="" evacuated.="" field="" field.="" for="" for:="" from="" g="" g0="" g0.="" g0.2,="" g0.2.39="" had="" had.="" high="" high-grade="" i,607="" i,607.="" if="" in="" including="" inc="" inc="" inc]="" inc]="" inc]).="" including="" inc]="" inc]).="" including<="" inc]="" inc]).="" including<sup="" inc]="" inc]).="" including<sup.="" inc]="" inc]).="" including<sup.0.2,="" inc]="" inc]).="" including<sup.0.2.39="" inc]="" inc]).="" including<sup.0.2.39="" inc]="" inc]).="" including<sup.0.2.39,="" inc]="" including<sup.}="" data="" de="" de.="" de.="" de.="" de.2.39,="" de.2.41="" de.2.41="" de.2.41.="" de.2.41.0.14,="" de.2.41.0.2.39="" de.2.41.0.2.41.0.2.41.0.2.41.0.2.41.0.2.41.0.2.41.0.2.41.0

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Table 20: Performance of the BD Onclarity HPV Assay in the ASC-US Population (≥21 years)

| Performance | ≥CIN2 | ≥CIN3 |
| --- | --- | --- |
| | Central Pathology Review Panel Diagnosis | |
| Sensitivity (%)
(95% CI) | 85.7 | 91.4 |
| | 90/105 | 32/35 |
| | (77.8, 91.1) | (77.6, 97.0) |
| Specificity (%)
(95% CI) | 64.1 | 62.0 |
| | 963/1502 | 975/1572 |
| | (61.7, 66.5) | (59.6, 64.4) |
| PPV (%)
(95% CI) | 14.3 | 5.1 |
| | 90/629 | 32/629 |
| | (13.0, 15.5) | (4.3, 5.6) |
| NPV (%)
(95% CI) | 98.5 | 99.7 |
| | 963/978 | 975/978 |
| | (97.6, 99.0) | (99.2, 99.9) |
| PLR
(95% CI) | 2.39 | 2.41 |
| | (2.13, 2.63) | (2.03, 2.64) |
| NLR
(95% CI) | 0.22 | 0.14 |
| | (0.14, 0.35) | (0.05, 0.36) |
| Disease Prevalence (%) | 6.5 | 2.2 |
| | 105/1607 | 35/1607 |

Out of the 15 subjects with ≥CIN2 who were negative by the BD Onclarity, 9 were negative by both the FDA approved test and PCR/sequencing, 3 were negative by the FDA approved test and positive by PCR/sequencing for HR HPV, and 3 were positive by the FDA approved test and negative by PCR/sequencing. These latter three subjects were identified by sequencing as positive for low-risk HPV types 67 and/or 82.

Out of the 3 subjects with ≥CIN3 that were negative by the BD Onclarity, 1 was negative by both the FDA approved test and PCR/sequencing, 1 was negative by the FDA approved test and positive by PCR/sequencing for HR HPV, and 1 was positive by the FDA approved test and negative by PCR/sequencing. This latter subject was identified by sequencing as positive for low risk HPV type 67.

The performance of the BD Onclarity HPV Assay in detecting high-grade cervical disease (≥CIN2 and ≥CIN3) and the performance of the FDA approved HPV test is presented in Table 21. The sensitivity for detecting ≥CIN2 histology was 85.7% (90/105) for the BD Onclarity HPV Assay and 82.9% (87/105) for the FDA approved HPV test. The specificity for detecting ≥CIN2 histology was 64.1% (959/1,496) for the BD Onclarity HPV Assay and 61.4% (919/1,496) for the FDA approved HPV test.

The sensitivity for detecting ≥CIN3 histology was 91.4% (32/35) for the BD Onclarity HPV Assay and 85.7% (30/35) for the FDA approved HPV test. The specificity for detecting ≥CIN3 histology was 62.0% (971/1,566) for the BD Onclarity HPV Assay and 59.5% (932/1,566) for the FDA approved HPV test.

Table 21: Performance of the BD HPV Assay and an FDA Approved HPV Test in the ASC-US Population (≥21 years)

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| Performance Metrics | BD Onclarity HPV Assay | | FDA Approved HPV Test | |
| --- | --- | --- | --- | --- |
| | Estimate | 95% CI | Estimate | 95% CI |
| ≥CIN2; Prevalence 6.6% (105/1601) | | | | |
| Sensitivity (%) | 85.7 (90/105) | (77.8, 91.1) | 82.9 (87/105) | (74.5, 88.9) |
| Specificity (%) | 64.1 (959/1496) | (61.6, 66.5) | 61.4 (919/1496) | (58.9, 63.9) |
| PPV (%) | 14.4 (90/627) | (13.0, 15.6) | 13.1 (87/664) | (11.8, 14.3) |
| NPV (%) | 98.5 (959/974) | (97.6, 99.0) | 98.1 (919/937) | (97.2, 98.7) |
| PLR | 2.39 | (2.13, 2.63) | 2.15 | (1.90 2.37) |
| NLR | 0.22 | (0.14, 0.35) | 0.28 | (0.18, 0.42) |
| ≥CIN3; Prevalence 2.2% (35/1601) | | | | |
| Sensitivity (%) | 91.4 (32/35) | (77.6, 97.0) | 85.7 (30/35) | (70.6, 93.7) |
| Specificity (%) | 62.0 (971/1566) | (59.6, 64.4) | 59.5 (932/1566) | (57.1, 61.9) |
| PPV (%) | 5.1 (32/627) | (4.3, 5.6) | 4.5 (30/664) | (3.7, 5.0) |
| NPV (%) | 99.7 (971/974) | (99.2, 99.9) | 99.5 (932/937) | (98.9, 99.8) |
| PLR | 2.41 | (2.03, 2.64) | 2.12 | (1.73, 2.37) |
| NLR | 0.14 | (0.05, 0.36) | 0.24 | (0.11, 0.49) |

Note: This table is a paired analysis of specimens with a valid BD Onclarity HPV assay and FDA approved HPV test result. Six women (&lt;CIN2) with a BD Onclarity result but no FDA approved HPV test result were excluded in this analysis.

The performance of the BD Onclarity HPV Assay for detecting $\geq$ CIN2 and $\geq$ CIN3 evaluated by age group is presented in Table 22 below. The sensitivity for detecting $\geq$ CIN2 was $93.6\%$ (44/47) in the 21-29 age group, $83.3\%$ (35/42) in the 30-39 age group, and $68.8\%$ (11/16) in the 40 age group. The specificity was highest in the $\geq$ 40 age group, with an estimate of $78.1\%$ (447/572).

The sensitivity for detecting $\geq$ CIN3 was $92.9\%$ (13/14) in the 21-29 age group, $92.9\%$ (13/14) in the 30-39 age group, and $85.7\%$ (6/7) in the $\geq$ 40 age group. Specificity was highest in the $\geq$ 40 age group, with an estimate of $77.6\%$ (451/581).

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Table 22: Performance of the BD Onclarity HPV Assay and an FDA Approved HPV Test by Age Group in the ASC-US (≥21 years) Population

| Performance Metrics | BD HPV | FDA Approved HPV Test | BD HPV | FDA Approved HPV Test | BD HPV | FDA Approved HPV Test |
| --- | --- | --- | --- | --- | --- | --- |
| | 21 - 29 Years | | 30 - 39 Years | | ≥40 Years | |
| ≥CIN2 | | | | | | |
| Sensitivity (%)95% CI | 93.644/47(82.8%, 97.8%) | 91.543/47(80.1%, 96.6%) | 83.3(35/42)(69.4%, 91.7%) | 78.6(33/42)(64.1%, 88.3%) | 68.8(11/16)(44.4%, 85.8%) | 68.8(11/16)(44.4%, 85.8%) |
| Specificity (%)95% CI | 49.5260/525(45.3%, 53.8%) | 45.9241/525(41.7%, 50.2%) | 63.2(254/402)(58.4%, 67.8%) | 60.7(244/402)(55.8%, 65.3%) | 78.2(445/569)(74.6%, 81.4%) | 76.3(434/569)(72.6%, 79.6%) |
| PPV (%)95% CI | 14.244/309(12.6%, 15.6%) | 13.1(43/327)(11.5%, 14.4%) | 19.1(35/183)(16.0%, 21.9%) | 17.3(33/191)(14.2%, 20.0%) | 8.1(11/135)(5.3%, 10.6%) | 7.5(11/146)(4.9%, 9.7%) |
| NPV (%)95% CI | 98.9(260/263)(97.0%, 99.6%) | 98.4(241/245)(96.2%, 99.4%) | 97.3(254/261)(95.2%, 98.6%) | 96.4(244/253)(94.1%, 98.0%) | 98.9(445/450)(98.0%, 99.5%) | 98.9(434/439)(98.0%, 99.5%) |
| PLR(95% CI) | 1.85(1.61, 2.06) | 1.69(1.46, 1.88) | 2.26(1.82, 2.69) | 2.00(1.59, 2.39) | 3.15(1.99, 4.20) | 2.90(1.83, 3.84) |
| NLR(95% CI) | 0.13(0.04, 0.35) | 0.19(0.07, 0.44) | 0.26(0.13, 0.49) | 0.35(0.19, 0.60) | 0.40(0.18, 0.71) | 0.41(0.19, 0.73) |
| ≥CIN2prevalence (%) | 8.2(47/572) | | 9.5(42/444) | | 2.7(16/585) | |
| ≥CIN3 | | | | | | |
| Sensitivity (%)95% CI | 92.9(13/14)(68.5%, 98.7%) | 92.9(13/14)(68.5%, 98.7%) | 92.9(13/14)(68.5%, 98.7%) | 85.7(12/14)(60.1%, 96.0%) | 85.7(6/7)(48.7%, 97.4%) | 71.4(5/7)(35.9%, 91.8%) |
| Specificity (%)95% CI | 47.0(262/558)(42.8%, 51.1%) | 43.7(244/558)(39.7%, 47.9%) | 60.5(260/430)(55.8%, 65.0%) | 58.4(251/430)(53.7%, 62.9%) | 77.7(449/578)(74.1%, 80.9%) | 75.6(437/578)(71.9%, 78.9%) |
| PPV (%)95% CI | 4.2(13/309)(3.1%, 4.7%) | 4.0(13/327)(2.9%, 4.4%) | 7.1(13/183)(5.3%, 8.1%) | 6.3(12/191)(4.4%, 7.4%) | 4.4(6/135)(2.5%, 5.5%) | 3.4(5/146)(1.7%, 4.6%) |
| NPV (%)95% CI | 99.6(262/263)(98.3%, 99.9%) | 99.6(244/245)(98.2%, 99.9%) | 99.6(260/261)(98.3%, 99.9%) | 99.2(251/253)(97.8%, 99.8%) | 99.8(449/450)(99.2%, 100.0%) | 99.5(437/439)(99.0%, 99.9%) |
| PLR(95% CI) | 1.75(1.28, 1.96) | 1.65(1.21, 1.84) | 2.35(1.71, 2.72) | 2.06(1.42, 2.45) | 3.84(2.15, 4.80) | 2.93(1.45, 3.99) |
| NLR(95% CI) | 0.15(0.03, 0.67) | 0.16(0.03, 0.72) | 0.12(0.02, 0.52) | 0.24(0.07, 0.69) | 0.18(0.03, 0.66) | 0.38(0.11, 0.85) |
| ≥CIN3prevalence (%) | 2.4(14/572) | | 3.2(14/444) | | 1.2(7/585) | |

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

Table 23 below summarizes the CPR adjudicated histology diagnoses reported by all possible BD Onclarity HPV Assay results for the ASC-US (≥21 years) population. The BD Onclarity HPV result is categorized hierarchically based on detected genotype (positive) in the order of HPV 16, HPV 18, HPV 45, 11 Other HPV HR, and HPV negative. Women with multiple genotypes detected were categorized in the earliest genotype listed (i.e., women positive for HPV 16 and HPV 18 was categorized as HPV 16).

PMA P160037: FDA Summary of Safety and Effectiveness Data

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Table 23: Summary of BD Onclarity HPV Assay Results and Adjudicated Histology Diagnoses in the ASC-US (≥21 years) Population

| BD Onclarity HPV Assay Genotyping Results | Central Pathology Review Panel Diagnosis | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | NEG | CIN1 | CIN2 | ≥CIN3 | Undetermined | Total |
| HPV16 Pos, HPV18 Pos, HPV45 Neg, Other HPV Pos | 1 | 2 | 0 | 0 | 1 | 4 |
| HPV16 Pos, HPV18 Pos, HPV45 Neg, Other HPV Neg | 1 | 0 | 0 | 0 | 1 | 2 |
| HPV16 Pos, HPV18 Neg, HPV45 Pos, Other HPV Pos | 0 | 1 | 0 | 0 | 0 | 1 |
| HPV16 Pos, HPV18 Neg, HPV45 Pos, Other HPV Neg | 1 | 0 | 0 | 0 | 0 | 1 |
| HPV16 Pos, HPV18 Neg, HPV45 Neg, Other HPV Pos | 29 | 8 | 4 | 5 | 13 | 59 |
| HPV16 Pos, HPV18 Neg, HPV45 Neg, Other HPV Neg | 31 | 5 | 11 | 13 | 17 | 77 |
| HPV16 Neg, HPV18 Pos, HPV45 Neg, Other HPV Pos | 5 | 5 | 3 | 0 | 2 | 15 |
| HPV16 Neg, HPV18 Pos, HPV45 Neg, Other HPV Neg | 17 | 3 | 2 | 1 | 4 | 27 |
| HPV16 Neg, HPV18 Neg, HPV45 Pos, Other HPV Pos | 9 | 6 | 1 | 1 | 1 | 18 |
| HPV16 Neg, HPV18 Neg, HPV45 Pos, Other HPV Neg | 18 | 4 | 1 | 0 | 4 | 27 |
| HPV16 Neg, HPV18 Neg, HPV45 Neg, Other HPV Pos | 311 | 82 | 36 | 12 | 91 | 532 |
| HPV16 Neg, HPV18 Neg, HPV45 Neg, Other HPV Neg | 888 | 75 | 12 | 3 | 212 | 1190 |
| Total | 1311 | 191 | 70 | 35 | 346a | 1953 |

a 340 women did not return or were no longer eligible for the colposcopy procedure. Three women had unsatisfactory histology results and three women had biopsy specimen collection errors.

The likelihood ratios, along with 95% confidence intervals, for a given HPV type by the Onclarity HPV Assay test result and histologic determination are presented in Table 24.

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Table 24: Likelihood Ratios by BD Onclarity HPV Assay Result in the ASC-US (≥21 years) Population

| BD Onclarity HPV Assay Test Results | Likelihood Ratio (95% CI) | |
| --- | --- | --- |
| | ≥ CIN2 vs. < CIN2 | ≥ CIN3 vs. < CIN3 |
| HPV HR Positive | 2.39
(2.13, 2.63) | 2.41
(2.03, 2.64) |
| HPV 16 Positive | 5.98
(4.15, 8.42) | 8.60
(5.69, 12.08) |
| HPV 18 Positive | 2.86
(1.24, 6.45) | 1.28
(0.22, 6.74) |
| HPV 45 Positive | 1.16
(0.38, 3.42) | 1.15
(0.20, 6.03) |
| HPV 16 and/or HPV 18 and/or HPV 45 Positive | 4.12
(3.07, 5.38) | 5.35
(3.72, 7.07) |
| 11 Other HPV HR Positive | 1.75
(1.38, 2.15) | 1.26
(0.76, 1.88) |
| HPV HR Negative | 0.22
(0.14, 0.35) | 0.14
(0.05, 0.36) |

For the ≥CIN2 endpoint, the likelihood ratio for an HPV HR positive result was 2.39 (95% CI 2.13, 2.63), indicating that an HPV positive result was 2.39 times more likely to be associated with a woman with ≥CIN2. Of the individual genotypes identified and reported by the BD Onclarity HPV Assay, a positive HPV 16 result had the highest positive likelihood ratio of 5.98 (95% CI 4.15, 8.42), indicating that a positive result is 5.98 times more likely to come from a subject with ≥CIN2 than without. The likelihood ratio of a combined HPV 16/18/45 test outcome was 4.12 (95% CI 3.07, 5.38). The likelihood ratio for an 11 other result was 1.75 (95% CI 1.38, 2.15). For an HPV HR negative result, the likelihood ratio was 0.22 (95% CI 0.14, 0.35), indicating that the negative result was 4.55 (1/0.22) times more likely to come from a subject without disease (i.e., &lt;CIN2) than with disease.

For the ≥CIN3 endpoint, the likelihood ratio for an HPV HR positive result was 2.41 (95% CI 2.03, 2.64). Of the individual genotypes identified and reported by the BD Onclarity HPV Assay, a positive HPV 16 result had the highest positive likelihood ratio of 8.60 (95% CI 5.69, 12.08). The likelihood ratio of a combined HPV 16/18/45 test outcome was 5.35 (95% CI 3.72, 7.07). For an HPV HR negative result, the likelihood ratio was 0.14 (95% CI 0.05, 0.36), indicating that the negative result was 7.1 (1/0.14) times more likely to come from a subject without disease &lt;CIN3, than with disease.

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

The risk of disease is the probability of having disease given an HPV test result. Table 25 summarizes the absolute risk of disease for each BD Onclarity HPV result. For the ≥CIN2 disease endpoint, the pre-…