Human Papillomavirus Oncogenic mRNA Testing for Cervical Cancer Screening

Jennifer L. Reid, PhD; Thomas C. Wright Jr, MD; Mark H. Stoler, MD; Jack Cuzick, PhD; Philip E. Castle, PhD; Janel Dockter; Damon Getman, PhD; Cristina Giachetti, PhD

Disclosures

Am J Clin Pathol. 2015;144(3):473-483.

Abstract and Introduction

Abstract

Objectives: This study determined the longitudinal clinical performance of a high-risk human papillomavirus (HR-HPV) E6/E7 RNA assay (Aptima HPV [AHPV]; Hologic, San Diego, CA) compared with an HR-HPV DNA assay (Hybrid Capture 2 [HC2]; Qiagen, Gaithersburg, MD) as an adjunctive method for cervical cancer screening.

Methods: Women 30 years or older with a negative result for intraepithelial lesions or malignancy cytology (n = 10,860) positive by AHPV and/or HC2 assays and randomly selected women negative by both assays were referred to colposcopy at baseline. Women without baseline cervical intraepithelial neoplasia (CIN) grade 2 or higher (CIN2+) continued into the 3-year follow-up.

Results: The specificity of AHPV for CIN2 or lower was significantly greater at 96.3% compared with HC2 specificity of 94.8% (P < .001). Estimated sensitivities and risks for detection of CIN2+ were similar between the two assays. After 3 years of follow-up, women negative by either human papillomavirus test had a very low risk of CIN2+ (<0.3%) compared with CIN2+ risk in women with positive AHPV results (6.3%) or positive HC2 results (5.1%).

Conclusions: These results support the use of AHPV as a safe and effective adjunctive cervical cancer screening method.

Introduction

Cervical cancer is one of the most frequent cancers in women worldwide, accounting for approximately 530,000 new cases and 275,000 deaths annually.^[1] Countries with well-organized screening programs using conventional Papanicolaou (Pap) stain cytology have experienced substantially reduced mortality from the disease in the past 5 decades.^[2–4] Despite this advance, the relatively low sensitivity and reproducibility of both conventional Pap smear and liquid-based cytology screening methods have prompted investigation into identifying adjunctive methods with Pap cytology for improving detection of cervical neoplasia.^[5–9]

Infection with 14 high-risk human papillomavirus (HR-HPV) genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) is associated with almost all cases of cervical precancer, defined as cervical intraepithelial neoplasia (CIN) grade 2 (CIN2), grade 3 (CIN3), and cancer.^[10] Addition of HR-HPV nucleic acid testing to a cervical cytology screening regimen offers higher sensitivity and negative predictive value (NPV) for detection of cervical precancer and cancer compared with cytology alone, especially in older women.^[11–15] For this reason, HR-HPV nucleic acid testing is recommended as an adjunctive test to cytology to assess the presence of HR-HPV types in women 30 years of age or older.^[16] In this context, HR-HPV testing guides patient management by identifying women at elevated risk for CIN2 or higher (CIN2+) but, importantly, also reassures women who are negative for HR-HPV of their extremely low cancer risk.^[17–19]

First-generation HR-HPV molecular tests used for adjunctive cervical cancer screening function by detecting viral genomic DNA in cellular samples from the uterine cervix. However, because the presence of HR-HPV in the female genital tract is common and often transient in nature,^[20,21] and most cervical HPV infections resolve without becoming cancerous,^[22,23] HR-HPV DNA-based test methods yield only moderate specificity for detection of high-grade cervical disease.^[12,24] This leads to unnecessary follow-up and referral of patients to colposcopy, increasing the physical and emotional burdens on patients and elevating health care costs.

A test approved by the US Food and Drug Administration (FDA) for detection of HR-HPV E6/E7 messenger RNA (mRNA) (Aptima HPV [AHPV]; Hologic, San Diego, CA) has shown higher specificity with similar sensitivity for detection of CIN2+ compared with HPV DNA-based tests in patients referred for colposcopy due to an abnormal Pap smear result as well as in a screening setting.^[25–30] Expression of mRNA from viral E6 and E7 oncogenes is highly associated with the development of CIN,^[31,32] and extensive investigation into the role of E6 and E7 oncoproteins in the human papillomavirus (HPV) life cycle has revealed that the expression of the corresponding oncogenes is necessary and sufficient for cell immortalization, neoplastic transformation, and the development of invasive cancer.^[33–35]

To confirm and extend the previous evidence on the clinical utility of HR-HPV oncogenic mRNA testing in a US population-based setting, the clinical performance of AHPV was evaluated as an adjunctive method for cervical cancer screening in women aged 30 years or older with negative for intraepithelial lesions or malignancy (NILM) cytology results from routine Pap testing in a pivotal, prospective, multicenter US clinical study including 3 years of follow-up (the Clinical Evaluation of Aptima mRNA [CLEAR] study). We report herein the results from this study.

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Abstract and Introduction
Materials and Methods
Results
Discussion
References

Table 1. Demographics of Evaluable Participants (n = 10,860)
Characteristic	Value
Age, y
Mean	44.2
Median	43
Minimum-maximum	30–89
IQR	15
Age groups, No. (%), y
30 to <40	4,192 (38.6)
≥40	6,668 (61.4)
Race/ethnicity, No. (%)
Non-Hispanic white	4,774 (44.0)
Hispanic white	1,814 (16.7)
Black	1,354 (12.5)
Asian	622 (5.7)
Other^a	488 (4.5)
Unknown	1,808 (16.6)

IQR, interquartile range.
^aOther includes American Indian, Alaska Native, Native Hawaiian, Pacific Islander, and multiple races.

Table 2. Disease Status at Baseline and After 3 Years of Follow-up and Corresponding AHPV and HC2 Test Results at Baseline
	Participants at Baseline, No.	AHPV+, No.			AHPV–, No.
	Participants at Baseline, No.	HC2+	HC2–	HC2 Missing^a	HC2+	HC2–	HC2 Missing^a
Disease status at baseline^b
Total No.	10,860	383	97	32	282	9,467	599
Verified
Normal	769	211	19	12	170	353	4
CIN1	29	12	0	1	7	9	0
CIN2	9	4	0	0	2	2	1
CIN3	8	7	0	0	1	0	0
AIS	3	2	1	0	0	0	0
CIN2+	20	13	1	0	3	2	1
CIN3+	11	9	1	0	1	0	0
Unverified	10,042	147	77	19	102	9,103	594
Disease status after 3-y follow-up^c
Total No.	10,843d	383	97	32	281	9,452	599
Normal	6,098	161	48	10	123	5,440	316
CIN1	56	10	0	0	6	36	4
CIN2	24	7	0	1	3	12	1
CIN3	20	14	0	1	2	3	0
AIS	3	2	1	0	0	0	0
CIN2+	47	23	1	2	5	15	1
CIN3+	23	16	1	1	2	3	0
Missing	4,378	167	44	17	130	3,756	264
Indeterminate	264	21	4	3	17	205	14

AHPV, Aptima HPV (Hologic, San Diego, CA); AIS, adenocarcinoma in situ; CIN1, cervical intraepithelial neoplasia grade 1; CIN2, cervical intraepithelial neoplasia grade 2; CIN2+, CIN2 or higher; CIN3, cervical intraepithelial neoplasia grade 3; CIN3+, CIN3 or higher; HC2, Hybrid Capture 2 (Qiagen, Gaithersburg, MD).
^aIn total, 631 women with AHPV assay results did not have HC2 test results primarily due to insufficient volume of the cytology specimen.
^bVerified disease status was determined for women who attended colposcopy at baseline and had a consensus histology result. Women without a consensus histology result have an unverified disease status.
^cDisease status after 3-year follow-up is based on completing 3-year follow-up with cytology performed at least once and colposcopy attendance for ASC-US or higher results during the first 2 years and during the third year.
^dSeventeen women were determined ineligible after completion of baseline; results are excluded from follow-up analyses.

Table 3. Absolute and Relative Risk of CIN2+ and CIN3+ Disease at Baseline (Verification Bias Adjusted)
Disease Status/Assay Result	AHPV Assay			HC2 Test
Disease Status/Assay Result	Absolute Risk (95% CI), %	Relative Risk (95% CI)	Prevalence, %	Absolute Risk (95% CI), %	Relative Risk (95% CI)	Prevalence, %
CIN2+
Positive	4.5 (2.7–7.4)	7.5 (2.1–26.3)	0.9	3.7 (2.3–6.1)	7.3 (1.6–33.5)	0.9
Negative	0.6 (0.2–1.9)			0.5 (0.1–2.1)
CIN3+
Positive	3.0 (1.6–5.5)	24.9 (2.0–307.0)	0.4	2.3 (1.3–4.1)	21.0 (1.0–423.8)	0.4
Negative	0.1 (0.0–1.7)			0.1 (0.0–2.4)

AHPV, Aptima HPV (Hologic, San Diego, CA); CI, confidence interval; CIN2+, cervical intraepithelial neoplasia grade 2 or higher; CIN3+, cervical intraepithelial neoplasia grade 3 or higher; HC2, Hybrid Capture 2 (Qiagen, Gaithersburg, MD).

Table 4. Cumulative Absolute and Relative Risk of CIN2+ and CIN3+ Disease by Age Group After 3-Year Follow-up (Life Table Analysis)
Disease Status/Age Group, y	Assay Result	AHPV Assay			HC2 Test
Disease Status/Age Group, y	Assay Result	Absolute Risk (95% CI), %	Relative Risk (95% CI)	Prevalence, %	Absolute Risk (95% CI), %	Relative Risk (95% CI)	Prevalence, %
CIN2+
Overall	Positive	6.32 (4.29–9.27)	23.94 (13.59–42.18)	0.55	5.12 (3.53–7.41)	22.39 (12.19–41.12)	0.55
	Negative	0.26 (0.17–0.41)			0.23 (0.14–0.38)
30–39	Positive	7.76 (4.81–12.40)	31.11 (13.04–74.21)	0.76	6.46 (3.99–10.39)	27.36 (10.88–68.80)	0.79
	Negative	0.25 (0.12–0.53)			0.24 (0.10–0.54)
≥40	Positive	4.51 (2.34–8.63)	16.57 (7.26–37.82)	0.42	3.77 (2.10–6.71)	16.85 (7.21–39.35)	0.40
	Negative	0.27 (0.16–0.46)			0.22 (0.12–0.42)
CIN3+
Overall	Positive	4.42 (2.76–7.03)	67.87 (25.32–181.88)	0.27	3.43 (2.14–5.48)	59.14 (20.09–74.12)	0.28
	Negative	0.07 (0.03–0.16)			0.06 (0.02–0.16)
30–39	Positive	5.74 (3.22–10.11)	102.84 (23.17–456.51)	0.44	4.78 (2.67–8.48)	171.50 (22.39–1,313.63)	0.45
	Negative	0.06 (0.01–0.22)			0.03 (0.00–0.20)
≥40	Positive	2.81 (1.27–6.16)	41.80 (10.53–166.00)	0.16	2.05 (0.93–4.52)	28.46 (7.15–113.20)	0.17
	Negative	0.07 (0.02–0.21)			0.07 (0.02–0.22)

Table 5. Clinical Sensitivity and Specificity for CIN2+ and CIN3+ Disease After 3-Year Follow-up ^a
HC2	AHPV, No.			Sensitivity/Specificity, % [No./Total No.] (95% CI)^b		Difference (95% CI)	P Value
HC2	Positive	Negative	Total	AHPV	HC2	Difference (95% CI)	P Value
CIN2+				55.3 [26/47] (41.2 to 68.6)	63.6 [28/44] (48.9 to 76.2)	–9.1 (–21.9 to 3.8)	.219
Positive	23	5	28
Negative	1	15	16
Missing/equivocal	2	1	3
Total	26	21	47
<CIN2				96.3 [5,925/6,154] (95.8 to 96.7)	94.8 [5,524/5,824] (94.3 to 95.4)	1.4 (0.9 to 1.9)	<.001
Positive	171	129	300
Negative	48	5,476	5,524
Missing/equivocal	10	320	330
Total	229	5,925	6,154
CIN3+				78.3 [18/23] (58.1 to 90.3)	81.8 [18/22] (61.5 to 92.7)	–4.5 (–24.4 to 15.3)	1.000
Positive	16	2	18
Negative	1	3	4
Missing/equivocal	1	0	1
Total	18	5	23
<CIN3				96.2 [5,941/6,178] (95.5 to 96.5)	94.7 [5,536/5,846] (94.1 to 95.2)	1.4 (1.0 to 1.9)	<.001
Positive	178	132	310
Negative	48	5,488	5,536
Missing/equivocal	11	321	332
Total	237	5,941	6,178

AHPV, Aptima HPV (Hologic, San Diego, CA); CI, confidence interval; CIN2+, cervical intraepithelial neoplasia grade 2 or higher; <CIN2, cervical intraepithelial neoplasia grade 2 or lower; CIN3+, cervical intraepithelial neoplasia grade 3 or higher; <CIN3, cervical intraepithelial neoplasia grade 3 or lower; HC2, Hybrid Capture 2 (Qiagen, Gaithersburg, MD).
^aDifferences (95% CI) and P values (McNemar exact test) are calculated including only women with both AHPV and Digene HC2 assay results (excluding samples with missing or equivocal Digene HC2 results).
^bSensitivity is reported for CIN2+ and CIN3+; specificity is reported for <CIN2 and <CIN3.