A Comparison of Linear Array and Hybrid Capture 2 for Detection of Carcinogenic HPV, and Cervical Precancer in ALTS

Abstract

Background

We were interested in comparing the performance of Linear Array (LA; Roche Molecular Systems, Alameda, CA) to Hybrid Capture 2 (hc2; Digene Corporation, Gaithersburg, MD) for the detection of carcinogenic HPV and cervical precancer.

Methods

LA and hc2 results were compared on baseline specimens collected from women with an atypical squamous cells of undetermined significance (ASCUS) Pap referred into ALTS (n = 3,488). Hybrid Capture 2 was conducted at the time of the study on liquid cytology specimens. LA was conducted retrospectively on aliquots from a second, stored cervical specimen masked to the hc2 results and clinical data. Paired LA and hc2 results (n = 3,289, 94%) were compared for the detection of carcinogenic HPV (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) and 2-year cumulative ≥CIN3 as diagnosed by the quality control pathology review.

Results

LA was more likely to test positive for carcinogenic HPV than hc2 (55% vs. 53%, p = 0.001). For two-year cumulative ≥CIN3, LA and hc2 had similar sensitivities (93.3% vs. 92.6%, respectively, p = 1.0), and LA was marginally less specific than hc2 (48.1% vs. 50.6%, respectively, p = 0.05). LA and hc2 had similar negative predictive values (98.70% vs. 98.64% respectively, p = 0.4), and LA had a slightly lower PPV than hc2 (14.6% vs. 15.1%, respectively, p < 0.0001).

Conclusion

We observed that LA and hc2 performed similarly in the detection of carcinogenic HPV and identification of CIN3 among women with an ASCUS Pap.

INTRODUCTION

It is now generally recognized and accepted that approximately 15 cancer-associated (carcinogenic) human papillomavirus (HPV) genotypes cause virtually all cervical cancer and its immediate precursor lesions, most importantly cervical intraepithelial neoplasia grade 3 (CIN3) (“cervical precancer”). HPV infections are common and typically resolve within 1-2 years. Uncommonly, carcinogenic HPV infections can persist, and this persistence is strongly associated with cervical precancer, which if left untreated can lead to invasive disease.

Based on knowledge of the central role for persistent, carcinogenic HPV in the development of cervical cancer, carcinogenic HPV testing offers several advantages over cytology including: [1] greater sensitivity for the detection of CIN3 or cancer (≥CIN3) (1-7); [2] as a consequence of greater sensitivity, higher negative predictive value i.e., testing negative for carcinogenic HPV DNA implies an extremely low risk of prevalent or incipient cancer/CIN3 (1,2,8), permitting an extension of screening intervals; and [3] greater reproducibility (9,10). Accordingly, carcinogenic HPV testing has now been approved in the United States as an adjunct to cytology for triage of equivocal cytology at all ages and for general screening in women ≥30 years old (11).

One pooled probe test for detection of the carcinogenic genotypes of HPV DNA (as a group), Hybrid Capture 2 (hc2, Digene Corporation, Gaithersburg, MD, USA), has already been FDA-approved. Other tests are in development or being validated and are expected to become widely available soon. One of these tests, Linear Array (LA; Roche Molecular Systems, Alameda, CA, USA), is a PCR-based assay that detects 37 HPV genotypes individually, including the main 14 carcinogenic HPV genotypes. LA is the commercialized (research use-only) version of the Line Blot Assay (Roche) that has been used in numerous epidemiological and clinical studies (12-17), including the Atypical Squamous Cells of Undetermined Significance (ASCUS) and Low-Grade Squamous Intraepithelial Lesion (LSIL) Triage Study (ALTS) (14,17,18). Using baseline specimens from women referred into ALTS because of an ASCUS (equivocal) Pap, we sought to evaluate the performance of LA, compared to the referent standard of hc2, for detection of carcinogenic HPV and triage of ASCUS Paps for identifying women with ≥CIN3.

SUBJECTS AND METHODS

Study Design and Population

ALTS was a randomized trial comparing three management strategies for 5,060 women with ASCUS (n = 3,488) or LSIL (n = 1,572) Pap (19): 1) immediate colposcopy (IC arm) (referral to colposcopy regardless of enrollment test results); 2) HPV triage (HPV arm) (referral to colposcopy if enrollment HPV result was positive by hc2 or missing or if the enrollment cytology was high-grade squamous intraepithelial lesion [HSIL] for patient safety); or 3) conservative management (CM arm) (referral to colposcopy at enrollment if cytology was HSIL). At enrollment, all women received a pelvic examination with collection of two cervical specimens; the first specimen in PreservCyt for ThinPrep cytology (Cytyc Corporation, Marlborough, MA, USA) and hc2 testing and the second in specimen transport medium (STM; Digene Corporation). Women in all 3 arms of the study were reevaluated by cytology every 6 months for 2 years of follow-up and sent to colposcopy if cytology was HSIL. An exit examination with colposcopy was scheduled for all women, regardless of study arm or prior procedures, at the completion of the follow-up. We refer readers to other references for details on randomization, examination procedures, patient management, and laboratory and pathology methods (19-23). The National Cancer Institute and local institutional review boards approved the study and all participants provided written informed consent. This evaluation was restricted to women referred for an ASCUS Pap because previous work indicated that carcinogenic HPV testing was not clinically useful in young women with LSIL Paps, as more than two-thirds of the women were positive (20).

HPV DNA Testing

Residual PreservCyt specimens, after being used for liquid-based cytology, were tested by hc2 (23), a pooled-probe, signal-amplification DNA test that targets a group of 13 HPV genotypes (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68) (24). Per the manufacturer's instructions, a protocol for converting the liquid cytology samples into an STM equivalent was used. In addition, hc2 cross-reacts strongly with a 14^th carcinogenic HPV genotype, HPV66 (25-27), as well as other untargeted, non-carcinogenic HPV genotypes including HPV53. Hybrid Capture 2 signal strength (relative light units per positive control [RLU/CO]) was used as semi-quantitative measure of HPV viral load (“HPV semi-quantitative viral load”)(28). An equivalent of 10% of each original specimen was used for hc2 testing

Aliquots of the archived, enrollment STM specimens were retrospectively tested by LA (29), which employs L1 consensus primers PGMY09/11 for amplification, and amplicons were subjected to reverse-line blot hybridization for detection of 37 HPV individual genotypes (HPV 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51-56, 58, 59, 61, 62, 64, 66-73, 81, 82, 82v, 83, 84, and 89). Because of intellectual property rights, LA does not directly detect HPV52 but combines a set of probes that detects HPV33, 35, 52 and 58 combined (HPVmix). Specimens that test negative for HPV33, 35, and 58 individually, but are positive for the HPVmix, are considered to be HPV52 positive. The specimens that test positive for the HPVmix and HPV33, 35, and/or 58 have an uncertain HPV52 status, and for this analysis these specimens were considered to be HPV52 negative. LA was used according to the manufacturer's instructions in the product insert, which includes DNA extraction using the QIAamp MinElute Media Kit (QIAGEN, Inc., Valencia, CA). The only deviation from the LA product insert protocol was to implement an automated sample preparation for extraction of up to 96 specimens at a time on the QIAGEN MDx platform (using the MinElute Media MDx Kit and manufacturer's instructions) rather than processing 24 specimens per batch with the manual vacuum method (30,31). An equivalent of 2.8% of each original specimen was used for LA testing. Specimens were tested by LA a median of 104 months after collection, masked to the hc2 results and clinical data.

HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 were considered the primary carcinogenic genotypes (32,33). We included HPV66 in our definition because it was recently re-classified as a carcinogenic HPV genotype (33), and it is well-known that hc2 strongly detects HPV66 although it is not one of the 13 genotypes directly targeted by hc2 (25,26,34). In fact, in a previous analysis, we found hc2 to be more likely to be positive in the presence of HPV66 than in the presence of HPV68, one of the hc2-targeted HPV genotypes [Schiffman]. LA results were assigned a HPV risk group according to a priori established cervical cancer risk (e.g., HPV16 causes 50%-60% of all cancers, HPV18 causes 15%-20% of all cancers, and the other 12 carcinogenic HPV genotypes cause the remaining cases of cancer): (1) positive for HPV16; (2) else positive for HPV18 and negative for HPV16; (3) else positive for any carcinogenic HPV genotypes and negative for HPV16 and HPV18 (carcinogenic HPV exc. HPV16 & HPV18); (4) else positive for any non-carcinogenic HPV genotypes and negative for all carcinogenic genotypes; or (5) LA negative (HPV16>HPV18> carcinogenic HPV exc. HPV16 & HPV18>non-carcinogenic HPV>PCR negative).

Pathology and Treatment

Clinical management was based on the clinical center pathologists' cytologic and histologic diagnoses. In addition, all referral smears, ThinPreps, and histology slides were sent to the Pathology Quality Control Group (QC pathology) based at the Johns Hopkins Hospital for independent review and diagnosis. CIN2 or worse (≥CIN2) diagnosis based on the clinical center pathology or a CIN3 or worse (≥CIN3) diagnosis based on the QC pathology review triggered treatment by Loop Electrosurgical Excision Procedure (LEEP). In addition, women with persistent LSIL or carcinogenic HPV-positive ASCUS at the time of the exit from the study were offered LEEP.

Statistical Analysis

Detection of carcinogenic HPV by LA and hc2 was compared by calculating kappa values, percent total agreement, and percent positive agreement (i.e., agreement among paired tests in which at least one was positive). Differences in detection were tested for statistical significance (p < 0.05) using an exact McNemar's χ² (2 categories). Results of paired tests were also compared following stratification on enrollment ThinPrep cytology results (negative vs. ASCUS or more severe; alternatively, negative, ASCUS, LSIL, and high-grade squamous intraepithelial lesion [HSIL] including atypical squamous cells, cannot rule out HSIL [ASC-H]) (n.b., because of the variability of cytologic interpretation (35), the enrollment ThinPrep cytology results differed from the referral cytology). LA results were also stratified on hc2 status (negative, positive) and hc2-derived log units of the HPV semi-quantitative viral load (1.00-9.99, 10.00-99.99, 99.99-999.99, and 1000+ RLU/CO).

Sensitivity, specificity, positive (PPV) and negative (NPV) predictive values, and Youden's Index (YI) were calculated for histologically-confirmed CIN3 or worse (≥CIN3) as diagnosed by QC pathology at enrollment restricted to the IC arm in which all women were referred to colposcopy (and therefore there was no bias in referral) and over the 2-year duration of ALTS in all arms combined since the 2-year cumulative ≥CIN3 did not differ by study arm, only in the timing of diagnosis. In secondary analyses, the clinical performance was also calculated for histologically-confirmed CIN2 or worse (≥CIN2) as diagnosed by the clinical center pathologist at enrollment (restricted to the IC arm) and over the 2-year duration of ALTS, restricted to IC and HPV arms of the trial because of the evidence of regression of CIN2 in the CM arm of the trial (21,22). Differences in test sensitivity and specificity for ≥CIN3 and ≥CIN2 were tested for statistical significance using an exact McNemar's χ². Differences in PPV and NPV were tested for statistical significance using a method developed by Leisenring et al. (36). Differences in YI were tested for statistical significance as previously described (3). Finally, the 2-year cumulative absolute risks of histologically-confirmed ≥CIN3 as diagnosed by QC pathology were calculated for testing positive or negative at enrollment for carcinogenic HPV as detected by LA or hc2. These risks were also calculated for LA-defined subgroups of HPV genotypes and for log-unit categories of semi-quantitative viral load as measured by hc2.

RESULTS

Carcinogenic HPV Detection

Among the women referred into ALTS because of an ASCUS Pap, 3,289 (94%) had valid baseline results for hc2 using PreservCyt specimens (missing hc2 results were primarily the result of insufficient specimen volume [<4 mL] for testing), 3,446 (99%) had valid results for LA testing of archived, baseline STM specimens, and 3,289 (94%) had valid results for both tests. Restricting our analyses to paired results, LA was more likely to test positive for carcinogenic HPV than hc2 (55% vs. 53%, p = 0.001), with a percent agreement of 84%, a percent positive agreement of 74%, and a kappa of 0.68 (Table 1). Both tests were more likely to test positive for carcinogenic HPV among women with an enrollment cytology of ASCUS or worse (72% for LA vs. 74% for hc2, p = 0.003) than women with negative cytology (38% for LA vs. 30% for hc2, p < 0.0001). There was also better agreement between tests among women with an enrollment cytology of ASCUS or worse compared to women with negative cytology. Among women with enrollment LSIL and HSIL cytology, LA was less likely to test positive compared to hc2 (p < 0.0001 and p = 0.03, respectively). Among hc2-positive women, the proportion of LA tests positive for carcinogenic HPV increased with increasing semi-quantitative viral load categorized in log units (p_trend < 0.0001).

Table 1.

A comparison of and agreement for detection of carcinogenic HPV by Linear Array (LA) and Hybrid Capture 2 (hc2) for all paired tests, stratified by cytologic interpretations, and stratified by Hybrid Capture 2 status (negative, positive) and by log units of hc2 signal strength (RLU/CO), semi-quantitative measure of viral load, for women referred into ALTS for an ASCUS Pap. Row percentages are shown beneath number in each cell. Differences were tested for statistical significance using an exact McNemar's χ² test.

	N	hc2+	LA+	LA-/hc2-	LA-/hc2+	LA+/hc2-	LA+/hc2+	%Agreement	%Positive Agreement	Kappa	P
All hc2	3,289	1,749
		53%
All LA	3,446		1,914
			56%
All Pairs	3,289	1,749	1,824	1,238	227	302	1,522	84%	74%	0.68	0.001
		53%	55%	38%	7%	9%	46%
Cytology Negative	1,535	465	580	862	93	208	372	80%	55%	0.57	<0.0001
		30%	38%	56%	6%	14%	24%
Cytology Positive (≥ASC-US)	1,707	1,266	1,222	354	131	87	1,135	87%	84%	0.68	0.003
		74%	72%	21%	8%	5%	66%
ASC-US	822	456	465	295	62	71	394	84%	75%	0.67	0.4
		55%	57%	36%	8%	9%	48%
LSIL	581	535	493	37	51	9	484	90%	89%	0.50	<0.0001
		92%	85%	6%	9%	2%	83%
HSIL (including ASC-H)	304	275	264	22	18	7	257	92%	91%	0.59	0.03
		90%	87%	7%	6%	2%	85%
hc2-Positive (RLU/CO: 1.00-9.99)	409		287		122		287
			70%		30%		70%
hc2-Positive (RLU/CO: 10.00-99.99)	478		418		60		418
			87%		13%		87%
hc2-Positive (RLU/CO: 100.00-999.99)	621		582		39		582
			94%		6%		94%
hc2-Positive (RLU/PC: 1000.00+)	241		235		6		235
			98%		2%		98%

Clinical Performance

There were no significant differences between LA and hc2 in clinical sensitivity (89.1% vs. 90.9%, respectively, p = 1) and specificity (46.5% vs. 47.8%, respectively, p = 0.3) for detection of enrollment QC pathology-diagnosed ≥CIN3 (n = 55) in the IC arm (Table 2). Using Youden's Index (YI) (sensitivity + specificity -100%) as a measure of accuracy, LA appeared very slightly less accurate than hc2 (35.6% vs. 38.7%) but the difference was not significant (p = 0.4). LA had a slightly lower PPV than hc2 (8.1% vs. 8.5%, p = 0.0009), which was statistically but perhaps not clinically significant. The NPV for both assays were similar (98.77% for LA vs. 99.00% for hc2). Similar estimates for clinical performance parameters for both assays and similar differences between assays were observed when diagnoses of ≥CIN2 by the clinical center pathologists in the IC arm were used as the endpoint.

Table 2.

Clinical performance of carcinogenic HPV detection by Linear Array (LA) and Hybrid Capture 2 (hc2) for enrollment ≥CIN3 as diagnosed by QC pathology and ≥CIN2 as diagnosed by clinical center pathologists in women referred into ALTS for an ASCUS Pap and restricted to women participating in the IC arm (n = 1,089). Differences in sensitivity, specificity, and referral were tested for statistical significance using an exact McNemar's χ² test. Statistical differences in positive (PPV) and negative (NPV) predictive values were a method developed by Leisenring et al. (36), a score statistic derived from a marginal regression model and bears some relation to McNemar's statistic.

	Linear Array		hc2
	Estimate	95%CI	Estimate	95%CI	P
QC Pathology ≥CIN3 (n = 55)
Sensitivity	89.1%	77.8%-95.9%	90.9%	80.0%-97.0%	1
Specificity	46.5%	43.4%-49.6%	47.8%	44.7%- 50.9%	0.3
PPV	8.1%	6.1%-10.6%	8.5%	6.4%-11.0%	0.0009
NPV	98.77%	97.34%-99.55%	99.00%	97.68%-99.67%	0.8
Referral	55.3%	52.3%-58.3%	54.2%	51.2%-57.2%	0.4
CC Pathology ≥CIN2 (n = 113)
Sensitivity	87.6%	80.1%-93.1%	89.5%	82.3%-94.4%	0.8
Specificity	48.2%	45.0%-51.3%	50.0%	46.8%-53.2%	0.3
PPV	16.3%	13.4%-19.5%	17.3%	14.3%-20.6%	<0.0001
NPV	97.12%	95.22%-98.42%	97.60%	95.84%-98.75%	0.8
Referral	55.5%	52.5%-58.5%	54.1%	51.1%-57.1%	0.4

We also considered the clinical performance of both assays for two-year cumulative QC pathology-diagnosed ≥CIN3 (in all arms) (Table 3), thereby correcting for the differences in sensitivity by study arm and initial clinical management (37). LA and hc2 had similar sensitivities (93.3% vs. 92.6%, respectively, p = 1) and LA was marginally less specific (48.1% vs. 50.6%, respectively, p = 0.05). The YI for LA, 42.2%, was similar to the YI for hc2, 43.2%, (p = 0.4). The PPV for LA was lower than for hc2 (14.6% vs. 15.1%, respectively, p < 0.0001) and there was no statistically significant difference in NPV between LA and hc2 (98.70% vs. 98.64%, respectively, p = 0.4). LA was less sensitive (87.8% vs. 91.0%, p = 0.09) and less specific (50.4% vs. 53.1%, p = 0.007) than hc2 for clinical center pathology-diagnosed ≥CIN2. The YI was lower for LA (38.2%) than for hc2 (44.1%) (p = 0.003).

Table 3.

Clinical performance of carcinogenic HPV detection by Linear Array (LA) and Hybrid Capture 2 (hc2) for two-year cumulative ≥CIN3 as diagnosed by QC pathology (n = 3,289) and ≥CIN2 as diagnosed by clinical center pathologists (n = 2,189, restricted to women in the IC and HPV arms) referred into ALTS for an ASCUS Pap. Differences in sensitivity, specificity, and referral were tested for statistical significance using an exact McNemar's χ² test. Statistical differences in positive (PPV) and negative (NPV) predictive values were determined by Statistical differences in positive (PPV) and negative (NPV) predictive values were a method developed by Leisenring et al. (36), a score statistic derived from a marginal regression model and bears some relation to McNemar's statistic.

	Linear Array		hc2
	Estimate	95%CI	Estimate	95%CI	P
QC Pathology ≥CIN3 (n = 285)
Sensitivity	93.3%	89.8%-95.9%	92.6%	89.0%-95.4%	1
Specificity	48.1%	46.3%-49.9%	50.6%	48.8%-52.4%	0.05
PPV	14.6%	13.0%-16.3%	15.1%	13.4%-16.9%	<0.0001
NPV	98.70%	97.98%-99.22%	98.64%	97.92%-99.15%	0.4
Referral	55.5%	53.7%-57.2%	53.2%	51.5%-54.9%	0.001
CC Pathology ≥CIN2 (n = 354)
Sensitivity	87.8%	83.9%-91.1%	91.0%	87.5%-93.8%	0.09
Specificity	50.4%	48.1%-52.7%	53.1%	50.8%-55.4%	0.007
PPV	24.8%	22.4%-27.3%	26.5%	24.0%-29.2%	<0.0001
NPV	95.68%	94.20%- 96.87%	96.93%	95.67%-97.91%	0.8
Referral	55.6%	53.5%-57.7%	53.9%	51.7%-56.0%	0.04

The 2-year cumulative risk of ≥CIN3 diagnosed by QC pathology for women with and without carcinogenic HPV considering the results of the two tests jointly is shown in Table 4. The risk of ≥CIN3 was <1.5% for women who tested negative for carcinogenic HPV and approximately 15% for women who tested positive by either test. Women who tested positive for HPV16 by LA (n = 563) were at a higher 2-year cumulative risk (28.6%) of ≥CIN3 than women with HPV18 (9.5%) or other carcinogenic HPV genotypes (8.2%). Women positive for any α9 genotype excluding HPV16 (n = 729) (HPV31, 33, 35, 52, 58, and 67) were at an 11.4% 2-year cumulative risk of ≥CIN3; women positive for any α7 genotype excluding HPV18 (n = 732) (HPV39, 45, 59, 68, and 70) were at a 12.4% 2-year cumulative risk of ≥CIN3 (data not shown). For comparison, we consider risk stratification for 2-year cumulative risk of ≥CIN3 using hc2-measured semi-quantitative HPV viral load. Women with higher semi-quantitative viral loads (10 RLU/CO or greater) were at about a 2-fold higher 2-year cumulative risk for ≥CIN3 than women with the lowest viral load (1-9.99 RLU/CO) (7.6%), but there was no trend with increasing viral load (17.4% for 10-99.99 RLU/CO, 18.2% for 100-999.99 RLU/CO, and 15.4% for 1000+ RLU/CO).

Table 4.

Two-year cumulative absolute risk of histologically-confirmed cervical intraepithelial neoplasia grade 3 or more severe (≥CIN3) as diagnosed by QC pathology for carcinogenic HPV detection by Linear Array vs. Hybrid Capture 2 referred into ALTS because of an ASCUS Pap. LA results were also stratified on HPV risk group, and hc2 results were stratified on log units of signal strength (RLU/CO), a semi-quantitative measure of viral load. Carc., positive for any carcinogenic HPV genotype other than HPV16 or HPV18; Non-carc., positive for any of non-carcinogenic HPV genotypes

		QC Pathology
	N	N≥CIN3	%≥CIN3
Linear Array
Carcinogenic HPV
Negative	1,465	19	1.3%
Positive	1,824	266	14.6%
total	3,289	285	8.7%
HPV Risk Group
Negative	1,030	11	1.1%
Non-Carc.	435	8	1.8%
Carc.	1,103	90	8.2%
HPV18	158	15	9.5%
HPV16	563	161	28.6%
total	3,289	285	8.7%
Hybrid Capture 2
Carcinogenic HPV
Negative	1,540	21	11.4%
Positive	1,749	264	15.1%
total	3,289	285	8.7%
Semi-Quantitative Viral Load
1-9.99 (RLU/CO)	410	31	7.6%
10-99.99 (RLU/CO)	477	83	17.4%
100-999.99 (RLU/CO)	621	113	18.2%
1000+(RLU/CO)	241	37	15.4%
Total	3,289	285	8.7%

DISCUSSION

We compared detection of oncogenic HPV genotypes by LA to hc2, as a benchmark of performance, and found the two assays very similar for detection of carcinogenic HPV and ≥CIN3. Each assay offers different advantages over the other. Hybrid Capture 2 demonstrated slightly greater clinical specificity and therefore marginally better PPV than LA. Conversely, LA offers the advantage of HPV genotyping for identifying women at the greater risk of ≥CIN3 among carcinogenic HPV-positive women. These high-risk groups include women with HPV16 infection as shown here and in previous reports (18,38-41). Additionally, LA may confer an advantage of potentially identifying those women with genotype-specific persistent carcinogenic HPV infections that are at the greatest risk of having or getting cervical precancer or cancer (42-44).

When we used two-year cumulative, clinical center pathology diagnoses of ≥CIN2 as our endpoint, the differences between hc2 and LA were more pronounced, with hc2 showing marginally greater sensitivity than LA due to the detection of CIN2 caused by non-carcinogenic HPV genotypes. Eleven of the 22 CIN2 diagnoses that tested hc2 positive and LA negative for carcinogenic HPV were positive for non-carcinogenic HPV genotypes as detected by LA, and 10 of 11 were confirmed as having non-carcinogenic HPV genotypes (and no carcinogenic HPV genotypes) by Line Blot Assay, the prototype assay for LA (17,45) (data not shown). This is consistent with previous reports that hc2 cross-reacts with some non-carcinogenic HPV genotypes (25,26), and as shown here, these genotypes occasionally cause CIN2. In addition, some of these untargeted HPV genotypes, such as HPV73 and HPV82, are possibly carcinogenic (46). Although CIN2 is the threshold diagnosis for treatment, CIN2 is equivocal precancer (47), representing a mix of HPV infections of carcinogenic HPV and non-carcinogenic HPV that cause histopathologic changes such as CIN1, most likely to regress, and CIN3, which is precancerous and may invade if left undetected and untreated. It is also a poorly reproducible diagnosis (35) partly as a consequence of its ambiguous nature and because of the technical difficulties in mounting biopsies. It is therefore important when interpreting clinical performance for detection of ≥CIN2 to account for clinically-irrelevant diagnoses of CIN2. For these reasons, we emphasized ≥CIN3 as our primary and more rigorous endpoint.

We previously has shown that LA was more analytically sensitive for any carcinogenic HPV and HPV risk groups compared to its prototype assay, Line Blot Assay (LBA)(48). This is the likely explanation of why LA was more likely to test positive and was less clinically specific than hc2, the converse of what we observed in our comparison of LBA with hc2 (17). LA was more likely to test positive among women with normal cytology, which led to greater overall likelihood of testing positive compared to hc2. Conversely, hc2 was more likely than LA to test positive among women with LSIL and HSIL cytology. It was previously observed hc2 was more likely than another PCR assay, SPF₁₀LiPA₂₅, to test positive among women with LSIL cytology, which is indicative of a productive HPV infection producing higher viral loads compared with normal cytology. We suggest that viral load of the cervical specimen may have a greater influence on hc2 test performance than on PCR-based assay performance.

One additional advantage of LA has over hc2 is the internal β-globin control to verify the adequacy of the specimen. Although specimen inadequacy is probably an uncommon occurrence, the lack of internal control for hc2 is a concern. It is also noteworthy that LA uses a smaller fraction of the specimen than hc2 so that its use as a co-test with liquid cytology specimens is also less apt to have a missing test result due to insufficient specimen volume.

One limitation of our analysis was LA and hc2 were conducted on different specimens and in different manners. LA was conducted on STM specimens, a specimen genotype for which it has not been optimized, a median of 104 months after collection whereas hc2 was conducted on PreservCyt specimens in real time after cytology slides were made. We did not conduct the LA testing on the archived PreservCyt specimens, which had been stored at ambient temperature since 1997-8, because we found LA performed less sensitively on the PreservCyt specimens than the STM specimens in a pilot study (data not shown). Yet, the performance of LA was very similar to hc2, indicating that LA may be a robust assay as performed in an expert laboratory.

Although we found similar clinical performance between the two assays, we point out that clinical performance is only one of several important considerations for introducing a molecular test into clinical practice. Other criteria include reproducibility and capacity for high through-put. Hybrid Capture 2 testing has been shown to be reproducible (9,10,49,50) and using the automation provided by Rapid Capture® System (Digene) (http://www.digene.com/labs/labs_rapid_01.html) can achieve high-volume through-put. LA has neither demonstrated reproducibility nor high-volume capacity, both of which will be needed before LA can be widely used in cervical cancer screening programs.

LA has been approved for use in Europe and is currently in clinical trials, and it will be important to verify its clinical performance and establish reproducibility as criteria for clinical use. We compared LA to Hybrid Capture, which has repeatedly demonstrated both good clinical performance (1,7,51,52) and reliability (9,10,49,50), and found the two tests comparable. If and when it is FDA-approved and has high through-put capacity, LA may be another valuable screening tool for the secondary prevention of cervical cancer.