SUMMARY Although most cervical human papillomavirus type 16 (HPV16) infections become undetectable within 1–2 years, persistent HPV16 causes half of all cervical cancers. We used a novel HPV whole-genome sequencing technique to evaluate an exceptionally large collection of 5,570 HPV16-infected case-control samples to determine whether viral genetic variation influences risk of cervical precancer and cancer. We observed thousands of unique HPV16 genomes; very few women shared the identical HPV16 sequence, which should stimulate a careful re-evaluation of the clinical implications of HPV mutation rates, transmission, clearance, and persistence. In case-control analyses, HPV16 in the controls had significantly more amino acid changing variants throughout the genome. Strikingly, E7 was devoid of variants in precancers/cancers compared to higher levels in the controls; we confirmed this in cancers from around the world. Strict conservation of the 98 amino acids of E7, which disrupts Rb function, is critical for HPV16 carcinogenesis, presenting a highly specific target for etiologic and therapeutic research.

Specific HPV16 variant sublineages strongly influence risk of histologic types of precancer and cancer, and viral genetic variation may help explain its unique carcinogenic properties.

Objective In 2012, the United States Preventive Services Task Force (USPSTF) and a consensus of 25 organizations endorsed concurrent cytology and HPV testing (“cotesting”) for cervical cancer screening. Past screening and management guidelines were implicitly based on risks defined by Pap-alone, without consideration of HPV test results. To promote management that is consistent with accepted practice, new guidelines incorporating cotesting should aim to achieve equal management of women at equal risk of cervical intraepithelial neoplasia grade 3 and cancer (CIN3+). Methods We estimated cumulative 5-year risks of CIN3+ for 965,360 women aged 30–64 undergoing cotesting at Kaiser Permanente Northern California 2003–2010. We calculated the implicit risk thresholds for Pap-alone and applied them for new management guidance on HPV and Pap cotesting, citing 2 examples: HPV-positive/ASC-US and HPV-negative/Pap-negative. We call this guidance process “benchmarking”. Results LSIL, for which immediate colposcopy is prescribed, carries 5-year CIN3+ risk of 5.2%, suggesting that test results with similar risks should be managed with colposcopy. Similarly, ASC-US (2.6% risk) is managed with 6–12 month follow-up and Pap-negative (0.26% risk) is managed with 3-year follow-up. The 5-year CIN3+ risk for women with HPV-positive/ASC-US was 6.8% (95%CI 6.2% to 7.6%). This is greater than the 5.2% risk implicitly leading to referral to colposcopy, consistent with current management recommendations that HPV-positive/ASC-US should be referred for immediate colposcopy. The 5-year CIN3+ risk for women with HPV-negative/Pap-negative was 0.08% (95%CI 0.07% to 0.09%), far below the 0.26% implicitly required for a 3-year return and justifying a longer (e.g., 5-year) return. Conclusions Using the principle of “equal management of equal risks,” benchmarking to implicit risk thresholds based on Pap-alone can be used to achieve safe and consistent incorporation of cotesting.

Infection with a group of high-risk human papillomaviruses (HPV) has been identified as the cause of cervical cancer; thus, high-risk HPV testing is being incorporated into cervical cancer screening to improve cervical cancer prevention. Recently released U.S. guidelines recommend cotesting with HPV assays and cytology (1) as an alternative to the use of cytology alone. Moreover, primary stand-alone HPV testing is being introduced in multiple regions (2), including the United States (3). As a major advantage, HPV testing is more sensitive than cytology alone, and a negative HPV test result provides prolonged reassurance against cervical cancer, permitting the safe lengthening of screening intervals (4, 5).As a disadvantage, HPV testing is less specific than cytology, and optimal management is unclear for some of the nonnormal cytology/HPV combined results that occur with HPV testing, whether in the context of cotesting or primary HPV testing followed by cytology triage of HPV-positive women (6, 7). One prominent issue, that of HPV-positive/cytology-negative results, i.e., the finding of positive HPV test results when cytology result is negative, is common in absolute terms. For example, HPV-positive/cytology-negative results were found in nearly 4% of the cotest results in a recent large series of approximately 1 million women age 30 to 64 years at Kaiser Permanente Northern California (KPNC) (8, 9).The decision of how to manage HPV-positive/cytology-negative results is not straightforward. Recently, U.S. consensus guideline groups have issued recommended management strategies based on comparisons of the risks of cervical intraepithelial neoplasia grade 3 (CIN3) or cancer (CIN3ϩ, including adenocarcinoma in situ). These consensus guidelines form the basis for the consistent management of women with similar risks. Specifically, for women with HPV-positive/cytology-negative results, the attendant risk is not quite high enough for immediate colposcopy (1, 10). In comparison, immediate colposcopy is recommended for cytologically evident HPV infection (HPV-positive atypical squamous cells of undetermined significance [ASC-US] or lowgrade squamous intraepithelial lesion [LSIL]) (10). Cytologically evident HPV infection confers a 5-year risk of CIN3ϩ that is only slightly higher than that for HPV-positive/cytology-negative results. However, referring all women with HPV-positive/cytologynegative results would more than double the number of colposcopic procedures, and many of those women would not yet have colposcopically diagnosable lesions (8).Thus, the guidelines recommend managing HPV-positive/cy-

Objective New screening guidelines recommend that HPV-negative/ASC-US results be considered as equivalent to HPV-negative/Pap-negative results, leading to rescreening in 5 years. However, despite ample research data, the routine clinical performance of HPV testing of women with ASC-US has not been adequately documented. Methods We estimated 5-year risks of CIN3+ and cancer for 2 groups between 2003-2010 at Kaiser Permanente Northern California: 27,050 women aged 30-64 who underwent HPV and Pap cotesting and had an ASC-US Pap result, and 12,209 women aged 25-29 who underwent HPV triage of ASC-US. Results Five-year risks of CIN3+ and of cancer for women aged 30-64 testing HPV-negative/ASC-US and for 923,152 women testing Pap-negative alone were similar although statistically distinguishable (CIN3+: 0.43% vs. 0.26% (p=0.001); Cancer: 0.050% vs. 0.025% (p=0.1, respectively)). The cancer risk increase for HPV-negative/ASC-US versus Pap-negative alone was confined to women aged 60-64 (0.26% vs. 0.035%, p=0.3). Five-year risks of CIN3+ and of cancer for women with HPV-negative/ASC-US were substantially higher than those for women testing HPV-negative/Pap-negative (CIN3+: 0.43% vs. 0.08% (p<0.0001); Cancer: 0.050% vs. 0.011% (p=0.003, respectively)). For women aged 30-64 testing HPV-positive/ASC-US, 5-year risks of CIN3+ and cancer were slightly higher than for the 9,374 women with LSIL (CIN3+: 6.8 % vs. 5.2% (p=0.0007); Cancer: 0.41% vs. 0.16% (p=0.04)). Similar patterns were seen for women aged 25-29. Conclusions Women with HPV-negative/ASC-US had similar risk as women testing Pap-negative alone, but had higher risk than women testing HPV-negative/Pap-negative. Based on the principle of “equal management of equal risks”, our findings support equal management of women with HPV-negative/ASC-US and those with Pap-negative alone, except for exiting women from screening because cancer risks at ages 60-64 may be higher for HPV-negative/ASC-US. Our findings also support managing HPV-positive/ASC-US and LSIL similarly. Précis Women testing HPV-negative/ASC-US have similar risk of CIN3+ or cancer as women testing Pap-negative alone, but have higher risk than women testing HPV-negative/Pap-negative.

Background: HPV testing is replacing cytology for cervical cancer screening because of greater sensitivity and superior reassurance following negative tests for the dozen HPV genotypes that cause cervical cancer. Management of women testing positive is unresolved. The need for identification of individual HPV genotypes for clinical use is debated. Also, it is unclear how long to observe persistent infections when precancer is not initially found. Methods: In the longitudinal NCI-Kaiser Permanente Northern California Persistence and Progression (PaP) Study, we observed the clinical outcomes (clearance, progression to CIN3+, or persistence without progression) of 11,573 HPV-positive women aged 30À65 yielding 14,158 type-specific infections. Findings: Risks of CIN3+ progression differed substantially by type, with HPV16 conveying uniquely elevated risk (26% of infections with seven-year CIN3+ risk of 22%). The other carcinogenic HPV types fell into 3 distinct seven-year CIN3+ risk groups: HPV18, 45 (13% of infections, risks >5%, with known elevated cancer risk); HPV31, 33, 35, 52, 58 (39%, risks >5%); and HPV39, 51, 56, 59, 68 (23%, risks <5%). In the absence of progression, HPV clearance rates were similar by type, with 80% of infections no longer detected within three years; persistence to seven years without progression was uncommon. The predictive value of abnormal cytology was most evident for prevalent CIN3+, but less evident in follow-up. A woman's age did not modify risk; rather it was the duration of persistence that was important.

HPV16 causes half of cervical cancers worldwide; for unknown reasons, most infections resolve within two years. Here, we analyze the viral genomes of 5,328 HPV16-positive case-control samples to investigate mutational signatures and the role of human APOBEC3-induced mutations in viral clearance and cervical carcinogenesis. We identify four de novo mutational signatures, one of which matches the COSMIC APOBEC-associated signature 2. The viral genomes of the precancer/cancer cases are less likely to contain within-host somatic HPV16 APOBEC3-induced mutations (Fisher’s exact test, P = 6.2 x 10−14), and have a 30% lower nonsynonymous APOBEC3 mutation burden compared to controls. We replicate the low prevalence of HPV16 APOBEC3-induced mutations in 1,749 additional cases. APOBEC3 mutations also historically contribute to the evolution of HPV16 lineages. We demonstrate that cervical infections with a greater burden of somatic HPV16 APOBEC3-induced mutations are more likely to be benign or subsequently clear, suggesting they may reduce persistence, and thus progression, within the host.

For unknown reasons, there is huge variability in risk conferred by different HPV types and, remarkably, strong differences even between closely related variant lineages within each type. HPV16 is a uniquely powerful carcinogenic type, causing approximately half of cervical cancer and most other HPV-related cancers. To permit the large-scale study of HPV genome variability and precancer/cancer, starting with HPV16 and cervical cancer, we developed a high-throughput next-generation sequencing (NGS) whole-genome method. We designed a custom HPV16 AmpliSeq™ panel that generated 47 overlapping amplicons covering 99% of the genome sequenced on the Ion Torrent Proton platform. After validating with Sanger, the current “gold standard” of sequencing, in 89 specimens with concordance of 99.9%, we used our NGS method and custom annotation pipeline to sequence 796 HPV16-positive exfoliated cervical cell specimens. The median completion rate per sample was 98.0%. Our method enabled us to discover novel SNPs, large contiguous deletions suggestive of viral integration (OR of 27.3, 95% CI 3.3–222, P=0.002), and the sensitive detection of variant lineage coinfections. This method represents an innovative high-throughput, ultra-deep coverage technique for HPV genomic sequencing, which, in turn, enables the investigation of the role of genetic variation in HPV epidemiology and carcinogenesis.