HPV16 variant lineage, clinical stage, and survival in women with invasive cervical cancer

Patient Demographics

Clinico-pathologic variables

Of 149 women with definite clinical outcome, the overwhelming majority, 91.3%, were diagnosed with SCC. 16-E variants were identified in 119 (87.5%) SCC lesions. These accounted for 93.7% of all 16-E variant cases in this population. Although SCC cancers harboring 16-E variants dominated in this population, and 16-E cases were more frequent in all other histologic categories as well, the percentage of 16-NE variants ranged from 44.4% (4 of 9) of CAC compared with 25% (1 of 4) for ASC and 12.5% (17 of 136) for SCC. The distribution of individual 16-NE variant categories for histologic cell type is as follows: AA = 12 SCC and 4 CAC; NA-1 = 3 SCC; AF1 = 1 SCC and AF2 = 1 SCC and 1 ASC. Despite the disparity in case numbers, the differences in the distribution of HPV16-variants among the histologic cell types were statistically significant (p = 0.03).

There was no significant difference in the distribution of HPV16 variants according to tumor size, depth of invasion, vascular space invasion, lymph node involvement, parametrial involvement, or status of surgical margins. Similarly, none of the clinical parameters showed a significant difference associated with 16-variant status including race/ethnicity, age group, FIGO stage and number of pregnancies.

Although significant differences were not identified between the 16-E and 16-NE variant groups for the above prognostic variables affecting survival in cervical cancer patients, there were interesting trends among 16-NE cases for some parameters that may reveal significance in a larger study with a larger population of 16-NE cases. These variables include younger age, early stage at presentation, three or fewer pregnancies, race/ethnicity other than white, depth of stromal invasion greater than 5 mm, and the presence of lymphovascular space invasion for 16-NE patients. While not statistically significant (p_trend = 0.13), the modal peak for 16-NE cases was the 31-40 age group (mean = 41.4 ± 2.6 years; median 38 years) compared with the 41-50 group for the 16-E cases (mean = 45.3 ± 1.0; median 45 years). Figure 1 illustrates that the majority of women with 16-NE lesions presented at stage IB1 while those with 16-E lesions presented at a more variable stage (p_trend = 0.02).

Because of the uneven distribution of cases in this study, a second analysis was performed that was restricted to the 123 cases of SCC associated with European variants. This population had a mean age of 45.9 years ± 1.1 (SEM), ranging from 20 to 76 years of age. Median follow-up was 28 months ± 22.4 (mean 33.0 ± 2.0). This population was divided into three categories as follows based on subgroups of European variants: European prototype (16-EP) (N = 53), European variants with a small number of nucleotide changes compared to prototype (16-EV) (N = 21), and those European variants harboring the 350 T>G nucleotide change (N = 49).

Cross-sectional analysis restricted to the subgroup of 123 women with SCC harboring European variants failed to demonstrate significant differences among the various clinical and pathologic traits for the different variant categories (Table 2). The 16-EP and 16-EV-G categories had similar characteristics. The 16-EV group was somewhat divergent but the numbers were small and the differences not significant. A comparison of 16-EV-G cases relative to all other 16-E SCC also showed no significant differences (data not shown).

Clinical Follow-up

After a median follow-up of 29.1 months ± 23.3, death due to cervical cancer was highly associated with 16-E variant status (p < 0.01). While 31% (41/127) of women harboring tumors with 16-E variants died from cervical cancer, only 1 of 23 (4.4%) 16-NE cases died of cervical cancer. There were no differences in follow-up status or total follow-up time within the subgroups of 16-E squamous cancers.

We attempted to explain the survival differences by adjustment (by stratification or restriction) for possible factors such as stage at diagnosis or age, which showed a relationship both to variant status and outcome. Even within Stage 1 cancers, however, for which survival was favorable, tumors with 16-E variants tended to have somewhat worse outcomes than those with 16-NE variants (data not shown).

Patient Population

From 1999-2009, 319 unselected cases of primary, metastatic and recurrent invasive cervical carcinoma were prospectively entered into this study based on the collection of a liquid-based cytologic sample that was tested for HPV DNA and a histologic diagnosis of invasive cervical cancer. The population seen at our institution is variably screened for cervical cancer so that some cancer patients were identified by routine screening while others presented with clinical signs and symptoms related to cervical cancer. Included in this total were 164 cases from the SUCCEED study [19] and 85 cases that were included in previous reports [20]. These women were staged according to FIGO (International Federation of Gynecology and Obstetrics, 2000) and treated at the OU Medical Center in Oklahoma City using standard therapies of surgery, radiation, and chemotherapy as indicated by individual patient characteristics. Of the 167 HPV16-positive cancer cases, 155 were available for variant analysis. Because 80 (51.6%) women did not undergo total hysterectomy and 40 (25.8%) did not have a lymphadenectomy, some pathologic variables were not evaluable in all cases. Follow-up was determined by chart review and death records for the state of Oklahoma. In addition to clinico-pathologic parameters, we compared over-all follow-up taking into account clinical stage at diagnosis, race, age, and censoring due to loss-to-follow-up. Statistical analyses reported below were performed using the population of women (n = 149) with defined outcome (i.e., no evidence of disease or dead of cervical cancer). This study was performed with the approval of the Institutional Review Board for the University of Oklahoma Health Sciences Center.

Pathologic Categorization

All cancer diagnoses were confirmed histologically and the cell type was determined using routine hematoxylin and eosin staining supplemented by any special stains performed at the time of initial diagnosis. The tumor cell types were categorized using standard histologic criteria [21]. Squamous cell carcinomas (SCC) were designated as "large cell keratinizing" when at least one well-formed keratin pearl was identified [22] and "large cell non-keratinizing" when no pearls were identified. Squamous tumors that were very small or for which only small biopsies were available, were listed as "SCC, not otherwise specified".

The major histologic categories included SCC (n = 141), both keratinizing and non-keratinizing patterns, adenocarcinoma (CAC) (n = 10), and adenosquamous carcinoma (ASC) (n = 4). There were no HPV16-positive small cell neuroendocrine carcinomas in this population.

HPV Testing Using L1 Consensus Primers

Liquid-based cytologic samples were collected in PreservCyt^® (Hologic, Malborough, MA) at the time of clinical evaluation and the cellular DNA was extracted using the QIAamp^® DNA Blood and Tissue Mini Kit (Qiagen, Valencia, CA). HPV genotyping was performed using the reverse line blot/Linear Array® HPV Genotyping Test (Roche Molecular Systems, Alameda, CA), as previously described [20].

Variant Analysis

Aliquots of cellular DNA from HPV16-positive tumor samples were analyzed for HPV16 E6 variant category using PCR amplification and bidirectional PCR-based fluorescent dideoxy chain termination sequencing using the same primers used for initial amplification, as previously described [11].

Sequence alignments were performed using CLUSTALW [23]. Only nucleotide changes verified as occurring on both strands were accepted.

An HPV16-variant category was assigned for each case using the prototype nucleotide sequence for HPV16 [24] (modified as HPV16R [25]) as well as published HPV16 E6 sequence patterns that define the different variants [3]. The 16-E category included the prototype sequence and related patterns showing only a small number of nucleotide changes, while the 16-NE categories included Asian-American (AA), Native-American (NA-1), and African (AF-1 and AF-2).

In the secondary analysis restricted to 16-E SCC cases, the variants were subdivided into 16-EP (prototype), 16-EV (European prototype sequence with a small number of additional nucleotide differences), and 16-EV-G (European sequence with the common 350T>G substitution; cases with nucleotide changes in addition to 350T>G were also included).

Statistics

Tabular analyses were performed using chi square and Fisher's exact and Cochran-Mantel-Haenszel trend tests. Statistical significance was assigned to 2-sided probability values < .05.