The science of epigenetics -- how genes are regulated and expressed -- is informing new and potentially more accurate tests for cervical cancer.
"In contrast to what most researchers and clinicians are saying, we are seeing more and more evidence that it is in fact epigenetics, and not DNA mutations, that drives a whole range of early cancers, including cervical, anal, oropharyngeal, colon, and prostate," Atilla Lorincz, PhD, of Queen Mary University in London, said in a statement. Lorincz helped develop the first FDA-approved human papilloma virus (HPV) test in 1988 and is currently working on a new epigenetic-based test.
Although cervical cytology (Pap smears) and high-risk HPV testing are the current standards for testing, according to the 2018 update on cervical cancer screening from the U.S. Preventive Services Task Force, these tests still have important limitations, Megan Clarke, PhD, of the National Cancer Institute, and colleagues wrote in >JAMA Oncology: "Cytology requires frequent re-testing at shorter intervals because the risk of pre-cancer in cytology-negative women is not low enough to provide long-term assurance. Efficient management of HPV-positive women requires triage markers that distinguish those at high risk who need colposcopy from those who can safely return to routine screening."
Dual-Stain Testing for p16/Ki-67
Clarke and colleagues are studying one such marker: the co-expression of the tumor-suppressor protein p16 and the cell-proliferation marker Ki-67, as detected by dual-stain testing.
"We and others have shown that p16 and dual-stain testing have better performance than cytology for detection of precancers in HPV-positive women," the team wrote. "However, longitudinal studies are needed to determine the long-term risk of precancer following a negative dual-stain result."
To answer this question, Clarke's group conducted a prospective cohort study of approximately 1,500 HPV-positive women who underwent routine cervical cancer screening in 2012 with HPV and cytology co-testing, and then conducted additional p16/Ki-67 dual-stain testing on participants' cytology specimens. The women were followed through 2017. The researchers estimated the 5-year cumulative risk of cervical intraepithelial neoplasia grade 2 or worse (≥CIN2) or grade 3 or worse (≥CIN3) by baseline dual-stain testing and cytology, and compared these risks with clinical management thresholds for colposcopy referral and a 1-year return interval.
Positive dual-stain results were associated with significantly higher cumulative 5-year risks of ≥CIN2 compared with abnormal cytology (31% vs 25%; P = 0.03).Women with dual-stain-negative findings had significantly lower 5-year risks of ≥CIN2 compared with women with normal cytology (8.5% vs 12.3%; P = 0.04). In dual-stain-negative women, the risks of both ≥CIN2 and ≥CIN3 remained below the colposcopy referral threshold for all 5 years, crossing the 1-year return threshold at 3 years.
"Triage with p16/Ki-67 dual-stain provides better long-term risk-stratification than cytology over 5 years. The low risk of cervical precancer in p16/Ki-67 dual-stain negative women permits safe extension of follow-up intervals for 3 years," Clarke and colleagues wrote.
DNA Methylation Testing
Lorincz and colleagues, writing in the >International Journal of Cancer, acknowledged that p16/Ki-67 immunostaining is more sensitive than standard cytology and identifies women at higher risk. However, this technique requires interpretation of samples by subjective microscopy: "An objective triage strategy which could be automated and incorporated as a reflex molecular test following HPV screening would be advantageous," the researchers said. "DNA methylation assays targeting host and/or HPV genes may meet this requirement as they have been shown to have higher sensitivity and similar specificity to liquid-based cytology for identifying CIN2+."
DNA methylation is a key regulator of gene expression. The team studied the S5 DNA methylation classifier, which is based on late regions of HPV16, HPV18, HPV31, and HPV33 combined with the promoter region of the human tumor-suppressor gene EPB41L3. The researchers compared S5 methylation testing with an algorithm combining cytology and HPV 16/18-positive genotyping in 257 HPV-positive women from the HPV FOCAL cervical cancer screening trial.
The relative sensitivity of S5 for detecting CIN3 was 93.2% (95% CI, 81%-98%), and the relative specificity was 41.8% (95% CI, 35.2%-48.8%), compared with a sensitivity of 86.4% (95% CI, 75%-95.7%) and a specificity of 49.8% (95% CI, 43.1%-56.6%) for combined abnormal cytology and HPV genotyping. The positive predictive value of S5 was comparable to cytology + HPV testing (18% vs 19%).
"DNA methylation assessed by the S5 classifier correlates strongly with aggressive cervical disease, showing high sensitivity for CIN3 and cancer, the raison d'être for a cervical screening program," Lorincz's group said. "S5 positive predictive value for CIN3 is compatible with both U.S. and European colposcopy referral thresholds. Methylation tests have the potential to simplify triage by more quickly identifying HPV-infected women in need of colposcopy."
Non-coding microRNA (miRNA) sequences are another potential biomarker for detecting cervical cancer, a research team led by Iris Babion, a PhD candidate at VU University Medical Center in Amsterdam, noted in >Clinical Epigenetics. "Primary testing for high-risk HPV (hrHPV) is increasingly implemented in cervical cancer screening programs. Many hrHPV-positive women, however, harbor clinically irrelevant infections, demanding additional disease markers to prevent over-referral and over-treatment."
MicroRNAs are another important regulator of gene expression, Babion and colleagues said. "Altered expression of miRNAs has been shown to contribute to human malignancies, including cervical cancer, by influencing expression of oncogenes and tumor-suppressor genes and subsequent deregulation of important intracellular pathways."
The group analyzed cervical samples from 58 HPV-positive women, including normal samples, those with CIN2 and CIN3 lesions, and those with cervical cancer. The researchers identified five miRNAs whose expression correlated highly with CIN3 and cancer. These miRNAs were tested in samples from 66 hrHPV-positive women and 121 women with CIN3. The team identified a classifier consisting of two miRNAs (miR-15b-5p and miR-375) that had a sensitivity of 55% and a specificity of 70% for detecting CIN3. When this 2-miRNA classifier was combined with HPV 16/18 genotyping, the sensitivity increased to 63% and the specificity to 77%.
"Our data suggest that miRNA expression analysis offers a promising alternative molecular tool to triage hrHPV-positive women," the researchers said. "Further optimization of the marker panel and validation in an independent cohort of hrHPV-positive cervical scrapes will reveal whether triage of hrHPV-positive women by miRNA expression analysis offers an objective and economical alternative to cytology."
The study by Lorincz et al. was supported by Cancer Research UK and the Canadian Institutes of Health Research. Lorincz reported having no conflicts of interest; co-authors reported relationships with companies including Roche Molecular Systems, Merck, and Hologic Inc.
The study by Clarke et al. was supported by the National Institutes of Health; no authors reported relevant conflicts of interest.
The study by Babion et al. was supported by the Dutch Cancer Society and the European Research Council. Babion reported having no conflicts of interest; co-authors reported relationships with companies including Pfizer, Qiagen, and Roche.
Source : https://www.medpagetoday.com/reading-room/asco/gynecological-cancers/77409