Improving Cervical Screening Through Multiplex Detection and Self-Sampling

Cervical cancer, is the result of infection by sexually transmitted, high risk strains of the human papillomavirus (hrHPV)[1]. Viral persistence and subsequent integration into the human genome induces a number of molecular and cellular changes in cervical epithelial cells, which override normal growth control mechanisms, leading to sustained cell cycle progression and subsequent transformation. Determination of the molecular mechanisms engaged in malignant transformation of the cervical mucosa has led to the discovery of a range of candidate cervical cancer biomarkers that could be used to identify HPV-positive patients at the greatest risk for developing cervical cancer and most likely to benefit from further intervention. Accordingly, evidence continues to accumulate that adjunct testing with single or multiplex cervical cancer biomarkers improves screening; however, these practices have been difficult to deploy in low resource settings, requiring additional approaches such as self sampling, and high throughput analyses to facilitate screening and model validation. Further discovery of the mechanisms that drive the pathogenesis of cervical cancer have the potential to improve adjunct testing, which ! 1 OncoGenesis 385 Woodview Ave, Suite 150 Morgan Hill, CA 95037 could one day supplant normal cytology, and focus the development of novel therapies ultimately improving cervical screening and patient survival in low resource settings where need is greatest. Cervical Screening – Disease and Guidelines The American Cancer Society (ACS) screening guidelines for detection of cervical cancer, cosponsored by the American Society for Colposcopy and Cervical Pathology (ASCCP) and the American Society for Clinical Pathology (ASCP), recommend HPV co-testing with cytology every 5 years in women ≥30 years of age, and emphasize several priorities for future research[2], among them: • Strategies that improve screening in unscreened or under-screened women; • Novel strategies utilizing HPV testing and other molecular approaches, such as self collection of cervico-vaginal specimens for co testing with HPV and cytology; • Research on the use of novel biomarkers Detection of precancerous cervical lesions or invasive cancer is typically achieved through a subjective, program of cytological screening using Papanicolaou (PAP) stained cervical specimens. Based upon morphology, cervical specimens are stratified into diagnostic categories, including, normal, atypical squamous cells of undetermined significance (ASC-US), low-grade intraepithelial lesion (LSIL), high-grade intraepithelial lesions (HSIL), and cancer. Although effective in reducing cancer deaths in developed countries, due to the lack of qualified cytopathologists and required infrastructure in many third world settings, it has been challenging ! 2 OncoGenesis 385 Woodview Ave, Suite 150 Morgan Hill, CA 95037 to implement slide-based screening programs for women with the greatest need, forcing clinical care providers to rely on less sensitive and specific methods, such as in situ visualization with acetic acid (VIA). As with many cancers, early detection can improve outcome however, identifying the subset of patients with precancerous lesions that pose the greatest risk can be difficult. This is particularly challenging for women with cytology results that are suspicious (ASC-H) or diagnostic of HSIL, as these are usually flat lesions that may be very difficult to visualize. Similarly, potential premalignant findings of the endocervical glandular mucosa, including atypical glandular cells (AGC) and adenocarcinoma in situ (AIS) may be extremely hard to detect by clinical examination. With potentially greater numbers of women stratified as HPV-positive/cytology negative, or ASC-US, which may harbor underlying HSIL or high-grade endocervical glandular lesions, there is a call to implement methodologies that will improve diagnostic/prognostic accuracy[3]. The challenge in applying differentiating factors as part of cytological testing was evident in an ASCUS-LSIL triage study where patients with LSIL or ASCUS were randomized to 1 of 3 study arms including immediate colposcopy, HPV triage testing using Hybrid Capture 2 or conservative follow-up with cytology only[4]. Although HPV triage proved useful for women with ASCUS, the high incidence of HPV invalidated its use as a triage for women with LSIL. While patients with a cytological diagnosis of ASCUS have a 5% 17% chance of an underlying ! 3 OncoGenesis 385 Woodview Ave, Suite 150 Morgan Hill, CA 95037 HSIL, the majority of ASCUS and AGC patients were not found to have clinically significant lesions. Patients with LSIL referred for colposcopy, revealed HISL on biopsy in ~ 25% of cases upon further examination; LSIL in 45% of cases, but no dysplasia in >25%. Furthermore, even patients deemed to have HSIL on cytology have a 24% 94% chance of high grade cervical intraepithelial neoplasia(CIN2+/3+) upon biopsy[5]. This variability underlies problems of both limited specificity and sensitivity of cervical cytology, reported to range from 1.6 to as much as 28%[5]. Co-Testing – Impact on screening recommendations The molecular mechanisms underlying HPV-induced cervical transformation support using analyses of additional targets in the diagnosis of high-risk premalignant lesions. Foremost is the inclusion of tests detecting hrHPV strains to provide an additional basis for identifying patients that should be further evaluated with colposcopy/biopsy. Although HPV infection, is a common STD, affecting nearly 79 million Americans with ~ 14 million new cases identified each year[6] [7] most infections clear spontaneously without medical intervention[8]. Thus, the detection of hrHPV has generally poor specificity for underlying premalignant or malignant lesions that require intervention. Although there is evidence that increased HPV copy number is a risk factor for disease progression, the highest levels of HPV are associated with LSIL lesions that harbor productive infections[5] while HPV copy number can be quite low in most HSILs and SCCs [9], precluding its use as a reliable differentiating factor for CIN[10]. Furthermore, the detection of hrHPV DNA or RNA in a cytologic specimen fails to provide information on events such as viral ! 4 OncoGenesis 385 Woodview Ave, Suite 150 Morgan Hill, CA 95037 integration, loss/gain-of-function that drive oncogenic transformation, or that potentially guide intervention. Current ACS guidelines support HPV and cytology co-testing of women aged 30-65 years of age, every 5 years; however, due to the high rates of infection in younger women, HPV testing is not recommended for women < 30 years of age, nor is it recommended as a standalone assay. Evidence from this approach demonstrates improved sensitivity for HSIL, reducing patient cancer risk, but a lack of specificity, potentially leads to unnecessary colposcopy/biopsy [11,12]. The limited specificity for high-grade lesions is due in part to the very high rates of transient HPV infections that are cleared by the host’s immune system[13]. Although results are encouraging, conclusions on the effectiveness of HPV testing for primary screening will require further evidence, including large-scale, long-term clinical studies. A potential problem with adding HPV detection to cytology is that greater numbers of women may undergo otherwise unnecessary colposcopic examinations, particularly patients that test positive for HPV on repeated screenings, even in the absence of abnormal cytological evidence to support transformation. It is imperative then to also increase the reassurance provided by negative colposcopy findings by adding metrics that improve colposcopic sensitivity or ideally improve the selection of women forwarded for colposcopy/biopsy. For this group of patients, testing for additional ! 5 OncoGenesis 385 Woodview Ave, Suite 150 Morgan Hill, CA 95037 biomarkers that more closely reflect cellular changes indicative of HPV-induced transformation would provide increased confidence. Secondary Targets for Triage of HPV (+) Patients Screening guidelines recognize that secondary testing of cervical cancer biomarkers can improve specificity and disease management, however, the limited data regarding test performance has hindered adoption. Until such time that additional biomarkers can be clinically validated as both safe and effective adjuncts or alternatives to HPV testing, the ACS has recommended continued research into the use of novel biomarkers to improve the management of cervical cancer screening/diagnosis, particularly in women with HPV-positive/cytology-negative co-test results[14]. Examination of mRNA species that may be altered in CIN has been proposed as a tool to provide greater of cellular changes underlying transformation, however, studies examining viral mRNA levels in CIN were inconsistent and not strongly supportive of using HPV mRNA as a marker to gauge malignant transformation[15,16]. Recent evidence suggests that microRNA expression in HPV-induced cervical lesions could provide alternative biomarkers for evaluation[17] however, as with the analysis of mRNA, qPCR processes for microRNA are demanding. Without unique implementation approaches, the expense, time and technical requirements do not favor their adoption in low resources settings, yet they remain powerful tools for collecting data necessary for accurately modeling HPV-induced transformation. 6 OncoGenesis 385 Woodview Ave, Suite 150 Morgan Hill, CA 95037 Expression of both host and viral genes is influenced by CpG methylation patterns and several candidate host genes, which function as negative regulators of cell growth and motility, display altered methylation frequency, which correlates with increase severity of cervical dysplasia[18]. Looking beyond the presence of nucleic acids indicative of high-risk HPV, the methylation status of specific genes may serve as an effectiv