**Cervical Cancer Screening at a Crossroads: Learnings from the Past Driving Change for the Future**

Laurence M. Vaughan, Brian R. Faherty, Erin C. Gutierrez, James M. Harris, William A. Nussbaumer and Ryan J. Schwab

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/63334

#### **Abstract**

Cervical cancer screening has been one of the most impactful human interventions in medical history, saving the lives of countless thousands of women since the introduction of organized cytology screening programs. Today, we stand at a crossroads in the fight against cervical cancer, with several countries actively engaged in introducing primary human papillomavirus (HPV) testing and vaccination as more effective means of prevention. This chapter discusses the history of organized screening and how this led to HPV test methods to detect cervical cancer. We go on to examine the technologies used to screen for high-risk HPV types and how they affect clinical performance. We examine the evidence for primary HPV screening and review recent self-collection initiatives to reach underserved women, including the use of urine as novel sample type. In addition, we critically examine the evolution of HPV test methods and make the case for the use of extended genotyping as an improved risk stratification tool for guiding clinical management. Finally, we look to the future of cervical cancer screening and consider options for future management programs.

**Keywords:** genotyping, HPV screening tests/strategies, Pap, risk stratification, selfsampling

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

Cervical cancer screening has advanced considerably since the introduction of the Pap smear in the 1960s: organized cytology screening programs have successfully reduced the burden of disease associated with human papillomavirus (HPV) and sensitive new molecular methods have been developed to detect the virus. Perhaps more importantly, safe and efficacious HPV vaccines are now widely available and offer the prospect of greatly reducing the incidence of cervical cancer if sufficient numbers of the target population can be vaccinated. Vaccines offer the best hope for developing countries, which lack the resources to implement an effective screening program. Despite the introduction of vaccination, there is still a need for improved screeningmodalitiesasbothscreeningandvaccinationwillnecessarilycoexistfor somedecades to come. It is against this backdrop that we briefly review the history of the Pap smear and the development of organized cytology screening programs and then go on to discuss the devel‐ opment of molecular methods. We discuss the pros and cons of the different molecular assay design approaches and review the case for primary HPV screening. Despite the effective tools at our disposal, reaching underserved women remains the biggest challenge to controlling cervicaldisease.However,newself-samplingmethodsoffertheprospectofaneffectiveoutreach program to reach women most in need. Finally, we discuss the benefits of extended genotyp‐ ing and how this might influence future screening algorithms. We conclude that the biggest hurdle to preventing and detecting cervical disease may lie in our inability to adapt to change andeffectivelyimplementnewstrategies.Thishistoryof cervical cancer screeningsuggests that the pace of change is slow but we must respond more quickly to the global threat posed by cervical cancer and make efficient use of the tools at our disposal.

## **2. The history of organized screening programs**

George Nicholas Papanicolaou is almost single-handedly credited with developing cervical cancer screening. He was encouraged to leave his native Greece by the renowned geneticist, Thomas Hunt Morgan, who helped him gain a position in the Department of Pathology and Bacteriology at New York Hospital as an assistant, and later at the Department of Anatomy at Cornell Medical School. Dr. Papanicolaou began to study vaginal secretions in women, beginning with a case study of his wife, Mary [1]. Later work led to the observation of cancer cells while studying smears of women in the New York Hospital, which led him to propose that a systematic study of smears could lead to early cancer detection. His "New Cancer Diagnosis" theory was initially very poorly received by his peers [2]. It was not until over 10 years later that a successful collaboration with Herbert Traut (1894–1963), a renowned gynecologist and pathologist at Cornell University, resulted in a more widely received manuscript "The diagnostic value of vaginal smears in carcinoma of the uterus." This work was presented to the New York Gynecological Society in 1941 and published in the prestigious *American Journal of Obstetrics and Gynecology* [3]. In 1947, Dr. Papanicolaou and the head of the Anatomy Department, Dr. Joseph Hinsey, began focusing on training other physicians on the new technique. This led to a gradual adoption of the screening method, and by 1960 it was being adopted across the United States. However, it was not until just before Papanicolaou's death that the impact of his discovery was becoming apparent and he finally began to get the recognition he deserved. Today, he is remembered as "the father of exfoliative cytology" and cervical cytology stands as one of the most successful clinical interventions of the twentieth century. Organized cervical cancer screening has led to an over two-fold reduction in the incidence of cervical cancer in the United States in the period 1975–2012. This success in the United States has been replicated in other countries with organized screening programs such as the United Kingdom, which has seen a similar reduction in mortality from 1985 to 2012. However, over the last decade it has become evident that cytology screening may have reached the limits of its effectiveness in terms of reducing the incidence rate, with both the US and UK trend lines reaching a plateau (**Figure 1**).

**1. Introduction**

44 Human Papillomavirus - Research in a Global Perspective

Cervical cancer screening has advanced considerably since the introduction of the Pap smear in the 1960s: organized cytology screening programs have successfully reduced the burden of disease associated with human papillomavirus (HPV) and sensitive new molecular methods have been developed to detect the virus. Perhaps more importantly, safe and efficacious HPV vaccines are now widely available and offer the prospect of greatly reducing the incidence of cervical cancer if sufficient numbers of the target population can be vaccinated. Vaccines offer the best hope for developing countries, which lack the resources to implement an effective screening program. Despite the introduction of vaccination, there is still a need for improved screeningmodalitiesasbothscreeningandvaccinationwillnecessarilycoexistfor somedecades to come. It is against this backdrop that we briefly review the history of the Pap smear and the development of organized cytology screening programs and then go on to discuss the devel‐ opment of molecular methods. We discuss the pros and cons of the different molecular assay design approaches and review the case for primary HPV screening. Despite the effective tools at our disposal, reaching underserved women remains the biggest challenge to controlling cervicaldisease.However,newself-samplingmethodsoffertheprospectofaneffectiveoutreach program to reach women most in need. Finally, we discuss the benefits of extended genotyp‐ ing and how this might influence future screening algorithms. We conclude that the biggest hurdle to preventing and detecting cervical disease may lie in our inability to adapt to change andeffectivelyimplementnewstrategies.Thishistoryof cervical cancer screeningsuggests that the pace of change is slow but we must respond more quickly to the global threat posed by

George Nicholas Papanicolaou is almost single-handedly credited with developing cervical cancer screening. He was encouraged to leave his native Greece by the renowned geneticist, Thomas Hunt Morgan, who helped him gain a position in the Department of Pathology and Bacteriology at New York Hospital as an assistant, and later at the Department of Anatomy at Cornell Medical School. Dr. Papanicolaou began to study vaginal secretions in women, beginning with a case study of his wife, Mary [1]. Later work led to the observation of cancer cells while studying smears of women in the New York Hospital, which led him to propose that a systematic study of smears could lead to early cancer detection. His "New Cancer Diagnosis" theory was initially very poorly received by his peers [2]. It was not until over 10 years later that a successful collaboration with Herbert Traut (1894–1963), a renowned gynecologist and pathologist at Cornell University, resulted in a more widely received manuscript "The diagnostic value of vaginal smears in carcinoma of the uterus." This work was presented to the New York Gynecological Society in 1941 and published in the prestigious *American Journal of Obstetrics and Gynecology* [3]. In 1947, Dr. Papanicolaou and the head of the Anatomy Department, Dr. Joseph Hinsey, began focusing on training other physicians on the new technique. This led to a gradual adoption of the screening method, and by 1960 it was

cervical cancer and make efficient use of the tools at our disposal.

**2. The history of organized screening programs**

**Figure 1.** (A) US age-standardized incidence and mortality rates per 100,000 women (1975–2012). Source: http:// seer.cancer.gov/statfacts/html/cervix.html (accessed January 2016). (B) United Kingdom age-standardized incidence rates per 100,000 women (1979–2012). Source: Cancer Research UK, http://www.cancerresearchuk.org/health-professio‐ nal/cancer-statistics/statistics-by-cancer-type/cervical cancer/incidence#heading-Two (accessed January 2016).

## **3. The development of molecular screening tools**

The development of molecular biology techniques in the late 1970s and early 1980s paved the way for the discovery that the HPV was both necessary and responsible for cervical disease. zur Hausen played a pivotal role in establishing the link between papillomaviruses and cervical cancer [4]. As interest intensified, novel HPV types began to be isolated from genital warts (HPV6 [5–7] and laryngeal papillomas (HPV11) [8]). The detection of additional typespecific viral sequences followed: [9, 10], HPV16 [11], HPV18 [12–14], and high-risk types HPV31 and HPV45, which were first described by Lorincz et al. [15] in the United States [16]. These discoveries laid the foundation for the development of cervical cancer diagnostic tests and the development of the first HPV cancer vaccines. Harald zur Hausen went on to win the Nobel Prize in Physiology or Medicine in 2008 for this work on the association of papilloma‐ viruses with human cancers.

While the work of Harald zur Hausen and others set the stage for modern cervical cancer diagnostics, it took another wave of development to advance HPV detection to the point that it could be widely adopted in the clinical laboratory. The early work led to rapid development of basic laboratory tests to detect various HPV types. However, the techniques employed involved the use of 32P-labeled probes that were hybridized using Southern blotting or slot blot methods, which meant they were labor intensive. These early methods (ViraPap and ViraType) were commercialized by Life Technologies Inc. and later sold to the Digene Corporation but they did not achieve widespread adoption, primarily because they did not detect all oncogenic HPV types and also lacked sensitivity. This period also saw the develop‐ ment of *in situ* hybridization (ISH) techniques by Enzo Diagnostics and Life Technologies Inc., and additional methods were later automated by Ventana Medical Systems (now a Roche Company), the Dako Corporation, and others [17]. These tests also suffered from lack of sensitivity [18] and were ultimately not adopted as screening tools. However, ISH methods may prove to be useful in assisting in the diagnosis of Cervical Intraepithelial Neoplasia cases (CIN) [19].

### **4. The** *digene***hybrid capture 2 assay**

The Digene Corporation (now part of QIAGEN) under the scientific leadership of another early HPV molecular pioneer, Dr. Attila Lorincz [15, 16], developed the modern market for HPV cervical cancer screening. Lorincz et al. developed a technology called hybrid capture that fused traditional immunoassay techniques with the newer nucleic acid isolation technology [20]. The core principle leveraged the fact that DNA–RNA hybrids have a distinct shape different than a DNA helix that is detectable using antibodies. By generating *in vitro* RNA transcripts of each of the target HPV types and pooling them into a single probe cocktail, the method was capable of detecting as many as 13 high-risk HPV types at once. The capture antibody is bound to a solid surface that binds cognate target hybrids which in turn are then detected using a secondary antibody coupled with alkaline phosphatase using a chemilumi‐ nescent substrate. The first generation hybrid capture assay utilized two mixtures of singlestranded RNA probes: one for low-risk HPV types 6, 11, 42, 43, and 44, and one for high-risk HPV types 16, 18, 31, 33, 35, 45, 51, 52, and 56. The analytic sensitivity of the first generation test was estimated at 50,000 copies of HPV16 DNA [21, 22], and this was not found to be sensitive enough versus polymerase chain reaction (PCR) and histology [23]. Digene research‐ ers then launched a second generation assay (Hybrid Capture 2) whose analytical sensitivity was increased to 1000 HPV DNA copies by the reformulation of hybridization reagents and by the addition of new probes for four high-risk HPV types, 39, 58, 59, and 68. This assay was launched in Europe in 1998 and was subsequently approved by the US Food and Drug Administration (FDA) in 2003, for the triage of Atypical Squamous Cells of Undetermined Significance (ASC-US) cytology patients and as an adjunctive test with cytology for cervical cancer screening [23]. The assay went on to achieve worldwide acceptance and became the *de facto* clinical standard for all subsequent assay development. It has the largest body of clinical evidence supporting its use, and it has been used in numerous clinical trials across the globe [24, 25].

## **5. PCR- and other amplification-based technologies**

**3. The development of molecular screening tools**

46 Human Papillomavirus - Research in a Global Perspective

viruses with human cancers.

(CIN) [19].

**4. The** *digene***hybrid capture 2 assay**

The development of molecular biology techniques in the late 1970s and early 1980s paved the way for the discovery that the HPV was both necessary and responsible for cervical disease. zur Hausen played a pivotal role in establishing the link between papillomaviruses and cervical cancer [4]. As interest intensified, novel HPV types began to be isolated from genital warts (HPV6 [5–7] and laryngeal papillomas (HPV11) [8]). The detection of additional typespecific viral sequences followed: [9, 10], HPV16 [11], HPV18 [12–14], and high-risk types HPV31 and HPV45, which were first described by Lorincz et al. [15] in the United States [16]. These discoveries laid the foundation for the development of cervical cancer diagnostic tests and the development of the first HPV cancer vaccines. Harald zur Hausen went on to win the Nobel Prize in Physiology or Medicine in 2008 for this work on the association of papilloma‐

While the work of Harald zur Hausen and others set the stage for modern cervical cancer diagnostics, it took another wave of development to advance HPV detection to the point that it could be widely adopted in the clinical laboratory. The early work led to rapid development of basic laboratory tests to detect various HPV types. However, the techniques employed involved the use of 32P-labeled probes that were hybridized using Southern blotting or slot blot methods, which meant they were labor intensive. These early methods (ViraPap and ViraType) were commercialized by Life Technologies Inc. and later sold to the Digene Corporation but they did not achieve widespread adoption, primarily because they did not detect all oncogenic HPV types and also lacked sensitivity. This period also saw the develop‐ ment of *in situ* hybridization (ISH) techniques by Enzo Diagnostics and Life Technologies Inc., and additional methods were later automated by Ventana Medical Systems (now a Roche Company), the Dako Corporation, and others [17]. These tests also suffered from lack of sensitivity [18] and were ultimately not adopted as screening tools. However, ISH methods may prove to be useful in assisting in the diagnosis of Cervical Intraepithelial Neoplasia cases

The Digene Corporation (now part of QIAGEN) under the scientific leadership of another early HPV molecular pioneer, Dr. Attila Lorincz [15, 16], developed the modern market for HPV cervical cancer screening. Lorincz et al. developed a technology called hybrid capture that fused traditional immunoassay techniques with the newer nucleic acid isolation technology [20]. The core principle leveraged the fact that DNA–RNA hybrids have a distinct shape different than a DNA helix that is detectable using antibodies. By generating *in vitro* RNA transcripts of each of the target HPV types and pooling them into a single probe cocktail, the method was capable of detecting as many as 13 high-risk HPV types at once. The capture antibody is bound to a solid surface that binds cognate target hybrids which in turn are then detected using a secondary antibody coupled with alkaline phosphatase using a chemilumi‐ nescent substrate. The first generation hybrid capture assay utilized two mixtures of singlestranded RNA probes: one for low-risk HPV types 6, 11, 42, 43, and 44, and one for high-risk

The development of PCR technology [26, 27] meant that small quantities of DNA or RNA could now be amplified and detected either directly in endpoint PCR [28] or through the use of accumulating fluorescence that could be monitored continuously in real-time (RT) PCR [29– 31]. As with most new techniques, refinements and improvements overcame some of the early difficulties such as assay robustness and the potential for contamination/false positives [32, 33]. The use of PCR to detect HPVs mirrored that of other methods, with HPV initially being detected using gel analysis of PCR products [34] and subsequently using real-time methods [35, 36]. Type-specific primer amplification methods eventually gave way to consensus PCR approaches where multiple HPV types could be detected in one reaction [37]. Most PCR approaches targeted the L1 gene region, and included the MY09–MY11 (MY09/MY11) and GP51–GP61 (GP51/GP61) primer systems. The MY primers were later redesigned to improve performance (PGMY09/PGMY11 [38]) as were the GP primers, to GP5+/GP6+, which were subsequently widely adopted [39, 40]. PCR-based tests are intrinsically more analytically sensitive than Hybrid Capture 2 technology, detecting as little as 10–100 copies of target. However, the Hybrid Capture 2 assay established that approximately 1 pg/ml or 5000 copies of HPV16 target DNA per reaction equated to an actionable clinical result (CIN2+ histology) [41]. Therefore, assays that are clinically more sensitive than this would have poor specificity, referring low level infections unnecessarily to follow colposcopy and possible biopsy or treatment. This has been the subject of discussion in cervical cancer screening since the introduction of highly sensitive molecular methods. Key opinion leaders have cautioned against the use of analytically validated assays whose clinical performance has not been well established in large longitudinal studies, emphasizing the need to strike the proper balance between sensitivity and specificity [42]. In one case, even an FDA approved test was considered clinically unacceptable due to its high HPV positivity [43]. The issue is compounded by the fact that even large reference laboratories and smaller commercial companies cannot afford to fund large studies with a minimum of 3-year follow up of enrolled patients. This led a group of international HPV experts to propose acceptance criteria for HPV tests that wish to be used in primary HPV screening [44]. These criteria, commonly known as "Meijer Criteria" after the lead author, were founded on the principle that "candidate high-risk HPV tests to be used for screening should reach an optimal balance between clinical sensitivity and specificity for detection of high-grade CIN and cervical cancer to minimize redundant or excessive followup procedures for high-risk HPV positive women without cervical lesions" and set forth the following guidelines for an acceptable HPV screening test:


Meijer criteria have been well accepted as a critical litmus test in the absence of large-scale longitudinal trial data and have become a clinical benchmark for validation of tests, especially in the European Union. Despite the use of this leaner approach where the disease samples can be sourced retrospectively, only a small number of existing commercially available assays have actually met the criteria. A 2015 review of currently available commercial assays "identified 193 distinct commercial HPV tests" [45]. However, Arbyn et al. [46], also reported in the same year that only five commercially available tests fully met the Meijer criteria and could be considered suitable for use in primary HPV screening: PapilloCheck® HPV-Screening test; Abbott RealTime hrHPV test; cobas® 4800 HPV test; BD Onclarity™ HPV assay; HPV-Risk assay and the Aptima assay, targeting E6/E7 mRNA. The authors stated that the Cervista® assay should also be added to the list, despite one report of a lack of noninferiority for specificity (see also reference [104]). Three other assays met the sensitivity/specificity criteria but did not disclose accuracy and reproducibility data and were thus considered to partially meet the criteria (an in-house quantitative RT-PCR targeting E6/E7DNAsequences, a GP5+/ GP6+ PCR with Luminex identification of high-risk types and a MALDITOF assay). The authors also concluded that the Aptima assay, while fully meeting the criteria, needed further longitudinal validation of its long-term negative predictive value (NPV) because it was an RNA-based assay and to date this had only been established for DNA assays [46].

E6 and E7 gene RNA-based assays have been extensively tested in cervical cancer screening. The impetus for using RNA- versus a DNA-based targets, is likely based on the observation that both the E6 and E7 genes encode oncogenes that are involved in the development of cancer and are upregulated as disease progresses [47]. E6 and E7 viral oncoproteins bind and modulate cellular gene products (p53 and pRb) that play a key role in cell cycle control and DNA repair. The resulting genomic instability caused by E6 and E7 oncoproteins is a necessary condition for cell transformation and immortalization [48, 49]. Thus, it is reasonable to of international HPV experts to propose acceptance criteria for HPV tests that wish to be used in primary HPV screening [44]. These criteria, commonly known as "Meijer Criteria" after the lead author, were founded on the principle that "candidate high-risk HPV tests to be used for screening should reach an optimal balance between clinical sensitivity and specificity for detection of high-grade CIN and cervical cancer to minimize redundant or excessive followup procedures for high-risk HPV positive women without cervical lesions" and set forth the

**1.** The sensitivity of the candidate test for ≥CIN2 should be at least 90% of the sensitivity of the HC2 (i.e., relative sensitivity of at least 90%) as assessed by a noninferiority score test.

**2.** The specificity of the candidate test for ≥CIN2 should be at least 98% of the specificity of

**3.** The intralaboratory reproducibility in time and interlaboratory agreement should be determined by evaluation of at least 500 samples, 30% of which tested positive in a reference laboratory using a clinically validated assay. This should result in a percentage of agreement with a lower confidence bound not less than 87% (kappa value of at least

Meijer criteria have been well accepted as a critical litmus test in the absence of large-scale longitudinal trial data and have become a clinical benchmark for validation of tests, especially in the European Union. Despite the use of this leaner approach where the disease samples can be sourced retrospectively, only a small number of existing commercially available assays have actually met the criteria. A 2015 review of currently available commercial assays "identified 193 distinct commercial HPV tests" [45]. However, Arbyn et al. [46], also reported in the same year that only five commercially available tests fully met the Meijer criteria and could be considered suitable for use in primary HPV screening: PapilloCheck® HPV-Screening test; Abbott RealTime hrHPV test; cobas® 4800 HPV test; BD Onclarity™ HPV assay; HPV-Risk assay and the Aptima assay, targeting E6/E7 mRNA. The authors stated that the Cervista® assay should also be added to the list, despite one report of a lack of noninferiority for specificity (see also reference [104]). Three other assays met the sensitivity/specificity criteria but did not disclose accuracy and reproducibility data and were thus considered to partially meet the criteria (an in-house quantitative RT-PCR targeting E6/E7DNAsequences, a GP5+/ GP6+ PCR with Luminex identification of high-risk types and a MALDITOF assay). The authors also concluded that the Aptima assay, while fully meeting the criteria, needed further longitudinal validation of its long-term negative predictive value (NPV) because it was an

RNA-based assay and to date this had only been established for DNA assays [46].

E6 and E7 gene RNA-based assays have been extensively tested in cervical cancer screening. The impetus for using RNA- versus a DNA-based targets, is likely based on the observation that both the E6 and E7 genes encode oncogenes that are involved in the development of cancer and are upregulated as disease progresses [47]. E6 and E7 viral oncoproteins bind and modulate cellular gene products (p53 and pRb) that play a key role in cell cycle control and DNA repair. The resulting genomic instability caused by E6 and E7 oncoproteins is a necessary condition for cell transformation and immortalization [48, 49]. Thus, it is reasonable to

following guidelines for an acceptable HPV screening test:

48 Human Papillomavirus - Research in a Global Perspective

0.5 in this series of samples including 30% positives).

HC2.

postulate that an E6/E7 RNA target might offer an advantage over DNA in that it should be overexpressed in high- versus low-grade disease and that it might offer both a sensitivity and specificity advantage. There are two commercially based E6/E7 RNA assays: the Proofer Assay (Norchip, Klokkarstua, Norway) is a real-time multiplex nucleic acid sequence-based ampli‐ fication assay(NASBA) for isothermal amplification and detection of E6/E7 mRNA from five high-risk oncogenic types, HPV16, 18, 31, 33, and 45, using molecular beacon probes, and the Aptima® HPV Assay (Hologic—GenProbe) that is a qualitative nucleic acid amplification test that detects HPV E6/E7 mRNA from 14 high-risk HPV types [50]. The Aptima assay uses target amplification using transcription-mediated amplification (TMA) [51] and detection of the amplification products (amplicon) by the hybridization protection assay (HPA) [52]. HPV mRNA is captured on magnetic particles and then amplified using TMA that is a transcriptionbased nucleic acid amplification method that utilizes two enzymes, Moloney Murine Leukemia Virus (MMLV) reverse transcriptase and T7 RNA polymerase. The reverse transcriptase is used to generate a DNA copy of the target mRNA sequence containing a promoter sequence for T7 RNA polymerase. T7 RNA polymerase produces multiple copies of RNA amplicon from the DNA copy template. Detection of the amplicon is achieved by HPA using single-stranded nucleic acid probes with chemiluminescent labels that are complementary to the amplicon. The labeled nucleic acid probes hybridize specifically to the amplicon. The selection reagent differentiates between hybridized and unhybridized probes by inactivating the label on the unhybridized probes. During the detection step, light emitted from the labeled RNA–DNA hybrids is measured as photon signals called relative light units (RLU) in a luminometer. Final assay results are interpreted based on the analyte signal-to-cutoff (S/CO) [53]. The Norchip Proofer assay has been shown to be substantially less sensitive but more specific when compared to other clinically validated assays, including Roche cobas and Hybrid Capture 2 [54–56]. Arbyn et al. [57] performed a meta-analysis of the performance of the Aptima HPV assay versus Hybrid Capture 2 and found that for both triage of ASC-US and Low-Grade Squamous Intraepithelial Lesion (LSIL), Aptima is as sensitive but more specific than HC2 for detecting cervical precancer. In head to head tests, Aptima had a substantially higher sensi‐ tivity than Proofer in both a screening and a referral population, but Proofer showed improved specificity [55, 56]. The Aptima test has broader clinical application and relevance because it detects all 14 high-risk types versus just five high-risk types in the Proofer assay. However, as noted above, it needs further confirmation of its long-term NPV over at least a 5-year period, as recommended by Arbyn et al. [46]. A recent report with a 3-year longitudinal follow up found that the NPV is similar to that of Hybrid Capture 2 and again that its specificity was significantly better (96.3% compared with HC2 specificity of 94.8%; *P* < 0.001 [58]. A compre‐ hensive review of the clinical performance of the assay versus HC2 also concluded that the NPV was sufficient to justify a 3-year interval and that the specificity of the assay was consistently higher [59]. The increase in specificity is similar to that previously reported [57] and is relatively modest. It could also result from a slight clinical cut-point bias toward the specificity axis of the receiver operating characteristic (ROC) plot of sensitivity versus 1– specificity for this assay. Whatever the driver, it seems clear that there is no significant difference in sensitivity when comparing RNA- and DNA-based assays and the specificity differences are not as high as one might have predicted from the upregulation of E6/E7 RNA during oncogenesis. The latter may reflect that one does not see the full potential of upregu‐ lation when screening for less severe cellular abnormalities versus cancer. The answer may also lie in the observation that neither assay exclusively targets RNA: both assays have been reported to detect cognate DNA sequences present in endocervical specimens at levels where one might expect the DNA signal alone to be sufficient to record a positive clinical result [60, 61]. Thus, it is conceivable that this could render otherwise RNA-negative specimens positive, reducing the specificity of the assay.

### **6. How target regions impact assay performance**

### **6.1. Which target region to choose?**

HPV is double-stranded DNA virus whose circular genome is approximately 8000 base pairs long. It encodes eight open reading frames (ORFs) that are divided into early and late genes (**Figure 2**) involved in replication (i.e., E1 and E2) and packaging (i.e., L1 and L2) with the remaining genes (E6, E7, E5, and E4), playing roles in driving cell cycle entry, immune evasion, and virus release (reviewed in [49]). Papillomaviruses are ancient in origin, believed to have arisen in reptiles approximately 350 million years ago. They have evolved in their various host lineages (including humans) over the millennia and they have relatively stable genomes given their (redundant) double-stranded DNA structure. The rate of nucleotide substitution for HPV18 has been estimated at ~4.5 × 10−7 subs/site/year [62]. This means that assay developers can look across the entire genome for conserved target regions in which to design gene probes.

**Figure 2.** Physical map of the HPV16 genome.

Given the aforementioned large number of commercial assays available on the market, not surprisingly most ORFs have been targeted by one or more assays, including L1 [63], E1 [64], and E6/E7 [65, 66]. However, most commercial assays to date have been developed using the L1 gene as a target region [45, 63], including the Roche cobas assay that has received FDA approval [67].

The question one must then ask is: does it matter which region of the genome you use—are all genomic regions created equal? While a detailed phylogenetic and evolutionary analysis (beyond the scope of the current work) would be required to fully address this question, the following sections discuss some important topics with respect to target analyte performance in clinical diagnostics and how this can influences clinical results.

### **6.2. Cross-reactivity with nontarget HPV types**

during oncogenesis. The latter may reflect that one does not see the full potential of upregu‐ lation when screening for less severe cellular abnormalities versus cancer. The answer may also lie in the observation that neither assay exclusively targets RNA: both assays have been reported to detect cognate DNA sequences present in endocervical specimens at levels where one might expect the DNA signal alone to be sufficient to record a positive clinical result [60, 61]. Thus, it is conceivable that this could render otherwise RNA-negative specimens positive,

HPV is double-stranded DNA virus whose circular genome is approximately 8000 base pairs long. It encodes eight open reading frames (ORFs) that are divided into early and late genes (**Figure 2**) involved in replication (i.e., E1 and E2) and packaging (i.e., L1 and L2) with the remaining genes (E6, E7, E5, and E4), playing roles in driving cell cycle entry, immune evasion, and virus release (reviewed in [49]). Papillomaviruses are ancient in origin, believed to have arisen in reptiles approximately 350 million years ago. They have evolved in their various host lineages (including humans) over the millennia and they have relatively stable genomes given their (redundant) double-stranded DNA structure. The rate of nucleotide substitution for HPV18 has been estimated at ~4.5 × 10−7 subs/site/year [62]. This means that assay developers can look across the entire genome for conserved target regions in which to design gene probes.

reducing the specificity of the assay.

50 Human Papillomavirus - Research in a Global Perspective

**6.1. Which target region to choose?**

**Figure 2.** Physical map of the HPV16 genome.

**6. How target regions impact assay performance**

There is general agreement that only high-risk types cause cervical cancer and that low-risk types should not be screened for as a part of routine cervical cancer prevention [68]. Previously, there was consensus that 14 high-risk types caused the majority of cancers. However, more recently the evidence for HPV66 in cervical cancers was considered too weak to keep it in the group of 14 high-risk types and it was recommended to remove it [69]. However, all FDA tests approved to date detect HPV66 as part of their 14 high-risk panel (Roche cobas, Hologic-Genprobe Aptima, and Hologic Cervista) and the Hybrid Capture 2 assay detects it via crossreactivity [70]. In addition, a number of possible/probably carcinogenic types (including HPV66) have been shown to have oncogenic potential and "are biologically active and affect the same cellular pathways as any of the fully recognized carcinogenic HR-HPV types" [71]. Thus, it is difficult to draw an absolute line between known carcinogenic types and those that have that potential but very infrequently result in cancers [69].

Whether you take the view that there are 14 high-risk types or 13 high‐risk types (omitting HPV66), there is little doubt that screening for additional HPV types will not increase sensi‐ tivity for cancer detection and will reduce specificity. In addition, it may do harm to patients both psychologically and potentially physically if they are treated for lesions that will not result in cancer [68, 72]. Thus, it is important that clinically validated assays perform inclusivity and exclusivity studies to ensure that the assay detects only the intended HPV types for which they have claims. They Hybrid Capture 2 test has been shown to have excellent clinical sensitivity for CIN2+ endpoints and as described earlier, has been used as a clinical benchmark for the last decade. However, it does have a well-documented cross-reactivity with non-high-risk HPV types [70, 73, 74]. This results from the fact that the assay detects the entire HPV genome (**Figure 2**) and thus there is more potential for closely related nononcogenic sequences to be detected. One study found that "some 20% of HC2-positive samples did not contain the targeted HPV types. About two-thirds of them resulted from cross-hybridization, especially with HPV53, HPV66, and HPV70" [75]. When split sample testing is performed with HC2 and a second assay, one typically sees a larger proportion of HC2+/other assay–results which, if they are resolved by third-party genotyping or sequencing method, shows both low-risk and no HPV present results, confirming the negative result as truth with respect to high-risk HPV [66, 76]. In another recent study (*n* = 6172) comparing Roche cobas assay with Hybrid Capture 2, where discordants were resolved using the Roche Linear Array genotyping assay, the authors reported that "HC2+/COBAS− were less likely to contain hrHPV genotypes (12.3 versus 68.9%; *P* < 0.0001) and more likely to contain only lrHPV genotypes (52.8 versus 12.1%; *P* < 0.0001) than those HC2−/COBAS+" and they found "lower CIN2+ rates among women with HC2+/COBAS− results" [77].

The clinical impact of this cross-reactivity has not been widely discussed in the literature. Castle et al. used a more stringent dual reference assay method to determine the level of HC2 crossreactivity and concluded that about 8% of all HPV positives were due to cross-reactivity with non-high-risk HPV types in the referred (ALTS Study) population. They concluded that crossreactivity would likely be further reduced in a screening population where there is less overall infection. However, they cautioned that these non-high-risk HPV "cases of CIN2 might be treated by excisional procedures, which can cause iatrogenic morbidity and adversely affect reproductive outcomes" [70]. The previously cited study of Gillio-Tos et al. [75] concluded "If only the samples containing HC2-targeted types tested positive, the positive predictive value (PPV) would have increased from 7.0% (95% CI, 6.1–8.0%) to 8.4% (95% CI, 7.3–9.6%), although 4.9% (95% CI, 2.4–8.8%) of cervical intraepithelial neoplasia grade 2+ (CIN2+) cases would have been missed". Regarding the latter point, "missing" CIN2+ disease caused by non-high-risk HPV types should be the goal of future assays since it has an extremely low risk of resulting in cancer. In summary, there is an 8–20% false-positivity associated with the Hybrid Capture 2 assay that unfortunately is correlated with bona fide abnormal cytology and histology (CIN2+). However, it is important to recognize that since these abnormalities are *not* caused by high-risk HPV types, they have a very low cancer risk and should be managed accordingly. While this phenomenon is well documented, it tends to be largely ignored and considered noise in the overall clinical performance of an assay. It does however have implications for benchmarking studies and in our view should also be taken into account in any future revisions of the Meijer criteria, since it has a direct impact on the maximum sensitivity and specificity of any assay which only detects high-risk HPV.

### **6.3. L1 consensus-based versus gene-specific PCR approaches**

All molecular assays require some sort of amplification technology in order for them to have the required sensitivity to detect small quantities of target nucleic acid. This can take the form of signal amplification such as that described for the Hybrid Capture 2 assay (where the signal rather than the target is amplified using a chemiluminescent substrate), or in the case of the Hologic Cervista assay, where signal amplification is achieved through the use of a unique isothermal Invader® chemistry that leverages a reusable universal flap and fluo‐ rescence energy transfer (FRET) to amplify the signal [78, 79] or it can use RNA amplifica‐ tion (Proofer and Aptima assays described earlier) or PCR that accounts for the majority of HPV assays on the market [45]. Traditional PCR methods where the amplicon is detected in a secondary process such as on an agarose gel or via hybridization on strips have gradually been replaced by real-time detection methods for clinical use, although the Roche Linear Ar‐ ray and InnoLIPA line blot assays have remained popular where researchers are interested in determining which genotypes are present in clinical samples or for discordant result anal‐ ysis [76, 80, 81]. With PCR one can choose to selectively amplify each viral gene target indi‐ vidually (gene-specific PCR) or utilize a consensus (broad spectrum) primer approach. The latter typically uses degenerate primer sequences to detect conserved protein coding regions of multiple HPV types [82]. This has the advantage that multiple (e.g., all high-risk) HPV types can be detected in a single reaction but it means that you can only record a pooled type result, unless you use a secondary method such as a line blot or bead approach to sub‐ sequently identify the specific genotypes present [83–85]. Gene-specific detection ap‐ proaches offer the possibility of direct individual genotype detection but require the use of multiple reactions [86] or the combining of one or more genotypes in different fluorescent channels using real-time PCR technology [66, 87]. **Table 1** lists the major approaches to HPV DNA detection and details some of their advantages and disadvantages. Head to head test‐ ing suggests that the top performing assays have broadly similar clinical sensitivity and spe‐ cificity [55, 56, 88, 89] so clinical laboratories have a choice in the assay they use. The decision on which assay to adopt may be made based on the needs of a particular laborato‐ ry. However, it is instructive to consider the following assay characteristics when choosing an HPV test:

2, where discordants were resolved using the Roche Linear Array genotyping assay, the authors reported that "HC2+/COBAS− were less likely to contain hrHPV genotypes (12.3 versus 68.9%; *P* < 0.0001) and more likely to contain only lrHPV genotypes (52.8 versus 12.1%; *P* < 0.0001) than those HC2−/COBAS+" and they found "lower CIN2+ rates among women

The clinical impact of this cross-reactivity has not been widely discussed in the literature. Castle et al. used a more stringent dual reference assay method to determine the level of HC2 crossreactivity and concluded that about 8% of all HPV positives were due to cross-reactivity with non-high-risk HPV types in the referred (ALTS Study) population. They concluded that crossreactivity would likely be further reduced in a screening population where there is less overall infection. However, they cautioned that these non-high-risk HPV "cases of CIN2 might be treated by excisional procedures, which can cause iatrogenic morbidity and adversely affect reproductive outcomes" [70]. The previously cited study of Gillio-Tos et al. [75] concluded "If only the samples containing HC2-targeted types tested positive, the positive predictive value (PPV) would have increased from 7.0% (95% CI, 6.1–8.0%) to 8.4% (95% CI, 7.3–9.6%), although 4.9% (95% CI, 2.4–8.8%) of cervical intraepithelial neoplasia grade 2+ (CIN2+) cases would have been missed". Regarding the latter point, "missing" CIN2+ disease caused by non-high-risk HPV types should be the goal of future assays since it has an extremely low risk of resulting in cancer. In summary, there is an 8–20% false-positivity associated with the Hybrid Capture 2 assay that unfortunately is correlated with bona fide abnormal cytology and histology (CIN2+). However, it is important to recognize that since these abnormalities are *not* caused by high-risk HPV types, they have a very low cancer risk and should be managed accordingly. While this phenomenon is well documented, it tends to be largely ignored and considered noise in the overall clinical performance of an assay. It does however have implications for benchmarking studies and in our view should also be taken into account in any future revisions of the Meijer criteria, since it has a direct impact on the maximum sensitivity and specificity

All molecular assays require some sort of amplification technology in order for them to have the required sensitivity to detect small quantities of target nucleic acid. This can take the form of signal amplification such as that described for the Hybrid Capture 2 assay (where the signal rather than the target is amplified using a chemiluminescent substrate), or in the case of the Hologic Cervista assay, where signal amplification is achieved through the use of a unique isothermal Invader® chemistry that leverages a reusable universal flap and fluo‐ rescence energy transfer (FRET) to amplify the signal [78, 79] or it can use RNA amplifica‐ tion (Proofer and Aptima assays described earlier) or PCR that accounts for the majority of HPV assays on the market [45]. Traditional PCR methods where the amplicon is detected in a secondary process such as on an agarose gel or via hybridization on strips have gradually been replaced by real-time detection methods for clinical use, although the Roche Linear Ar‐ ray and InnoLIPA line blot assays have remained popular where researchers are interested in determining which genotypes are present in clinical samples or for discordant result anal‐

with HC2+/COBAS− results" [77].

52 Human Papillomavirus - Research in a Global Perspective

of any assay which only detects high-risk HPV.

**6.3. L1 consensus-based versus gene-specific PCR approaches**



Cervical Cancer Screening at a Crossroads: Learnings from the Past Driving Change for the Future http://dx.doi.org/10.5772/63334 55


**Table 1.** HPV detection methods.

**Assay name HPV**

Hologic Cervista (screening)

Hologic Cervista (genotyping)

Abbott Realtime high-risk HPV

Greiner Papillocheck

Sonic Laboratories (formerly RIATOL) E6/E7 realtime PCR test

BD Onclarity HPV Assay

Cepheid Xpert® HPV **target region**

E6/E7/L 1

E6/E7/L 1

**Nucleic acid type**

54 Human Papillomavirus - Research in a Global Perspective

E1 DNA Consensus

**Consensus or gene-specific**

DNA Gene-specific 2—HPV16,

(genotypes resolved using

array hybridization)

E6/E7 DNA Gene-specific 14 + 3

L1 DNA Consensus 14 Single reaction that

**High-risk type coverage**

HPV18

24 includes 14 high-risk Or 14 highrisk only

moderate risk types

E6/E7 DNA Gene-specific 14 Extended genotyping

E6/E7 DNA Gene-specific 14 Single reaction that

DNA Gene-specific 14 Single reaction

**Advantages Disadvantages References**

No genotyping High positivity rate (clinical specificity has been questioned)

Additional test required for limited genotyping Limited genotyping

Limited genotyping

Labor-intensive workflow requiring three separate rooms

17 individual PCR reactions plus controls required Laboratory developed test (LDT)

Three-well assay design reduces throughput

Limited genotyping capability, Meijer criteria not yet established

[43, 78]

[79]

[92, 93]

[64, 94]

[86]

[66, 95, 96]

[87]

Clinically validated. Internal control CE marked/FDA approved

Single reaction Clinically validated. Internal control CE marked/FDA approved

includes HPV16/18 genotyping. Internal

Semiautomated, CE

Full genotyping CE marked

Full genotyping Partially validated per Meijer criteria Semiautomated

HPV16, 18, 45, 31, 51,

Clinically validated Fully automated CE marked

includes HPV16/18+45 genotyping. Internal

52

control

control

marked


The first decision one may want to make is whether to choose an assay that targets RNA or DNA. Clinical performance is very similar so it may come down to a practical decision on workflow and laboratory suitability. At the current time, the long-term NPV of DNA-based assays is well established and enshrined in consensus guidelines whereas the evidence for RNA assays is still accumulating [58, 59, 98]. DNA assays also have the advantage of improved target stability which imposes stricter adherence to laboratory cleaning methods to avoid degrading the more labile RNA targets [53]. As mentioned above, real-time PCR approaches now predominate in the market so one has the choice of either a consensus-based primer design or a gene-specific primer approach (**Table 1**). Consensus approaches yield a pooled high-risk result with HPV16/HPV18 genotype identification from a single well whereas gene-specific approaches can offer more extended genotyping information which can limit throughput. The clinical benefits of extended genotyping will be discussed later in this chapter but here we will focus on the analytical performance of the two assay design approaches.

#### **6.4. The link between analytical and clinical assay performance**

Good analytical assay performance is a requirement but not a guarantee of clinical assay performance. The viral load that correlates well with prediction of CIN2+ risk is approximately 5000 copies per reaction as established by the Hybrid Capture 2 test [41]. Thus, it is important to determine the clinical cutoff (a level of positivity in the assay that best correlates with histologically confirmed disease) for each individual assay using standard receiver operating characteristic plots of sensitivity versus 1–specificity so as to provide the highest possible sensitivity and an optimal specificity [55, 56, 88]. Assays that are analytically too sensitive will refer an unacceptably high number of women to colposcopy for possible biopsy and treatment [43]. **Table 2** describes some of the unique diagnostic challenges associated with cervical cancer screening that make this task a complicated one. This is reflected in the reports in the literature on viral load measurements, which are mixed at best. While HPV16 viral load has been shown to correlate with disease progression [99, 100], non-HPV16 types have been reported to show less correlation [101]. A recent well controlled study that focused exclusively on single infections found that HPV16/HPV18/HPV31/HPV45 viral load was correlated with abnormal cytology. This study is likely to have benefited by excluding the complication of mixed infections and the use of a high quality E6 gene-specific real-time PCR assay [102]. A positive correlation for these types (and HPV33) with disease severity was also confirmed in an independent study with histological endpoints [103]. These apparent discrepancies may be explained by analytical performance differences in the methods used to measure viral load. As mentioned above, most commercial assays use consensus L1 primers because of the ease of use offered by a single reaction that can detect 14 high-risk viruses. However, consensus primer designs exhibit poor detection of mixed infections due to HPV-type suppression or restriction, where the overabundance of one HPV type in a mixed infection can lead to failure to detect lower levels of a coinfecting virus. Van Doorn et al. were one of the first to demonstrate this phenomenon using spiking experiments where detectable levels of HPV18 were elimi‐ nated using an increasing concentration of a competing HPV16 target [104]. This result was later confirmed in cervical smear and biopsy specimens where the L1 SPF10 consensus primer assay was compared to a gene-specific E6 assay where significantly more genotypes (*P* < 0.0001) were identified by the E6 assay, especially for HPV types 16, 35, 39, 45, 58, and 59 and the authors concluded "that broad-spectrum PCRs are hampered by type competition when multiple HPV genotypes are present in the same sample" [105]. Similar results have been RNA assays is still accumulating [58, 59, 98]. DNA assays also have the advantage of improved target stability which imposes stricter adherence to laboratory cleaning methods to avoid degrading the more labile RNA targets [53]. As mentioned above, real-time PCR approaches now predominate in the market so one has the choice of either a consensus-based primer design or a gene-specific primer approach (**Table 1**). Consensus approaches yield a pooled high-risk result with HPV16/HPV18 genotype identification from a single well whereas gene-specific approaches can offer more extended genotyping information which can limit throughput. The clinical benefits of extended genotyping will be discussed later in this chapter but here we will

Good analytical assay performance is a requirement but not a guarantee of clinical assay performance. The viral load that correlates well with prediction of CIN2+ risk is approximately 5000 copies per reaction as established by the Hybrid Capture 2 test [41]. Thus, it is important to determine the clinical cutoff (a level of positivity in the assay that best correlates with histologically confirmed disease) for each individual assay using standard receiver operating characteristic plots of sensitivity versus 1–specificity so as to provide the highest possible sensitivity and an optimal specificity [55, 56, 88]. Assays that are analytically too sensitive will refer an unacceptably high number of women to colposcopy for possible biopsy and treatment [43]. **Table 2** describes some of the unique diagnostic challenges associated with cervical cancer screening that make this task a complicated one. This is reflected in the reports in the literature on viral load measurements, which are mixed at best. While HPV16 viral load has been shown to correlate with disease progression [99, 100], non-HPV16 types have been reported to show less correlation [101]. A recent well controlled study that focused exclusively on single infections found that HPV16/HPV18/HPV31/HPV45 viral load was correlated with abnormal cytology. This study is likely to have benefited by excluding the complication of mixed infections and the use of a high quality E6 gene-specific real-time PCR assay [102]. A positive correlation for these types (and HPV33) with disease severity was also confirmed in an independent study with histological endpoints [103]. These apparent discrepancies may be explained by analytical performance differences in the methods used to measure viral load. As mentioned above, most commercial assays use consensus L1 primers because of the ease of use offered by a single reaction that can detect 14 high-risk viruses. However, consensus primer designs exhibit poor detection of mixed infections due to HPV-type suppression or restriction, where the overabundance of one HPV type in a mixed infection can lead to failure to detect lower levels of a coinfecting virus. Van Doorn et al. were one of the first to demonstrate this phenomenon using spiking experiments where detectable levels of HPV18 were elimi‐ nated using an increasing concentration of a competing HPV16 target [104]. This result was later confirmed in cervical smear and biopsy specimens where the L1 SPF10 consensus primer assay was compared to a gene-specific E6 assay where significantly more genotypes (*P* < 0.0001) were identified by the E6 assay, especially for HPV types 16, 35, 39, 45, 58, and 59 and the authors concluded "that broad-spectrum PCRs are hampered by type competition when multiple HPV genotypes are present in the same sample" [105]. Similar results have been

focus on the analytical performance of the two assay design approaches.

**6.4. The link between analytical and clinical assay performance**

56 Human Papillomavirus - Research in a Global Perspective

reported by Mori et al. who found that "three consensus primers frequently caused incorrect genotyping in the selected clinical specimens containing HPV16 and one or two of HPV18, 31, 51, 52, and 58" and went on to conclude "that PCR with consensus primers is not suitable for genotyping HPV in specimens containing multiple HPV types" [106]. Given that approxi‐ mately one-third or more of clinical specimens can harbor more than one HPV type [107], this should be considered carefully when designing vaccine monitoring or other studies requiring genotyping. This has recently been underlined by a *post-hoc* analysis of the PATRICIA vaccine trial where Struyf et al. found that an E6-based multiplex type-specific PCR and reverse hybridization assay showed improved sensitivity versus the L1-based SPF10 PCR-DNA enzyme immunoassay (DEIA)/line probe assay (LiPA25) used in the original trial, resulting in higher vaccine efficacy estimates for nonvaccine oncogenic HPV types [108]. The E6-assay was developed by Van Doorn et al. and had previously been shown to increase genotype detection by 14.3% [105]. Another negative impact of HPV-type suppression was reported by Cornall et al. who hypothesized that the sensitivity of consensus-based PCR approaches could be altered in highly vaccinated populations such as Australia. They confirmed their hypothesis using the Roche HPV linear array genotype assay by simulating samples containing 1000 copies of one or two high-risk HPV DNA genomes in the presence and the absence of 10,000 copies of the HPV16 genome. HPV16 alone did not affect detection of other high-risk genotypes; however, when HPV16 and an additional genotype were present, detection of HPV31, 33, 51, or 59 was impeded, indicating potential for misrepresentation of population-based prevalence of these genotypes and false evidence for type replacement following vaccination [109]. A nextgeneration sequencing (NGS) method also found that consensus MY09/MY11 primers had "lower sensitivity for some HPV types than LiPA, conceivably due to the poor sensitivity of the MY09/MY11-based primers"[110]. The results from the WHO LabNet genotyping panel broadly support these findings. The panel consists of approximately 43 DNA standardized DNA samples with single and mixed infections of the 14 high-risk HPV types (and low-risk types HPV6 and HPV11) and three cell line extraction controls. Candidate assays are consid‐ ered proficient if they can detect 50 international units (IU) of HPV type 16 (HPV16) and HPV18 DNA and 500 genome equivalents (GE) for the other 14 HPV types. The 2010 panel results reported the data from 98 laboratories who submitted 132 datasets, only ~20% of which were deemed proficient for all HPV types. In addition, approximately 35% of the test panels had multiple false positive results and were considered nonproficient. Virtually all of the assays that submitted test results were L1 consensus-primer based [63]. The results from the 2014 panel from 119 participating global laboratories (146 datasets) were recently reported and the overall results showed an improvement, with 59% of the test results deemed 100% proficient and 20% nonproficient [111]. Nevertheless, there is little room for complacency when one considers that this implies that 40% of current assays do not accurately detect HPV types. In summary, assay design has a direct impact on clinical performance and the ability to accurately genotype both single and mixed infections will play an ever-increasing role, especially in a postvaccination era.


#### Cervical Cancer Screening at a Crossroads: Learnings from the Past Driving Change for the Future http://dx.doi.org/10.5772/63334 59


**Table 2.** Diagnostic challenges associated with cervical cancer screening.

**Diagnostic challenge**

Most infections do no result in cancer

Younger women are more likely to be infected with

more likely to develop cancer

Analytical positive ≠ clinical positive

Results impacted by quality of specimen collection

Large number of normal versus abnormal cells present in sample leads to sampling variation

Most endocervical samples are collected in preservative which lead to clumping of exfoliated cells

HPV

[42]

Cancer incidence can be as low as 5–8 per 100,000 women screened [112]

58 Human Papillomavirus - Research in a Global Perspective

Screening of women < 25–30 may

to overdiagnosis

Overtesting may cause unnecessary anxiety in younger

lead

[113]

patients

Over- and underreferral possible

Poorly collected or expressed samples

Sampling errors can occur and lead to false negative results [116]

increases unsatisfactory rate and can lead to false negative results if no cellular control present

**Impact Mitigation Current status Comments**

50% of cancers still occur in women who have not been screened

Primary screening approved in US for >25, with >30 more common for cotesting with cytology

Meijer guidelines and FDA approval provide reassurance but in-country real-world

Performance of different collection devices should be considered [114, 115]

Most of the newer assays

Semiautomated or fully automated methods improve reliability (**Table**

have a cellular processing control which helps mitigate this

risk

**1**)

validation is also informative Need to continue to expand programs to reach underserved women (self-sampling)

Ongoing countrywide programs will shed more light on the correct age to start screening (which could be population

Need more research on the impact of crossreactivity and molecular mimics as well as mixed

infections

Physicians and laboratory personnel need to adhere to recommended

procedures for collection and processing of specimens

Most manufacturers have adapted their methods for the two common liquid-based-cytology media on the market

Organized screening programs need to reach as many women

Current guidelines recommend not screening women <25 or <30

specific) Older women are

Establish appropriate clinical cutoff in target

population

Include cellular versus PCR

the assay

processing control in

Assay needs to be robust to differences in pipetting volumes

recommended test

Samples need to be vortexed adequately and aliquots removed promptly prior to robust extraction and

around

volume

Adhere to expert guidelines and screen appropriately

as possible

### **6.5. HPV integration-induced deletions: impact on screening programs?**

It is now well established that the HPV viral genome can integrate into the host genome during disease progression. The ability to integrate varies by high-risk HPV type with approximately 70% of HPV16-associated cervical cancers containing integrated HPV16 sequences, rising to almost 100% for HPV18 [122, 123]. Next-generation sequencing methods are now shedding new light on this phenomenon and the emerging picture suggests the following:


Thus, it appears that viral integration HPV integration is an unintended consequence of HPV replication and that following integration, the virus life cycle is aborted. Nevertheless, virus integration is a routine consequence of infection and the observation that it can occur earlier in the disease progression prompts the question whether it has a measureable effect on the ability of HPV assays to detect the virus. Several authors have expressed concern that L1-based assays can fail to detect late-stage cancers due to target deletion [128–130] and there is no doubt that L1-deleted cancers occur as evidenced by case studies [129, 131]. Several studies have reported that the E6/E7 assays have increased sensitivity for cancer versus L1-based assays [132–134]. Some have dismissed this as due to earlier methodological differences due to the difficulty of detecting longer L1 amplicons in formalin fixed paraffin embedded (FFPE) tissue. However, this is not supported by more recent literature where even using gold standard FFPE processing methods, one group reported only a 91% detection rate in cancer specimens with two different L1 primer assays [135]. Another recent study of cervical adenocarcinomas reported that PCR using a very sensitive L1-based SPF10-PCR resulted in 482 cases (67%) positive for HPV DNA, but that testing using type-specific E6 PCR added 53 HPV-positive cases (a 7% increase in detection). The study design also accounted for DNA adequacy in the samples [136]. Adenocarcinomas represent about 25% of all cervical cancers [137, 138] and are known to be prone to HPV DNA integration, so this increase in sensitivity for detection of adenocarcinomas could potentially translate to the detection of another 1–2% of total cancer cases (assuming an increase in just adenocarcinoma cases). Large-scale studies will be needed to address the question of whether or not the above reported difference in assay performance (due to increased maintenance of intact target regions postintegration) has a measureable impact on clinical sensitivity in cervical cancer screening. This warrants further study, especially given the reported ability of the virus to integrate in approximately 50% of cervical smears from patients with CIN [124]. Finally, the Roche cobas assay has recently been reported to have a 94% detection rate in the smears from women with histologically confirmed invasive cervical cancer [139].

## **7. The evolution of HPV screening**

**1.** Viral integration appears to be a random (nontargeted) event with disruption of a wide

**2.** Integration is associated with fragile sites in human DNA and is likely opportunistic, with the HPV virus taking advantage of exposed DNA to integrate into the host genome. Regions of microhomology between the viral and human sequences are enriched near integration sites suggesting that integration may be driven by the host DNA repair

**4.** Contrary to what was previously believed E6 and E7 oncogenes can also be deleted during integration. However, this occurs less frequently than in other viral genes such as L1, L2, E1, and E2 and there appears to be significant enrichment of (intact) viral gene E7 > E4 > E5 > E6 reads among the cervical tumor samples [124, 125, 127].(It is possible that this may simply due to the random nature of the integration process and reflect the fact that L1, L2,

**5.** Viral integration appears to occur at a higher frequency than perhaps previously under‐ stood and can also be detected in Pap smears, one study reporting integration in 53% of

Thus, it appears that viral integration HPV integration is an unintended consequence of HPV replication and that following integration, the virus life cycle is aborted. Nevertheless, virus integration is a routine consequence of infection and the observation that it can occur earlier in the disease progression prompts the question whether it has a measureable effect on the ability of HPV assays to detect the virus. Several authors have expressed concern that L1-based assays can fail to detect late-stage cancers due to target deletion [128–130] and there is no doubt that L1-deleted cancers occur as evidenced by case studies [129, 131]. Several studies have reported that the E6/E7 assays have increased sensitivity for cancer versus L1-based assays [132–134]. Some have dismissed this as due to earlier methodological differences due to the difficulty of detecting longer L1 amplicons in formalin fixed paraffin embedded (FFPE) tissue. However, this is not supported by more recent literature where even using gold standard FFPE processing methods, one group reported only a 91% detection rate in cancer specimens with two different L1 primer assays [135]. Another recent study of cervical adenocarcinomas reported that PCR using a very sensitive L1-based SPF10-PCR resulted in 482 cases (67%) positive for HPV DNA, but that testing using type-specific E6 PCR added 53 HPV-positive cases (a 7% increase in detection). The study design also accounted for DNA adequacy in the samples [136]. Adenocarcinomas represent about 25% of all cervical cancers [137, 138] and are known to be prone to HPV DNA integration, so this increase in sensitivity for detection of adenocarcinomas could potentially translate to the detection of another 1–2% of total cancer cases (assuming an increase in just adenocarcinoma cases). Large-scale studies will be needed to address the question of whether or not the above reported difference in assay performance (due to increased maintenance of intact target regions postintegration) has a measureable impact on clinical sensitivity in cervical cancer screening. This warrants further study, especially given the reported ability of the virus to integrate in approximately 50% of cervical

**3.** Viral sequences are frequently deleted during the integration process [124, 125].

E1, and E2 are the largest ORFs in the HPV genome, **Figure 2**).

cytology samples with histology grades CIN1–3 [124].

variety of host genes across the human genome [124–126].

machinery [124, 125].

60 Human Papillomavirus - Research in a Global Perspective

### **7.1. Cotesting with cytology versus primary HPV screening**

It has been known for 10 years that HPV testing is substantially more sensitive in detecting CIN2+ than cytology (96.1 versus 53.0%) but less specific (90.7 versus 96.3%) [140]. In the intervening period, large randomized controlled trials (RCTs) have continued to reaffirm this point. Pileggi et al. [141] recently performed a meta-analysis of global RCTs which included trials from Italy (NTCC trials I+II [142, 143]), the UK (ARTISTIC Trial [144]), Finland [145], India [146], Canada [147], and the Netherlands (POBASCAM [148]). The analysis from this larger more comprehensive dataset showed a significantly higher detection of both CIN2+ and CIN3+ by HPV testing versus cytology and that the relative specificity of cytology was higher. However, in women greater than 30 years of age, the specificity was not statistically different ("almost overlapping"). In addition, the pooled relative PPV was not significantly lower for HPV compared with cytology. These results suggest that the difference in specificity is actually less than previously thought, at least in women over 30. A recent US *post-hoc* analysis of a large group of women from multiple practices reported that positive Pap + HPV (cotest) was more sensitive than either a positive HPV alone or Pap alone for detection of CIN3+ and suggested that HPV primary screening might miss cancers if not combined with the Pap [149]. However, the study design has come under criticism from an independent expert, because the short follow up time of 1 year biases results in favor of cytology and all women were not followed equally [150]. Thus, large global studies support the use of primary HPV testing as a means to improve cervical cancer detection and it explains why many countries are now in the process of implementing either pilot or countrywide programs to replace cytology and/or cotesting [151, 152]. In the United States, one HPV test has received FDA approval for primary screening in women >25 years of age [153] and interim guidelines for its use have been issued [113]. The reaction to this decision has been mixed with some arguing that cotesting detects more disease [154]. However, data continue to accumulate both in the United States and in Europe that supports the long-term NPV of a primary HPV negative result, thus permitting safe interval extension [155, 156]. A study comparing primary HPV versus cotesting versus primary cytology concluded that "primary HPV testing every 3 years might provide as much, if not more, reassurance against precancer and cancer, compared to primary Pap testing every 3 years and cotesting every 5 years"[156]. Thus, the choice of which screening paradigm to adopt will likely be influenced by the resources available together with the desired screening interval and risk tolerance of the medical community and the patients they serve. Despite the slow pace of change, it seems clear that HPV primary screening will gradually be adopted worldwide, especially in countries which do not have current cytology infrastructure. HPV testing is considerably more reproducible than cytology [157] and with the advent of automation, highly skilled personnel are no longer required to implement IVD-qualified tests. It should be noted, however, that primary HPV screening cannot be used in isolation to refer women to colpo‐ scopy. Biomarkers such as p16 [121] or cytology or HPV16/HPV18 genotyping need to be considered to increase specificity and reduce the number of unnecessary colposcopies [72, 153, 158].

### **7.2. Reaching underserved women: self-sampling methods**

It is well established that the effectiveness of cervical cancer screening programs is limited by the number of women who do not participate—with 50% or more of disease being detected in women who have not been screened [159–162]. While there may be many reasons (cultural, socioeconomic, and religious) why women choose not to participate in screening programs available to them, it is clear that self-collection offers an effective outreach tool with which to increase participation. For example, in Finland the participation rate was <70% but was increased to 72.6–79.9% by sending reminder letters to nonattendees in 22 municipalities, and to 83.4% by sending self-collection kits to those who did not attend after receiving the initial invitation letter [163]. Similar finding on outreach were reported in Sweden where telephone invitations to long-term nonattendees increased the participation rate within the following 12 months to 18.0% versus 10.6% in a control group [164]. In Canada, reminder letters were compared head to head with sending a self-sampling kit and standard screening and women receiving the self-collected HPV kit were 3.7 times more likely to undergo screening compared with the standard of care [165]. Finally, a recent Danish outreach program where 5000 women were invited to "opt-in" and receive a self-collection kit resulted in the detection of nine cancers. This translates to yield of 1.8 per 1000, a dramatic enrichment for disease detection versus the Danish population rate, estimated at 12.9 per 100,000 women [166, 167]. Thus, selfsampling is emerging as a very important tool in national screening programs and one that continues to generate a high degree of interest [168]. Two principal self-collection methods that have been described in the literature: cervico-vaginal collection and urine collection.

### *7.2.1. Cervico-vaginal collection methods*

A number of different cervico-vaginal methods have been described in the literature (reviewed in [169, 170]) and range from simple brush devices currently used by physicians to custom designed self-collection devices such as the Rovers® Evalyn® brush (Rovers Medical Devices BV, the Netherlands) and HerSwab™ (Eve Medical, Canada). Earlier literature reported mixed results versus physician collected endocervical samples in terms of sensitivity and specificity. However, more recent studies have confirmed that self-collected specimens can have the same sensitivity for CIN2+ as physician-collected specimens [171]. A recent meta-analysis concluded that signal amplification methods were not sensitive enough for self-collection use but that certain PCR methods had similar sensitivity to physician-collected samples [172]. This has been confirmed in a number of small studies using clinically validated HPV tests where sensitivities for CIN2+ were similar to physician-collected samples [173–176]. It is likely that both the quality of the instructions used and the sample workflow/assay play a role in the ability to accurately detect infections. Further large-scale studies are needed to further demonstrate the utility of these methods in screening populations.

### *7.2.2. HPV testing from urine*

however, that primary HPV screening cannot be used in isolation to refer women to colpo‐ scopy. Biomarkers such as p16 [121] or cytology or HPV16/HPV18 genotyping need to be considered to increase specificity and reduce the number of unnecessary colposcopies [72, 153,

It is well established that the effectiveness of cervical cancer screening programs is limited by the number of women who do not participate—with 50% or more of disease being detected in women who have not been screened [159–162]. While there may be many reasons (cultural, socioeconomic, and religious) why women choose not to participate in screening programs available to them, it is clear that self-collection offers an effective outreach tool with which to increase participation. For example, in Finland the participation rate was <70% but was increased to 72.6–79.9% by sending reminder letters to nonattendees in 22 municipalities, and to 83.4% by sending self-collection kits to those who did not attend after receiving the initial invitation letter [163]. Similar finding on outreach were reported in Sweden where telephone invitations to long-term nonattendees increased the participation rate within the following 12 months to 18.0% versus 10.6% in a control group [164]. In Canada, reminder letters were compared head to head with sending a self-sampling kit and standard screening and women receiving the self-collected HPV kit were 3.7 times more likely to undergo screening compared with the standard of care [165]. Finally, a recent Danish outreach program where 5000 women were invited to "opt-in" and receive a self-collection kit resulted in the detection of nine cancers. This translates to yield of 1.8 per 1000, a dramatic enrichment for disease detection versus the Danish population rate, estimated at 12.9 per 100,000 women [166, 167]. Thus, selfsampling is emerging as a very important tool in national screening programs and one that continues to generate a high degree of interest [168]. Two principal self-collection methods that have been described in the literature: cervico-vaginal collection and urine collection.

A number of different cervico-vaginal methods have been described in the literature (reviewed in [169, 170]) and range from simple brush devices currently used by physicians to custom designed self-collection devices such as the Rovers® Evalyn® brush (Rovers Medical Devices BV, the Netherlands) and HerSwab™ (Eve Medical, Canada). Earlier literature reported mixed results versus physician collected endocervical samples in terms of sensitivity and specificity. However, more recent studies have confirmed that self-collected specimens can have the same sensitivity for CIN2+ as physician-collected specimens [171]. A recent meta-analysis concluded that signal amplification methods were not sensitive enough for self-collection use but that certain PCR methods had similar sensitivity to physician-collected samples [172]. This has been confirmed in a number of small studies using clinically validated HPV tests where sensitivities for CIN2+ were similar to physician-collected samples [173–176]. It is likely that both the quality of the instructions used and the sample workflow/assay play a role in the ability to accurately detect infections. Further large-scale studies are needed to further demonstrate the

**7.2. Reaching underserved women: self-sampling methods**

62 Human Papillomavirus - Research in a Global Perspective

*7.2.1. Cervico-vaginal collection methods*

utility of these methods in screening populations.

158].

HPV testing from urine has been performed for over 20 years and similar to cervico-vaginal methods, early studies were somewhat discouraging with decreased sensitivity versus physician-collected endocervical samples being reported [177]. This was compounded by the demonstration that male urine samples were a poor sample type for HPV detection [178], which has been confirmed in more recent studies and likely reflects a true biological difference between men and women [179, 180]. The general interest in self-sampling and the introduction of HPV vaccines, which can benefit from noninvasive population-based monitoring, has resulted in a renewed interest in urine as an alternative sample type for cervical cancer screening [181]. There is now a growing body of evidence that urine, in particular first void urine, has a lot of potential for detecting HPV infections [182, 183]. The basic premise of HPV urine testing is founded on the hypothesis that "at the start of the void, urine gets contaminated by debris and impurities lining the urethra opening, including mucus and debris of exfoliated cells from the vagina, cervix and uterus" [181]. Recent research has also demonstrated that optimal sample workflows and sensitive detection methods improve clinical performance:


as real-time PCR should be used to ensure optimal results and clinical cutoff adjustments may be required versus endocervical specimens [193].

## **8. "Safety in numbers": time for extended genotyping in cervical cancer screening**

The majority of (clinically validated) HPV screening tests have the ability to provide geno‐ typing information on only two types, HPV16 and HPV18 [46]. There is now a growing body of evidence that extended genotyping (beyond types 16 and18) better stratifies a woman's risk for subsequent disease: A recent Danish study looked at the long-term absolute risk for CIN3+ in women following a baseline HPV genotyping result and found that HPV31 and HPV33 had the same or greater longitudinal risk as HPV18 over a 10-year period [194]; Schiffman et al. [195] confirmed this finding for HPV31 in a US cohort over a 15-year follow up period; Cuzick et al. [196] looked at the positive predictive value of genotyping in a referred UK population and found that HPV33 had a higher PPV than HPV16 (59.8 versus 57.8) and that HPV31 was higher than HPV18 (39.5 versus 29.3). Similar trends were reported in a much larger New Mexico study of 21,297 women of whom 77% had biopsies. Among women with CIN3+ (*n* = 1880), 14.9% were attributable to HPV31, 5.2% to HPV33 and 4.9% to HPV18, with HPV16 responsible for 54.1% [197]. Roche Linear Array genotyping analysis of patients from the ATHENA trial also support these findings: among 40,901 women aged ≥25 years HPV16 conferred the greatest absolute risk of ≥CIN3 both in women aged 25–29 and ≥30 years (14.2% and 15.1%, respectively) followed by HPV31 (8.0% and 7.9%), HPV52 (6.7% and 4.4%), and HPV18 (2.7% and 9.0%) [198].

While the majority of cancers are caused by HPV16 and HPV18, presumably due to the increased ability of these genotypes to persist and induce oncogenic changes, one must equally concede that HPV31, 33, 45, 52, and 58 are the next most potent carcinogens of the high-risk HPV types [199]. This is supported by the introduction of the nine-valent HPV vaccine, where the addition of these types is estimated to provide protection against an additional ~20% of cancers [200]. The clinical paradigm of equal management of equal risk [201] strongly suggests that knowledge of HPV31 and HPV33 infections has clinical value and can aid in patient management. In addition, the widespread introduction of the new nine-valent vaccine is expected to have a profound effect on the incidence of CIN3+ disease, further underlining the need for a more comprehensive genotype profile of pre- and postvaccinated women [202]. Finally, the use of gene-specific versus consensus PCR genotyping approaches can alleviate the issue of apparent type replacement in vaccinated women where HPV types such as HPV31 and HPV33 may appear to be increasing in prevalence but are simply being unmasked due to the reduced incidence of HPV16 and HPV18 in the postvaccination era [109].

Extended genotyping also offers a potential solution to key emerging issues in cervical cancer screening:

**I.** Accurate risk stratification—absolute risk calculations *a priori* are underestimated when two or more HPV types with different CIN3 risks are pooled together (e.g., the 10-year longitudinal study of Kahn et al. attributes a very low risk to pooled non-16/18 types [203] which has been consistently found *not* to be the case in subsequent studies where individual genotypes have been detected [194, 196–198]). Precise risk stratifi‐ cation is essential for consistent and informed patient management. This is especially true in cytology-negative HPV-positive women where genotyping can provide clinicians with a much more informed assessment of future risk versus a pooled HPV positive result.

**II.** On the other hand, extended genotyping allows one to group HPV types with similar risk, thereby simplifying patient management algorithms. Cuzick et al. [196] pro‐ posed a three tiered risk group approach to patient stratification based on the positive predictive value of different genotypes for CIN3+ disease in a referred population. Schiffman et al. recently published the largest longitudinal ASC-US population analysis to date (Persistence and Progression cohort with 13,890 women aged 21+with HC2 (QIAGEN)-positive ASC-US at enrollment and median follow-up of 3.0 years). The authors used the concept of equal management of equal risk approach and calculated the 3-year CIN3+ risk for all HC2-positive women with ASC-US (5.2%), using this as the "benchmark" risk for colposcopic referral. They concluded that the 3-year risk for developing CIN3+ associated with high-risk HPV types 35, 39, 51, 56, 59, 66, and 68 (2.7% for HPV51, 1.6% for HPV39/HPV68/HPV35, and 1.3% for HPV59/ HPV56/HPV66) "might be low enough to recommend 1-year retesting, permitting viral clearance. This strategy would defer colposcopy for 40% of women with HPVpositive ASC-US, half of whom would be cotest-negative at 1-year return" [96].

Thus, extended genotyping offers the potential of improved risk stratification and simpler patient management, helping to improve patient outcomes with reduced intervention.

## **9. Future perspectives**

as real-time PCR should be used to ensure optimal results and clinical cutoff adjustments

**8. "Safety in numbers": time for extended genotyping in cervical cancer**

The majority of (clinically validated) HPV screening tests have the ability to provide geno‐ typing information on only two types, HPV16 and HPV18 [46]. There is now a growing body of evidence that extended genotyping (beyond types 16 and18) better stratifies a woman's risk for subsequent disease: A recent Danish study looked at the long-term absolute risk for CIN3+ in women following a baseline HPV genotyping result and found that HPV31 and HPV33 had the same or greater longitudinal risk as HPV18 over a 10-year period [194]; Schiffman et al. [195] confirmed this finding for HPV31 in a US cohort over a 15-year follow up period; Cuzick et al. [196] looked at the positive predictive value of genotyping in a referred UK population and found that HPV33 had a higher PPV than HPV16 (59.8 versus 57.8) and that HPV31 was higher than HPV18 (39.5 versus 29.3). Similar trends were reported in a much larger New Mexico study of 21,297 women of whom 77% had biopsies. Among women with CIN3+ (*n* = 1880), 14.9% were attributable to HPV31, 5.2% to HPV33 and 4.9% to HPV18, with HPV16 responsible for 54.1% [197]. Roche Linear Array genotyping analysis of patients from the ATHENA trial also support these findings: among 40,901 women aged ≥25 years HPV16 conferred the greatest absolute risk of ≥CIN3 both in women aged 25–29 and ≥30 years (14.2% and 15.1%, respectively) followed by HPV31 (8.0% and 7.9%), HPV52 (6.7% and 4.4%), and

While the majority of cancers are caused by HPV16 and HPV18, presumably due to the increased ability of these genotypes to persist and induce oncogenic changes, one must equally concede that HPV31, 33, 45, 52, and 58 are the next most potent carcinogens of the high-risk HPV types [199]. This is supported by the introduction of the nine-valent HPV vaccine, where the addition of these types is estimated to provide protection against an additional ~20% of cancers [200]. The clinical paradigm of equal management of equal risk [201] strongly suggests that knowledge of HPV31 and HPV33 infections has clinical value and can aid in patient management. In addition, the widespread introduction of the new nine-valent vaccine is expected to have a profound effect on the incidence of CIN3+ disease, further underlining the need for a more comprehensive genotype profile of pre- and postvaccinated women [202]. Finally, the use of gene-specific versus consensus PCR genotyping approaches can alleviate the issue of apparent type replacement in vaccinated women where HPV types such as HPV31 and HPV33 may appear to be increasing in prevalence but are simply being unmasked due to

the reduced incidence of HPV16 and HPV18 in the postvaccination era [109].

Extended genotyping also offers a potential solution to key emerging issues in cervical cancer

**I.** Accurate risk stratification—absolute risk calculations *a priori* are underestimated

when two or more HPV types with different CIN3 risks are pooled together (e.g., the 10-year longitudinal study of Kahn et al. attributes a very low risk to pooled non-16/18

may be required versus endocervical specimens [193].

64 Human Papillomavirus - Research in a Global Perspective

**screening**

HPV18 (2.7% and 9.0%) [198].

screening:

While we have made great strides in both detecting and preventing cervical cancer over the last 20 years, it still represents a significant challenge in both developed and developing settings. In countries with a high disease burden such as Mexico, Ecuador, Samoa, and Colombia, it is the number one cancer in young women and the number two cancer in women of all ages, whereas in several areas in Africa and in Cambodia, it is primary cause of cancer and death [112]. With 50% of cancers occurring in underserved women, it is clear that the biggest remaining challenges are in implementation, not technology development. Self-collection methods offer new hope of reaching women who are currently not captured in traditional screening programs. Ongoing studies should shed further light on the ability of these methods to equal those used in the physician's office or at least provide a means to target these women for follow up in the medical system (analogous to current home pregnancy test kits). HPV vaccines have been shown to be safe and efficacious but here again the biggest hurdle is achieving the required vaccine coverage in the target population. It is hoped that the successful implementation models such as those in the UK and Australia will be adopted elsewhere to boost global vaccine coverage. It should also not go unmentioned that the other barrier to improved disease prevention and detection is resistance to change. While medicine can be understandably conservative, waiting for substantial bodies of evidence to accumulate prior to changing clinical management, we should also constantly challenge ourselves on this point, given the severity of the disease burden imposed by HPV. With the benefit of hindsight, one can justifiably argue that even the Pap smear itself took 30 years too long to be implemented, HPV primary screening could have been adopted 10 years ago based on the available evidence, and vaccine coverage is suffering today due to the emergence of vaccine opposition which is not supported by scientific evidence [204]. We should also continue to challenge the *status quo* on cervical cytology and histopathology while they have an important role to play, their future role should be evaluated in the context of comprehensive screening strategies [205, 206]. There is an urgent need to eradicate cervical cancer for the sake of both the current generation of women and the men and women who follow them. At the same time, there is an ever increasing pressure on global healthcare systems to do more with less. Ironically, this will likely serve as a catalyst for change as health economic studies identify which screening and vaccination strategies are the most cost-effective and offer the highest adoption rate. Ongoing and future research should focus on answering key questions in cervical cancer program implementation such as the effect of vaccination on HPV prevalence, and the ability of extended genotyping to better stratify a patient's risk. Optimal triage strategies should also be investi‐ gated together with new studies on the long-term effects on the physical and psychological health of patients who are overtested and overtreated. As we settle into the twenty-first century of cervical cancer screening, one thing is clear, we have effective means to detect and prevent disease. The real question is: do we have the resolve to deploy these tools in an effective manner? We join others in the hope that we will learn from the past and quicken the pace of change in response to the global threat posed by cervical disease [207].

Trademarks are the property of their respective owners.

© 2016 BD. BD, the BD Logo and BD Onclarity are trademarks of Becton, Dickinson and Company..

## **Author details**

Laurence M. Vaughan\* , Brian R. Faherty, Erin C. Gutierrez, James M. Harris, William A. Nussbaumer and Ryan J. Schwab

\*Address all correspondence to: laurence.vaughan@bd.com

Diagnostics Systems, BD Life Sciences, Sparks, MD, USA

## **References**

models such as those in the UK and Australia will be adopted elsewhere to boost global vaccine coverage. It should also not go unmentioned that the other barrier to improved disease prevention and detection is resistance to change. While medicine can be understandably conservative, waiting for substantial bodies of evidence to accumulate prior to changing clinical management, we should also constantly challenge ourselves on this point, given the severity of the disease burden imposed by HPV. With the benefit of hindsight, one can justifiably argue that even the Pap smear itself took 30 years too long to be implemented, HPV primary screening could have been adopted 10 years ago based on the available evidence, and vaccine coverage is suffering today due to the emergence of vaccine opposition which is not supported by scientific evidence [204]. We should also continue to challenge the *status quo* on cervical cytology and histopathology while they have an important role to play, their future role should be evaluated in the context of comprehensive screening strategies [205, 206]. There is an urgent need to eradicate cervical cancer for the sake of both the current generation of women and the men and women who follow them. At the same time, there is an ever increasing pressure on global healthcare systems to do more with less. Ironically, this will likely serve as a catalyst for change as health economic studies identify which screening and vaccination strategies are the most cost-effective and offer the highest adoption rate. Ongoing and future research should focus on answering key questions in cervical cancer program implementation such as the effect of vaccination on HPV prevalence, and the ability of extended genotyping to better stratify a patient's risk. Optimal triage strategies should also be investi‐ gated together with new studies on the long-term effects on the physical and psychological health of patients who are overtested and overtreated. As we settle into the twenty-first century of cervical cancer screening, one thing is clear, we have effective means to detect and prevent disease. The real question is: do we have the resolve to deploy these tools in an effective manner? We join others in the hope that we will learn from the past and quicken the pace of change in

66 Human Papillomavirus - Research in a Global Perspective

response to the global threat posed by cervical disease [207].

© 2016 BD. BD, the BD Logo and BD Onclarity are trademarks of Becton, Dickinson and

, Brian R. Faherty, Erin C. Gutierrez, James M. Harris,

Trademarks are the property of their respective owners.

\*Address all correspondence to: laurence.vaughan@bd.com

Diagnostics Systems, BD Life Sciences, Sparks, MD, USA

William A. Nussbaumer and Ryan J. Schwab

Company..

**Author details**

Laurence M. Vaughan\*


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http://dx.doi.org/10.5772/62729

### **Abstract**

The purpose of this paper is to describe the updated biotechnologies approved to be used for identifying precancerous cervical lesions in clinical practice. The paper focuses on the new biotechnologies able to detect human papillomavirus (HPV) such as nucleic acid hybridization assays, signal amplification assays, and nucleic acid amplification. Particular attention is given to the discussion regarding the differences among the biotechnologies used, such as Digene Hybrid Capture test using Hybrid Capture 2 technology and the Cervista HR-HPV assay. The scientific progress is emphasized by new markers such as cycline p16INK4a, viral oncoproteins E6 and E7, high-risk (HR) HPV genotyping, and dual test p16/Ki67. The results of the large ongoing studies conduct‐ ed worldwide highlight these markers' capacity to disclose the differences between transient and transforming HPV infection and mild abnormal cytologies which could spontaneously regress or develop into cancer. Although both screening programs and opportunistic screening concerning cervical cancer are used worldwide, major geographic differences exist nowadays as regards the access to these programs. Finally, to achieve the objective of this study, the recommendations of various guidelines available across Europe, United States, and Australia, as well as the diagnosis tests accessible to women in low-resource countries are presented.

**Keywords:** Pap's smear, HPV genotyping test, E6/E7 mRNA, p16INK4a, dual test p16INK4/Ki67

### **1. Introduction**

According to the data reported by GLOBOCAN 2012 (IARC), cervical cancer is included among the leading causes of cancer worldwide. As regards the incidence of cervical cancer, GLOBO‐

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CAN report underlines that this type of cancer is the fourth most common cancer in women, and the seventh overall. The estimated number of new cases for 2012 is 528,000 [1]. For the same year, the registered number of deaths from cervical cancer was assessed at 266,000 cases. In terms of cervical cancer incidence and death by this disease, there is a major difference between the demographic regions of the world, strongly corresponding with the resource-setting level. Mortality varies 18-fold between various world regions, with rates ranging from less than 2 per 100,000 (in Western Asia, Western Europe, and Australia/New Zealand) to more than 20 per 100,000 (in Melanesia (20.6), Middle Africa (22.2), and Eastern Africa (27.6)) as stated by the GLOBOCAN 2012 report. A valuable consequence of the cervical cancer screening programs' implementation is that the incidence of cervical cancer rate dropped to half or more than half over the past 30 years, especially in countries with high levels of resources. For 2016, accord‐ ing to the American Cancer Society's estimates for cervical cancer in the United States, approx‐ imately 12,990 new cases of invasive cervical cancer will be diagnosed and 4120 women will die from cervical cancer. Cervical cancer is extremely rare in women younger than 20 and women over 65 years of age; only 15% of cervical cancer was reported to be identified at these ages. The improvement of collected data quality (statistics, information range) as concerns the occur‐ rence of cervical cancer and the death by this disease is due to the implementation of national screening programs in many countries. Nowadays, more types of cervical cancer screening programs are in place, and a larger number of different biotechnologies are available across the world, which allow to identify early precancerous conditions of the cervix and therefore to obviate progression to cancer. On the other hand, it is very important that clinicians know the usable benefits and potential harms of biotechnologies able to achieve the triage of women with abnormal cytology or to identify cases with high-risk human papilloma virus in the stage of transforming infection. Due to a noteworthy scientific progress, clinicians now have many possibilities for early detection of a cervical lesion that might evolve into cervical cancer. The aim of the new biotechnology procedures is to achieve both high sensitivity and specificity in order to differentiate cervical lesions that may develop into cancer from those which sponta‐ neously regress. Within the frame of this paper are included both the principal methods recommended by clinicians and researchers in cervical cancer field and the benefits and disadvantages of each biotechnology and marker. Our approach is designed so as to be useful especially to gynecologists with a view to a better management of the diagnosis and treat‐ ment of precancerous lesions. Hence, this paper explains what attempts should be made in the framework of each chosen biotechnology and what kind of tests increases the accuracy of an early diagnosis with regard to precancerous cervical lesions.

The data collection was performed by literature search, using PubMed, EMBASE, and the Cochrane Library (covering the 2000–2015 time frame), the main subject being detection of Human Papillomavirus Infection and cellular markers for early detection of precancerous cervical lesions. This study makes reference to the results of large studies published world‐ wide, such as in ATHENA, HERMES, PALMS, KPNC studies, Compass Trial, and the Newsletter on Human Papillomavirus––HPV Today (2015). This paper is structured in three sections. The first one covers different types of biotechnologies able to uncover precancerous and cancerous cervical lesions which are approved for use in medical clinical practice. The second section is dedicated to discussions about the benefits and drawbacks of each biotech‐ nology reported to detect precancerous lesions. At last, attention is paid to recommendations of the overall current guidelines.

## **2. Biotechnologies able to detect the precancerous and cancerous cervical lesions**

A strong contribution to the early detection of precancerous cervical lesions belongs to Papanicolou. The Pap's test has been in place since 1950, when George Papanicolou introduced this method as a cytological test able to detect morphological changes of abnormal cells. Generally, there are three kinds of markers for cervical cancer screening: viral markers, cellular markers, and epigenetic markers, which can identify, alone or in combination, early precan‐ cerous cervical lesions.

The laboratory tests able to reveal precancerous and cancerous cervical lesions use cytololog‐ ical, viral, cellular, and genetic markers such as Pap's test, HPV genotyping test, cellular markers, epigenetic markers, and other markers.

### **2.1. Pap's test: cytological diagnosis**

CAN report underlines that this type of cancer is the fourth most common cancer in women, and the seventh overall. The estimated number of new cases for 2012 is 528,000 [1]. For the same year, the registered number of deaths from cervical cancer was assessed at 266,000 cases. In terms of cervical cancer incidence and death by this disease, there is a major difference between the demographic regions of the world, strongly corresponding with the resource-setting level. Mortality varies 18-fold between various world regions, with rates ranging from less than 2 per 100,000 (in Western Asia, Western Europe, and Australia/New Zealand) to more than 20 per 100,000 (in Melanesia (20.6), Middle Africa (22.2), and Eastern Africa (27.6)) as stated by the GLOBOCAN 2012 report. A valuable consequence of the cervical cancer screening programs' implementation is that the incidence of cervical cancer rate dropped to half or more than half over the past 30 years, especially in countries with high levels of resources. For 2016, accord‐ ing to the American Cancer Society's estimates for cervical cancer in the United States, approx‐ imately 12,990 new cases of invasive cervical cancer will be diagnosed and 4120 women will die from cervical cancer. Cervical cancer is extremely rare in women younger than 20 and women over 65 years of age; only 15% of cervical cancer was reported to be identified at these ages. The improvement of collected data quality (statistics, information range) as concerns the occur‐ rence of cervical cancer and the death by this disease is due to the implementation of national screening programs in many countries. Nowadays, more types of cervical cancer screening programs are in place, and a larger number of different biotechnologies are available across the world, which allow to identify early precancerous conditions of the cervix and therefore to obviate progression to cancer. On the other hand, it is very important that clinicians know the usable benefits and potential harms of biotechnologies able to achieve the triage of women with abnormal cytology or to identify cases with high-risk human papilloma virus in the stage of transforming infection. Due to a noteworthy scientific progress, clinicians now have many possibilities for early detection of a cervical lesion that might evolve into cervical cancer. The aim of the new biotechnology procedures is to achieve both high sensitivity and specificity in order to differentiate cervical lesions that may develop into cancer from those which sponta‐ neously regress. Within the frame of this paper are included both the principal methods recommended by clinicians and researchers in cervical cancer field and the benefits and disadvantages of each biotechnology and marker. Our approach is designed so as to be useful especially to gynecologists with a view to a better management of the diagnosis and treat‐ ment of precancerous lesions. Hence, this paper explains what attempts should be made in the framework of each chosen biotechnology and what kind of tests increases the accuracy of an

88 Human Papillomavirus - Research in a Global Perspective

early diagnosis with regard to precancerous cervical lesions.

The data collection was performed by literature search, using PubMed, EMBASE, and the Cochrane Library (covering the 2000–2015 time frame), the main subject being detection of Human Papillomavirus Infection and cellular markers for early detection of precancerous cervical lesions. This study makes reference to the results of large studies published world‐ wide, such as in ATHENA, HERMES, PALMS, KPNC studies, Compass Trial, and the Newsletter on Human Papillomavirus––HPV Today (2015). This paper is structured in three sections. The first one covers different types of biotechnologies able to uncover precancerous and cancerous cervical lesions which are approved for use in medical clinical practice. The second section is dedicated to discussions about the benefits and drawbacks of each biotech‐ Papanicolau stain––commonly known as Pap's test––is the best-performing method used for cervical screening. The cytologist analyses, via this test, the exfoliated cervical cells to detect the morphological changes characteristic to neoplasic alterations. The cervical cell samples must be taken within the squamocolumnar junction of the cervix. This area is relatively accessible, making sampling easy, but it is unable to provide information about the lesions within the endocervical canal such as adenocarcinoma precursors [2, 3]. For the same Papa‐ nicolou stain, there are two methods that differ in terms of collecting technology: one is used to collect cervical cells (being known as conventional type), and the other is liquid-based (cytology type).

The conventional type uses the fixation of cervical cells of the sample, followed by classic Pap staining (EA 50, Hematoxilina Harris, Orange G, and different concentrations of ethanol). The duration of this procedure is about 45 min.

The liquid-based collection medium biotechnology is deemed more advanced because it is more versatile. Cervical cells are collected by using a cytobrush, which is then introduced into a collection medium (e.g., Cytofast). This method gives the opportunity both to keep the physiological structure and morphology of any kind of cells for 24 months at room temperature and to perform more investigations (e.g., HPV oncotypes, cyclin immunomarkers) [4, 5]. Since 2013, Hospitex Diagnostics highlights that the monolayer slides from liquid-based collection medium are safer, faster and fully representative, in comparison with the conventional smear screening procedure. The interpretation of the cytological results is done according to the Bethesda System Criteria established in 2001. During the past 15 years, a cytological diagnosis which included medical recommendation was possible due to the Bethesda System Criteria.

### **2.2. Viral markers**

The viral markers validated for usage in cervical cancer detection are HPV DNA and HPV genotyping, E6/E7 mRNA, and HPV proteins [6].

First of all, this paper describes, in line with its purpose, the biotechnologies which can be used to become aware of human papillomavirus infection (HPV) and identify the high-risk HPV genotypes (HR-HPV). There are several other technologies able to identify human papilloma‐ virus infection. These methods have different benefits and drawbacks, for which reason a good option should be made to properly choose the individual test or screening program.

One of the following methods should be used to accomplish HPV detection: nucleic acid hybridization assays, signal amplification assays, and nucleic acid amplification [7, 8].

The techniques able to employ radiolabeled nucleic acid hybridization assay to identify HPV infection are Southern blotting, in situ hybridization, or dot blot hybridization.

Signal amplification assays pertain to another biotechnology consisting in two tests known as the Digene Hybrid Capture test which utilizes Hybrid Capture 2 (hc2) technology (by Qiagen) and the Cervista HR-HPV assay (by Hologic) [9].

Nucleic acid amplification methods involve many kinds of methodologies based on microar‐ ray analysis, PapilloCheck, polymerase chain reaction (PCR), real-time PCR, Abbott real-time PCR, COBAS 4800 HPV test, HPV genome sequencing, the Linear Array, CLART human papillomavirus, INNO-LiPA, clinical array HPV, Microplate colorimetric hybridization assay, HPV-mRNA detection, HPV viral load quantification and integration.

Many technologies approved in the Unites States and Europe, relying on large populationbased studies and randomized trial, recommend using Hybrid Capture 2 test and Cervista test to spot HPV infection. Hybrid Capture 2 test was endorsed by the US Food and Drug Admin‐ istration (FDA) in 2003 and is able to detect 13 HR-HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) or 5 low risk (LR)-HPV types (6, 11, 42, 43, and 44), using specific antibodies' signal amplification and chemoluminescent detection. In 2009, FDA approved the Cervista HR-HPV, which detects 14 HR-HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) by using a signal amplification method and a fluorescent signal for detecting specific nucleic acid sequences. In 2009, FDA approved another HPV DNA test, Cervista HPV 16/18, which uncovers HPV 16 and 18 oncogenotypes [10].

In April 2014, FDA approved Cobas HPV test, which is a PCR-based HPV DNA test using the same fluorescent label for the fluorescent signal from 12 HR-HPV and simultaneous recogni‐ tions with three separate fluorescent labels of HPV 16, HPV18, and beta-globulin signals.

When applied alone, the detection of HR-HPV DNA does not discriminate between the transient and transforming HPV infections. This differentiation is possible by discerning viral oncoproteins E6 and E7 mRNA and protein expression. The progression from a transient to a transforming HPV infection is identified by the high increase of E6/E7 mRNA expression [11, 12]. These oncoproteins interfere with key cellular cycles that control cell proliferation and apoptosis. E7 disrupts pRb from its binding to E2F, and E6 interferes with p53. Therefore, viral oncoprotein E7 triggers uncontrolled cell cycling and E6 abrogates apoptosis. Two of the most widespread tests of the commercial assays designed to detect HPV E6/E7 mRNA are Pre Tect Proofer (Norchip), which detects five oncotypes of HR-HPV (16, 18, 31, 33, and 45), and APTIMA (GenoProbe) which covers 14 HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68).

Another HPV assay––known as Qiagen assay (United States)––is an adoption of Digene HC2 assay. The signal amplification assay perceives 14 different HR-HPV oncotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 66). The particularity of Qiagen HPV assay consists in the fact that this platform does not require electricity or water, but only needs a limited work space (25 × 50 cm2 ). The result is obtained shortly (2.5 h only).

The main methods used to detect HPV integration are PCR, fluorescence in situ hybridization, and Real-Time PCR. The latter two methods allow calculating the ratio between the levels of E2 and E6/E7 HPV genes. When the HPV is integrated, the viral genome shows a 1:1 ratio between the E2 and E6/E7 genes [13].

### **2.3. Cellular markers**

**2.2. Viral markers**

genotyping, E6/E7 mRNA, and HPV proteins [6].

90 Human Papillomavirus - Research in a Global Perspective

and the Cervista HR-HPV assay (by Hologic) [9].

which uncovers HPV 16 and 18 oncogenotypes [10].

The viral markers validated for usage in cervical cancer detection are HPV DNA and HPV

First of all, this paper describes, in line with its purpose, the biotechnologies which can be used to become aware of human papillomavirus infection (HPV) and identify the high-risk HPV genotypes (HR-HPV). There are several other technologies able to identify human papilloma‐ virus infection. These methods have different benefits and drawbacks, for which reason a good

One of the following methods should be used to accomplish HPV detection: nucleic acid hybridization assays, signal amplification assays, and nucleic acid amplification [7, 8].

The techniques able to employ radiolabeled nucleic acid hybridization assay to identify HPV

Signal amplification assays pertain to another biotechnology consisting in two tests known as the Digene Hybrid Capture test which utilizes Hybrid Capture 2 (hc2) technology (by Qiagen)

Nucleic acid amplification methods involve many kinds of methodologies based on microar‐ ray analysis, PapilloCheck, polymerase chain reaction (PCR), real-time PCR, Abbott real-time PCR, COBAS 4800 HPV test, HPV genome sequencing, the Linear Array, CLART human papillomavirus, INNO-LiPA, clinical array HPV, Microplate colorimetric hybridization assay,

Many technologies approved in the Unites States and Europe, relying on large populationbased studies and randomized trial, recommend using Hybrid Capture 2 test and Cervista test to spot HPV infection. Hybrid Capture 2 test was endorsed by the US Food and Drug Admin‐ istration (FDA) in 2003 and is able to detect 13 HR-HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) or 5 low risk (LR)-HPV types (6, 11, 42, 43, and 44), using specific antibodies' signal amplification and chemoluminescent detection. In 2009, FDA approved the Cervista HR-HPV, which detects 14 HR-HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) by using a signal amplification method and a fluorescent signal for detecting specific nucleic acid sequences. In 2009, FDA approved another HPV DNA test, Cervista HPV 16/18,

In April 2014, FDA approved Cobas HPV test, which is a PCR-based HPV DNA test using the same fluorescent label for the fluorescent signal from 12 HR-HPV and simultaneous recogni‐ tions with three separate fluorescent labels of HPV 16, HPV18, and beta-globulin signals.

When applied alone, the detection of HR-HPV DNA does not discriminate between the transient and transforming HPV infections. This differentiation is possible by discerning viral oncoproteins E6 and E7 mRNA and protein expression. The progression from a transient to a transforming HPV infection is identified by the high increase of E6/E7 mRNA expression [11, 12]. These oncoproteins interfere with key cellular cycles that control cell proliferation and apoptosis. E7 disrupts pRb from its binding to E2F, and E6 interferes with p53. Therefore, viral

option should be made to properly choose the individual test or screening program.

infection are Southern blotting, in situ hybridization, or dot blot hybridization.

HPV-mRNA detection, HPV viral load quantification and integration.

The cell markers approved by the World Health Organization (WHO) to be used in clinical practice are p16INK4a and dual test p16INK4a/Ki67. Another possible option arises from the ongoing research about a new clinical cell marker concerning the detection of topoisomerase 2*α*, in cervical cytology slides.

## *2.3.1. Cellular markers: cyclin p16INK4a*

Uncovering of p16INK4a is tightly correlated with HPV integration. In a normal cell, p16INK4a blocks cyclin-dependent kinases (CDK) 4/6. Increased expression of the E6 and E7 oncogenes disrupt cell cycle regulation. In the normal cell, cell cycle progression is activated by CDK 4/6 and partially regulated by p16INK4a. However, because in HPV-transformed cells, cell cycle activation is caused by E7 and not by CDK 4/6, p16INK4a has no effect on the cell cycle activation. Increased expression of p16INK4a in cells driven by viral oncogene-mediated cell cycle dysfunc‐ tion can be distinguished through cellular immunostaining by immunocytology or immuno‐ histology tests [14]. In brief, p16INK4a is a tumor-suppressor protein and cyclin-dependent kinase (cdk) inhibitor that blocks CDK 4/6-mediated pRb phosphorylation to inhibit E2Fdependent transcription and cell cycle progression. It is obvious that the progression of dysplastic lesions to cancer is highlighted by increased expression of two viral oncogenes, E6 and E7. The last-mentioned oncoprotein, E7, inactivates retinoblastoma gene product (pRB) that inhibits transcription of the cyclin-dependent kinase inhibitor gene p16INK4a. This explains why the overexpression of p16INK4a is similar to the increased activity of E7, and so the overexpression of both p16INK4a and E7 are markers of HPV integration in the genome of the host cell [15]. The cyclin p16INK4a must be evaluated as a stand-alone test and as an adjunct to cytology or HPV testing [16, 17].

The cervical cells are collected similar to those for Pap's smear test. The liquid-based collection medium has the advantage of being able to collect more cervical cells both for Pap's smear, immunocytochemistry tests and for HPV genotyping test. In the current activity, the clinician has the possibility to demand two tests used to identify specific immunocytomarkers, such as p16INK4a alone or the dual test which evaluates, on the same cervical cell, two immunocyto‐ markers consisting in p16INK4a and Ki67.

Over the past 10 years, the cyclin inhibitor kinase p16INK4a has been identified by immunocy‐ tochemistry staining using a CINtec p16INK4a ready-to-use cytological kit (clone E6H4) manu‐ factured by mtm laboratories AG (Heidelberg, Germany). Wentzensen published the morphological characteristics necessary to evaluate the immunocytological expressions of p16INK4a within the cervical cell, both for nucleus and cytoplasm. Therefore, the criteria which must be analyzed are increased nucleus size or increased nucleus/cytoplasmic ratio, irregular nuclear shape, granular or hyperchromatic chromatin with variable cellular morphology, together with the intensity of cytoplasmic staining [18].

There are many models in the published literature that allow identifying the intensity of the immunocytoexpression of p16INK4a. One method used for a correct interpretation of the immunoexpression classified samples as p16INK4a-negative and p16INK4a-positive when posi‐ tivity was observed for at least two of the aforementioned criteria. On the other hand, to obtain greater accuracy in the slides' interpretation, a scoring system was introduced, which scored the absence and the gradual intensity of the immunocytological expression staining. Therefore, the scoring system assigns 0 point for cases without p16-positive cells (p16−), 1 point for p16 positive cells with changed morphology in the absence of nuclear abnormalities (p16+), 2 points for p16-positive cells with a single nuclear abnormality (p16++), and 3 points for the presence of at least two nuclear alterations within the same cell (p16+++) [18]. In this manner, the physician can establish a correct cytodiagnosis much more accurately.

### *2.3.2. Cellular markers: p16INK4a/Ki67 dual-stained*

The CINtec PLUS assay (mtm laboratories AG, Heidelberg, Germany) is currently gaining increased credibility; it is a commercially available test which combines the immunostaining of p16INK4a with the immunostaining of Ki67 within the same cervical cell [19].

This methodology allows the cytologist to see, within the same epithelial cell, the nucleus stained red for Ki67 immunoexpression, and both the nucleus and the cytoplasm stained brown for p16INK4a immunoexpression. The test has predictive power to identify the progres‐ sive evolution to a higher degree of dysplasia and cancer. Positive dual staining is significant only for cells with modified morphology (atypical cells). Schmidt and other researchers [20] highlight the fact that for a better cytological diagnosis, the most important aspect is the positive dual staining within the nucleus of atypical cells. The evaluation of the p16/Ki67 dual staining could be better ascertained by an automatic reader system that obviates subjectivism.

### *2.3.3. Other cellular markers*

Other cellular markers expressed in the S-phase of uncontrolled cellular cycle due to the activity of HPV oncoproteins in the stage of transforming infection include Topoisomerase 2α (TOP2A) and minichromosome maintenance protein 2 (MCM2). These markers could be noticed in SurePath cervical cytology specimens by an indirect polymer-based immunoper‐ oxidase method (ProEx C, TriPath Oncology, Burlington, NC). The cytologist is interested in evaluating the presence of nuclear stain in epithelial cells, and the combination of nuclear staining and abnormal morphology. ProEx C is an immunocytochemical test, able to identify the possibility of proliferative process in women with low-grade cytological abnormalities [21].

There are also many other studies that recommend the immunocytochemical test ProEx C for TOP2A and MCM2 as adjunct test to conventional ASC-US cytology test.

### **2.4. Epigenetic markers**

immunocytochemistry tests and for HPV genotyping test. In the current activity, the clinician has the possibility to demand two tests used to identify specific immunocytomarkers, such as p16INK4a alone or the dual test which evaluates, on the same cervical cell, two immunocyto‐

Over the past 10 years, the cyclin inhibitor kinase p16INK4a has been identified by immunocy‐ tochemistry staining using a CINtec p16INK4a ready-to-use cytological kit (clone E6H4) manu‐ factured by mtm laboratories AG (Heidelberg, Germany). Wentzensen published the morphological characteristics necessary to evaluate the immunocytological expressions of p16INK4a within the cervical cell, both for nucleus and cytoplasm. Therefore, the criteria which must be analyzed are increased nucleus size or increased nucleus/cytoplasmic ratio, irregular nuclear shape, granular or hyperchromatic chromatin with variable cellular morphology,

There are many models in the published literature that allow identifying the intensity of the

immunoexpression classified samples as p16INK4a-negative and p16INK4a-positive when posi‐ tivity was observed for at least two of the aforementioned criteria. On the other hand, to obtain greater accuracy in the slides' interpretation, a scoring system was introduced, which scored the absence and the gradual intensity of the immunocytological expression staining. Therefore, the scoring system assigns 0 point for cases without p16-positive cells (p16−), 1 point for p16 positive cells with changed morphology in the absence of nuclear abnormalities (p16+), 2 points for p16-positive cells with a single nuclear abnormality (p16++), and 3 points for the presence of at least two nuclear alterations within the same cell (p16+++) [18]. In this manner, the

The CINtec PLUS assay (mtm laboratories AG, Heidelberg, Germany) is currently gaining increased credibility; it is a commercially available test which combines the immunostaining

This methodology allows the cytologist to see, within the same epithelial cell, the nucleus stained red for Ki67 immunoexpression, and both the nucleus and the cytoplasm stained brown for p16INK4a immunoexpression. The test has predictive power to identify the progres‐ sive evolution to a higher degree of dysplasia and cancer. Positive dual staining is significant only for cells with modified morphology (atypical cells). Schmidt and other researchers [20] highlight the fact that for a better cytological diagnosis, the most important aspect is the positive dual staining within the nucleus of atypical cells. The evaluation of the p16/Ki67 dual staining could be better ascertained by an automatic reader system that obviates subjectivism.

Other cellular markers expressed in the S-phase of uncontrolled cellular cycle due to the activity of HPV oncoproteins in the stage of transforming infection include Topoisomerase 2α (TOP2A) and minichromosome maintenance protein 2 (MCM2). These markers could be

One method used for a correct interpretation of the

markers consisting in p16INK4a and Ki67.

92 Human Papillomavirus - Research in a Global Perspective

immunocytoexpression of p16INK4a.

*2.3.2. Cellular markers: p16INK4a/Ki67 dual-stained*

*2.3.3. Other cellular markers*

together with the intensity of cytoplasmic staining [18].

physician can establish a correct cytodiagnosis much more accurately.

of p16INK4a with the immunostaining of Ki67 within the same cervical cell [19].

Nowadays there are ongoing studies about the chromosomal imbalances involved in the development of cervical cancer. Researchers pay attention to the chromosomal changes occurring early in the proliferative process. Epigenetic markers consist in DNA methylation, chromosomal abnormalities, miRNA abnormalities, and proteomics. The literature data reveal that it is about host methylation and viral methylation. Many genes are frequently methylated and remain in a silent stage during carcinogenesis, acting as negative regulators of cellular cell cycle. To identify the increased frequency of DNA, methylation of many genes (e.g., *DAPK, CADM1, TERT, CDH13, MAL*) in the early stage of carcinogenesis may hint that these could be biomarkers for early detection of cervical cancer [22]. Studies were not focused on a single gene only, and therefore, a gene panel was developed (e.g., *CADM/MAL; RAS ß/TWST/ MGMT*), targeting to improve the possibilities to attain earlier the best triage of HPV-infected cervical lesions. The analysis of viral methylation suggests that the promoter regions of oncoprotein E5 and E7 are more frequently methylated in the later stages of carcinogenesis, and as a consequence these tests allow to detect HGCIN [23].

Cervical cancer recognizes genomic instability manifested by amplification of the same regions, especially 3q/TERC or loss of other regions such as 6q and 11q. These abnormalities in the chromosomal architecture could be used in the triage of HPV-positive women with ASC-US or LSIL conventional cytology [24]. Other biotechnologies including miRNAs with increased or decreased expression, proteomics within cervical–vaginal mucus arise; their target is to discover the best predictive markers to discover, at an early stage, the risk of progress from mild to severe dysplasia and cancer.

## **3. Biotechnologies and test results: advantages and limitations**

### **3.1. Pap's test: advantages and limitations**

Researchers agreed that the advantage of Pap's test consists in its high specificity concerning the detection of women who do not prove to be positive for cervical lesions. On the other hand, Pap's test is limited due to its low sensitivity in identifying women with dysplastic cervical lesion prone to develop into cancer. The studies conducted on women of Europe and North America showed that the sensitivity of the cytology in detecting CIN2+ lesions is only 53%, while other studies reveal a modest sensitivity (60–70%) [25, 26].

An advantage of Pap's test consists in its use for both opportunistic cervical cancer screening and national screening programs. The clinician must know that neither the changes in the terminology of cytology by Bethesda reporting system, nor the novel techniques such as liquidbased cytology do not prove more efficient in terms of the improvement of the sensitivity and specificity of Pap's test [27, 28]. On the contrary, Hospitex Diagnostics' report (2013) highlights that the monolayer slides from liquid-based collection medium are safer, faster, and fully representative in comparison with conventional smears screening procedure. We must admit that the introduction of liquid-based cytology has decreased the number of inadequate slides and allowed to advocate a reflex testing for other viral or molecular markers [29].

A disadvantage of Pap's test is the need to be interpreted by two or more specialists trained in cytology for a more accurate cytologic diagnosis; thereby, the test implies subjectivity in the evaluation of morphology cells [30]. Also, the costs of Pap's test are not negligible by two reasons, the first is the training cost of the specialist reader and the second is the fact that screening based on cytology frequently needs repeated Pap's test during the lifetime. Initially, the Pap's test was recommended at 1-year interval, and this is why Pap testing brings sub‐ stantial cost burden on the health system [31]. The method of cytological screening is able to perceive the lesions that have a high risk of progression, but this prospect is limited by the fact that when used alone, the method can distinguish neither atypical squamous cells of unde‐ termined significance (ASC-US) lesion, nor low-grade squamous intraepithelial (L-SIL) lesions able to spontaneously go into remission against lesions able to progress. Statistical data have shown that 10–15% cases with ASC-US and LSIL cytology develop CIN3 [32, 33].

### **3.2. HPV test: advantages and limitations**

The disadvantages of Pap's test justify why it is necessary to identify the HR-HPV types. With this aim in mind, researchers are trying to define new marks in order to get more powerful characterizations for cervical cancer prevention.

As regards the time necessary to achieve a result concerning HR-HPV assay, an advantage can be seen in Qiagen platform which needs only 2.5 h, while Digene Hybrid Capture HR-HPV testing needs about 6 h. Therefore, the former platform allows both diagnosis and treatment in a single day.

The disadvantage of HR-HPV assay is that it exposes women to overtreatment, as, when applied alone, HR-HPV assay does not reveal the difference between transient and transform‐ ing HPV infections. These features do not make it possible to differentiate women with spontaneous remission of cervical lesions, from women with progressive cervical lesions that develop into cervical cancer. Hence, this triage of women with remission or progress of cervical lesion with HPV infection requires a larger number of investigations such as detection of E6 and E7 mRNA markers whose immunoexpression is a real proof of HPV integration in host cells.

The inconveniences of the three techniques which use radiolabeled nucleic acid hybridization assays to detect HPV infection consist in relatively large amounts of purified DNA, more timeconsuming procedures, and low sensitivity of the results of test [8].

The Hybrid Capture 2 system has the added advantage of detecting 13 HR-HPV types and 5 LR-HPV types [34].

Cervista assay shows a high sensitivity and specificity to HPV 16/18 genotyping and 100% sensitivity in the detection of CIN3 [35, 36].

A deep analysis of the published reviews underlines that Hybrid Capture 2 test and Cervista HR-HPV have two limitations concerning both the lack of differentiation between single HPV genotype infections and multiple concurrent HPV genotype infections, and the shortage of a test in relation to quantitative viral load [10]. It is very important for the gynecologists to obtain details about the existence of HPV infection with type 16 or 18, because these types allow for a stratification of the risk with reference to the possibility of developing cervical cancer. Thereby, the detection of HPV 16 or 18 has both the advantage of stratifying the oncologic risk of HPV infection and of providing clinicians with information which is useful in managing the precursors of cervical lesions, taking into account that in situations with persistent infections, the risk of precancerous lesions' progression to cancer is between 10 and 15% in cases with HR-HPV 16/18, and below 3% for all other combined HR types [7].

Abreu and colleagues [7] put forward a concept aiming to classify the necessity of DNA HPV testing. These authors concluded that there are six circumstances which require the test, as follows:


### **3.3. Cellular markers: advantages and limitations**

### *3.3.1. Cycline p16INK4a*

An advantage of Pap's test consists in its use for both opportunistic cervical cancer screening and national screening programs. The clinician must know that neither the changes in the terminology of cytology by Bethesda reporting system, nor the novel techniques such as liquidbased cytology do not prove more efficient in terms of the improvement of the sensitivity and specificity of Pap's test [27, 28]. On the contrary, Hospitex Diagnostics' report (2013) highlights that the monolayer slides from liquid-based collection medium are safer, faster, and fully representative in comparison with conventional smears screening procedure. We must admit that the introduction of liquid-based cytology has decreased the number of inadequate slides

A disadvantage of Pap's test is the need to be interpreted by two or more specialists trained in cytology for a more accurate cytologic diagnosis; thereby, the test implies subjectivity in the evaluation of morphology cells [30]. Also, the costs of Pap's test are not negligible by two reasons, the first is the training cost of the specialist reader and the second is the fact that screening based on cytology frequently needs repeated Pap's test during the lifetime. Initially, the Pap's test was recommended at 1-year interval, and this is why Pap testing brings sub‐ stantial cost burden on the health system [31]. The method of cytological screening is able to perceive the lesions that have a high risk of progression, but this prospect is limited by the fact that when used alone, the method can distinguish neither atypical squamous cells of unde‐ termined significance (ASC-US) lesion, nor low-grade squamous intraepithelial (L-SIL) lesions able to spontaneously go into remission against lesions able to progress. Statistical data have

and allowed to advocate a reflex testing for other viral or molecular markers [29].

shown that 10–15% cases with ASC-US and LSIL cytology develop CIN3 [32, 33].

The disadvantages of Pap's test justify why it is necessary to identify the HR-HPV types. With this aim in mind, researchers are trying to define new marks in order to get more powerful

As regards the time necessary to achieve a result concerning HR-HPV assay, an advantage can be seen in Qiagen platform which needs only 2.5 h, while Digene Hybrid Capture HR-HPV testing needs about 6 h. Therefore, the former platform allows both diagnosis and treatment

The disadvantage of HR-HPV assay is that it exposes women to overtreatment, as, when applied alone, HR-HPV assay does not reveal the difference between transient and transform‐ ing HPV infections. These features do not make it possible to differentiate women with spontaneous remission of cervical lesions, from women with progressive cervical lesions that develop into cervical cancer. Hence, this triage of women with remission or progress of cervical lesion with HPV infection requires a larger number of investigations such as detection of E6 and E7 mRNA markers whose immunoexpression is a real proof of HPV integration in host

The inconveniences of the three techniques which use radiolabeled nucleic acid hybridization assays to detect HPV infection consist in relatively large amounts of purified DNA, more time-

consuming procedures, and low sensitivity of the results of test [8].

**3.2. HPV test: advantages and limitations**

94 Human Papillomavirus - Research in a Global Perspective

in a single day.

cells.

characterizations for cervical cancer prevention.

The p16INK4a positivity shows that the cervical cells are HPV-infected but could not clearly detect a real progress to cancer. The p16INK4a positivity is correlated with HPV infection but could not allow to discriminate between a HR-HPV or a LR-HPV oncotype infection. On the other hand, the p16INK4a expression is independent of the HPV type, and therefore genotyping is unnecessary. In the context of disruption of cell cycle regulation, the p16INK4a expression by cycling cells is a specific marker of HPV-E7 overexpression or other events that inactivate Rb [15].

The immunoexpression of p16INK4a exists both in the nucleus and cytoplasm, but its intensity is in line with the degree of cervical dysplasia. Only the p16INK4a overexpression within the nucleus of the cell shows a high degree of cervical dysplasia. The presence of low p16INK4a immunoexpression within the cytoplasm could not be related to cervical dysplasia's progress to cancer. Another limitation of this marker is the subjectivism in immunostaining slides' evaluation, despite the above-mentioned criteria and the scoring system proposed by Went‐ zensen, Samarawardana, and Denton. The evaluation of slides requires special cytologist abilities to detect the morphological abnormalities of cells and interpret the immunoexpression degree p16INK4a. The clinician must take into account the possibility that sometimes the cytologist notices the physiological presence of p16INK4a. In this context, it is necessary to require a morphological evaluation of p16-immunostained cells with the purpose of distinguishing HPV-transformed cells from metaplastic cells [37, 38].

Along with the aforementioned features of cycline p16INK4a, and with the benefits and draw‐ backs of this test, the conclusion is that it must be evaluated both as a stand-alone test and as an adjunct to cytology or HPV testing. In line with the overall opinion, cyclin p16INK4a is a specific biomarker able to identify dysplastic cervical epithelia in sections of cervical biopsy samples or cervical smears [34]. The meta-analysis of reviews published shows that there is a major heterogeneity in the methods used to identify p16INK4a in samples. As regards the statistical value in detecting CIN2+ lesions, cycline p16INK4a has a sensitivity between 0.59 and 0.96, and a specificity flanked by 0.41–0.96 [17].

### *3.3.2. Dual test p16INK4a/Ki67: advantages and limitations*

Dual test p16INK4a/Ki67 has a better interobserver reproducibility and accuracy in cervical cancer screening compared to stand-alone p16INK4a. As indicated in Kaise Permanente Northern California (KPCN) study, where cancer screening was performed based on HPV and cytology co-testing by p16/Ki67 dual staining in 2400 HPV-positive women, the recommendation to use p16/Ki67 cytologies is feasible in routine cytology laboratories with minimal time of training and easy reproducibility [39, 40].

The evaluation of p16/Ki67 dual staining could be better conducted by an automatic reader system that reduces subjectivism to the highest possible extent.

p16INK4a/Ki67 dual test has a higher sensitivity than Pap's test in identifying high-grade cervical lesions associated with HR-HPV infection and the same specificity as the above-mentioned test.

### **3.4. Epigenetic markers: advantages and limitations**

Despite some extended studies investigating the utility of epigenetic markers, such markers are not yet applied in clinical practice and guidelines do not refer to samples of these biotech‐ nologies. However, the researchers' discoveries as regards this solution proved the utility of epigenetic markers for the triage of HPV-positive women with mild abnormal cytologies. There seems to be an opportunity in the future to self-sample from cervical mucus or vaginal fluid, and such samples could theoretically be investigated with reference to epigenetic markers.

## **4. Discussion: recommendations of the large studies and worldwide guidelines**

cycling cells is a specific marker of HPV-E7 overexpression or other events that inactivate Rb

The immunoexpression of p16INK4a exists both in the nucleus and cytoplasm, but its intensity is in line with the degree of cervical dysplasia. Only the p16INK4a overexpression within the nucleus of the cell shows a high degree of cervical dysplasia. The presence of low p16INK4a immunoexpression within the cytoplasm could not be related to cervical dysplasia's progress to cancer. Another limitation of this marker is the subjectivism in immunostaining slides' evaluation, despite the above-mentioned criteria and the scoring system proposed by Went‐ zensen, Samarawardana, and Denton. The evaluation of slides requires special cytologist abilities to detect the morphological abnormalities of cells and interpret the immunoexpression degree p16INK4a. The clinician must take into account the possibility that sometimes the cytologist notices the physiological presence of p16INK4a. In this context, it is necessary to require a morphological evaluation of p16-immunostained cells with the purpose of distinguishing

Along with the aforementioned features of cycline p16INK4a, and with the benefits and draw‐ backs of this test, the conclusion is that it must be evaluated both as a stand-alone test and as an adjunct to cytology or HPV testing. In line with the overall opinion, cyclin p16INK4a is a specific biomarker able to identify dysplastic cervical epithelia in sections of cervical biopsy samples or cervical smears [34]. The meta-analysis of reviews published shows that there is a major heterogeneity in the methods used to identify p16INK4a in samples. As regards the statistical value in detecting CIN2+ lesions, cycline p16INK4a has a sensitivity between 0.59 and

Dual test p16INK4a/Ki67 has a better interobserver reproducibility and accuracy in cervical cancer screening compared to stand-alone p16INK4a. As indicated in Kaise Permanente Northern California (KPCN) study, where cancer screening was performed based on HPV and cytology co-testing by p16/Ki67 dual staining in 2400 HPV-positive women, the recommendation to use p16/Ki67 cytologies is feasible in routine cytology laboratories with minimal time of training

The evaluation of p16/Ki67 dual staining could be better conducted by an automatic reader

p16INK4a/Ki67 dual test has a higher sensitivity than Pap's test in identifying high-grade cervical lesions associated with HR-HPV infection and the same specificity as the above-mentioned

Despite some extended studies investigating the utility of epigenetic markers, such markers are not yet applied in clinical practice and guidelines do not refer to samples of these biotech‐ nologies. However, the researchers' discoveries as regards this solution proved the utility of

HPV-transformed cells from metaplastic cells [37, 38].

96 Human Papillomavirus - Research in a Global Perspective

0.96, and a specificity flanked by 0.41–0.96 [17].

and easy reproducibility [39, 40].

test.

*3.3.2. Dual test p16INK4a/Ki67: advantages and limitations*

system that reduces subjectivism to the highest possible extent.

**3.4. Epigenetic markers: advantages and limitations**

[15].

Pap's test has limited sensitivity to detect precancerous cervical lesions but has high specificity. Compared to cytological diagnosis by HPV DNA testing, it offers a higher sensitivity and a long-term reassurance as regards the minimal risk of developing cervical cancer in women who were proven to be negative in HPV-DNA testing. Hence, these women are safe as regard a progressive dysplasia and could benefit from a large interval between cytological tests, with extension to 2–3 years of the screening interval [29].

In keeping with the ASC-US/LSIL Triage Study (ALTS)––1998, HPV DNA testing is a viable strategy able to clarify especially ASC-US cytology. In fact, ALTS Study counsels clinicians to manage ASC-US cytologies using three different options: triage by HPV testing as an adjunct to cytology, immediate referral to colposcopy, and conservative management with repeating Pap's test. The triage of ASC-US cytologies has relevant significance, because approximately 10–15% of women with ASC-US cytologies proved to be CIN3 at histopathological exam. The relevance of ALTS study consists in the fact that researchers deem that HPV DNA testing could be achieved only by triage of ASC-US cytologies, while the triage of mildly abnormal cytologies is not possible due to the high number of HR-HPV belonging to this female population [32].

Numerous studies have confirmed that HPV testing demonstrates high sensitivity and lower specificity for detecting high-grade cervical intraepithelial lesions. This poor specificity is explained by the fact that most HPV infections are transient and only a lower number of cases develop transforming infections.

Due to the fact that HPV DNA testing offers higher sensitivity and Pap's testing has higher specificity, researchers have progressed in line with the necessity of developing a new marker able to corroborate high sensitivity and specificity.

Berjeron and colleagues (2010) demonstrated on 500 cases, by using H&E-stained slides, as well as p16INK4a-immunostained slides analyzed by the same 12 pathologists, that the diagnosis was improved and the sensitivity increased by about 13% after adding p16INK4a immunostain‐ ing. The research results recommend using p16INK4a for clinical practice, because this marker was proved able to identify both CIN1 lesions associated with HR-HPV types and CIN2+ lesions [41].

The papers published in 2010 by Samarawardana and Denton have shown that p16INK4a immunostaining on cytology provides significantly better specificity than HR-HPV for the triage of ASC-US and LSIL cytology cases [42, 43].

In conclusion, the diagnosis accuracy is higher when clinicians suggest one of these options: HPV DNA testing in conjunction with Pap's test or p16INK4a associated with Pap's test for the triage of ASC-US cytologies.

In 2014, ATHENA study results demonstrated that COBAS HPV technology contributes to the ASC-US triage. The researchers' remarks were useful for a new approach of the cervical cancer screening. Hence, ATHENA study highlights the fact that negative intraepithelial lesions (NIEL) on cytological exam proved to be ≥CIN3 on histopathological exam. The careful data analysis of ATHENA study has shown that more than 57% of women aged 25–29 years whose cytological diagnosis is negative for intraepithelial cervical lesion proved to have a histopa‐ thological diagnosis ≥ CIN3. This evidence justifies why HR-HPV testing for genotypes 16/18 is more efficient in the primary screening of the cervical cancer compared to the Papanicolou cytology test. Cervical lesions able to progress to cancer could be prevented by the early detection of 16 and 18 HPV genotypes by COBAS HPV technology [44]. This technology is able to simultaneously identify 16 and 18 HPV genotypes (the HPV types that are the most responsible for the progress of dysplastic cervical lesions) associated with other 12 oncogeno‐ types.

The major contribution of COBAS HPV technology consists in the information that individu‐ alization of the 16 or 18 HPV oncoproteins underlines the necessity to change the management of an abnormal middle dysplasia from follow-up strategy with Pap's test repeated in 1 year, to other strategies involving, for example, colposcopy, biopsies.

However, HR-HPV testing alone is not able to distinguish between transient and proliferative HPV infections. In view of the gains and weaknesses proven by research as regards the usefulness of Pap's test and HR-HPV in screening women in order to discover those cervical lesions able to evolve into cancer, it is obvious that attention should also be paid to other biomarkers for increased credibility. Therefore, the progress of research led to the necessity of highlighting an immunomarker capable to increase the sensitivity of Pap's test. The cyclin p16INK4a, both in cytology exam and histology exam, is able to identify the HPV-infected cells and therefore to provide a more accurate diagnosis. As a matter of fact, it is acknowledged that the positivity of p16INK4a is an important feature of high-risk HPV infected cells.

Women tested HPV DNA-positive and with p16INK4a ICC-negative could be safely managed with follow-up HPV DNA testing in 2 to 3 years.

Conversely, the positivity for Ki67 in the cell nucleus is a marker of nuclear proliferation. The intensity of Ki67 immunoexpression increases more strongly in abnormal cells. The advantage of p16INK4a test used alone or within a dual test (p16INK4a and Ki67) is that the high intensity of p16INK4a and Ki67 immunoexpression in the nucleus warns that the cell will develop into cancer. If p16INK4a is positive in the cytoplasm only, there is a transient HPV infection, and the cervical lesion is able to spontaneously regress.

As regards the significance of p16INK4a/ Ki67 dual staining immunohistochemical test, Samar‐ awardana and colleagues showed as early as 2011 that this test has increased its capacity to identify high-grade cervical lesions [45].

The novel data established that the p16INK4a/ Ki67 dual staining by immunocytochemical (ICC) method is a better solution to identify the high-grade cervical lesions associated with HR-HPV infection [46].

In conclusion, the diagnosis accuracy is higher when clinicians suggest one of these options: HPV DNA testing in conjunction with Pap's test or p16INK4a associated with Pap's test for the

In 2014, ATHENA study results demonstrated that COBAS HPV technology contributes to the ASC-US triage. The researchers' remarks were useful for a new approach of the cervical cancer screening. Hence, ATHENA study highlights the fact that negative intraepithelial lesions (NIEL) on cytological exam proved to be ≥CIN3 on histopathological exam. The careful data analysis of ATHENA study has shown that more than 57% of women aged 25–29 years whose cytological diagnosis is negative for intraepithelial cervical lesion proved to have a histopa‐ thological diagnosis ≥ CIN3. This evidence justifies why HR-HPV testing for genotypes 16/18 is more efficient in the primary screening of the cervical cancer compared to the Papanicolou cytology test. Cervical lesions able to progress to cancer could be prevented by the early detection of 16 and 18 HPV genotypes by COBAS HPV technology [44]. This technology is able to simultaneously identify 16 and 18 HPV genotypes (the HPV types that are the most responsible for the progress of dysplastic cervical lesions) associated with other 12 oncogeno‐

The major contribution of COBAS HPV technology consists in the information that individu‐ alization of the 16 or 18 HPV oncoproteins underlines the necessity to change the management of an abnormal middle dysplasia from follow-up strategy with Pap's test repeated in 1 year,

However, HR-HPV testing alone is not able to distinguish between transient and proliferative HPV infections. In view of the gains and weaknesses proven by research as regards the usefulness of Pap's test and HR-HPV in screening women in order to discover those cervical lesions able to evolve into cancer, it is obvious that attention should also be paid to other biomarkers for increased credibility. Therefore, the progress of research led to the necessity of highlighting an immunomarker capable to increase the sensitivity of Pap's test. The cyclin p16INK4a, both in cytology exam and histology exam, is able to identify the HPV-infected cells and therefore to provide a more accurate diagnosis. As a matter of fact, it is acknowledged that

Women tested HPV DNA-positive and with p16INK4a ICC-negative could be safely managed

Conversely, the positivity for Ki67 in the cell nucleus is a marker of nuclear proliferation. The intensity of Ki67 immunoexpression increases more strongly in abnormal cells. The advantage of p16INK4a test used alone or within a dual test (p16INK4a and Ki67) is that the high intensity of p16INK4a and Ki67 immunoexpression in the nucleus warns that the cell will develop into cancer. If p16INK4a is positive in the cytoplasm only, there is a transient HPV infection, and the cervical

As regards the significance of p16INK4a/ Ki67 dual staining immunohistochemical test, Samar‐ awardana and colleagues showed as early as 2011 that this test has increased its capacity to

the positivity of p16INK4a is an important feature of high-risk HPV infected cells.

to other strategies involving, for example, colposcopy, biopsies.

with follow-up HPV DNA testing in 2 to 3 years.

lesion is able to spontaneously regress.

identify high-grade cervical lesions [45].

triage of ASC-US cytologies.

98 Human Papillomavirus - Research in a Global Perspective

types.

Another aspect which must be thoroughly discussed consists in the careful measurement of the sensitivity and specificity of each biotechnology used for the diagnosis of cervical lesions [43].

A meta-analysis performed by Jolien Roelens (2012) highlights that p16INK4a ICC has more accuracy than the HR-HPV test concerning the triage of ASC-US cytology samples. Both tests have similar sensitivities, but p16INK4a ICC has higher specificity than HR-HPV test. In LSIL samples, p16INK4a was more specific, but less sensitive than HR-HPV in the detection of ≥CIN2+ [20]. Over the past 10 years, similar results have been published by well-known researchers such as Izaaks, Denton, Holladay, Wentzensen [14, 43, 48, 49].

The positive p16INK4a ICC test rises Pap's test sensitivity to identify the cervical lesions prone to develop into cervical cancer. Therefore, the intensity of p16INK4a immunoexpression in the nucleus is in relation to the degree of cervical lesion.

In the study conducted by Denton and Bergeron (2010), it was shown that p16INK4a cytology provides significantly better specificity than HR-HPV for the triage of ASC-US and LSIL. Both HPV-testing and p16INK4a immunocytomarker test have similar sensitivity percentage rates, capable to detect high-grade cervical lesions at histopathological exam. In conclusion, the specialists involved in this research field have shown that p16INK4a cytology has the potential of being used as a triage of abnormal cytology LSIL [43].

Beginning with 2010, Samarawardana et al. have demonstrated by statistical analysis that the sensitivity (Se) and specificity (Sp) of p16INK4a for the detection of underlying CIN ≥ 2+ are 81.7% and 83.3%, respectively (*p* = 0.81). They underline that the Se and Sp of HR-HPV are lower than those of p16INK4a, albeit statistically significant: 78.1% and 50.9%, respectively (*p* < 0.01) [42].

There are many research studies recommending the usage of p16INK4a as a supplemental triage biomarker for ASC-US and LSIL cytologies, which have already been assigned as "high-risk" after HR-HPV detection [49, 50].

Large studies approached the comparison between the sensitivity and specificity of immuno‐ cytomarker p16 INK4a /Ki67dual stain versus HPV test in order to reveal if one of these tests is statistically more powerful to detect high-grade histopathological lesions ≥CIN2+. So, PALMS study is a bulky study which has enrolled 27,349 women from five European countries. All women aged 18 and older were tested by conventional cytology and p16/Ki67 dual test, while women aged 30 or older were tested by HPV DNA. The comparison between Se of p16INK4a / Ki67 dual stain cytology versus Pap's test with regard to the detection of high-grade cervical intraepithelial neoplasia (HGCIN) has shown higher values for p16INK4a /Ki67 test (86.7 vs 68.5%). As regards the Sp of the tests, it was comparable (95.2 vs 95.4%). By comparing Se and Sp of HPV DNA test versus p16INK4a /Ki67 dual stain, the study remarks that HPV DNA test has a higher Se to detect CIN2+, 93.3 versus 84.7%, but a low drop in specificity, 93.0 versus 96%. On the other hand, the dual test p16/Ki67 sets the idea that the test offers the same high sensitivity and specificity to identify HGCIN (see **Table 1**). The conclusions of researchers published on August 2015 within the Specialist Forum concerning PALMS study underline that the immunoexpression of dual-stained p16/Ki67 biomarkers is a novel approach to scrutinize efficiently for HGCIN and to achieve the same specificity conferred by Pap's test.


p16INK4a/Ki67 = dual test CINTech Plus; Se = sensitivity; Sp = specificity; HGCIN = high-grade cervical intraepithelial neoplasia.

**Table 1.** Cellular markers able to detect HGCIN sensitivity and specificity––results of PALMS study.

Similar attention is given to the cervical cancer screening of vaccinated women. In this regard, the trial known as Compass Trial is being conducted today in Australia. This trial enrolled 121,000 HPV-vaccinated women who are analyzed from the point of view of cervical screening programs using HPV test versus cytology tests [52].


**Table 2.** Cervical cancer screening programmes and guideline recommendations––update.

In line with the recent discoveries made by researches and the large studies mentioned above, the guidelines approved in the United States recommend HPV vaccination, HPV primary screening and co-testing with Pap's test, with major benefits in terms of sorting abnormally low cytologies and extending the screening interval. The ATHENA study prescribes the circumstances for applying the HPV test: triage of ASC-US in women over 21 years of age, HPV test with reflex Pap's cytology in women between 25 and 65 years, and HPV test with HPV 16/18 genotyping for primary screening in women older than 25. As to the accepted screening interval, this is 3–5 years for women negative at both intraepithelial malign cervical lesions (NIEML) and HPV infection, and 12 months in cases with HPV-positive test, but cytology-negative for NIEML, followed up by classic advices when the tests are constantly changed. Positive test for HPV 16/18 genotyping push the screening directly to colposcopy due to the presence of an absolute risk for CIN3+ progression. The interval for screening is prolonged to 5 years when women are negative at co-testing. Access to this screening program is given only to women with healthcare insurance [53]. The present guideline recommenda‐ tions and cervical cancer screening programs existing nowadays are summarized in **Table 2**.

In Europe, there are many differences as regards the modality of carrying out the screening of cervical cancer, because health policies and financial resources differ among Europe's demo‐ graphic regions. The most commonly used national screening program is mainly Pap's test; however, it is complemented by other new markers and biotechnologies prescribed by physicians with the goal of increasing diagnosis accuracy.

The Australian National Screening Program recommends only the screening using HPV test, and encompasses ages between 25 and 74 (applicable if women were screened in the past). The program includes both vaccinated and unvaccinated women. If the HPV test is positive, the follow-up program will be in line with cervical screening pathway [52].

## **5. Conclusions**

sensitivity and specificity to identify HGCIN (see **Table 1**). The conclusions of researchers published on August 2015 within the Specialist Forum concerning PALMS study underline that the immunoexpression of dual-stained p16/Ki67 biomarkers is a novel approach to scrutinize efficiently for HGCIN and to achieve the same specificity conferred by Pap's test.

**Se Sp Se Sp**

Se Sp Se Sp

**Detection of HGCIN p16/Ki67 Pap's test**

**Table 1.** Cellular markers able to detect HGCIN sensitivity and specificity––results of PALMS study.

**Methods Unites States Europe Australia** HPV vaccine NSP NSP

Co-test PAP NSP NO NSP

P16 GR––Triage GR––Triage GR––Triage P16/Ki67 GR––Triage GR––Triage GR––Triage E6/E7 mRNA GR––Triage GR––Triage GR––Triage NSP = National Screening Programme; GR––Triage = Guideline Recommendations––Triage; NO = negative.

In line with the recent discoveries made by researches and the large studies mentioned above, the guidelines approved in the United States recommend HPV vaccination, HPV primary screening and co-testing with Pap's test, with major benefits in terms of sorting abnormally low cytologies and extending the screening interval. The ATHENA study prescribes the circumstances for applying the HPV test: triage of ASC-US in women over 21 years of age, HPV test with reflex Pap's cytology in women between 25 and 65 years, and HPV test with

Pap's test Conjunction to HPV DNA testing NSP

**Table 2.** Cervical cancer screening programmes and guideline recommendations––update.

HPV DNA testing NSP primary test GR-triage NSP primary test

programs using HPV test versus cytology tests [52].

100 Human Papillomavirus - Research in a Global Perspective

neoplasia.

p16/Ki67 dual test versus Pap's test 86.7% 95.2% 68.5% 95.4% Detection of HGCIN p16/Ki67 HPV DNA test

p16INK4a /Ki67 vs. HPV DNA 84.7% 96% 93.3% 93.00%

p16INK4a/Ki67 = dual test CINTech Plus; Se = sensitivity; Sp = specificity; HGCIN = high-grade cervical intraepithelial

Similar attention is given to the cervical cancer screening of vaccinated women. In this regard, the trial known as Compass Trial is being conducted today in Australia. This trial enrolled 121,000 HPV-vaccinated women who are analyzed from the point of view of cervical screening

> The detection of carcinogenetic HPV DNA, and especially of HPV 16/18 genotyping, stands for approved tests to be used as primary screening and for triage of equivocal cytology equally on vaccinated and unvaccinated women.

> Recognition of E6/E7 mRNA is largely applied in adjunct to primary HPV screening to select cases with HPV integrated in the host cell. The result of E6/E7 mRNA is able to achieve the triage of equivocal or mildly abnormal cytology.

> Uncovering of HPV protein by cytology and histology immunostaining underlines the accuracy of diagnosis and can be used together with primary HPV screening or for the triage of equivocal or mildly abnormal cytology.

> The success that was achieved in researches across the world showed that the identification of immnuocytomarkers inside the same cervical cell––dual stain p16INK4a and Ki67––warns more accurately about the possibility of progression to cervical cancer.

> Correctly performed and interpreted, the results of approved tests for cervical screening programs allow to obtain an extended interval screening between 2 and 5 years, with a larger number of advantages in terms of diagnosis accuracy and healthcare costs.

## **Author details**

Ruxandra Stanculescu

Address all correspondence to: ruxandra\_v\_stanculescu@yahoo.com

Obstetrics and Gynaecology Department, "Carol Davila" University of Medicine, "St. Pantelimon"Clinical Emergency Hospital Bucharest, Romania

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Address all correspondence to: ruxandra\_v\_stanculescu@yahoo.com

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## **The Diagnostic of Cervical Carcinoma: From Theory to Practice**

J. Rajčáni, K. Kajo, O. el Hassoun, M. Adamkov and M. Benčat

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/62834

### **Abstract**

Human papillomaviruses (HPV) are naked particles composed of 72 subunits, each formed by 2 structural proteins designated L1 and L2 (L = late). HPV does not grow outside of squamous epithelium cells, in which it infects the suprabasal prickle cell layer. The viral double-stranded DNA (vDNA) has about 8 kilobase pairs (kbp) and also encodes several non-structural polypeptides, designated E1–E7 (E = early). At least 3 early oncoproteins (E5, E6, and E7) induce host cell proliferation, driving them into permanent division. During long-term latency, the circularized HPV DNA may get integrated into the host cell DNA molecule. The circular HPV DNA is then interrupted, usually within the E2 open reading frame (ORF), which then cannot exert its regulatory (feedback) effect on the early gene expression. The increased expression of E6/E7 proteins seriously affects the regulation of host cell division mainly via dysregulation of the functions of p53 and Rp proteins. HPV infects the female genital tract representing the main cause of cervical dysplasia and subsequent squamous cell carcinoma (SCa). The HPV isolates exist mainly in the form of amplified DNAs; based on the similarity and/or variations (dissimilarity) of their L1 capsid polypeptide sequence, 96 human genotypes were included into five genera of the *Papillomaviridae* family. The clinically most important genotypes that cause lesions at mucosal membranes and/or on the skin, belong mainly to the Alphapapillo‐ mavirus genus. The genotypes, associated with severe dysplastic changes and/or cervical cancer, were designated as high risk (HR-HPV). The prevalence of the integrated HPV DNA sequence over the episomal molecules appears in a proportion smears-graded LSIL (low-grade squamous intraepithelial lesion). Later on, carrier cells revealing the integrated HPV genome expression the oncoproteins (E6/E7) clearly prevail especially in HSIL (highgrade squamous intraepithelial lesion) smears and in the cervical cancer itself. What is crucial for the modern diagnostic of cervical dysplasia, is the p16/INK4A (inhibitor kinase) polypeptide, which itself represents a form of cell defense against the viral oncogenic proteins. The p16 antigen shows a continuous parabasal staining in the CIN I lesion. If dysplastic cells occupy at least one half (or two thirds) of squamous epithelium, the

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

designation CIN II/HSIL is correct, and at the stage of CIN III/HSIL, dysplastic cells replace the entire squamous epithelium. Another frequently used immunohistochemical marker of intraepithelial cervical dysplasia so far is the Ki-67 antigen, which occurs in the nuclei of proliferating and/or repeatedly dividing (immortalized) cells. Women revealing p16 positive ASCUS (atypical smear cells of unknown significance) as well as those showing LSIL (low-grade cytological changes) should be examined for the presence of the HPV DNA. The detection of HPV DNA alone, that is, in the absence of cytological screening, has a relatively lower prediction value, though the HR HPV positive DNA test in the absence of morphological alterations may in part predict the possible progression into malignancy. Nevertheless, only the combined cytological as well as molecular followup (cervical smear examined for cytology as well as for HPV DNA) is regarded for the most reliable diagnostic approach.

**Keywords:** Human papillomavirus, Cervical intraepithelial neoplasia (CIN), Cervical dysplasia, Squamous cell carcinoma, p16/INK4A Polypeptide, Ki-67 polypeptide, Lab‐ oratory diagnostic, PA smears, Squamous intraepithelial lesion (SIL), Liquid-based cy‐ tology (lbc), Cervical biopsy, Histology, HPV DNA testing

### **1. Introduction**

Virus infection had been for a long time anticipated in the pathogenesis of cervical cancer. The suspected role of herpes simplex virus 2 (HSV 2) early protein as suggested by Aurelian et al. [1] was not confirmed, since several carefully designed prospective and/or serological studies following the significance of elevated anti-HSV 2 antibody levels did not confirm any associa‐ tion. Although certain isolated DNA fragments coming from the HSV genome may transform the tissue culture cells of rodent origin, their relevance in the induction of human cervical carcinoma has not met any further approval [2, 3]. Contemporarily, the human papillomavi‐ rus (HPV) has been accepted to represent the most probable infectious agent in respect of cervical cancer [4]. During the last decades, a great bulk of convincing data has accumulated in favor of the latter assumption, which has become widely accepted. Thus, based on substantial clinical and biological evidence, strong relationship exists between HPV infection and the develop‐ ment of cervical cancer. Nevertheless, in line with a few but independent observations, a hypothesis has repeatedly emerged saying that HSV 2 co-infection might represent an acceler‐ ating cofactor for cervical carcinoma formation [5]. Novel reports from Brazil and/or some other comparative studies on the given issue, which had been performed simultaneously among Indonesian and Swedish population, confirmed the possible role of HSV 2 in the former but not the in the latter geographical area [6].

Our paper reconciles the role of latent HPV infection (mainly based on the detection of corresponding DNA sequences and/or their fragments) in the stratified squamous epithelium cells of oral, vaginal, and/or anal mucosa (especially at their transition sites to cylindrical epithelium, such as uterine cervix) and/or in the keratinized squamous skin epithelium cells. Regarding to the role of the HPV genome in the pathogenesis of cervical cancer, we shall point at the importance of a complex diagnostic approach overcoming the possible barriers between scientific disciplines such as virology, pathology, and molecular biology on one hand and explain the need of the rapid application of novel achievements in diagnostic pathology, biochemistry, cytology as well as in the clinical practice. Despite of the difficulties of any interdisciplinary approach, the introduction of molecular virology (such the HPV DNA test) and immunohistochemical procedures (p16 and Ki-67 antigen detection) into diagnostic pathology and/or cytology when done in close cooperation with gynecologists has led to a dramatic decrease of the frequency of cervical cancer in several European countries, Slovakia not excluding. Reporting the frequency of positive cervical smears was registered in the terms of the conventional Papanicolaou (PA) test, which is still most suitable for large-scale screen‐ ing. **Table 1** summarizes six annual reports of one of several Alpha medical Company Ltd Diagnostic Centres, which were destined for domestic insurance companies. The categories of classification of cervical dysplasia in given statistics do not correspond to the Bethesda classification system [7, 8]. According to the latter, atypical squamous cells of unknown significance (ASCUS) should be distinguished from non-neoplastic reactive changes of cervical squamous epithelium cells using the criteria summarized in **Table 2**. The squamous intraepi‐ thelial lesion (SIL) can be recognized by the presence of dysplastic cells, which show either mild (low grade) or more severe (high grade) alterations (**Table 3**). The original cytological nomenclature used to describe the appearance of cells in conventional PA smears before the introduction of Bethesda classification system has been repeatedly compared by several authors [9, 10] with the recently adopted definitions. Based on these data, we proved the individual categories of the so-called MKCH classification system (Slovak abbreviation for the expression International Classification of Diseases). To achieve an upmost precise interpreta‐ tion, we used terms essentially resembling to the CIN (cervical intraepithelial lesion) classifi‐ cation, namely a similar three-degree scale (I—low, II—medium, and III—high). Evaluating in detail each protocol from the archives of the Pathology Diagnostic Centre of Alpha medical Company in Martin coming from a single-year period, that is, the year 2015 (compare **Table 4**), we found that the medium-grade cervical dysplasia category from the MKCH statistics in fact encompassed as many as 64% of smears-graded LSIL, while the proportion smears scored HSIL in the same category was approximately 6%. Noteworthy, another 6% fall into the group of patients with negative smears and the rest protocols revealed the diagnosis of ASCUS and/ or ASC-H (in 24%). It should be mentioned that out of 516 samples of the given MKCH category, 110 had been repeatedly tested patients (these women were re-examined within 4– 6 months intervals, i.e., at least twice during the same year). Thus, the MKCH category "medium-grade cervical dysplasia" consisted of 516 smears coming from 457 women. In the group of repeatedly examined patients, 10% underwent spontaneous healing during the relatively short 1-year follow-up period (data not shown). The repeatedly negative smears came mainly from patients, which were at first examination scored ASCUS. Unfortunately, their HPV DNA status is not always determined, even though in this group ("medium-grade cervical dysplasia") during the year 2015, altogether 162 of smears were tested HPV DNA, from which 74 were positive (a proportion of 39.5%), while 13 were HPV type 16 positive and 6 were HPV type 18 positive. While only 32% of screened patients were examined by cytology as well as for HPV, many HPV tests were made in the absence of cytology and/or *vice versa* (compare paragraphs 4 and 5, see also **Table 12**).

designation CIN II/HSIL is correct, and at the stage of CIN III/HSIL, dysplastic cells replace the entire squamous epithelium. Another frequently used immunohistochemical marker of intraepithelial cervical dysplasia so far is the Ki-67 antigen, which occurs in the nuclei of proliferating and/or repeatedly dividing (immortalized) cells. Women revealing p16 positive ASCUS (atypical smear cells of unknown significance) as well as those showing LSIL (low-grade cytological changes) should be examined for the presence of the HPV DNA. The detection of HPV DNA alone, that is, in the absence of cytological screening, has a relatively lower prediction value, though the HR HPV positive DNA test in the absence of morphological alterations may in part predict the possible progression into malignancy. Nevertheless, only the combined cytological as well as molecular followup (cervical smear examined for cytology as well as for HPV DNA) is regarded for the

**Keywords:** Human papillomavirus, Cervical intraepithelial neoplasia (CIN), Cervical dysplasia, Squamous cell carcinoma, p16/INK4A Polypeptide, Ki-67 polypeptide, Lab‐ oratory diagnostic, PA smears, Squamous intraepithelial lesion (SIL), Liquid-based cy‐

Virus infection had been for a long time anticipated in the pathogenesis of cervical cancer. The suspected role of herpes simplex virus 2 (HSV 2) early protein as suggested by Aurelian et al. [1] was not confirmed, since several carefully designed prospective and/or serological studies following the significance of elevated anti-HSV 2 antibody levels did not confirm any associa‐ tion. Although certain isolated DNA fragments coming from the HSV genome may transform the tissue culture cells of rodent origin, their relevance in the induction of human cervical carcinoma has not met any further approval [2, 3]. Contemporarily, the human papillomavi‐ rus (HPV) has been accepted to represent the most probable infectious agent in respect of cervical cancer [4]. During the last decades, a great bulk of convincing data has accumulated in favor of the latter assumption, which has become widely accepted. Thus, based on substantial clinical and biological evidence, strong relationship exists between HPV infection and the develop‐ ment of cervical cancer. Nevertheless, in line with a few but independent observations, a hypothesis has repeatedly emerged saying that HSV 2 co-infection might represent an acceler‐ ating cofactor for cervical carcinoma formation [5]. Novel reports from Brazil and/or some other comparative studies on the given issue, which had been performed simultaneously among Indonesian and Swedish population, confirmed the possible role of HSV 2 in the former but not

Our paper reconciles the role of latent HPV infection (mainly based on the detection of corresponding DNA sequences and/or their fragments) in the stratified squamous epithelium cells of oral, vaginal, and/or anal mucosa (especially at their transition sites to cylindrical epithelium, such as uterine cervix) and/or in the keratinized squamous skin epithelium cells. Regarding to the role of the HPV genome in the pathogenesis of cervical cancer, we shall point at the importance of a complex diagnostic approach overcoming the possible barriers between

most reliable diagnostic approach.

110 Human Papillomavirus - Research in a Global Perspective

the in the latter geographical area [6].

**1. Introduction**

tology (lbc), Cervical biopsy, Histology, HPV DNA testing

#### 112 Human Papillomavirus - Research in a Global Perspective


1 Corresponds to LSIL (88%) and/or ASCUS (12%).2 Corresponds to LSIL (60%), ASC-H/ASCUS (18%) and/or to HSIL (10%).3 Corresponds to HSIL.4 Modified according to above mentioned1,2,3, remarks (see text for details).\* Designation of given statistical categories according to author´s translation (from Slovak).\*\*Positive smears after excluding the negative ones or those referred to as reactive changes.\*\*\*Including 5 cases showing invasive growth into distant into (surrounding) tissues.# From report of Pathology Diagnostic Centre, Alpha medical Company Ltd, Martin (Slovakia) for domestic authorities (with permission).

**Table 1.** Frequency of cervical dysplasia and/or cervical cancer by screening of cytological smears from 1 January 2010 to 31 December 2015# .


**Table 2.** Definition of ASCUS versus reactive changes in cervical smears\* .


**Table 3.** Definition of low-grade versus high-grade squamous intraepitelial lesion (SIL)\* .

**Diagnosis Patient number Per cent Modified4**

Medium-grade cervical dysplasia2 1456 0.7% Cancelled High-grade cervical dysplasia3 263 0.13% 466 (0.23%)

Carcinoma *in situ* 36 0.02% No change

given statistical categories according to author´s translation (from Slovak).\*\*Positive smears after excluding the negative ones or those referred to as reactive changes.\*\*\*Including 5 cases showing invasive growth into distant into

Atypical cells of unknown significance (ASCUS) Reactive changes due to inflammation

Moderately dense chromatin staining Slight hyperchromatic staining only

Size of nuclei increased significantly (three times) Slightly enlarged nuclei (up most two times)

**Table 1.** Frequency of cervical dysplasia and/or cervical cancer by screening of cytological smears from 1 January 2010

Cells revealing doubled nuclei may be present

The fine granular chromatin is dispersed throughout nucleus Nuclei may be slightly elliptic, but regularly are nearly round shaped The nuclear/cytoplasm ratio (N/C) in moderately increased

Low-grade cervical dysplasia\*,1

Cervical (spinocellular)

(surrounding) tissues.#

to 31 December 2015#

carcinoma

(10%).3

1

ASCUS (includes also ASC-H, from low and medium dysplasias)1,2

Corresponds to HSIL.4

for domestic authorities (with permission).

.

Koilocytes (proving HPV) can be recognized Hyperchomatosis, dense chromatin granules. The nuclear membrane has irregular appearance

A faint perinuclear ring may be visible

\* Based on the data from corresponding manuals [11, 12].

**Table 2.** Definition of ASCUS versus reactive changes in cervical smears\*

Corresponds to LSIL (88%) and/or ASCUS (12%).2

112 Human Papillomavirus - Research in a Global Perspective

**Positive Total examined**

Modified according to above mentioned1,2,3, remarks (see text for details).\*

From report of Pathology Diagnostic Centre, Alpha medical Company Ltd, Martin (Slovakia)

Atrophic or shrink nuclei

Bacteria may be present

.

The cytoplasm may be vacuolated

Polymorphonuclear leukocytes are present

3070\*\* 206,317 1.5% 3943 (1.9%)

16\*\*\* 0.007% No change

Corresponds to LSIL (60%), ASC-H/ASCUS (18%) and/or to HSIL

230 (0.13%)

Designation of


**Table 4.** The frequency of cervical dysplasia and/or cervical cancer by screening of cytological smears from 1 January 2015 to 31 December 2015.

Alternatively, we analyzed a proportion of still available original protocols from a limited number of women (300 at random chosen patients were re-evaluated out of 1425, i.e., 20.6%), in order to assess the estimate number of LSIL and HSIL cases in result of cancelling the undesired category of "medium-grade dysplasia". Despite of some doubts concerning the precision of such calculations, we could demonstrate the decreased incidence as well as a significantly lower morbidity for cervical carcinoma within the last 6-years period. Regarding to the frequency of 7.5 positive smears out of 100,000 samples enrolled, and comparing this number with the overall morbidity rate of 15.4/100,000 as reported for Slovakia in 1999 [13], the estimated decrease of cervical carcinoma cases should be much higher than 50%. Namely, the former number reflects the relative proportion of positive samples out of the total enrolled, while the latter represents the frequency of disease in whole women population (either in the fertile and/or post-fertile age). The estimated proportion of women examined in comparison with the total woman population in given age might range from 10 to 20%. Even if this proportion of followed may not be correct, the decreased morbidity for cervical cancer in Slovakia during the last decade might be at least tenfold. Furthermore, **Table 1** also shows that at least 6% (by minimal rate 263/3070) but not more than 12% (by maximal rate 466/3943) of smears which had been scored LSIL progressed into the stage of HSIL. For the above-men‐ tioned minimal rate, the intermediate MKCH category was not taken into account; for the maximum rate, it has been split (modified) into corresponding Bethesda categories as ex‐ plained above. Thus, regardless to any imperfections of both calculations, which may arise from comparison of the Tables 1 and 4, our data point at an increased incidence of newly emerging LSIL cases. Namely, the average positive rate among the smears, which was in the range of 1.5–1.9% during the last 6-years period, has reached 3.5% in the year 2015 (an increase of 180%). As already suggested in our previous paper [14], and confirmed by others, the probability of transition from LSIL and/or ASC-H cases into HSIL strongly depends on the presence of HPV as detected by one of the available HPV DNA tests [15, 16]. The great majority of the ASCUS cases and up to 85% of LSIL cases (especially in the absence of HPV DNA) might undergo spontaneous healing as shown in next paragraph.

## **2. The role of human papillomavirus (HPV) in the pathogenesis of cervical dysplasia and cancer**

The first mammalian papillomavirus (PV) was described by Shope and Hurst [17], who characterized the transmissible nature of cutaneous papillomas arising in wild cottontail rabbits. The narrow host range of PVs in culture to sites with stratified squamous epithelia that is either cornified (skin) or non-cornified (mucosa) was overcome by introducing molec‐ ular technics such as the DNA extraction, the polymerase chain reaction (PCR), and vDNA sequencing allowing to identify the genes of the PV DNA regarding to the function of corresponding proteins. The papillomaviruses (PVs) comprise a group of non-enveloped epitheliotropic DNA viruses that predominantly induce benign lesions of the skin (warts) and mucous membranes (condylomas). Some PVs have also been implicated in the development of epithelial malignancies, especially in the cancer of uterine cervix, certain tumors of urogen‐ ital tract, and upper airway cancers [18]. Due to given relationships, an increasing amount of information has accumulated from sequencing results of various PV DNAs. Later on, the PVs were classified according to the host species they infect, so that the PVs of human origin were designated as human PVs (HPV). Officially recognized by the International Committee on the Taxonomy of Viruses (ICTV), the former *Papovaviridae* family now falls into two separate families, *Papillomaviridae* and *Polyomaviridae* [19]. The reason being the missing helicase motif in the HPV E1 protein, a domain stretching longer than about 230 amino acids (aa) within the analogous non-structural HPV polypeptide, which otherwise has some similarity with the SV40 T-antigen, the parvovirus NS1 protein, and with a planarian virus-like element [20].

to the frequency of 7.5 positive smears out of 100,000 samples enrolled, and comparing this number with the overall morbidity rate of 15.4/100,000 as reported for Slovakia in 1999 [13], the estimated decrease of cervical carcinoma cases should be much higher than 50%. Namely, the former number reflects the relative proportion of positive samples out of the total enrolled, while the latter represents the frequency of disease in whole women population (either in the fertile and/or post-fertile age). The estimated proportion of women examined in comparison with the total woman population in given age might range from 10 to 20%. Even if this proportion of followed may not be correct, the decreased morbidity for cervical cancer in Slovakia during the last decade might be at least tenfold. Furthermore, **Table 1** also shows that at least 6% (by minimal rate 263/3070) but not more than 12% (by maximal rate 466/3943) of smears which had been scored LSIL progressed into the stage of HSIL. For the above-men‐ tioned minimal rate, the intermediate MKCH category was not taken into account; for the maximum rate, it has been split (modified) into corresponding Bethesda categories as ex‐ plained above. Thus, regardless to any imperfections of both calculations, which may arise from comparison of the Tables 1 and 4, our data point at an increased incidence of newly emerging LSIL cases. Namely, the average positive rate among the smears, which was in the range of 1.5–1.9% during the last 6-years period, has reached 3.5% in the year 2015 (an increase of 180%). As already suggested in our previous paper [14], and confirmed by others, the probability of transition from LSIL and/or ASC-H cases into HSIL strongly depends on the presence of HPV as detected by one of the available HPV DNA tests [15, 16]. The great majority of the ASCUS cases and up to 85% of LSIL cases (especially in the absence of HPV DNA) might

**2. The role of human papillomavirus (HPV) in the pathogenesis of cervical**

The first mammalian papillomavirus (PV) was described by Shope and Hurst [17], who characterized the transmissible nature of cutaneous papillomas arising in wild cottontail rabbits. The narrow host range of PVs in culture to sites with stratified squamous epithelia that is either cornified (skin) or non-cornified (mucosa) was overcome by introducing molec‐ ular technics such as the DNA extraction, the polymerase chain reaction (PCR), and vDNA sequencing allowing to identify the genes of the PV DNA regarding to the function of corresponding proteins. The papillomaviruses (PVs) comprise a group of non-enveloped epitheliotropic DNA viruses that predominantly induce benign lesions of the skin (warts) and mucous membranes (condylomas). Some PVs have also been implicated in the development of epithelial malignancies, especially in the cancer of uterine cervix, certain tumors of urogen‐ ital tract, and upper airway cancers [18]. Due to given relationships, an increasing amount of information has accumulated from sequencing results of various PV DNAs. Later on, the PVs were classified according to the host species they infect, so that the PVs of human origin were designated as human PVs (HPV). Officially recognized by the International Committee on the Taxonomy of Viruses (ICTV), the former *Papovaviridae* family now falls into two separate families, *Papillomaviridae* and *Polyomaviridae* [19]. The reason being the missing helicase motif

undergo spontaneous healing as shown in next paragraph.

**dysplasia and cancer**

114 Human Papillomavirus - Research in a Global Perspective

Among the most extensively studied HPV genomes, nearly 100 genotypes were described based on the at least 90% nucleotide homology of sequence encoding one of 2 structural capsid proteins (the L1 protein sequence). The L1 ORF is the most conserved gene within the genome and has therefore been used for the identification and classification of new HPV types corresponding to later characterized species is shown in **Table 5**. It should be mentioned that sorting into species and genus has some theoretical and/or scientific importance, but for practical reasons, the old genotype classification is still in use (**Table 6**). A new HPV isolate is recognized as such, if its complete genome has been cloned and sequenced to determine in which extent the L1 ORF differs from the closest known HPV genotype. If a more than 10% bp difference can be found that is a distinct genotype. Differences between 2 and 10% homology define a subtype and <2% difference reveals a variant. The closely related HPV types HPV-2 and 27, HPV-6 and 11, and HPV-16 and 31, which cause common warts, genital warts, and cervical cancer, respectively, are excellent examples of numerous consistencies between phylogeny and pathology. Furthermore, the HPV genotypes could have been distinguished as high risk (HR) and low risk (LR) according to their ability to transform cells, due to their relationship to cervical dysplasia and/or cancer, and according to their frequency. The squamous carcinoma (SCa) cells and/or the cells in *epidermodysplasia veruciformis* (EV) harbor multiple genome copies of specific HPV types, especially HPV5 and HPV8, but also of HPV14, HPV20, and a few others [21–23]. Their transcripts have been described in several the so-called EV-associated SCa cells [24]. In 2009, HPV5 and HPV8 were classified by IARC as "possibly carcinogenic" in EV patients [25].




1 According to deVilliers et al. [30]; 2 epidermodysplasia verruciformis. \* ERL = the DNA segment between E and L ORFs.

**Table 5.** Classification of papillomaviruses and the list of their important genotypes.


\* Congenital skin lesion with high sensitivity to HPV infection.

\*\* Mainly in CIN I/LSIL, CIN II and/or non-invasive forms CINIII/HSIL and/or CIN III+/HSIL.
