Current Pharmaceutical Design - Volume 19, Issue 8, 2013

Current diagnostic approaches for primary cervical cancer screening, work-up of equivocal or positive screening results or follow- up after treatment of precancerous lesions primarily rely on the morphologic interpretation of squamous epithelial cells (Pap cytology), in some setting accompanied by the detection of human papillomavirus DNA and have largely contributed to remarkable reduction of disease incidence in countries with implemented screening programs. However, these approaches are limited by a poor sensitivity and reproducibility of Pap cytology and low specificity for high grade cervical intraepithelial neoplasia of HPV DNA detection assays. Early detection might be improved by complementing or even replacing these tests by markers which are more directly related to molecular events triggering HPV-induced carcinogenesis and thereby might deliver more accurate diagnostic performance. The delineation of molecular changes which occur during different stages of HPV infections and the identification of changes which induce neoplastic alterations allow for the detection of markers that specifically highlight the transforming stage of the infection where viral oncogenes are overexpressed and therefore allow for a more specific diagnosis of lesions that require treatment. The evaluation of such markers in clinical studies revealed that some indeed show an improved diagnostic performance compared to Pap cytology or HPV DNA tests only.

Cervical cancer is a major health problem and almost all of these cancers are related to a high risk Human Papillomavirus cervical infection. The Human Papillomavirus transforming potential has long been probed and is mainly characterized by the integration of the virus in the host genomes and the expression of the viral oncoproteins such as the E6 and E7 mRNAs. The vast majority of the HPV infections are transient and resolve and just a few percentages of patients are noted to develop a persistent infection. Therefore, there is the need of identifying appropriate biomarkers that can predict and differentiate transient vs persistent and clinical relevant HPV cervical infections. To date, two categories of commercial assays have been introduced, mainly aiming to the detection of the E6 and E7 mRNAs and of the surrogates of the E7 activity (p16ink4A protein). The aim of the present study is to analyse the basis of the HPV-related carcinogenesis and to discuss the commercial biomarkers developed to-date, reviewing those studies that used the above-mentioned assays in a clinical setting.

Cervical cancer is the second most common malignancy among women worldwide. While more than 90% of cervical cancers carry one or more high risk HPV types, the exact relationship between HPV oncoproteins and signaling pathways alterations in cervical cancer remains to be clarified. Vaccines and targeted therapies are today considered of great potential for the improvement of patients' outcome. This review will focus on the interaction of HPV with cervical cancer pathway activations. Next generation high throughput screening technologies for molecular and protein profiling as well as innovative logistics, necessary for the implementation of the personalized care of cervical cancer patients will be discussed.

Persistence of human papillomavirus infection (HPV) of the cervix after treatment for cervical intraepithelial neoplasia predisposes to lesion recurrence. Given the weakness of natural immunity against HPV it has been suggested that certain anatomical sites could act as viral reservoirs though which the infection could be retransmitted to the cervix even if the initial HPV tests after treatment are negative. This review examined the possible role of various reservoirs such as the oral cavity, the anus, the fingers and the partner's penis. The available data are insufficient to confirm a significant risk of cervical re-infection from any site other than the penis. It seems that the risk of transmission by the male partner can be reduced by consistent condom use; therefore this should be included in the patient's counselling. Further studies are required to elucidate the role of the other sites especially the anus where some reports imply a possibility for transmission to the cervix. In this context expanding the indications for HPV vaccination to include women who have been treated for CIN should be considered.

Background: In delayed HPV triage women with atypical squamous cells of uncertain significance (ASC-US) cytology are retested after 6-12 months in order to decide whether they should be referred for colposcopy, further follow-up cytology or routine screening in three years. Triage using a specific HPV E6/E7 mRNA test may reduce referrals for colposcopy of women with ASC-US cytology compared to HPV DNA testing. We explored whether HPV mRNA triaging could reduce the time from ASC-US index cytology to biopsy compared with repeat cytology, and whether the positive predictive value (PPV) of the HPV mRNA test for high grade cervical intraepithelial neoplasia (CIN2+) was comparable with the PPV of repeat cytology. Material and methods: We used repeat cytology and the HPV mRNA test PreTect HPV-Proofer, which detects E6/E7 mRNA from HPV subtypes 16, 18, 31, 33 and 45, in the triage of women with ASC-US. We included all women from the two northernmost counties of Norway with a first ASC-US cytology during the period 2004-2008. Two triage methods were evaluated 1) only repeat cytology (n=964) and 2) both HPV mRNA testing and cytology (n=542). Histologically confirmed CIN2+ was the study endpoint. Results: Among 1506 women with an ASC-US index cytology, 59 women (3.9%) had biopsy taken, of whom 49 women had CIN2+ (PPV 83.1%). The mean time from index ASC-US cytology until the case was resolved (biopsy or return to screening) was 10.6 months in the repeat cytology group and 7.3 months in the HPV group (P<0.001). Of the 964 women in the group with repeat cytology only, 35 women (3.6%) had biopsy and 30 had CIN2+ (PPV 85.7%). Of the 542 women in the group with both HPV test and cytology, 24 women (4.4%) had biopsy and 19 had CIN2+ (PPV 79.2%). Conclusion: In triage of women with ASC-US, the HPV mRNA test significantly reduced the time from the first abnormal cytology until biopsy and had predictive values comparable with those of repeat cytology.

Objective: Liquid-based cytology with supplementary human papillomavirus triage (LBC+HPV triage) of low-grade cytological abnormalities may improve the detection of cervical intraepithelial neoplasia (CIN) compared with conventional cytology. To investigate this subject, LBC+HPV triage and conventional cytology were alternated in a population-based screening setting. Cases with abnormal cytology were referred for colposcopy. Methods: We compared the performance of LBC+HPV triage [n=4059] and conventional cytology [n=4261] in detecting CIN2 or worse [CIN2+] and CIN3 or worse [CIN3+]. We used logistic regression to assess CIN detection rates and abnormal cytology rates, which yielded unadjusted odds ratios (OR) and corresponding 95% confidence intervals (CI). We computed adjusted ORs from a multivariate logistic regression model that included potential confounders such as age, screening centre and time period. Results: We found similar detection rates of CIN2+ by LBC+HPV triage and conventional cytology; the adjusted OR for the comparison of CIN detection rates was 0.87 (95% CI: 0.60-1.26) for CIN2+ and 1.00 (95% CI: 0.64-1.58) for CIN3+. We also found similar positive predictive values between methods. Thus, there was no advantage in using LBC+HPV triage as compared to conventional cytology in terms of sensitivity, specificity and positive and negative predictive value to detect histologically confirmed CIN2+ and CIN3+. Conclusions: LBC+HPV triage may lead to a reduction in unnecessary work-ups for women with abnormal cytological lesions who are negative for high-risk HPV. It is important to continuously monitor abnormal cytology rates, both when testing a new method, and after the new method has become routine.

Cervical cancer (CC) is caused by a persistent infection by certain human papillomavirus (HPV) genotypes. Although Papanicolaou (Pap) Test is considered the most cost-effective test for reducing CC mortality, a considerable number of high-grade precursor lesions of CC could pass unnoticed with the Pap. The addition of high-risk human papillomavirus (HPV) genotype detection in cervical cytology has improved the sensitivity, but due to its low specificity, further biomarkers of malignancy have been searched for. Given the fact that the oncogenic role of HPV is exerted primarily by affecting cell cycle control it is not surprising that most of the useful biomarkers of HPV-related uterine lesions are cell cycle proteins, with p16 and Ki67 the most widely used. More recently, molecular profiling and marker combination tests have identified the utility of antibody cocktails such as p16/Ki67 dual and ProEx C, which detect both TOP2A and MCM2 cell cycle proteins. In this article we revise the rationale for the use of the most common cell cycle biomarkers, also including p53 and cyclin D1, and their clinical utility drawing attention to novel biomarkers and how HPV vaccination could influence their use.

Invasive cervical cancer is a common problem worldwide and while rates of squamous cell carcinoma of the cervix (SCC) have been declining with the implementation of community screening programs, adenocarcinoma of the cervix (ADC) has not shown a similar response to screening. At this time, the two entities are tested for and treated with the same clinical algorithms, namely gynecologic cytology and molecular testing for HPV DNA. However, ADC arises more proximally within the cervical canal and frequently occurs below the superficial mucosal lining, precluding it from being easily diagnosed with cytologic examination. Furthermore, ADC has a more aggressive course than SCC and is significantly less responsive to radiotherapy. These factors combine to produce a lesion that is diagnosed at a higher stage and has an overall poorer prognosis than SCC. While both lesions have pathogenic origins with the high-risk species of Human papilloma viruses (HPV), the high association of ADC with HPV 18, a virus with a higher genome integration rate, suggests that the transformation pathway of ADC differs from SCC. Modern methods of molecular analysis can produce gene expression profiles of various cell types and preliminary studies have shown that SCC and ADC do, indeed, produce a distinct genetic signature and can be reproducibly segregated. This technology has promise for clinical applicability in the diagnosis, the prognostic stratification, formation of a treatment care plan and post-therapeutic monitoring of ADC.

Although several epidemiologic studies have confirmed the association between high-risk human papillomavirus (hr-HPV) and adenocarcinoma of the cervix, there are few papers focusing on the molecular immunophenotype of the HPV related cervical adenocarcinoma and its precursor lesions. The present study is aimed to assess the immunohistochemical expression of p16, p53, cyclin D1, EGFR, and COX-2 in benign and malignant lesions of cervical glandular components, and consequently the identification of the relationship between these markers and the HPV L1 capsid protein. We investigated 7 cases of endocervical adenocarcinoma in situ (AIS), 8 cases of adenosquamous carcinoma, 15 cases of invasive adenocarcinoma of endocervical type, and 5 cases without malignant lesions (normal and/or benign endocervical epithelium). The tissue fragments underwent standard laboratory procedures for the histopathological and immunohistochemical exams. For each marker, the semi-quantitative assessment was performed using appropriate scoring systems. Our results showed that: (i) the combination of L1 capsid protein and p16 can predict the progression risk of precursor lesion of endocervical adenocarcinomas; (ii) p53 - COX2 - p16 co-assessment is useful as a panel of relevant biomarkers for L1 – p16 association; (iii) EGFR increases according to the progression in lesions severity; (iv) cyclin D1 is a reliable marker for the invasive capacity. Further studies are necessary to quantify the value of these markers, as prognostic factors in HPV related cervical adenocarcinoma.

Human Papillomavirus (HPV) testing has been extensively studied in randomized controlled trials of primary cervical screening. Based on encouraging results concerning its high detection rates and a high negative predictive value for high-grade cervical intraepithelial neoplasia (CIN), HPV testing will probably replace cytology in future primary cervical screening. However, HPV testing is associated with more frequent false-positive tests compared to cytology. False-positive tests are defined as positive screening tests which are not subsequently confirmed with high-grade CIN. Several authors have claimed that the frequency of false-positive HPV tests could be reduced if an additional test was used to decide on referral for colposcopy of HPV-positive women. Data from the trials, however, do not support this claim. In fact, when compared to standard cytology screening and triage procedures, HPV testing leads to more screenpositive women being referred for colposcopy without having high-grade CIN, and to more women undergoing repeated testing. The only reasonable solution to the problem of false-positive tests appears to be a revised definition of a positive HPV screening test. However, further studies are needed to determine how this definition could be revised while at the same time keeping the high negative predictive value of HPV testing.

Background: Loop Electrosurgical Excision Procedure (LEEP) represents the mainstay technique for CIN2+ removal. The major concern in conservative treatment is to verify whether CIN eradication was complete, since incomplete excision is associated with an increased risk of cervical cancer. The histopathologic evaluation of resection margins status is far from perfect, since cervical lesions may recur in 5-15% of patients who had conisation specimens with clean margins. Current follow-up protocol of patients treated by conisation for high grade CIN is manly based on the combination of cytology-plus- HPV-DNA testing. This approach showed high sensitivity but low specificity level in detecting recurrence. The consequence were overdiagnosis and overtreatment, especially in youngest women, in which spontaneous regression rate of CIN is substantial. In this longitudinal study we investigated whether patient's age, cone depth and pre-conisation HPV-load level, may be used as predictive markers for residual/recurrent CIN after conisation. Then we aimed to examined the role of E6/E7 mRNA testing during post-conization follow-up. Methods: The study, focused on the outcome of 116 patients treated for CIN by LEEP, included three consecutive steps. Firstly, the authors analysed the prevalence of residual/recurrence disease after conization; then, they investigated which factors may influence treatment failure even when resection margins were clean; finally, they evaluated the diagnostic accuracy of E6/E7 mRNA test as predictive marker of recurrence. Results: HPV infection was detected in 31% of patients at 6-month follow-up and in 11.2% of patients, at 24-month follow-up. Younger women showed higher rate of recurrence than older ones. The risk of residual/recurrent infection did not correlate with cone-depth. Recurrence is higher in patients with low viral load level than in those having high load levels. mRNA test showed higher specificity and positive predictive value than the combination cytology-plus-HPV-DNA test. Conclusion: The inclusion of mRNA test within the current protocol of follow-up would efficiently and earlier predict the risk of residual/ recurrent cervical abnormalities after conisation. This molecular strategy would also reduce overtreatment, particularly in patients above 30 years of age.

The biomolecular follow up of Human Papilloma Virus (HPV) is widely investigated in patients treated for HPV related cervical lesions, since the HPV-mRNA test is more specific and have a higher positive predictive value for CIN2-3 in triage of high risk (HR) women and in follow-up of women treated for CIN2/3. Material & Methods: we investigated, during a 5 years' study, a cohort of patients divided in: group 1, patients at high risk for HPVinfections, and group 2, women diagnosed for CIN2/3, Cervicocarcinoma in situ (CIS) and Adenocarcinoma in situ (AIS) and surgically treated. The overall scheduled follow up was repeated each 6 months by: Pap Test, HPV-DNA test, m-RNA-HPV test and, in case of CIN2/3, CIS and AIS, also by colposcopy and biopsy. Results: The follow up involved a total of 203 women: 85 women with mRNA-HPV positive test and 118 patients surgically treated for CIN2/3, CIS and AIS. In the group 1, the long term follow up detected, after one year, 32 positive mRNA-HR HPV women and, of these, after more than 2 years, 37.5% developed CIN1 and 21.8% developed CIN2/3. Similarly, in the follow up of group two, women with abnormal Pap test showed positivity of mRNA HR-HPV in 71.4% of cases even after 6 months; 65% of these developed a CIN1 within 2.5 years and 20% had CIN2/3 after 2.3 years. Conclusions: Study results indicate either that patients with mRNA HR-HPV positive controls, on average, after 12 months are all at risk of progression to CIN1 and CIN2/3, or the higher specificity of mRNA-HPV test than Pap Test in follow up of surgical treated patients. This investigation confirmed a strong association between HR mRNA-HPV presence and risk of neoplastic progression.

We carried out a systematic review of HPV vaccine pre- and post-licensure trials to assess the evidence of their effectiveness and safety. We find that HPV vaccine clinical trials design, and data interpretation of both efficacy and safety outcomes, were largely inadequate. Additionally, we note evidence of selective reporting of results from clinical trials (i.e., exclusion of vaccine efficacy figures related to study subgroups in which efficacy might be lower or even negative from peer-reviewed publications). Given this, the widespread optimism regarding HPV vaccines long-term benefits appears to rest on a number of unproven assumptions (or such which are at odd with factual evidence) and significant misinterpretation of available data. For example, the claim that HPV vaccination will result in approximately 70% reduction of cervical cancers is made despite the fact that the clinical trials data have not demonstrated to date that the vaccines have actually prevented a single case of cervical cancer (let alone cervical cancer death), nor that the current overly optimistic surrogate marker-based extrapolations are justified. Likewise, the notion that HPV vaccines have an impressive safety profile is only supported by highly flawed design of safety trials and is contrary to accumulating evidence from vaccine safety surveillance databases and case reports which continue to link HPV vaccination to serious adverse outcomes (including death and permanent disabilities). We thus conclude that further reduction of cervical cancers might be best achieved by optimizing cervical screening (which carries no such risks) and targeting other factors of the disease rather than by the reliance on vaccines with questionable efficacy and safety profiles.

It will likely be more than 20 years before there is unequivocal evidence available that HPV vaccination decreases the incidence of invasive cervical cancer. However, existing data strongly suggests that as many as 440,000 cervical cancer cases and 220,000 deaths due to this malignancy will be prevented with the establishment of an effective worldwide HPV immunization program.

The identification of HPV as the cause of cervical cancer has changed the information required for surveillance. HPV prevalence and screening uptake worldwide are now necessary to describe the epidemiology of this cancer. In low-income countries cervical cancer incidence varies from low to very high levels and is strongly influenced by virus prevalence. In high-income countries, instead, incidence is low and little influenced by the virus prevalence, thanks to screening. The European Guidelines currently recommend Pap test every 3-5 years starting at age 22-30 and stopping at age 60-65. HPV testing is now limited to triage of borderline lesions? and to post-CIN follow up. Pilot studies evaluating HPV testing as primary test are recommended. Future governmental agency Guidelines in Europe will probably include HPV testing as viable for primary screening, with cytology triage for HPV-positive women (those positive to cytology will be directly referred to colposcopy, those negative will repeat HPV at one year. Persistence of HPV positivity will refer to colposcopy directly, clearance to regular screening). The interval after an HPV-negative test should be at least 5 years and starting age should be age 30/35. Pap test is still recommended for women under 30.

Persistent infection of High Risk (HR) Human papillomavirus (HPV) infection can lead to cervical cancer. The HPV genotypes are found worldwide, but important regional variations have been found. For a population-based HPV type prevalence study to assess the effect of existing and new prevention methods, frequently updated information on the burden of cervical cancer is essential. We evaluated the prevalence of HPV genotypes in a volunteer population screened for cervical cancer at the Local Health Unit (LHU) of Lecce. A total of 9,720 women were studied. The tests were performed by INNO-Lipa HPV Genotyping and LINEAR ARRAY HPV Genotyping Test. The overall HPV prevalence was 29.7% (95% CI, 28.8-30.6) for any HPV DNA. The prevalent type for all age groups was HPV 16 (7.4%; CI, 6.9-7.9) followed by HPV 31 (3.4%; CI, 3.0-3.7), 51 (3.0%; CI, 2.6-3.3), 52 (2.7%; CI, 2.3-3.0) and 58 (2.4%; CI, 2.1-2.7). HPV 53 was the most common low-risk HPV type with prevalence rate of 3.5 (CI, 3.1-3.8), followed by HPV 66 (3.0; CI, 2.6-3.3), 6 (2.9; CI, 2.6-3.2) and 42 (2.5; CI, 2.2-2.8). Multiple infections were present in 13.6% of HPV-tested women (CI, 12.9-14.3). Among these, the most common combination was of HPV 16 and HPV 52 genotypes. This study reports high prevalence of HPV infection and may serve as a valuable reference for assessing the impact of HPV vaccination programs. Furthermore, it supports the need for new vaccines that contain the most common HPV genotypes present in the population.

We estimated the frequency of detection of different human papillomavirus (HPV) types in women with atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesion (LSIL) cytology in a population-based primary screening programme. 247 liquid-based cytology (LBC) samples with ASCUS/LSIL results were tested using the LINEAR ARRAY HPV Genotyping Test (LA; Roche Diagnostics), which detects 37 HPV types. 79.4% of samples were positive by LA, and 60.7% were positive for high-risk HPV types (ASCUS: 41.2%; LSIL: 71.0%). 34 of the 37 HPV types included in LA were detected in our samples. HPV16 was detected in 18.6% of samples, and 66.8% of samples contained more than one HPV type, with a maximum of nine types observed in one LSIL sample. A random subset of 47 samples from the 247 samples tested by LA, was also analysed using the AMPLICOR HPV Test (Amplicor, Roche Diagnostics). A separate set of 42 samples, which were positive by LA for the five high-risk HPV types included in the PreTect HPV-Proofer (Proofer, NorChip AS), was also analysed for E6/E7 mRNA expression using Proofer. Concordance between LA and Amplicor was 91.5% (kappa=0.83). One false-negative and three false-positives were recorded for Amplicor, using LA as the “gold standard”. Concordance between LA and Proofer was 88.0%; 100% of Proofer samples that were HPV18- positive by LA, and 75.0% of HPV16-positive samples, expressed E6/E7 mRNA. In the present study using LBC samples in a triage situation, where negative predictive value is paramount, LA gave the most reliable results.

In recent years, targeted ultrasound contrast agents (UCA) have emerged with the rapid development of contrast-enhanced ultrasound technology and bio-nanotechnology, showing good prospects for applications in molecular imaging and targeted therapy. The non-invasive treatment modality can reduce the drug dosage, toxicity and side effects, providing a new kind of methodology and approach for treatments, which will greatly expand the research and its applications. Targeted UCA have been developed rapidly in recent years, and a variety of new UCA appear, such as multi-functional UCA, multi-modal UCA, UCA with double ligands or more ligands, long-circulating UCA and immune type UCA, which meet growing clinical and research needs. The novel UCA have made a great progress and play an increasingly important role in molecular imaging. In this article, we reviewed the concepts of ultrasound molecular imaging, and summarized the applications of targeted UCA in molecular imaging and targeted therapy.

Pyrrolo[2,1-f][1,2,4]triazine template, a unique bridgehead nitrogen heterocycle, certainly deserves the title of "privileged scaffold" in the drug discovery field because of the versatility and potential to yield derivatives with a wide range of biological activities, such as anti-anaplastic lymphoma kinase (ALK), Janus kinase 2 (JAK2), VEGFR-2, EGFR and/or HER2, Met kinase, p38α mitogenactivated protein (MAP) kinase and insulin-like growth factor receptor (IGF-1R) kinase activities, etc. These different biological properties of pyrrolo[2,1-f][1,2,4]triazine derivatives have motivated new studies in searching for novel derivatives with improved activity and also other applications in pharmaceutical field. However, no systematic review is available in the literature on the pyrrolo[2,1- f][1,2,4]triazine derivatives concerning the design of potent drug-like compounds. Owing to the importance of this heterocyclic system, the present paper is an attempt to the pharmacological activities, structural modifications and the structure-activity relationship (SAR) reported for bridgehead nitrogen heterocycles in the current literature, making an effort to highlight the importance and therapeutic potentials of the pyrrolo[2,1-f][1,2,4]triazine scaffold and its bridgehead nitrogen bioisosters as heterocyclic privileged medicinal scaffolds.