Periodic Reporting for period 1 - SixthSense (Early diagnosis of cervical cancer through the validation of Human Papilloma Virus viral load and expression of oncogenic viral proteins E6/E7 as risk indicators)
Reporting period: 2015-04-01 to 2015-09-30
Summary of the context and overall objectives of the project
Cervical cancer (CC) is the fourth leading cause of cancer deaths in women worldwide, causing more than 275.000 deaths annually. CC has been shown to be preventable, by means of population-based screening programmes and follow-up, and curable, if diagnosed and treated early. In countries where cervical screening programmes are in place, these have resulted in a marked decrease in cervical cancer incidence. The incidence of cervical cancer can be reduced by as much as 80% if the quality, coverage and follow-up of screening are high. It is now understood that persistent cervical infection with high-risk (hr) Human Papilloma virus (HPV) genotypes is necessary for the development of cervical cancer and its immediate precursor lesion (i.e. cervical intraepithelial neoplasia grade 3, CIN3). In particular, genotypes 16 (HPV16) and 18 (HPV18) are the most carcinogenic, accounting for approximately 60% and 15% of all cervical cancers respectively. Therefore, it’s now a fact that the HPV persistence of genotypes HPV16 and HPV18 can be very useful as predictive of the risk of developing cancer. In this respect, recent confirmations arrived from the latest results of the ATHENA trial: by using the FDA approved Cobas HPV test (Roche Diagnostics) they showed that 1-year and 2-year HPV persistence strongly predicts CIN3 or more severe diagnoses in the subsequent years (a 20%-30% risk of CIN3+ over 5 years) while the untreated CIN3 has a 30% probability of becoming an invasive cancer. hrHPV testing has been more recently introduced in population-based screening programmes for the prevention of cervical cancer, as hrHPV DNA tests have been demonstrated to improve sensitivity to over 95% for the detection of high-grade lesions, as compared to cervical cytology (Pap test), which had been long considered the gold standard in cervical cancer screening but which has a sensitivity as low as 50%. Furthermore, recently HPV testing of women’s self-taken vaginal samples has been advocated in order to further improve screening coverage. In this respect, the recent guidelines that concern the development of high quality HPV screening programmes will foster the replacement of Pap testing with the HPV testing as primary screening tool for CC screening.
However, the major drawback of using HPV testing as stand-alone in CC screening is due to its poor clinical specificity. This turns into extra avoidable costs for the National Health System (NHS) due to the unnecessary further investigations and medical follow-ups. In this current scenario, there is a strong need for novel biomarkers with improved clinical specificity able to:
• distinguish clinically relevant and irrelevant hrHPV infections in order to improve cervical screening programmes;
• reduce avoidable costs to the NHS, associated with unnecessary multiple sampling, further investigations and medical follow-ups;
• reduce anxiety and overtreatment associated with over-diagnosis in the vast majority of sexually active women with commonly occurring transient HPV infections.
The solution we envisaged relies on the collection of a single cervical sample which could undergo: a) first line screening for the qualitative detection of hrHPV DNA using highly sensitive nucleic acid amplification methods (i.e. Real time PCR); b) a second line screening using 2 more clinically specific second line biomarkers only for positive samples from step a). The second line test will be able to identify more accurately women with persistent hrHPV infection who are at a higher risk of developing cervical cancer.
In particular, the 2 novel biomarkers offer a measure of increased viral replication (viral load) and/or the production of viral oncogenic proteins (oncogenic transcripts) responsible for transforming cervical cell into CC by means of:
i) genotype-specific quantification of oncogenic/high risk HPV DNA (Viral Load) normalized to the number of cells present in the sample;
ii) genotype-specific detection of oncogenic transcripts (E6/E7 mRNA).
Furthermore, evaluation of cervical sample cellularity for viral load normalization would also allow avoiding false negative results associated with inadequate sample collection which could be responsible of failure during the follow up of women at increased risk of CC.
In this respect, the objectives of the SixthSense project Phase 1 were:
To design and implement an HPV viral load and oncogenic transcript diagnostic kit using the PathoFinder proprietary technology, to allow sensitive genotype-specific Real Time PCR based multiplex assays development;
To determine genotype-specific viral load cut-off values for the quantitative Real time PCR assays which will enable to improve clinical specificity of the biomarker distinguishing infections likely to progress to pre-cancerous and cancerous lesions from transient ones;
To analytically and clinically validate the biomarker and the associated analytical device through a multicentre screening study.
However, the major drawback of using HPV testing as stand-alone in CC screening is due to its poor clinical specificity. This turns into extra avoidable costs for the National Health System (NHS) due to the unnecessary further investigations and medical follow-ups. In this current scenario, there is a strong need for novel biomarkers with improved clinical specificity able to:
• distinguish clinically relevant and irrelevant hrHPV infections in order to improve cervical screening programmes;
• reduce avoidable costs to the NHS, associated with unnecessary multiple sampling, further investigations and medical follow-ups;
• reduce anxiety and overtreatment associated with over-diagnosis in the vast majority of sexually active women with commonly occurring transient HPV infections.
The solution we envisaged relies on the collection of a single cervical sample which could undergo: a) first line screening for the qualitative detection of hrHPV DNA using highly sensitive nucleic acid amplification methods (i.e. Real time PCR); b) a second line screening using 2 more clinically specific second line biomarkers only for positive samples from step a). The second line test will be able to identify more accurately women with persistent hrHPV infection who are at a higher risk of developing cervical cancer.
In particular, the 2 novel biomarkers offer a measure of increased viral replication (viral load) and/or the production of viral oncogenic proteins (oncogenic transcripts) responsible for transforming cervical cell into CC by means of:
i) genotype-specific quantification of oncogenic/high risk HPV DNA (Viral Load) normalized to the number of cells present in the sample;
ii) genotype-specific detection of oncogenic transcripts (E6/E7 mRNA).
Furthermore, evaluation of cervical sample cellularity for viral load normalization would also allow avoiding false negative results associated with inadequate sample collection which could be responsible of failure during the follow up of women at increased risk of CC.
In this respect, the objectives of the SixthSense project Phase 1 were:
To design and implement an HPV viral load and oncogenic transcript diagnostic kit using the PathoFinder proprietary technology, to allow sensitive genotype-specific Real Time PCR based multiplex assays development;
To determine genotype-specific viral load cut-off values for the quantitative Real time PCR assays which will enable to improve clinical specificity of the biomarker distinguishing infections likely to progress to pre-cancerous and cancerous lesions from transient ones;
To analytically and clinically validate the biomarker and the associated analytical device through a multicentre screening study.
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far
The aim of the SixthSense business idea will be to validate a diagnostic algorithm for HPV screening to detect the presence of oncogenic/high-risk types HPV DNA, and to measure their viral load and expression of oncogenic viral proteins E6/E7, to be used as risk indicators (biomarkers) for the development of cervical cancer. The approach proposed and developed along the Phase 1 project is based on the combination of the technology and expertise coming from the two main partners:
• PathoFinder B.V.: the SME coordinator of the project and proprietary of the SMARTFinder technology that is the ground for the assay development.
• Hiantis: owner of the European Patent (EP 2150629) describing the new biomarkers proposed within a novel diagnostic algorithm for first line and second line HPV testing with the participation of Clementina Cocuzza, Associate Professor of Clinical Microbiology at University of Milano Bicocca, as patent’s inventor and member of Hiantis.
The initial HPV test developed by PathoFinder is the PapillomaFinder® SMART 20 assay. This assay is based on multiplex ligation-dependent probe amplification (MLPA), a detection method which allows high multiplexing, and uses Single tube Multiplex Amplification in Real Time (SMART) to distinguish reaction products by fluorescent melt curve analysis. This method can be performed on a standard real-time PCR platform. The assay detects and distinguishes the E6 genes of hrHPV types 16, 18, 31, 33, 35, 39, 45, 52, 56 and 58, which together account for >95% of CC. The hrHPV types 51, 59, 66 and 68, causing less than 3% of the cervical cancer cases are detected in a hr-pool and the low risk (lr) HPV types 6, 11, 40, 42, 43 and 44 in a lr-pool . Human β-globin is detected to monitor sampling efficiency and DNA quality.
The assay was tested on plasmid models, proficiency panels and cytological samples (the latter in comparison with a clinically validated quantitative real-time PCR) and found to be highly sensitive, specific, rapid and easy to use. Post-market information of the SMARTFinder method showed that the high multiplexing capacity was much appreciated, but also showed the need for reduced hands-on time and time-to-result. In addition, the SMARTFinder protocol is not easily incorporated into unidirectional PCR workflow that many Molecular Diagnostic laboratories adhere to. Therefore, the 2SMART method was developed, which has the following advantages over the SMARTFinder method:
o a reduction of the time-to-result (2 ½ hrs, versus 4 hrs for the original SMARTFinder);
o a reduction of the hands-on-time because the hybridization and ligation steps are omitted;
o a smooth incorporation into a unidirectional workflow because all the reaction mixes used within one working day can be prepared in the morning in the pre-PCR environment.
Given the abovementioned advantages of the 2SMART method, the assay to be developed in the context of the SixthSense project will therefore be based on this method.
• PathoFinder B.V.: the SME coordinator of the project and proprietary of the SMARTFinder technology that is the ground for the assay development.
• Hiantis: owner of the European Patent (EP 2150629) describing the new biomarkers proposed within a novel diagnostic algorithm for first line and second line HPV testing with the participation of Clementina Cocuzza, Associate Professor of Clinical Microbiology at University of Milano Bicocca, as patent’s inventor and member of Hiantis.
The initial HPV test developed by PathoFinder is the PapillomaFinder® SMART 20 assay. This assay is based on multiplex ligation-dependent probe amplification (MLPA), a detection method which allows high multiplexing, and uses Single tube Multiplex Amplification in Real Time (SMART) to distinguish reaction products by fluorescent melt curve analysis. This method can be performed on a standard real-time PCR platform. The assay detects and distinguishes the E6 genes of hrHPV types 16, 18, 31, 33, 35, 39, 45, 52, 56 and 58, which together account for >95% of CC. The hrHPV types 51, 59, 66 and 68, causing less than 3% of the cervical cancer cases are detected in a hr-pool and the low risk (lr) HPV types 6, 11, 40, 42, 43 and 44 in a lr-pool . Human β-globin is detected to monitor sampling efficiency and DNA quality.
The assay was tested on plasmid models, proficiency panels and cytological samples (the latter in comparison with a clinically validated quantitative real-time PCR) and found to be highly sensitive, specific, rapid and easy to use. Post-market information of the SMARTFinder method showed that the high multiplexing capacity was much appreciated, but also showed the need for reduced hands-on time and time-to-result. In addition, the SMARTFinder protocol is not easily incorporated into unidirectional PCR workflow that many Molecular Diagnostic laboratories adhere to. Therefore, the 2SMART method was developed, which has the following advantages over the SMARTFinder method:
o a reduction of the time-to-result (2 ½ hrs, versus 4 hrs for the original SMARTFinder);
o a reduction of the hands-on-time because the hybridization and ligation steps are omitted;
o a smooth incorporation into a unidirectional workflow because all the reaction mixes used within one working day can be prepared in the morning in the pre-PCR environment.
Given the abovementioned advantages of the 2SMART method, the assay to be developed in the context of the SixthSense project will therefore be based on this method.
Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)
There are approximately 1.6 billion women globally who should be screened for cervical cancer, but only 10% -14% of them are tested and the bulk of these tests are performed within the developed countries of the world. Obtaining widespread coverage of the target population at the correct screening interval is achieved most easily through organized, as opposed to opportunistic, screening programs. Despite the schema adopted for screening, the current guidelines for cervical cancer prevention recommend performing a cytology test (i.e. the Pap test) from a starting age of 25-30 years, repeated with a time interval of 3-5 years. The further follow-ups usually depend on the level of severity that is recognized from the cellular sample observation:
• Negative result: no alterations are evaluated and the woman is sent back to routinely screening.
• Low severity infections (L-SIL) and ASC-US (Atypical Squamous Cell-Undermined Significance): the patient can be recalled for repeating the Pap test (within 6-12 months) or referred directly to colposcopy.
• Moderate to high severity infections (CIN2, CIN3): the patient is subjected to biopsy and medical treatment with further follow-ups.
The key factor that affects the performance of cervical cancer screening is that HPV infections are very common in sexually active women and very often they spontaneously regress within 1-2 years (approximately 90% of them are transient). To this respect, the increased understanding of the association between HPV and cervical cancer risk has led to the development of molecular tests for a correct HPV evaluation. Incorporation of HPV testing into cervical cancer screening strategies has the potential to allow both increased disease detection (improving benefits) and increased length of screening intervals by decreasing harms such as: a) the psychosocial impact of screening positive, b) additional clinical visits and procedures, c) and treatment of lesions destined to resolve.
Given the current scenario, we want to evaluate the business opportunity that the SixthSense project will represent through the development and clinical validation of a real-time PCR assay able to increase the sensitivity of the screening performance (through the viral load evaluation) and improving the capability to detect earlier the HPV infections thanks to the E6/E7 evaluation and viral load determination. In fact, the SixthSense value proposition is to ensure timely and accurate HPV screening, early identifying those cases of HPV infection that will ultimately lead to cervical cancer, through novel biomarkers based on the measurement of the HPV viral load, oncogenic transcripts and the relating analytical device. These results will be made possible through the validation of an assay based on:
A first line screening, aimed at the rapid and “low cost” (< 30€/test) viral DNA detection, to determine the presence of one or more of hrHPV genotypes in the sample, combined with the human CC-Chemokine 5 (CCR5) gene quantification to check for sample quality (to avoid false negative results), and to allow subsequent normalization of viral load in the case of hrHPV DNA positivity at screening.
Two combined second line biomarkers assays to be applied to all positive samples (at first line screening), aiming at determining whether hrHPV infection detected is clinically relevant by evaluating the normalized hrHPV viral load and the presence of HPV’s oncogenic transcripts E6/E7 based on the European Patent EP 2150629, and combined with PathoFinder proprietary technology.
Thanks to this approach we will have longer time interval of screening (thanks to high sensitivity) and a reduced number of false positives (thanks to higher specificity) that will all contribute to reduce the cost for National Health systems (less unnecessary follow-ups mainly).
In addition, as there is a great need for a low cost, easy to use cervical cancer screening test in the emerging markets, where significant numbers of women die each year from cervical cancer, one of the competitive advantage of the SixthSense assay will derive from the clinical validation for self-collecting samples. In fact, in emerging countries only 14% of the target population receives any screening given the lack of necessary laboratory infrastructures, and the possibility to reach the non-attendees will help to decrease the mortality due to cervical cancer thanks to the early and more diffused prevention.
The SixthSense solution will turn into the following advantages for the stakeholders involved in cervical cancer diagnosis and treatment:
• Advantages for the patients: Achievement of a complete diagnostic algorithm comprising hrHPV tests whose positive results are informative about the presence or absence of CIN2+, will allow women to undergo only one gynaecological sampling/visit for both first line and second line testing, as well as avoiding unnecessary and excessive follow up procedures. This procedure will avoid worry and anxiety as well as unpleasant and embarrassing medical tests for women who are likely to have transient hrHPV infections or hrHPV positive women without cervical lesions. The possibility of the new hrHPV test prototype to check for sample quality will also favour the now more widely accepted practice of self-sampling.
• Advantages for the clinical laboratories: Diagnostic laboratories will be processing only one cervical sample in order to perform screening, followed up by second line testing only in case of positivity. This will reduce time and costs.
• Advantages for the National Healthcare Systems: The National Healthcare Systems will benefit from the use of such system by reducing costs of unnecessary follow up visits and of more invasive investigations. Gynaecologists will also be favoured in the management of patients with low-grade dysplasia, as well as saving clinical practice time by being able to reassure women with clinically irrelevant infection. Moreover it is believed that the use of more clinically specific biomarkers of risk may encourage HPV testing, as clinicians are presently faced with having to explain to anxious HPV positive women the results of test of debatable clinical value.
• Negative result: no alterations are evaluated and the woman is sent back to routinely screening.
• Low severity infections (L-SIL) and ASC-US (Atypical Squamous Cell-Undermined Significance): the patient can be recalled for repeating the Pap test (within 6-12 months) or referred directly to colposcopy.
• Moderate to high severity infections (CIN2, CIN3): the patient is subjected to biopsy and medical treatment with further follow-ups.
The key factor that affects the performance of cervical cancer screening is that HPV infections are very common in sexually active women and very often they spontaneously regress within 1-2 years (approximately 90% of them are transient). To this respect, the increased understanding of the association between HPV and cervical cancer risk has led to the development of molecular tests for a correct HPV evaluation. Incorporation of HPV testing into cervical cancer screening strategies has the potential to allow both increased disease detection (improving benefits) and increased length of screening intervals by decreasing harms such as: a) the psychosocial impact of screening positive, b) additional clinical visits and procedures, c) and treatment of lesions destined to resolve.
Given the current scenario, we want to evaluate the business opportunity that the SixthSense project will represent through the development and clinical validation of a real-time PCR assay able to increase the sensitivity of the screening performance (through the viral load evaluation) and improving the capability to detect earlier the HPV infections thanks to the E6/E7 evaluation and viral load determination. In fact, the SixthSense value proposition is to ensure timely and accurate HPV screening, early identifying those cases of HPV infection that will ultimately lead to cervical cancer, through novel biomarkers based on the measurement of the HPV viral load, oncogenic transcripts and the relating analytical device. These results will be made possible through the validation of an assay based on:
A first line screening, aimed at the rapid and “low cost” (< 30€/test) viral DNA detection, to determine the presence of one or more of hrHPV genotypes in the sample, combined with the human CC-Chemokine 5 (CCR5) gene quantification to check for sample quality (to avoid false negative results), and to allow subsequent normalization of viral load in the case of hrHPV DNA positivity at screening.
Two combined second line biomarkers assays to be applied to all positive samples (at first line screening), aiming at determining whether hrHPV infection detected is clinically relevant by evaluating the normalized hrHPV viral load and the presence of HPV’s oncogenic transcripts E6/E7 based on the European Patent EP 2150629, and combined with PathoFinder proprietary technology.
Thanks to this approach we will have longer time interval of screening (thanks to high sensitivity) and a reduced number of false positives (thanks to higher specificity) that will all contribute to reduce the cost for National Health systems (less unnecessary follow-ups mainly).
In addition, as there is a great need for a low cost, easy to use cervical cancer screening test in the emerging markets, where significant numbers of women die each year from cervical cancer, one of the competitive advantage of the SixthSense assay will derive from the clinical validation for self-collecting samples. In fact, in emerging countries only 14% of the target population receives any screening given the lack of necessary laboratory infrastructures, and the possibility to reach the non-attendees will help to decrease the mortality due to cervical cancer thanks to the early and more diffused prevention.
The SixthSense solution will turn into the following advantages for the stakeholders involved in cervical cancer diagnosis and treatment:
• Advantages for the patients: Achievement of a complete diagnostic algorithm comprising hrHPV tests whose positive results are informative about the presence or absence of CIN2+, will allow women to undergo only one gynaecological sampling/visit for both first line and second line testing, as well as avoiding unnecessary and excessive follow up procedures. This procedure will avoid worry and anxiety as well as unpleasant and embarrassing medical tests for women who are likely to have transient hrHPV infections or hrHPV positive women without cervical lesions. The possibility of the new hrHPV test prototype to check for sample quality will also favour the now more widely accepted practice of self-sampling.
• Advantages for the clinical laboratories: Diagnostic laboratories will be processing only one cervical sample in order to perform screening, followed up by second line testing only in case of positivity. This will reduce time and costs.
• Advantages for the National Healthcare Systems: The National Healthcare Systems will benefit from the use of such system by reducing costs of unnecessary follow up visits and of more invasive investigations. Gynaecologists will also be favoured in the management of patients with low-grade dysplasia, as well as saving clinical practice time by being able to reassure women with clinically irrelevant infection. Moreover it is believed that the use of more clinically specific biomarkers of risk may encourage HPV testing, as clinicians are presently faced with having to explain to anxious HPV positive women the results of test of debatable clinical value.