Final Report Summary - PIPAVIR (Detection of persistent infections by human papillomaviruses)
Executive Summary:
Early detection of cervical precancerous lesions is a prerequisite for successful secondary prevention of cervical cancer. To improve current technology used for the clinical diagnosis of cervical precancer, the PIPAVIR consortium developed two test systems based on an ELISA and a Rapid test in order to detect E7 oncoproteins of 12 major high-risk HPV-types in cervical smears. Both tests have been clinically evaluated for future use in patient screening, triage and therapy monitoring. In both cases industrial prototypes have been developed, which were used for the clinical evaluation. The available data suggest that the hrE7 ELISA has sufficient specificity and sensitivity to detect patients with ongoing disease in the 5 studies mentioned above. Concerning the E7 Rapid-test, the available data suggest that further improvements of this test format will be required before clinical use can be considered. During the project lifetime, the concept and progress of the PIPAVIR project has been broadly disseminated to opinion leaders, the scientific community and the general public. In conclusion, the PIPAVIR project has generated a solid basis for commercial exploitation of the hrE7 ELISA, which is expected to take place in the next couple of years.
Project Context and Objectives:
Early detection of cervical pre-cancerous lesions is a prerequisite for successful secondary prevention of cervical cancer. Current technology used in the clinical setting to achieve this goal suffers from the problem, that conventional cytological diagnostic tests are imprecise and characterized by a large rate of false-positive and false-negative results. On the other hand, molecular detection of HPV DNA, considered as an alternative diagnostic regime in several countries, has the inherent problem that the presence of a high-risk HPV DNA in a clinical specimen cannot discriminate between transient infections (in the majority of cases) and ongoing tumorigenesis in a given patient. To improve clinical diagnosis of cervical pre-cancer, the PIPAVIR consortium developed two test systems, based on an ELISA and a rapid test in order to detect E7 oncoproteins of 12 major high-risk HPV types in cervical smears. The key objectives were to clinically validate a novel ELISA test for patient screenings, triage and therapy monitoring in a clinical setting, to develop and validate a rapid test for the detection of E7 in cervical smears and to produce two separate diagnostic kits based on the results of this work. The project started with experiments aimed at assay optimisation, i.e. to improve the sensitivity and specificity of pre-existing laboratory formats. Another important objective was to adapt the hrE7 ELISA to routine laboratory requirements and perform a technical validation. The next objective was to launch prototypes for the hrE7 ELISA and the hrE7 rapid test IVDs. These prototypes were used to measure E7 levels in clinical samples that were generated in five separate clinical studies, aiming at a proof of concept that detection of E7 is suitable for improved diagnosis in the context of a screening population, as a triage test, and as test of cure for cervical cancer patients. The results obtained with the technical procedures described above have been and will be used to broadly disseminate the information about the new diagnostic concept and actual progress in its realisation in the scientific community and in the general public. This is very important to prepare key opinion leaders for the adaptation of the new diagnostic technology in cervical cancer early detection.
A sandwich ELISA test was developed, based on proprietary rabbit monoclonal antibodies and goat polyclonal antibodies, with the potential to recognize E7 proteins of the most important 12 high-risk HPV types. Currently, two different formats are available: On the one hand, three different measurements are performed per clinical sample to separately asses the concentration in cervical smears of E7 proteins from three groups of hrHPV viruses, based on immunological cross-reactivity of the respective E7 proteins. In a second attempt, a sandwich ELISA for the simultaneous detection of all E7 proteins in a single measurement has been generated. Both formats differ to some extent in their analytical precision and sensitivity and we envisage that final clinical validation will provide a basis for the selection of the final setting of the ELISA test. In terms of the E7 rapid test, the first test that has been developed is focused on the E7 proteins of HPV 16, HPV 18 and HPV 45, which are the clinically most relevant HPV types. Here a detection system has been developed and technically validated. For both the ELISA and the rapid test, commercial prototypes have been produced. Due to some unexpected problems with primary biological materials and the production of rabbit monoclonal antibodies, the completion of the prototypes has been delayed in both cases. At the clinical sites contributing to PIPAVIR, the collection of the cervical smears for the five clinical studies mentioned above has been completed and E7 protein levels have been determined in all samples. A major achievement at the analytical laboratory was the development of a protocol which allows to perform E7 tests on cervical swabs prepared by the method of liquid based cytology (LBC), a modern and wide-spread sample collection tool with many advantages. The development of a LBC-compatible ELISA SOP is considered a major break-through concerning the implementation of E7 ELISA in clinical routine, based on successful clinical validation. We performed all the measurements with the prototype diagnostic kits as foreseen. Preliminary analysis of the available data provided clinical proof-of-concept for PIPAVIR.
After successful validation of the clinical prototypes we expect to have two clinically validated E7 detection systems which can be used for significantly improved detection of pre-cancerous lesions of the cervix uteri in routine screening as well as in clinical management of patients with high-grade cervical lesions. The here described in vitro diagnostic kits along with the scientific technical evidence for their clinical validation, will provide the basis for a significant global improvement of cervical cancer screening and the diagnosis of cervical pre-cancers. The availability of these validated tests will contribute significantly to the quality of life of women who are enrolled in screening programmes and/or effected by high-grade cervical lesions and boost the economic capacities of the participating SME partners.
Project Results:
4.1.3 Description of the main S&T results/foregrounds
4.1.3.1 Overview
In Work Package 1 (WP1) the hrE7 ELISA was optimized by using coating with different RabMab concentrations, in combination with polyclonal goat antibodies used for detection (Task 1.1). This was followed by the transfer of the hrE7 ELISA protocol to the clinical scale. Here, a major achievement is the development of a protocol, which allows to carry out the ELISA on material collected by liquid based cytology (LBC), which will make the final in vitro diagnostic device very attractive for clinicians all over the world (Task 1.2). This was followed by a technical validation of the hrE7 ELISA, using cervical carcinoma cells and tumour biopsies (Task 1.3). This resulted in the establishment of a hrE7 ELISA, which was technically validated for the detection of 12 hrE7 proteins (see Figure 1), and hence a timely and successful conclusion of WP1.
In Work Package 2 (WP2), a hrE7 rapid test has been set up, which allows the detection of E7 proteins of the three most relevant HPV types, HPV 16, HPV 18 and HPV 45 (Task 2.1) and this hrE7 rapid test has been technically validated as foreseen (Task 2.2). Although the work was slightly delayed into Period 2. The development of the self-sampling test has been postponed, due to a delay experienced in the development of prototype test kits (see below, WP3).
In Work Package 3 (WP3), prototypes have been launched for the hrE7 ELISA as well as the hrE7 rapid test (Task 3.2). The development of the industrial prototype test kits required the availability of several independent batches of all biological materials to be used. Here a bottleneck was the production of the rabbit monoclonal antibodies. Initially, it was thought that these monoclonal antibodies can be produced by recombinant overexpression of cDNAs encoding the relevant rabbit immunoglobulins. However, this procedure yielded unsatisfactory results and the consortium decided to refer to the classical production of monoclonal antibodies from hybridoma cell lines. This step turned out to be more complex than expected, since the yield of rabbit monoclonal antibodies produced by hybridoma was found to be three to five fold lower as compared to mouse monoclonal antibodies. In addition, the establishment of the hybridoma cell lines by a commercial supplier took longer than expected. As a result of these, the development of the prototypes has been delayed by approximately 6 months.
In Work Package 4 (WP4) the preparation of the clinical study including the preparation of study documents, as well as teaching doctors and nurses (Task 4.1) has been performed as foreseen. In addition, the collection of samples has been completed for the test of the hrE7 ELISA prototype (Task 4.2) as well as the sample collection for the test of the hrE7 rapid test prototype (Task 4.3). It should be noted, that the delayed development of the prototype had no direct impact on the timing of the clinical WPs (WP4-WP6). A protocol has been established, that allows the retrospective sampling of cervical smears due to the development of a technology that can use liquid based cytology samples (of note, it was established in WP1 that liquid based cytology samples can be stored for several years without any loss of signal). The HPV screening study has been performed with 1.600 samples obtained from 1.311 women. Experiments to validate the hrE7 rapid test with self-sampling have been performed with 574 samples from 384 women.
In Work Package 5 (WP 5), the E7 tests were validated as triage test. Collection of clinical samples for validation of the E7 test for triage of 281 hrHPV positive women (Task 5.2) as well as for the validation of E7 tests for triage of 181 women with abnormal Pap-test (Task 5.3) have been completed; E7 protein content in all samples was measured with the prototype hrE7 ELISA and the prototype hrE7 rapid test.
In Work Package 6 (WP6) the E7 tests have been validated as tests of cure. The validation of E7 tests for test of cure in patients with HSIL (Task 6.2) and validation of the E7 tests as a test of cure in cervical cancer patients (Task 6.3) has been completed on samples from a total of 220 patients.
Work in Work Package 7 (WP7) has been performed as foreseen. Dissemination of the concept, progress and results of PIPAVIR has been performed via classic media (Task 7.3) and by presentations on scientific meetings (Task 7.4). For reasons of IPR protection the consortium has so far not published any detailed project results in scientific journals, because such publications would potentially jeopardize protection of intellectual property rights. P4 and P5 collaborated in existing initiatives to promote women’s health and started the development of recommendations for physicians (Task 7.5 Task 7.6). The long-term success will critically depend on the validation of the assay with the finalized prototypes. Similarly, the exploitation activities including preparation for EC approval and product launch (Task 7.8 Task 7.7) have been initiated.
In the following section, a detailed description of work performed in WP1 through WP7 is provided.
4.1.3.2. Detailed description of Work
Work Package No: WP1
Plan Start: M01
Plan End: M12
Lead Participant: MIKROGEN
Actual Start: M01
Actual End: M12
Work package title: Final steps of hrE7-ELISA development
Activity Type: Research activities
Participants involved: UIBK, MIKROGEN, BIOSYNEX, CHARITE
Project objectives for the period
• Assay optimization: improve sensitivity and specificity
• Adaption of hrE7-ELISA to routine lab requirements
• Technical validation of the hrE7-ELISA assay
Work progress and achievements during the period
Background information
High sensitivity is crucial for the detection of low levels of cellular E7 proteins in lysates of cervical smears. The current E7-sandwich-ELISA is based on the coating of a mixture of four different RabMabs as capture antibodies, and a mixture of two goat polyclonal antibodies as detection antibodies. This combination was shown in preliminary experiments by P1 to recognize E7 proteins of 12 hrHPV types (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59) with a detection limit of 1 picogram E7 protein or less, providing sufficient sensitivity. However, additional experiments are warranted to find the optimal configuration of RabMabs as capturing antibodies. Moreover, it is known that the transfer of new diagnostic tests from lab scale to industrial scale requires a set of adaptation measures, which are in the focus of this WP.
Task 1.1: hrE7-ELISA optimization (month 1-12)
Goal: P1 and P2 will further optimize the existing hr E7-ELISA test by coating up to ten different RabMab combinations at various concentrations. Modifications of incubation conditions and reagents will be tested to improve technical performance in terms of specificity and sensitivity, using recombinant E7 proteins. Different RabMabs chosen for optimal capture performance and subsequent detection will be combined in a way that E7 proteins of the 12 high-risk HPV genotypes can be detected with comparable sensitivity.
Result: After detailed characterization of 16 different RabMabs and 3 polyclonal goat antibodies, two different strategies for detection of 12 hr E7 proteins were developed by P1 and P2, as outlined below. Whereas the detection system of P1 contains three different kinds of wells (i.e. well 1: detection of 16, 18, and 45 E7; well 2: detection of 39, 51, 56, and 59 E7; and well 3: detection of 16, 31, 33, 35, 52, and 58 E7), the strategy of P2 is based on the combination of one screening well, which detects 12 hr E7 types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 E7) and a well for detection of the HPV types with highest carcinogenicity (16, 18, and 45 E7). Exemplary results of Task 1.1 are provided in Figure 1.
Additionally, different types of samples (cervical sample swabs in different transportation systems / buffers) were tested to ensure an easy and common procedure of sampling, transportation, and storage of the samples. Here is to emphasize that the usage of samples stored in ThinPrep™ was enabled by development of a lysis protocol by P2, which is based on a buffer system provided by P3. Advantages of the ThinPrep system are the following: it is a common and well known system used by gynecologists, the samples can be shipped at ambient temperature, stored at 4°C and the stability of a surrogate (cervix carcinoma cell lines such as HeLa and Caski) was shown to be greater than 12 months. Hence, nearly all kinds of analyses (PCR, histology and ELISA testing) scheduled for the clinical studies of the PIPAVIR project can be done from one and the same sample.
Task 1.2: hrE7-ELISA transfer to clinical scale (ongoing)
Goal: P2 will transfer assay design from laboratory scale to clinical scale, i.e. further modifications of assay design will be tested to assure compliance with routine laboratory requirements, such as assay robustness, reproducibility of assay
results and stability of assay components.
Results: The entire assay protocol was tested and optimized. To receive highest possible specificity and sensitivity, different types of conjugates, substrates, buffers and plates were tested. Also, the concentrations of all components were titrated to assure optimal assay performance (ongoing process due to current production of the RabMabs and goat antibodies). Furthermore, a buffer system of P2 was adapted to the assay to achieve least possible unspecific binding and maximum stability of the assay. Stability was shown by stress tests at 37°C up to 6 weeks for all antibody containing components. Also, robustness regarding incubation times and temperatures was checked. Reproducibility was shown by repetition of experiments with at least two operators and performance of the assays at the two development sites (test of the P1 assay strategy at P2 and vice versa, see also Figure 1).
Task 1.3: technical validation of hrE7-ELISA (ongoing)
Goal: For technical validation, E7 RabMabs will be used to stain 30 established cancer biopsies by immunohistochemistry with E7 antibodies (P1). 30 biopsies obtained from hysterectomies (performed due to benign diseases AND HPV DNA-negative) will be used as negative control. In addition, tissue of 30 HPV typed and fully diagnosed cancers will be lysed and lysates will be measured for E7 content. Clinical material will be provided by P4 and P5.
Results: For technical validation, cervical samples were collected at P4 and P5. Partner 4 produced cell line lysates and collected clinical samples (>800) of patients with known clinical status for technical validation of the hrE7-ELISA assay. These samples were not part of the clinical studies but characterized by PCR for HPV-positivity and histologically for progression of the disease. Measurement of the samples showed good correlation between the ELISA result and the status of the disease, i.e. HPV positive and Pap IVa and higher grades for Cytology classified samples were positive for hrE7 protein in the ELISA. Interestingly, measurement of one HPV-PCR negative but histologically abnormal sample resulted in a positive signal for expression of hr E7 protein in the ELISA test. One explanation could be that by integration of the viral DNA into the host genome the PCR target gene was possibly deleted. On the contrary, one HPV-PCR positive but Cytology negative sample gave a positive result in hr E7 protein detection. Hence, the advantage of E7 protein detection became obvious since patients were identified which are not found by testing with PCR or cytology alone.
Deviations from the work plan:
During optimization of the assay it became clear that the critical biological material was not available in quantity and quality as needed. Hence, immunization of goats for polyclonal antibodies was mandatory as well as an industrial production of the RabMabs. The immunization of the animals for production of the polyclonal antibodies was finalized in November 2013 with a final bleeding. Purification and modification of the antibodies (i.e. biotinylation and/or direct conjugation of HRP) was completed in May 2014. For production of the RabMabs, in a first step an evaluation study of the hybridomas was performed (from February to September 2013), subsequently followed by production of two batches of each RabMab in industrial scale (completed f April 2014).
Work Package No: WP2
Plan Start: M01
Plan End: M12
Lead Participant: BIOSYNEX
Actual Start: M01
Actual End: M24
Work package title: Development of the hrE7-Rapid test
Activity Type: Research activities
Participants involved: UIBK, BIOSYNEX, CHARITE
Objectives:
The objectives were to develop, manufacture and validate the performances of the hrHPV-E7 Rapid Test. The aim was to have two test lines: the first one to detect three hrHPV types (HPV-16,-18,-45) and the second one to detect the nine other hrHPV types (HPV-31, -33, -35, -39, -51, -52, -56, -58, -59).
Progress of work:
The work package 2 was divided in three tasks.
Task 2.1 : Setup of hrE7-Rapid test (month 1-6):
Month 1-15: selection of antibodies and test format
The development began with the selection of antibodies for the detection of hr-HPV. Twenty-six different antibodies were tested: twenty-one monoclonal anti HPV-E7 (designated as RabMab) and five polyclonal anti HPV-E7 (designated as Goat). The RabMab Klon 42-3 and 143-7 gave the best performances and were selected to continue the development. Both antibodies were conjugated with various labeling: gold particles, biotine, latex particles, HRP-magnetic particles. Gold particles were selected because they gave the best sensitivity. The polyclonal anti HPV-E7 (Goat 1 and Goat 2) are sprayed on the membrane in the single test line.
An “uncommon” immunochromatography format was selected. A basic rapid test is divided in three parts: the lower part composed of a sample and a conjugate pad, the middle part of a membrane and the upper part of an absorbent pad. Antibodies conjugate with gold particles are usually sprayed and dried on the conjugate pad. In our selected format a liquid one is used instead of a dried one.
The liquid gold conjugate particle is added to the sample prior to its migration on the test. This conjugate/ sample incubation time increases the sensitivity but will probably also reduce the shelf life of the rapid test.
Month 1-6: selection of membrane
Three membranes with different porosity were tested: 5 µm, 8 µm and 15 µm. The membrane regulates the sample migration flux. The porosity of 8 µm was selected: the contact time between test line and the sample is optimum on the recombinant antigens.
Month 11-18: sample buffer formulation
The rapid test buffer is divided in two parts: buffer A and buffer B. The vaginal cervical sample taken on a brush is first put in buffer A which is very corrosive and lyses the cells. The buffer B is then added to neutralize the pH.
The main difficulty of cervical samples is their heterogeneous viscosities. Some samples didn’t migrate totally on the rapid test membrane. The sample buffer B formulation had to be improved.
This point had been discussed at the Munich meeting of August 2013. Due to the time needed to improve the sample buffer formulation, it was decided to start the sampling with the standard buffer used for PCR and PAP testing (named Thinprep buffer). The Thinprep buffer is composed of chemicals which conserves the cells. Therefore, a centrifugation step has to be performed to remove the buffer and concentrate the cells. The use of Thinprep buffer was validated on rapid test. However this option is not user-friendly as it is not correlated with the usual rapid testing which is to be a point of care technique with little to no equipment.
The adding of two chemicals into buffer B improved the sample migration. This new formulation is currently validated on further clinical samples.
It was also decided that the development of the second test line detecting the nine others hrHPV was postponed.
Month 18-24: sensitivity improvement
Discussion with the other partners pointed to a possible lack of sufficient sensitivity. Decision was therefore made to add a secondary conjugate detecting the RabMab conjugate with Goat anti-Rabbit antibodies coupled to gold particles. This secondary conjugate is added immediately before adding the test strip into the conjugate/sample mixture.
Running time has also been increased to 30 minutes, further improving the sensitivity.
Task 2.2: Technical validation of hrE7-Rapid test (month 6-12):
Month 18-24: internal technical validation on improved test version, prototype production and clinical study samples testing
Determination of new detection limits were performed to validate the use of the new, improved, protocol for the clinical study.
Two batches were produced for this purpose: one used for the 1srt smears and one used for the second smears.
In total, 4491 samples were tested.
Task 2.3: Development of self-sampling test (month 6-12):
The Delphi sample buffer was not tested on hrE7-R rapid test
Results: A rapid test prototype detecting HPV-16, -18 and -45 was produced in March 2014.
During the development, the sensitivity and specificity of the hrE7-Rapid test for detection of HPV-16, -18 and -45 E7 were determined on recombinant E7 proteins and cervical cancer cell lines. The result are resumed in the table in the attachement
Preliminary-stability results: R&D prototype was stable six months at +4°C and room temperature. However, a decrease of signal was observed for HPV16-E7. At the end of the project R&D prototype (1rst version) stability was of 16 months at a storage temperature of 2°C.
Work Package No: WP3
Plan Start: M12
Plan End: M36
Lead Participant: MIKROGEN
Actual Start: M12
Actual End: M36
Work package title: Launch of the PIPAVIR prototype kits
Activity Type: Demonstration
Participants involved: MIROGEN, BIOSYNEX, CHARITE, AUTH
Project objectives
• Prototype launch for the hrE7 ELISA
• Prototype launch for the rapid test
• Preparation of a prospective study for the rapid test as screening test in underserved regions
Background information
It is clear that from the initial idea of an in vitro diagnostic device (IVD) to its broad routine application often more than one decade is necessary. Said this, the PIPAVIR consortium is fully aware of the challenges related to the translation of its expertise into a new medical device in the field of persistent HPV infections. Hence, the aim of this workpackage is to launch a prototype IVD for subsequent application in large European trials in cooperation with key opinion leaders (KOLs) and the relevant scientific societies, such as the International Federation of Gynecology and Obstetrics(http://www.figo.org/(se abrirá en una nueva ventana)) International Gynecologic Cancer Society (IGCS)(http://www.igcs.org(se abrirá en una nueva ventana)) Society of Gynecologic Oncology (http://www.sgo.org/ ), European Society of Gynecologic Oncology (http://www.esgo.org/Pages/default.aspx(se abrirá en una nueva ventana)) Deutsche Gesellschaft für Gynäkologie und Geburtshilfe (DGGG), Deutsche Krebsgesellschaft, and the Bundesverband der Frauenärzte, Studiengruppe Kolposkopie (SGK; G-CONE).
Task 3.1: Prototype launch for hrE7- ELISA (ongoing)
Results: Since the critical material was not available in quantity and quality as needed, immunization of goats for polyclonal antibodies as well as industrial production of RabMabs was mandatory.
The immunization of the goats was finished in November 2013, purification and further processing of the goat antibodies including testing of different labeling methods such as direct conjugation with HRP and biotinylation was finished by February 2015 with a total of 4 independent batches for each goat antibody.
Industrial production of the RabMabs was started in September 2013 and finished in June 2015 with a total of 4 independent batches for each RabMab.
During assay optimization (WP1) two different strategies for detection of hrE7 protein of 12 different HPV subtypes were developed. Whereas the detection system of P1 contains three different kinds of wells (i.e. well 1: detection of 16, 18, and 45 E7; well 2: detection of 39, 51, 56, and 59 E7; and well 3: detection of 16, 31, 33, 35, 52, and 58 E7), the strategy of P2 is based on the combination of one screening well, which detects 12 hr E7 types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 E7) and a well for detection of the HPV types with highest carcinogenicity (16, 18, and 45 E7).
In a first step, pre-prototypes of both strategies for the hrE7 ELISA were produced in M27 and M28 in an industrial scale to ensure that an automated production is possible. Thereafter three batches of both, RabMabs and goat antibodies, were used to produce the prototypes of both hrE7 ELISA systems, i.e. three Lots of each system with a total of 15 prototypes. This work was started in M28 and finished in M31.
All produced prototypes were adjusted and tested for validity. Thereafter one prototype of each system was choosen for measurement of hrE7 in the clinical samples of WP4-6.
Deviations from the work plan:
During technical validation of the hrE7 ELISA it became clear that due to the heterogeneity of the cervical smears validity testing for the samples is mandatory. For this purpose, an additional ELISA test system for validity testing was developed. This test system was produced (three prototypes) and tested in parallel with the hrE7 ELISA.
Task 3.2: Prototype launch for hrE7- Rapid test (month 12-15)
Result: Due to the delay in WP1 (reasons discussed in the corresponding chapter), the product launch for the hrE7 rapid test will be postponed. Approximately 5384 ThinPrep samples were collected combining all studies and patient visits (pre-study: 1542; HPV negative validation set: 500; Screening: 1600; Self-sampling: 574; HPVTriage: 344; CytoTriage: 234; HSILCure: 574; CxCa:16). All samples were tested for HPV DNA and genotyped. Study samples were subjected to E7 ELISA when the prototype assay became available.
Task 3.3: Preparation for a prospective study for hrE7 rapid test validation as “Screening test in underserved regions” (Month 24-36)
Instead of Ghana samples were obtained from Ethiopia (Black Lion Hospital, Addis Ababa) from cervical dysplasia patients (n=144) and tested by HPV genotyping and hrE7 rapid test when the prototype became available. This study showed feasibility of the approach.
The planned studies require existing prototypes; accordingly, these studies have to be postponed.
Work Package No: WP4
Plan Start: M01
Plan End: M36
Lead Participant: CHARITE
Actual Start: M01
Actual End: M36
Work package title: Clinical validation of E7 tests as screening tools
Activity Type: Research activities
Participants involved: ALL
Partners 4 and 5 designed a clinical study to investigate and validate the tools developed in PIPAVIR with respect to laboratory-based hrE7 ELISA test and hrE7 Rapid-Test. Full ethical and data safety board approval for the clinical study covered in this WP (screening population) was obtained at both clinical sites. The logistics for sample shipment from/to Partners 4 and 5 and to the partners 1, 2 and 3 was established. Clinical trials according to the follow-up time were initiated. Recruitment finished of all 1200 patients necessary for the first trial (Screening study). The following sub-tasks are ongoing: i) Collection and asservation of clinical material, ii) compilation of anamnestic and clinical data of the recruited patients in a data capture website (designed by Partner 5), iii) characterization of clinical material for cytology and HPV genotyping results.
Task 4.1: Preparation of study documents materials, teaching of doctors and nurses ( M1-M5)
During this period the following activities were performed:
• In collaboration with Partner 4 two Screening Studies protocols were defined by Partner 5. The outcome has been reported in the PIPAVIR protocol internal document.
• The application to Bioethics Committee of the Medical School of Aristotle University of Thessaloniki was prepared and submitted. Approval was received.
• The AUTH team was established and trained. The team consists of the principal investigator, 2 gynecologists, 1 cytopathologist, 1 lab technician, 1 assistant and three informatics scientists. A step-by-step document was created and distributed to each member of the team in order to help them know exactly the tasks they have to do.
• Materials for cytopathology evaluation, sample collection and transfer were purchased; patient forms were created; A google document for monitoring sample tracking was created and was shared with Partner 4 and the other partners.
• The first version of the Data Capture System (DCS) was developed and tested by the informatics scientists of Partner 5. DCS is a multi-user, multi-role web application allowing for registration of the subjects and reporting of the results of cytology, colposcopy, histology, HPV-DNAg test, hrE7-ELISA and hrE7-Rapid. Data visualization and export are also supported. All data is anonymous. Instructions of use were provided by Partner 5 to all PIPAVIR users.
Task 4.2 : Prospective study for hrE7-ELISA validation as screening test
During this period the following activities were performed by Partner 5:
• April 2014 - May 2014: Completion of subject recruitment for the screening study. 146 subjects were recruited, resulting to a total of 1068 from the start of the project.
• September 2014 – June 2015: All subjects were called for their follow-up visits. 269 subjects came for a second visit.
• Sept 2014- Jan 2015: 190 subjects were recruited for the self-sampling study. In this case, in addition to the physician sample, women collected a self-sample using the Evalyn device.
• All women were recruited at the Family Planning Center of the “Hippokratio” Hospital of Thessaloniki, where the 4th Department of Obstetrics and Gynecology of AUTH, is placed. For each subject the AUTH team performed the following tasks:
• Sample collection and subject registration to the DCS
• Cytology evaluation and reporting of results to the DCS
• Preparation of samples for HPV-DNAg and hrE7-ELISA. These samples were sent for evaluation to Partner 4 and Partner 2 respectively.
• Review of test results. For the subjects with positive cytology and/or HPV-DNAg result a colposcopy examination was conducted. Colposcopy results and follow-up data was reported to the DCS.
For all subjects an additional sample was collected to evaluate the hrE7-Rapid test. These samples were sent to Partner 3 for analysis.
Cytology evaluation for additional samples
In addition to the samples collected at Partner 5, cytology evaluation was conducted also for the pre-study and prospective studies samples from Partner 4.
Detection of E7 with the hrE7 ELISA
All samples were shipped to P2. For each sample, the total volume and the volume of the cell pellet including color was determined. All samples were measured from M34 to M36 at P2 for presence of hr E7 with both systems and additional validity testing was performed. Hence, each sample was measured six times and the results were transferred into the DCS. The following E7 tests were performed: Well1 (16/18/45), Well2, Well3, screen, 16/18/45.
Data extraction, validation and Statistical analyses
Upon completion of data collection from both partners 4 and 5, and reporting of results from partners 1, 2 and 3, the appropriate datasets were extracted from the DCS. In total, since the beginning of the project, 1311 women were recruited in the screening study and 1600 samples were collected and analyzed. For the self-sampling study, 384 women were recruited and 574 samples were collected and analyzed. Following data extraction, data were validated to ensure consistency with the protocol and completeness. Finally, statistical analyses was conducted to assess: thresholds, sensitivity, specificity, PPV and NPV for each one of the following E7 tests: Well1 (16/18/45), Well2, Well3, screen, 16/18/45.
Statistically significant associations between E7 positivity in the test formats Well 1, Well 3, and 16/18/45 could be detected, providing proof-of-principle for the E7 test. The calculation of sensivity, specificity, PPV and NPV has been performed for these tests in the screening study which yielded preliminary data. Currently, a more detailed data evaluation is being performed by partners 1, 2, 4 and 5, involving model calculations with different cut-off values for E7 protein concentrations. The results of the more extended data analysis will be available by the end of December 2015.
Deviation from work plan
Partner 5 recruited more subjects than originally planned. According to the original plan, AUTH would recruit approximately 500 subjects for the screening study, and 65 subjects for the self-sampling study. However, due to the high number of women coming for screening to the Family Planning Center, it was decided that AUTH would focus on recruitment for the screening study instead of recruitment for the triage and treatment studies.
During this period the following activities were performed by partner 4:
For the prospective Screening study 243 patients were recruited at MVZ Im Mare, Kiel Germany in a cooperation with Charite Berlin. Of those 123 returned for a planned follow up visit. For the self-sampling feasibility study 193 samples were obtained at Charite and also collected in Ethiopia as a feasibility for low resource settings. Sample sets were combined with the screening study samples and self-sampling samples, respectively, collected by partner 5 (see below).
Partner 4 was responsible for HPV genotyping (by WHO reference test MPG-Luminex) for 18 high-risk and 9 low-risk HPV types. HPV determinations were performed for all ThinPrep samples collected during all studies.
Task 4.3 : Prospective study for hrE7-Rapid validation as screening test
During this period the following activities were performed by Partner 5:
• April 2014 - May 2014: Completion of subject recruitment for the screening study. 146 subjects were recruited, resulting to a total of 1068 from the start of the project.
• September 2014 – June 2015: All subjects were called for their follow-up visits. 269 subjects came for a second visit.
• Sept 2014- Jan 2015: 190 subjects were recruited for the self-sampling study. In this case, in addition to the physician sample, women collected a self-sample using the Evalyn device.
• All women were recruited at the Family Planning Center of the “Hippokratio” Hospital of Thessaloniki, where the 4th Department of Obstetrics and Gynecology of AUTH, is placed. For each subject the AUTH team performed the following tasks:
• Sample collection and subject registration to the DCS
• Cytology evaluation and reporting of results to the DCS
• Preparation of samples for HPV-DNAg and hrE7-ELISA. These samples were sent for evaluation to Partner 4 and Partner 2 respectively.
• Review of test results. For the subjects with positive cytology and/or HPV-DNAg result a colposcopy examination was conducted. Colposcopy results and follow-up data was reported to the DCS.
• For all subjects an additional sample was collected to evaluate the hr-E7-Rapid test. These samples were analyzed by Partner 3.
Cytology evaluation for additional samples
In addition to the samples collected at Partner 5, cytology evaluation was conducted also for the pre-study and prospective studies samples from Partner 4.
Data extraction, validation and Statistical analyses
Upon completion of data collection from both partners 4 and 5, and reporting of results from partners 1, 2 and 3, the appropriate datasets were extracted from the DCS. In total, since the beginning of the project, 1311 women were recruited in the screening study and 1600 samples were collected and analyzed. For the self-sampling study, 384 women were recruited and 574 samples were collected and analyzed. Following data extraction, data were validated to ensure consistency with the protocol and completeness. Finally, statistical analyses was conducted to assess: thresholds, sensitivity, specificity, PPV and NPV for the hr-E7 Rapid test.
The clinical sensitivity of the E7 rapid test was lower than expected and the initial evaluation of the data did not provide significant associations between E7 positivity and the clinical data. Currently a more detailed data evaluation is performed by partners 3, 4 and 5. The results of the more extended data analysis will be available by the end of December 2015.
Deviation from work plan
Partner 5 recruited more subjects than originally planned. According to the original plan, AUTH would recruit approximately 500 subjects for the screening study, and 65 subjects for the self-sampling study. However, due to the high number of women coming for screening to the Family Planning Center, it was decided that AUTH would focus on recruitment for the screening study instead of recruitment for the triage and treatment studies.
Work Package No: WP5
Plan Start: M01
Plan End: M34
Lead Participant: AUTH
Actual Start: M01
Actual End: M34
Work package title: Validation of E7 tests as triage test
Activity Type: Research Activities
Participants involved: All
Partners 4 and 5 designed two clinical studies to investigate and validate the tools developed in PIPAVIR with respect to laboratory-based hrE7 ELISA test and the hrE7 Rapid-Test. Full ethical and data safety board approval for the clinical studies covered in this WP (triage tests) was obtained at both clinical sites. Clinical trials for testing in a triage setting for HPV positive and cytologically positive women were initiated with first recruitment and ongoing recruitment. The following sub-tasks are ongoing: i) Collection and asservation of clinical material, ii) compilation of anamnestic and clinical data of the recruited patients in a data capture website (designed by Partner 5), iii) characterization of clinical material for cytology and HPV genotyping results. The logistics for sample shipment from/to Partners 4 and 5 and to the partners 1, 2 and 3 was established.
Task 5.1: Preparation of study documents materials, teaching of doctors and nurses
Τhis task is similar to T4.1. During this period the following activities were performed:
• Definition of the two Triage Studies protocols, in collaboration with Partner 4. The outcome has been reported in the internal “PIPAVIR protocol“ document.
• The application to Bioethics Committee of the Medical School of Aristotle University of Thessaloniki was prepared and submitted. Approval was received.
• Deviations from the sample collection procedure of T4.2 and T4.3 were identified and the AUTH team (see Task 4.1) was informed accordingly.
• The DCS (see Task 4.1) was updated to incorporate the design of the triage studies.
Task 5.2: validation of E7 tests for triage of hrHPV-positive women & Task 5.3: validation of E7 tests for triage of women with abnormal PAP test
The following activities were performed by Partner 4:
Patient recruitment was further performed for the validation sample sets and the prospective studies. For validation purposes of technical developments lysates from different HPV positive and negative cultured cell lines were generated as positive control samples. Altogether 1540 pre-study samples from patients with different dysplasia grades and 500 ThinPrep samples that tested HPV-negative were collected as technical validation sets. The HPV negative samples were used for technical cutoff determination by partner 2. Pre-study samples are still under investigation.
Recruitment was done mainly at Charité Campuses Berlin, and at a primary clinical center in Kiel (MVZ Im Mare). Validation set HPV negative samples were obtained from a gynecology clinic in Wolfsburg, Germany. Some samples for triage and cure studies were also recruited by partner 5.
In the HPVTriage study 229 patients were recruited and 32 returned for a follow-up visit because of of negative clinical findings. In the CytoTriage study 171 patients were recruited and 52 returned for follow up due to negative findings for verification. These samples were combined for the analysis of results with the corresponding samples collected by partner 5 (see below).
All samples were sent to partner 2 for hrE7 ELISA testing. For all samples a second smear sample was collected and sent to partner 3 for analysis by hrE7 rapid test. For self-sampling studies and for independent control testing hrE7 test was also performed by partner 4 in a good proportion of study samples and pre-study samples.
Partner 4 was responsible for HPV genotyping (by WHO reference test MPG-Luminex) for 18 high-risk and 9 low-risk HPV types. This amounted to more than 5384 HPV determinations in all samples collected during all studies.
For each patient the following tasks were performed:
• Sample collection and subject registration to the DCS
• HPV testing, evaluation of sample adequacy, and reporting of results to the DCS
• Preparation of cytological slides for reading at AUTH and aliquot sending to partner 1, 2 and 3 for hrE7-ELISA and rapid test, respectively.
• Review of test results. For the subjects with positive cytology and/or HPV-DNAg result a colposcopy examination was conducted if not done at inclusion. Colposcopy results and follow-up data were reported to the DCS.
• Retesting of samples with non-concordant result in the HPV and hrE7 tests.
Recruitment of 56 subjects in the TriageHPV+ and of 10 subjects into the Triage Cyto+ studies. These subjects were called for a second follow-up visit after 6 months. All women were recruited at the Family Planning Center of the “Hippokratio” Hospital of Thessaloniki, where the 4th Department of Obstetrics and Gynecology of AUTH, is placed. For each subject the AUTH team performed the following tasks:
o Sample collection and subject registration to the DCS
o Cytology evaluation and reporting of results to the DCS
o Preparation of samples for HPV-DNAg and hrE7-ELISA. These samples were sent for evaluation to Partner 4 and Partner 2 respectively.
o Review of test results. For certain subjects, according to the specific study protocol, a colposcopy examination was conducted. Colposcopy results and follow-up data were reported to the DCS.
o For all subjects an additional sample was collected to evaluate the hrE7-Rapid test. These samples were sent to Partner 3 for analysis.
• Cytology evaluation for samples from Partner 4 Maintenance and support of the DCS
Detection of E7 with the hrE7 ELISA
All samples were shipped to P2. For each sample, the total volume and the volume of the cell pellet including color was determined. All samples were measured from M34 to M36 at P2 for presence of hr E7 with both systems and additional validity testing was performed. Hence, each sample was measured six times and the results were transferred into the DCS.
Data extraction, validation and Statistical analyses: Upon completion of data collection from both partners 4 and 5, and reporting of results from partners 1, 2 and 3, the appropriate datasets were extracted from the DCS. In total, since the beginning of the project, 281 women were included into the TriageHPV study and a total of 346 samples were collected and analysed. For the TriageCyto study 181 women were recruited and 232 samples were collected and analysed. Following data extraction, data were validated to ensure consistency with the protocol and completeness. Finally, statistical analyses was conducted by Partner 5 to assess: thresholds, sensitivity, specificity, PPV and NPV for each one of the following tests: Well1 (16/18/45) , Well2, Well3, screen, 16/18/45 and hrE7 Rapid Test.
Statistically significant associations between E7 positivity in the test formats Well 1, Well 3, and 16/18/45 could be detected, providing proof-of-principle for the E7 test. The calculation of sensivity, specificity, PPV and NPV has been performed for these tests which yielded preliminary data. Currently, a more detailed data evaluation is being performed by partners 1, 2, 4 and 5 involving model calculations with different cut-off values for E7 protein concentrations. The clinical sensitivity of the E7 rapid test was lower than expected and the initial evaluation of the data did not provide significant associations between E7 positivity and the clinical data. Currently a more detailed data evaluation is performed by partners 3, 4 and 5. The results of the more extended data analysis will be available by the end of December 2015.
Deviation from the work plan: The number of subjects that were recruited by Partner 5 for the TriageCyto+ study were less than originally planned (~60). The Family Planning Center receives mostly women for screening and not referral. For this reason partner 5 focused merely to recruitment of subjects for the screening and self-sampling studies, as reported in Wp4.
Work Package No: WP6
Plan Start: M01
Plan End: M34
Lead Participant: AUTH
Actual Start: M01
Actual End: M34
Work package title: Validation of hrE7 tests as “test of cure”
Activity Type: Research activities
Participants involved: All
Partners 4 and 5 designed two clinical studies to investigate and validate the tools developed in PIPAVIR with respect to laboratory-based hrE7 ELISA test and the hrE7 Rapid-Test. Full ethical and data safety board approval for the clinical studies covered in this WP (test of cure) was obtained at both clinical sites. Clinical trials for test of cure in patients with HSIL or cervical cancer were initiated with first recruitment and recall of patients in the cure HSIL trial. Problematic recruitment of patients for the cure CxCa trial due to lack of accessibility. The logistics for sample shipment from/to Partners 4 and 5 and to the partners 1, 2 and 3 was established.
Task 6.1: Preparation of study documents, materials, teaching of doctors and nurses
Τhis task is similar to T4.1 and T5.1. During this period the following activities were performed:
• Definition of the two Test of Cure studies protocols, in collaboration with Partner 4. The outcome has been reported in the internal “PIPAVIR protocol“ document.
• The application to Bioethics Committee of the Medical School of Aristotle University of Thessaloniki was prepared and submitted. Approval was received.
• The Data Capture System (DCS) was updated to incorporate the design of the triage studies.
Task 6.2 : Validation of E7 tests as “Test of cure” in patients with HSIL & Task 6.3 Prospective study for hrE7-ELISA validation as “Test of cure” in cervical cancer patients
The following activities were performed by Partner 4:
Recruitment of patients into the HSILCure study was done mainly at Charité Campuses Berlin, and fewer at a primary clinical center in Kiel (MVZ Im Mare). Some HSILcure study patients were also recruited by partner 5 (see below).
For the HSILCure study 206 patients were recruited of whom 106 returned for Visit 2, 103 for Visit 3, 64 for Visit 4 (an interim visit due to common practice) and 42 for Visit 5). Follow up is still ongoing in this study in some patients.
For CxCaCure the available patient numbers (n=14, n=2 for visit 2) for recruitment were too low due to a change in referral practices to our clinics.
For each patient the following tasks were performed:
• Sample collection and subject registration to the DCS
• HPV testing, evaluation of sample adequacy, and reporting of results to the DCS
• Preparation of cytological slides for reading at AUTH and aliquot sending to partner 1, 2 and 3 for hrE7-ELISA and rapid test, respectively.
• Review of test results. Subjects treated by conization were followed up by colposcopy, cytology and HPVg Data were reported to the DCS.
• Retesting of samples with non-concordant result in the HPV and hrE7 tests.
All samples were sent to partner 2 for hrE7 ELISA testing. For all samples a second smear sample was collected and sent to partner 3 for analysis by hrE7 rapid test.
Partner 4 was responsible for HPV genotyping (by WHO reference test MPG-Luminex) for 18 high-risk and 9 low-risk HPV types. This amounted to more than 5384 HPV determinations in all samples collected during all studies.
Deviation from work plan
The number of subjects recruited by partner 4 for the HSILCure study was higher than initially planned. This was to compensate for lower recruitment by partner 5 and to enhance the power of the analysis.
The CxCaCure study had to be cancelled because due to restructuring of the Gynecology department only very few patients were referred to the clinic during the recruitment period.
• Recruitment of 14 women for the CureHSIL study. All women were recruited at the Family Planning Center of the “Hippokratio” Hospital of Thessaloniki, where the 4th Department of Obstetrics and Gynecology of AUTH, is placed. For each subject the AUTH team performed the following tasks:
o Sample collection and subject registration to the DCS
o Cytology evaluation and reporting of results to the DCS
o Preparation of samples for HPV-DNAg and hrE7-ELISA. These samples were sent for evaluation to Partner 4 and Partner 2 respectively.
o Review of test results. For certain subjects, according to the specific study protocol, a colposcopy examination was conducted. Colposcopy results and follow-up data was reported to the DCS.
o For all subjects an additional sample was collected to evaluate the hrE7-Rapid test. These samples were sent to Partner 3 for analysis.
• Cytology evaluation for the samples from Partner 4
• Maintenance and support of the DCS
Detection of E7 with the hrE7 ELISA and hrE7 rapid test
All samples were shipped to P2. For each sample, the total volume and the volume of the cell pellet including color was determined. All samples were measured from M34 to M36 at P2 and P3 for presence of hr E7 with both hrE7 Rapid test, hrE7 ELISA systems and additional validity testing was performed. Hence, each sample was measured six times at P2 and once at P3 the results were transferred into the DCS.
Data extraction, validation and Statistical analyses: Upon completion of data collection from both partners 4 and 5, and reporting of results from partners 1, 2 and 3, the appropriate datasets were extracted from the DCS. In total, since the beginning of the project, 220 women were recruited in the study and 595 samples were collected and analysed. Following data extraction, data were validated to ensure consistency with the protocol and completeness. Finally, statistical analysis was conducted to assess: thresholds, sensitivity, specificity, PPV and NPV for each one of the following hrE7 Rapid tests: Well1 (16/18/45), Well2, Well3, screen, 16/18/45.
Statistically significant associations between E7 positivity in the test formats Well 1, Well 3, and 16/18/45 could be detected, providing proof-of-principle for the E7 test. The calculation of sensivity, specificity, PPV and NPV has been performed for these tests in the screening study which yielded preliminary data. Currently, a more detailed data evaluation is being performed by partners 1, 2, 4 and 5 involving model calculations with different cut-off values for E7 protein concentrations. The clinical sensitivity of the E7 rapid test was lower than expected and the initial evaluation of the data did not provide significant associations between E7 positivity and the clinical data. Currently a more detailed data evaluation is performed by partners 3, 4 and 5. The results of the more extended data analysis will be available by the end of December 2015.
Deviation from the work plan: The number of subjects that were recruited for the Cure studies by partner 5 was less than originally planned. As the Family Planning Center receives high number of women for screening and not referral, it was decided early in the project, that partner 5 focuses in the recruitment of subjects for the screening and self-sampling studies (see WP4) instead of the triage (WP5) and cure (WP6) studies.
Work Package No: WP7
Plan Start: M01
Plan End: M36
Lead Participant: UIBK
Actual Start: M01
Actual End: M36
Work package title: Dissemination and Exploitation
Activity Type: OTHER
Participants involved: ALL
The Dissemination of the PIPAVIR concept and of preliminary project results has been carried out at several levels in the reporting period. Of particular importance is the participation of PIPAVIR scientists in national and international symposia, giving topical presentations focused on the role of papillomaviruses in cervical carcinoma and on clinical procedures for cervical cancer screening. It can be noted, that the presentations by PIPAVIR scientists at those occasions (see attached list of meetings attended) created a high level of interest by national and international key opinion leaders and has been used to organize collaborations with a couple of new clinical sites, who are interested in using the PIPAVIR in vitro diagnostic systems as soon as they are available. This will enable a massive spread of the technology. The acceptance of the new diagnostic principle in the field will be considerably speeded up by these activities. On the other hand, PIPAVIR scientists, in particular of the coordinator, have spread the information about the PIPAVIR concept and preliminary results in the lay press, where several featured articles have been published. These activities will make the general public familiar with the development of a new and efficient test for rational diagnosis of cervical pre-cancer, which is a topic of considerable public health interest and is also of immediate interest to a large part of the female population. Taken together, the dissemination activities of PIPAVIR have met all the expectations as set out in the description of work and we expect a further enhancement of these activities, once the clinical validation of the test has been achieved. Exploitation activities are currently still in the phase of prototype development and we expect more substantial exploitation activities, once the prototypes are available and clinically validated.
Task 7.1: Preparation of a communication plan for dissemination of the project
Achieved
Task 7.2: Implement a project design, information materials, and a public interest website
Achieved.
Task 7.3: Disseminate progress and results via classic and new media
Achieved
Task 7.4: Disseminate progress and final results on scientific meetings and through publications
The PIPAVIR trials programme was presented at workshops of the German consortium for colposcopy and several international conferences (as indicated in the attached dissemination table). Preliminary results were presented at Eurogin 2015 and different scientific meetings, such as. ECCMID 2014 and 2015, ESCV 2014, Eurogin 2015, and IPV 2014.
• there have been several articles in the public press concerning the PIPAVIR-Project (mostly in Austria)
• there have been Workshops, in which partner Partner 4 participated and represented the PIPAVIR Consortium
• Our project web-site can be found under www.pipavir.com. This site provides information about the project itself, the partners involved, the planned work and the progress achieved. The homepage is also used as a platform for publications (press releases as well as scientific publications) and as a browser-based communication platform (partners are able to log in and work in a password-secured internal area). On this web space, presentations of the various meetings are stored, so all PIPAVIR partners have access to all information. The interim reports and the final reports will be made available to the consortium also throughout this site. Besides this, this tool helps coordinating the information flow among the partners, specifically regarding the project meetings.
• P1 and P2 presented the results of the hrE7 ELISA development as poster presentations or oral presentations at different meetings, i.e. ECCMID 2014 and 2015, ESCV 2014, Eurogin 2015, and IPV 2014
Task 7.5: Collaboration with existing initiatives to promote women’s health
Achieved.
Task 7.6: Development of recommendations for physicians
Achieved.
Task 7.7: Preparation for CE-approval and filing (month 18-36)
Based on the results of the clinical studies according to WP5 & WP6, P2 and P3 will file a CE-registration to get the CE-approval for both products. This is a must for the commercialization of the assay as IVD in Europe. To promote commercial exploitation of project results, the cost effectiveness of the diagnostic kit will be evaluated.
Task 7.8: Product Launch and evaluation of perception of hrE7 screening by patients and doctors (month 3-36)
After CE-marking IVD will be launched. The IVDs will be installed at reference centers, key opinion leaders and first customers. P1, P4 and P5 jointly will measure the acceptability of this new test format by patients and doctors. Each medical diagnostic method need to be accepted by the patients and physicians. In order to evaluate the perception of the test by the patients and the interpretation by the doctors we will use a standardized questionnaire. Patients and doctors will be requested to answer the questionnaire after use of the hrE7 tests. A score for patient and doctor acceptability of the test will be calculated. P2 has started with the preparation of all material needed for CE-registration in M20. A business plan was prepared as well as the cost effectiveness of the diagnostic kit was evaluated. By participation of P2 at scientific meetings and symposia from M4 on, key opinion leaders and potential customers were introduced to the novel hr E7 ELISA system and the acceptability of the test system was assessed.
Deviations from the work plan:
Due to the delay in WP1, the measurement of the clinical samples from WP4-6 and the analysis of the clinical study results were also delayed. Hence, the preparation and documentation for CE-filing was not completed by M36. Since the material has to be accurate prepared and verified the product lunch was postponed to beginning of 2016.
Potential Impact:
4.1.4 Impact
The potential impact (including the socio-economic impact and the wider societal implications of the project so far) and the main dissemination activities and exploitation of results (not exceeding 10 pages).
4.1.4.1 Summary
MIKROGEN is well established in the field of diagnostic tests for infectious diseases in humans and performs active research into innovative products. The aim of the current proposal is to clinically validate a new biomarker test (referred to as PIPAWELL E7 test i.e. the selected brand name for the hrE7-ELISA developed in the PIPAVIR project) for early detection of cervical precancerous lesions and cancers. Currently used molecular diagnostic tools can detect human papillomavirus (HPV) DNA or RNA in cervical smears but fall short of discriminating between transient HPV infections, which can spontaneously regress, and persistent infections, which have a high chance to progress to high-grade squamous intraepithelial lesions (HSIL). MIKROGEN has developed a new diagnostic test that allows detection, by sandwich-ELISA, of HPV E7 proteins in cervical smears, as specific biomarkers for persistent HPV infections with unfavourable prognosis. The E7-ELISA developed by MIKROGEN uses a robust and appropriate analytical method to determine expression levels of viral E7 oncoproteins, which are causally linked to CINIII/cervical carcinoma as a pertinent clinical endpoint. The validation procedure has been curtailed to provide evidence for high analytical validity, appropriate sensitivity and specificity, as well as clinical validity and utility, and therefore will allow to develop new diagnostics with exceptional prognostic and predictive power; the clinical performance of the new diagnostic device will be validated and tested against existing standards, such as cytology, HPV testing and p16 immunohistochemistry. The HPV oncoproteins are disease-related biomarkers and as such will allow an assessment of the risk to develop cervical cancer. The biomarkers can also be used for disease monitoring and prognosis. The PIPAWELL E7 test will enable improved clinical decisions which will lead to better health outcomes, such as a reduction in overtreatment. Accordingly, the HPV oncoprotein biomarkers proposed here have a high potential for short term uptake into clinical practice. Due to the high cost efficiency of the PIPAWELL E7 test, in combination with the reduced requirement for repeated testing, the project will contribute to the sustainability of health care systems. MIKROGEN has achieved appropriate IP protection for the E7-ELISA technology and owns all rights to use it, through both licensing agreements and own patent applications, providing full freedom to operate. The longstanding (>25 years) expertise of MIKROGEN in the field of producing and marketing diagnostic devices for infectious diseases provides excellent conditions for successful commercial exploitation. Based on the potential of the technology to meet a real clinical need, it can be anticipated that the ability of MIKROGEN to provide and market such superior diagnostic tools will create great business opportunities. With these objectives, PIPAWELL corresponds fully to the topic description in the work programme topic PHC 12.
4.1.4.2 Impact on health and welfare of European citizens
To date, screening for persistent HPV infections is based on cytological or smear samples that are sent to specialized laboratories for processing and microscopical and molecular testing. This means a time delay for further measures and clinical treatment. A second appointment is necessary for the patient for triage of equivocal cytology results, or for treatment. Results from standard screening methods suffer from low sensitivity (as in cytology) or low specificity (as in HPV DNA testing). A combination of both methods is regarded to be necessary to reach high sensitivity and specificity. However this also means higher cost and effort. A test based on E7 protein would have intrinsically a higher specificity for high grade disease since this is related to upregulation of E7 expression. In addition, a format of a rapid test may be used to an onsite testing and treatment in the same visit ("see and treat") reducing the number of visits & uncertainty on the result, and thereby accelerating treatment. This will lead to a reduction of cancer cases due to earlier detection of relevant premalignant lesions, and thereby confront a major threat to public health. The physician may decide on treatment or not, according to the result of the E7 test. That means also a reduction of overtreatment in cases that are not progressed and thus do not overexpress E7 although an infection is prevalent. Less invasive treatment and avoidance of overtreatment will lead to a reduction in perinatal problems, because women who have lost cervical tissue after deep or repeated conisations have a 5 fold higher risk for premature birth. In these aspects, the project meets real clinical and public health needs. Use of an E7-based test will also reduce follow-up appointments when no relevant lesion is found in spite of a persistent infection. In the case of uncertain colposcopical findings, a E7 rapid test can clarify if there is a high grade lesion present or not. Taken together, the impact on clinical management will be impressive, reducing number of visits, treatment procedures, and stress and anxiety. It will in turn lead to less overtreatment and less perinatal problems due to cervical insufficiency.
4.1.4.3 Markets and potential users
The total addressable HPV diagnostic market has a potential of 350 Mio. € world-wide with an annual increase of 6% as shown in Table 1. The biggest market represents the US (60%), followed by Europe (25%) and the rest of the world (15%) (see Figure 1). The HPV business creates new and huge market opportunities for MIKROGEN and provides a step forward to rational and focused diagnostic procedures.
Europe has no integrative public health system. Unfortunately, each country has its own public health and re-imbursement system. In Germany as well as in other European countries (e.g. Netherlands) reorganization from traditional Pap Screen to Primary HPV Screening concerning cervical cancer prevention takes places in near future (beginning of 2016). This will be a door opener into the HPV diagnostic market for the PIAWELL E7 test.
Due to MIKROGENs' long tradition in the foreign markets, there are good and reliable contacts to health authorities which will help to overcome reimbursement hurdles. As an example, the reimbursement of HPV related diagnostics in Germany differs considerably for patients enrolled in public health insurance (EBM) and patients enrolled in private health insurance (GOÄ, see Table 2). For the PIAWELL E7 test no reimbursement code exists to date, but due to the upcoming reorganization of cervical cancer screening new reimbursement codes will be generated. These facts will be considered in the PIPAWELL commercialisation strategy, facilitated by the long standing sales experience of MIKROGEN in the most important health markets.
Current testing methods, while reliable in detecting the presence of HPV infection, are unreliable in terms of predicting the likelihood of a patient developing cervical cancer. Neither Pap nor HPV tests provide definitive results for the actual risk for cervical cancer. The diagnostic market for cervical carcinoma detection is characterised by economically strong players, pushing HPV DNA testing as primary diagnostic measure in screening populations. This is contrasted by key opinion leaders in cervical cytology, who insist that cytology should remain the first-line diagnostic test. At the same time, conventional cytology is being questioned in an increasing number of European countries, leaving a vacuum for new tests, such as the PIPAWELL E7 test that provide identification of patients at risk beyond HPV DNA positivity.
Cervical cancer early diagnosis and prevention reflects an unmet medical need throughout Europe
Whereas the traditional way of cervical cancer screening is based on cytological analysis (PapSmear) followed by more laborious follow-up procedures for women with abnormal cytology, the introduction of HPV DNA testing (with its higher sensitivity but lower specificity) has necessitated a readjustment of treatment regimes and screening algorithms. Some European countries have opted for HPV testing as primary screening tool and consider to abandon cytological analysis altogether, due to inherent lack of precision of conventional cytology. Germany has initiated to re-organize towards regular invitation and optional HPV testing 5 yearly instead of cytology yearly. On the other hand, the inherent inability of HPV DNA testing to discriminate between clinically irrelevant transient infections and ongoing tumor progression has led key opinion leaders to state the urgent need for new molecular tests with higher clinical relevance, i.e. tests which can at the same time inform about HPV infection and the presence of a high grade lesion, such as CIN2+. Since the expected high rate of referral of HPV positive patients may overload current triage systems like colposcopy, better biomarkers will be urgently needed.
Many competitors in Europe and worldwide develop additional assays aiming at the detection of persistent HPV infections by detection of viral DNA, like HC-II adding an extra HPV16, 18, 45 probe (Qiagen), Amplicore of Roche using PCR, or chip-based DNA analysis (Greiner bio-one). Other companies try to enhance specificity by using mRNA as target (Aptima Assay by Gen-Probe and the HPV Proofer), by using E6 protein as target (prototype test by Arbor Vita) or try to enhance rapidness of assays (multiplexed RT-PCR, in development). In addition, identification of progression markers for differentiation of regressing and potentially progressing lesions is done. Detection of L1 in cytology smears is tested as a regression marker (Cytoimmune), detection of cell cycle proteins like p16/Ki-67 and MCM5 (Roche), and methylation patterns in viral and cellular genes (in development) are means to evaluate progressive potential of lesions. The HPV-related diagnostic tests competing with PIPAWELL E7 test are summarized in Table 3. All mentioned current testing methods, while reliable in detecting the presence of HPV infection, are unreliable in terms of predicting the likelihood of a patient developing cervical cancer. Neither Pap nor all mentioned HPV tests provide definitive results for risk for cervical cancer. The PIPAWELL E7 test solves this issue and provides a new and superior tool in HPV diagnostics.
PIPAWELL marketing strategy
MIKROGEN’s PIPAWELL E7 test includes a new diagnostic technique which improves efficiency and effectiveness of HPV testing radically. Instead of detecting viral DNA/RNA it identifies the existence of oncoprotein E7. Overexpression of this oncoprotein is a critical and necessary step toward HPV-related cancer. As false-positive results are prevented and self-resolving HPV types are not tested as positive, the number of patients in need for additional treatment can be dramatically reduced. The main outcome of the project is an innovative and cost efficient diagnostic test kit for the diagnosis of persistent infection with high risk HPVs. Using a cost-efficient, easy-to-use and highly informative diagnostic tool will allow entry into a market, which is already highly competitive, although competitors so far rely on HPV DNA/RNA testing or cytological analysis (Pap test etc.), which are indirect, less informative, more laborious and much more expensive than the technology described here. This provides MIKROGEN with a unique selling proposition for the PIPAWELL test (Figure 2). Instead of a complex matrix of complementary yet unprecise and uncertain diagnostic procedures, an objective quantification of a disease-related predictive biomarker (i.e. the E7 oncoproteins detected by the PIPAWELL E7 test) will save cost and time.
User groups and how to convince them
In contrast to the indirect tests provided by the state of the art (see above, Table 3), PIPAWELL follows a novel approach and uses proprietary technology not available anywhere else in the world. This unique combination of expertise will allow the development of a highly competitive diagnostic tool. This provides a unique selling position. The combination of two by MIKROGEN developed assay systems represents a diagnostic breakthrough for the cervical cancer screening which allows a highly sensitive detection of cervical cancer and pre-cancerous stages. This reflects a great milestone in cervical cancer prevention and results in a dramatic reduction of cervical cancer cases world-wide.
This situation provides clear opportunities for market introduction of the PIPAWELL E7 test. The final users of the PIPAWELL product will be private laboratories, clinics, universities, pathologists/cytologists, reference labs and key opinion leaders. Different user groups will be convinced to buy the test based on distinct advantages of the PIPAWELL E7 test, as follows:
• Patients will be convinced to pay for the test out of their own pocket, because the test gives a more conclusive result discriminating low-grade from high-grade disease, thereby helping to decide for treatment or watchful waiting. Further arguments for patients to pay for the test are related to taking away uncertainty from both patient and physician and a reduction of overtreatment.
• Gynecologists will be convinced to recommend and prescribe the test as it may generate income when cytology is not recommended anymore and HPV testing is done by diagnostic labs. At the same time they will have something to offer to patients who are anxious because of a positive HPV result. At the same time the test will offer an improved triaging method for pregnant women. Additionally, it will be easy for gynecologists to explain to patient “no oncoprotein – no cancer”. This is different with DNA due to low specificity or cytology due to low sensitivity. Moreover, the PIPAWELL E7 test may be run in the office and generate direct income to the gynecologist.
• To convince key opinion leaders (KOL) to promote the test to colleagues, conclusive data showing higher specificity as compared to HPV and cytology will be needed, ideally derived of a longitudinal trial testing progressive potential of E7 positive vs negative intermediate grade dysplasia, similar to the studies described in this proposal (see below, Work Package 3). Moreover, the number of clinical interventions can be reduced by safely watching E7 negative lesions.
• Health insurance companies will be convinced to reimburse the test by the opportunity to have, at the same time, cost savings, less interventions, better care to customers, less follow-up visits and less costly triaging methods. This will require cost efficiency studies in comparison to current screening system and intervals, which will be performed by PIPAWELL. The potential of the test to advance women’s’ health will be used as marketing tool for the insurance provider.
• Private and public diagnostic laboratories will create significant additional income from the possibility to offer PIPAWELL E7 test as a conclusive screening or reflex test in conjunction with HPV testing. Additional advantages for the user are easy follow –up testing from same sample material (Thinprep) like cytology or DNA / RNA based assays. In addition, further increases of income can be expected from the use of the PIPAWELL E7 test in other indications like head and neck squamous cell carcinoma, anal disease, penile disease etc.
In order to reach the highest possible impact, MIKROGEN is aware of the importance to keep the public, policy makers and all other research and health stakeholders informed about project development and achievements. This will be done by MIKROGEN’s marketing and sales force, via new media such as special PIPAWELL websites, where different target groups can get the information they need. Additionally the key opinion leaders working with MIKROGEN on this project will meet on a regular basis at advisory boards and congresses in order to disseminate data on PIPAWELL. Further key opinion leaders (laboratory physicians and gyneacologists) will be selected and trained on PIPAWELL by MIKROGEN in order to use them in terms of cascading their knowledge on PIPAWELL to their peers. The objective of this premarketing phase is to make physicians more and more familiar with the scientific background, USP and practical advantages of PIPAWELL. Additional participants in these advisory boards will be the representatives of reimbursement authorities. Having the key opinion leaders convinced to use PIPAWELL, MIKROGEN will start a training concept for private gynaecologicsts and laboratory physicians. This will create a market willing to pay for the innovation with clients having a genuine interest to buy the product. Although primary care physicians are not yet performing HPV-related testing, this could possibly change in future, in particular using the PIPAWELL E7 test. Today the diagnosis must be accepted and also requested primarily by gynaecologists. Further on all customer segments must be split up into tier 1 to tier 3 customers - who will be the early adopters, who the followers? MIKROGEN will provide a clear segmentation for every country together with the distribution partners. The gynaecologists have to be convinced of the PIPAWELL E7 test, they are the decision makers to test patient samples by this test. User recommendations/guidelines for the assay will be developed (when to test, interpretation of results). HPV related diagnostics is subject to massive market-entry barriers imposed by regulatory agencies, such as the EMA and the FDA in the USA. Currently, HPV-related diagnostic solutions are being extensively evaluated by these authorities, providing new business opportunities for tests that identify patients at risk beyond their HPV DNA status. The market entry barriers and opportunities are summarized in Figure 3.
4.1.4.4 Exploitation Strategy (This section contains confidential information and MUST NOT be published)
MIKROGEN is an internationally working company with focus on the European market, US, Asia and Latin America, so with the best requirements to successfully launch PIPAWELL in the most important market. Specialized sales teams in the US, Europe (by own sale force in Germany and by distributors in rest of Europe), Asia (own MIKROGEN subsidiary in Beijing) and Latin America (MIKROGEN sales consultants) assure the professional roll out and marketing of PIPAWELL. Besides the scientific excellence and reputation that derives from the project for MIKROGEN, this is internally seen as the most important blockbuster/business chance of the company in the coming years and will dramatically boost the company sales from actually 12 Mio. € per year up to 35 Mio. € in 2027 (see also Figure 6 below). MIKROGEN has the commercial and management experience required to develop the current prototype of the PIPAWELL E7 test in a marketable product. A prerequisite for commercialisation is the clinical validation of the test for which third parties are needed who have the required clinical expertise and access to patients. This key step forward is attempted by the current proposal. The overall MIKROGEN business strategy is to further on succeed in serology with the four platforms LINE, ELISA, PCR and BEAD. Within this market segment MIKROGEN is market leader with a high scientific expertise in developing and marketing serological assays. MIKROGEN was founded 25 years ago and is a constantly growing SME biotech company with a broad ELISA portfolio and know how. The ELISA expertise in development and production together with a long experience in sales and marketing will be very helpful in successfully implementing PIPAWELL E7 test in the market. The strategic business model of MIKROGEN is based on defending the market leader position for serological assays in Germany, gain market leader position in all European countries, the US, Asia and Latin America and to identify and occupy profitable new sources of business like oncology in future. Therefore, PIPAWELL is fully in line with the overall strategy of MIKROGEN, and is seen as one of the most interesting and important new business fields for the company.
Strategy plan for commercialisation
Introduction of novel products is a great challenge. This situation is especially true for medical products that are research intensive even after the product was clinically validated and launched, since acceptance of a medical device is related to a "critical mass" of high quality research data, convincing public health authorities, insurances and scientific societies of clinical and cost effectiveness as well as product safety. In this respect, PIPAWELL products have a very good background to be successful in European and other markets with the scientific know-how, the results of the clinical studies with CE-marked products as well as the strong marketing and sales power of MIKROGEN. Consideration of a novel medical diagnostic test in diagnostic and/or therapeutic guidelines is a prerequisite for its broad application.
Within the pre-marketing phase in 2016 MIKROGEN will segment the potential customers which are known for every country via sales force/distributor information into early adopters, early majority, late majority, (laggards) with the objective to focus the marketing mix accordingly. Depending on the target group the physicians belong to different marketing mix instruments will be implemented. Starting with the already mentioned advisory boards, scientific study projects post launch, beta site testings and RUO projects will follow. All relevant international congresses will be used to present data on PIPAWELL, satellites, poster and publications will be planned and organized. In order to meet customer needs professional market research will be conducted per country and strategy and marketing mix will be adapted accordingly. This marketing mix will primarily contain launch meetings for physicians in the targeted countries, local trainings for physicians led by key opinion leaders, sales trainings, promotional material for sales force, promotional material for physicians, website incl. FAQ, PR plan, plan for poster and publication, mailings, concept for social media, congress plan, pricing policy per country and post launch scientific study plan for the coming years. A detailed marketing plan will be developed by MIKROGEN in 2016. Assuming an average market share of roughly 1%, total PIPAWELL sales will amount to roughly 208 Mio. € in the next 14 years (see Figure 5, above).
Figure 5 shows the significantly increasing sales until 2027 which will allow MIKROGEN to become a much more important player within the market. The projected sales will exceed the current marketing, sales, and production capacities of MIKROGEN resulting in the need to significantly extend the business activities in order to adopt to the growing sales volume. Thereby, the PIPAWELL project has a strong potential to boost the growth of MIKROGEN. The adaptations of the company to the expected growing sales are shown in Figure 5, below. The PIPAWELL project will drive our company in a new diagnostic area and will increase our revenues significantly. This is a great opportunity for MIKROGEN to get an access into the field of cancer diagnostics and to further diversify the company.
Actually MIKROGEN is one of the most important employers in the Biotech Cluster of south Munich and will then turn into an even more important employer for the whole region. In the past years, MIKROGEN has constantly invested in R&D and employees. The investment in personnel for PIPAWELL will be increased as shown in Figure 6 above. In summary, the business case HPV will reach a net present value of roughly 23 Mio. €, interest rate of 18%, internal rate of return of 66.8%, and a cash flow of 160 Mio. € in 14 years (see Figure 7 below). This results in an increase of MIKROGEN’s current total revenues of approx. 12 Mio. € to 220 Mio. €.
List of Websites:
The public website address is www.pipavir.com
Here are the relevant contact details:
1 UIBK, Universitaet Innsbruck, PI: PIdder Jansen-Dürr, pidder.jansen-duerr@uibk.ac.at
2 MIKROGEN, Mikrogen GmbH, PI: Oliver Böcher, boecher@mikrogen.de
3 BIOSYNEX, Biosynex SA, PI: Thierry Paper, paper@biosynex.com
4 CHARITE, Charite - Universitaetsmedizin Berlin, PI: Andreas Kaufmann, andreas.kaufmann@charite.de
5 AUTH, Aristotelio Panepistimio Thessalonikis, PI: Thodoros Agorastos, agorast@auth.gr
For detailed information about the contact information, please see section 4.1.5 in the attachement.
Early detection of cervical precancerous lesions is a prerequisite for successful secondary prevention of cervical cancer. To improve current technology used for the clinical diagnosis of cervical precancer, the PIPAVIR consortium developed two test systems based on an ELISA and a Rapid test in order to detect E7 oncoproteins of 12 major high-risk HPV-types in cervical smears. Both tests have been clinically evaluated for future use in patient screening, triage and therapy monitoring. In both cases industrial prototypes have been developed, which were used for the clinical evaluation. The available data suggest that the hrE7 ELISA has sufficient specificity and sensitivity to detect patients with ongoing disease in the 5 studies mentioned above. Concerning the E7 Rapid-test, the available data suggest that further improvements of this test format will be required before clinical use can be considered. During the project lifetime, the concept and progress of the PIPAVIR project has been broadly disseminated to opinion leaders, the scientific community and the general public. In conclusion, the PIPAVIR project has generated a solid basis for commercial exploitation of the hrE7 ELISA, which is expected to take place in the next couple of years.
Project Context and Objectives:
Early detection of cervical pre-cancerous lesions is a prerequisite for successful secondary prevention of cervical cancer. Current technology used in the clinical setting to achieve this goal suffers from the problem, that conventional cytological diagnostic tests are imprecise and characterized by a large rate of false-positive and false-negative results. On the other hand, molecular detection of HPV DNA, considered as an alternative diagnostic regime in several countries, has the inherent problem that the presence of a high-risk HPV DNA in a clinical specimen cannot discriminate between transient infections (in the majority of cases) and ongoing tumorigenesis in a given patient. To improve clinical diagnosis of cervical pre-cancer, the PIPAVIR consortium developed two test systems, based on an ELISA and a rapid test in order to detect E7 oncoproteins of 12 major high-risk HPV types in cervical smears. The key objectives were to clinically validate a novel ELISA test for patient screenings, triage and therapy monitoring in a clinical setting, to develop and validate a rapid test for the detection of E7 in cervical smears and to produce two separate diagnostic kits based on the results of this work. The project started with experiments aimed at assay optimisation, i.e. to improve the sensitivity and specificity of pre-existing laboratory formats. Another important objective was to adapt the hrE7 ELISA to routine laboratory requirements and perform a technical validation. The next objective was to launch prototypes for the hrE7 ELISA and the hrE7 rapid test IVDs. These prototypes were used to measure E7 levels in clinical samples that were generated in five separate clinical studies, aiming at a proof of concept that detection of E7 is suitable for improved diagnosis in the context of a screening population, as a triage test, and as test of cure for cervical cancer patients. The results obtained with the technical procedures described above have been and will be used to broadly disseminate the information about the new diagnostic concept and actual progress in its realisation in the scientific community and in the general public. This is very important to prepare key opinion leaders for the adaptation of the new diagnostic technology in cervical cancer early detection.
A sandwich ELISA test was developed, based on proprietary rabbit monoclonal antibodies and goat polyclonal antibodies, with the potential to recognize E7 proteins of the most important 12 high-risk HPV types. Currently, two different formats are available: On the one hand, three different measurements are performed per clinical sample to separately asses the concentration in cervical smears of E7 proteins from three groups of hrHPV viruses, based on immunological cross-reactivity of the respective E7 proteins. In a second attempt, a sandwich ELISA for the simultaneous detection of all E7 proteins in a single measurement has been generated. Both formats differ to some extent in their analytical precision and sensitivity and we envisage that final clinical validation will provide a basis for the selection of the final setting of the ELISA test. In terms of the E7 rapid test, the first test that has been developed is focused on the E7 proteins of HPV 16, HPV 18 and HPV 45, which are the clinically most relevant HPV types. Here a detection system has been developed and technically validated. For both the ELISA and the rapid test, commercial prototypes have been produced. Due to some unexpected problems with primary biological materials and the production of rabbit monoclonal antibodies, the completion of the prototypes has been delayed in both cases. At the clinical sites contributing to PIPAVIR, the collection of the cervical smears for the five clinical studies mentioned above has been completed and E7 protein levels have been determined in all samples. A major achievement at the analytical laboratory was the development of a protocol which allows to perform E7 tests on cervical swabs prepared by the method of liquid based cytology (LBC), a modern and wide-spread sample collection tool with many advantages. The development of a LBC-compatible ELISA SOP is considered a major break-through concerning the implementation of E7 ELISA in clinical routine, based on successful clinical validation. We performed all the measurements with the prototype diagnostic kits as foreseen. Preliminary analysis of the available data provided clinical proof-of-concept for PIPAVIR.
After successful validation of the clinical prototypes we expect to have two clinically validated E7 detection systems which can be used for significantly improved detection of pre-cancerous lesions of the cervix uteri in routine screening as well as in clinical management of patients with high-grade cervical lesions. The here described in vitro diagnostic kits along with the scientific technical evidence for their clinical validation, will provide the basis for a significant global improvement of cervical cancer screening and the diagnosis of cervical pre-cancers. The availability of these validated tests will contribute significantly to the quality of life of women who are enrolled in screening programmes and/or effected by high-grade cervical lesions and boost the economic capacities of the participating SME partners.
Project Results:
4.1.3 Description of the main S&T results/foregrounds
4.1.3.1 Overview
In Work Package 1 (WP1) the hrE7 ELISA was optimized by using coating with different RabMab concentrations, in combination with polyclonal goat antibodies used for detection (Task 1.1). This was followed by the transfer of the hrE7 ELISA protocol to the clinical scale. Here, a major achievement is the development of a protocol, which allows to carry out the ELISA on material collected by liquid based cytology (LBC), which will make the final in vitro diagnostic device very attractive for clinicians all over the world (Task 1.2). This was followed by a technical validation of the hrE7 ELISA, using cervical carcinoma cells and tumour biopsies (Task 1.3). This resulted in the establishment of a hrE7 ELISA, which was technically validated for the detection of 12 hrE7 proteins (see Figure 1), and hence a timely and successful conclusion of WP1.
In Work Package 2 (WP2), a hrE7 rapid test has been set up, which allows the detection of E7 proteins of the three most relevant HPV types, HPV 16, HPV 18 and HPV 45 (Task 2.1) and this hrE7 rapid test has been technically validated as foreseen (Task 2.2). Although the work was slightly delayed into Period 2. The development of the self-sampling test has been postponed, due to a delay experienced in the development of prototype test kits (see below, WP3).
In Work Package 3 (WP3), prototypes have been launched for the hrE7 ELISA as well as the hrE7 rapid test (Task 3.2). The development of the industrial prototype test kits required the availability of several independent batches of all biological materials to be used. Here a bottleneck was the production of the rabbit monoclonal antibodies. Initially, it was thought that these monoclonal antibodies can be produced by recombinant overexpression of cDNAs encoding the relevant rabbit immunoglobulins. However, this procedure yielded unsatisfactory results and the consortium decided to refer to the classical production of monoclonal antibodies from hybridoma cell lines. This step turned out to be more complex than expected, since the yield of rabbit monoclonal antibodies produced by hybridoma was found to be three to five fold lower as compared to mouse monoclonal antibodies. In addition, the establishment of the hybridoma cell lines by a commercial supplier took longer than expected. As a result of these, the development of the prototypes has been delayed by approximately 6 months.
In Work Package 4 (WP4) the preparation of the clinical study including the preparation of study documents, as well as teaching doctors and nurses (Task 4.1) has been performed as foreseen. In addition, the collection of samples has been completed for the test of the hrE7 ELISA prototype (Task 4.2) as well as the sample collection for the test of the hrE7 rapid test prototype (Task 4.3). It should be noted, that the delayed development of the prototype had no direct impact on the timing of the clinical WPs (WP4-WP6). A protocol has been established, that allows the retrospective sampling of cervical smears due to the development of a technology that can use liquid based cytology samples (of note, it was established in WP1 that liquid based cytology samples can be stored for several years without any loss of signal). The HPV screening study has been performed with 1.600 samples obtained from 1.311 women. Experiments to validate the hrE7 rapid test with self-sampling have been performed with 574 samples from 384 women.
In Work Package 5 (WP 5), the E7 tests were validated as triage test. Collection of clinical samples for validation of the E7 test for triage of 281 hrHPV positive women (Task 5.2) as well as for the validation of E7 tests for triage of 181 women with abnormal Pap-test (Task 5.3) have been completed; E7 protein content in all samples was measured with the prototype hrE7 ELISA and the prototype hrE7 rapid test.
In Work Package 6 (WP6) the E7 tests have been validated as tests of cure. The validation of E7 tests for test of cure in patients with HSIL (Task 6.2) and validation of the E7 tests as a test of cure in cervical cancer patients (Task 6.3) has been completed on samples from a total of 220 patients.
Work in Work Package 7 (WP7) has been performed as foreseen. Dissemination of the concept, progress and results of PIPAVIR has been performed via classic media (Task 7.3) and by presentations on scientific meetings (Task 7.4). For reasons of IPR protection the consortium has so far not published any detailed project results in scientific journals, because such publications would potentially jeopardize protection of intellectual property rights. P4 and P5 collaborated in existing initiatives to promote women’s health and started the development of recommendations for physicians (Task 7.5 Task 7.6). The long-term success will critically depend on the validation of the assay with the finalized prototypes. Similarly, the exploitation activities including preparation for EC approval and product launch (Task 7.8 Task 7.7) have been initiated.
In the following section, a detailed description of work performed in WP1 through WP7 is provided.
4.1.3.2. Detailed description of Work
Work Package No: WP1
Plan Start: M01
Plan End: M12
Lead Participant: MIKROGEN
Actual Start: M01
Actual End: M12
Work package title: Final steps of hrE7-ELISA development
Activity Type: Research activities
Participants involved: UIBK, MIKROGEN, BIOSYNEX, CHARITE
Project objectives for the period
• Assay optimization: improve sensitivity and specificity
• Adaption of hrE7-ELISA to routine lab requirements
• Technical validation of the hrE7-ELISA assay
Work progress and achievements during the period
Background information
High sensitivity is crucial for the detection of low levels of cellular E7 proteins in lysates of cervical smears. The current E7-sandwich-ELISA is based on the coating of a mixture of four different RabMabs as capture antibodies, and a mixture of two goat polyclonal antibodies as detection antibodies. This combination was shown in preliminary experiments by P1 to recognize E7 proteins of 12 hrHPV types (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59) with a detection limit of 1 picogram E7 protein or less, providing sufficient sensitivity. However, additional experiments are warranted to find the optimal configuration of RabMabs as capturing antibodies. Moreover, it is known that the transfer of new diagnostic tests from lab scale to industrial scale requires a set of adaptation measures, which are in the focus of this WP.
Task 1.1: hrE7-ELISA optimization (month 1-12)
Goal: P1 and P2 will further optimize the existing hr E7-ELISA test by coating up to ten different RabMab combinations at various concentrations. Modifications of incubation conditions and reagents will be tested to improve technical performance in terms of specificity and sensitivity, using recombinant E7 proteins. Different RabMabs chosen for optimal capture performance and subsequent detection will be combined in a way that E7 proteins of the 12 high-risk HPV genotypes can be detected with comparable sensitivity.
Result: After detailed characterization of 16 different RabMabs and 3 polyclonal goat antibodies, two different strategies for detection of 12 hr E7 proteins were developed by P1 and P2, as outlined below. Whereas the detection system of P1 contains three different kinds of wells (i.e. well 1: detection of 16, 18, and 45 E7; well 2: detection of 39, 51, 56, and 59 E7; and well 3: detection of 16, 31, 33, 35, 52, and 58 E7), the strategy of P2 is based on the combination of one screening well, which detects 12 hr E7 types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 E7) and a well for detection of the HPV types with highest carcinogenicity (16, 18, and 45 E7). Exemplary results of Task 1.1 are provided in Figure 1.
Additionally, different types of samples (cervical sample swabs in different transportation systems / buffers) were tested to ensure an easy and common procedure of sampling, transportation, and storage of the samples. Here is to emphasize that the usage of samples stored in ThinPrep™ was enabled by development of a lysis protocol by P2, which is based on a buffer system provided by P3. Advantages of the ThinPrep system are the following: it is a common and well known system used by gynecologists, the samples can be shipped at ambient temperature, stored at 4°C and the stability of a surrogate (cervix carcinoma cell lines such as HeLa and Caski) was shown to be greater than 12 months. Hence, nearly all kinds of analyses (PCR, histology and ELISA testing) scheduled for the clinical studies of the PIPAVIR project can be done from one and the same sample.
Task 1.2: hrE7-ELISA transfer to clinical scale (ongoing)
Goal: P2 will transfer assay design from laboratory scale to clinical scale, i.e. further modifications of assay design will be tested to assure compliance with routine laboratory requirements, such as assay robustness, reproducibility of assay
results and stability of assay components.
Results: The entire assay protocol was tested and optimized. To receive highest possible specificity and sensitivity, different types of conjugates, substrates, buffers and plates were tested. Also, the concentrations of all components were titrated to assure optimal assay performance (ongoing process due to current production of the RabMabs and goat antibodies). Furthermore, a buffer system of P2 was adapted to the assay to achieve least possible unspecific binding and maximum stability of the assay. Stability was shown by stress tests at 37°C up to 6 weeks for all antibody containing components. Also, robustness regarding incubation times and temperatures was checked. Reproducibility was shown by repetition of experiments with at least two operators and performance of the assays at the two development sites (test of the P1 assay strategy at P2 and vice versa, see also Figure 1).
Task 1.3: technical validation of hrE7-ELISA (ongoing)
Goal: For technical validation, E7 RabMabs will be used to stain 30 established cancer biopsies by immunohistochemistry with E7 antibodies (P1). 30 biopsies obtained from hysterectomies (performed due to benign diseases AND HPV DNA-negative) will be used as negative control. In addition, tissue of 30 HPV typed and fully diagnosed cancers will be lysed and lysates will be measured for E7 content. Clinical material will be provided by P4 and P5.
Results: For technical validation, cervical samples were collected at P4 and P5. Partner 4 produced cell line lysates and collected clinical samples (>800) of patients with known clinical status for technical validation of the hrE7-ELISA assay. These samples were not part of the clinical studies but characterized by PCR for HPV-positivity and histologically for progression of the disease. Measurement of the samples showed good correlation between the ELISA result and the status of the disease, i.e. HPV positive and Pap IVa and higher grades for Cytology classified samples were positive for hrE7 protein in the ELISA. Interestingly, measurement of one HPV-PCR negative but histologically abnormal sample resulted in a positive signal for expression of hr E7 protein in the ELISA test. One explanation could be that by integration of the viral DNA into the host genome the PCR target gene was possibly deleted. On the contrary, one HPV-PCR positive but Cytology negative sample gave a positive result in hr E7 protein detection. Hence, the advantage of E7 protein detection became obvious since patients were identified which are not found by testing with PCR or cytology alone.
Deviations from the work plan:
During optimization of the assay it became clear that the critical biological material was not available in quantity and quality as needed. Hence, immunization of goats for polyclonal antibodies was mandatory as well as an industrial production of the RabMabs. The immunization of the animals for production of the polyclonal antibodies was finalized in November 2013 with a final bleeding. Purification and modification of the antibodies (i.e. biotinylation and/or direct conjugation of HRP) was completed in May 2014. For production of the RabMabs, in a first step an evaluation study of the hybridomas was performed (from February to September 2013), subsequently followed by production of two batches of each RabMab in industrial scale (completed f April 2014).
Work Package No: WP2
Plan Start: M01
Plan End: M12
Lead Participant: BIOSYNEX
Actual Start: M01
Actual End: M24
Work package title: Development of the hrE7-Rapid test
Activity Type: Research activities
Participants involved: UIBK, BIOSYNEX, CHARITE
Objectives:
The objectives were to develop, manufacture and validate the performances of the hrHPV-E7 Rapid Test. The aim was to have two test lines: the first one to detect three hrHPV types (HPV-16,-18,-45) and the second one to detect the nine other hrHPV types (HPV-31, -33, -35, -39, -51, -52, -56, -58, -59).
Progress of work:
The work package 2 was divided in three tasks.
Task 2.1 : Setup of hrE7-Rapid test (month 1-6):
Month 1-15: selection of antibodies and test format
The development began with the selection of antibodies for the detection of hr-HPV. Twenty-six different antibodies were tested: twenty-one monoclonal anti HPV-E7 (designated as RabMab) and five polyclonal anti HPV-E7 (designated as Goat). The RabMab Klon 42-3 and 143-7 gave the best performances and were selected to continue the development. Both antibodies were conjugated with various labeling: gold particles, biotine, latex particles, HRP-magnetic particles. Gold particles were selected because they gave the best sensitivity. The polyclonal anti HPV-E7 (Goat 1 and Goat 2) are sprayed on the membrane in the single test line.
An “uncommon” immunochromatography format was selected. A basic rapid test is divided in three parts: the lower part composed of a sample and a conjugate pad, the middle part of a membrane and the upper part of an absorbent pad. Antibodies conjugate with gold particles are usually sprayed and dried on the conjugate pad. In our selected format a liquid one is used instead of a dried one.
The liquid gold conjugate particle is added to the sample prior to its migration on the test. This conjugate/ sample incubation time increases the sensitivity but will probably also reduce the shelf life of the rapid test.
Month 1-6: selection of membrane
Three membranes with different porosity were tested: 5 µm, 8 µm and 15 µm. The membrane regulates the sample migration flux. The porosity of 8 µm was selected: the contact time between test line and the sample is optimum on the recombinant antigens.
Month 11-18: sample buffer formulation
The rapid test buffer is divided in two parts: buffer A and buffer B. The vaginal cervical sample taken on a brush is first put in buffer A which is very corrosive and lyses the cells. The buffer B is then added to neutralize the pH.
The main difficulty of cervical samples is their heterogeneous viscosities. Some samples didn’t migrate totally on the rapid test membrane. The sample buffer B formulation had to be improved.
This point had been discussed at the Munich meeting of August 2013. Due to the time needed to improve the sample buffer formulation, it was decided to start the sampling with the standard buffer used for PCR and PAP testing (named Thinprep buffer). The Thinprep buffer is composed of chemicals which conserves the cells. Therefore, a centrifugation step has to be performed to remove the buffer and concentrate the cells. The use of Thinprep buffer was validated on rapid test. However this option is not user-friendly as it is not correlated with the usual rapid testing which is to be a point of care technique with little to no equipment.
The adding of two chemicals into buffer B improved the sample migration. This new formulation is currently validated on further clinical samples.
It was also decided that the development of the second test line detecting the nine others hrHPV was postponed.
Month 18-24: sensitivity improvement
Discussion with the other partners pointed to a possible lack of sufficient sensitivity. Decision was therefore made to add a secondary conjugate detecting the RabMab conjugate with Goat anti-Rabbit antibodies coupled to gold particles. This secondary conjugate is added immediately before adding the test strip into the conjugate/sample mixture.
Running time has also been increased to 30 minutes, further improving the sensitivity.
Task 2.2: Technical validation of hrE7-Rapid test (month 6-12):
Month 18-24: internal technical validation on improved test version, prototype production and clinical study samples testing
Determination of new detection limits were performed to validate the use of the new, improved, protocol for the clinical study.
Two batches were produced for this purpose: one used for the 1srt smears and one used for the second smears.
In total, 4491 samples were tested.
Task 2.3: Development of self-sampling test (month 6-12):
The Delphi sample buffer was not tested on hrE7-R rapid test
Results: A rapid test prototype detecting HPV-16, -18 and -45 was produced in March 2014.
During the development, the sensitivity and specificity of the hrE7-Rapid test for detection of HPV-16, -18 and -45 E7 were determined on recombinant E7 proteins and cervical cancer cell lines. The result are resumed in the table in the attachement
Preliminary-stability results: R&D prototype was stable six months at +4°C and room temperature. However, a decrease of signal was observed for HPV16-E7. At the end of the project R&D prototype (1rst version) stability was of 16 months at a storage temperature of 2°C.
Work Package No: WP3
Plan Start: M12
Plan End: M36
Lead Participant: MIKROGEN
Actual Start: M12
Actual End: M36
Work package title: Launch of the PIPAVIR prototype kits
Activity Type: Demonstration
Participants involved: MIROGEN, BIOSYNEX, CHARITE, AUTH
Project objectives
• Prototype launch for the hrE7 ELISA
• Prototype launch for the rapid test
• Preparation of a prospective study for the rapid test as screening test in underserved regions
Background information
It is clear that from the initial idea of an in vitro diagnostic device (IVD) to its broad routine application often more than one decade is necessary. Said this, the PIPAVIR consortium is fully aware of the challenges related to the translation of its expertise into a new medical device in the field of persistent HPV infections. Hence, the aim of this workpackage is to launch a prototype IVD for subsequent application in large European trials in cooperation with key opinion leaders (KOLs) and the relevant scientific societies, such as the International Federation of Gynecology and Obstetrics(http://www.figo.org/(se abrirá en una nueva ventana)) International Gynecologic Cancer Society (IGCS)(http://www.igcs.org(se abrirá en una nueva ventana)) Society of Gynecologic Oncology (http://www.sgo.org/ ), European Society of Gynecologic Oncology (http://www.esgo.org/Pages/default.aspx(se abrirá en una nueva ventana)) Deutsche Gesellschaft für Gynäkologie und Geburtshilfe (DGGG), Deutsche Krebsgesellschaft, and the Bundesverband der Frauenärzte, Studiengruppe Kolposkopie (SGK; G-CONE).
Task 3.1: Prototype launch for hrE7- ELISA (ongoing)
Results: Since the critical material was not available in quantity and quality as needed, immunization of goats for polyclonal antibodies as well as industrial production of RabMabs was mandatory.
The immunization of the goats was finished in November 2013, purification and further processing of the goat antibodies including testing of different labeling methods such as direct conjugation with HRP and biotinylation was finished by February 2015 with a total of 4 independent batches for each goat antibody.
Industrial production of the RabMabs was started in September 2013 and finished in June 2015 with a total of 4 independent batches for each RabMab.
During assay optimization (WP1) two different strategies for detection of hrE7 protein of 12 different HPV subtypes were developed. Whereas the detection system of P1 contains three different kinds of wells (i.e. well 1: detection of 16, 18, and 45 E7; well 2: detection of 39, 51, 56, and 59 E7; and well 3: detection of 16, 31, 33, 35, 52, and 58 E7), the strategy of P2 is based on the combination of one screening well, which detects 12 hr E7 types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 E7) and a well for detection of the HPV types with highest carcinogenicity (16, 18, and 45 E7).
In a first step, pre-prototypes of both strategies for the hrE7 ELISA were produced in M27 and M28 in an industrial scale to ensure that an automated production is possible. Thereafter three batches of both, RabMabs and goat antibodies, were used to produce the prototypes of both hrE7 ELISA systems, i.e. three Lots of each system with a total of 15 prototypes. This work was started in M28 and finished in M31.
All produced prototypes were adjusted and tested for validity. Thereafter one prototype of each system was choosen for measurement of hrE7 in the clinical samples of WP4-6.
Deviations from the work plan:
During technical validation of the hrE7 ELISA it became clear that due to the heterogeneity of the cervical smears validity testing for the samples is mandatory. For this purpose, an additional ELISA test system for validity testing was developed. This test system was produced (three prototypes) and tested in parallel with the hrE7 ELISA.
Task 3.2: Prototype launch for hrE7- Rapid test (month 12-15)
Result: Due to the delay in WP1 (reasons discussed in the corresponding chapter), the product launch for the hrE7 rapid test will be postponed. Approximately 5384 ThinPrep samples were collected combining all studies and patient visits (pre-study: 1542; HPV negative validation set: 500; Screening: 1600; Self-sampling: 574; HPVTriage: 344; CytoTriage: 234; HSILCure: 574; CxCa:16). All samples were tested for HPV DNA and genotyped. Study samples were subjected to E7 ELISA when the prototype assay became available.
Task 3.3: Preparation for a prospective study for hrE7 rapid test validation as “Screening test in underserved regions” (Month 24-36)
Instead of Ghana samples were obtained from Ethiopia (Black Lion Hospital, Addis Ababa) from cervical dysplasia patients (n=144) and tested by HPV genotyping and hrE7 rapid test when the prototype became available. This study showed feasibility of the approach.
The planned studies require existing prototypes; accordingly, these studies have to be postponed.
Work Package No: WP4
Plan Start: M01
Plan End: M36
Lead Participant: CHARITE
Actual Start: M01
Actual End: M36
Work package title: Clinical validation of E7 tests as screening tools
Activity Type: Research activities
Participants involved: ALL
Partners 4 and 5 designed a clinical study to investigate and validate the tools developed in PIPAVIR with respect to laboratory-based hrE7 ELISA test and hrE7 Rapid-Test. Full ethical and data safety board approval for the clinical study covered in this WP (screening population) was obtained at both clinical sites. The logistics for sample shipment from/to Partners 4 and 5 and to the partners 1, 2 and 3 was established. Clinical trials according to the follow-up time were initiated. Recruitment finished of all 1200 patients necessary for the first trial (Screening study). The following sub-tasks are ongoing: i) Collection and asservation of clinical material, ii) compilation of anamnestic and clinical data of the recruited patients in a data capture website (designed by Partner 5), iii) characterization of clinical material for cytology and HPV genotyping results.
Task 4.1: Preparation of study documents materials, teaching of doctors and nurses ( M1-M5)
During this period the following activities were performed:
• In collaboration with Partner 4 two Screening Studies protocols were defined by Partner 5. The outcome has been reported in the PIPAVIR protocol internal document.
• The application to Bioethics Committee of the Medical School of Aristotle University of Thessaloniki was prepared and submitted. Approval was received.
• The AUTH team was established and trained. The team consists of the principal investigator, 2 gynecologists, 1 cytopathologist, 1 lab technician, 1 assistant and three informatics scientists. A step-by-step document was created and distributed to each member of the team in order to help them know exactly the tasks they have to do.
• Materials for cytopathology evaluation, sample collection and transfer were purchased; patient forms were created; A google document for monitoring sample tracking was created and was shared with Partner 4 and the other partners.
• The first version of the Data Capture System (DCS) was developed and tested by the informatics scientists of Partner 5. DCS is a multi-user, multi-role web application allowing for registration of the subjects and reporting of the results of cytology, colposcopy, histology, HPV-DNAg test, hrE7-ELISA and hrE7-Rapid. Data visualization and export are also supported. All data is anonymous. Instructions of use were provided by Partner 5 to all PIPAVIR users.
Task 4.2 : Prospective study for hrE7-ELISA validation as screening test
During this period the following activities were performed by Partner 5:
• April 2014 - May 2014: Completion of subject recruitment for the screening study. 146 subjects were recruited, resulting to a total of 1068 from the start of the project.
• September 2014 – June 2015: All subjects were called for their follow-up visits. 269 subjects came for a second visit.
• Sept 2014- Jan 2015: 190 subjects were recruited for the self-sampling study. In this case, in addition to the physician sample, women collected a self-sample using the Evalyn device.
• All women were recruited at the Family Planning Center of the “Hippokratio” Hospital of Thessaloniki, where the 4th Department of Obstetrics and Gynecology of AUTH, is placed. For each subject the AUTH team performed the following tasks:
• Sample collection and subject registration to the DCS
• Cytology evaluation and reporting of results to the DCS
• Preparation of samples for HPV-DNAg and hrE7-ELISA. These samples were sent for evaluation to Partner 4 and Partner 2 respectively.
• Review of test results. For the subjects with positive cytology and/or HPV-DNAg result a colposcopy examination was conducted. Colposcopy results and follow-up data was reported to the DCS.
For all subjects an additional sample was collected to evaluate the hrE7-Rapid test. These samples were sent to Partner 3 for analysis.
Cytology evaluation for additional samples
In addition to the samples collected at Partner 5, cytology evaluation was conducted also for the pre-study and prospective studies samples from Partner 4.
Detection of E7 with the hrE7 ELISA
All samples were shipped to P2. For each sample, the total volume and the volume of the cell pellet including color was determined. All samples were measured from M34 to M36 at P2 for presence of hr E7 with both systems and additional validity testing was performed. Hence, each sample was measured six times and the results were transferred into the DCS. The following E7 tests were performed: Well1 (16/18/45), Well2, Well3, screen, 16/18/45.
Data extraction, validation and Statistical analyses
Upon completion of data collection from both partners 4 and 5, and reporting of results from partners 1, 2 and 3, the appropriate datasets were extracted from the DCS. In total, since the beginning of the project, 1311 women were recruited in the screening study and 1600 samples were collected and analyzed. For the self-sampling study, 384 women were recruited and 574 samples were collected and analyzed. Following data extraction, data were validated to ensure consistency with the protocol and completeness. Finally, statistical analyses was conducted to assess: thresholds, sensitivity, specificity, PPV and NPV for each one of the following E7 tests: Well1 (16/18/45), Well2, Well3, screen, 16/18/45.
Statistically significant associations between E7 positivity in the test formats Well 1, Well 3, and 16/18/45 could be detected, providing proof-of-principle for the E7 test. The calculation of sensivity, specificity, PPV and NPV has been performed for these tests in the screening study which yielded preliminary data. Currently, a more detailed data evaluation is being performed by partners 1, 2, 4 and 5, involving model calculations with different cut-off values for E7 protein concentrations. The results of the more extended data analysis will be available by the end of December 2015.
Deviation from work plan
Partner 5 recruited more subjects than originally planned. According to the original plan, AUTH would recruit approximately 500 subjects for the screening study, and 65 subjects for the self-sampling study. However, due to the high number of women coming for screening to the Family Planning Center, it was decided that AUTH would focus on recruitment for the screening study instead of recruitment for the triage and treatment studies.
During this period the following activities were performed by partner 4:
For the prospective Screening study 243 patients were recruited at MVZ Im Mare, Kiel Germany in a cooperation with Charite Berlin. Of those 123 returned for a planned follow up visit. For the self-sampling feasibility study 193 samples were obtained at Charite and also collected in Ethiopia as a feasibility for low resource settings. Sample sets were combined with the screening study samples and self-sampling samples, respectively, collected by partner 5 (see below).
Partner 4 was responsible for HPV genotyping (by WHO reference test MPG-Luminex) for 18 high-risk and 9 low-risk HPV types. HPV determinations were performed for all ThinPrep samples collected during all studies.
Task 4.3 : Prospective study for hrE7-Rapid validation as screening test
During this period the following activities were performed by Partner 5:
• April 2014 - May 2014: Completion of subject recruitment for the screening study. 146 subjects were recruited, resulting to a total of 1068 from the start of the project.
• September 2014 – June 2015: All subjects were called for their follow-up visits. 269 subjects came for a second visit.
• Sept 2014- Jan 2015: 190 subjects were recruited for the self-sampling study. In this case, in addition to the physician sample, women collected a self-sample using the Evalyn device.
• All women were recruited at the Family Planning Center of the “Hippokratio” Hospital of Thessaloniki, where the 4th Department of Obstetrics and Gynecology of AUTH, is placed. For each subject the AUTH team performed the following tasks:
• Sample collection and subject registration to the DCS
• Cytology evaluation and reporting of results to the DCS
• Preparation of samples for HPV-DNAg and hrE7-ELISA. These samples were sent for evaluation to Partner 4 and Partner 2 respectively.
• Review of test results. For the subjects with positive cytology and/or HPV-DNAg result a colposcopy examination was conducted. Colposcopy results and follow-up data was reported to the DCS.
• For all subjects an additional sample was collected to evaluate the hr-E7-Rapid test. These samples were analyzed by Partner 3.
Cytology evaluation for additional samples
In addition to the samples collected at Partner 5, cytology evaluation was conducted also for the pre-study and prospective studies samples from Partner 4.
Data extraction, validation and Statistical analyses
Upon completion of data collection from both partners 4 and 5, and reporting of results from partners 1, 2 and 3, the appropriate datasets were extracted from the DCS. In total, since the beginning of the project, 1311 women were recruited in the screening study and 1600 samples were collected and analyzed. For the self-sampling study, 384 women were recruited and 574 samples were collected and analyzed. Following data extraction, data were validated to ensure consistency with the protocol and completeness. Finally, statistical analyses was conducted to assess: thresholds, sensitivity, specificity, PPV and NPV for the hr-E7 Rapid test.
The clinical sensitivity of the E7 rapid test was lower than expected and the initial evaluation of the data did not provide significant associations between E7 positivity and the clinical data. Currently a more detailed data evaluation is performed by partners 3, 4 and 5. The results of the more extended data analysis will be available by the end of December 2015.
Deviation from work plan
Partner 5 recruited more subjects than originally planned. According to the original plan, AUTH would recruit approximately 500 subjects for the screening study, and 65 subjects for the self-sampling study. However, due to the high number of women coming for screening to the Family Planning Center, it was decided that AUTH would focus on recruitment for the screening study instead of recruitment for the triage and treatment studies.
Work Package No: WP5
Plan Start: M01
Plan End: M34
Lead Participant: AUTH
Actual Start: M01
Actual End: M34
Work package title: Validation of E7 tests as triage test
Activity Type: Research Activities
Participants involved: All
Partners 4 and 5 designed two clinical studies to investigate and validate the tools developed in PIPAVIR with respect to laboratory-based hrE7 ELISA test and the hrE7 Rapid-Test. Full ethical and data safety board approval for the clinical studies covered in this WP (triage tests) was obtained at both clinical sites. Clinical trials for testing in a triage setting for HPV positive and cytologically positive women were initiated with first recruitment and ongoing recruitment. The following sub-tasks are ongoing: i) Collection and asservation of clinical material, ii) compilation of anamnestic and clinical data of the recruited patients in a data capture website (designed by Partner 5), iii) characterization of clinical material for cytology and HPV genotyping results. The logistics for sample shipment from/to Partners 4 and 5 and to the partners 1, 2 and 3 was established.
Task 5.1: Preparation of study documents materials, teaching of doctors and nurses
Τhis task is similar to T4.1. During this period the following activities were performed:
• Definition of the two Triage Studies protocols, in collaboration with Partner 4. The outcome has been reported in the internal “PIPAVIR protocol“ document.
• The application to Bioethics Committee of the Medical School of Aristotle University of Thessaloniki was prepared and submitted. Approval was received.
• Deviations from the sample collection procedure of T4.2 and T4.3 were identified and the AUTH team (see Task 4.1) was informed accordingly.
• The DCS (see Task 4.1) was updated to incorporate the design of the triage studies.
Task 5.2: validation of E7 tests for triage of hrHPV-positive women & Task 5.3: validation of E7 tests for triage of women with abnormal PAP test
The following activities were performed by Partner 4:
Patient recruitment was further performed for the validation sample sets and the prospective studies. For validation purposes of technical developments lysates from different HPV positive and negative cultured cell lines were generated as positive control samples. Altogether 1540 pre-study samples from patients with different dysplasia grades and 500 ThinPrep samples that tested HPV-negative were collected as technical validation sets. The HPV negative samples were used for technical cutoff determination by partner 2. Pre-study samples are still under investigation.
Recruitment was done mainly at Charité Campuses Berlin, and at a primary clinical center in Kiel (MVZ Im Mare). Validation set HPV negative samples were obtained from a gynecology clinic in Wolfsburg, Germany. Some samples for triage and cure studies were also recruited by partner 5.
In the HPVTriage study 229 patients were recruited and 32 returned for a follow-up visit because of of negative clinical findings. In the CytoTriage study 171 patients were recruited and 52 returned for follow up due to negative findings for verification. These samples were combined for the analysis of results with the corresponding samples collected by partner 5 (see below).
All samples were sent to partner 2 for hrE7 ELISA testing. For all samples a second smear sample was collected and sent to partner 3 for analysis by hrE7 rapid test. For self-sampling studies and for independent control testing hrE7 test was also performed by partner 4 in a good proportion of study samples and pre-study samples.
Partner 4 was responsible for HPV genotyping (by WHO reference test MPG-Luminex) for 18 high-risk and 9 low-risk HPV types. This amounted to more than 5384 HPV determinations in all samples collected during all studies.
For each patient the following tasks were performed:
• Sample collection and subject registration to the DCS
• HPV testing, evaluation of sample adequacy, and reporting of results to the DCS
• Preparation of cytological slides for reading at AUTH and aliquot sending to partner 1, 2 and 3 for hrE7-ELISA and rapid test, respectively.
• Review of test results. For the subjects with positive cytology and/or HPV-DNAg result a colposcopy examination was conducted if not done at inclusion. Colposcopy results and follow-up data were reported to the DCS.
• Retesting of samples with non-concordant result in the HPV and hrE7 tests.
Recruitment of 56 subjects in the TriageHPV+ and of 10 subjects into the Triage Cyto+ studies. These subjects were called for a second follow-up visit after 6 months. All women were recruited at the Family Planning Center of the “Hippokratio” Hospital of Thessaloniki, where the 4th Department of Obstetrics and Gynecology of AUTH, is placed. For each subject the AUTH team performed the following tasks:
o Sample collection and subject registration to the DCS
o Cytology evaluation and reporting of results to the DCS
o Preparation of samples for HPV-DNAg and hrE7-ELISA. These samples were sent for evaluation to Partner 4 and Partner 2 respectively.
o Review of test results. For certain subjects, according to the specific study protocol, a colposcopy examination was conducted. Colposcopy results and follow-up data were reported to the DCS.
o For all subjects an additional sample was collected to evaluate the hrE7-Rapid test. These samples were sent to Partner 3 for analysis.
• Cytology evaluation for samples from Partner 4 Maintenance and support of the DCS
Detection of E7 with the hrE7 ELISA
All samples were shipped to P2. For each sample, the total volume and the volume of the cell pellet including color was determined. All samples were measured from M34 to M36 at P2 for presence of hr E7 with both systems and additional validity testing was performed. Hence, each sample was measured six times and the results were transferred into the DCS.
Data extraction, validation and Statistical analyses: Upon completion of data collection from both partners 4 and 5, and reporting of results from partners 1, 2 and 3, the appropriate datasets were extracted from the DCS. In total, since the beginning of the project, 281 women were included into the TriageHPV study and a total of 346 samples were collected and analysed. For the TriageCyto study 181 women were recruited and 232 samples were collected and analysed. Following data extraction, data were validated to ensure consistency with the protocol and completeness. Finally, statistical analyses was conducted by Partner 5 to assess: thresholds, sensitivity, specificity, PPV and NPV for each one of the following tests: Well1 (16/18/45) , Well2, Well3, screen, 16/18/45 and hrE7 Rapid Test.
Statistically significant associations between E7 positivity in the test formats Well 1, Well 3, and 16/18/45 could be detected, providing proof-of-principle for the E7 test. The calculation of sensivity, specificity, PPV and NPV has been performed for these tests which yielded preliminary data. Currently, a more detailed data evaluation is being performed by partners 1, 2, 4 and 5 involving model calculations with different cut-off values for E7 protein concentrations. The clinical sensitivity of the E7 rapid test was lower than expected and the initial evaluation of the data did not provide significant associations between E7 positivity and the clinical data. Currently a more detailed data evaluation is performed by partners 3, 4 and 5. The results of the more extended data analysis will be available by the end of December 2015.
Deviation from the work plan: The number of subjects that were recruited by Partner 5 for the TriageCyto+ study were less than originally planned (~60). The Family Planning Center receives mostly women for screening and not referral. For this reason partner 5 focused merely to recruitment of subjects for the screening and self-sampling studies, as reported in Wp4.
Work Package No: WP6
Plan Start: M01
Plan End: M34
Lead Participant: AUTH
Actual Start: M01
Actual End: M34
Work package title: Validation of hrE7 tests as “test of cure”
Activity Type: Research activities
Participants involved: All
Partners 4 and 5 designed two clinical studies to investigate and validate the tools developed in PIPAVIR with respect to laboratory-based hrE7 ELISA test and the hrE7 Rapid-Test. Full ethical and data safety board approval for the clinical studies covered in this WP (test of cure) was obtained at both clinical sites. Clinical trials for test of cure in patients with HSIL or cervical cancer were initiated with first recruitment and recall of patients in the cure HSIL trial. Problematic recruitment of patients for the cure CxCa trial due to lack of accessibility. The logistics for sample shipment from/to Partners 4 and 5 and to the partners 1, 2 and 3 was established.
Task 6.1: Preparation of study documents, materials, teaching of doctors and nurses
Τhis task is similar to T4.1 and T5.1. During this period the following activities were performed:
• Definition of the two Test of Cure studies protocols, in collaboration with Partner 4. The outcome has been reported in the internal “PIPAVIR protocol“ document.
• The application to Bioethics Committee of the Medical School of Aristotle University of Thessaloniki was prepared and submitted. Approval was received.
• The Data Capture System (DCS) was updated to incorporate the design of the triage studies.
Task 6.2 : Validation of E7 tests as “Test of cure” in patients with HSIL & Task 6.3 Prospective study for hrE7-ELISA validation as “Test of cure” in cervical cancer patients
The following activities were performed by Partner 4:
Recruitment of patients into the HSILCure study was done mainly at Charité Campuses Berlin, and fewer at a primary clinical center in Kiel (MVZ Im Mare). Some HSILcure study patients were also recruited by partner 5 (see below).
For the HSILCure study 206 patients were recruited of whom 106 returned for Visit 2, 103 for Visit 3, 64 for Visit 4 (an interim visit due to common practice) and 42 for Visit 5). Follow up is still ongoing in this study in some patients.
For CxCaCure the available patient numbers (n=14, n=2 for visit 2) for recruitment were too low due to a change in referral practices to our clinics.
For each patient the following tasks were performed:
• Sample collection and subject registration to the DCS
• HPV testing, evaluation of sample adequacy, and reporting of results to the DCS
• Preparation of cytological slides for reading at AUTH and aliquot sending to partner 1, 2 and 3 for hrE7-ELISA and rapid test, respectively.
• Review of test results. Subjects treated by conization were followed up by colposcopy, cytology and HPVg Data were reported to the DCS.
• Retesting of samples with non-concordant result in the HPV and hrE7 tests.
All samples were sent to partner 2 for hrE7 ELISA testing. For all samples a second smear sample was collected and sent to partner 3 for analysis by hrE7 rapid test.
Partner 4 was responsible for HPV genotyping (by WHO reference test MPG-Luminex) for 18 high-risk and 9 low-risk HPV types. This amounted to more than 5384 HPV determinations in all samples collected during all studies.
Deviation from work plan
The number of subjects recruited by partner 4 for the HSILCure study was higher than initially planned. This was to compensate for lower recruitment by partner 5 and to enhance the power of the analysis.
The CxCaCure study had to be cancelled because due to restructuring of the Gynecology department only very few patients were referred to the clinic during the recruitment period.
• Recruitment of 14 women for the CureHSIL study. All women were recruited at the Family Planning Center of the “Hippokratio” Hospital of Thessaloniki, where the 4th Department of Obstetrics and Gynecology of AUTH, is placed. For each subject the AUTH team performed the following tasks:
o Sample collection and subject registration to the DCS
o Cytology evaluation and reporting of results to the DCS
o Preparation of samples for HPV-DNAg and hrE7-ELISA. These samples were sent for evaluation to Partner 4 and Partner 2 respectively.
o Review of test results. For certain subjects, according to the specific study protocol, a colposcopy examination was conducted. Colposcopy results and follow-up data was reported to the DCS.
o For all subjects an additional sample was collected to evaluate the hrE7-Rapid test. These samples were sent to Partner 3 for analysis.
• Cytology evaluation for the samples from Partner 4
• Maintenance and support of the DCS
Detection of E7 with the hrE7 ELISA and hrE7 rapid test
All samples were shipped to P2. For each sample, the total volume and the volume of the cell pellet including color was determined. All samples were measured from M34 to M36 at P2 and P3 for presence of hr E7 with both hrE7 Rapid test, hrE7 ELISA systems and additional validity testing was performed. Hence, each sample was measured six times at P2 and once at P3 the results were transferred into the DCS.
Data extraction, validation and Statistical analyses: Upon completion of data collection from both partners 4 and 5, and reporting of results from partners 1, 2 and 3, the appropriate datasets were extracted from the DCS. In total, since the beginning of the project, 220 women were recruited in the study and 595 samples were collected and analysed. Following data extraction, data were validated to ensure consistency with the protocol and completeness. Finally, statistical analysis was conducted to assess: thresholds, sensitivity, specificity, PPV and NPV for each one of the following hrE7 Rapid tests: Well1 (16/18/45), Well2, Well3, screen, 16/18/45.
Statistically significant associations between E7 positivity in the test formats Well 1, Well 3, and 16/18/45 could be detected, providing proof-of-principle for the E7 test. The calculation of sensivity, specificity, PPV and NPV has been performed for these tests in the screening study which yielded preliminary data. Currently, a more detailed data evaluation is being performed by partners 1, 2, 4 and 5 involving model calculations with different cut-off values for E7 protein concentrations. The clinical sensitivity of the E7 rapid test was lower than expected and the initial evaluation of the data did not provide significant associations between E7 positivity and the clinical data. Currently a more detailed data evaluation is performed by partners 3, 4 and 5. The results of the more extended data analysis will be available by the end of December 2015.
Deviation from the work plan: The number of subjects that were recruited for the Cure studies by partner 5 was less than originally planned. As the Family Planning Center receives high number of women for screening and not referral, it was decided early in the project, that partner 5 focuses in the recruitment of subjects for the screening and self-sampling studies (see WP4) instead of the triage (WP5) and cure (WP6) studies.
Work Package No: WP7
Plan Start: M01
Plan End: M36
Lead Participant: UIBK
Actual Start: M01
Actual End: M36
Work package title: Dissemination and Exploitation
Activity Type: OTHER
Participants involved: ALL
The Dissemination of the PIPAVIR concept and of preliminary project results has been carried out at several levels in the reporting period. Of particular importance is the participation of PIPAVIR scientists in national and international symposia, giving topical presentations focused on the role of papillomaviruses in cervical carcinoma and on clinical procedures for cervical cancer screening. It can be noted, that the presentations by PIPAVIR scientists at those occasions (see attached list of meetings attended) created a high level of interest by national and international key opinion leaders and has been used to organize collaborations with a couple of new clinical sites, who are interested in using the PIPAVIR in vitro diagnostic systems as soon as they are available. This will enable a massive spread of the technology. The acceptance of the new diagnostic principle in the field will be considerably speeded up by these activities. On the other hand, PIPAVIR scientists, in particular of the coordinator, have spread the information about the PIPAVIR concept and preliminary results in the lay press, where several featured articles have been published. These activities will make the general public familiar with the development of a new and efficient test for rational diagnosis of cervical pre-cancer, which is a topic of considerable public health interest and is also of immediate interest to a large part of the female population. Taken together, the dissemination activities of PIPAVIR have met all the expectations as set out in the description of work and we expect a further enhancement of these activities, once the clinical validation of the test has been achieved. Exploitation activities are currently still in the phase of prototype development and we expect more substantial exploitation activities, once the prototypes are available and clinically validated.
Task 7.1: Preparation of a communication plan for dissemination of the project
Achieved
Task 7.2: Implement a project design, information materials, and a public interest website
Achieved.
Task 7.3: Disseminate progress and results via classic and new media
Achieved
Task 7.4: Disseminate progress and final results on scientific meetings and through publications
The PIPAVIR trials programme was presented at workshops of the German consortium for colposcopy and several international conferences (as indicated in the attached dissemination table). Preliminary results were presented at Eurogin 2015 and different scientific meetings, such as. ECCMID 2014 and 2015, ESCV 2014, Eurogin 2015, and IPV 2014.
• there have been several articles in the public press concerning the PIPAVIR-Project (mostly in Austria)
• there have been Workshops, in which partner Partner 4 participated and represented the PIPAVIR Consortium
• Our project web-site can be found under www.pipavir.com. This site provides information about the project itself, the partners involved, the planned work and the progress achieved. The homepage is also used as a platform for publications (press releases as well as scientific publications) and as a browser-based communication platform (partners are able to log in and work in a password-secured internal area). On this web space, presentations of the various meetings are stored, so all PIPAVIR partners have access to all information. The interim reports and the final reports will be made available to the consortium also throughout this site. Besides this, this tool helps coordinating the information flow among the partners, specifically regarding the project meetings.
• P1 and P2 presented the results of the hrE7 ELISA development as poster presentations or oral presentations at different meetings, i.e. ECCMID 2014 and 2015, ESCV 2014, Eurogin 2015, and IPV 2014
Task 7.5: Collaboration with existing initiatives to promote women’s health
Achieved.
Task 7.6: Development of recommendations for physicians
Achieved.
Task 7.7: Preparation for CE-approval and filing (month 18-36)
Based on the results of the clinical studies according to WP5 & WP6, P2 and P3 will file a CE-registration to get the CE-approval for both products. This is a must for the commercialization of the assay as IVD in Europe. To promote commercial exploitation of project results, the cost effectiveness of the diagnostic kit will be evaluated.
Task 7.8: Product Launch and evaluation of perception of hrE7 screening by patients and doctors (month 3-36)
After CE-marking IVD will be launched. The IVDs will be installed at reference centers, key opinion leaders and first customers. P1, P4 and P5 jointly will measure the acceptability of this new test format by patients and doctors. Each medical diagnostic method need to be accepted by the patients and physicians. In order to evaluate the perception of the test by the patients and the interpretation by the doctors we will use a standardized questionnaire. Patients and doctors will be requested to answer the questionnaire after use of the hrE7 tests. A score for patient and doctor acceptability of the test will be calculated. P2 has started with the preparation of all material needed for CE-registration in M20. A business plan was prepared as well as the cost effectiveness of the diagnostic kit was evaluated. By participation of P2 at scientific meetings and symposia from M4 on, key opinion leaders and potential customers were introduced to the novel hr E7 ELISA system and the acceptability of the test system was assessed.
Deviations from the work plan:
Due to the delay in WP1, the measurement of the clinical samples from WP4-6 and the analysis of the clinical study results were also delayed. Hence, the preparation and documentation for CE-filing was not completed by M36. Since the material has to be accurate prepared and verified the product lunch was postponed to beginning of 2016.
Potential Impact:
4.1.4 Impact
The potential impact (including the socio-economic impact and the wider societal implications of the project so far) and the main dissemination activities and exploitation of results (not exceeding 10 pages).
4.1.4.1 Summary
MIKROGEN is well established in the field of diagnostic tests for infectious diseases in humans and performs active research into innovative products. The aim of the current proposal is to clinically validate a new biomarker test (referred to as PIPAWELL E7 test i.e. the selected brand name for the hrE7-ELISA developed in the PIPAVIR project) for early detection of cervical precancerous lesions and cancers. Currently used molecular diagnostic tools can detect human papillomavirus (HPV) DNA or RNA in cervical smears but fall short of discriminating between transient HPV infections, which can spontaneously regress, and persistent infections, which have a high chance to progress to high-grade squamous intraepithelial lesions (HSIL). MIKROGEN has developed a new diagnostic test that allows detection, by sandwich-ELISA, of HPV E7 proteins in cervical smears, as specific biomarkers for persistent HPV infections with unfavourable prognosis. The E7-ELISA developed by MIKROGEN uses a robust and appropriate analytical method to determine expression levels of viral E7 oncoproteins, which are causally linked to CINIII/cervical carcinoma as a pertinent clinical endpoint. The validation procedure has been curtailed to provide evidence for high analytical validity, appropriate sensitivity and specificity, as well as clinical validity and utility, and therefore will allow to develop new diagnostics with exceptional prognostic and predictive power; the clinical performance of the new diagnostic device will be validated and tested against existing standards, such as cytology, HPV testing and p16 immunohistochemistry. The HPV oncoproteins are disease-related biomarkers and as such will allow an assessment of the risk to develop cervical cancer. The biomarkers can also be used for disease monitoring and prognosis. The PIPAWELL E7 test will enable improved clinical decisions which will lead to better health outcomes, such as a reduction in overtreatment. Accordingly, the HPV oncoprotein biomarkers proposed here have a high potential for short term uptake into clinical practice. Due to the high cost efficiency of the PIPAWELL E7 test, in combination with the reduced requirement for repeated testing, the project will contribute to the sustainability of health care systems. MIKROGEN has achieved appropriate IP protection for the E7-ELISA technology and owns all rights to use it, through both licensing agreements and own patent applications, providing full freedom to operate. The longstanding (>25 years) expertise of MIKROGEN in the field of producing and marketing diagnostic devices for infectious diseases provides excellent conditions for successful commercial exploitation. Based on the potential of the technology to meet a real clinical need, it can be anticipated that the ability of MIKROGEN to provide and market such superior diagnostic tools will create great business opportunities. With these objectives, PIPAWELL corresponds fully to the topic description in the work programme topic PHC 12.
4.1.4.2 Impact on health and welfare of European citizens
To date, screening for persistent HPV infections is based on cytological or smear samples that are sent to specialized laboratories for processing and microscopical and molecular testing. This means a time delay for further measures and clinical treatment. A second appointment is necessary for the patient for triage of equivocal cytology results, or for treatment. Results from standard screening methods suffer from low sensitivity (as in cytology) or low specificity (as in HPV DNA testing). A combination of both methods is regarded to be necessary to reach high sensitivity and specificity. However this also means higher cost and effort. A test based on E7 protein would have intrinsically a higher specificity for high grade disease since this is related to upregulation of E7 expression. In addition, a format of a rapid test may be used to an onsite testing and treatment in the same visit ("see and treat") reducing the number of visits & uncertainty on the result, and thereby accelerating treatment. This will lead to a reduction of cancer cases due to earlier detection of relevant premalignant lesions, and thereby confront a major threat to public health. The physician may decide on treatment or not, according to the result of the E7 test. That means also a reduction of overtreatment in cases that are not progressed and thus do not overexpress E7 although an infection is prevalent. Less invasive treatment and avoidance of overtreatment will lead to a reduction in perinatal problems, because women who have lost cervical tissue after deep or repeated conisations have a 5 fold higher risk for premature birth. In these aspects, the project meets real clinical and public health needs. Use of an E7-based test will also reduce follow-up appointments when no relevant lesion is found in spite of a persistent infection. In the case of uncertain colposcopical findings, a E7 rapid test can clarify if there is a high grade lesion present or not. Taken together, the impact on clinical management will be impressive, reducing number of visits, treatment procedures, and stress and anxiety. It will in turn lead to less overtreatment and less perinatal problems due to cervical insufficiency.
4.1.4.3 Markets and potential users
The total addressable HPV diagnostic market has a potential of 350 Mio. € world-wide with an annual increase of 6% as shown in Table 1. The biggest market represents the US (60%), followed by Europe (25%) and the rest of the world (15%) (see Figure 1). The HPV business creates new and huge market opportunities for MIKROGEN and provides a step forward to rational and focused diagnostic procedures.
Europe has no integrative public health system. Unfortunately, each country has its own public health and re-imbursement system. In Germany as well as in other European countries (e.g. Netherlands) reorganization from traditional Pap Screen to Primary HPV Screening concerning cervical cancer prevention takes places in near future (beginning of 2016). This will be a door opener into the HPV diagnostic market for the PIAWELL E7 test.
Due to MIKROGENs' long tradition in the foreign markets, there are good and reliable contacts to health authorities which will help to overcome reimbursement hurdles. As an example, the reimbursement of HPV related diagnostics in Germany differs considerably for patients enrolled in public health insurance (EBM) and patients enrolled in private health insurance (GOÄ, see Table 2). For the PIAWELL E7 test no reimbursement code exists to date, but due to the upcoming reorganization of cervical cancer screening new reimbursement codes will be generated. These facts will be considered in the PIPAWELL commercialisation strategy, facilitated by the long standing sales experience of MIKROGEN in the most important health markets.
Current testing methods, while reliable in detecting the presence of HPV infection, are unreliable in terms of predicting the likelihood of a patient developing cervical cancer. Neither Pap nor HPV tests provide definitive results for the actual risk for cervical cancer. The diagnostic market for cervical carcinoma detection is characterised by economically strong players, pushing HPV DNA testing as primary diagnostic measure in screening populations. This is contrasted by key opinion leaders in cervical cytology, who insist that cytology should remain the first-line diagnostic test. At the same time, conventional cytology is being questioned in an increasing number of European countries, leaving a vacuum for new tests, such as the PIPAWELL E7 test that provide identification of patients at risk beyond HPV DNA positivity.
Cervical cancer early diagnosis and prevention reflects an unmet medical need throughout Europe
Whereas the traditional way of cervical cancer screening is based on cytological analysis (PapSmear) followed by more laborious follow-up procedures for women with abnormal cytology, the introduction of HPV DNA testing (with its higher sensitivity but lower specificity) has necessitated a readjustment of treatment regimes and screening algorithms. Some European countries have opted for HPV testing as primary screening tool and consider to abandon cytological analysis altogether, due to inherent lack of precision of conventional cytology. Germany has initiated to re-organize towards regular invitation and optional HPV testing 5 yearly instead of cytology yearly. On the other hand, the inherent inability of HPV DNA testing to discriminate between clinically irrelevant transient infections and ongoing tumor progression has led key opinion leaders to state the urgent need for new molecular tests with higher clinical relevance, i.e. tests which can at the same time inform about HPV infection and the presence of a high grade lesion, such as CIN2+. Since the expected high rate of referral of HPV positive patients may overload current triage systems like colposcopy, better biomarkers will be urgently needed.
Many competitors in Europe and worldwide develop additional assays aiming at the detection of persistent HPV infections by detection of viral DNA, like HC-II adding an extra HPV16, 18, 45 probe (Qiagen), Amplicore of Roche using PCR, or chip-based DNA analysis (Greiner bio-one). Other companies try to enhance specificity by using mRNA as target (Aptima Assay by Gen-Probe and the HPV Proofer), by using E6 protein as target (prototype test by Arbor Vita) or try to enhance rapidness of assays (multiplexed RT-PCR, in development). In addition, identification of progression markers for differentiation of regressing and potentially progressing lesions is done. Detection of L1 in cytology smears is tested as a regression marker (Cytoimmune), detection of cell cycle proteins like p16/Ki-67 and MCM5 (Roche), and methylation patterns in viral and cellular genes (in development) are means to evaluate progressive potential of lesions. The HPV-related diagnostic tests competing with PIPAWELL E7 test are summarized in Table 3. All mentioned current testing methods, while reliable in detecting the presence of HPV infection, are unreliable in terms of predicting the likelihood of a patient developing cervical cancer. Neither Pap nor all mentioned HPV tests provide definitive results for risk for cervical cancer. The PIPAWELL E7 test solves this issue and provides a new and superior tool in HPV diagnostics.
PIPAWELL marketing strategy
MIKROGEN’s PIPAWELL E7 test includes a new diagnostic technique which improves efficiency and effectiveness of HPV testing radically. Instead of detecting viral DNA/RNA it identifies the existence of oncoprotein E7. Overexpression of this oncoprotein is a critical and necessary step toward HPV-related cancer. As false-positive results are prevented and self-resolving HPV types are not tested as positive, the number of patients in need for additional treatment can be dramatically reduced. The main outcome of the project is an innovative and cost efficient diagnostic test kit for the diagnosis of persistent infection with high risk HPVs. Using a cost-efficient, easy-to-use and highly informative diagnostic tool will allow entry into a market, which is already highly competitive, although competitors so far rely on HPV DNA/RNA testing or cytological analysis (Pap test etc.), which are indirect, less informative, more laborious and much more expensive than the technology described here. This provides MIKROGEN with a unique selling proposition for the PIPAWELL test (Figure 2). Instead of a complex matrix of complementary yet unprecise and uncertain diagnostic procedures, an objective quantification of a disease-related predictive biomarker (i.e. the E7 oncoproteins detected by the PIPAWELL E7 test) will save cost and time.
User groups and how to convince them
In contrast to the indirect tests provided by the state of the art (see above, Table 3), PIPAWELL follows a novel approach and uses proprietary technology not available anywhere else in the world. This unique combination of expertise will allow the development of a highly competitive diagnostic tool. This provides a unique selling position. The combination of two by MIKROGEN developed assay systems represents a diagnostic breakthrough for the cervical cancer screening which allows a highly sensitive detection of cervical cancer and pre-cancerous stages. This reflects a great milestone in cervical cancer prevention and results in a dramatic reduction of cervical cancer cases world-wide.
This situation provides clear opportunities for market introduction of the PIPAWELL E7 test. The final users of the PIPAWELL product will be private laboratories, clinics, universities, pathologists/cytologists, reference labs and key opinion leaders. Different user groups will be convinced to buy the test based on distinct advantages of the PIPAWELL E7 test, as follows:
• Patients will be convinced to pay for the test out of their own pocket, because the test gives a more conclusive result discriminating low-grade from high-grade disease, thereby helping to decide for treatment or watchful waiting. Further arguments for patients to pay for the test are related to taking away uncertainty from both patient and physician and a reduction of overtreatment.
• Gynecologists will be convinced to recommend and prescribe the test as it may generate income when cytology is not recommended anymore and HPV testing is done by diagnostic labs. At the same time they will have something to offer to patients who are anxious because of a positive HPV result. At the same time the test will offer an improved triaging method for pregnant women. Additionally, it will be easy for gynecologists to explain to patient “no oncoprotein – no cancer”. This is different with DNA due to low specificity or cytology due to low sensitivity. Moreover, the PIPAWELL E7 test may be run in the office and generate direct income to the gynecologist.
• To convince key opinion leaders (KOL) to promote the test to colleagues, conclusive data showing higher specificity as compared to HPV and cytology will be needed, ideally derived of a longitudinal trial testing progressive potential of E7 positive vs negative intermediate grade dysplasia, similar to the studies described in this proposal (see below, Work Package 3). Moreover, the number of clinical interventions can be reduced by safely watching E7 negative lesions.
• Health insurance companies will be convinced to reimburse the test by the opportunity to have, at the same time, cost savings, less interventions, better care to customers, less follow-up visits and less costly triaging methods. This will require cost efficiency studies in comparison to current screening system and intervals, which will be performed by PIPAWELL. The potential of the test to advance women’s’ health will be used as marketing tool for the insurance provider.
• Private and public diagnostic laboratories will create significant additional income from the possibility to offer PIPAWELL E7 test as a conclusive screening or reflex test in conjunction with HPV testing. Additional advantages for the user are easy follow –up testing from same sample material (Thinprep) like cytology or DNA / RNA based assays. In addition, further increases of income can be expected from the use of the PIPAWELL E7 test in other indications like head and neck squamous cell carcinoma, anal disease, penile disease etc.
In order to reach the highest possible impact, MIKROGEN is aware of the importance to keep the public, policy makers and all other research and health stakeholders informed about project development and achievements. This will be done by MIKROGEN’s marketing and sales force, via new media such as special PIPAWELL websites, where different target groups can get the information they need. Additionally the key opinion leaders working with MIKROGEN on this project will meet on a regular basis at advisory boards and congresses in order to disseminate data on PIPAWELL. Further key opinion leaders (laboratory physicians and gyneacologists) will be selected and trained on PIPAWELL by MIKROGEN in order to use them in terms of cascading their knowledge on PIPAWELL to their peers. The objective of this premarketing phase is to make physicians more and more familiar with the scientific background, USP and practical advantages of PIPAWELL. Additional participants in these advisory boards will be the representatives of reimbursement authorities. Having the key opinion leaders convinced to use PIPAWELL, MIKROGEN will start a training concept for private gynaecologicsts and laboratory physicians. This will create a market willing to pay for the innovation with clients having a genuine interest to buy the product. Although primary care physicians are not yet performing HPV-related testing, this could possibly change in future, in particular using the PIPAWELL E7 test. Today the diagnosis must be accepted and also requested primarily by gynaecologists. Further on all customer segments must be split up into tier 1 to tier 3 customers - who will be the early adopters, who the followers? MIKROGEN will provide a clear segmentation for every country together with the distribution partners. The gynaecologists have to be convinced of the PIPAWELL E7 test, they are the decision makers to test patient samples by this test. User recommendations/guidelines for the assay will be developed (when to test, interpretation of results). HPV related diagnostics is subject to massive market-entry barriers imposed by regulatory agencies, such as the EMA and the FDA in the USA. Currently, HPV-related diagnostic solutions are being extensively evaluated by these authorities, providing new business opportunities for tests that identify patients at risk beyond their HPV DNA status. The market entry barriers and opportunities are summarized in Figure 3.
4.1.4.4 Exploitation Strategy (This section contains confidential information and MUST NOT be published)
MIKROGEN is an internationally working company with focus on the European market, US, Asia and Latin America, so with the best requirements to successfully launch PIPAWELL in the most important market. Specialized sales teams in the US, Europe (by own sale force in Germany and by distributors in rest of Europe), Asia (own MIKROGEN subsidiary in Beijing) and Latin America (MIKROGEN sales consultants) assure the professional roll out and marketing of PIPAWELL. Besides the scientific excellence and reputation that derives from the project for MIKROGEN, this is internally seen as the most important blockbuster/business chance of the company in the coming years and will dramatically boost the company sales from actually 12 Mio. € per year up to 35 Mio. € in 2027 (see also Figure 6 below). MIKROGEN has the commercial and management experience required to develop the current prototype of the PIPAWELL E7 test in a marketable product. A prerequisite for commercialisation is the clinical validation of the test for which third parties are needed who have the required clinical expertise and access to patients. This key step forward is attempted by the current proposal. The overall MIKROGEN business strategy is to further on succeed in serology with the four platforms LINE, ELISA, PCR and BEAD. Within this market segment MIKROGEN is market leader with a high scientific expertise in developing and marketing serological assays. MIKROGEN was founded 25 years ago and is a constantly growing SME biotech company with a broad ELISA portfolio and know how. The ELISA expertise in development and production together with a long experience in sales and marketing will be very helpful in successfully implementing PIPAWELL E7 test in the market. The strategic business model of MIKROGEN is based on defending the market leader position for serological assays in Germany, gain market leader position in all European countries, the US, Asia and Latin America and to identify and occupy profitable new sources of business like oncology in future. Therefore, PIPAWELL is fully in line with the overall strategy of MIKROGEN, and is seen as one of the most interesting and important new business fields for the company.
Strategy plan for commercialisation
Introduction of novel products is a great challenge. This situation is especially true for medical products that are research intensive even after the product was clinically validated and launched, since acceptance of a medical device is related to a "critical mass" of high quality research data, convincing public health authorities, insurances and scientific societies of clinical and cost effectiveness as well as product safety. In this respect, PIPAWELL products have a very good background to be successful in European and other markets with the scientific know-how, the results of the clinical studies with CE-marked products as well as the strong marketing and sales power of MIKROGEN. Consideration of a novel medical diagnostic test in diagnostic and/or therapeutic guidelines is a prerequisite for its broad application.
Within the pre-marketing phase in 2016 MIKROGEN will segment the potential customers which are known for every country via sales force/distributor information into early adopters, early majority, late majority, (laggards) with the objective to focus the marketing mix accordingly. Depending on the target group the physicians belong to different marketing mix instruments will be implemented. Starting with the already mentioned advisory boards, scientific study projects post launch, beta site testings and RUO projects will follow. All relevant international congresses will be used to present data on PIPAWELL, satellites, poster and publications will be planned and organized. In order to meet customer needs professional market research will be conducted per country and strategy and marketing mix will be adapted accordingly. This marketing mix will primarily contain launch meetings for physicians in the targeted countries, local trainings for physicians led by key opinion leaders, sales trainings, promotional material for sales force, promotional material for physicians, website incl. FAQ, PR plan, plan for poster and publication, mailings, concept for social media, congress plan, pricing policy per country and post launch scientific study plan for the coming years. A detailed marketing plan will be developed by MIKROGEN in 2016. Assuming an average market share of roughly 1%, total PIPAWELL sales will amount to roughly 208 Mio. € in the next 14 years (see Figure 5, above).
Figure 5 shows the significantly increasing sales until 2027 which will allow MIKROGEN to become a much more important player within the market. The projected sales will exceed the current marketing, sales, and production capacities of MIKROGEN resulting in the need to significantly extend the business activities in order to adopt to the growing sales volume. Thereby, the PIPAWELL project has a strong potential to boost the growth of MIKROGEN. The adaptations of the company to the expected growing sales are shown in Figure 5, below. The PIPAWELL project will drive our company in a new diagnostic area and will increase our revenues significantly. This is a great opportunity for MIKROGEN to get an access into the field of cancer diagnostics and to further diversify the company.
Actually MIKROGEN is one of the most important employers in the Biotech Cluster of south Munich and will then turn into an even more important employer for the whole region. In the past years, MIKROGEN has constantly invested in R&D and employees. The investment in personnel for PIPAWELL will be increased as shown in Figure 6 above. In summary, the business case HPV will reach a net present value of roughly 23 Mio. €, interest rate of 18%, internal rate of return of 66.8%, and a cash flow of 160 Mio. € in 14 years (see Figure 7 below). This results in an increase of MIKROGEN’s current total revenues of approx. 12 Mio. € to 220 Mio. €.
List of Websites:
The public website address is www.pipavir.com
Here are the relevant contact details:
1 UIBK, Universitaet Innsbruck, PI: PIdder Jansen-Dürr, pidder.jansen-duerr@uibk.ac.at
2 MIKROGEN, Mikrogen GmbH, PI: Oliver Böcher, boecher@mikrogen.de
3 BIOSYNEX, Biosynex SA, PI: Thierry Paper, paper@biosynex.com
4 CHARITE, Charite - Universitaetsmedizin Berlin, PI: Andreas Kaufmann, andreas.kaufmann@charite.de
5 AUTH, Aristotelio Panepistimio Thessalonikis, PI: Thodoros Agorastos, agorast@auth.gr
For detailed information about the contact information, please see section 4.1.5 in the attachement.
