Case Studies

Case study 1. HPV biomarker research – PROTOTYPE OF HPV SEROTYPE-SPECIFIC DNA-BASED DIAGNOSTIC RAPID TEST INNOVATION

Research background

Overview

The human papillomaviruses (HPV) is small DNA tumour virus. HPV types are classified into “low- or high-risk” types, dependent upon their oncogenic potential. Persistent infection with high-risk HPVs can lead to cervical cancer, the second most common cancer in women. The HPVs gene can manipulate cell cycle control to help viral persistence and replication. The majority of these cancers contain HPV DNA integrated into the infected host cell genome. Moreover the HPV induced genomic instability of the genome of host cells plays a key role in cervical cancer, “easing” the progression of tumour and the formation of its intense heterogeneity.

Aim

The aim of the project was to develop the applied methods (HPV-specific quick test) in the diagnosis of the HPV-infection and to identify biomarkers, which able to indicate the presence of the virus oncogene in the sample and to provide information about the existence/absence of HPV-induced genomic lesions in the cells.

Experiment

Biospecimens and data collected by Pharmaster

  1. Our client requested Pharmaster to collect different types of DNA biospecimens isolated from the subsequent human clinical samples as described below. Cervical and vaginal mucosa samples containing human cells from 200 patients. Distribution of patients after HPV genotyping:
    • 150 sample proven high risks,
    • 25 genotyped low risks,
    • 25 genotyped negative.

    According to the required biospecimens in the course of experiments, the samples were divided into three groups:

    • The first group/control group was proven negative for HPV infection.
    • The low-risk HPV infected biospecimens were in the second group.
    • In the third group contained the high-risk HPV infected samples.

     

    Classification of the collected clinical samples

     

  2. The client then asked Pharmaster to formulate a workflow for the required biospecimen collection.
      • The workflow of the biospecimen collection

     

  3. Next, Pharmaster helped ethical approval process.
    • Contacting two research hospitals and sharing the project details with the PI-s clinicians and the leaders of the laboratory involved in the project.
    • Submitting a request to the competent ethics committee
      • Filling and submitting the adequate documents.
    • Expert opinion, decision making
      • The ethics committee (Scientific and Research Ethics Committee of the Scientific Counsel of Health), and the Scientific Counsel of Health Human Reproductuion Comity as reviewer and professional licensing authorities examined the submitted request and made a decision.
    • Approval of request
      • Based on the opinion of the Research Ethics Committee the permission is given by the Policy Administration Services of Public Health the locally competent county government office of Public Health’s services.
  4. Implementation of the study.
      • Provided controlled and specific conditions during the biospecimen storage at the location of sampling and during the transportation process to the laboratory. (The samples were quickly frozen and stored on the spot, and then transported deep frozen).
      • Obtained the necessary special tools for biospecimen collection, compilation of sample collection kit, which included the following documents and tools:
        • Research consent form
        • Project information for patients
        • Sample collection protocol
        • Patient Questionnaire (for collecting project-relevant data belongs to the patients, anonymously)
        • Sample Questionnaire (for collecting sample data and information of circumstances of sampling)
        • Sampling devices (DNA PAP Cervical Sampler – QIAGEN)
      • After obtaining the ethical approval, Pharmaster contacted the clinical specialists (gynecologists), visiting the hospital staff (assistants, nurses), the involvement of specimen collection, arrangements and made an agreement.
      • Sampling and DNA extraction: Cervical and vaginal sampling was performed with DNA PAP Cervical Sampler (QIAGEN). All utensils were disposable and were discarded immediately after each use (single use for each specimen). The total DNA was purified with QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer’s recommendations.

     

  5. Storage of biospecimens: The samples were stored at -80°C at the collection site until transportation.
  6. Transportation of samples:
    • The freshly isolated DNA samples and recorded clinical data (e.g. the age of the patient, short epicrisis, colposcopic and cytologic results and furthermore the results of HPV genotyping) were transported to the client on-time.
  7. Data management of experimental and clinical data:
    • As an additional service, the experimental and clinical data were stored and managed in a research data management software the SmartBiobank.

Case study 2. Cervical cancer tumor research – DEVELOPMENT OF DIAGNOSTIC TECHNOLOGY FOR CERVICAL CANCER SCREENING

Research background

Overview

Cervical cancer is the second most common cause of cancer and the fourth most common cause of cancer death in women. Human papillomavirus (HPV) infection appears to be involved in the development of more than 90% of cases. In fact, cervical cancer is caused by pre-existing human papilllomavírus (HPV) infection, infection of cervical epithelial cells.

 

Aim

The aim of the project was to develop an epigenetic based-diagnostic method which can be applied for screening of the cervical cancer. The advantage of this test compared to the currently widespread testing procedures is that this method can detect the early lesions before the appearance of cell abnormality, preventing the development of cervical cancer at an earlier stage.

Biospecimens and data collected by Pharmaster

Experiment

  1. The client was requested Pharmaster to collect DNA samples isolated human clinical biospecimens diagnosed with different stages of cervical cancer.
    • Description of tissue samples
      • non-tumor tissue samples
        • the non-tumor tissue samples were taken from the same patient as the tumor tissue samples and used as a control.
      • tumor tissue samples, 7-8 slides/ sample
    • Distribution of biospecimens
      • 20 FFPE (formalin-fixed paraffin-embedded) tissue samples from cervical conization origin (7-8 pieces of section in case of cancer and healthy tissues, respectively)
      • 20 FFPE (formalin-fixed paraffin-embedded) tissue samples from metastatic cervical cancer tissue (7-8 pieces of section in case of tumor, metastatic and healthy tissues, respectively).
  2. The client then asked Pharmaster to formulate a workflow for the required biospecimen collection.
      • The workflow of the biospecimen collection

     

  3. Next, Pharmaster helped ethical approval process.
    • Contacting two research hospitals and sharing the project details with the PI-s clinicians and the leaders of the laboratory involved in the project.
    • Submitting a request to the competent ethics committee
      • Filling and submitting the adequate documents.
    • Expert opinion, decision making
      • The ethics committee (Scientific and Research Ethics Committee of the Scientific Counsel of Health), and the Scientific Counsel of Health Human Reproductuion Comity as reviewer and professional licensing authorities examined the submitted request and made a decision.
    • Approval of request
      • Based on the opinion of the Research Ethics Committee the permission is given by the Policy Administration Services of Public Health the locally competent county government office of Public Health’s services.
  4. Implementation of the study.
      • After obtaining the ethical approval, Pharmaster contacted the professional relations and all the relevant biobanks, involving of biospecimen collection, arrangements and made an agreement.
      • Banked FFPE biospecimen collections (formalin fixed paraffin embedded cervical sections) from biobanks having the required specific tissue samples.

     

  5. Transportation of samples
    • Transportation of the needed FFPE samples under controlled storage conditions to the processing genetics laboratory.
  6. DNA extraction
    • During the sample processing, after the tissue preparation, total DNA was purified from the collected specific FFPE tissue samples by QIAamp DNA FFPE Tissue Kit (QIAGEN) according to the manufacturer’s recommendations.
  7. Storage of biospecimens
    • The samples were stored at -80oC until transportation. The isolated DNA samples and collected clinical data were transported to the client on-time.
  8. Data management of experimental and clinical data:
    • As an additional service, the experimental and clinical data were stored and managed in a research data management software the SmartBiobank.

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