A Comprehensive Evaluation of Circulating Tumor DNA and Circulating Tumor Cells as a Predictive Marker in Lung Cancer
Condition: Lung Cancer
MeSH terms: Lung Neoplasms; Neoplastic Cells, Circulating
Study type: Observational [Patient Registry]

Summary: 
      This study evaluates the use of ctDNA and CTCs in predicting disease activity and drug
      response in lung cancer patients and serves to complement existing methods to achieve a
      non-invasive and accurate means to guide treatment decisions.
    

Detailed description: 
      Lung cancer is not only amongst the top 3 most common cancer type in both men and women, but
      also the leading cause of cancer mortality in Singapore and the United States. Platinum-based
      chemotherapy is currently the mainstay in drug treatment for lung cancer patients although it
      only relieves disease-related symptoms temporarily. Recently, the use of molecularly targeted
      therapy has led to improved survival in subsets of patients. In particular, the presence of
      EGFR or ALK mutations from lung biopsies predicts improved response rate and progression-free
      survival from the use of tyrosine kinase inhibitors or crizotinib respectively. Importantly,
      EGFR mutations are more common in tumors of Asian patients (about 35%) versus non-Asians
      (about 10%).

      The main cause of mortality in patients with solid cancers is metastases from dissemination
      via blood circulation. The ability to detect the presence of tumor cells in circulation even
      before overt metastases occur has evolved rapidly in recent years. The prognostic importance
      of circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) in several cancers has
      been found and this technique is widely recognized to be of major clinical and biologic
      importance. This study evaluates the use of ctDNA and CTCs in predicting disease activity and
      drug response in lung cancer patients and serves to complement existing methods to achieve a
      non-invasive and accurate means to guide treatment decisions.

      Indeed, the use of ctDNA and CTCs as a marker for risk assessment and treatment monitoring is
      advantageous as it is accessible may be monitored on a regular basis on a non-invasive basis.
      This will better tailor our surveillance programmes and treatment to optimize disease
      outcomes.

      The Investigators have developed an EGFR mutation detection kit that detects the 3 main EGFR
      mutations at ultrasensitive levels. The Investigators have applied this to a variety of
      clinical materials including formalin-fixed paraffin embedded DNA, pleural effusions,
      circulating DNA as well as circulating tumor cells and found that the kits can pick up these
      mutations with high sensitivity and specificity.

      A microsieve membrane filter device to effectively isolate circulating tumor cells from whole
      blood sample has been developed by the Institute of Bioengineering and Nanotechnology. The
      device exploits the size difference between cancer cells and blood cells to achieve the
      cancer cell capture on a microsieve filter with > 85% recovery rate from 4.5-ml whole blood
      within 10 min, demonstrated with cancer cells spiked into whole blood samples. The device
      contains a densely packed pore array (> 8000 pores/mm^2) with an optimized pore diameter of
      10 µm. Fluid regulation and cell counting are achieved by a simple vacuum pumping and
      laboratory fluorescence microscope. The captured cells are stained with anti-EpCAM-PE for
      CTCs, anti-CD45-FITC for white blood cells, and 4',6-diamidino-2-phenylindole (DAPI) for cell
      nuclei. The CTCs are identified with anti-EpCAM positive, anti-CD45 negative and DAPI
      positive, characterized using a fluorescence microscope.

      Hence, from our preliminary studies, the investigators hypothesize that our technology is
      useful for isolating circulating tumour cells and that these circulating tumor cells likely
      correlate with disease activity and are not found in health individuals.

      The monitoring of circulating tumor DNA and circulating tumour cells may provide a clinically
      valuable lead time in determining response, compared to conventional radiologic methods.

      A cohort of patients will be recruited for a longitudinal cohort study where tissue and blood
      are collected for biomarkers. No intervention is envisioned. The study will be conducted over
      three years.

      Patient Samples and Consent

      Prospectively over six years(August 2014 to August 2020), about 150 patients diagnosed with
      lung cancer who provide full informed consent to this study will have tumor and/or blood
      and/or effusions collected. Patients from other locations may be referred to these sites.

      If patients do not consent to collection of blood for clinical studies but have consented and
      signed informed operative consent for collection of frozen or formalin-fixed paraffin
      embedded material for research purposes including biopsies, cytology or surgical resections
      as part of the standard surgical consent form, tumor tissue and normal tissue will be
      collected from Changi General Hospital. Tumor staging and grading were obtained from review
      of the pathology reports and evaluation of the case notes by individual clinician
      investigators. Written informed consent for analysis of prospectively collected clinical
      samples will be obtained from all patients, including the consent to obtain a biopsy lung
      tissue specimen for research purpose. No other additional procedures are needed other than
      the recommended procedure to remove the patient's tissue for assessment of the tumour, be it
      bronchoscopy, CT-guided biopsy or open surgery, depending on physician's assessment. Once
      routine procedure is completed, one of the tissues will be selected for research purposes.
      The "research tissue blocks" will be anonymized and the slides batched for subsequent
      examination/verification of tumor presence by a pathologist.

      Each prospective patient will provide baseline 3 EDTA tubes of blood (total approximately 30
      ml). Urine (~100mL) will be collected at the same time for study of urine DNA.

      These blood samples will be centrifuged for 10 minutes at 3000 rpm, for collection of serum
      and plasma for validation of biomarkers identified by microarray analysis, selecting for
      candidate secreted proteins in the blood.

      Effusions (pleural or peritoneal) will be obtained as part of routine therapeutic procedures
      as recommended by the treating clinician, where the fluids would otherwise have been
      discarded. At no point will effusion fluid be obtained for the sole purpose of this study.

      Downstream work in terms of evaluation of the CTCs, peripheral blood mononuclear cells, DNA,
      RNA, protein, and other genetic and molecular materials will be performed. Techniques applied
      will include quantitative PCR, sequencing, flow cytometry and immunophenotyping,
      immunohistochemistry, fluorescent in situ-hybridization, culture, etc.

      Mutational analysis:

      Genetic material will be extracted from 3-5 mL of materials trapped on sieve and from blood
      plasma, as well as relevant material such as effusions or urine. This will be analysed by
      quantitative PCR using custom designed primers and/or sequencing. Genetic materials will also
      be profiled using next-generation sequencing techniques to identify clinically actionable
      mutations such as BRAF, ALK, ROS, RET, etc.

      Samples will be processed fresh. Any remaining sample will be separated into buffy coats and
      plasma and stored in liquid nitrogen and/or -80C.

      Data and specimens are de-identified for laboratory processing. Each site will retain
      information linking patient to specimen. This is necessary for determination of follow-up.

      Identified data will be located on a networked PC, physically located in Changi General
      Hospital and behind institutional firewalls. Individually identifiable data will be retained
      in the hospital and will not be shared with the laboratory.

      The data will be archived for downstream projects once the study is completed.
    

Intervention name(s): EGFR mutation detection kit
Intervention type(s): Diagnostic Test
Intervention description(s): A microsieve membrane filter device to effectively isolate circulating tumor cells from whole blood sample has been developed by the Institute of Bioengineering and Nanotechnology. The device exploits the size difference between cancer cells and blood cells to achieve the cancer cell capture on a microsieve filter with > 85% recovery rate from 4.5-ml whole blood within 10 min, demonstrated with cancer cells spiked into whole blood samples. The device contains a densely packed pore array (> 8000 pores/mm^2) with an optimized pore diameter of 10 µm. Fluid regulation and cell counting are achieved by a simple vacuum pumping and laboratory fluorescence microscope. The captured cells are stained with anti-EpCAM-PE for CTCs, anti-CD45-FITC for white blood cells, and 4',6-diamidino-2-phenylindole (DAPI) for cell nuclei. The CTCs are identified with anti-EpCAM positive, anti-CD45 negative and DAPI positive, characterized using a fluorescence microscope.
Observational model: Cohort
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        150 patients (aged 21-99) suspected to have lung cancer will be recruited from Changi
        General Hospital. There are no restrictions based on the race of subject. We will exclude
        study participants who are unable to provide informed consent, such as children and
        mentally incapable individuals.