Cell free Deoxy Ribonucleic Acid
Circulating Tumor Cell
Cell-free circulating tumor DNA
Clinical Trials Registry India
Datar Cancer Genetics Limited
Good Clinical Practice
Good Laboratory Practice
Hepatitis B Virus
Hepatitis C Virus
Human Immunodeficiency Virus
Informed Consent Form
International Conference on Harmonisation
Indian Council of Medical Research
Institutional Review Board
Magnetic Resonance Imaging
Positron Emission Tomography/Computed Tomography
Standard of Care
Standard Operating Procedure
1. Background and Introduction:
Cancer is one of the leading causes of deaths in India and over 630,000 people die of cancer each year. According to the most recent predictions by the International Agency for Research on Cancer GLOBOCAN project, India’s cancer burden will nearly double in the next 20 years, from a million new cases in 2012 to more than 1·7 million by 2035. (Ferlay J et al., Mallath MK et al.)
First step in cancer management is establishing cancer diagnosis with histopathological typing of tumor tissue sample. Tissue sampling is achieved either through Fine Needle Aspiration Cytology (FNAC) or tissue biopsies or other specialized cytology procedures e.g. bronchoscopy, ultrasound-guided bronchoscopy, transthoracic needle biopsy, and thoracoscopy in lung cancer. The diagnostic yield of these procedures varies. In lung cancer the diagnostic yield varies between 58-97% among various tissue sampling procedures. (Ofiara LM et al.)
These techniques are invasive and may not always feasible in all patients due to various factors like associated comorbidities or localization of tumor close to vital organs.
Additionally, these techniques may be associated with complications due to their invasive nature. In tissue sampling procedures for lung cancer, various complications like pneumothorax, hypoxemia, hemorrhage etc. may occur. The rates of these complications vary with type of procedure used like major complication rates between 0.08 and 5% for fiberoptic bronchoscopy (Simpson FG et al) whereas rate of major complications like bleeding and pneumothorax may occur in 10% and up to 20% of cases, respectively with transthoracic needle aspirate. (Klein JS et al.)
In certain percentage, tissue sampling may be inadequate or may not representative leading to repetition of these invasive procedures. For example, in case of lung cancer up to 80% of patients receiving chemotherapy for advanced disease will have only a small biopsy and/or cytology samples available for diagnosis. (Kerr et al.)
Also, with advances in molecular testing and targeted therapies, in addition to routine histopathological and immunohistochemical examination, molecular testing has become imperative in certain malignancies. The recent discovery of driver mutations and their association with greater responsiveness to specific targeted agents led to a paradigm shift in cancer treatment from conventional chemotherapy to biomarker-driven targeted therapy. This new era of personalized therapy brings an increasing demand for characterization of tumour genotypes and phenotypes.
Tissue samples are routinely formalin fixed for further examination. The accuracy of molecular tests depends on sample quality as much as the molecular analyses themselves. The formaldehyde initiates DNA and RNA denaturation, formalin fixed tissue exhibit a high frequency of nonreproducible sequence alteration because of formalin cross-linking cytosine nucleotides on either strand, creating an artificial C-T or G-A mutation. (Srinivasan M et al.) All these fixation induced artefacts can finally impact the quality of molecular analysis and its results.
Tissue sampling also faces challenge of tumor heterogeneity. Intratumor heterogeneity and heterogeneity between tumors at different sites, can lead to underestimation of the tumor genomics landscape portrayed from single tumor-biopsy samples and may present major challenges to personalized-medicine and biomarker development. (Gerlinger M et al.)
Due to invasive nature of these tissue sampling modalities, it is difficult to repeat them for longitudinal disease monitoring for therapy response evaluation, detection of molecular evidence of acquired resistance or tumor evolution.
Tissue biopsy from cancer of internal organs including brain are invasive to various extent and may at times require multiple attempts to get representative tissue sample to arrive at a diagnosis which not only delays starting treatment but also necessitates the patient to undergo several invasive procedures along with its antecedent complication. In cases of recurrence, repeat invasive biopsy will have to be undertaken to establish whether it is recurrence or new malignancy.
Primary Central Nervous System (CNS) malignancies including high grade gliomas pose a clinical challenge as MRI at times can be equivocal. Repeated brain biopsy especially of deep seated and eloquent structures to evaluate disease progression is both invasive and impractical given the cumulative surgical risk.
All these limitations of invasive tissue sampling modalities urge us to find a non-invasive modality which can provide information about cancer diagnosis and molecular evaluation necessary to plan treatment protocols in cancer patients.
2. Study Rationale:
Liquid biopsy involves the analysis of biomarkers like circulating tumor cells (CTCs), cell-free nucleic acids - circulating free DNA (cfDNA), exosomal messenger RNA (mRNA) and micro RNA (miRNA) in bodily fluids such as blood. The obtaining of specimens for liquid biopsy is easier and less invasive than tissue biopsy.
Current liquid biopsy approaches focus on CTC enumeration for disease prognostication and cfDNA analysis for cancer genotyping for selection of certain targeted therapies e.g. Osimertinib in EGFR T790M NSCLC.
There is no data available for utility or potential of liquid biopsy for morphological typing of malignancies.
A preliminary trial has suggested that a single blood test could one day be used to detect a variety of cancers. (Joshua D Cohen et al.) However, development of methods for noninvasive detection of cancer has still not been standardized. (Jillen Phallen et al.) Biopsy of tumour tissue and tumour markers from tumour tissue and blood remains the gold standard for diagnosis and prognostication.
Analysis of cell free DNA and mRNA in the peripheral blood (Liquid Biopsy) has shown high degree of concordance both in terms of diagnosis and grading of tumour even in CNS malignancies. (Lee I et al.)
However liquid biopsy based on cfDNA is currently unable to identify underlying tissue of origin in a large number of cases due to the fact that most somatic alterations are not cancer type specific. (Joshua D Cohen et al.)
Circulating tumor cells (CTCs) are the tumor cells which have detached from primary tumor site and have gained access to peripheral circulation. These may potentially lodge in distant organs giving rise to metastasis. Thus, CTCs are a pre-requisite for distant disease spread and thus are detectable before late stage/metastatic disease develops. Also, CTCs have additional advantage of expressing tissue-of-origin specific markers in their cytoplasm/nucleus/membrane giving rise to possibility of identification of primary tumor site. In certain cases, morphological classification into probable cancer type e.g. squamous vs adenocarcinoma may be feasible, giving better access to patient management in addition to the diagnosis. Thus, the evaluation of CTCs in cancer management, has potential to extend beyond prognostication. As technologies emerge to analyze CTCs at the molecular level, biological behavior of the tumour can be obtained in real time, with the promise of CTCs eventually acting as a ‘surrogate tumour biopsy’. (Mathew G Krebs et al.)
All in all, comprehensive blood based biomarkers based liquid biopsy has potential to replace tissue biopsy and to pave a way for non-invasive, accurate blood based diagnostic modality to improve patient care and quality of life.
Current study is undertaken to evaluate the feasibility of tissue biopsy replacement with unique evaluation of circulating bio-markers at Datar Cancer Genetics Limited for morphological evaluation and clinically relevant molecular typing of malignancies from blood sample.
3. Study Objectives:
Utility of TrueBlood test in establishing cancer diagnosis in all patients of suspected or newly diagnosed cancer or in cases of recurrence, irrespective of stage of the disease
i.) Utility of TrueBlood test for identification of morphological/histopathological type of the malignancy and its comparison with histopathology of the tumor obtained through tissue biopsy
ii.) Utility of TrueBlood test for molecular evaluation of malignancy
Primary Outcome Measure
Specificity of TrueBlood for establishing cancer diagnosis
Secondary Outcome Measures
i.) Sensitivity of TrueBlood for establishing cancer diagnosis
ii.) Specificity of TrueBlood for morphological/histopathological evaluation of malignancy
iii.) Sensitivity of TrueBlood for morphological/histopathological evaluation of malignancy
iv.) Specificity of TrueBlood for molecular evaluation of malignancy
v.) Sensitivity of TrueBlood for molecular evaluation of malignancy
4. Study Design:
Study Type: Observational
Study Design: Prospective Observational study
Study Population: Patients above 18 years of age, suspected to have malignancy or newly diagnosed for malignancy or recurrence of malignancy (excluding Hematolymphoid malignancy), who are therapy naïve at the time of inclusion in the study ( in case of recurrence/relapsed/refractory malignancy, participant should be therapy naïve for current recurrence or should be off therapy at least for last 90 days prior to sample collection). Effort will be made to have a healthy mix of both genders to account for gender specific malignancies
Estimated Enrollment: 40,000
Masking: None (Open Label)
Actual Study Start Date: After ethics committee approval and CTRI registration (Likely March 06, 2019)
Estimated Primary Completion Date: 12 months
Estimated Study Completion Date: Likely March 06, 2020
5. Study Population
Patients above 18 years of age, suspected to have malignancy or newly diagnosed for malignancy or recurrence of malignancy (excluding Hematolymphoid malignancy), who are therapy naïve at the time of inclusion in the study (in case of patients with relapsed/refractory/recurrent malignancy, who have taken last cancer therapy before 3 months of study participation i.e. they have not received any cancer therapy in last 90 days ). Effort will be made to have a healthy mix of both genders to account for gender specific malignancies.
6. Study Eligibility
For Inclusion, an individual must meet all of the following criteria:
1. Age more than 18 years. Effort will be made to have a healthy mix of both genders to account for gender specific malignancies
2. Suspected/ newly diagnosed malignancy or recurrence
3 Therapy naïve for newly diagnosed malignancy and patients with relapsed/refractory/recurrent malignancy, who have taken last cancer therapy before 3 months of study participation i.e. they have not received any cancer therapy in last 90 days
4 Willingness to provide blood sample (mandatory) and tissue sample (tissue sample - preferable but not mandatory) as per study protocol
5 Provision of signed informed consent form and expresses understanding of the protocol and its requirements, risks, and discomforts.
6 Stated willingness to comply with all study procedures
7 No history of blood transfusion in last 21 days.
8 No history of PET-CT scan in last 21 days.
For Exclusion, an individual may meet any of the following criteria:
1. Age – < 18 years
2. Received any form of cancer therapy (radiation/ chemotherapy/surgery/
Endocrine / immunotherapy etc.). In case of recurrence/refractory/relapsed malignancy, received any form of
cancer therapy (radiation/ chemotherapy/surgery/ endocrine etc.) within 90 days prior to sample collection.
3. Unable to provide informed consent
4. Unable to comply with all project screening procedures
5. Unable/unwilling to provide blood sample as per study requirement.
6. Oral or IV corticosteroid use in past 14 days prior to blood draw
7. Current febrile illness
8. Acute exacerbation or flare of an inflammatory condition requiring escalation in medical therapy within 14 days prior to blood draw.
9. History of blood transfusion/ PET-CT scan/CT-scan in last 21 days
10. HIV/HBV/HCV infection.
7. Study Assessment
After eligibility determination, detailed information sharing about the nature of study and answering all concerns and queries of study participants, study enrollment will be done and written informed consent will be obtained.
All patients will undergo tissue histopathological diagnosis as a part of their standard of care (SoC) management. In case of molecular analysis required as part of SoC, same may be conducted. All such SoC investigations are not being conducted as part of TrueBlood study but as part of routine management, and hence DCGL or others working on sponsor’s behalf will not be liable for any financial or legal implications arising out of these SoC investigations. Study participant will not undergo any additional procedure exclusively for TrueBlood project except for peripheral 15-20 ml blood sample collection which is not associated with significant health hazard. Tissue sample will be provided to DCGL only when patient is undergoing tissue sampling for SoC management and providing additional tissue sample to TrueBlood project does not impose any significant additional risk to study participant.
The clinical records and results of SoC investigations like HPE, IHC and molecular analysis reports along with relevant clinical data will be collected as a part of TrueBlood project.
Patient will provide blood sample for the study before (Preferably) or after the invasive biopsy, subject to adherence to the inclusion criteria enumerated above. Effort will be made to obtain tissue sample whenever feasible.
8. Study Conduct
a. STEP 1 (Recruitment and Consent):
Patients fulfilling eligibility criteria are recruited after providing detailed information about study protocol, its utility and limitations. Participant enters study only after providing written informed consent.
b. STEPS 2: (Sample Collection) :
After consent, participants submit blood sample as proposed in study assessment, before starting treatment. Whenever feasible tissue sample will be provided.
c. STEP 3 (Sample Processing):
Samples will be processed at DCGL as per TrueBlood protocol.
d. STEP 4 (Data Evaluation):
The data from study analytes of Cell free DNAs ± RNAs ± CTCs will be compared with available histopathogical diagnosis and molecular analysis reports to determine sensitivity and specificity TrueBlood and analyze whether it can replace invasive tissue biopsy.