Concordance study of Circulating Tumor Cells (CTCs) with Biopsy in Patients with newly diagnosed Lung Cancer

BACKGROUND AND RATIONALE

The management of lung cancer necessitates availability of tissue not only for histopathologic testing but for subtyping and evaluation of multiple actionable molecular targets and immunotherapeutic targets. It may not always be feasible to perform an invasive lung biopsy especially in inaccessible sites, anatomically challenging locations, bedridden or very sick patients, patients in remote areas where access to trained doctors in interventional procedures is not feasible. Further knowledge gaps and optimal utilization of precious lung cancer tissue may deter the physicians from providing optimal management for want of an accurate diagnosis.

Circulating tumor cells (CTCs) enumeration offers potential utility as prognostic, predictive and/or pharmacodynamic biomarker, and it may fulfil the criteria for a surrogate diagnostic biomarker where gold standard biopsies are not feasible in the diagnosis of lung malignancies. The comprehensive molecular profiling liquid biopsy in plasma of  lung cancers is now incorporated in lung cancer guidelines as a decisive tool when positive results are obtained

CTCs or Circulating Tumor Associated Cells (CTACs) are the cells shaded by tumor enters in the bloodstream and can survive in the blood environment often by epithelial mesenchymal transition (EMT) Lung cancer is known to be a heterogeneous disease and sometimes a challenge to diagnose on small biopsy as the sampled tissue might represent only one type of differentiation viz adenosquamous carcinoma. We hypothesize that CTC or CTACs may overcome this limitation as they can address spatial heterogeneity.

A sample of sufficient CTACs is comparable to a micro-biopsy of tumor tissue; CTTACs and CTCs retain the antigenic features of the primary tumor and can be profiled by Immunocytochemistry (ICC) for Lung cancer- such as Napsin-A, TTF-1, EMA D2-40 Calretinin CD56, Synaptophysin, CK7, p40, CD45. Thus, ICC profiling of CTCs can non-invasively provide diagnostic guidance in suspected Lung cancer epithelial malignancies.

TruBloodTM test incorporates characterization of CTCs and CTACs and detect presence of lung cancer. The performance evaluation and characterization of TruBloodTM has been established and validated by Datar Cancer Genetics Pvt Ltd in compliance with the requirements of CAP/CLIA as applicable for Single Laboratory Developed Test. The results of applicability of this in different cancers have been hitherto published in the International Journal of Cancer, Cancer Cytopathology and Cancer Prevention Research (1,2,3).   CTCs and CTACs have been reported to include Cancer Stem Cells (CSCs), Tumor Associated Leucocytes (TAL) and Tumor Associated Macrophages (TAM) etc.

STUDY DESIGN

Study Type:                  Observational

Observational Model: Cohort

Time Perspective:       Prospective

Target numbers:         138 (to achieve 90% accuracy @95% confidence interval)

Start Date:                   6th July 2021

Subjects:                      Patients above 18 years of age, suspected to have lung malignancy who are therapy naïve at the time of inclusion in the study.

 Study Procedures: Collection of peripheral blood at diagnosis along with biopsy (Histopathology processing and diagnosis will be performed at the site of PI)

 Study outline:

Prior to Enrollment

•       Screen potential participants by Inclusion and Exclusion Criteria.

·       Obtain Clinical History Documents

On Enrollment

•         Obtain Informed Consent (Patient information leaflet and informed consent form)

•         Collect Blood sample 25 ml before diagnostic biopsy is being done (Annexure 2)

 Data Analysis- Following completion of sample collection of entire study cohort, perform analysis of samples and data.

 Methods :  Peripheral blood mononuclear cells (PBMCs) are isolated from  peripheral blood

Samples. The PBMCs are treated with an epigenetically activating medium which is cytotoxic towards non-malignant hematolymphoid cells and allows survival of apoptosis resistant

malignant cells of tumorigenic origin, i.e., CTCs and their clusters, C-ETACs

C-ETACs are gently dissociated into single CTCs, aspirated transferred to coated slides,

where they are fixed and permeabilized treated with primary and secondary

fluorophore conjugated antibodies, and visualized using high content fluorescence

imaging.

PDL1 22C3 ICC staining on CTC will be done.

Corresponding blocks of tissue also will be processed for PDL1 IHC.

Cell free DNA/ RNA and tissue will be sequenced for 12 genes related to lung cancer therapy indications by Next Generation Sequencing.

 ENDPOINTS

Immuno-concordance rate (ICR) is the proportion of samples where diagnostic and findings on ICC were concordant with histopathologic and IHC findings (where available) on matched tumor tissue.