P59.28 Protocol Adaptation for gDNA Extraction of FFPE Samples of Lung Adenocarcinoma Tissue and Related Mediastinal Lymph Nodes

Abstract

Lung adenocarcinoma, the most common subtype of non-small-cell lung carcinoma (NSCLC), is characterized by low response to treatment with 50% rate of metastases at diagnosis and low five-year survival. To associate the molecular evaluation of mediastinal lymph nodes (MLN) obtained by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) to the histological examination could improve TNM staging and diagnosis. Our objective is to adapt and optimize the genomic DNA (gDNA) extraction protocol to be used in formalin-fixed paraffin-embedded (FFPE) samples from tumor tissue and mediastinal lymph nodes for molecular analysis. The gDNA from 28 paired samples of primary tumor (PT) obtained by transthoracic biopsy or tumor resection, and MLN obtained by EBUS-TBNA were extracted with GeneRead DNA FFPE kit (Qiagen). Fourteen paired samples were submitted to the manufacturer's protocol (MP- one 10 μm cut of FFPE samples incubated in deparafinization solution for 3 minutes at 56° C, followed by incubation in proteinase K, RNAse free water and FTB buffer for 1 hour at 56°C); eight paired FFPE samples were submitted to an adapted protocol (AP-no use of deparafinization solution, 3-4 10 μm cuts of directly incubated in a mix containing proteinase K, RNAse free water and FTB buffer for 3 hours at 56ºC), and 6 paired samples were submitted to both protocols (MP-AP). The gDNA quantification was performed in the Qubit 3.0 fluorometer (Thermo Fisher Scientific). Paired t test was applied, considering statistically significant p≤0.05. The range of gDNA yield obtained in the MP was 0.121-8.51 ng/μL for tumor samples and 0.025-1.14 ng/μL for MLN samples, and 1.04-26.0 ng/μL and 0.065-3.11 ng/μL, respectively, in the AP. In the paired samples extracted using both protocols, the range of gDNA obtained was 0.121-2.65 ng/μL and 1.04-4.95 ng/μL for PT samples (p=0.030) and 0.043-0.054 ng/μL and 0.065-3.11 ng/μL for MLN samples (p = 0.071). To obtain purified gDNA from FFPE samples is a laborious process, especially in archived samples over 5 years old and in samples with scarce cellularity, as are the small fragments of lung tissue obtained by transthoracic biopsy, or MLN fragments obtained by EBUS/TBNA. We observed a significant improving in the gDNA extraction for tumor samples using the AP protocol and a trend in improving for MLN samples, probably due to the small sample size. Changes in commercial protocols are frequently required to ensure better molecular results in these types of samples.