Detection of Targetable Alterations in Non-small Cell Lung Cancer using Next-generation Sequencing

My research discovers genetic mutations in cancer to improve diagnosis and treatment. To begin, register the sample information in the Laboratory Information Management System and fill out the informed consent form for gene mutation testing. Using disposable microtome blades, cut sections from the non-small cell lung cancer, formal and fixed paraffin embedded sample.

Then perform routine hematoxylin and eosin staining to evaluate the tumor cell content. For nucleic acid extraction, using a microtome, section the formal and fixed paraffin embedded block to obtain a ribbon, and place the ribbon into a 1.5-milliliter microcentrifuge tube. Transfer the tube to the designated sample input location on the automated extraction instrument.

Next, use an automatic extraction instrument with an automatic extraction kit. Replace manual pipetting steps with an automatic magnetic bead nucleic acid extraction system. Then select the C1102 program on the instrument.

Choose the sample de-waxing incubation time as 16 hours, and set the elucian volume to 100 microliters. Use spectrophotometry to verify DNA purity. Then use a detection kit with sequence-specific fluorescent dyes to perform a fluorometric assay for precise DNA concentration measurement.

To prepare fragmented genomic DNA using ultrasonication, mix 200 nanograms of genomic DNA in 50 microliters of low tris-EDTA buffer. Place the sample tube in a pre-cooled sample rack, maintained at eight degrees Celsius. Set the instrument parameters according to the protocol and initiate DNA fragmentation.

To start automated library preparation, press the power button to activate the system and wait for the initialization to complete. Navigate to program setting, select run protocol, and choose protocol BurningRock HS.Set the sample type to high quality DNA, input amount nanograms to 50, pre-PCR cycles to 12, and post-PCR cycles to 12. Then click Run to proceed.

Then insert the prepackaged reagent cartridge into the designated slot of the instrument, which will automatically validate reagent placement and status. After loading the samples, press start to initiate the autonomous run. Observe the LED indicators on the system to monitor the progress.

After the program ends, click OK to confirm and the system will automatically transfer the final library into the library tube, completing library preparation. Measure the concentration of both pre-library and total library using fluorometry. Dilute the sequencing library to 1.6 picomolar and 1300 microliters for sequencing operations.

Control the sequencing environment by maintaining an indoor temperature and indoor humidity. Complete quality control of the instrument output data by verifying base quality above Q 30 and cluster density passing filter. The optimized detection workflow reliably identified several clinically actionable genomic alterations in representative tumor samples.

The sequencing results demonstrated a high frequency EGFR p. L858R missense mutation, accompanied by a significant EGFR gene copy number amplification. Moderate amplification of the MET gene was observed and low level amplification of the BRAF gene was observed.

The sample exhibited an intermediate tumor mutation burden and microsatellite stable status. All targeted gene loci, including ALK, BRAF, KRAS, and ROS1 achieved a 100%detection rate across four replicate experiments. Each mutation was consistently detected in all four repeated experiments, with expected abundance values remaining within the normal fluctuation range, NJS provides a compressive genetic profile for non-small cell lung cancer by detecting common, rare, and novel genes at the same time.

Our future research will focus on combining molecular pathology with artificial intelligence to explore new biomarkers for lung cancer.