A Blood-based Test for the Detection of ROS1 and RET Fusion Transcripts from Circulating Ribonucleic Acid Using Digital Polymerase Chain Reaction

The manufacturers' instructions are followed for the reagents listed below, unless otherwise described. The PCR assays are commercially available products designed to detect ROS1 and RET fusions.

1. Working with RNA in Preparation for Reverse Transcription (RT)-dPCR: Laboratory Best Practices

1. Create an RNase-free environment when working with RNA.
   1. Use commercially available sprays designed to inactivate contaminating RNases.
   2. Use certified RNase-free reagents, tips, and tubes. Use barrier tips for pipettors to prevent introduction of RNases or cross-contamination of samples.
2. Always wear a laboratory coat to prevent particulates from falling from clothing into your sample. Designate a lab coat specific to use with RNA processing.
3. Wear gloves to prevent sample contamination from RNases in skin. Change gloves frequently.
   Note: Assume laboratory surfaces are contaminated with RNase since they are exposed to the environment. Gloves that contact skin, hair, doorknobs, freezer handles, pens/markers, etc. are assumed to no longer be RNase-free.
4. Decontaminate pipettors, benchtops, centrifuges, and other work surfaces with a RNase inactivation spray prior to use.
5. If possible, maintain a set of equipment for use with RNA only.
6. Minimize disruption of air flow in lab areas when working with RNA samples to prevent particulates from falling into samples or contaminating the work area.
7. Store purified RNA at -80 ˚C.
8. Avoid multiple freeze-thaws of RNA samples, as this can cause degradation.

2. Generation of Analytic RNA Material for the Positive Controls

1. Design synthetic DNA using published mRNA sequences for fusion variants of interest¹⁰.
   1. For a given fusion variant, select an mRNA fusion sequence that includes the fusion site plus sufficient length flanking on each side to cover the PCR amplicon.
   2. Select nucleotide sequences between 50 – 250 nt to mimic the size of circulating RNA captured using platelet-enriched plasma.
   3. Add a T7 promoter sequence (5'-CAGAGATGCATAATACGACTCACTATAGGGAGA-3') to the 5' end of the target sequence.
2. Order synthetic sequences as double stranded deoxyribonucleic acid (DNA) fragments.
3. Reconstitute synthetic DNA in Tris-EDTA (TE) buffer to a final concentration of 10 ng/µL.
4. Convert 60 ng synthetic DNA to RNA using in vitro transcription.
5. Purify RNA transcripts using phenol/guanidine-based reagent¹².
   1. Include DNase I, RNase-free to remove residual template DNA.
6. Measure concentration of purified in vitro RNA using a commercially available fluorometer with RNA-specific dyes and standards. Ensure the RNA is within the range acceptable for the chosen standards. Dilution may be required.
7. Confirm successful transcription by gel electrophoresis using a 2% agarose gel mixed with RNA gel stain and a high range RNA ladder including 50 – 250 nt size range.
   1. Load 500 ng of each in vitro RNA onto a gel.
   2. Run the gel at 5 V/cm.
   3. Visualize single bands using illumination, and document the results.
   4. Confirm expected transcript size for each of the fusion variants (based on design in step 2.1.2).
8. Confirm detection of each in vitro RNA by RT-dPCR using matched variant-specific PCR assay (see steps 5 – 8 of this protocol).
9. Optional: Prepare an equimolar mixture of in vitro RNA that contains each of the fusion variants and the control gene GUSB.
10. If Step 2.9 is performed: Confirm detection of each of the fusion variants included in the control mixture by dPCR using variant-specific PCR assays (see steps 5 – 8 of this protocol).
11. Determine desired input concentration for analytic positive controls by testing concentrations ranging from 0.25 to 2.5 fg¹⁰. Choose concentration based on desired copy number output.
12. Following confirmation, prepare 10 µL single-use aliquots of analytic RNA for use in the positive control (step 4.4) and store at -80 ˚C.

3. Donor Specimens

1. Collect 10 mL human whole blood specimens in 10 mL blood collection tubes (BCT) containing a cell-free RNA preservative.
   Note: All human donors shall consent to research use and no donor-specific identifying information shall be collected or used during testing.
2. Process whole blood samples within the timeframe specified by the BCT manufacturer.
3. Pooled normal human plasma can be purchased from a commercial source for use within the analytic positive control. Prepare single-use, 1 mL aliquots of the pooled normal human plasma and store at -80 ˚C for use with the positive control (step 4.4).

4. Recovery of Circulating RNA from Plasma

Note: It is important to work quickly during this procedure.

1. Centrifuge whole blood tubes at 200 x g for 20 min.
2. Collect up to 4 mL of plasma from centrifuged blood collection tube using a serological pipette. Be careful not to disturb or aspirate the buffy coat layer.
3. Isolate circulating RNA using a commercially available kit that can capture exosomes, platelets and cell-free RNA from plasma. Isolate RNA from positive control sample alongside each batch.
4. Prepare the Positive Control for each batch of clinical samples as follows:
   1. Thaw 1 mL pooled normal human plasma aliquot (Step 3.3).
   2. Thaw 10 µL analytic RNA aliquot (Step 2.12).
   3. Prepare positive control by adding 10 µL analytic RNA into the normal human plasma sample once ethanol has been added to the plasma lysate.
5. Elute samples with 100 µL nuclease-free water. Proceed immediately with RNA clean up and concentration.
   1. Samples may be stored on wet ice, and covered, for up to one hour.
6. Concentrate RNA using column based method and elute in 9 µL of RNase-free water.
   1. Proceed immediately to Step 5, or keep samples on wet ice for up to one hour.

5. Reverse Transcription of RNA to cDNA

1. Convert concentrated circulating RNA sample to cDNA using a commercially available reverse transcription reaction kit, including random primers (see Table 1 for components).
   Note: Gene specific primers are optional and can be designed for test variants. Primers are designed based on the target RNA sequence. Use fusion variant sequences from Step 2.1.
   1. Include no reverse transcriptase control sample and no RNA control sample (see Table 1).
2. Isolate cDNA from reverse transcription reaction using a commercially available DNA concentrator spin column.
   Note: This step facilitates removal of enzymes, primers, and free deoxynucleotide triphosphates (dNTPs).
3. Use cDNA immediately in PCR reactions or store at -80 ˚C.

6. Digital PCR

Note: This PCR is specific to droplet digital PCR (See Table of Materials).

1. PCR mix precautions.
   1. Wear a disposable lab coat and nitrile gloves.
   2. Use PCR mix reagents in a dedicated reagent preparation area. Do not handle cDNA in the reagent-only preparation area.
   3. Cover probes while working to protect them from light. Excessive light can photo bleach the fluorescent dye attached to the probe.
   4. Transport mixes, covered and protected from light, into a separate pre-amplification area before cDNA is to be added.
   5. Add cDNA to be tested to PCR mix in a PCR clean hood located in pre-amplification area.
2. Prepare PCR mixes for a final reaction volume of 20 µL according to Table 2.
3. Distribute PCR mixes + cDNA to PCR plates.
   Note: Use of a plate layout as a guide is recommended.
4. Cover the plate using a removable plate sealer.
5. Centrifuge plates briefly to collect samples at the bottom of the wells.
6. Mix on plate shaker on a low setting for 10 s.
7. Centrifuge plates briefly to collect sample at the bottom of the well.
8. Remove plate sealer. Perform droplet generation for PCR-cDNA mix with either a manual or automated droplet generation system.
   1. For manual droplet generation, transfer 20 µL PCR mix to sample wells on the droplet generation cartridge. Add 70 µL of droplet generation oil. Cover with rubber gasket and transfer cartridge to manual droplet generator to initiate droplet generation. Following droplet generation, transfer droplets to a fresh PCR plate using tips recommended by the manufacturer. Aspirate and dispense the droplets slowly, over 5 to 6 s each, without touching the opening of the tip to the droplet cartridge or plate.
   2. For automated droplet generation, seal the plate with a foil seal and transfer to the droplet generator. Ensure all tips, cartridges, and plates are in place before starting droplet generation.
9. Following droplet generation and transfer of droplets to a fresh PCR plate, seal with a foil plate sealer, and thermal cycle plates using the settings in Table 3.
10. After the thermal cycler run is complete, read the plate using a droplet reader. Create a plate layout for reader software that identifies the location of controls, samples, etc., and load into software to start read.

7. Data Analysis and Review, and Generation of Results

1. Analyze plate read results using commercially available software.
2. Navigate to the Analyze menu to view two-dimensional (2D) amplitude plots.
3. Evaluate the overall quality of the data by examining the droplet data.
   1. Evaluate data for total accepted event numbers using the Events menu. If there are fewer than 10,000 events per well, carefully evaluate the data for additional problems.
   2. Check data for aberrant fluorescence amplitudes. Significant amplitude differences and concentration differences between replicate samples indicate poor handling or mixing of samples.
   3. Make notes of droplet clusters with spray patterns on a 45-degree axis, which is indicative of poor quality droplets or problematic samples.
   4. Examine Positive Control, No Reverse Transcriptase (No RT), and No RNA Control (NRC) data first. Select all control samples and examine cluster quality by 2D plot. For proper thresholding, a clear separation between droplet clusters should be apparent.
4. For each assay variant, set the threshold based on control wells.
   1. Set thresholds on 2D plots using the crosshairs tool to separate the double negative droplet population from the control gene population (labeled with 5'-hexachloro- fluorescein-CE phosphoramidite probe), y-axis, and variant gene population, if present (labeled with fluorescein amidite OR 6-carboxyfluorescein probe), x-axis.
   2. Sum copies from each replicate well for a single sample.
   3. Express test results as the number of variant copies detected.
      Note: To determine the analytic cutoff value for calling a positive or negative sample, run a normal healthy donor sample set (at least 10 individual samples) through the finalized process and establish the cutoff above any detectable background signal for the mutation of interest. Additionally, establish the number of control gene copies required to call a positive or negative result. This control gene cut-off functions as an internal quality control (QC) to evaluate the quantity and quality of each RNA sample that is processed.

8. Verification of RT-dPCR Reaction Conditions Using Cell-lines (Optional)

1. To verify detection of fusion variants, use commercially available cell-lines expressing the ROS1 or RET fusion mRNA of interest. Proceed as follows:
   1. Homogenize flash-frozen cells in a guanidinium-based lysis solution directly from the frozen state. Even brief thawing prior to homogenization can cause RNA degradation and loss.
   2. Isolate RNA using silica-membrane spin columns designed for RNA.
   3. Measure the concentration of RNA samples using a fluorometer with RNA-specific reagents and standards.
   4. Dilute isolated RNA into a background of wild-type RNA from plasma or another commercial source.
2. Carry out steps from Reverse Transcription of RNA to cDNA, Digital PCR, and Data Analysis and Review, and Generation of Results listed in this protocol to confirm detection of the desired variant.