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

Summary

Detection of circulating ribonucleic acid (cRNA) from blood is an unmet need in clinical diagnostics. Here we describe methods that characterize cRNA from non-small cell lung cancer patients using sensitive and specific digital polymerase chain reaction. The tests meet design requirements to detect fusion variants within 72 hours.

Abstract

We have developed novel methods for the isolation and characterization of tumor-derived circulating ribonucleic acid (cRNA) for blood-based liquid biopsy. Robust detection of cRNA recovered from blood represents a solution to a critical unmet need in clinical diagnostics. The test begins with the collection of whole blood into blood collection tubes containing preservatives that stabilize cRNA. Cell-free, exosomal, and platelet-associated RNA is isolated from plasma in this test system. The cRNA is reverse transcribed to complementary DNA (cDNA) and amplified using digital polymerase chain reaction (dPCR). Samples are evaluated for both the target biomarker as well as a control gene. Test validation included limit of detection, accuracy, and robustness studies with analytic samples. The method developed as a result of these studies reproducibly detect multiple fusion variants for ROS1 (C-Ros proto-oncogene 1; 8 variants) and RET (rearranged during transfection proto-oncogene; 8 variants). The sample processing workflow has been optimized so that test results can consistently be generated within 72 hours of sample receipt.

Introduction

Up to 25% of non-small cell lung cancer (NSCLC) patients may not have sufficient tissue available for testing at the time of diagnosis. Even in cases where tissue is available, it may not be of sufficient quantity or quality to perform recommended molecular tests^1,2. In cases where there is enough tissue from a biopsy for molecular profiling, patients may have to wait several weeks or longer for results, or begin treatment without molecular results^3,4. However, it is critical that informative molecular diagnosis be available given the advent of multiple targeted treatment options for patients diagnosed with NSCLC. Testing of circulating cell-free DNA (cfDNA) from liquid biopsy is a solution to the challenges of traditional tissue testing^4,5,6. Current testing options for actionable mutations in NSCLC using cfDNA and a similar dPCR-based workflow for rapid result generation, include the epidermal growth factor receptor (EGFR) sensitizing mutations ΔE746-A750 and L858R, EGFR resistance mutation T790M, KRAS Proto-Oncogene (KRAS) variants, and B-Raf Proto-Oncogene (BRAF) variant V600E. Although not as broadly adopted by the field, circulating tumor-derived messenger RNA (mRNA) isolated from liquid biopsy can also provide important clinical information^7,8,9. We have previously developed and reported on methods of multiplexed detection of the Echinoderm Microtubule Associated Protein Like 4-Anaplastic Lymphoma Receptor Tyrosine Kinase (EML4-ALK) fusion variants from blood plasma¹⁰. In this study, we extended these methods to include higher-order multiplexed RNA targets for ROS1 and RET, covering eight fusion variants within each assay. The objective was to develop a rapid, sensitive, specific, and reproducible technique for the detection of these fusion variants from the plasma of patients previously diagnosed with NSCLC.

The test process is initiated in a physician's office using RNA stabilizing blood collection tubes¹¹. These tubes contain a cell preservative as well as RNase inhibitors. Samples are shipped priority overnight to the centralized College of American Pathologists (CAP)-accredited/Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory (Clinical Laboratory) for processing by competent personnel. Once received by the Clinical Laboratory, each step of processing is conducted under approved Standard Operating Procedures (SOP). Whole blood is centrifuged to recover plasma, which is then used to isolate circulating RNA that is either free in the blood or within encapsulating moieties, such as exosomes and platelets^7,8,9. To isolate RNA from these compartments, we selected the system for RNA recovery based on comparisons of several extraction methods. The isolated RNA is concentrated and reverse transcribed to cDNA. Several reverse transcriptase enzymes and gene-specific primers were evaluated during the optimization of the cDNA synthesis method to maximize ROS1 and RET target transcript conversion¹⁰. This is critical for low abundance circulating transcripts, such as tumor-derived fusion variants. Finally, we optimized dPCR primer and probe concentrations to allow for multiplexed detection of RET or ROS1 fusion variants and the control gene, glucuronidase-β (GUSB). We then combined the best conditions from each of the optimization studies into a final locked protocol before performing the analytic validation studies described in this report. This protocol and these results provide the basis for a rapid and sensitive workflow for the routine detection of rare fusion variants in circulation.

Protocol

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.

Representative Results

This protocol describes a test system developed for the detection of RNA fusion variants for use in the measurement of driver mutations within the plasma of NSCLC patients (Figure 1A). Fusion mRNA products from the expression of the most common RET and ROS1 rearrangements in the NSCLC population were identified^{13,14,15,16,17}. Multiplexed PCR assays were then designed to detect the eight most common transcript variants for each target in NSCLC within a single reaction. The most common translocations at the ROS1 locus generate associations with the 5' portions of the CD74, SDC4, SLC34A2, EZR or TPM3 genes (Figure 1B). The most common translocations at the RET locus lead to juxtaposition with KIF5B, for which the assay covers six exon junctions. Additional RET partners that are covered include those with CCDC6 and TRIM33 (Figure 1C). In total, the assays cover approximately 88% of ROS1 and 99% of RET alterations known to occur in the NSCLC patient population¹⁷.

The specificity of the assay components was first evaluated using eight individual in vitro RNAs that contain the mRNA sequence for the fusion transcripts covered by the ROS1 or RET multiplexed assays. Each RNA species was tested against each individual variant assay that comprises the multiplexed version. There was no cross-reactivity of these assays, thus demonstrating 100% analytic specificity within the designed multiplexed assays (data not shown). To determine the lower limit of detection of the test protocol, total RNA derived from cell lines expressing a fusion variant included in the assay were mixed into a background of normal RNA at 5%, 1%, 0.2%, and 0.04% concentrations. The multiplexed RET and ROS1 variant PCR assays detected as little as 0.2% fusion variant (Figure 2A-B). Additionally, a preparation of 5% off-target cell line derived RNA (expressing an EML4-ALK fusion transcript) was not detected with the multiplexed ROS1 and RET assays, further demonstrating specificity (Figure 2A-B).

Precision testing of the RT-dPCR process was performed for both ROS1 and RET. Analytic control material comprised of equimolar in vitro RNAs was processed at three concentrations (High, Medium, and Low) through reverse transcription and dPCR on three different occasions within the same day (intra-day), on three consecutive days (inter-day), and with two operators (inter-operator). Results from precision testing demonstrated accurate detection of both the fusion transcript of interest, as well as a control gene, GUSB, which is included as an internal QC metric (Figure 2C-D).

In addition to the GUSB internal control, each batch of clinical samples was run with a set of batch controls. A positive control was developed from a mixture of analytic in vitro RNA that represented each of the fusion variants tested in RT-dPCR, as well as analytic in vitro RNA for GUSB. This RNA was spiked into normal human plasma lysate during RNA extraction and was processed alongside the clinical samples throughout the protocol. The no reverse transcriptase (No RT) control was a negative control to confirm the absence of contaminating material in the RNA extraction workflow and demonstrate the specificity of the primers for RNA. The No RT control was generated using the same material as the positive control, but it does not include enzyme within the cDNA synthesis reaction. The no RNA control (NRC) is a negative control to confirm the absence of contaminating transcripts in the reverse transcription reaction components. This control was introduced into the workflow at the cDNA synthesis step, and water was added in the reaction instead of an RNA template. The No RT and NRC controls must be negative in both channels, if accurate results are to be delivered. Table 1 lists the reverse transcription reaction components for each control. Examples of the 2D plots for each of these controls are shown for the ROS1 (Figure 3 A-C) and RET (Figure 3 E-G) multiplex assays. Fusion variants were detected using a fluorescein amidite (FAM) probe and are represented along the y-axis, while the control gene, GUSB, was detected using a 5'-hexachloro-fluorescein-CE phosphoramidite (HEX) probe and is on the x-axis. These batch controls were assessed over the course of 21 days to determine assay robustness. Fusion positive droplets and GUSB control gene droplets were observed for ROS1 and RET in all 21 runs executed over the course of the study (Figure 3D,H). All negative controls (No RT and NRC) yielded negative results across the entire 21 days (data not shown).

The ability to troubleshoot is a critical component of any test protocol to be run in the clinical laboratory setting. Here, we provide real world examples of sub-optimal results using the RT-dPCR protocol. The first is an example 2D plot demonstrating the importance of the no reverse transcriptase control (Figure 4A). In this example, mutant positive droplets were present even though there was no cDNA conversion due to lack of enzyme. This outcome was likely due to dPCR primers amplifying off-target genomic DNA. In this case, design of an intron-spanning assay will prevent amplification of genomic DNA. Alternatively, an RNase-free DNase enzyme can be used to eliminate the contaminating DNA, but this is not recommended for detection of rare targets, as some RNA degradation may occur during incubation with the enzyme. The next example 2D plot was a NRC with positive droplets in both channels (Figure 4B). This indicated contamination at some point in the RT-dPCR setup. In this case, the recommendation is to discard any potentially contaminated reagents used in testing, thoroughly decontaminate all equipment, and re-test with fresh reaction components. The third example 2D plot presented as a spray of droplets along a 45° line (Figure 4C). This is often caused by shearing and coalescing of droplets. Careful droplet handling prior to thermal cycling is essential, as droplets are prone to damage. We recommend the use of automated droplet generation, when available. If transferring manually generated droplets, be certain to choose the recommended wide-bore tips and employ careful pipetting technique. Droplet transfer requires slow aspiration and dispensing, with each taking place over 5-6 seconds, and it is essential that the pipette tip opening does not touch the droplet cartridge or well. When dispensing, keep the pipette tip at the liquid level and raise it slowly as droplets are dispensed (view video for demonstration). The final 2D plot example demonstrates a lack of separation between the positive and negative droplet populations (Figure 4D). This can have several causes. Strong PCR inhibitors, such as detergents used in lysis buffers and an excess of highly degraded DNA, can cause loss of separation. In this case, consider adding a clean-up step between cDNA synthesis and dPCR (such as described within Step 5 of this protocol). Finally, lack of separation can also be due to sub-optimal amplification conditions, and optimization of the PCR step should also be considered.

Data within Figure 5 represent 984 real world patient sample turn-around times and demonstrates the rapid nature of this test workflow. Results were reported to the treating physician as early as within 48 hours (79% of cases) of sample receipt and in 95% of cases, within 72 hours. In conclusion, the use of stabilized circulating RNA blood collection tubes, optimized RNA extraction procedures from blood, and RT-dPCR run according to an optimized protocol with the appropriate internal and batch controls, can provide a rapid test system for the accurate detection of fusion RNA variants relevant in NSCLC.

Figure 1: Overview of Blood Sample Processing Steps for Fusion Variant Detection using Assays Specific to the Most Prevalent RET and ROS1 Variants in NSCLC. (A) Sample testing is initiated when whole blood is drawn and a BCT is shipped within the specimen collection kit to the Clinical Laboratory. Circulating RNA is recovered from multiple sources within the platelet-enriched plasma, reverse transcribed with gene specific priming, and purified for use in dPCR. Samples are processed using a commercially available system which consists of droplet generation (emulsion), amplification, and droplet counting. Data is analyzed using commercially available software. The test results are then documented and reported back to the test-requesting physician. The process is designed to work within a timeframe of 72 hours from sample receipt to result release. Eight variants for (B) ROS1 and (C) RET are covered within the multiplexed assays. Adapted from Biodesix website with permission. Please click here to view a larger version of this figure.

Figure 2: Analytic Validation. Cell-lines expressing (A) SDC4-ROS1 fusion and (B) CCDC6-RET fusion were diluted in a background of total human wild-type RNA (WT RNA). With each fusion variant, the limit of detection was established at 0.2% variant frequency using pre-defined criteria for each variant assay. All samples above this threshold also contained at least 21 copies of control gene. 5% EML4-ALK (ALK) standard in a background of wild-type RNA was tested to demonstrate assay specificity, which was confirmed by a negative result. Analytic multiplexed RNA standards were measured at high, medium, and low concentrations for (C) ROS1 and (D) RET. Precision was evaluated over three runs on the same day (Intra-day), three runs on three consecutive days (Inter-day), and with two independent operators (Inter-operator). The means of copy number and standard deviations are shown. Adapted from Biodesix website with permission. Please click here to view a larger version of this figure.

Figure 3: Batch Processing Control Examples and Robustness Data. 2D plot of the ROS1 multiplexed assay dPCR results for (A) positive control, (B) no reverse transcriptase control, and (C) no RNA template control. (D) Controls were run on 21 consecutive days (excluding weekends and holidays). Mean copy number +/- standard deviations for ROS1 positive control were 439 +/- 141. No reverse transcriptase and no template controls were also run on each day, and these were all negative (data not shown). 2D plot of the RET multiplexed assay dPCR results for (E) positive control, (F) no reverse transcriptase control and (G) no RNA template control. (H) Controls were run on 21 consecutive days (excluding weekends and holidays). Mean copies +/- standard deviations for RET positive control were 586 +/- 182. Not shown are no reverse transcriptase and no template controls that were also run on each day and were all negative. Adapted from Biodesix website with permission. Please click here to view a larger version of this figure.

Figure 4: Troubleshooting RT-dPCR. 2D plots representing sub-optimal dPCR results obtained when there is (A) contamination within the no reverse transcriptase control, (B) contamination within the no RNA control, (C) shearing and coalescing of droplets, and (D) poorly optimized PCR conditions or PCR inhibition. Please click here to view a larger version of this figure.

Figure 5: Turnaround time (TAT). TAT (in hours) was compiled for tests requesting an RNA variant (n = 984). Data excludes weekends, holidays, and samples held for >24 h due to incomplete clinical information on the laboratory Test Request Forms. Please click here to view a larger version of this figure.

Table 1: Preparation of Reverse Transcription Reagents for Process Controls.

Component	Volume
2x dPCR supermix for probes (no 2’-deoxyuridine 5’-triphosphate)	10 µL
20x variant target primers/probes set  (450 nmol/L primers, 250 nmol/L FAM probe)	1 µL
20x control target primers/probe set (450 nmol/L primers, 250 nmol/L HEX probe)	1 µL
nuclease-free water	1 µL
cDNA	7 µL

Table 2: Preparation of the master mix for dPCR.

Cycling Step	Temperature	Time	# Cycles	Ramp Rate
Enzyme activation	95 °C	10 min	1	~2 oC/s
Denaturation	94 °C	30 s	40
Annealing/extension	55 °C	1 min
Enzyme deactivation	98 °C	10 min	1
Hold (optional)	4 °C	infinite	1	~1 oC/s

Table 3: Thermal Cycling Conditions.

Discussion

RET and ROS1 rearrangements together make up ~3% of the driver mutations within the NSCLC population¹⁸. Although rare, the detection of these genetic alterations is vital. NSCLC patients with these alterations may benefit from targeted therapeutics that specifically inhibit the aberrant kinase activity that results from the onco-protein¹³. Some such therapies are already approved by the FDA for use in ROS1 positive NSCLC, while others have been demonstrated to be efficacious against RET in clinical trials¹⁹.

Digital PCR technology provides sensitivity that is ideal for liquid biopsy applications²⁰. There has been significant adoption of this technology for use with circulating cell-free DNA for the measurement of tumor mutations in patients with NSCLC^4,6,21,22,23. In addition to cfDNA, we developed a protocol designed for robust measurement of the most prevalent fusion variants in patients with NSCLC from circulating tumor RNA (Figure 1A)¹⁰.

Our established protocol allows for analytic limits of detection down to 0.2% (Figure 2). While the RT-dPCR is exceptionally specific and sensitive, the assays are limited to the panel of known fusion variants that are chosen and multiplexed for detection in the PCR assay. Thus, fusions to be included in multiplexed assays must be carefully selected to ensure proper coverage within the population of patients with NSCLC. We have successfully designed assays for RET and ROS1 that simultaneously detect eight fusion variants resultant from rearrangements of the RET or ROS1 loci and cover 99% and 88% of the RET and ROS1 positive population, respectively (Figure 1B-C)¹⁷.

The final test workflow as described in this study includes batch controls to ensure consistency of results. These controls include both a positive analytic standard as well as two negative controls, which together ensure there is no contamination or PCR inhibition occurring within the batch (Figure 3). To ensure robustness of the assay, a study was performed using the batch controls over a 21-day period (Figure 3D,H). These data demonstrate the consistency of the RNA process as established within this protocol.

Good laboratory practices and appropriate RNA handling are key components of ensuring robust and accurate results. Laboratory space and equipment dedicated to use with RNA, cleaning of equipment after each use, using RNase-free reagents and consumables, and applying an RNase inactivation spray to the work space all help to reduce contaminating RNases. Conscientious handling of RNA samples by technicians, including a dedicated lab coat, frequent glove changes, working quickly through the RNA extraction procedure, and keeping samples on ice are of utmost importance to preserving sample integrity. Once RNA has been reverse transcribed to cDNA, the sample is in a more stable form that is less prone to degradation. In addition to practices that support RNA integrity, PCR components and samples should be maintained in segregated areas to prevent cross-contamination that could lead to false positive results. The stock PCR reagents and preparation of PCR master mixes should be kept separate from PCR templates and great care should be taken to segregate amplified template (post-PCR) from all pre-amplified materials including reagents, RNA, and cDNA samples. Finally, proper generation and handling of emulsified PCR mixes prior to amplification is central to maintaining droplet integrity and optimal dPCR conditions. Precautions like these are critical during execution of this protocol to obtain consistent and accurate results. All data should be examined by trained personnel before the release of results to be sure that all QC metrics have been met. In the case of suboptimal results (Figure 4), the batch must be reviewed by technical staff and the Laboratory Director and may require re-processing.

RT-dPCR results can be produced as early as 24 hours from sample receipt and 95% of sample results within the test set used in this study (n = 984) were reported to the ordering physician in less than 72 hours from the time of receipt (Figure 5). This turn-around time provides physicians with much needed molecular information in a time frame that allows initiation of the appropriate therapy. These results are typically available earlier than those obtained using a conventional tissue biopsy. Additional biomarkers for NSCLC and other cancers could be developed using similar circulating RNA-based approaches, and would benefit from the same rapid time-to-results. For example, measurement of the Programmed Death Ligand 1 (PD-L1) mRNA transcript using RT-dPCR could inform physicians about immunotherapy options. There is also a growing interest in the utility of liquid biopsy and dPCR in monitoring for therapeutic efficacy. Earlier indications of resurgence of the tumor using genomic testing for specific variants could allow physicians to adjust treatment regimens before patients are symptomatic by standard of care measures such as imaging²⁴. Protocols such as the one reported in this study are ideal for monitoring due to their non-invasiveness, sensitivity, rapid turn-around time, and cost effectiveness. The assay described herein provides results within 72 hours from sample receipt, with minimal false-positive detection rates, which facilitates rapid treatment decisions and circumvents some limitations experienced with tissue-based testing⁴.

Our protocol and data demonstrate a robust test system for identifying low abundance RNA variants as well as the potential for blood-based mutation testing in clinical practice. For those patients who do not have an actionable driver mutation identified by rapid targeted liquid biopsy approaches like this one, the addition of more extensive genome and proteome testing from both tissue and blood may provide even broader clinical information to support treatment planning.

Materials

Name	Company	Catalog Number	Comments
Ultrapure Distilled Water (DNAse, RNAse Free) (500 mL)	Life Technologies	10977-015	1604071
Ultrapure Distilled Water (DNAse, RNAse Free) (500 mL)	Life Technologies	10977-015	1809353
Nuclease-free water (molecular grade)	Ambion	AM9938	1604071
Nuclease-free water (molecular grade)	Ambion	AM9938	1606077
Phosphate Buffered Saline 1X, Sterile	Amresco	K812-500mL	1446C189
Phosphate Buffered Saline 1X, Sterile – 500 mL	Invitrogen	10010023	1916C092
RNase Zap (Life Tech) (250 mL)	Ambion	AM9780	353952
Beta-Mercaptoethanol (BME)  (250 mL)	CalbioChem	6050	W105B
OmniPur Ethyl Alcohol	CalbioChem	4455-4L	56054611
OmniPur Ethyl Alcohol	CalbioChem	4455-4L	56238638
Isopropyl Alcohol	VWR	0918-4L	2116C416
TranscriptAid T7 High Yield Transcription Kit	Thermo Scientific	K0441	403648
TranscriptAid T7 High Yield Transcription Kit	Thermo Scientific	K0441	288461
DNase I	Thermo	K0441	371299
QIAzol Lysis Reagent	Qiagen	79306	54809699
20x TE buffer pH 8.0	Alfa Aesar	J62388	R13C548
UltraPure Agarose	Invitrogen	16500-100	552730
10x TBE buffer	Invitrogen	AM9863	353065
Cell-Free RNA BCT	Streck	218976	60110327
Cell-Free RNA BCT	Streck	218976	61900327
Cell-Free RNA BCT	Streck	218976	61480327
Cell-Free RNA BCT	Streck	218976	62320327
Plasma/Serum Circulating and Exosomal RNA Purification Kit (Slurry Format) 50 preps	Norgen	42800	585849
Plasma/Serum Circulating and Exosomal RNA Purification Kit (Slurry Format) 50 preps	Norgen	42800	588308
Lysis Buffer	Norgen	21205	A5F61E
RNA Cleanup and Concentration Micro-Elute Kit (Norgen) 50 preps	Norgen	61000	585848
RNA Cleanup and Concentration Micro-Elute Kit (Norgen) 50 preps	Norgen	61000	588309
DNA Clean and ConcentratorTM- 5  200 preps (samples)	Zymo	D4014	ZRC186976
DNA Clean and ConcentratorTM- 5  200 preps (samples)	Zymo	D4014	ZRC188077
DNA Clean and ConcentratorTM- 5  200 preps (samples)	Zymo	D4014	ZRC188413
Collection Tubes 500 pack	Zymo	C1001-500	N/A
SuperScript IV First Strand Synthesis System 200 rxn (samples)	Life Technologies	18091200	391657
SuperScript IV First Strand Synthesis System 200 rxn (samples)	Life Technologies	18091200	392504
SuperScript IV First Strand Synthesis System 200 rxn (samples)	Life Technologies	18091200	448001
SuperScript IV Reverse Transcriptase	Life Technologies	18090200	451702
Qubit HS RNA Assay Kit (500)	Life Technologies	Q32854	1745264
Qubit assay tubes (500)	Life Technologies	Q32856	13416Q311
ddPCR Supermix for Probes (no dUTP)	Bio-Rad	1863023	64031651
ddPCR Supermix for Probes (no dUTP)	Bio-Rad	1863023	64063941
ddPCR Supermix for Probes (no dUTP)	Bio-Rad	1863023	64065740
ddPCR Supermix for Probes (no dUTP)	Bio-Rad	1863023	64065741
ddPCR Supermix for Probes (no dUTP)	Bio-Rad	1863023	64079083
ddPCR Buffer Control for Probes	Bio-Rad	1863052	64025320
ddPCR Buffer Control for Probes	Bio-Rad	1863052	64052358
gBlock KIF5B-RET K15:R12	IDT	151004172	4-Oct-16
gBlock KIF5B-RET K16:R12	IDT	151004173	4-Oct-16
gBlock KIF5B-RET K22:R12	IDT	151004174	4-Oct-16
gBlock KIF5B-RET K23:R12	IDT	151004175	4-Oct-16
gBlock KIF5B-RET K24:R11	IDT	151004176	4-Oct-16
gBlock KIF5B-RET K24:R8	IDT	151004177	4-Oct-16
gBlock CCDC6-RET C1:R12	IDT	151004178	4-Oct-16
gBlock TRIM33-RET T14:R12	IDT	151004179	4-Oct-16
RET exon 8 RT Gene Specific Primer	IDT	150554385	28-Sep-16
5’-CTCCACTCACACCTG-3’	IDT	150554385	28-Sep-16
RET exon 11 RT Gene Specific Primer	IDT	150554384	28-Sep-16
5’-GCAAACTTGTGGTAGCAG-3’	IDT	150554384	28-Sep-16
RET exon 12 RT Gene Specific Primer	IDT	150554383	28-Sep-16
5’-CTGCCTTTCAGATGGAAG-3’	IDT	150554383	28-Sep-16
gBlock CD74-ROS1 C6:R34	IDT	152324366	15-Nov-16
gBlock CD74-ROS1 C6:R32	IDT	152324367	15-Nov-16
gBlock SDC4-ROS1 S2:R32	IDT	152324368	15-Nov-16
gBlock SDC4-ROS1 S2:R34	IDT	152324369	15-Nov-16
gBlock S13del2046-ROS1 S13del2046:R32	IDT	152324370	15-Nov-16
gBlock S13del2046-ROS1 S13del2046:R34	IDT	152324371	15-Nov-16
gBlock EZR-ROS1 E10:R34	IDT	152324372	15-Nov-16
gBlock TPM3-ROS1 T8:R35	IDT	152324373	15-Nov-16
ROS1 exon 34 RT Gene Specific Primer	IDT	152704983	21-Nov-16
5’-CCTTCCTTGGCACTTT-3’	IDT	152704983	21-Nov-16
ROS1 exon 35 RT Gene Specific Primer	IDT	152704985	21-Nov-16
5’-CTCTTGGGTTGGAAGAGTATG-3’	IDT	152704985	21-Nov-16
ALK Gene Specific Primer	IDT	140035422	26-Aug-16
5’-CAGTAGTTGGGGTTGTAGTCG-3’	IDT	140035422	26-Aug-16
EML4-ALK Cell line pellet	Horizon Discovery	N/A	11-Jun-15
SLC34A2-ROS1 Cell line pellet	Horizon Discovery	N/A	11-Jun-15
CCDC6-RET Cell line pellet	Horizon Discovery	N/A	11-Jun-15
Human Brain Total RNA	Ambion	AM7962	1703548
PrimePCR ddPCR Expert Design Assay: K15:R12	Bio-Rad	N/A	17-Aug-16
PrimePCR ddPCR Expert Design Assay: K16:R12	Bio-Rad	N/A	17-Aug-16
PrimePCR ddPCR Expert Design Assay: K22:R12	Bio-Rad	N/A	17-Aug-16
PrimePCR ddPCR Expert Design Assay: K23:R12	Bio-Rad	N/A	17-Aug-16
PrimePCR ddPCR Expert Design Assay: K24:R11	Bio-Rad	N/A	17-Aug-16
PrimePCR ddPCR Expert Design Assay: K24:R8	Bio-Rad	N/A	17-Aug-16
PrimePCR ddPCR Expert Design Assay: C1:R12	Bio-Rad	N/A	17-Aug-16
PrimePCR ddPCR Expert Design Assay: T14:R12	Bio-Rad	N/A	17-Aug-16
PrimePCR ddPCR Expert Design Assay: C6:R34	Bio-Rad /Biodesix	N/A	6-Dec-16
PrimePCR ddPCR Expert Design Assay: C6:R32	Bio-Rad /Biodesix	N/A	6-Dec-16
PrimePCR ddPCR Expert Design Assay: S2:R32	Bio-Rad /Biodesix	N/A	6-Dec-16
PrimePCR ddPCR Expert Design Assay: S2:R34	Bio-Rad /Biodesix	N/A	6-Dec-16
PrimePCR ddPCR Expert Design Assay: S13del2046:R32	Bio-Rad /Biodesix	N/A	6-Dec-16
PrimePCR ddPCR Expert Design Assay: S13del2046:R34	Bio-Rad /Biodesix	N/A	6-Dec-16
PrimePCR ddPCR Expert Design Assay: E10:R34	Bio-Rad /Biodesix	N/A	6-Dec-16
PrimePCR ddPCR Expert Design Assay: T8:R35	Bio-Rad /Biodesix	N/A	6-Dec-16
(version 2)	Bio-Rad	12003909	213939881
PrimePCR ddPCR Expert Design Assay: ROS1 Multiplex (version 3.2)	Bio-Rad	N/A	13-Dec-16
PrimePCR ddPCR Expert Design Assay: ROS1 Multiplex (version 3.2)	Bio-Rad	N/A	20170112v3.2
PrimePCR ddPCR Gene Expression Probe Assay: GUSB, Human	Bio-Rad	10031257	212851151
PrimePCR ddPCR Gene Expression Probe Assay: GUSB, Human	Bio-Rad	10031257	207383915
PrimePCR ddPCR Gene Expression Probe Assay: GUSB, Human	Bio-Rad	10031257	195995635
PrimePCR ddPCR Gene Expression Probe Assay: GUSB, Human	Bio-Rad	10031257	212851152
PrimePCR ddPCR Gene Expression Probe Assay: GUSB, Human	Bio-Rad	10031257	213949301
PrimePCR ddPCR Expert Design Assay: EML4-ALK	Bio-Rad	12003909	20160914
PrimePCR ddPCR Expert Design Assay: EML4-ALK	Bio-Rad	12003909	211383227
Droplet Generation Oil for Probes	Bio-Rad	186-3005	1065C220
Droplet Generation Oil for Probes	Bio-Rad	186-3005	64052953
Droplet Generation Oil for Probes	Bio-Rad	186-3005	64052358
Automated Droplet Generation Oil for Probes (20x96)	Bio-Rad	186-4110	1065C320
Automated Droplet Generation Oil for Probes (20x96)	Bio-Rad	186-4110	64052952
Automated Droplet Generation Oil for Probes (20x96)	Bio-Rad	186-4110	64064127
DG8 Cartridges for QX100/QX200 Droplet Generator	Bio-Rad	186-4008	C000065883
DG8 Cartridges for QX100/QX200 Droplet Generator	Bio-Rad	186-4008	C000084276
DG8 Cartridges for QX100/QX200 Droplet Generator	Bio-Rad	186-4008	C000079928
DG8 Cartridges for QX100/QX200 Droplet Generator	Bio-Rad	186-4008	C000084395
DG8 Cartridges for QX100/QX200 Droplet Generator	Bio-Rad	186-4008	C000084634
Droplet Generator DG8 Gasket	Bio-Rad	186-3009	20160627
Droplet Generator DG8 Gasket	Bio-Rad	186-3009	20161107
Droplet Generator DG8 Gasket	Bio-Rad	186-3009	20161206
Droplet Generator DG8 Gasket	Bio-Rad	186-3009	20161216
Droplet Generator DG8 Gasket	Bio-Rad	186-3009	20170125
Pipet Tips for Automated Droplet Generator	Bio-Rad	1864120	PR125340
DG32 Cartridge for Automated Droplet Generator (10-96 well plates)	Bio-Rad	186-4108	206894
DG32 Cartridge for Automated Droplet Generator (10-96 well plates)	Bio-Rad	186-4108	206893
Pierceable Foil Heat Seal	Bio-Rad	1814040	1409850
Pierceable Foil Heat Seal	Bio-Rad	1814040	100402
Pierceable Foil Heat Seal	Bio-Rad	1814040	145851
Microseal 'B' seals	Bio-Rad	MSB1001	BR00428490
ddPCR Droplet Reader Oil	Bio-Rad	186-3004	64039089
ddPCR Droplet Reader Oil	Bio-Rad	186-3004	64049253
ddPCR Droplet Reader Oil	Bio-Rad	186-3004	64049255
ddPCR Droplet Reader Oil	Bio-Rad	186-3004	64081870
DNA Lo Bind Tube 0.5 mL	Eppendorf	22431005	E1629620
DNA Lo Bind Tube 1.5 mL	Eppendorf	22431021	F16698K
DNA Lo Bind Tube 2 mL	Eppendorf	22431048	E160610I
50 mL Conicals, Polypropylene (25)	Thermo	339652	G5ZF5W8118
TempAssure PCR 8-Strips, Optical Caps, Natural, polypropylene (120)	USA Scientific	1402-4700	16202
For Rainin LTS Pipettors 0.5-20 µL tips	Pipette.com	LF-20	40155-642C4-642C
For Rainin LTS Pipettors 5-200 µL tips	Pipette.com	LF-250	40154-642C4-642B
Tips LTS 200 ul Filter 960/10 RT-L200F (10 boxes)	Rainin	17002927	1635
Pipet Tips, 10 ul TipOne RPT ultra low retention filter tip refill cassette, sterile	USA Scientific	1181-3710	F1175551-1108
Pipet Tips, 10 ul TipOne RPT ultra low retention filter tip refill cassette, sterile	USA Scientific	1181-3710	F118054L-1720
Pipet Tips, 100 ul TipOne RPT ultra low retention filter tip refill cassette, sterile (10x96)	USA Scientific	1180-1740	0014961Q-2501
Pipet Tips, 200 ul TipOne RPT ultra low retention filter tip refill cassette, sterile	USA Scientific	1180-8710	E116684P-1540
Pipet Tips, 1000 ul XL TipOne RPT ultra low retention filter tip refill cassette, sterile (10x96)	USA Scientific	1182-1730	F118815P
5 mL Standard Racked Gilson-fit Reference Tips	Scientific Specialties	4411-00	14312
Combitips advanced, 0.1 mL Biopur	Eppendorf	003 008 9618	F165414H
Combitips advanced, 0.2 mL Biopur	Eppendorf	0030 089.626	F166689J
Combitips advanced, 5 mL Biopur	Eppendorf	0030.089 669	F166054J
Combitips advanced, 50 mL Biopur	Eppendorf	003.008.9693	F166055I
Reagent Reservoir	VWR	89094-680	141500
Twin tec PCR Plate 96, semi-skirted, Clear	Eppendorf	951020303	E163697P
Twin tec PCR Plate 96, semi-skirted, Clear	Eppendorf	951020303	F165029I
Twin tec PCR Plate 96, semi-skirted, Clear	Eppendorf	951020303	F165028G
Twin tec PCR Plate 96, semi-skirted, Clear	Eppendorf	951020303	E163697P
Twin tec PCR Plate 96, semi-skirted, Green	Eppendorf	951020346	F166183K
Equipment Type		Equipment ID
Analytical Balance		EQP0125
Cryogenic Freezer 1, -80oC		EQP0095
Refrigerator 6.1 cu ft GP06W1AREF		EQP0139
-20oC Freezer		EQP0140
Beckman Coulter Microfuge 22R		EQP0025
Beckman Coulter Microfuge 22R		EQP0124
Thermo Scientific Hereaus Megafuge 8		EQP0104
Mini Centrifuge		EQP0131
Mini Centrifuge		EQP0136
Mini Centrifuge		EQP0134
Mini Centrifuge		EQP0235
Mini Centrifuge		EQP0216
Thermo Scientific HeraTherm Incubator		EQP0105
Pipette 0.1 - 2.5 μL		EQP0182
Pipette 0.1 - 2.5 μL		EQP0072
Pipette 0.1 - 2.5 μL		EQP0070
Pipette 0.5-10 μL		EQP0218
Pipette 0.5-10 μL		EQP0075
Pipette 0.5-10 μL		EQP0169
Pipette 0.5-10 μL		EQP0074
Pipette 0.5-10 μL		EQP0147
Pipette 2 - 20 μL		EQP0128
Pipette 2 - 20 μL		EQP0160
Pipette 2 - 20 μL		EQP0018
Pipette 2 - 20 μL		EQP0146
Pipette 10 - 100 μL		EQP0079
Pipette 10 - 100 μL		EQP0181
Pipette 10 - 100 μL		EQP0085
Pipette 10 - 100 μL		EQP0077
Pipette 20 - 200 μL		EQP0088
Pipette 20 - 200 μL		EQP0087
Pipette 20 - 200 μL		EQP0231
Pipette 100 - 1000 μL		EQP0050
Pipette 100 - 1000 μL		EQP0158
Pipette 100 - 1000 μL		EQP0217
Pipette 100 - 1000 μL		EQP0082
Pipette 100 - 1000 μL		EQP0183
Pipette 100 - 1000 μL		EQP0083
Pipette 5 mL		EQP0153
Timer		S/N 140623950
Hamilton SafeAire VAV Fume Hood		EQP0206
Biosafety Cabinet		EQP0205
Biosafety Cabinet		EQP0204
Qubit 3.0		EQP0102
Benchmark Digital Heat Block		EQP0108
Benchmark Digital Heat Block		EQP0231
Polaroid Z2300 Instant Print Digital Gel Camera with WiFi and 16GB SDHC memory card		EQP0111
Electrophoresis Power Unit		EQP0113
Electrophoresis Small Gel Box		EQP0116
Maestro Transilluminator		EQP0118
Microwave		EQP0215
Multichannel 8-well Pipette 2 -  20 μL		EQP0207
Multichannel 8-well Pipette 10 - 100 μL		EQP0090
Rainin Multichannel 8-well Pipette 50 μL		EQP0094
Rainin Multichannel 8-well Pipette 50 μL		EQP0161
Rainin Multichannel 8-well Pipette 50 μL		EQP0162
Rainin Multichannel 8-well Pipette 50 μL		EQP0163
Vortex Genie 2		EQP0052
Vortex Genie 2		EQP0007
Vortex Genie 2		EQP0132
Vortex Genie 2		EQP0137
Vortex Genie 2		EQP0135
Air Clean PCR Workstation		EQP0203
Air Clean PCR Workstation		EQP0096
Air Clean PCR Workstation		EQP0148
Air Clean PCR Workstation		EQP0097
QX200 Droplet Generator		EQP0202
QX200 Droplet Generator		EQP0121
Automated Droplet Generator		EQP0179
PX1 PCR Plate Sealer		EQP0123
PX1 PCR Plate Sealer		EQP0186
C1000 Touch Cycler w/96W FS RM		EQP0120
S1000 Cycler w/96W FS RM		EQP0174
S1000 Cycler w/96W FS RM		EQP0173
T100 Thermal Cycler		EQP0180
T100 Thermal Cycler		EQP0175
QX200 Droplet Reader		EQP0194
QX200 Droplet Reader		EQP0122