Seung Eun Lee and Mi-Sook Lee contributed equally to this work.
Tropomyosin receptor kinase (TRK) inhibitors are approved for the treatment of neurotrophic receptor tyrosine kinase (
A total of 18 samples, including a positive standard reference and eight positive and nine negative clinical samples, were validated using the VENTANA pan-TRK (EPR17341) and TruSight Oncology 500 assays. These samples were then tested using four different NGS panels currently being used at the six participating institutions.
This study is the first to test the proficiency of NGS-based
The neurotrophic receptor tyrosine kinase (
Several different TRK inhibitors are currently investigated for clinical application (larotrectinib, entrectinib, selitrectinib, and repotrectinib). Of these agents, Larotrectinib is a highly selective TRK inhibitors [
To qualify for the former treatment, tumors must have an
This study aimed to compare the ability of four different targeted RNA and DNA sequencing assays to reliably detect
FFPE tumor tissue blocks were cut into 5 μm thick sections and stained with hematoxylin and eosin. To detect the expression of TRK, all samples were stained using the VENTANA pan-TRK (EPR17341) assay kit (Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s instructions. Although there is no scoring algorithm or criteria to determine immunohistochemistry (IHC) positivity, positive staining has been defined as at least 1% of tumor cells in any pattern including cytoplasmic, membranous, perinuclear and nuclear staining, as described in recent studies [
For the NGS assays, institution A—the organizing institution—prepared a total of 18 samples, including eight positive clinical samples with the
Three different RNA panels and one DNA panel used to diagnose patients at each hospital were tested to detect the fusion of the
DNA (40 ng) was quantified using the Qubit dsDNA HS Assay (Thermo Fisher Scientific, Waltham, MA) on a Qubit 2.0 Fluorometer (Thermo Fisher Scientific), then sheared using a Covaris E220 Focused-ultrasonicator (Woburn, MA) and the 8 microTUBE–50 Strip AFA Fiber V2 following manufacturer’s instructions. The treatment time was optimized for FFPE material. The treatment settings were as follows: peak incident power (W), 75; duty factor, 15%; cycles per burst, 500; treatment time (seconds), 360; temperature (°C), 7; water level, 6. The DNA library was prepared and enriched using the TSO 500 Kit (Illumina); manufacturer’s instructions were followed. Post-enriched libraries were quantified, pooled, and sequenced using NextSeq 500 (Illumina). The quality of the NextSeq 500 sequencing runs was assessed using Illumina Sequencing Analysis Viewer (Illumina). Sequencing data were analyzed using the TSO 500 Local App ver. 1.3.0.39 (Illumina). The TSO 500 is a comprehensive tumor profiling assay designed to identify known and emerging tumor biomarkers, including small variants, splice variants, and fusions. Importantly, the TSO 500 measures tumor mutational burden (TMB) and microsatellite instability, which are potentially key biomarkers for immunotherapy. TMB was reported as mutations per megabase (Mb) sequenced, and a high TMB was defined as mutations of more than 10 per Mb (≥ 10 Mut/Mb).
Genomic DNA was isolated from each section of FFPE tissue using a NEXprep FFPE Tissue kit (NexK-9000, Genes Laboratories, Seongnam, Korea), according to the manufacturer’s protocol. Tissue pellets were lysed completely by overnight incubation with proteinase K in lysis buffer at 56°C, followed by additional incubation for 3 minutes with magnetic beads and solution A at 37°C. After incubation for 5 minutes on a magnetic stand, the supernatants were removed and washed 3 times with ethanol. After the beads were air-dried for 5 minutes, DNA was eluted in 50 μL of nuclease-free water and quantified using a Qubit dsDNA HS Assay kit (Thermo Fisher Scientific). Targeted NGS was performed using the NextSeq platform (Illumina) with OncoPanel v.4.3, designed in-house by the Center for Cancer Genome Discovery at institution D to target a total of 328 genes (808 kb), including a complete exonic sequence of 225 genes, 105 hot spots, and a partial intronic sequence of six genes. A DNA library was prepared by fragmenting gDNA (200 ng) to an average of 250 bp using S1 enzyme, followed by sequential reactions of end repair, A tailing, and ligation of 50 ng of purified DNA using a TruSeq adaptor, using a SureSelectXT Reagent kit (Agilent Technologies, Santa Clara, CA). Each library was addressed with sample-specific barcodes of 6 bp and quantified using Qubit. Eight libraries were pooled to a total of 750 ng for hybrid capture using an Agilent SureSelectXT custom kit (OncoPanel ver. 4.3 RNA bait, Agilent Technologies). The concentration of the enriched target was measured using a quantitative polymerase chain reaction (Kapa Biosystems, Woburn, MA) and the sample was loaded onto the NextSeq platform (Illumina) for paired-end sequencing.
Lung Cancer Panel v3.0 is a laboratory-developed DNA- and RNA-based NGS assay to target 75 DNA genes (DNA) and 21 genes (RNA), respectively, including
For library preparation, the multiplex PCR-based Ion Torrent AmpliSeq technology (Life Technologies, Thermo Fisher Scientific) with the Oncomine Comprehensive Assay v3 (IonTorrent, Thermo Fisher Scientific) was used. The OCA v3 allows concurrent analysis of DNA and RNA to detect multiple types of variants across 161 solid tumor–related genes simultaneously in a single workflow. Amplicon library preparation was performed using the Ion AmpliSeq Library Kit v2.0 (Thermo Fisher Scientific). Briefly, 20 ng of genomic DNA was mixed with the two primer pools and the AmpliSeq HiFi Master Mix before they were transferred to a PCR cycler (Bio-Rad, Munich, Germany). For detection of gene–fusions, RNA was reverse transcribed using the SuperScript VILO cDNA Synthesis Kit according to the manufacturer’s instructions (Thermo Fisher Scientific). The amplicon libraries were prepared from 20 ng RNA, which were mixed with two primer pools and the AmpliSeq HiFi Master Mix before transferring them to a PCR cycler (Bio-Rad). Subsequently, DNA respectively RNA pools were combined and primer end sequences were partially digested using FuPa reagent, followed by the ligation of barcoded sequencing adapters using the Ion Xpress Barcode Adapters 1–48 Kit (Thermo Fisher Scientific). The purified libraries were quantified using the Ion Library TaqMan Quantitation Kit (Thermo Fisher Scientific).
All five participating institutions, apart from the organizing institution (institution A), were blinded to the
The samples used in the test were surgical tissues from solid cancer patients diagnosed from 2013–2020 without oncogenic driver alterations (
Reference standards for monitoring the analytical performance of the assay were incorporated in the analysis. The Seraseq FFPE
No fusion reads were observed in any of the nine
The
Unfortunately, two cases (BN04 and BN07) failed RNA quality control in the OCA v3 assay (institution C). The cutting order of these two samples was curl numbers 1 and 2. Curl numbers 1 and 2 are the uppermost sections of FFPE tissue.
The OCA v3 panel did not include the
The OncoPanel v.4.3 assay (institution D) was the only DNA-based sequencing panel included in this study.
The
Lastly, OncoPanel v.4.3 (institution D) could not be tested using the
This was a multi-centric comparative study for NGS-based detection of
Analytical sensitivity indicates how well a test can detect specific molecules, namely it can only evaluate technical performance, whereas clinical sensitivity is affected by pre-analytical factors other than technical performance. We analyzed separately clinical and analytical sensitivity, since this ring study have pre-analytical factors that may not occur when applied in under real diagnostics. RNA-based targeted NGS assays showed 100% analytical sensitivity and specificity, regardless of test panels and superior clinical sensitivity than DNA-based targeted NGS assays.
The majority of
Furthermore,
Sample types included both DNA and RNA; similar to previous reports, the highest detection rate was obtained with NGS using RNA [
All DNA- and RNA-based NGS assays used in this ring study are currently being used in routine diagnostics. Even if all tests were conducted according to their respective protocols, the detection rates might have varied depending on factors known to affect NGS assay results, such as sample preparation, processing method, and data analysis. NGS testing utilizing RNA, in particular, can be more complicated and demanding than other single-gene tests; therefore, they would require a careful procedure by the institution conducting the test.
The sensitivity of the targeted NGS assay can be limited by pre- and post-analytical factors. Pre-analytical tissue selection plays a key role in the success of NGS analysis. To ensure the selection of appropriate tissue, most institutions must select appropriate surgical tissue sections that best meet the requirements of the RNA-based NGS assay. It is necessary to optimize RNA inputs based on tumor cellularity or withhold poor quality samples from library preparation, because it is inevitable that FFPE samples would be used for RNA sequencing in a routine clinical setting. Thus NGS analysis of
Exposure of FFPE sections to light and air may negatively influence RNA quality. This is another concern that is different from that related to low tumor purity in the pre-analytic phase. However, storing entire FFPE blocks either in the open or protected from air and light will ensure that RNA quality is not affected as long as the uppermost section is discarded. Unfortunately, two cases (BN04 and BN07) failed RNA quality control in the OCA v3 assay (institution C). The cutting order of these two samples was curl numbers 1 and 2, which were the uppermost sections of FFPE tissue; therefore, the exposure of the FFPE tissue section to air, resulting in “oxidation,” has been assumed to be related to the failure [
The next issue is related to post-analytical factors such as sophisticated bioinformatics. In the OCA v3 assay used at institution F, several variants with non-targeted fusion and a low abundance of transcripts were absent in the initial output from the software Ion Reporter. Adjusting the analysis parameters and conducting a re-analysis resulted in the identification of the variants, indicating that these variants had been successfully sequenced but filtered out by the bioinformatics pipeline. In OCA v3, non-targeted fusions with either novel fusion partners or novel breakpoints were also reported by assessing the significance of supporting mapped sequencing read information [
Furthermore, at least 40 reads of a specific fusion were required—in the OCA v3 assay—to call the fusion a variant. Initially, several variants with fewer than 40 read counts were filtered out because they were not annotated because of the low number of transcript reads. The minimum number of fusion supporting reads for positive calling differed for each assay. Therefore, to detect rare but clinically significant RNA fusions, the count of unique supporting reads for the fusion–given the detection limit based on tumor purity–should be considered as making a positive fusion call via an adjustment of analysis parameters.
In conclusion, the RNA-based targeted NGS assay showed an overall high success rate of identification of
Supplementary materials are available at Cancer Research and Treatment website (
A total of six institutes participated in the ring trial and analyses were conducted on four different NGS panels. This retrospective cohort study was approved by the Institutional Review Board of each institution (institution A: 2020-05-095; institution B: H-2009-056-1155; Institution C: KC20SIDI0651; institution D: 2020-1386; institution E: 4-2020-0721; institution F: KUMC 2020-09-031) and performed in accordance with the principles of the Declaration of Helsinki. The requirement for informed consent was waived.
Conceived and designed the analysis: Lee SE, Lee MS, Choi YL.
Collected the data: Lee SE, Lee MS, Choi YL.
Contributed data or analysis tools: Lee SE, Lee MS, Jeon YK, Shim
HS, Kang J, Kim J, Choi YL.
Performed the analysis: Lee SE, Lee MS, Jeon YK, Shim HS, Kang J, Kim J, Choi YL.
Wrote the paper: Lee SE, Lee MS, Kim J, Choi YL.
This study was supported by Bayer Korea Ltd.
This study was supported by National Research Foundation of Korea (NRF) grants funded by the Korean government (Ministry of Science, ICT and Future Planning) (NRF-2016R1A5A2945889 and 2019R1A2B5B0269979).
Overview cutting of the tumor tissue slices with formalin-fixed paraffin-embedded (FFPE) blocks. Seventeen FFPE tissue samples were cut with a 5 μm curl for next-generation sequencing analysis and curls were numbered according to the order of cut followed by placed in a microcentrifuge tube. Two vials were assigned to each institution. IHC, immunohistochemistry; TRK, tropomyosin receptor kinase.
Tumor histology and pan–tyrosine receptor kinase (TRK) staining of ring trial samples. Resected formalin-fixed paraffin-embedded (FFPE) samples were stained with VENTANA pan-TRK (EPR17341) assay kits; cases determined to be positive for staining were further validated for neurotrophic receptor tyrosine kinase (
Tissue cuts assigned at each institution
Curl No. | Hospital | |||||
---|---|---|---|---|---|---|
A | B | C | D | E | F | |
BN01 | 1, 2 | 3, 4 | 7, 8 | 5, 6 | 9, 10 | 11, 12 |
BN02 | 1, 2 | 5, 6 | 9, 10 | 7, 8 | 11, 12 | 3, 4 |
BN03 | 1, 2 | 3, 4 | 7, 8 | 5, 6 | 9, 10 | 11, 12 |
BN04 | 9, 10 | 3, 4 | 1, 2 | 11, 12 | 5, 6 | 7, 8 |
BN05 | 1, 2 | 3, 4 | 7, 8 | 5, 6 | 9, 10 | 11, 12 |
BN06 | 1, 2 | 11, 12 | 5, 6 | 3, 4 | 7, 8 | 9, 10 |
BN07 | 9, 10 | 11, 12 | 1, 2 | 3, 4 | 5, 6 | 7, 8 |
BN08 | 1, 2 | 11, 12 | 3, 4 | 9, 10 | 5, 6 | 7, 8 |
BN09 | 1, 2 | 9, 10 | 7, 8 | 11, 12 | 3, 4 | 5, 6 |
BN10 | 5, 6 | 7, 8 | 11, 12 | 9, 10 | 3, 4 | 1, 2 |
BN11 | 3, 4 | 5, 6 | 7, 8 | 1, 2 | 9, 10 | 11, 12 |
BN12 | 1, 2 | 7, 8 | 11, 12 | 9, 10 | 5, 6 | 3, 4 |
BN13 | 11, 12 | 3, 4 | 7, 8 | 5, 6 | 1, 2 | 9, 10 |
BN14 | 1, 2 | 5, 6 | 9, 10 | 7, 8 | 11, 12 | 3, 4 |
BN15 | 1, 2 | 3, 4 | 7, 8 | 5, 6 | 9, 10 | 11, 12 |
BN17 | 1, 2 | 11, 12 | 9, 10 | 7, 8 | 3, 4 | 5, 6 |
BN18 | 1, 2 | 11, 12 | 5, 6 | 3, 4 | 7, 8 | 9, 10 |
The numbers represent the cut order from top (1) to bottom (12).
Clinicopathological characteristics in 17 clinical samples
ID | Age (yr) | Sex | Collected date | Specimen type | Tumor site | Final histologic diagnosis | Tumor cells (%) | Pan-TRK IHC | Confirmed NGS panel | NGS |
Fusion type | Fusion supporting reads count |
---|---|---|---|---|---|---|---|---|---|---|---|---|
BN01 | 51 | F | 2020-03-23 | Resection | Lung | Inflammatory myofibroblastic tumor | 20 | Positive | Lung Cancer Panel v3.0 | Positive | 12 | |
BN02 | 33 | M | 2016-07-25 | Resection | Brain | Glioblastoma, |
60 | Positive | TruSight Oncology 500 | Negative | ||
BN03 | 69 | F | 2020-06-25 | Resection | Peritoneum | Mucinous adenocarcinoma of uncertain origin | 60 | Positive | TruSight Oncology 500 | Positive | 128 | |
BN04 | 23 | M | 2017-05-22 | Resection | Salivary gland | Mammary analogue secretory carcinoma | 70 | Positive | Lung Cancer Panel v3.0 | Positive | 8 | |
BN05 | 50 | F | 2020-08-18 | Resection | Lung | Metastatic leiomyosarcomas | 90 | Negative | TruSight Oncology 500 | Negative | ||
BN06 | 39 | M | 2018-12-27 | Resection | Brain | Glioblastoma, |
30 | Positive | TumorSCAN level II | Negative | ||
BN07 | 63 | M | 2013-11-04 | Resection | Colon | Adenocarcinoma | 20 | Positive | OncoPanel v.4.3 | Positive | 13 | |
BN08 | 57 | M | 2020-07-17 | Resection | Small intestine | Dedifferentiated liposarcoma | 30 | Negative | TruSight Oncology 500 | Negative | ||
BN09 | 56 | M | 2020-07-15 | Resection | Ureter | Infiltrating urothelial carcinoma | 40 | Negative | TruSight Oncology 500 | Negative | ||
BN10 | 72 | M | 2019-06-18 | Resection | Brain | Glioblastoma, |
90 | Positive | TruSight Tumor 170 | Positive | 795 | |
BN11 | 58 | F | 2020-04-07 | Resection | Colon | Adenocarcinoma | 30 | Positive | OncoPanel v.4.3 | Positive | 16 | |
BN12 | 71 | M | 2020-07-07 | Resection | Ureter | Infiltrating urothelial carcinoma | 90 | Negative | TruSight Oncology 500 | Negative | ||
BN13 | 35 | M | 2018-01-11 | Resection | Prostate | Prostate stromal sarcoma | 80 | Positive | TruSight Tumor 170 | Positive | 1,390 | |
BN14 | 44 | M | 2020-07-15 | Resection | Lung | Mucinous adenocarcinoma | 40 | Negative | TruSight Oncology 500 | Negative | ||
BN15 | 29 | M | 2017-07-06 | Resection | Brain | Glioblastoma, |
90 | Positive | TruSight Oncology 500 | Negative | ||
BN17 | 67 | M | 2020-06-24 | Resection | Colon | Adenocarcinoma | 80 | Positive | TruSight Oncology 500 | Positive | 1,269 | |
BN18 | 53 | M | 2020-01-30 | Resection | Abdominal wall | Leiomyosarcoma | 60 | Negative | TruSight Oncology 500 | Negative |
IHC, immunohistochemistry; NGS, next-generation sequencing; NTRK, neurotrophic receptor tyrosine kinase; TRK, tyrosine receptor kinase.
Sample ID | Fusion type | Exon No. of BP | Supporting read count | Inframe/Out-of-frame |
---|---|---|---|---|
BN01 | E(5)N(15) | 9 | Inframe | |
| ||||
BN03 |
T(4)N(10) | 128 | Inframe | |
| ||||
BN04 | E(5)N(15) | 24 | Inframe | |
E(5)N(14) | 15 | Intronic fusion | ||
E(6)N(14) | 17 | Inframe | ||
| ||||
BN07 | T(7)N(10) | 80 | Inframe | |
| ||||
BN10 |
H(13)N(14) | 290 | Inframe | |
H(2)N(14) | 81 | Out-of-frame | ||
H(6)N(14) | 6 | Inframe | ||
H(2)N(14) | 71 | Inframe | ||
| ||||
BN11 | T(int7)N(8) | 29 | Intronic fusion | |
T(7)N(10) | 524 | Inframe | ||
| ||||
BN13 | R(5)N(14) | 939 | Inframe | |
| ||||
BN17 | T(7)N(10) | 1,269 | Inframe | |
T(8)N(10) | 346 | Out-of-frame |
Exon numbers correspond to NM_002529.4 for
Genes not in OCA v3.
Analytical sensitivity of NGS assays for
Sample ID | Fusion type | Fusion supporting reads counts (curl number of sample) | |||||
---|---|---|---|---|---|---|---|
A (TSO 500) | B (Lung Cancer Panel v3.0) | C (OCA v3) | D (OncoPanel v.4.3) | E (TSO 500) | F (OCA v3) | ||
BN01 | Not detected (1, 2) | 12 (3, 4) | 2,082 (7, 8) | Not detected (5, 6) | 7 (9, 10) | Not detected (11, 12) | |
BN02 | - | - | - | - | - | - | - |
BN03 | 128 (1, 2) | 406 (3, 4) | 36,852 (7, 8) | 37 (5, 6) | 172 (9, 10) | 94,492 (11, 12) | |
BN04 | 24 (9, 10) | 8 (3, 4) | QC fail (1, 2) | Not detected (11, 12) | 122 (5, 6) | 218,059 (11, 12) | |
BN05 | - | - | - | - | - | - | - |
BN06 | - | - | - | - | - | - | - |
BN07 | 80 (9, 10) | 54 (11, 12) | QC fail (1, 2) | 13 (3, 4) | 321 (5, 6) | 447,970 (7, 8) | |
BN08 | - | - | - | - | - | - | - |
BN09 | - | - | - | - | - | - | - |
BN10 | 290 (5, 6) | 220 (7, 8) | Not covered (11, 12) | Not detected (9, 10) | 795 (3, 4) | Not covered (1, 2) | |
BN11 | 524 (3, 4) | 929 (5, 6) | 612,303 (7, 8) | 16 (1, 2) | 1,309 (9, 10) | 302,550 (11, 12) | |
BN12 | - | - | - | - | - | - | - |
BN13 | ? (9, 10) | 155 (3, 4) | 2,569,146 (7, 8) | Not detected (5, 6) | 1,390 (1, 2) | 530,524 (9, 10) | |
BN14 | - | - | - | - | - | - | - |
BN15 | - | - | - | - | - | - | - |
BN17 | 1,269 (1, 2) | 251 (11, 12) | 744,925 (11, 12) | 110 (7, 8) | 2,843 (3, 4) | 21,468 (5, 6) | |
BN18 | - | - | - | - | - | - | - |
NGS, next-generation sequencing; NTRK, neurotrophic receptor tyrosine kinase; OCA, Oncomine Comprehensive Assay; TSO, TruSight Oncology.
Detected but filtered out because of non-targeted fusion.
Fusion Supporting Reads counts | ||||||
---|---|---|---|---|---|---|
A (TSO 500) | B (Lung Cancer Panel v3.0) | C (OCA v3) | D (OncoPanel v.4.3) | E (TSO 500) | F (OCA v3) | |
234 | 44 | 39,211 | NA | 278 | 21,468 | |
324 | 29 | - | NA | 426 | - | |
188 | 35 | 112 | NA | 341 | - | |
184 | 14 | 46,023 | NA | 174 | 114,933 | |
266 | 31 | 7,200 | NA | 418 | 33 | |
133 | 53 | 17,371 | NA | 205 | 16 | |
191 | 5 | 38,997 | NA | 229 | 1,611,524 | |
163 | - | 5,791 | NA | 172 | 28 | |
168 | - | 16,377 | NA | 199 | 743,802 | |
152 | 32 | NC | NA | 320 | NC | |
147 | 70 | 28,094 | NA | 268 | 489 | |
97 | 68 | 16,823 | NA | 224 | 1,955 | |
153 | 18 | 1,024 | NA | 194 | 3 | |
192 | 16 | 2,391 | NA | 212 | 4 | |
100 | 58 | 40,086 | NA | 167 | 29,051 |
Detected but filtered out because < 40 reads.