The EML4-ALK fusion gene, formed by chromosomal rearrangement of the echinoderm microtubule-associated protein 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene in echinoderms, is an oncogene found in non-small cell lung cancer (NSCLC). The discovery of the EML4-ALK fusion gene provides a new target for the treatment of NSCLC1. These fusion genes are constituted by the inversion of two genes located on the broken arm of chromosome 2, which is a continuously active structural kinase with highly stimulating activity and potent carcinogenic activity in vivo and in vitro, and this activity can be effectively blocked by small molecular TKIs targeting ALK2,3. Various types of fusions have been reported to date, all of which are mainly based on the splicing of the exon of dissimilar lengths after the breakage of the EML4 gene, and the fusion between exon 20 of the ALK gene which is relatively conservative in the insertion position. The most common fusion types are EML4 exon 13 fusion to ALK exon 20 (variant 1, V1) and EML4 exon 6 fusion to ALK exon 20 (variant 3, V3), accounting for about 62% of the total types4.

EML4-ALK can be detected by a variety of techniques such as fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), or reverse transcription polymerase chain reaction (RT-PCR) and next generation sequencing (NGS)5,6,7. These methods vary in sensitivity, specificity, and applicability for different types of samples6. According to Molecular Testing Guidelines, both FISH and IHC can be used to detect ALK rearrangements, and most diagnostic laboratories use FISH with ALK-lysis probes as the “gold standard”, which has the advantages of low cost and relative ease of operation compared with IHC6,8. However, their judgment signals can be subtle, difficult to identify, and highly subjective in diagnosis, limiting accurate quantification. For this reason, two to three experienced and skilled experts are required to rate the test9,10. The advantage of NGS lies in its ability to effectively detect both known and unknown types of fusion genes, addressing the limitations of conventional detection methods, which may miss detections or fail to identify fusion partner genes. However, it also presents drawbacks, including high costs, complex data analysis, and significant technical barriers11,12. Of the four methods routinely used to detect ALK gene rearrangements, FISH and ICH are effective diagnostic techniques for preliminary assessment of ALK rearrangement status. The sensitivity of RT-PCR and NGS assay is comparable, while RT-PCR can be used to identify known fusion variants of ALK gene rearrangements with high sensitivity but relies on external standard to quantify13,14. Recently, quantification of EML4-ALK gene rearrangements by digital PCR (dPCR) was evaluated to harmonize results between laboratory and clinical assays14,15,16. Wang et al.14 used dPCR to detect EML4-ALK gene rearrangement in clinical lung adenocarcinoma FFPE samples with high sensitivity and specificity. They further discussed the potential benefits of crizotinib in patients with low ALK rearrangements at copy number. Damm-Welk et al.15 compared minimal residual disease (MRD) measurement of NPM1-ALK fusion gene transcripts using the RT-dPCR and RT-qPCR, and the RT-dPCR showed more precise detection.

To ensure the detection results from different methods, platforms and laboratories are comparable and consistent, it is necessary to establish the metrological traceability for gene fusion measurement and this is also required by ISO1751117. However, the lack of system international (SI) traceable reference materials (RMs) or primary calibrators hinders the establishment of metrological traceability. Currently, no RMs for EML4-ALK fusion measurement are available due to a lack of reference measurement procedure (RMP) which can be used to assign the value of higher order calibrators. Digital PCR (dPCR), as a counting-based method, can provide absolute quantification results with no calibration needed, which makes the measurement result traceable to unite one, symbol “1” within the SI system, agreed by Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM) Nucleic Acid Analysis Working Group (NAWG) in our previous international comparison of CCQM P15418,19. Thus, it has the potential to be a RMP to assign the value of RMs. Previous studies have demonstrated that dPCR is a SI traceable RMP for assignment of copy number concentration and fractional abundance values of KRAS G12D, BRAF V600E DNA and EGFR reference materials20,21,22. All the above established RMPs are fit for the purpose of DNA measurement, an RMP is urgently needed for RNA measurement.

The aims of this study are (1) to establish and validate a potential RMP for EML4-ALK fusion measurement at RNA level based on reverse transcription dPCR (RT-dPCR), (2) to develop RMs for EML4-ALK fusion variant measurement and (3) to evaluate the interlaboratory performance of dPCR as a potential RMP for quantification of EML4-ALK.

Materials and methods

Study design

A study route to develop EML4-ALK RMs was designed, as shown in Fig. 1. The study route consists of the following steps: (1) establishing and validating the RT-dPCR by in vitro transcribed RNA; (2) preparing the genomic RNA (gRNA) candidate RMs from two cell lines; (3) assessing the homogeneity and stability of the gRNA RMs by the established RT-dPCR; (4) evaluating the interlaboratory reproducibility by using the gRNA RMs.

Figure 1
figure 1

Study route of development of EML4-ALK reference materials. (A) Validation of the one-step reverse transcription dPCR (RT-dPCR) including identification and quantification of EML4-ALK in two cell lines genomic RNA (gRNA), the dynamic range, and false positive rate, quantitative consistency among different digital PCR platforms, and reverse transcription efficiency assessment by isotope dilution mass spectrometry (IDMS). (B) Preparation of gRNA RMs including cell culture, genome extraction and purification, sequence confirmation and bottling. (C) Characterization of gRNA RMs consisting assessment of homogeneity and stability and determining the reference value. (D) Evaluation of interlaboratory repeatability and reproducibility by using the RMs.

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Cell line, plasmid and in vitro transcribed RNA

Cell lines H3122 (containing EML4-ALK V1) and H2228 (containing EML4-ALK V3) were purchased from Meisen Chinese Tissue Culture Collections (Meisen CTCC). The cell line of 293T is stored at the National Institution of Metrology (NIM). The RPMI 1640 medium with 10% Fetal bovine serum (FBS, Invitrogen, #10099–141 C) and 1% Penicillin–Streptomycin Solution (PS, Invitrogen, #15140-122) for each cell line was used according to the information provided from Meisen CTCC and cultured by NIM. Cell lines were cultured in a humidified atmosphere of 5% CO2 at 37℃. Subcultures were performed at a ratio of 1:2 or 1:3 when the cell density reached 80% and 90% every 3 or 4 days.

A pBluescrip II SK (+) plasmid containing the EML4-ALK (containing V1 and V3) and partial ALK (NM_004304.5, domain at the junction of exons 20 and 21) was synthesized from BGI (Beijing, China) to generate in vitro transcribed RNA molecules. The plasmid was linearized with 15 U/µL Xba I (Takara, Code No. 1093 S), 10×M buffer (Takara, 1093 S) and ddH2O in a final reaction volume of 200 µL for 1.5 h at 37 °C, for 65 °C inactivated at 30 min. The corresponding band was purified using a commercial DNA Clean-up Kit (CW2301M, CWBIO, Jiangsu, China) with 50 µL of elution buffer. The linearized plasmid was checked using 2% agar gel electrophoresis (Supplemental Fig. 1a) and NanoDrop 2000 (Thermo Fisher Science, USA). The MEGAscript™T7 transcription kit (Thermo Fisher Science, USA) was used for the in vitro transcription reaction, and 1 µL of Turbo DNase was added after transcription to remove the template DNA according to the manufacturer’s instructions. The transcribed RNA was then purified with the MEGAclear™ kit (Thermo Fisher Science, USA). The quantity and quality of in vitro transcribed RNA were assessed using the Nanodrop 2000 (Thermo Fisher Science, USA) and Bioanalyzer (Agilent 2100, USA) with RNA 6000 Nano Reagents Part I kit (Agilent, USA) (Supplemental Fig. 1b), respectively. And then in vitro transcribed RNA was analyzed to check the nucleotide impurity by mass spectrometry and the result showed that no nucleotide impurity existed (Supplemental Fig. 1c).

Preparation of genomic RNA reference material

The two-cell line total RNA was extracted by using an RNeasy® Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. The quantity and quality of total RNA were assessed using Nanodrop 2000 and Bioanalyzer (Agilent 2100 with an RNA 6000 Nano Reagents Part I kit), respectively. High purity and integrity of total RNA were confirmed by A260/280 (1.8-2.0) and A260/230 (> 2.0) and RNA integrity number (RIN = 10.0). Then the two RNA stock solutions were prepared in RNA storage solution (AM7001, Thermo Fisher Science, USA). To prepare the candidate RNA RMs, the two stocks of EML4-ALK total RNA were diluted to a concentration of 50 ng/µL by the RNA storage solution with 5 ng/µL of yeast total RNA (sigma, Code No.63231630, USA). The candidate RNA RMs (labeled as RM1 and RM2, respectively) were packaged in 30 µL per vial in 0.5 mL micro tubes and stored at -80 °C until needed.

Sanger sequencing

cDNA of the two candidate RMs and the in vitro transcribed RNA were transcribed by using a commercial kit (Takara, Code No.6215 A) and were sequenced by Sanger sequencing (ABI 3730XL, USA) to confirm the presence of EML4-ALK fusion variant by Sangon Biotech Co., Ltd (Shanghai, China) and to verify the bases sequence of in vitro transcribed RNA by BGI Tech Solutions (Beijing Liu He) Co., Limited. The Sequence data was analyzed by SeqMan v12.2 software (DNASTAR). No mismatch was observed in the in vitro transcribed RNA sequencing results (Supplementary Fig. 2).

Reverse transcription digital PCR (RT-dPCR)

The sequence of the optimized TaqMan probe PCR assays23 targeting the fusion variant (V1 and V3) and ALK reference gene (ALK-ref) were shown in Supplemental Table 1. The RT-dPCR reactions were optimized and performed on a QX200 (Bio-Rad) (Supplemental Fig. 3a-3c). The reaction mixture was in a volume of 20 µL and comprised 5 µL of supermix (Bio-Rad, USA), 2 µL of reverse transcriptase, 1 µL of 300 mM DTT, 1 µL of mixture of primers and probe, 9 µL of RNase-free water and 2 µL of template. The final concentrations of primers and probes were 400 nM and 200 nM for V1, 500 nM and 400 nM for V3 and ALK-ref, respectively.

The optimized PCR thermal profile was conducted on a conventional PCR machine (VeritiPro, Applied Biosystems) (Supplemental Fig. 3d-3f). Thermal cycling consisted of a 60 min reverse transcription at 45℃ and 10 min activation period at 95 ℃ followed by 40 cycles of a two-step thermal profile of 30 s at 95 ℃ denaturation and 30 s at 54℃/60 ℃ for combined annealing-extension and 1 cycle of 98 ℃ for 10 min.

All samples were analyzed in three replicates. Results were analyzed with the QuantaSoft v.1.7.4.0917 software (Bio-Rad). The workflow and data analysis were described in our previous report24.

Limit of blank (LoB), detection (LoD) and quantification (LoQ)

To determine the dynamic range of the dPCR assays for the two fusion variants of EML4-ALK and ALK-ref target, serial dilutions of the above in vitro transcribed RNA were prepared gravimetrically. Each dilution was measured in three to six replicates with the three singleplex dPCR assays. The LoD is determined to be the lowest concentration and the detected number of positive was greater than 95% in 20 tests17. The coefficient of variation (CV) of each concentration was analyzed to determine the LoQ according to CV < 25%. 60 measurements were performed on blank samples to determine the LoB according to EP17-A25.

Isotope dilution mass spectrometry (IDMS) analysis

In vitro transcribed RNA was quantified using isotope dilution mass spectrometry (IDMS) to assess the reverse transcription efficiency (RTE) of our established RT-dPCR method, which was published in our previous report26. Five concentration standard solutions were prepared, consisting of certified reference materials (CRMs) of pure adenosine 5’-monophosphate (AMP, GBW(E)100,154), guanosine 5’-monophosphate disodium salt (GMP, GBW(E)100,068), cytidine 5’-monophosphate (CMP, GBW(E)100,067), and uridine 5’-monophosphate disodium salt (UMP, GBW(E)100,069) (NIM, China) mixtures and isotope–labeled (13C, 15N) nucleotide monophosphates (LNMPs) (Silantes, Germany) mixtures with a mass ratio of NMPs to LNMP of 0.4, 0.8, 1.2, 1.6, and 2.0. The concentrations of the four NMPs and LNMPs in the mixture were estimated from the native NMPs of in vitro transcribed RNA sample, and the molar ratio of NMPs to LNMPs in the sample was guaranteed to be 1. The digestive reaction mixture was in a volume of 56 µL and comprised 5 µL of LNMPs mix solution, 1 µL of SVP (0.00023U/µL) and 50 µL of RNA sample (approximately 1 mg/g). The digestion mixture was incubated at 37 °C for 15 min, inactivated at 80 °C for 10 min, and centrifuged at 13,000 rpm for 2 min. The detail optimization information of IDMS was described in our previous report26. Two tubes of samples were taken for analysis at a time, each tube was digested twice, and each digestion reaction was analyzed with three replicates. The average of NMPs measured in four digestion reactions was taken as a measure of in vitro transcribed RNA.

Interlaboratory assessment

Two RMs were analyzed in eight different laboratories according to ISO 5725 − 127 to evaluate the repeatability and reproducibility of RT-dPCR. Six labs were measured on the QX200 (Bio-Rad, CA, USA) platform, one lab on the DQ24 (Sniper, Suzhou, China), and one lab on the TD-1 ddPCR (TargetingOne Corporation, Beijing, China) platform. All specific details on laboratory performance are listed in Supplemental Table 2. Dixon’s and Grubbs’ tests were used to test for the presence of any outliers. Mandel’s h and k statistics were used to assess the consistency of between and within laboratories, according to ISO 5725 − 228.

Result

Validation of RT-dPCR

Three singleplex dPCR assays (EML4-ALK V1, EML4-ALK V3 and ALK-ref) were optimized and established. The two-dimensional scatter plots are shown in Supplemental Fig. 4. Good separation of positive and negative partition was obtained for all three targets.

Cross reaction between V1, V3 and ALK-ref were assessed by a combination of each variant targeted assay with WT RNA template extracted from 293T, candidate RMs and in vitro transcribed RNAs (Fig. 2). No positive droplet clusters appeared for V1 assay when amplifying WT and RM2 (containing V3 variant) template (Fig. 2a). Similarly, no positive droplet clusters occurred for the V3 variant assay when amplifying WT and RM1 (containing V1 variant) template (Fig. 2b). Additionally, no cross-reaction was observed for the ALK-ref assay when amplifying in vitro transcribed RNAs of only mutation (MU, containing V1 and V3 variant) and RMs. This indicates that there was no cross-reaction between the two variants and the reference gene (Fig. 2c).

Figure 2
figure 2

Evaluation of cross reaction of three assays including EML4-ALK V1, V3 and ALK-ref. (A) amplification of V1 assay with sample of RM1 (A05, B05), RM2 (A02, B02), WT RNA (C02, D02); (B) amplification of V3 assay with sample of RM2 (C06, D06), RM1 (C01, D01), WT RNA (E01, F01); (C) amplification of ALK-ref assay with sample of RM1 (D01, E01), RM2 (D02, E02), in vitro transcribed V1 + V3 (D03, E03).

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The false-positive rate (FPR) of EML4-ALK fusion mutations measured on different dPCR platforms using the candidate EML4-ALK measurement method was investigated to identify possible biases affecting the authenticity of the measurement results. Five commercial dPCR platforms were selected to detect the non-target-template for comparison, which was divided into two categories: water-in-oil droplet dPCR platform (QX200, Microdrop-100, DQ24) and microfluidic chip dPCR platform (QIAcuity One, BioDigital Yan) (Fig. 3). FPR of V1, V3 and WT on QX200 was 0%, less than 0.02% on Microdrop-100 and DQ24, indicating no positive bias for quantification on QX200, and insignificant positive bias on Microdrop-100 and DQ24. It is notable that a high FPR of 0.02%, 0.06% and 0.03% for V1, V3 and WT on the QIAcuity One platform was observed. It is speculated that contamination occurred during the automatic operation of this micro-fluidic chip dPCR platform.

Figure 3
figure 3

Comparison of false-positive rate using a non-targeted template for different digital PCR platforms.

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Dynamic range of the potential RMP

The precision of dPCR relies on the absolute measurement of the number of partitions referred to as lambda (λ)29. Thus, the dynamic range of potential RMP was investigated by a serial dilution shown in Fig. 4. The upper limit of the dynamic range was 1.2 × 105 copies/partition tests by the V1, V3 and ALK-ref assays, corresponding to λ of 7.69, 8.49 and 7.62, respectively (Fig. 4a). As a general feature of the three assays, the measured RNA concentrations matched the expected ones within a range. An excellent linearity (both R2 ≧ 0.99) between the measured value and the prepared value in the tested interval was observed for all three assays over the range from approximately 104 to 101 copies/reactions (Fig. 4b). The consistency between three RT-dPCR assays and gravimetric method was close to 1 (0.92 < slope < 1.06), indicating that RT-dPCR is highly accurate in detecting EML4-ALK fusion gene at a concentration of higher than 10 copies/reaction. Reactions containing a mean of 37 V1, 49 V3 or 13 ALK-ref copies fulfilled the criterion for an LOQ with a CV lower than 25%. LoD of EML4-ALK V1, V3 and ALK-ref were determined to be 2 copies/reaction, 2 copies/reaction and 1 copy/reaction in which more than 19 of the 20 measurement results were positive (Supplemental Tables 3–5). Since the distribution of the 60 blank measurements was non-normally distributed, the LoB was estimated by the non-parametric as the 95th percentile of the measurements, which corresponds to 57.5 ordinal observations (= 60*(95/60 + 0.5). Due to the 60 blank measurements being mostly 0, only the 15 highest blank values of the LoB are displayed in Supplemental Table 6. The LoB of EML4-ALK V1, V3 and ALK-ref were determined to be 0.84 copy/reaction, 0.90 copy/reaction and 0.20 copy/reaction.

Figure 4
figure 4

Validated the range of the candidate’s primary reference measurement procedure. Evaluation of the linearity of RNA samples containing EML4-ALK V1 and V3 variant over the (A) extended λ range (0.02 < λ < 8.5) and (B) low λ range (λ < 1.3) in a background of 105 WT molecules per reaction.

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Quantification of in vitro-transcribed RNA by IDMS

Given that the accuracy of our analytical methods relies on calibrators, prepared using gravimetric methods and quantified with reference materials traceable to NIMC. The concentration of native NMP digested from the in-vitro transcribed RNA including EML4-ALK (containing V1 and V3) and partial ALK (NM_004304.5, domain at the junction of exons 20 and 21) was calculated by establishing standard curves. The four standard curves of AMP/LAMP, CMP/LCMP, UMP/LUMP and GMP/LGMP showed a good linear relationship with R2, which were 0.9989, 0.9989, 0.9989 and 0.9991, respectively (Supplemental Fig. 5). Then, NMPs digested from in-vitro transcribed RNA samples were detected and analyzed by standard curves. Based on RNA samples with known sequences could be used to calculate the concentration of intact RNA based on the mass concentration of each NMP detected by IDMS (Table 1). The calculated concentrations of the in-vitro transcribed RNA samples from the measured concentrations of AMP, UMP, CMP, and GMP were 0.88 µg/g, 1.00 µg/g, 0.91 µg/g, and 0.95 µg/g, respectively.

Table 1 Quantitative results for the in-vitro transcribed RNA mass concentration calculated from four NMPs measurements by IDMS (mg/g).
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According to the Avogadro constant and the molecular weight of RNA, the in vitro transcribed RNA mass fraction obtained by IDMS was converted into copy number concentration, which was used as the absolute copy number concentration of RNA molecule (denoted as \(\:{C}_{RNA}\)). The copy number concentration of the cDNA molecule was reversed as determined by the established RT-dPCR (named \(\:{C}_{cDNA}\)). From this, the efficiency of reverse transcription of the RNA template into cDNA was determined and expressed as the ratio of the RNA copy number concentration of RT-dPCR and IDMS, expressed by the formula RTE=\(\:{C}_{RNA}/{C}_{cDNA}\). The RTE of V1, V3 and ALK genes was 1.07, 0.949 and 0.995 (Supplemental Table 7). These results showed that the RNA concentrations measured by the two methods were the same.

Confirmation of EML4-ALK fusion variant in the candidate RMs

The fusion variant of EML4-ALK in the candidate RMs was confirmed by Sanger sequencing and shown in Supplemental Fig. 6. The result demonstrated that the candidate RMs prepared from H3122 and H2228 cell lines genomic RNA carries the fusion mutation of EML4-ALK V1 and EML4-ALK V3, which are EML4 exon 13 fusion to ALK exon 20 and EML4 exon 6 fusion to ALK exon 20.

Homogeneity and stability of the candidate RMs

The homogeneity and stability of EML4-ALK RMs were assessed according to ISO 17,034 and ISO Guide 3530,31 and provided in the supplemental material (Supplemental Tables 8–11, Supplemental Figs. 7–8). Eleven vials were randomly selected from the batch, and each was tested in triplicate by the established RT-dPCR method. The results of the homogeneity test of the RMs and their corresponding CVs are shown in Supplemental Tables 8 and 9. The result showed that the F values of the copy number of EML4-ALK each fusion variant, ALK-ref and the ratio of EML4-ALK/ALK were less than the critical value, satisfying the F test32. The stability test results indicated that the RMs showed no significant changes in the copy number of EML4-ALK in each fusion variant, ALK-ref and the ratio of EML4-ALK/ALK at -20 ℃ and 4 ℃ for 2 weeks and − 80 ℃ for at least 12 months (P > 0.05, Supplemental Figs. 7 and 8, Supplemental Tables 10 and 11). However, we have other similar whole transcriptome RNA reference material that can be stable for 2 years under − 80 ℃ according to our previous data33. Thus, we speculate the total RNA RM for EML4-ALK developed in this study can be stable for at least 2 years under − 80 ℃. The detail of measurement uncertainty of the reference material value was provided in the supplemental information and the combined uncertainty value was shown in Supplemental Tables 12 and 13.

Interlaboratory of repeatability and reproducibility

The interlaboratory repeatability and reproducibility of the potential RMP were evaluated by using the two candidate RMs. Each EML4-ALK fusion variant RM was analyzed in eight different laboratories. All the data generated by the laboratory was submitted to NIM. Wells with RT-dPCR assay results in less than 10,000 acceptable droplets were excluded from the assessment.

The interlaboratory results of the eight laboratories agreed well with the property values within the expanded uncertainty range (Supplemental Fig. 9). Six participating laboratories analyzed the RM with the QX200, one with the DQ24 chip digital PCR platform and one with the TD-1 platform. This allows more digital PCR-based platforms to further evaluate the performance criteria of candidate RMP20,21. Thus, overall repeatability was calculated with an interlaboratory CV of less than 7.3% for the copy number of EML4-ALK each fusion variant, ALK-ref and the ratio of EML4-ALK/ALK, indicating good repeatability of the candidate reference measurement procedure (Supplemental Tables 14 and 15).

The Mandel’s h and k statistics are plotted to check for consistency between and within laboratories (Fig. 5). All testing laboratories agreed well in the copy number concentration of V1 and ALK-ref as well as the mutant abundance shown by the Mandel’s h plot at 5% significance level (Fig. 5a), which indicates that EML4-ALK V1 RM has good repeatability. Mandel’s k plot of EML4-ALK V1 RM showed that the V1 copy number of Lab 3 has poor repeatability at a significant level of 1%, followed by the copy number of the V1 in Lab 6 and the ALK-ref in Lab 2 at a significant level of 5% (Fig. 5b). For EML4-ALK V3 RM, Mandel’s h plot showed that 8 laboratories achieved agreement at 1% significance level across all tests, however, Lab 4 had poor repeatability in the ALK-ref copy number concentration measurement at a significance level of 5% (Fig. 5c). Reproducibility for each laboratory represented by the Mandel’s k plot was satisfactory at a significant level of 1%, except for a significant variation in the V3 copy number of Lab 7 (Fig. 5d). The V3 copy number of laboratory 4 and 5 and the ALK-ref copy number of laboratory 6 had poor repeatability at a significant level of 5%. Overall, 7 participating laboratories had good repeatability and consistency for both V1 and V3 measurements.

Figure 5
figure 5

Consistency assessment by Mandel’s between laboratory statistic, h, (a and c) and within laboratory statistic, k, (b and d), for the average copy number concentration of each EML4-ALK fusion variant, ALK-ref and the ratio of EML4-ALK/ALK of EML4-ALK reference material. The solid and dash lines are the 1% and 5% significance levels, respectively.

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Discussion

In clinical studies, procedures vary widely from sample collection to selection of an internal control and to reporting of results, has prohibited the comparability of test results in interlaboratories. Therefore, there is a need for a standardized procedure to ensure consistent and comparable test results to assist clinicians in making appropriate decisions about disease diagnosis and treatment. NGS and RT-PCR are currently the most clinically recognized methods for the detection of fusion genes. RNA-based NGS technology has been recommended by clinical practice guidelines and expert consensus as one of method for detecting fusion genes in NSCLC, but there is still a lack of norms and standards regarding the timing, application scenarios, and quality control of RNA based NGS for detecting fusion genes in China34. A series of experimental studies by Malapelle et al.35,36,37 have developed and validated a reference standard for gene fusion-positive artificial cytological reference slides based on NGS assays (Oncomine Focus Assay (Thermo Fisher Scientific)), as well as multi-gene mutation quantitative cytology molecular reference slides with varying mutation frequencies. They analyzed the results of different detection methods, platforms, and validation parameters, thereby promoting enhanced standardization of molecular cytopathology procedures. However, Goto K et al.38 demonstrated a direct comparison between the Amoy PCR-9 gene product and Oncomine™ Comprehensive Assay (OCA, 161 genes NGS-panel, Thermo Fisher) v3 in the detection of variants in NSCLC driver genes. Due to Oncomine™ also detects fusions at the RNA level, the study is highly comparable. The results showed that in terms of fusion gene detection ability, the Amoy PCR-9 gene products showed better detection ability. There are two reasons, one is that RT-PCR is better than NGS for the detection of short fragments and end fusions; Second, RT-PCR has a higher detection sensitivity, which is a lower minimum detection limit. Although RT-PCR is comparable in sensitivity to NGS based on RNA-based detection of fusion genes, compared with NGS, RT-PCR has significant advantages in detection success rate, detection time and technology popularity38. As a third-generation PCR technology, digital PCR can achieve absolute quantification of endpoint measurement without standard curves, providing guidance for further genetic analysis and molecular diagnosis. In this study, we successfully established and validated a one-step reverse transcription digital PCR (RT-dPCR) method that combines reverse transcription and digital PCR for measuring potential RMPs related to fusion gene at the RNA level.

With the discovery of an increasing number of rare fusion subtypes of ALK genes and the elucidation of the mechanisms behind acquired resistance to ALK inhibitors, there is a growing demand for ALK gene testing in clinical practice. It is clinically significant to select accurate, rapid, and appropriate ALK detection methods based on patient needs and to screen the target population suitable for ALK inhibitors. In non-small cell lung cancer patients diagnosed with EML4-ALK fusion gene positivity, ALK targeted therapy is preferred rather than TKI. Therefore, detecting the EML4-ALK fusion gene is crucial for the personalized treatment of tumors. In this study, a potential RMP based on RT-dPCR quantitative detection of EML4-ALK V1 and V3 fusion variants, along with a reference gene (ALK -ref) in NSCLC was established. We also examined specificity and analytical sensitivity. The results indicate that potential RMPs exhibit good specificity and high sensitivity, capable of detecting rare targets with low copy numbers (2 copies/reaction). Additionally, two homogenous and stable EML4-ALK fusion gene reference materials have been developed, serving as valuable resources for verifying various methods and reagents for detecting EML4-ALK, which is of great significance in improving the comparability and accuracy.

Nonetheless, this study also has its shortcomings. When using established RT-dPCR for RNA quantification, cDNA must first be obtained through reverse transcription (RT) before PCR amplification can be performed. However, previous researches have shown that such reverse transcription efficiency (RTE) varies significantly depending on the RT reagents and reaction conditions24,39. Consequently, we propose a correction of the RTE of in vitro transcribed RNA based on isotope-dilution mass spectrometry (IDMS)26. This method has no specificity for RNA sequences and is independent of the RNA reverse transcription process, which to some extent solves the problem of large deviations in RNA amplification quantification between different laboratories and methods. However, further research is necessary to ascertain whether the RTE from short transcripts to mRNA in the genome is consistent. In addition, the RT-dPCR based RMP established in this study has not been validated on clinical samples such as tissue biopsy. Given that the reference measurement procedures (RMPs) in ISO 15,19340 play a crucial role in science, technology, and daily services, they are primarily used for the following purposes: first, to quantify higher-order reference materials and to verify the authenticity of calibration or routine measurement procedures. Second, to evaluate and compare the performance characteristics of the measurement system as both a reference method and a daily method. Especially for medical laboratory measurements, the measurement results reported to doctors and patients have sufficient comparability, reproducibility, and accuracy, which is crucial for patient care and health screening. The purpose of this research is to first establish RMP to achieve the determination of RMs, and then to study its applicability to clinical samples, both of which require further improvement in future research.