Factors Impacting Clinically Relevant RNA Fusion Assays Using Next-Generation Sequencing

Retrospective review of the pathology database from January 1 through April 30, 2018, was performed to identify consecutive cases evaluated by a targeted RNA-based NGS assay in the Clinical Laboratory Improvement Amendments–certified molecular diagnostics laboratory at our institution. Our study was approved by the institutional review board. The specimens included in-house surgical and cytopathology samples as well as outside consultation/referral specimens from other institutions. All specimens were evaluated by board-certified pathologists for cellularity and tumor fraction as a prerequisite to assess suitability for NGS testing. Formalin-fixed, paraffin-embedded tissue sections (hematoxylin-eosin stained) and/or cytology smears (Diff-Quik and Papanicolaou stained) were reviewed and the tumor area was circled by the pathologist. Specimens with at least 20% tumor cellularity and 300 cells or more in the circled area of slides were considered adequate for testing. Tissue was extracted by scraping of circled regions of tumor on the matching unstained slides of the cell block sections or tissue sections or direct smears by macrodissection or microdissection using light microscopy.

RNA extraction was performed by means of AllPrep kit (catalog number 80204) on a QIAcube liquid handling platform (Qiagen, Germantown, Maryland). Following extraction, the RNA was quantified by Qubit fluorometer (Thermo Fisher, Waltham, Massachusetts) and diluted to 5 ng/μL to prepare complementary DNA (cDNA) from reverse transcription of messenger RNA. For cases where the RNA concentration was 5 ng/μL or lower, the elute was used as is without any dilution. Qualitative assessment of RNA was not performed on these samples. For library preparation, 10 μL of cDNA was treated with FuPa Reagent (Thermo Fisher) to partially digest the primers and phosphorylate the amplicons. The amplicons were then ligated to bar code adapters, using a separate bar code for each sample in the cDNA library. The cDNA libraries were prepared using Ion AmpliSeq Library kit plus polymerase chain reaction–based target amplification, templated using Ion Sphere Particles through emulsion polymerase chain reaction, loaded using Ion 540 Chef Kit on an Ion 540 chip (Thermo Fisher), and sequenced via a commercially available targeted amplicon-based NGS on the Ion S5XL platform, to amplify a set of expected control RNA sequences as well as targeted fusion sequences analogous to the clinically relevant known intragenic and intergenic fusions in 51 genes (Oncomine Comprehensive Assay v3M, Thermo Fisher). The genes included AXL, AR, ALK, AKT2, BRCA1, BRCA2, BRAF, CDKN2A, ERB84, ERBB2, EGFR, ETV1, ETV4, ETV5, ESR1, ERG, FGR, FGFR1, FGFR2, FGFR3, FLT3, JAK2, KRAS, MET, MYB, MDM4, MYBL1, NOTCH1, NOTCH4, NF1, NRG1, NTRK1, NTRK2, NTRK3, NUTM1, PIK3CA, PPARG, PRKACA, PRKACB, PTEN, PDGFRA, PDGFRB, RELA, RET, ROS1, RSPO2, RSPO3, RAD51B, RAF1, RB1, and TERT. Seraseq FFPE Tumor Fusion RNA reference material (Sera Care, Milford, Massachusetts) containing 16 clinically significant RNA fusions was used as a positive control and included with every run to confirm that the run could be analyzed for clinical reporting. The Seraseq fusion control library and the samples tested were required to be at least 50 pM for the sample to be included for template preparation and sequencing.

Sequences were analyzed using Torrent Suite and Ion Reporter (Thermo Fisher) servers using an in-house bioinformatics data pipeline (OncoSeek, MD Anderson Cancer Center). Parameters used to consider a successful sequencing included 1 000 000 or more overall reads, 500 000 or more total mapped fusion panel reads, mean RNA read length 50 or more, an AQ20 read depth of 2 million reads, and more than 10 000 fusion reads considered reproducible and reportable without any need for confirmation. Quality control steps (as defined above) were implemented at RNA extraction, library preparation, and sequencing steps to identify samples that failed.

In the event of a failure of a sample due to inadequate amplification or poor sequencing (low read length or coverage), a second attempt was made from repeat extraction of the RNA or repeat cDNA conversion. A sample was considered a failure when both attempts failed to pass quality control and provide adequate sequencing.

The pathology database was used to collect the information for the analyzed samples, including tumor type, specimen type, tumor percentage, paraffin block age, RNA yield, and RNA fusion results. Next-generation sequencing quality parameters and other clinical data were acquired and the RNA fusion testing results were correlated with these factors. Logistic regression model was used to identify the potential relationships among testing outcomes and all the clinical variables. Type II ANOVA tests were also applied to examine the statistical significance of each variable. Stepwise model selection was performed to achieve the final model. Contingency table and Fisher exact test were used to examine the associations between categorical variables. Test results were considered to be statistically significant at a P value of <.05.

A total of 767 consecutive cases that underwent RNA-based NGS fusion testing were evaluated. The median patient age was 65 years (range, 7–92 years), with a male to female ratio of 0.98:1. The specimens included 493 in-house cases and 274 outside consultations/referral cases. Of these, 422 of 767 (55%) were core needle biopsy (CNB) samples, 268 of 767 (35%) were resections, and 77 of 767 (10%) were cytology specimens. The clinicopathologic characteristics of the cases evaluated are summarized in Table 1.

The total success rate of RNA fusion testing was 90.9% (697 of 767) with a failure rate of 9.1% (n = 70). Of the 697 cases that were successful, 50 (7.2%) had clinically relevant fusions and 647 (92.8%) did not have fusions (Supplemental Table; see the supplemental digital content at https://meridian.allenpress.com/aplm in the November 2021 table of contents). The various factors that we identified as impacting RNA fusion testing are discussed further.

The success of NGS-based RNA fusion testing showed significant positive correlation with RNA yield (P < .001) (Table 2). The mean RNA yields for test success and failure groups were 0.10 and 0.05 μg/μL, respectively. The median RNA yields in the 2 groups, success versus failure, were 0.07 μg/μL (range, 0.002–0.53 μg/μL), and 0.02 μg/μL (range, 0.002–0.39 μg/μL), respectively. The median RNA yields of CNB and cytology samples were similar, each 0.04 μg/μL (ranges, 0.002–0.5 μg/μL and 0.002–0.4 μg/μL, respectively), whereas the median RNA yield of resection samples was 0.15 μg/μL (range, 0.003–0.5 μg/μL) (Figure 1).

RNA fusion testing success did not correlate with the size of the lesion sampled (P = .26) (Table 2). Although the number of slides used for RNA extraction varied based on the total cellularity of an individual case, testing success did not correlate with the number of slides used for RNA extraction (P = .49) (Table 2). The median estimated tumor percentage in the RNA fusion testing success was 50% (range, 20%–100%) versus 50% in the failed group (range, 20%–98%, respectively). The success of RNA fusion testing did not show a significant association with the estimated tumor percentage (P = .97), indicating that RNA fusion testing success/failure was independent of tumor percentage in the sample, provided the minimum threshold of 20% tumor was met (Table 2).

Core needle biopsy specimens were the largest group (n = 422) of cases tested using the RNA-based NGS fusion assay, and 31 of these cases (7.2%) failed testing. Of the 391 cases that were successfully tested, 367 (93.8%) did not have any detectable fusions, whereas a fusion was present in 24 cases (6.1%) (Figure 2). In contrast, resection specimens (n = 268) that were tested showed a failure rate of 12.7% (n = 34) and a fusion detection rate of 9.4% (n = 22). Of the 77 cytology samples that were tested, 5 cases (6.4%) failed testing. Of the remaining 72 cases that were successfully tested, 68 (94.4%) did not have any detectable fusions, whereas 4 (5.5%) had a fusion detected. The testing success rates and fusion detection rates were very similar between CNB and cytology samples (Figure 2, A and B). However, RNA fusion testing success negatively correlated with resection specimens when compared with CNB and cytology specimens (P = .007). Of the 77 cytology cases analyzed by NGS-based RNA fusion, 35 cases used cell block sections, 17 cases used direct smears, and 25 cases used both cell blocks and direct smears for tissue extraction. A total of 33 of 35 cell block cases (94%) were sequenced successfully, whereas 2 of 35 (6%) failed. Of direct smears, 11 of 17 cases (65%) were sequenced successfully, whereas 6 of 17 (35%) failed. All 25 cases in which combined cell block and smears were used for testing were successfully sequenced.

Of the 70 cases that failed NGS testing, 23 specimens (32.9%) were submitted from an outside institution. These included 10 CNBs and 13 resections; however, there was no statistically significant correlation between in-house versus outside specimens with respect to fusion testing success (P = .48). Other factors for test failure that were identified include necrosis (10% of failed cases) and decalcification (15.7% of failed cases). The success rate in necrotic specimens (15 of 22; 68%) was significantly lower than in nonnecrotic specimens (682 of 745; 91.5%) (P = .002; Table 2). The fusion success rate in decalcified specimens (13 of 24; 54%) was significantly lower than in specimens that did not undergo decalcification (684 of 743; 92%) (P < .001; Table 2).

The age of the paraffin block used for RNA-based fusion testing also impacted NGS success rates. RNA fusion success rate was significantly lower in the samples with paraffin block age more than 2 years (66 of 82; 80.5%) than in those samples with paraffin block age less than 2 years (631 of 685; 92.1%) (P = .001; Table 2). Most of the older cases (>2 years) that failed testing were resection specimens (14 of 16; 87.5%), and the rest were CNB specimens (2 of 16; 12.5%).

Overall, 249 of 697 samples (35.7%) that were successfully sequenced had concurrent FISH and/or IHC results for comparison, typically performed on the same tissue block. In 241 of 249 cases (96.8%), the results were concordant between the RNA NGS fusion assay and FISH or IHC assays. Eight cases had discordant results that are summarized in Table 3. All cases with discordant results between NGS and FISH were performed on the same tissue block. In 3 cases, the NGS assay was positive for a gene fusion, but FISH was negative. In 5 cases, the NGS assay was negative for a gene fusion, but FISH was positive; in 3 of these cases the FISH result was considered borderline/focal. There were 2 additional cases, 1 with an SS18 gene rearrangement and 1 with a 12p abnormality, both detected by FISH, but these abnormalities were not covered by the NGS fusion panel. There were 7 cases wherein a fusion was identified, but the corresponding FISH did not test for the specific target (Table 3). There were no discordant results between the NGS assay and IHC.

In recent years, there has been an increasing focus on the utility of gene fusions for guiding therapy in patients with solid organ malignancies. Drugs that target fusion genes (eg, ROS1, RET, ALK, NTRK, FGFR1, FGFR2, FGFR3) have gained importance and have generated great interest because of promising therapeutic results using these agents.^17–21  The capability of NGS-based methods to detect multiple fusion genes in a single high-throughput multiplexed analysis has expanded the need for testing for gene fusions in the routine clinical setting.

Recognizing preanalytic factors that potentially impact RNA-based NGS fusion testing may help in optimizing specimen processing and the selection of appropriate specimens to improve testing success rates. To our knowledge, this is the first study evaluating preanalytical factors affecting RNA-based NGS fusion testing success performed in clinical specimens. We evaluated several parameters, including size and site of lesion, tumor fraction, number of slides used for extraction, age of paraffin block, RNA yield, specimen type (resection versus biopsy versus cytology), type of case (in-house versus outside consultation/referral specimen), and the presence of necrosis (>50% of specimen) and decalcification to identify preanalytic factors that impact testing success rates.

Our results show that test success correlates positively with RNA yield (P < .001), indicating the overall cellularity of the specimen tested plays a role in testing success. However, it is independent of other factors including tumor site and tumor size, tumor fraction (given that minimum 20% tumor fraction threshold is met), and the number of slides used for RNA extraction.

Fusion testing success rates and fusion detection rates were similar between our CNB and cytology samples with no statistically significant differences. Interestingly, despite a higher RNA yield in resection specimens than in CNB and cytology samples, testing success rates were significantly lower (P = .007). In addition, specimens with paraffin block age of more than 2 years had significantly higher fusion failure rate (P = .001) compared with those with paraffin block age of less than 2 years. Interestingly, most of the cases that failed testing in the older blocks were resection specimens (14 of 16; 87.5%). These results are in keeping with the published literature.^22,23  One possible cause for the higher failure rates seen in resection specimens may be related to the poor RNA quality due to suboptimal fixation of larger surgical specimens. Unlike CNB specimens, which are put into fixative (eg, 10% formalin) immediately or soon after collection, resection specimens tend to remain unfixed for longer periods of time, and may be subject to underfixation due to thicker sections prepared during grossing, or to overfixation, as they are left longer in fixatives, leading to RNA degradation. Studies that have evaluated preanalytical causes that impact nucleic acid quality and proteins in tissue specimens have reported the importance of cold ischemic time, fixative volume, and duration of fixation as critical parameters.²⁴  The current guidelines for clinical samples that are suitable for predictive biomarker testing recommend a cold ischemic time of less than 30 minutes, a fixative volume of 10:1 using 10% neutral buffered formalin, and a fixation time of 6 to 48 hours for CNB specimens and 24 to 48 hours for resection specimens.^24,25  Further, degradation of RNA may also be attributable to tissue preanalytics related to long-standing storage in paraffin.^26,27  In this study we were unable to evaluate RNA quality for individual samples, as preanalytical quality measures such as RNA integrity number or DV200 index were not available. Our results, however, suggest that preanalytic factors related to RNA quality (eg, time to fixation, time in fixative, type of collection media/preservative solution/fixative) that were not assessed in this study likely contribute to testing success. Additional studies in a more controlled setting, such as a validation study where known positives from different specimen types can be tested, are required to address this issue.

Although some of these parameters can be reasonably optimized for specimens that we process in house, there is little or no control over preanalytical variables for specimens that are processed at an outside institution. These factors are somewhat reflected in the higher number of cases that failed testing in the specimens that were submitted from an outside referral. Other factors that are known to affect nucleic acid quality in tissue specimens include decalcification using harsh acids²⁴  and the presence of necrosis.²⁸  Although most of our in-house specimens are decalcified using EDTA, which is more compatible with molecular testing, the decalcification reagent used on specimens submitted from an outside institution is largely unknown in our cohort of cases. In our study, testing failed more often in specimens with extensive necrosis (>50% of specimens) and those that were decalcified (P = .002 and P < .001, respectively).

At our institution, all requests for molecular analysis are usually referred to histologic specimens; however, cytology material, including cell blocks and direct smears, is used if histologic specimens are unavailable or inadequate for testing.²⁹  In the 4-month period of this study, 10% of the cases used for RNA-based NGS fusion testing cases were cytology specimens. Of these 77 cytology cases, 62 (80.5%) had no available concurrent histologic specimen, and the remaining 15 (19.5%) had concurrent histologic material that was deemed inadequate for testing. The cases without available histologic material comprised predominantly endobronchial ultrasound-guided FNAs of lymph nodes and lungs, with a smaller subset of effusion fluids and computed tomography–guided FNAs of small deep lesions. Hence, the ability to use cytologic specimens for RNA-based NGS fusion testing increases the number of specimens that can be evaluated for a clinical RNA-based NGS assay. In this small subset of cytology cases, cell block preparations were used most often. Although our data demonstrate that testing cell blocks have a higher success rate than testing direct smears (94% versus 65%), it is conceivable that cases with adequate cell blocks that were selected for testing also had adequate smears, but the cell block was selected because it was easier to process for molecular testing (selection bias). The subset of cases where smears were submitted for fusion testing was likely of lower cellularity with an inadequate cell block, possibly predisposing these cases to a higher rate of failure. This conclusion is also highlighted by a 100% testing success rate in those cases where a combination of cell block and direct smears was used, likely because of borderline cellularity in both substrates. Further studies with a larger number of cytology cases will be needed to confirm our hypothesis. The addition of non–formalin-fixed cytologic specimens like direct smears can provide high-quality RNA and may improve the adequacy rates of specimens that can be successfully tested. However, additional validation for these substrates is required prior to clinical molecular testing that may not be readily available in most commercial settings or reference molecular laboratories, thus precluding their use in most clinical settings.

Of the cases with concurrent FISH/IHC results available for comparison with the RNA-based NGS fusion assay, 97% showed concordant results. Eight cases were discordant between NGS fusion analysis and FISH analysis. In 5 cases, the NGS assay was negative for a gene fusion, but FISH was positive; in 3 of these cases the FISH result was considered borderline/focal (Table 3). This highlights the need for using an alternate orthogonal platform in cases with borderline results to confirm the suspected genomic alteration and exclude potential false-positive/false-negative results.³⁰  The fourth case had a lower tumor fraction (estimated 20%) and showed a low-frequency KRAS mutation (<5%), indicating that the reported negative fusion assay was likely a false-negative result. This highlights the importance of ensuring a minimum threshold for tumor fraction in specimens submitted for RNA fusion analysis to avoid potential false-negative results. The last case with a positive FISH result that did not detect fusion by the NGS assay had an estimated tumor fraction of 50% and the RNA sequencing metrics appeared to be satisfactory. In 3 cases, the NGS assay was positive for a gene fusion, but FISH was negative. In 2 of these cases, FISH results were false-negative, secondary to the detected fusion signals below the reporting threshold (test sensitivity issue). The discordance between FISH and NGS fusion assay results could be attributable to several factors, including technical issues causing false-positive/false-negative results, complex rearrangements, microscopic insertions involving a particular portion of a gene inserted into another gene, and rearrangement with a variant fusion partner or known partner with variant breakpoints.^31–33  Other factors could include different analytical sensitivity and specificity of each method, the quality and preparation of the tested specimens, and misinterpretation of results.³⁴ 

The outcomes of this study provide some insight into improving the RNA-based NGS fusion testing success of clinical specimens. There are certain limitations of this study, which include its retrospective nature and variations in processing and handling of the specimens and downstream analytics that vary across institutions. Because RNA quality measurements were not available as part of the preanalytic quality metrics, suboptimal RNA quality may have contributed to some of the assay failures seen in this study. Further, in a subset of cases where the RNA yield was below the recommended threshold (<5 ng/μL), the RNA input for cDNA preparation may have been suboptimal and contributed to assay failure. Further studies in a more controlled setting with additional preanalytic quality metrics such as ischemic time, tissue fixation, and specimen processing that likely impact RNA quality will be required to address these issues. In addition, the AmpliSeq-based sequencing approach used in this study is unable to provide for a good sequencing quality control metric reflecting library complexity. Alternative approaches using unique molecular indexes that can provide a measure of library complexity (diversity of read coverage) are currently being evaluated for future studies.

In conclusion, the success of RNA-based NGS fusion testing depends on several factors. Therefore, identification of preanalytical factors that affect RNA yield and quality may provide better understanding of specimen adequacy, guide the selection of samples for RNA-based NGS testing, and improve the overall rate of testing success.