This retrospective study suggested that the sensitivity and specificity of NGS could make it an alternative to PCR-based MSI testing, the approved companion diagnostic for detecting MSI-H in Japan. To the best of our knowledge, this is the first study to evaluate the real-world concordance and discordance between PCR-based testing kit and NGS using clinical data in Japan. Four patients included herein had been identified as MSI-H by PCR-based testing or NGS. Among them, two patients were initially determined as MSS by PCR-based testing and subsequently re-evaluated to be MSI-H by NGS. IHC staining confirmed the deficiency in all MMRs among all four patients. Notably, false negatives in PCR-based MSI testing were suspected in such cases. Generally, reports have shown that low tumor burden or increase in tumor DNA degradation over time observed in the samples could be attributed to false-negative results. Moreover, several studies have reported that Lynch syndrome with germline mutations in MSH6 or PMS2 might not necessarily show MSI-H (26, 27). However, the current study used the same samples during PCR-based testing and NGS assay, with all four patients showing MSI-H in NGS, unlike PCR-based testing.
Our study highlights the accuracy of PCR-based MSI testing as well as the possible usefulness of the NGS assay in clinical practice. Vanderwalde et al. assessed the concordance between PCR-based testing and NGS in 2,189 matched cases of 26 cancer types, including 23 cases with MSI-H. In MSI-H detection, NGS had a sensitivity of 95.8% (95% CI, 92.24–98.08), specificity of 99.4% (95% CI, 98.94–99.69), positive predictive value of 94.5% (95% CI, 90.62–97.14), and negative predictive value of 99.2% (95% CI, 98.75–99.57) in comparison with PCR-based testing (19). Moreover, Middha et al. validated NGS against PCR-based testing and MMR immunohistochemistry for 138 colorectal cancers (CRCs), including 24 MSI-H CRCs, and 40 uterine endometrioid cancers (UECs), including 15 MSI-H UECs, and showed a concordance of 99.4% (20). Although these studies have reported concordance between MSI status assessed by NGS and PCR-based testing with a larger number of patients in a laboratory setting, our present study demonstrated the clinical utility of NGS to analyze MSI status using real-world data in Japan where PCR-based MSI testing is the only approved companion diagnostic for the use of immune checkpoint blockade as of now. Further, our study was performed in a situation close to real clinical practice where tissue samples are not abundant or inadequate for MSI testing analysis compared with previous reports in which tissue samples were obtained from another study.
NGS can investigate a large variety of gene alterations at one time. Generally, PCR-based MSI testing requires tumor specimens with FFPE block or 5–10 pieces of undyed 5-µm pathological specimens, which is equivalent to specimens required for NGS assay to perform genomic testing. The number of tumor samples had been either limited, particularly in patients with pancreatic carcinoma, prostate cancer, or cancer of unknown primary whose tumor burden is generally low or tumor is difficult to access, such as those obtained through biopsy. Hence, we often experienced problems related to the inability to order new genomic testing kits given the lack of available specimens. However, rebiopsy might be difficult for patients with poor conditions or without superficial metastasis. To address such problems, using NGS initially might be an ideal alternative.
One strength of the NGS assay is its ability to simultaneously detect gene alterations in MLH1, MSH2, MSH6, and PMS2.(22, 24) Furthermore, certain types of NGS can also analyze germline mutations associated with Lynch syndrome by assessing matched normal–tumor pairs (28). Although the current NGS assay could not analyze loss of function due to DNA methylation, such an approach could be realized in the near future (29).
By counting thousands of fractional mutations, NGS was able to evaluate TMB. TMB is known to be one of the novel predictive biomarkers for the efficacy of immunotherapy (30, 31). Despite the current lack of approval for the use of pembrolizumab among these populations in Japan, the FDA has approved pembrolizumab for patients with unresectable or metastatic TMB-H (≥ 10 mutations/megabase solid tumors by F1CDx in 2020. Although the cutoff of TMB-H still remains controversial, clinicians would greatly benefit from knowing TMB values when considering immunotherapy.
Conversely, NGS does have some problems that need to be considered. First, NGS costs approximately 560,000 yen (5,300 US dollars), whereas PCR-based MSI testing costs 20,000 yen (190 US dollars) under the health insurance system in Japan, placing considerable economic burden on the national insurance system. Second, the average turnaround time (TAT) for NGS is approximately 4–5 weeks following sample receipt in the laboratory, which might affect treatment decision-making. Furthermore, an expert panel discussion is needed to confirm whether gene alterations are meaningful after the results have returned.
The utility of NGS, including the identification of MSI-H/dMMR status, could supersede that of PCR-based MSI testing provided that cost reductions and shortening of TAT are addressed. Furthermore, the ability of NGS to be performed earlier and at a more adequate timing has made it an indispensable diagnostic strategy. Moreover, forthcoming advancement would allow MSI-H/dMMR to be evaluated using cell-free DNA from a patient’s blood sample. Accordingly, FoundationOne Liquid CDx (Foundation Medicine, Cambridge, MA) and Guardant360 CDx (Guardant Health, Redwood City, CA), which are types of novel platforms for liquid biopsy, can detect MSI status and have received approval from the FDA in 2020 (32). Though liquid biopsy, MSI/MMR status can be determined more quickly and effortlessly, allowing us to not only administer immunotherapy for patients with MSI-H/dMMR tumors, but also recruit patients with certain gene alterations into clinical trials (33). We believe that NGS should be performed diligently in patients suspected to be MSI-H/dMMR after considering their clinical course, past medical history, and family history, regardless of whether they have been determined as MSS by PCR-based testing.
Some limitations of the current study inherent to its retrospective nature are worth noting. First, this was a single-center study; the sample size was limited with potential bias in patient selection. However, we believe that determining the concordance between NGS and PCR-based MSI testing using data from clinical practice is crucial. Second, we did not perform IHC staining in all cases determined as MSS by PCR-based testing, potentially underestimating the number of patients with dMMR. However, the percentage of MSI-H/dMMR cases in our study was 10%, comparable to those reported in previous reports. Further, the facilities where NGS can be performed are limited, and the conditions for insurance coverage are strictly limited in Japan. Patients with good performance status who have completed the standard treatment or who are with rare cancers for which a standard treatment has not been yet established are candidates for NGS. Thus, patients who were included in this study might have been biased, particularly with respect to tumor types.
In conclusion, the NGS assay has comparable utility to PCR-based MSI testing for evaluating MSI/MMR status. Thus, NGS can be expected to become an important alternative method for detecting MSI-H/dMMR in the near future after reducing costs, shortening TAT, and improving accessibility at appropriate testing periods.