The past decade has seen major changes in the treatment paradigm of non-small cell lung cancer (NSCLC) with the development of tyrosine kinase inhibitors (TKIs) targeting driver mutations. The technological advancement and access to molecular genomic studies have propelled uptake of rapidly expanding molecular classification and made therapeutic intervention available for patients in the clinic.

Despite a high response rate with first-generation EGFR TKIs, the majority of patients progress within 12 months, with the most common acquired resistance mutation found in p.Thr790Met point mutation (T790M) in the EGFR gene.^{1, 2} Osimertinib, a third-generation EGFR-TKI inhibiting both EGFR-TKI-sensitizing and EGFR T790M resistance mutations, showed superior efficacy over chemotherapy in patients with secondary T790M mutation following progression with a first-generation TKI.³ This has prompted the clinical need to repeat biopsy in those patients.

However, the need for repeat biopsies to identify resistance mechanisms is often constrained by limited tumor access or intra-patient and intra-tumor heterogeneity. Liquid biopsies such as analysis of plasma circulating tumor DNA (ctDNA) are minimally invasive and allow repeat sampling, which becomes particularly attractive when treatment decisions need to be based on these biopsy findings.

Multiple mutation testing platforms for ctDNA have been used, such as Cobas EGFR mutation kit, digital droplet PCR (ddPCR), and Next Generation Sequencing (NGS). Hence, studies to identify the optimal platform for ctDNA testing is vital for further progress. Plasma ctDNA from the AURA3 study was retrospectively analyzed for EGFR mutations to compare the sensitivity of the Cobas EGFR mutation Test v2 with ddPCR and NGS assays. The study reported the positive percent agreement (PPA) of plasma T790M was comparable between ddPCR (58%) and NGS (66%), demonstrating their higher sensitivity compared to cobas assays (51%).⁴ Patients with positive T790M mutation determined by plasma ctDNA benefited from Osimertinib in this study, with subsequent studies examining the different platforms of assessing plasma T790M ctDNA mutation status and the efficacy of Osimertinib.⁵

In this issue, Lee et al. ⁶ report the analytical performances of real-time PCR assays for plasma EGFR test using various platforms including Cobas EGFR mutation Test v2 (Roche), PANA Mutyper-R-EGFR kit (PANAGENE) and ddPCR in 75 NSCLC patients. An EGFR mutation was identified in 54 patients; 52 of 54 detected in tissue and 43 of 54 in plasma.

The analytical performances of the three commonly found EGFR mutations were assessed using matched tissues and plasma samples in 68 patients who underwent simultaneous tissue and plasma analysis. The sensitivity of the Cobas was statistically higher (P = 0.031) than that of the PANAMutyper. The sensitivity of specific mutations exon 19 deletion/L585R/T790M was 95.7%/87.5%/75.0% for Cobas, 69.6%/75%/62.5% for PANAMutyper and (not evaluated)/75%/75% for ddPCR. The analytical performance of ddPCR for exon 19 deletion was not evaluated as it has different coverage for exon 19 deletions compared to other techniques. Importantly, the specificity was 100% across all technology and specific EGFR mutations except for one suspected false-positive case of an L858R mutation using Cobas. Furthermore, the authors demonstrated a good correlation in VAF between real-time PCR and ddPCR (Pearson's r = 0.830).

From this report, it is clear that there is variability in sensitivity between different assays, but real-time PCRs offer broader coverage compared to ddPCR as in evaluating Exon 19 deletion. However, we would argue that single-gene testing such as studied in the current study is becoming less relevant, particularly given that detection of secondary T790M mutation is not necessary with Osimertinib moving to the first line setting with the report of the FLAURA study. ⁷

Therefore, what is the current role of plasma ctDNA detection in the clinical setting? In the diagnostic setting, patients who have limited biopsy samples to allow molecular characterization may benefit from examination of the ctDNA. However, a platform that can detect a broader range of potentially targetable mutations would be ideal, including EGFR, ALK (Anaplastic Lymphoma kinase), ROS1 (ROS proto-oncogene1), RET (rearranged during transfection), MET (Mesenchymal-epithelial transition), HER2 (human epidermal growth factor receptor 2), BRAF (B-raf proto-oncogene), KRAS (KRAS proto-oncogene), and NTRK (Neurotrophic tyrosine receptor kinase). Liquid biopsy can also detect possible mechanism of resistance following acquired resistance after first-line Osimertinib in patients who do not have an accessible biopsy site at the time of progression. These can include appearance of MET, HER2 and PI3Kinase. Due to the need for broader mutation testing for NSCLC, we are advancing towards NGS as the primary testing modality. It is worth noting that while NGS was not tested in the current study, the analysis from the AURA3 study showed NGS has broader coverage than other methodologies.

Other emerging use of liquid biopsy is to monitor treatment response and resistance, as residual ctDNA and dynamic changes of detectable ctDNA may be detected earlier than radiological disease progression.⁸ For instance, the detection of T790M mutation in plasma in patients on EGFR-TKI predates radiological progression by up to 344 days, although its impact on treatment decision is yet to be determined.⁹

The current NCCN (National Comprehensive Cancer Network) guideline recommends using a broad, panel-based approach like NGS and considering RNA-based NGS if any driver oncogenes are not identified, especially in nonsmokers, to improve detection of fusion events. It does not support the use of ctDNA in lieu of tissue diagnosis due to higher false-negative rates of up to 30%. However, NCCN guideline does support the use of ctDNA if the patient is not medically fit for an invasive procedure, in case of insufficient material following pathological confirmation on the tissue sample, or incomplete diagnostic biomarker assessment due to limited tissue availability.^{10, 11}

The 2021 IASLC (International Association for the Study of Lung Cancer) consensus statement also recognizes plasma ctDNA as a valid tool for genotyping of advanced NSCLC. Low multiplex-based approaches are considered appropriate only when plasma NGS is unavailable. IASLC consensus statement also regarded single-gene testing as incomplete if oncogene driver mutation is not detected and recommends serial testing for additional actionable biomarkers in line with other guidelines.¹²

However, the adoption of NGS in liquid biopsy has been relatively slow, partly due to the lack of standardization of methodologies and commercially available assays. Affordability of using NGS in liquid biopsy also remains a key issue in slow adoption, even in developed countries such as Singapore.¹³

Overall, we believe liquid biopsy has a potential role in lung cancer management. However, single-gene platform such as the ones reported in the current issue is unlikely to be helpful, given the broad range of mutations of clinical interest in lung cancer, both at the time of diagnosis and progression following targeted therapy. As such, when liquid biopsy is being considered in instances where there is inadequate tissue biopsy, NGS appears to be the most advantageous in this setting. The real-world uptake will be determined by access and high-quality evidence from methodological studies and clinical outcome data before it can evolve into a practice-changing modality for better patient care.

在过去十年中，随着针对驱动突变的酪氨酸激酶抑制剂 (TKI) 的发展，非小细胞肺癌 (NSCLC) 的治疗模式发生了重大变化。技术进步和分子基因组研究的普及推动了迅速扩展的分子分类的采用，并使临床患者可以进行治疗干预。

尽管第一代 EGFR TKI 的反应率很高，但大多数患者在 12 个月内进展，最常见的获得性耐药突变是 EGFR 基因中的 p.Thr790Met 点突变 (T790M)。^{1, 2} Osimertinib 是第三代 EGFR-TKI，可同时抑制 EGFR-TKI 致敏突变和 EGFR T790M 耐药突变，在第一代 TKI 进展后出现继发性 T790M 突变的患者中显示出优于化疗的疗效。³这促使临床需要对这些患者进行重复活检。

然而，重复活检以确定耐药机制的需要通常受到有限的肿瘤进入或患者内和肿瘤内异质性的限制。液体活检，如血浆循环肿瘤 DNA (ctDNA) 分析，是微创的并且允许重复取样，当治疗决策需要基于这些活检结果时，这变得特别有吸引力。

已使用多种 ctDNA 突变检测平台，例如 Cobas EGFR 突变试剂盒、数字微滴 PCR (ddPCR) 和下一代测序 (NGS)。因此，确定 ctDNA 测试最佳平台的研究对于进一步取得进展至关重要。对来自 AURA3 研究的血浆 ctDNA 进行了 EGFR 突变的回顾性分析，以比较 Cobas EGFR 突变测试 v2 与 ddPCR 和 NGS 检测的敏感性。该研究报告称，ddPCR (58%) 和 NGS (66%) 血浆 T790M 的阳性一致性百分比 (PPA) 相当，表明与 cobas 检测 (51%) 相比，它们具有更高的灵敏度。^4个通过血浆 ctDNA 确定的阳性 T790M 突变患者在本研究中受益于奥希替尼，随后的研究检查了评估血浆 T790M ctDNA 突变状态和奥希替尼疗效的不同平台。^5个

在这个问题上，Lee 等人。^{图 6}报告了在 75 名 NSCLC 患者中使用 Cobas EGFR 突变测试 v2（Roche）、PANA Mutyper-R-EGFR 试剂盒（PANAGENE）和 ddPCR 等各种平台对血浆 EGFR 检测进行实时 PCR 检测的分析性能。在 54 名患者中发现了 EGFR 突变；在组织中检测到 54 个中的 52 个，在血浆中检测到 54 个中的 43 个。

使用匹配的组织和血浆样本对 68 名同时接受组织和血浆分析的患者进行了三种常见 EGFR 突变的分析性能评估。Cobas 的敏感性在统计学上更高（P = 0.031) 比 PANAMutyper。Cobas 的特定突变外显子 19 缺失/L585R/T790M 的敏感性为 95.7%/87.5%/75.0%，PANAMutyper 为 69.6%/75%/62.5%，ddPCR 为（未评估）/75%/75%。ddPCR 对外显子 19 缺失的分析性能未进行评估，因为与其他技术相比，它对外显子 19 缺失的覆盖率不同。重要的是，除使用 Cobas 的 L858R 突变疑似假阳性病例外，所有技术和特定 EGFR 突变的特异性均为 100%。此外，作者证明了实时 PCR 和 ddPCR 之间 VAF 的良好相关性（Pearson's r  = 0.830）。

从这份报告中可以清楚地看出，不同检测之间的灵敏度存在差异，但在评估外显子 19 缺失时，与 ddPCR 相比，实时 PCR 提供了更广泛的覆盖范围。然而，我们认为当前研究中研究的单基因检测正变得越来越不相关，特别是考虑到随着 FLAURA 研究报告将奥希替尼转移到一线设置，检测继发性 T790M 突变是不必要的。⁷

因此，血浆 ctDNA 检测目前在临床环境中的作用是什么？在诊断环境中，活检样本有限以允许进行分子表征的患者可能会受益于 ctDNA 检查。然而，一个能够检测更广泛潜在靶向突变的平台将是理想的，包括 EGFR、ALK（间变性淋巴瘤激酶）、ROS1（ROS 原癌基因 1）、RET（转染期间重排）、MET（间充质-上皮细胞转变） 、HER2（人表皮生长因子受体 2）、BRAF（B-raf 原癌基因）、KRAS（KRAS 原癌基因）和 NTRK（神经营养性酪氨酸受体激酶）。液体活检还可以检测在进展时没有可及的活检部位的患者在一线奥希替尼后获得性耐药后可能的耐药机制。这些可能包括 MET、HER2 和 PI3 激酶的出现。由于需要对 NSCLC 进行更广泛的突变检测，我们正在推进将 NGS 作为主要检测方式。值得注意的是，虽然 NGS 未在当前研究中进行测试，但 AURA3 研究的分析表明，NGS 比其他方法具有更广泛的覆盖范围。

液体活检的其他新兴用途是监测治疗反应和耐药性，因为残留的 ctDNA 和可检测的 ctDNA 的动态变化可能比放射学疾病进展更早被检测到。⁸例如，在接受 EGFR-TKI 治疗的患者中检测到血浆中的 T790M 突变比放射学进展早了 344 天，尽管它对治疗决策的影响尚待确定。⁹

当前的 NCCN（美国国家综合癌症网络）指南建议使用广泛的、基于 panel 的方法，如 NGS，如果未识别出任何驱动致癌基因，尤其是在非吸烟者中，则考虑基于 RNA 的 NGS，以改进融合事件的检测。它不支持使用 ctDNA 代替组织诊断，因为假阴性率高达 30%。但是，如果患者在医学上不适合进行侵入性手术，组织样本经病理证实后材料不足，或由于组织可用性有限导致诊断生物标志物评估不完整，NCCN 指南确实支持使用 ctDNA。^{10, 11}

2021 IASLC（国际肺癌研究协会）共识声明也承认血浆 ctDNA 是晚期 NSCLC 基因分型的有效工具。只有当血浆 NGS 不可用时，基于低多重性的方法才被认为是合适的。IASLC 共识声明还认为，如果未检测到致癌基因驱动突变，则单基因检测是不完整的，并建议根据其他指南对其他可操作的生物标志物进行系列检测。¹²

然而，NGS 在液体活检中的采用相对缓慢，部分原因是缺乏标准化的方法和商业化检测。即使在新加坡等发达国家，液体活检中使用 NGS 的可负担性也仍然是缓慢采用的关键问题。¹³

总的来说，我们认为液体活检在肺癌治疗中具有潜在作用。然而，考虑到肺癌临床上感兴趣的突变范围广泛，无论是在诊断时还是在靶向治疗后的进展中，单基因平台（如本期报道的平台）不太可能有帮助。因此，当在组织活检不充分的情况下考虑液体活检时，NGS 似乎是这种情况下最有利的。现实世界的吸收将取决于方法学研究和临床结果数据的访问和高质量证据，然后才能演变成一种改变实践的模式，以更好地为患者提供护理。