Advances and challenges in molecular analysis among patients with synchronous multiple primary lung cancers

With advanced radiographic imaging and the utilization of lung cancer screening, more than 20% of patients undergoing lung cancer resection are found to have two or more synchronous multiple primary lung cancers (SMPLCs) (1,2). However, distinguishing between SMPLC and intrapulmonary metastasis (IPM) remains a challenging and increasingly important task given the growing prevalence of SMPLC in clinical practice. The primary diagnostic approach is the modified Martini and Melamed criteria [1975], which initially focused on major histology and lymphatic involvement and later expanded to encompass comprehensive histologic assessment (CHA), considering minor histologic subtype in adenocarcinomas and cytological or stromal features in squamous cell carcinomas (3). While CHA has improved the accuracy of distinguishing SMPLC from IPM, this method lacks standardization and varies between institutions and clinicians. Further, challenges emerge in patients with identical major and minor histologic subtypes, a common scenario where more than 80% of patients with SMPLC have adenocarcinomas. According to the 9^th edition of the International Association for Staging Lung Cancer (IASLC), such tumors are classified as IPM (4). However, contemporary survival data suggests that in the absence of nodal involvement or extranodal evidence of metastatic disease, these tumors behave more like SMPLC than metastatic disease (2).

Lately, precision oncology and genomic profiling have triggered a paradigm shift in the evaluation of clonal relationships. A common technique involves examination of driver mutations in key oncogenes, which are recognized as early clonal mutations through whole-exome sequencing (WES) of early-stage non-small cell lung cancers (5). The most common driver mutations include EGFR (17.1–53.7%), KRAS (6.1–41.2%), and BRAF (2.4–3.7%) (6,7). In theory, the presence of different or “discordant” driver mutations implies distinct clonal origins, or SMPLC, whereas identical or “concordant” driver gene mutations is suggestive of IPM. In a retrospective study, Chen et al. [2018] assessed driver gene mutations in 39 patients with multiple primary lung cancers, all exhibiting a different minor histologic subtype and meeting the criteria set forth by the modified Martini Melamed criteria (7). The authors found discordant driver gene mutations in 94.6% (35 of 37) of cases where at least one driver mutation was identified. Patients with discordant (n=35) driver mutations had a significantly lower rate of tumor recurrence compared to those with concordant (n=2) mutations (5.7% vs. 100%).

The favorable prognosis observed in patients with discordant driver mutations reinforces the concept that primary tumors arise from separate clonal origins. As a result, this approach improves staging accuracy in patients with multiple lung cancers compared to solely relying on histologic assessment, thus facilitating stage-based treatment strategies. In another study of 18 patients previously treated for multifocal non-small cell lung cancer, Zheng et al. [2020] utilized a 4-gene next-generation sequencing (NGS) panel to retrospectively evaluate the staging accuracy of these patients (6). Prior to molecular analysis, all but one patient was diagnosed as having IPM. However, following molecular analysis, 55.6% (10 of 18) patients were re-classified as SMPLC due to discordant driver mutations, of which 80% (8 of 10) of cases were downstaged from their initial histologic staging. Of the patients who were downstaged, 50.0% (4 of 8) underwent neoadjuvant chemotherapy unnecessarily.

While molecular analysis of driver genes can distinguish clonal differences in multifocal tumors of similar histology, relying solely upon driver genes has its own limitations. Genomic instability is a hallmark of cancer, which casts doubt on the reliability of discordant mutations (8). The discordance rate between primary and metastatic non-small cell lung cancer can reach up to 75% of cases, with rare trunk driver mutations (e.g., EGFR, KRAS, BRAF) and branching driver genes (e.g., PIK3CA and TP53) being most frequently affected (9). However, factors such as laboratory error, assay sensitivity, tumor cell contents, and tumor-associated inflammation can also influence discordance (8,9). NGS appears to have the highest sensitivity thus far, and the presence of discordant trunk driver mutations in specimens with adequate tumor cellularity can provide reassurance (9).

On the other hand, concordant driver mutations may not always entail IPM. Similar mutations often manifest in patients with comparable risk factors. For example, KRAS mutations are more prevalent among smokers and in Western countries, whereas EGFR mutations are more prevalent among non-smokers and in Asian countries (1,6,7). Similarly, concordant mutations within the same individual can arise due to “field cancerization”, in which lung epithelium exposed to similar mutagens can become cancerous. In the case of shared oncogene driver mutations, studies by Chang et al. [2019] and Yang et al. [2023] have used large-panel NGS to show that SMPLC can exhibit diverse genomic profiles characterized by distinct somatic (non-oncogene) mutations (10,11). In this context, the probability of independently developing the same somatic mutation independently is minimal, as they occur in less than 2% of adenocarcinomas (12). Therefore, in patients with shared driver mutations, where the likelihood of SMPLC is high, large-panel NGS or WES can be used to assess somatic mutations and distinguish SMPLC from IPM (10). To improve the efficiency of this approach, colleagues at the Mayo Clinic have also demonstrated the utility of large-panel NGS and mate-pair sequencing technology to map somatic gene junctions from commonly recurring chromosomal rearrangements; however, this technique is not accessible in the routine clinical setting (13). Notably, assessment of somatic mutations is not indicated in lesions sharing rare driver gene mutations, where mutations are highly suggestive of IPM.

When combined with CHA, the application of a large-panel NGS can accurately distinguish SMPLC from IPM, resulting in significant improvements in survival rates. In the aforementioned study of 32 patients by Yang et al. [2023], CHA alone identified 18 patients with SMPLC and 14 patients with IPM, with an overall survival rate of approximately 75% and 50% at 5-year, respectively (10). With the addition of a large-panel NGS, 18.8% (6 of 32) of cases, including two patients with IPM histologically and four patients with SMPLC histologically, were reclassified as SMPLC and IPM, respectively. Following molecular-based classification, the overall 5-year survival rates for patients with SMPLC improved to approximately 90%, whereas patients with IPM had a lower overall survival rate at approximately 35%. Notably, this study excludes mucinous invasive adenocarcinomas or lepidic predominant tumor pairs and has less than five patients at risk at 5-year. While the application of large-panel NGS holds promise as a valuable tool in enhancing the management and outcomes of patients with SMPLC, it is also imperative to acknowledge that large-panel NGS is costly, and for the time being, it must be used judiciously.

Perhaps the most significant drawback of molecular analysis, as well as CHA for that matter, is its reliance on tissue quantity, rendering it impractical for biopsy specimens. This limitation poses a considerable risk to patients who are incorrectly diagnosed with IPM, and subsequently receive palliative chemoradiation. In contrast, surgical resection remains the gold standard treatment for patients with SMPLC, although it can be limited by lung preservation. For those who are eligible for surgical resection, multi-staged sub-lobar resection is often preferred, whereas stereotactic ablative radiotherapy may be considered for patients who are deemed unsuitable for surgery (14,15). While in certain instances, biopsy specimens may offer insights regarding the potential relationship between tumor pairs; histological comparisons should be considered provisional until definitive resection is performed, particularly in patients who are eligible for surgical resection.

As we advance in the field, it is essential to integrate traditional and molecular-based methods, while also recognizing their respective limitations. One of the most promising advancements in this field is the development of liquid biopsies. Particularly, circulating tumor DNA in peripheral blood has gained popularity as a promising tool for identifying lung cancer, particularly among those with advanced disease (16). However, this approach is significantly limited in patients with early-stage lung cancer, and the initial randomized controlled trials (NCT02833467 and NCT04326751) involving patients with multiple primary lung cancers are still ongoing. The value of other types of liquid biopsies (e.g., circulating tumor cells) are currently under investigation and may serve as potential alternatives.

Even in patients who undergo resection, inconclusive results are common. One scenario arises in cases where no driver gene is identified, reported in approximately 26% to 32% of cases using small-panel (~4 genes) NGS (6,7). In such instances, medium (~50 genes) and large-panel (~400 genes) NGS can be reflexively ordered. Conversely, nearly 30% of patients undergoing small-panel NGS have a driver gene identified in one tumor but not in another (11). Provided adequate tumor tissue was obtained, these cases are suggestive of SMPLC. However, it may be reasonable to perform large-panel NGS in tumors exhibiting histologic features that increase the risk of IPM, such as invasive mucinous adenocarcinomas or those with micropapillary and solid architecture (1,17).

Another glaring question is whether molecular analysis should be performed in all patients undergoing lung cancer resection. For patients presenting with radiographic evidence of multifocal ground-glass opacities and/or histological evidence of in-situ disease, minimally invasive adenocarcinoma, or non-mucinous lepidic-predominant adenocarcinoma, one could argue that molecular analysis is unnecessary due to the high likelihood of SMPLC (1,8,11,18). However, it is worth noting that assuming lepidic pattern is synonymous with in-situ disease may be flawed. A minor lepidic component can be seen in 14% to 61% of IPM, whereas extensive lepidic component is typically consistent with SMPLC (10,11).

While molecular analysis serves as a robust diagnostic tool for patients with multifocal lung cancer, many techniques discussed here are not routinely accessible in clinical practice. Existing literature also remains limited, encompassing only a small cohort of patients and exhibiting considerable heterogeneity in the methods of genomic profiling employed by different institutions (6,10,11). The lack of standardization is exacerbated by the absence of guidelines. Unfortunately, the current guidelines concerning the use of molecular analysis in these patients dates back to the 7^th edition of the American Joint Committee on Cancer (AJCC) staging manual published in 2010, which recommends breakpoint analysis—an assay that is rarely used in contemporary practice (19). As we continue to assess the merits and constraints of genomic analysis, the thoracic community and the care of our patients urgently require updated guidelines delineating the indications for molecular analysis and an algorithm detailing the classification of multifocal tumors.

Acknowledgments

Funding: None.

Footnote

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