Multiple primary lung cancer: Updates of clinical management and genomic features

In recent decades, multiple primary lung cancer (MPLC) has been increasingly prevalent in clinical practice. However, many details about MPLC have not been completely settled, such as understanding the driving force, clinical management, pathological mechanisms, and genomic architectures of this disease. From the perspective of diagnosis and treatment, distinguishing MPLC from lung cancer intrapulmonary metastasis (IPM) has been a clinical hotpot for years. Besides, compared to patients with single lung lesion, the treatment for MPLC patients is more individualized, and non-operative therapies, such as ablation and stereotactic ablative radiotherapy (SABR), are prevailing. The emergence of next-generation sequencing has fueled a wave of research about the molecular features of MPLC and advanced the NCCN guidelines. In this review, we generalized the latest updates on MPLC from definition, etiology and epidemiology, clinical management, and genomic updates. We summarized the different perspectives and aimed to offer novel insights into the management of MPLC.


Introduction
Lung cancer is the most fatal malignancy globally, significantly burdening the public health system (1). In recent decades, lung cancer risk has increased along with the prolonged lifespan of people (2). Meanwhile, the prevalence of low-dose computed tomography (LDCT) lung cancer screening programs boosted lung cancer diagnosis (3).
Multiple primary lung cancer (MPLC), indicating patients with multiple tumor sites of independent origins in the lung, which can be synchronous or metachronous (4), is becoming a common phenomenon in clinical practice. In 1975, Martini and Melamed outlined the first clinical and pathological criteria for MPLC (M-M criteria) based on 108 cases (5). Based on the M-M criterion, many researchers have put much effort into this kind of disease.
The definite etiology and epidemiology of this disease remain unclear, although previous studies have reported that smoking (6), field cancerization (7-9), and familial heredity (10) were factors tightly associated with the initiation of MPLC. Moreover, choosing the best therapy, studying genomic mutations and architectures (11), distinguishing intrapulmonary metastasis (IPM) from MPLC (12), and understanding the tumor microenvironment of MPLC (13) are still research hotspots.
What we know about the MPLC is far from enough. From the perspective of diagnosis and clinical management, distinguishing MPLC from IPM is vital and may seriously affect the treatment strategy. Nowadays, preoperative imaging evidence can no longer accurately distinguish these two diseases. Therefore, distinguishing MPLC from IPM always expects genomic technology (14-16). Besides, surgical resection is still the mainstream treatment choice for MPLC patients. Researchers spent years searching for the best surgical time and resection strategy for patients (17). However, for patients who are not suitable for surgery, non-operative therapy such as image-guided thermal ablation (IGAB) and stereotactic ablative radiotherapy (SABR) were considered (18)(19)(20). And in recent years, immunotherapy for multiple GGOs (ground glass opacities) attracted much attention, while the curative effect is not ideal (21,22). From the perspective of genomic profiling, several previous researchers have reported the enormous genetic heterogeneity among the multiple lesions of MPLC using nextgeneration sequencing (11,15,23). Moreover, exploring the origin and initiation mechanisms of MPLC will also need the help of genomic profiling or other technologies such as RNA sequencing and single-cell sequencing (24).
In this review, we will give a comprehensive scenario of MPLC. We tried to generalize the latest updates on MPLC from definition, etiology and epidemiology, clinical management, and genomic updates. And we hope our work can open novel avenues for the major concerns of MPLC and advance the management of this unsolved disease.

Definition of MPLC
The definition and staging process of MPLC started with Martini and Melamed. They outlined the first clinical and pathological criteria for MPLC (M-M criteria) (5). In the M-M criteria, for synchronous MPLC, tumors should be physically separate, histologically identical, or different. If the tumors were the same histologically, they should originate from carcinoma in situ or without metastases. For metachronous MPLC, the tumors could be histologically heterogeneous or the same. If they were of the same histology, the intervals should be at least two years, or the tumors were physically distinct and without metastases, or they should originate from carcinoma in situ. The M-M criteria were used for 20 years until the revised definition of MPLC was brought up by Antakli et al. (25) in 1995. The underlying logic of the M-M criteria and Antakli's standard were similar, while Antakli was more concise. In 2016, the International Association for the Study of Lung Cancer (IASLC) classified multifocal lung nodules into four types based on histology judgment, clinical information (including lesion location and radiologic features), and prognosis (26): Type I-Second primary lung cancers, Type II-Separate tumor nodules (intrapulmonary metastasis), Type III-Multifocal lung adenocarcinoma with ground glass/lepidic features, and Type IV-Pneumonic-type lung adenocarcinoma. Most clinicians classified second primary lung cancers (Type I) and Multifocal lung adenocarcinoma with ground glass/lepidic feature (Type III) into MPLC. In second primary lung cancers (Type I), each tumor is of independent biological origin. Thus, in clinical practice, separate T, N, and M stage should be determined for each lesion, like individual tumors. For multifocal lung adenocarcinoma with ground glass/ lepidic feature (Type III), which belongs to ground-glass opacity (GGO), IASLC suggested assigning the T stage according to the highest T lesions and the N and M stages for all lesions collectively, and we summarized the criteria identifying them in Table 1. So far, the current classification of MPLC from IASLC has been widely accepted, offering much support for establishing the diagnosis criteria for MPLC. In 2022, the NCCN guidelines (NSCLC Version 3.2022) emphasized that the MPLC diagnosis requires a multidisciplinary setting involving surgeons, pathologists, radiation oncologists, and medical oncologists. In addition, they suggested some approaches to distinguish MPLC and IPM, which will be discussed later in this review.

Distinguishing MPLC from IPM
In the process of defining MPLC, IPM diagnosis is often accompanied. Although the M-M criteria involve pathological diagnosis, it is still important to distinguish between MPLC and IPM before treatment. If MPLC is wrongly diagnosed as IPM, the patient may lose the opportunity for surgery or receive chemical and radiation damage caused by unnecessary doses (27). Conversely, if IPM is diagnosed as MPLC, the patient's survival will be affected (11). Previously, the differential diagnosis of MPLC and IPM was mainly based on histopathological evidence. However, the accuracy of pathological diagnosis is limited, and the accuracy of the M-M standard has been proven to be relatively low (28). The pathological diagnosis standard proposed by Girard et al. in 2009 is considered as accurate as 91% and received wide acceptance (28,29). Since the diffusion of next-generation sequencing, the diagnosis at the gene level is indispensable to distinguishing the two diseases. We will elaborate these topics more specifically in the following article.

Etiology and epidemiology
The hypotheses of MPLC etiology are various. Tobacco use (including passive smoking by never-smokers) (6) and familial inheritance (9, 10) are the two main points. Field cancerization was also reported to be a likely mechanism of multifocal lung cancer (30, 31). In addition, a previous study reported that lung cancer survivors were more vulnerable to subsequent pulmonary malignancies (32). In 2021, Chen et al. first demonstrated that microplastic exposure was associated with the etiology of multiple pulmonary GGOs (ground-glass nodules) (33), linking MPLC pathogenesis to environmental factors. Genetic variants, such as EGFR germline mutations, were also reported to be the driving force of MPLC (15,34). A SEER (Surveillance Epidemiology and End Results) analysis suggested that younger age, female gender, earlier stage, and white race are risk factors for MPLC (35). Previous studies reported that the incidence of multi-focal lung cancer ranges from 0.2% to 20% (36)(37)(38). We believe that the incidence of MPLC in the real world is far more than what has been reported. Since the COVID-19 (Corona Virus Disease-19) pandemic in 2019, the number of patients undergoing pulmonary CT (computerized tomography) increased, resulting in a wave of the rapid growth of MPLC (39). So far, the associations between COVID-19 and MPLC have not been illustrated.

Clinical management and prognosis
Nowadays, there are multiple treatments for MPLC, including surgery, stereotactic ablative radiation (SABR), immunotherapy, and ablation. If the multiple lung tumors were diagnosed as IPM, the treatment strategy should obey the principle of treating T3/T4 NSCLC. In this part, we collected the relevant studies on MPLC and compared the efficacies and limitations of different treatments, which are summarized in Table 2.
For surgery, previous researchers have devoted much effort to identifying the most appropriate resection strategy and risk factors of prognosis for MPLC (40). Both synchronous and metachronous MPLC can benefit from surgical resection (41)(42)(43). In 2019, Chen et al. suggested that sub-lobar resection can lead to an equivalent prognosis to standard resection (lobectomy) and was beneficial in preserving pulmonary function (44), and for both ipsilateral and

Clinical Criteria
There are multiple subsolid nodules (either pure ground glass or part solid), with at least one suspected (or proved) to be cancer.

This applies whether or not a biopsy has been performed on the nodules.
This applies if the other nodules(s) are found by biopsy to be AIS, MIA, or LPA.
This applies if a nodule has become >50% solid but is judged to have arisen from a GGN, provided that there are other subsolid nodules.
GGN lesions <5 mm or lesions suspected to be AAH are not counted.

Pathologic Criteria
There are multiple foci of LPA, MIA, or AIS.
This applies whether a detailed histologic assessment (i.e., proportion of subtypes, etc.) shows a matching or different appearance.
This applies if one lesion(s) is LPA, MIA, or AIS and there are other subsolid nodules of which a biopsy has not been performed.
This applies whether the nodule(s) are identified preoperatively or only on pathologic examination.
Foci of AAH are not counted.
A radiographically solid appearance and the specific histologic subtype of solid of adenocarcinoma denote different things. AAH, atypical adenomatous hyperplasia; AIS, adenocarcinoma in situ; GGN, ground glass nodule; LPA, lepidic-predominant adenocarcinoma; MIA, minimally invasive adenocarcinoma.
bilateral MPLC, surgery resection is as safe as single-nodule lung cancer (43,45). It is believed that radical pneumonectomy should be avoided, if possible, for it may lead to a poor prognosis (46,47). It's worth mentioning that the situation becomes more complicated when GGO/lepidic lesions coexist with solid or subsolid nodules. In clinical practice, removing solid nodules might be the best choice and at the same time, removing the other GGOs on the same side is more helpful for diagnosing and staging (48). For patients with MPLC, surgery is far from a single treatment. On the one hand, there are many high-risk patients of older age or with underlying cardiopulmonary comorbidities, for whom thoracic operations might lead to severe intraoperative and postoperative complications. On the other hand, some MPLC patients have lung nodules disseminated in more than one lobe, especially for multifocal lung adenocarcinoma with ground glass/ lepidic features (Type III), which are inert in nature compared to solid and subsolid nodules. Removing all nodules seems impossible for these people or will lead to much lung function loss. Besides, the selection logic of resection strategy is different among different surgeons, for removing tumors to the maximum extent is as essential as guaranteeing patients' post-treatment life quality.
For MPLC patients who are not suitable for surgery, stereotactic ablative radiation (SABR), and image-guided thermal ablation (IGTA) (18)(19)(20)49) are recommended. The ESMO guidelines for early and locally advanced NSCLC recommended that MPLC should be mainly assessed with curative intent (complete resection), while SABR was also an effective choice (50). Previous research showed that stereotactic ablative body radiotherapy achieved an 84% lesion control rate after two years in MPLC (19). And Nikittas et al. compared the effectiveness and safety between MPLC patients and single nodule patients. They suggested improved disease and survival outcomes in patients receiving both surgery and SBRT and multiple courses of SBRT alone for MPLC, though this could be due to selection bias (18). Image-guided thermal ablation is a method that has been proven to be applicable to the treatment of pulmonary nodules in recent years (20,51). And it contains radiofrequency ablation (RFA), microwave ablation (MWA), and cryoablation (CA) (51). Aufranc et al. (52) studied 115 patients with a total of 160 lung tumors to compare microwave (MWA) and radiofrequency (RFA) ablation in the percutaneous treatment of primary and secondary lung tumors. They found that MWA and RFA are effective and safe IGTA techniques for the treatment of lung cancer. Besides, there are other retrospective studies that improved the safety and efficiency of IGTA (53-55), but they are small-sized cohort studies.
Many factors are associated with better survival in MPLC, including small tumor size (size of the largest tumor and sum of tumor sizes, also presenting as SUVmax value in radiologic images) (56, 57), similar histology of multiple lesions, N0 stage (better prognosis) (58), T1 stage (42), female gender (59), young age, nonsmoker and FEV1/FVC ≥70% (42). Evidence showed that male gender, symptomatic disease, and lymph node involvement were independent factors for adverse prognosis (60). The prognosis of stage I synchronous multiple primary non-small cell lung cancer is similar to that of solitary primary NSCLC (61). To take precautions against metachronous primary lung cancers, regular CT examination after surgical resection is recommended for heavy smokers (62). Long-term follow-up for both synchronous and metachronous MPLC is necessary by which potentially curable secondary lung cancers can be identified when they are still under control (63).
Taken together, although people spent years finding the best treatment for MPLC, surgical resection remains the mainstream, while the non-surgical treatment approaches for MPLC are also indispensable.

Clinical trials about MPLC
To the best of our knowledge, nine registered clinical trials about MPLC have been launched so far ( Table 3) Notably, all nine clinical trials are launched by Chinese researchers. Chinese researchers have paid much attention to MPLC in the past five years, and the results of the most established trials will come out within the next five years. We hope their findings can offer constructive suggestions to the management of MPLC, though, among the current trials, the maximal number of recruiting patients is 172, which might not be enough for a comprehensive illustration of this disease in our recognition.
Overall, the attention MPLC received from clinical trials does not match the significance of this disease in lung cancer, considering its high incidence. We are especially looking forward to more trials exploring the efficacy of immunotherapy in MPLC since the introduction of checkpoint inhibitors opens new avenues for cancer therapy. In addition, we look forward to the trials

Genomic updates about MPLC
Genomic characteristics of multifocal lung cancer started over 25 years ago (64). With the development of science and technology and the progress of gene research methods, several molecular methods such as DNA microsatellite analysis, array comparative genomic hybridization (aCGH), targeted sequencing, nextgeneration sequencing (NGS), and single-cell sequencing have been applied to profiling the characteristics of MPLC. We summarized the representative research on MPLC's genomic profiling in recent years, as shown in Table 4.
Before the widespread of NGS, gene panels containing a few oncogenic/tumor-suppressor genes (usually 1 to 5 genes) and chromosome alterations in MPLC were the focus, which was far from enough for profiling the MPLC genome. Using target sequencing, many researchers tested genes such as EGFR, KRAS, and p53 in MPLC (65)(66)(67). Chromosome alterations such as copy number analysis (27,68), microsatellite instability (MSI) (69), loss of heterozygosity (LOH) analysis (69,70), and X chromosome inactivation (37) were also detected in MPLC by genomic hybridization (aCGH). Those previous researchers tried to find out the difference and similarities of lesions of MPLC (37, 40), but inefficient technology hinders our in-depth understanding of the MPLC genome.
In the era of NGS, various sequencing approaches and analysis methods have emerged, fueling a wave of genomic research on MPLC.

The distinction between MPLC and IPM
The precise differentiation between MPLC and IPM is one of the driving forces of the genomic exploration of MPLC. Genomic profiling information challenged the traditional clinicopathologic criteria of MPLC. In the era of next-generation sequencing (NGS), the application of large gene sequencing panels, whole-genome sequencing (WGS), and chromosome rearrangements helped us distinguish MPLC from IPM In 2018, Santamaria et al. (71) conducted a targeted 50-gene DNA sequencing panel on a lung cancer patient with three lesions. Their results supported that all three lesions were independent in mutation background, indicating they were MPLC rather than advanced lung cancer. In 2019, similar research was conducted by Chang et al. using an NGS assay covering up to 468 cancer-related genes in 60 patients, showing that the histologic classification was contrary to the judgment based on NGS in 22% of all cases, especially in the prediction of IPMs, in which the misdiagnosis rate was up to 44% (72).
Whole-genome sequencing (WGS) and whole-exome sequencing (WES) are more comprehensive in profiling genomic features than targeted sequencing. In 2016, Liu et al. (73) were the first to portray the genomic architecture of MPLC using WGS and WES. They collected 16 tumor samples from six patients. One patient was identified as having metastasis under histopathological standards and was classified as primary based on genomic sequencing results. In 2018, Li et al. (74) performed exome sequencing on two non-smoker patients with multiple GGOs (patient 1 had seven subsolid nodules and one pure GGO, and all the six nodules of patient 2 were pure GGOs). Results showed that two subsolid nodules in patient one and two pure GGOs in patient 2 were clonally-related, respectively, indicating that intrapulmonary metastasis could occur among GGOs, even in pure GGOs, which reshaped people's impression of multiple GGOs.
Recently, chromosome rearrangements were reported to be an effective tool for this discrimination (29,38). In 2020, Wang and his team (77) first integrated the radiological image data of mGGOs into their genomic analysis to investigate the intratumoral heterogeneity and clonal relationship of multifocal GGOs. They found that the differences between subsolid nodules (SSNs) and advanced-stage LUADs at a genomic level were unraveled. Although multicentric origin was predominant, they also detected early metastatic events among multifocal SSNs. Similarly, Yu et al. (78) performed a custom 1021-gene panel sequencing of 334 resected pulmonary nodules presenting as GGO from 262 Chinese patients. They compared gene pathways enriched in different GGOs (pure GGOs and mixed GGOs) through genomic profiling. They demonstrate a driver landscape of radiologically detectable GGO-associated pulmonary nodules in Chinese patients and support that different driver patterns in RTK/RAS pathway correspond to different radiologic features.
Single-cell sequencing technology has been a brilliant new technology for tumor analysis in recent years. It studied the changes in the multicellular microenvironment of the tumor by studying the cell composition in tumor tissue. In 2021, Zhang et al. (21) performed WES, Immunohistochemistry (IHC), single-cell RNA seq, TCR (T cell receptor repertoire)-seq in the multiple nodules of one MPLC case undergoing neoadjuvant pembrolizumab treatment. The genomic disparities among responding and non-responding nodules were detected at various levels, suggesting that neoadjuvant PD-1/PD-L1 inhibitors alone may not be optimal for MPLC. In 2022, 10x Genomics single-cell RNA-seq was used in GGOs by Li et al. (79), and they identified transcriptomic differences in the vital signaling pathways of tumor and immune cells within GGOs, implying the transcriptomic heterogeneity of this disease. Similarly, He et al. (80) macro-dissected the solid (S) components and ground-glass (GG) components of mGGO and performed singlecell sequencing analyses of six paired components from three mGGO patients. They found that cancer cells and macrophages were the dominant cell types in the S and GG components, respectively. Cancer cells in the S components, which showed relatively malignant phenotypes, were likely to originate from both the GG and S components and monitor the surrounding tumor microenvironment (TME) through an intricate cell interaction network.
In summary, with the progress of science and technology, our perceptions about MPLC are growing. Thanks to the initial exploration of the heterogeneity of MPLC, to the later identification of MPLC and IPM, and finally to the use of advanced sequencing technology to clarify the development m e c h a n i s m s / t u m o r i m m u n e m i c r o e n v i r o n m e n t , ou r understanding of MPLC is now deeper and it is expected to increase in the future. Mutation analysis of SP-and MP-LUAD patients could identified genomic alterations and evolutionary trajectories underlying MP-LUAD and will provide new insights into the oncogenesis of MP-LUAD and useful information for development novel approach to target MP-LUAD.

Summary
From clinical management to genomic profiling, this review gives a comprehensive description of MPLC, which is a highly focused malignancy by clinicians. We hope our work can enlighten more profound reflections about MPLC from the public.

Author contributions
JH, SG, and CL designed this study and provided funding support. HT, GB, and ZY drafted the manuscript and completed the tables. PC, JX, TL, TF, BW, and CX collected the references and completed the tables. All authors contributed to the article and approved the submitted version.

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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