Pathologic Assessment and Staging of Multiple Non-Small Cell Lung Carcinomas: A Paradigm Shift with the Emerging Role of Molecular Methods.

Non-small cell lung carcinomas (NSCLCs) commonly present as 2 or more separate tumors. Biologically, this encompasses 2 distinct processes: separate primary lung carcinomas (SPLCs), representing independently arising tumors, and intrapulmonary metastases (IPMs), representing intrapulmonary spread of a single tumor. The advent of computed tomography imaging has substantially increased the detection of multifocal NSCLCs. The strategies and approaches for distinguishing between SPLCs and IPMs have evolved significantly over the years. Recently, genomic sequencing of somatic mutations has been widely adopted to identify targetable alterations in NSCLC. These molecular techniques have enabled pathologists to reliably discern clonal relationships among multiple NSCLCs in clinical practice. However, a standardized approach to evaluating and staging multiple NSCLCs using molecular methods is still lacking. Here, we reviewed the historical context and provided an update on the growing applications of genomic testing as a clinically relevant benchmark for determining clonal relationships in multiple NSCLCs, a practice we have designated "comparative molecular profiling." We examined the strengths and limitations of the morphology-based distinction of SPLCs vs IPMs and highlighted pivotal clinical and pathologic insights that have emerged from studying multiple NSCLCs using genomic approaches as a gold standard. Lastly, we suggest a practical approach for evaluating multiple NSCLCs in the clinical setting, considering the varying availability of molecular techniques.

ImmunoPET Imaging of CD146 in Murine Models of Intrapulmonary Metastasis of Non-Small Cell Lung Cancer.

CD146 has been identified as an excellent biomarker for lung cancer as its overexpression in solid tumors has been linked to disease progression, invasion, and metastasis. Previously, our group described a positive correlation between 64Cu-labeled YY146 uptake and increased expression of CD146 in six human lung cancer cell lines using subcutaneous tumor models. In this study, we investigate a monoclonal antibody called YY146 for immunoPET imaging of CD146 in two intrapulmonary metastasis models of non-small cell lung cancer (NSCLC). The binding and immunoreactivity of the tracer were assessed by in vitro assays. Radiolabeling of YY146 with positron emitting Cu-64 (64Cu-NOTA-YY146) enabled PET imaging of intrapulmonary metastasis. Mice were intravenously injected with two million tumor cells, and CT imaging was used to verify the presence of lung metastases. 64Cu-NOTA-YY146 was injected into tumor-bearing mice, and animals were subjected to PET/CT imaging at 4, 24, and 48 h postinjection. Both the average and maximum lung PET signal intensities were quantified and compared between high and low CD146-expressing metastases. Further validation was accomplished through immunofluorescence imaging of resected tissues with CD31 and CD146. In flow cytometry, YY146 revealed strong binding to CD146 in H460 cells due to its high expression with minimal binding to CD146-low expressing H358 cells. Both YY146 and NOTA-YY146 showed similar binding, suggesting that NOTA conjugation did not elicit any negative effects on its binding affinity. Imaging of 64Cu-NOTA-YY146 in H460 tumor-bearing mice revealed rapid, persistent, and highly specific tracer accumulation. Uptake of 64Cu-NOTA-YY146 in the whole lung was calculated for H460 and H358 as 7.43 ± 0.38 and 3.95 ± 0.47% ID/g at 48 h postinjection (n = 4, p < 0.05), and the maximum lung signals were determined to be 13.85 ± 1.07 (H460) and 6.08 ± 0.73% ID/g (H358) at equivalent time points (n = 4, p < 0.05). To ensure the specificity of the tracer, a nonspecific antibody was injected into H460 tumor-bearing mice. Ex vivo biodistribution and immunofluorescence imaging validated the PET findings. In summary, 64Cu-NOTA-YY146 allowed for successful imaging of CD146-expressing intrapulmonary metastases of NSCLC in mice. This preliminary study provides evidence supporting the future clinical utilization of 64Cu-NOTA-YY146 for possible treatment monitoring of CD146-targeted therapy or improving patient stratification.

[Non-small Cell Lung Cancer with Metachronous Mutations of EGFR and ALK Genes: 
A Case Report and Literature Review].

Multiple primary lung cancer (MPLC) refers to patients with two or more primary lesions of lung cancer. It can be divided into synchronous MPLC (sMPLC) and metachronous MPLC (mMPLC) based on the timing of occurrence. In recent years, the detection rate of MPLC has gradually increased. However, considerable controversy exists in distinguishing MPLC from intrapulmonary metastasis (IM), especially when the histopathological types are identical. Given the significant differences in treatment strategies and prognosis in clinical practice currently, accurate diagnosis of MPLC is crucial for personalized precision therapy. Molecular genetics and sequencing technologies offer effective strategies for assessing the clonal origin of tumors. There have been reports of coexisting mutations in the epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) fusion genes in non-small cell lung cancer, but case of EGFR mutation following an ALK mutation has not been mentioned. This article accurately diagnoses and retrospectively analyzes the clinical data of a case of ALK mutant adenocarcinoma in a male patient who developed an EGFR mutation with multiple metastases four years after surgery, and reviews the relevant literature. This paper aims to deepen the understanding of mMPLC and provide clinical references for the diagnosis and treatment of such patients.
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Contemporary Incidence of Synchronous Multiple Primary Lung Cancers and Survival in the Era of Lung Cancer Screening.

OBJECTIVE: Up to 15% of lung cancer patients have multiple suspicious nodules. While some of these nodules may represent metastatic lung cancer, others represent synchronous multiple primary lung cancer (SMPLC). The incidence of SMPLC ranges from 0.8% to 8.4% and appears to be increasing. Inconsistent identification of SMPLC can be detrimental for patients who are misdiagnosed as having intrapulmonary metastasis and not offered stage-based treatment. We sought to identify the contemporary incidence of SMPLC at a tertiary institution. METHODS: From January 2018 to September 2019, patients who underwent lung cancer resection were retrospectively reviewed. Patients with SMPLC were identified using the modified Martini-Melamed criteria. RESULTS: During the 21-month period, 227 patients underwent lung cancer resection. There were 47 patients (20.7%) who had 119 pathologically confirmed SMPLC. Most patients had ipsilateral tumors (n = 24, 51.1%) with at least 1 adenocarcinoma (n = 40, 85.1%). Considering histologic subtyping, 38 (80.9%) had histologically distinct tumors. Overall and cancer-specific survival at 4 years was 86% and 90%, respectively. Only patients with 3 or more SMPLC had poor 4-year overall (P = 0.002) and cancer-specific survival (P = 0.043) compared with those with 2 SMPLC. Patient demographics, histology, tumor location, and highest pathologic staging did not affect survival outcomes. CONCLUSIONS: Using a strict inclusion criterion, the incidence of SMPLC is higher than previously reported. SMPLC patients have favorable survival outcomes, suggesting that they behave like primary lung cancer, not intrapulmonary metastasis. Awareness of SMPLC by thoracic surgeons is critical in optimizing outcomes in this patient population.

Favourable surgical outcomes for either second primary lung cancer or intrapulmonary metastasis after resection of non-small-cell lung cancer.

OBJECTIVES: Metachronous lung cancer arising after resection of non-small-cell lung cancer is either a second primary lung cancer (SPLC) or intrapulmonary metastasis (IPM) of the initial lung cancer; however, differential diagnosis is difficult. We evaluated the surgical outcomes of metachronous lung cancer in a combined population of patients with SPLC and IPM. METHODS: A retrospective study of 3534 consecutive patients with resected non-small-cell lung cancer between 1992 and 2016 was conducted at 4 institutions. RESULTS: A total of 105 patients (66 males; median age, 70 years) who underwent a second pulmonary resection for metachronous lung cancer were included. Most patients (81%) underwent sublobar resection, and there was no 30-day mortality. All metachronous lung cancers were cN0, 5 were pN1-2. The postoperative comprehensive histologic assessment revealed SPLC (n = 77) and IPM (n = 28). The 5-year overall survival rate after the second resection was 70.6% (median follow-up: 69.7 months). A multivariable analysis showed that age >70 years at the second resection (P = 0.013), male sex (P = 0.003), lymph node involvement in metachronous cancer (P < 0.001), pathological invasive size of metachronous cancer >15 mm (P < 0.001) and overlapping squamous cell carcinoma histology of the initial and metachronous cancers (P = 0.003) were significant prognostic factors for poor survival after the second resection, whereas histological IPM was not (P = 0.065). CONCLUSIONS: Surgery for cN0 metachronous lung cancer is safe and shows good outcomes. There were no statistically significant differences in the SPLC and IPM results. Caution should be exercised when operating on patients with overlapping squamous cell carcinoma.

Genomic heterogeneity of multiple synchronous lung cancers in Chinese population.

INTRODUCTION: It is clinically challenging to infer the phylogenetic relationship between different tumor lesions of patients with multiple synchronous lung cancers (MSLC), whether these lesions are the result of independently evolved tumor or intrapulmonary metastases. METHODS: We used the Illumina X10 platform to sequence 128 stage I lung cancer samples collected from 64 patients with MSLC. All samples were analyzed for mutation spectra and phylogenetic inference. RESULTS: We detected genetic aberrations within genes previously reported to be recurrently altered in lung adenocarcinoma including, EGFR, ERBB2, TP53, BRAF, and KRAS. Other putative driver mutations identified were enriched in RTK-RAS signaling, TP53 signaling, and cell cycle. Also, we found some interesting cases, two cases that carried EGFR L858R and T790M co-mutation in one tumor and another tumor with only EGFR 19del, and 1 case with two KRAS hotspots in the same tumor. Due to the short follow-up time and early stage, further investigation is needed to determine whether this unique mutation profile will affect their progression-free survival (PFS) and overall survival (OS). Regarding genetic evolution analysis among 64 tumor samples, 50 of them display distinct mutational profiles, suggesting these are independently evolved tumors, which is consistent with histopathological assessment. On the other hand, six patients were identified to be intrapulmonary metastasis as the mutations harbored in different lesions are clonally related. CONCLUSION: In summary, unlike intrapulmonary metastases, patients with MSLC harbor distinct genomic profiles in different tumor lesions, and we could distinguish MSLC from intrapulmonary metastases via clonality estimation.

The utility of next-generation sequencing in distinguishing between separate primary lung carcinomas and intrapulmonary metastasis: A case report.

The distinction between separate primary lung carcinomas (SPLCs) and intrapulmonary metastases (IPMs) is crucial to accurate cancer staging. Histopathology-based classification cannot always determine the relatedness of multiple tumors taken from the lung. Recently, next-generation sequencing (NGS) has been used for biomarker determination, but it also has the potential to inform clonality determination among multiple tumors. Here we present a patient with three lung tumors, each diagnosed as adenocarcinoma by histopathology with a differential diagnosis of SPLC versus IPM. We pursued molecular profiling by NGS, which revealed three unique mutational patterns ruling out the possibility of clonal relatedness among the cancers. Our case supports the utility of NGS in supplementing histopathological methods to distinguish between SPLCs and IPMs and to guide treatment decisions.

Clinical and pathologic staging accuracy in patients with synchronous multiple primary lung cancers.

Background: The incidence of synchronous multiple primary lung cancer (SMPLC) is increasing, occurring in up to 20% of lung cancer patients. Accurately identifying SMPLC can be challenging, and failure to recognize SMPLC results in poor outcomes. We sought to assess the staging accuracy of patients with SMPLC at our tertiary institution. Methods: We retrospectively reviewed all patients who were evaluated for lung cancer resection between January 2018 to September 2019. Patients with SMPLC were identified using the modified Martini-Melamed criteria. Preoperative imaging, clinical assessment, and pathologic interpretation were reviewed and compared to the final staging assigned by a multidisciplinary lung cancer tumor board to determine accuracy. Results: Out of 227 patients presenting for lung cancer resection, 47 patients with 119 SMPLC were identified, of which 38 (80.9%) were incorrectly staged by at least one report. Incorrect staging was most common by computed tomography (CT) reports (n=33/47, 70.2%), followed by positron emission tomography-CT (PET-CT) reports (n=28/45, 62.2%), surgeons' clinical assessment (n=10/47, 21.3%), and histopathology reports (n=8/47, 17.0%). CT reports, when incorrect, under-staged 97.0% (n=32) of patients. PET-CT reports, when incorrect, over-staged 25.0% (n=7) of patients by reporting the second primary nodule to be "consistent with metastasis". Histopathology reports, when incorrect, over-staged 87.5% (n=7) of patients despite lack of lymph node involvement. Conclusions: Patients with SMPLC are at risk of receiving incorrect treatment based on radiographic and histopathologic staging reports alone. The observed staging inaccuracies are concerning, necessitating increased awareness among physicians caring for lung cancer patients.

Genomic characteristics and immune landscape of super multiple primary lung cancer.

BACKGROUND: In recent years, a growing number of patients with multiple primary lung cancer (MPLC) are being diagnosed, and a subset of these patients is found to have a large number of lesions at the time of diagnosis, which are referred to as 'super MPLC'. METHODS: Here, we perform whole exome sequencing (WES) and immunohistochemistry (IHC) analysis of PD-L1 and CD8 on 212 tumor samples from 42 patients with super MPLC. FINDINGS: We report the genomic alteration landscape of super MPLC. EGFR, RBM10 and TP53 mutation and TERT amplification are important molecular events in the evolution of super MPLC. We propose the conception of early intrapulmonary metastasis, which exhibits different clinical features from conventional metastasis. The IHC analyses of PD-L1 and CD8 reveal a less inflamed microenvironment of super MPLC than that of traditional non-small cell lung cancer (NSCLC). We identify the potentially susceptible germline mutations for super MPLC. INTERPRETATION: Our study depicts the genomic characteristics and immune landscape, providing insights into the pathogenesis and possible therapeutic guidance of super MPLC. FUNDING: A full list of funding bodies that supported this study can be found in the Acknowledgements section.

Using molecular characteristics to distinguish multiple primary lung cancers and intrapulmonary metastases.

Objectives: Multiple lung cancers may present as multiple primary lung cancers (MPLC) or intrapulmonary metastasis (IPM) with variations in clinical stage, treatment, and prognosis. However, the existing differentiation criteria based on histology do not fully meet the clinical needs. Next-generation sequencing (NGS) may play an important role in assisting the identification of different pathologies. Here, we extended the relevant data by combining histology and NGS to develop detailed identification criteria for MPLC and IPM. Materials and Methods: Patients with lung cancer (each patient had ≥2 tumors) were enrolled in the training (n = 22) and validation (n = 13) cohorts. Genomic profiles obtained from 450-gene-targeted NGS were analyzed, and the new criteria were developed based on our findings and pre-existing Martini & Melamed criteria and molecular benchmarks. Results: The analysis of the training cohort indicated that patients identified with MPLC had no (or <2) trunk or shared mutations. However, 98.02% of mutations were branch mutations, and 69.23% of MPLC had no common mutations. In contrast, a higher percentage of trunk (33.08%) or shared (9.02%) mutations were identified in IPM, suggesting significant differences among mutated components. Subsequently, eight MPLC and five IPM cases were identified in the validation cohort, aligning with the independent imaging and pathologic distinction. Overall, the percentage of trunk and shared mutations was higher in patients with IPM than in patients with MPLC. Based on these results and the establishment of new determination criteria for MPLC and IPM, we emphasize that the type and number of shared variants based on histologic consistency assist in identification. Conclusion: Determining genetic alterations may be an effective method for differentiating MPLC and IPM, and NGS can be used as a valuable assisting tool.

Classification of multiple primary lung cancer in patients with multifocal lung cancer: assessment of a machine learning approach using multidimensional genomic data.

Background: Multiple primary lung cancer (MPLC) is an increasingly well-known clinical phenomenon. However, its molecular characterizations are poorly understood, and still lacks of effective method to distinguish it from intrapulmonary metastasis (IM). Herein, we propose an identification model based on molecular multidimensional analysis in order to accurately optimize treatment. Methods: A total of 112 Chinese lung cancers harboring at least two tumors (n = 270) were enrolled. We retrospectively selected 74 patients with 121 tumor pairs and randomly divided the tumor pairs into a training cohort and a test cohort in a 7:3 ratio. A novel model was established in training cohort, optimized for MPLC identification using comprehensive genomic profiling analyzed by a broad panel with 808 cancer-related genes, and evaluated in the test cohort and a prospective validation cohort of 38 patients with 112 tumors. Results: We found differences in molecular characterizations between the two diseases and rigorously selected the characterizations to build an identification model. We evaluated the performance of the classifier using the test cohort data and observed an 89.5% percent agreement (PA) for MPLC and a 100.0% percent agreement for IM. The model showed an excellent area under the curve (AUC) of 0.947 and a 91.3% overall accuracy. Similarly, the assay achieved a considerable performance in the independent validation set with an AUC of 0.938 and an MPLC predictive value of 100%. More importantly, the MPLC predictive value of the classification achieved 100% in both the test set and validation cohort. Compared to our previous mutation-based method, the classifier showed better κ consistencies with clinical classification among all 112 patients (0.84 vs. 0.65, p <.01). Conclusion: These data provide novel evidence of MPLC-specific genomic characteristics and demonstrate that our one-step molecular classifier can accurately classify multifocal lung tumors as MPLC or IM, which suggested that broad panel NGS may be a useful tool for assisting with differential diagnoses.

A pairwise radiomics algorithm-lesion pair relation estimation model for distinguishing multiple primary lung cancer from intrapulmonary metastasis.

Background: Distinguishing multiple primary lung cancer (MPLC) from intrapulmonary metastasis (IPM) is critical for their disparate treatment strategy and prognosis. This study aimed to establish a non-invasive model to make the differentiation pre-operatively. Methods: We retrospectively studied 168 patients with multiple lung cancers (307 pairs of lesions) including 118 cases for modeling and internal validation, and 50 cases for independent external validation. Radiomic features on computed tomography (CT) were extracted to calculate the absolute deviation of paired lesions. Features were then selected by correlation coefficients and random forest classifier 5-fold cross-validation, based on which the lesion pair relation estimation (PRE) model was developed. A major voting strategy was used to decide diagnosis for cases with multiple pairs of lesions. Cases from another institute were included as the external validation set for the PRE model to compete with two experienced clinicians. Results: Seven radiomic features were selected for the PRE model construction. With major voting strategy, the mean area under receiver operating characteristic curve (AUC), accuracy, sensitivity, and specificity of the training versus internal validation versus external validation cohort to distinguish MPLC were 0.983 versus 0.844 versus 0.793, 0.942 versus 0.846 versus 0.760, 0.905 versus 0.728 versus 0.727, and 0.962 versus 0.910 versus 0.769, respectively. AUCs of the two clinicians were 0.619 and 0.580. Conclusions: The CT radiomic feature-based lesion PRE model is potentially an accurate diagnostic tool for the differentiation of MPLC and IPM, which could help with clinical decision making.

Distinguishing multiple primary lung cancers from intrapulmonary metastasis using CT-based radiomics.

PURPOSE: To develop CT-based radiomics models that can efficiently distinguish between multiple primary lung cancers (MPLCs) and intrapulmonary metastasis (IPMs). METHOD: This retrospective study included 127 patients with 254 lung tumors pathologically proved as MPLCs or IPMs between May 2009 and January 2020. Radiomics features of lung tumors were extracted from baseline CT scans. Particularly, we incorporated tumor-focused, refined radiomics by calculating relative radiomics differences from paired tumors of individual patients. We applied the L1-norm regularization and analysis of variance to select informative radiomics features for constructing radiomics model (RM) and refined radiomics model (RRM). The performance was assessed by the area under the receiver operating characteristic curve (AUC-ROC). The two radiomics models were compared with the clinical-CT model (CCM, including clinical and CT semantic features). We incorporated both radiomics features to construct fusion model1 (FM1). We also, build fusion model2 (FM2) by combing both radiomics, clinical and CT semantic features. The performance of the FM1 and FM2 were further compared with that of the RRM. RESULTS: On the validation set, the RM achieved an AUC of 0.857. The RRM demonstrated improved performance (validation set AUC, 0.870) than the RM, and showed significant differences compared with the CCM (validation set AUC, 0.782). Fusion models further led prediction performance (validation set AUC, FM1:0.885; FM2:0.889). There were no significant differences among the performance of the FM1, the FM2 and the RRM. CONCLUSIONS: The CT-based radiomics models presented good performance on the discrimination between MPLCs and IPMs, demonstrating the potential for early diagnosis and treatment guidance for MPLCs and IPMs.

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.

Towards the molecular era of discriminating multiple lung cancers.

In the era of histopathology-based diagnosis, the discrimination between multiple lung cancers (MLCs) poses significant uncertainties and has thus become a clinical dilemma. However, recent significant advances and increased application of molecular technologies in clonal relatedness assessment have led to more precision in distinguishing between multiple primary lung cancers (MPLCs) and intrapulmonary metastasis (IPMs). This review summarizes recent advances in the molecular identification of MLCs and compares various methods based on somatic mutations, chromosome alterations, microRNAs, and tumor microenvironment markers. The paper also discusses current challenges at the forefront of genomics-based discrimination, including the selection of detection technology, application of next-generation sequencing, and intratumoral heterogeneity (ITH). In summary, this paper highlights an entrance into the primary stage of molecule-based diagnostics.

Case report: targeted sequencing improves the diagnosis of multiple synchronous lung cancers.

Background: The ability to distinguish satellite nodules, multiple primary lung cancers (MPLCs), and intrapulmonary metastases (IPM) is crucial for prognosis and treatment. The traditional diagnostic criteria for MPLC/IPM including the Martini and Melamed (MM) criteria and the comprehensive histologic assessment (CHA) criteria, mainly relies on histological comparison between multiple lesions. However, many challenges remain in distinguishing them in clinical practice. Case Description: We herein present a report of 3 lung adenocarcinoma cases who presented with 2 lesions, with improved diagnosis based on targeted sequencing covering driver genes. Based on histopathological features, patient 1 (P1) was classified as MPLC, whereas patients 2 and 3 (P2, P3) were classified as satellite nodules. However, targeted sequencing revealed the clonality status of these lesions and improved their diagnosis. The result of the molecular testing indicated that P1 is IPM and the other two patients (P2, P3) should be diagnosed with MPLC. Conclusions: Different lesions in the same case had different driver mutations, suggesting that the 2 lesions were driven by different molecular events. Therefore, targeted sequencing containing driver genes should be used for the diagnosis of multiple synchronous lung cancers. A limitation of this report is the short follow up period, and long-term outcomes of the patients require further follow up.

A novel NGS-based diagnostic algorithm for classifying multifocal lung adenocarcinomas in pN0M0 patients.

The classification of multifocal lung adenocarcinomas (MLAs), including multiple primary lung adenocarcinomas (MPLAs) and intrapulmonary metastases (IPMs), has great clinical significance in staging and treatment determination. However, the application of molecular approaches in pN0M0 MLA diagnosis has not been well investigated. Here, we performed next-generation sequencing (NGS) analysis in 45 pN0M0 MLA patients (101 lesion pairs) who were initially diagnosed as having MPLA by comprehensive histologic assessment (CHA). Five additional patients with intrathoracic metastases were used as positive controls, while 197 patients with unifocal lung adenocarcinomas (425 random lesion pairs) were used as negative controls. By utilizing a predefined NGS criterion, all IPMs in the positive control group could be accurately classified, whereas 13 lesion pairs (3.1%) in the negative control cohort were misdiagnosed as IPMs. Additionally, 14 IPM lesion pairs were diagnosed in the study group, with at least 7 misdiagnoses. We thus developed a refined algorithm, incorporating both NGS and histologic results, that could correctly diagnose all the known MPLAs and IPMs. In particular, all IPMs identified by the refined algorithm were diagnosed to be IPMs or suspected IPMs by CHA reassessment. The refined algorithm-diagnosed MPLAs patients also had significantly better progression-free survival than the refined algorithm-diagnosed IPMs (p < 0.0001), which is superior to conventional NGS or CHA diagnoses. Overall, we developed an NGS-based algorithm that could accurately distinguish IPMs from MPLAs in MLA patients. Our results demonstrate a promising clinical utility of NGS to complement traditional CHA-based MLA diagnosis and help determine patient staging and treatment.

Lineage tracing for multiple lung cancer by spatiotemporal heterogeneity using a multi-omics analysis method integrating genomic, transcriptomic, and immune-related features.

Introduction: The distinction between multiple primary lung cancer (MPLC) and intrapulmonary metastasis (IPM) holds clinical significance in staging, therapeutic intervention, and prognosis assessment for multiple lung cancer. Lineage tracing by clinicopathologic features alone remains a clinical challenge; thus, we aimed to develop a multi-omics analysis method delineating spatiotemporal heterogeneity based on tumor genomic profiling. Methods: Between 2012 and 2022, 11 specimens were collected from two patients diagnosed with multiple lung cancer (LU1 and LU2) with synchronous/metachronous tumors. A novel multi-omics analysis method based on whole-exome sequencing, transcriptome sequencing (RNA-Seq), and tumor neoantigen prediction was developed to define the lineage. Traditional clinicopathologic reviews and an imaging-based algorithm were performed to verify the results. Results: Seven tissue biopsies were collected from LU1. The multi-omics analysis method demonstrated that three synchronous tumors observed in 2018 (LU1B/C/D) had strong molecular heterogeneity, various RNA expression and immune microenvironment characteristics, and unique neoantigens. These results suggested that LU1B, LU1C, and LU1D were MPLC, consistent with traditional lineage tracing approaches. The high mutational landscape similarity score (75.1%), similar RNA expression features, and considerable shared neoantigens (n = 241) revealed the IPM relationship between LU1F and LU1G which were two samples detected simultaneously in 2021. Although the multi-omics analysis method aligned with the imaging-based algorithm, pathology and clinicopathologic approaches suggested MPLC owing to different histological types of LU1F/G. Moreover, controversial lineage or misclassification of LU2's synchronous/metachronous samples (LU2B/D and LU2C/E) traced by traditional approaches might be corrected by the multi-omics analysis method. Spatiotemporal heterogeneity profiled by the multi-omics analysis method suggested that LU2D possibly had the same lineage as LU2B (similarity score, 12.9%; shared neoantigens, n = 71); gefitinib treatment and EGFR, TP53, and RB1 mutations suggested the possibility that LU2E might result from histology transformation of LU2C despite the lack of LU2C biopsy and its histology. By contrast, histological interpretation was indeterminate for LU2D, and LU2E was defined as a primary or progression lesion of LU2C by histological, clinicopathologic, or imaging-based approaches. Conclusion: This novel multi-omics analysis method improves the accuracy of lineage tracing by tracking the spatiotemporal heterogeneity of serial samples. Further validation is required for its clinical application in accurate diagnosis, disease management, and improving prognosis.

Case report: Targeted sequencing facilitates the diagnosis and management of rare multifocal pure ground-glass opacities with intrapulmonary metastasis.

Introduction: Treatments for multiple ground-glass opacities (GGOs) for which the detection rate is increasing are still controversial. Next-generation sequencing (NGS) may provide additional key evidence for differential diagnosis or optimal therapeutic schedules. Case presentation: We first reported a rare case in which more than 100 bilateral pulmonary GGOs (91.7% of the GGOs were pure GGOs) were diagnosed as both multiple primary lung cancer and intrapulmonary metastasis. We performed NGS with an 808-gene panel to assess both somatic and germline alterations in tissues and plasma. The patient (male) underwent three successive surgeries and received osimertinib adjuvant therapy due to signs of metastasis and multiple EGFR-mutated tumors. The patient had multiple pure GGOs, and eight tumors of four pathological subtypes were evaluated for the clonal relationship. Metastasis, including pure GGOs and atypical adenomatous hyperplasia, was found between two pairs of tumors. Circulating tumor DNA (ctDNA) monitoring of disease status may impact clinical decision-making. Conclusions: Surgery combined with targeted therapies remains a reasonable alternative strategy for treating patients with multifocal GGOs, and NGS is valuable for facilitating diagnostic workup and adjuvant therapy with targeted drugs through tissue and disease monitoring via ctDNA.

Differential Diagnostic Value of Histology in MPLC and IPM: A Systematic Review and Meta-Analysis.

Background: The paramount issue regarding multiple lung cancer (MLC) is whether it represents multiple primary lung cancer (MPLC) or intrapulmonary metastasis (IPM), as this directly affects both accurate staging and subsequent clinical management. As a classic method, histology has been widely utilized in clinical practice. However, studies examining the clinical value of histology in MLC have yielded inconsistent results; thus, this remains to be evaluated. Here, we performed a meta-analysis to assess the differential diagnostic value of histology in MPLC and IPM and to provide evidence-based medicine for clinical work. Methods: PubMed, Embase, and Web of Science databases were searched to collect relevant literature according to PRISMA, and inclusion and exclusion criteria were set up to screen and assess the literature. The data required for reconstructing a 2 × 2 contingency table were extracted directly or calculated indirectly from the included studies, and statistical analysis was carried out by using Stata 15, Meta-DiSc 1.4, and Review Manager 5.4 software. Results: A total of 34 studies including 1,075 pairs of tumors were included in this meta-analysis. Among these studies, 11 were about the M-M standard and the pooled sensitivity and specificity were 0.78 (95% CI: 0.71-0.84) and 0.47 (95% CI: 0.38-0.55), respectively; 20 studies were about CHA and the pooled sensitivity and specificity were 0.76 (95% CI: 0.72-0.80) and 0.74 (95% CI: 0.68-0.79), respectively; and 3 studies were about the "CHA & Lepidic" criteria and the pooled sensitivity and specificity were 0.96 (95% CI: 0.85-0.99) and 0.47 (95% CI: 0.21-0.73), respectively. The combined pooled sensitivity, specificity, PLR, NLR, DOR, and the area under the SROC curve of the 34 studies were 0.80 (95% CI: 0.73-0.86), 0.64 (95% CI: 0.51-0.76), 2.25 (95% CI: 1.59-3.17), 0.31 (95% CI: 0.23-0.43), 7.22 (95% CI: 4.06-12.81), and 0.81 (95% CI: 0.77-0.84), respectively. Conclusion: The current evidence indicated that histology had a moderate differential diagnostic value between MPLC and IPM. Among the three subgroups, the "CHA & Lepidic" criteria showed the highest sensitivity and CHA showed the highest specificity. Further research is necessary to validate these findings and to improve clinical credibility. Systematic Review Registration: PROSPERO, identifier CRD42022298180.