DNA methylation biomarkers-based method for the differential diagnosis of multiple lung cancers.

ABSTRACTAccurately distinguishing between multiple primary lung cancers (MPLC) and intrapulmonary metastasis (IPM) is crucial for tailoring treatment strategies and improving patient outcomes. While molecular methods offer significant advantages over traditional clinical‐pathological evaluations, they lack standardized diagnostic protocols and validated prognostic value. This study systematically compared the diagnostic and prognostic performance of molecular methods versus clinical‐pathological evaluations in diagnosing multiple lung cancers (MLCs), specifically focusing on the impact of next‐generation sequencing (NGS) parameters on diagnostic accuracy. A review of 41 studies encompassing 1266 patients revealed that two molecular methods, Mole1 (manually counting shared mutations) and Mole2 (bioinformatics‐assisted clonal probability calculation), both demonstrated superior diagnostic accuracy and prognostic discrimination capabilities. Molecular assessment, particularly Mole1, effectively stratified prognosis for MPLC and IPM, leading to significantly improved disease‐free survival (DFS: HR = 0.24, 95% CI: 0.15–0.39) and overall survival (OS: HR = 0.33, 95% CI: 0.18–0.58). Further analysis suggests that a minimal panel of 30–50 genes may be sufficient to effectively differentiate prognoses. Compared to Mole1, Mole2 demonstrated greater specificity and stability across various panels, achieving AUC values from 0.962 to 0.979. Clinical‐pathological evaluations proved unreliable, not only failing to distinguish prognosis effectively but also exhibiting a potential misdiagnosis rate of 35.5% and 33.6% compared to the reference diagnosis. To improve both cost‐effectiveness and diagnostic accuracy, bioinformatics‐assisted molecular diagnostics should be integrated into multidisciplinary assessments, especially for high‐risk cases where diagnostic errors are common.

e21038 Background: Patients with multiple primary lung cancers (MPLC) and intrapulmonary metastasis (IPM) are considered as different clinical stages and require different treatment strategies. At present, the molecular identification criteria employed for distinguishing MPLC and IPM still need to be improved. Therefore, there is an unmet medical need in regards to a diagnostic standard that could effectively distinguish MPLC from IPM. Methods: Between December 2014 and April 2020, a total of 35 patients, with more than two lung lesions, were selected and divided into a training cohort (N = 22) and a validation cohort (N = 13). Imaging, anatomical evidence, pathological results, and histological examinations of the primary tumor, as well as histological subtypes, were used to differentiate MPLC from IPM. Genomic profiles obtained using a targeted sequencing with 450 cancer-related genes panel were analyzed. Modified diagnostic criteria for MPLC and IPM were established based on Martini and Melamed criteria, as well as molecular profiles. Results: We developed interpretation criteria using a flowchart that distinguishes MPLC from IPM, as follows: 1) MPLC can be determined using the following aspects: no common mutations are present; one lesion has no mutations (tumor = 2); two common high frequency driver gene mutations are present, or one driver gene mutation and one rare mutation comprising the p53 mutation are present; and only one common mutation is present. 2) IPM can be determined using the following aspects: one driver gene mutation and one rare mutation comprising the p53 mutation (concordance rare ≥ 50%) are present; two driver gene mutations (concordance rare ≥ 60%) are present; all mutations are common; and more than three common mutations are present. 3) Undetermined results are based on the following aspects: no mutations in one tumor are present (tumor > 2); no mutations are identified in all tumors; or two rare mutations are present. Undetermined patients were identified by combining histological identifications. Subsequently, to verify the criteria, 13 patients were recruited as a validation cohort. Eight MPLC and 5 IPM patients were identified, and the results were 100% consistent with independent imaging and pathological diagnoses. Conclusions: In this work, we proposed more comprehensive and detailed molecular identification criteria for MPLC and IPM, and it suggested that genetic alterations may be an effective method for diagnosing MPLC and IPM. We also determined that NGS may be a useful tool for assisting with differential diagnoses. Key words: Multiple lung cancer, Multiple primary lung cancer, Intrapulmonary metastasis, Next-generation sequencing, molecular classification.

Multiple lung cancers may be intrapulmonary metastases or multiple primaries. Management and prognosis of both entities are different and make the pathologist’s role challenging. In fact, distinguishing both entities may be difficult especially when the tumors present the same microscopic subtype. The microscopic diagnoses of these tumors have been improved based on the 2015 WHO classification. The aim of the authors was to assess the diagnostic value of morphologic features in comparison to the gold standard test represented by molecular testing in the distinction between intrapulmonary metastases and multiple lung primaries. To retrieve all eligible articles, PubMed and Embase databases and Cochrane Library were comprehensively searched from 1999 to 2020 with limitation to French andEnglish language. The Meta-Disc software 5.1.32 was used to conduct this meta-analysis. The pSEN, pSPE, NLR, PLR, and DOR with the 95% confidence intervals were calculated. The area under the SROC was calculated based on the SEN and SPE of each study. Q test and I2 statistics were carried out to explore the heterogeneity among studies. P value <.1 for q test or I2 value >50% represented substantial between-study heterogeneity. Meta-regressions were performed to explore the sources of heterogeneity if necessary. Twelve eligible articles with 309 patients were included. pSEN was estimated to 65% with I-square estimated to 53%. pSPE reached 49% with I-square estimated to 56%. PLR was estimated to 1.23 with I-square estimated to 33%. NLR was estimated to 0.65 with I-square estimated to 23.1%. dOR was estimated to 2.13 [1.07–4.25] with I-square estimated to 26.5%. AUC was estimated to 0.63. The meta-regression analysis showed non-significant effect of the WHO classification, next-generation sequencing, or nucleotide-specific sequencing with p reaching respectively 0.38, 0.06, and 0.36. These results highlight that morphologic features may be useful in the diagnosis of multiple lung cancers especially when dealing with surgical specimen. The mild heterogeneity observed in this meta-analysis may be due to other covariates that were not described in the different articles including the sample nature.

P1.03-080 The SUVmax Ratio of Two Tumors on PET/CT May Differentiate Separate Primary Lung Cancers and Intrapulmonary Metastases

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. 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. 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. Determining genetic alterations may be an effective method for differentiating MPLC and IPM, and NGS can be used as a valuable assisting tool.

[Background] In the treatment of synchronous or metachronous multiple lung cancer (MLC), determination whether multiple primary lung cancer (MP) or intrapulmonary metastases (IM) is very important to make an appropriate management. Clinical or pathological diagnoses have been adopted to distinguish whether MLC were MP or IM, however, the accuracy of these approaches seemed to be insufficient. On the other hand, recent evolution of high-throughput sequencing made it possible to perform comprehensive gene mutation analysis in cancer cells. The aims of this study were to investigate mutational profiles of synchronous or metachronous MLC, and to compare multiplex gene mutation analysis of MP or IM among paired tumors with clinical or pathological evaluations. [Methods] We performed targeted sequencing for 20 lung cancer related oncogenes using next-generation sequencing technology in 82 tumors from 37 patients (18 patients with synchronous MLC and 19 patients with metachronous MLC) who underwent surgical resection in our department from July 2002 to April 2013. Then, classification of MP or IM was made by clinical, pathological, and gene mutational evaluation. [Results] Among paired tumors, matching of mutation was observed in 20 (54%) cases (nine cases with completely matched and 11 cases with partially matched), which were diagnosed as IM by mutational evaluation. In pathologically suggested IM cases (n=7), six (86%) patients were interpreted as IM by mutational evaluation, and most of them (n=5) had multiple matched mutations, which suggested the clonality between paired tumors strongly. In pathologically suggested MP cases (n=17), the mutational diagnosis was discordant in eight (47%) patients. Among these cases, four paired tumors had multiple matched mutation, suggesting the pathological diagnosis in these cases might be incorrect. In addition, careful interpretation was required when the paired tumors harboring frequent mutation including TP53 or EGFR because such mutation may match accidentally. [Conclusion] Our findings suggest that multiplex mutational analysis of synchronous or metachronous MLC could complement the pathological diagnosis in differentiation whether MP or IM. In the cases with pathologically equivocal or those with discordant between pathological diagnosis and mutational evaluation, the frequency and the number of matched mutation may be helpful for the differentiation. Citation Format: Yuta Takahashi, Kazuhiko Shien, Shuta Tomida, Eisuke Kurihara, Yusuke Ogoshi, Kei Namba, Takahiro Yoshioka, Hidejiro Trigoe, Hiroki Sato, Hiromasa Yamamoto, Junichi Soh, Shinichi Toyooka. Comparative mutational evaluation for multiple lung cancer by multiplex oncogene mutation analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3680.

The present study aimed to elucidate the genetic features of multiple lung cancer (MLC) and identify effective molecular markers for diagnosis using next generation sequencing (NGS). The present data may also inform patient treatment and prognosis. A total of 35 lesions were obtained from 17 patients with MLC. Based on lesion histology and NGS, 13 cases of multiple primary lung cancer (MPLC) were identified and 4 cases were classified as intrapulmonary metastasis (IPM). All 4 patients with IPM exhibited an epidermal growth factor receptor (EGFR) mutation and synchronous mutation of at least one tumor suppressor gene. The frequency and percentage of EGFR mutations, accompanied with tumor suppressor genes, were significantly higher in patients with IPM compared with MPLC. Furthermore, a high EGFR-heterogeneity score and male sex were risk factors of IPM occurrence. There were significant differences in mean EGFR mutation abundance alone, mutations of tumor suppressor genes and mutations of EGFR combined with tumor suppressor genes between patients with adenocarcinoma (ADC) and adenocarcinoma in situ (AIS). In conclusion, histological characteristics combined with genetic alterations may be an effective method for the diagnosis of MPLC and IPM, and NGS may serve as a useful diagnostic tool. MLC exhibited unique molecular characteristics, including higher rates of EGFR mutations, EGFR driver mutations accompanied with tumor suppressor gene mutations and the absence of anaplastic lymphoma kinase mutations, which may help distinguish between patients with MPLC or IPM. The present study hypothesized that the mean frequency of EGFR mutations, mutations of tumor suppressor genes and mutations of both EGFR and tumor suppressor genes may serve an important role in the development of AIS to ADC. The results of the present study highlight the potential underlying mechanisms of lung ADC development, which may assist with future elucidation of effective treatments to prevent the progression of lung cancer.

BackgroundThere is no consensus on whether patients with synchronous multiple lung cancers (SMLC) who present with lymph node metastasis (LNM) but whose epidermal growth factor receptor (EGFR) mutations are different are considered to have intrapulmonary metastases or multiple primary lung cancers. Few studies on these patients have been reported.MethodsThe electronic medical records of patients with surgically resected multiple lung cancers between February 2016 and July 2019 were retrospectively reviewed, focusing on the clinical characteristics and prognosis of patients with LNM and different EGFR mutations.ResultsA total of 125 patients were diagnosed with SMLC, and only 8 patients had LNM and different EGFR mutations. Their mean age was 61.43 ± 8.08 years (range 47–69 years). EGFR detection suggested that 4 patients had completely different mutation types, and 4 patients had mutations in only 1 tumor. Only 1 of the 17 total lesions was squamous cell carcinoma, the rest were adenocarcinoma. All patients underwent adjuvant therapy after surgery. Except for 1 patient who underwent chemotherapy, the rest received tyrosine kinase inhibitor-targeted therapy. As at 15 October 2020, the average follow-up time was 28.68 ± 10.74 months (range 10.5–40.5 months), and all patients were alive except 1 who died from extensive pleural metastasis.ConclusionThe current study highlights the clinical importance of EGFR detection in SMLC, especially in patients with LNM. SMLC with LNM and different EGFR mutations should be considered multiple primary lung cancers rather than intrapulmonary metastases, and comprehensive treatment based on surgery may be preferable in these patients due to a good prognosis.

BackgroundThe 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.MethodsPubMed, 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.ResultsA 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.ConclusionThe 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 RegistrationPROSPERO, identifier CRD42022298180.

A Novel Algorithm to Differentiate Between Multiple Primary Lung Cancers and Intrapulmonary Metastasis in Multiple Lung Cancers With Multiple Pulmonary Sites of Involvement.

BackgroundPatients with multiple lung cancer are becoming more common. The optimal criterion to distinguish multiple primary lung cancer (MPLC) from intrapulmonary metastases (IPM) is still unclear. In this study, we try to distinguish between MPLC and IPM and investigate their prognosis and risk factors.MethodsThis study was a retrospective analysis of patients with at least two malignant resected nodules in three medical centers from January 2019 to December 2019. Fifty-three patients with 130 lesions were enrolled and tested with 10-gene and 116-gene panels using next-generation sequencing (NGS). Disease-free survival (DFS) was defined as the time from surgery to either the date of the first recurrence (local or distant) or the last follow-up. The follow-up period was up to October 31, 2024. Tumor mutations were identified for each gene using the 116-gene and 10-gene panels, and clonal relatedness was identified by mutational profiling. Univariate and multivariate Cox regression analyses were conducted to identify independent risk factors for DFS.ResultsFifty-three cases with 130 lesions met the inclusion criteria. A total of 16 recurrences were identified during follow-up. The 3- and 5-year DFS was 77.4% and 69.8%, respectively. According to the 116-gene panel, 35 (66.1%) cases favored MPLC, and 18 cases (33.9%) favored IPM on the basis of shared mutations. There was no difference in the 3-year DFS (82.9% vs. 66.7%, log-rank P=0.22), while there was an obvious difference in the 5-year DFS (80% vs. 60%, log-rank P=0.02). Univariate analysis showed alkaline phosphatase and forced expiratory volume in the first second percentage (FEV1%) as risk factors for metastasis (P=0.03 and P=0.003). Multivariate analysis showed that FEV1% was an independent factor (P=0.001). Cox regression analysis showed that the positive covariates were as follows: early stage [hazard ratio (HR) =4.192; 95% confidence interval (CI): 1.378 to 12.749; P=0.01] and MPLC (HR =0.187; 95% CI: 0.057 to 0.613; P=0.006).ConclusionsNGS-based 116-gene panel classification can improve the accuracy of diagnosing MPLC and IPM. The diagnosis of IPM was associated with poor prognosis in Asian population.

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. 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. 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. 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.

While there have been many attempts to differentiate multiple lung cancers (MLCs) using the clinicopathological presentation and molecular profile, there are still some controversies regarding the prognostic factors for MLCs with clinical-N0 status. Between 1996 and 2012, 131 patients were diagnosed as MLCs pathologically. The main lesion of MLCs was defined as follows: (1) among synchronous lesions, the radiologically denser or larger tumor on thin-section computed tomography (CT) or (2) the second tumor among metachronous lesions. (18)F-fluorodeoxyglucose uptake on positron emission tomography (PET) scan was examined to evaluate maximum standardized uptake value (SUVmax) of the main tumor. Among 131 patients with clinical-N0 status, 66 were men and 65 were women and they had an average age of 67 years. One-hundred nine patients were diagnosed as MPLCs, and 22 were intrapulmonary metastases (PMs). Based on multivariate analyses, SUVmax was a significant prognostic factor in both synchronous and metachronous clinical-N0 MLCs (p = 0.0060, 0.0451, respectively). Among the overall patients, while pathological diagnosis, maximum tumor dimension, consolidation status, and SUVmax were all significant prognostic factors by a univariate analysis, SUVmax (p = 0.0016) was superior to pathological diagnosis based on the Martini and Melamed classification (p = 0.2258) based on a multivariate analysis. The 5-year survival rate of MPLCs (78.7%) was significantly greater than that of PMs (30.5%) (p = 0.0036). Furthermore, the 5-year survival rate in patients with low SUVmax (91.1%) was far better than that in patients with high SUVmax (17.9%) (p = 0.0001). SUVmax on PET was a significant clinical factor that more precisely reflected the prognosis of MLCs with clinical-N0 status, and could be superior to a pathological diagnosis based on the Martini and Melamed classification.

Multidisciplinary expert consensus on diagnosis and treatment of multiple lung cancers.

BackgroundThe 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 DescriptionWe 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.ConclusionsDifferent 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.