The purpose of this study was to investigate the feasibility of multi-gene detection in the differential diagnosis of multi-focal lung cancer. In the 50 cases we studied, 33 cases (66%) had consistent genetic test results with ACCP standard diagnosis, while 10 cases (20%) were inconsistent, and 7 cases (14%) results could not be validated. Within those 10 cases, 6 were diagnosed with MPLC by the ACCP standard but the same type of mutation was found in different lesions, 2 were diagnosed with IM by the ACCP standard but the mutation types were different, and 2 were diagnosed as IM by ACCP standard but mutation was detected in one lesion while no mutation was detected in another lesion.The same result was observed by Nicolas Girard ET, where inconsistent results were observed for 7 (32%) of the 22 cases [11]. Multi-gene detection of multi-focal lung cancer is more scientific. The heat map indicates that the same clone source mutation is related to IM, and different clone source mutations are related to MPLC.
In general, if the results of gene detection of two lesions in the lungs are the same, we consider that one of the lesions was metastatic. However, if the results of gene detection of two lesions in the lungs are different, we consider that both lesions are primary. In special circumstances, if the results of gene detection of two lesions in the lungs are the same, it is also possible that both lesions are primary. This is related to the type of mutation detected. If the mutant type of mutation in the population is very low, both lesions have a very low probability of mutation, and the two lesions are considered to be intra-lung metastasis. In this study, one patient had two lesion mutation types of HER-2, while HER-2 had a 1–2% mutation rate in Asian lung cancer patients; therefore, the patient was considered IM. If a certain mutation type has a higher proportion in the population, it is also possible to have multiple primary lesions. In this study, two patients had two lesions with EGFR L858R mutations at the same time; three patients had EGFR 19th exon missing at the same time; the EGFR mutation rate in Asian lung cancer patients is 50%, of which 85%-90% EGFR was 19th exon missing and L858R mutation. Therefore, even if the two lesion mutations are consistent, it is possible to be independent of each other; at this time, it is necessary to combine clinical information to make a comprehensive judgment, such as the degree of similarity of histological type, the lobe in which the lesion is located, whether there is infiltration or lymph node metastasis. In addition, studies have shown that there is a certain inconsistency between the primary and metastatic lesions of lung cancer; hence, the genetic detection results of two lesions are inconsistent and may be IM, which is related to the type of gene mutation detected: if there are two different types of mutations and the transfer probability is very small, then the two lesions are considered to be independent of each other. If one of the lesions is detected as wild type and another is detected to be a mutant, it is also possible that these two lesions are metastatic (12). The researchers believe that this is because the primary focus has a small number of mutant cells with strong metastasis ability, and because of this small amount, we cannot detect mutations in the primary focus; however, in the metastases region, the cell proportion is higher so that mutations are detected[13]. In this case, we need to combine clinical information to make a judgment such as the lung lobe of the lesions, the condition of lymph node metastasis, and the condition of tissue infiltration. In this study, two lesions in one patient were in the lower right lung, and the tissue morphology was diagnosed as intra-lung metastatic carcinoma according to the ACCP Standard, but according to the results of genetic tests, one of the two lesions had NRAS mutation and the other lesion had wild-type gene, which was in line with the above findings.
There were seven cases with different histological types of cancer stove but the same types of gene mutations. This fining indicates that the two lesions of tumor originated from the same tumor stem cell. The difference in histological morphology of the lesions may be caused by the differentiation during the development of the tumor stem cell. It also reflects the heterogeneity of the primary lesion and the metastatic lesion. According to ACCP standard, most of the multi-focus lung cancers with different histological morphology are diagnosed as MPLC. The reason is that the clinicians may ignore the existence of tumor heterogeneity between primary and metastatic tumors. This may also be one of the reasons why the clinical diagnosis results that rely on ACCP standards in this study are inconsistent with the results of genetic testing.
The identification of MPLC and IM has important guiding significance for clinical decision-making. According to the eighth edition of the international staging of lung cancer, if a patient has two or more nodules in the same lung lobe, his lung cancer stage is T3, if a patient has two or more nodules in different lung lobes on the same side of the lung, then his lung cancer stage is T4, and if a patient has two or more nodules in different side lungs, then his lung cancer stage is M1a. Therefore, MPLC is often ⅢB to IV stage in clinical staging, while the treatment of lung cancer in ⅢB to Ⅳ stage is generally dominated by conservative radiotherapy and chemotherapy. Studies have shown that lymph node metastasis is a significant independent prognostic factor for MPLC patients, with five-year survival rates of 15.6% and 52.5%[14] in lymph node metastatic and non-metastatic patients, respectively. In this study, DFS of patients with MPLC was better than those with IM, DFS of genetically mutated patients was better than that of patients with wild-type gene, and DFS of patients without lymph node metastasis was better than that of patients with lymph node metastasis. Therefore, clinicians need to refer to the type of genetic mutations of each lesion when their patients have multi-focus lung cancer), rather than simply staging through nodule position, in order to provide better and more accurate treatment. Based on this study, we summarized the process of identifying MPLC and IM. The details are provided in Fig. 4.
The heterogeneity of tumor includes the heterogeneity of primary and metastatic lesion in the same patient, the heterogeneity within the same tumor, the heterogeneity of different primary lesions in the same patient, and the heterogeneity between different lesions in different patients. The heterogeneity of primary and metastatic lesion is mainly reflected in the difference of tissue morphology and the difference of gene mutation types or loci. Both the lesions in one patient were in the upper left lung, of which one was adenocarcinoma dominated by the glandular bubble type, and the other was adenocarcinoma dominated by the wall type. Thus, although the ACCP standard diagnosed MPLC, two lesions were have L858R mutations through genetic testing; hence, they were likely to have the same cloning origin. This reflects the heterogeneity of the histological of the primary lesion and the metastatic lesion. Both of the lesions in one patient were in the upper right lung, and the histological type was micro-invasive adenocarcinoma; thus, it was diagnosed as IM by the ACCP Standard, but the gene test revealed that one lesion was HER-2 positive and the other lesion was had missing EGFR 19th exon. This shows that there is an essential difference between the two in the origin of cloning, and this patient should be diagnosed to have MPLC. This reflects the heterogeneity between the primary lesion and the metastatic lesion. After discussion and analysis, we believe that the following reasons explain the heterogeneity between primary and metastatic lesions of lung cancer: sensitivity difference of the detection method[15]; heterogeneity of the internal variation of the sample itself[16]; evolution and development of the mutation state of the primary and metastatic lesion[17]; number of tumor cells and non-tumor cells in tumor samples[18].
In this study, one patient had a lesion that exhibited both the absence of EGFR 19th exon and the presence of KRAS mutations. Studies have shown that mutations in the KRAS gene and EGFR gene in NSCLC are often mutually exclusive[19], but other studies have also found cases where the two coexist (2/85), with limited benefits from TKI and poor prognosis[20]. We believe there is some rejection of the KRAS gene mutation and the EGFR gene mutation, but this exclusion is not absolute and may be related to genetic differences between races.
Because testing can guide the diagnosis of multi-focal lung cancer, it can guide clinicians in the targeted treatment of patients. The ten genes in this study had corresponding targeted drugs, of which the most studied and widely used were EGFR-TKIs for EGFR mutations. EGFR mutations (57.4%) were found in 58 of the 101 tumors in this study, which is slightly higher than the EGFR mutation rate (40–50%) in NSCLC in East Asian people, and the possibility of EGFR mutations was not ruled out in patients with multi-focal lung cancer. In addition, the data showed that the proportion of other mutations were as follows: HER-2 mutations in seven cases (6.9%); ALK mutations in four cases (4%); four cases of KNRAS mutations (4%); one case of NRAS mutations (1%); one case of RET mutations (1%); one case of BRAF mutations (1%); one case (1%) of ROS1 mutations. For patients with positive mutations, clinicians can recommend appropriate targeted drugs, which is significant for patients. Follow-up data showed that a total of five patients had targeted therapy and their condition was effectively controlled. We also found that in patients with two lesions with different EGFR mutations, the use of Gefitinib and Icotinib treatment was significant, and studies have shown that the first generation of EGFR targeted drugs, such as Gefitinib and Icotinib, are the best choice for patients with more than two mutations.
According to statistical analysis, 10 patients with gene mutations had longer DPS than wild-type patients, and DPS in patients with no metastasis of lymph nodes was longer than those with lymph node metastasis, suggesting that gene mutations and lymph nodes were important factors for good prognosis of multi-focal lung cancer. This helps to guide clinicians to adopt more appropriate treatment options for patients, thus maximizing their benefits.
To sum up, the differential diagnosis of MPLC and IM should not be confined to the analysis of clinical data, and the results of genetic testing should also be taken into account. The differential diagnosis of multi-focus lung cancer by traditional multi-gene detection methods such as ARMS-PCR has advantages of cheaper price, simpler process, easier interpretation of results, and higher sensitivity. More importantly, if a sensitive mutation is detected, targeted therapy can be carried out if necessary, which is of great help to the patient's prognosis.