Use of MicroRNA Expression Prole For The Discrimination of Multiple Primary Lung Cancers From Intrapulmonary Metastasis

Background: The increasing number of patients with multifocal lung cancers (MFLCs) complicates tumor, node, and metastasis (TNM) staging and treatment assignments. Clinical guidelines, comprehensive histologic subtyping (CHS) and molecular analyses are available to help distinguish multiple primary lung cancers (MPLCs) from intrapulmonary metastasis (IPM). However, these guidelines and criteria are dicult to apply and often lead to conicting results. Methods: We enrolled 44 patients with MFLCs, 32 of whom had tumors with the same histology. We assessed their statuses using existing guidelines, which led to integrated results. Twelve patients with lymph-node metastasis (LNM) were selected for comparison. Results: The sum of the differences of ve microRNAs (miRNAs) in matched tumors was evaluated by quantitative real-time polymerase chain reaction (RT-qPCR). All 12 patients with LNM showed a sum of differences of < 9 in terms of miRNA expression between primary tumors and LNM. According to the integrated analysis, seven patients were diagnosed with denite MPLCs, which were newly classied as MPLCs according to the miRNA criteria. Four patients with “no denite conclusion” according to the integrated analysis were newly classied by the miRNA criteria. The results between the integrated analysis and the miRNA criteria were contradictory in 7 patients and consistent in 14 patients. Conclusions: A gold standard has yet to be developed for MPLCs. However, currently, an analysis of the miRNA expression prole is a useful way to distinguish MPLCs from intrapulmonary metastasis.

Pathologic review was conducted using serial sections from the same FFPE blocks that were processed in a similar manner according to standard histologic procedures. All specimens were stained with hematoxylin and eosin (H&E) for histological diagnosis. Routine immunohistochemical (IHC) experiments were also performed with antibodies against thyroid transcription factor 1 (TTF-1), cytokeratin 7 (CK7), novel aspartic proteinase A (Napsin A), cytokeratin 5/6 (CK5/6), p63 and p40 for histological diagnosis.
We used the CHS criteria and molecular analysis [EGFR mutation testing/anaplastic lymphoma kinase (ALK) rearrangement testing/c-ros oncogene 1 receptor tyrosine kinase (ROS-1) rearrangement testing] to de ne lineage relationships among MFLCs of the same histological type. Tissue specimens from these patients were formally reviewed again in a blind manner by two pathologists for tumor histopathologic subtyping according to the 2015 World Health Organization Classi cation of Lung Tumors [42]. The CHS criteria of lung cancer were used to evaluate the relative area of each type of distinct morphology, including adenocarcinoma (ADC; lepidic, acinar, papillary and micropapillary, solid components) and squamous cell carcinoma (SCC; keratinizing, non-keratinizing, basaloid).
Patients with the same subtype according to CHS underwent further molecular testing (EGFR/ALK/ROS-1 status). EGFR status was tested by DNA sequencing, while ALK or EML4-ALK and ROS1 rearrangements were screened by IHC and were con rmed using the break-apart uorescence in situ hybridization assay (FISH).

RNA extraction
Total RNA fractions including miRNAs were extracted from the FFPE tissues. A representative section from each sample was stained with H&E and reviewed to identify regions that contained more than 70% malignant epithelial cells for macrodissection. Five 8-μm-thick sections from FFPE tumor tissues were collected by dissecting away the tumor stroma using a sterile scalpel for each case. Total RNA (including miRNAs) from FFPE was depara nized with 100% xylene, washed with 100% ethanol, then extracted using a miRNAprep pure FFPE Kit (Tiangen Biotech Co. Ltd., Beijing, China); nally, RNA was treated with DNase I according to the manufacturer's instructions.
Total RNA quantity and quality were determined using a Nanodrop 2000c spectrophotometer (Thermo Scienti c, Waltham, MA, USA). The optical density (OD) 260/280 and OD 260/230 ratios were used for quality control.
Reverse transcription and quantitative real-time polymerase chain reaction (RT-qPCR) Five miRNAs (hsa-miR-21-5p, hsa-miR-30a-5p, hsa-miR-126-3p, hsa-miR-129-5p, and hsa-miR-182-5p) were selected and used to evaluate the differential diagnosis of MPLCs and IPM. The levels of the ve miRNAs and hsa-U6 were detected by reverse transcription and RT-qPCR. The expressions of the ve miRNAs were normalized to the levels of the internal control, U6.
The rst-strand cDNA was synthesized using an Escherichia coli Poly(A) polymerase for Poly(A) addition and oligo(dT)-universal as the primer; the reaction was performed with a miRcute miRNA rst-strand cDNA synthesis kit (Tiangen Biotech).
RT-qPCR was performed to detect the levels of miRNAs using an SYBR Premix miRcute miRNA qPCR Detection kit (Tiangen Biotech) in a CFX96™ RT-PCR system (Bio-Rad, Hercules, CA, USA) using the following thermal cycle protocol: (1) 94 °C for 120 s; (2) 94 °C for 20 s, 60 °C for 34 s, for a total of 45 cycles.
The melting curve data were collected to verify the speci city of the PCR. All samples were analyzed in triplicate. The same reaction for U6 was set up in parallel. Negative control reactions were performed without reverse transcription and without template cDNA. The cycle threshold (Ct, the PCR cycle at which the probe signal reaches the threshold) was determined for each well. All samples presented qRT-PCR repeats within 1Ct. If the AvgCt miR-U6 was not between 20 and 32 Ct values, the assay was repeated.

Patient Characteristics
We identi ed 44 patients with MFLCs for which individual FFPE tumor samples were available. The characteristics of these 44 patients and the 12 patients with LNM are shown in Table 2. These 44 patients included 32 patients with tumors of the same histological type and 12 patients with tumors of different histological types; 31 patients had synchronous multiple lung cancers (SMLCs), while and 13 had metachronous multiple lung cancers (MMLCs). The 32 patients with tumors of the same histological type included 24 patients with SMLCs (10 men and 14 women; 47 to 80 years of age) and 8 patients with MMLCs (4 men and 4 women; 52 to 71 years of age). The time interval ranged from 15 to 76 months. The 12 patients with LNM included 7 men and 5 women who ranged in age from 50 to 80 years.
All 44 patients were evaluated according to the CHS criteria in order to determine the differential diagnosis of MPLCs and IPM. Patients with the same CHS result were further analyzed according to molecular criteria. The results of the patients with tumors of the same histological type are shown in Table 3. The patient numbers are ranked by miRNA criteria results in the next section. Among the 32 patients with tumors of the same histological type, 25 patients were diagnosed with adenocarcinoma(ADC), 4 were diagnosed with squamous cell carcinoma(SCC), 2 were diagnosed with adenosquamous carcinoma (ADC-SCC) and 1 was diagnosed with small cell lung carcinoma (SCLC). In regards to ADC, 44 specimens exhibited mixed morphologies and 6 specimens exhibited a unique morphology; the subtypes of these matched tumors were the same in 21 patients but were different in 4 patients. For SCC, 8 specimens were of a unique subtype; the tumor subtypes were the same in the matched tumors from all 4 patients. For ADC-SCC, 4 specimens exhibited characteristics unique to the ADC and SCC subtypes; the characteristics of the tumor subtypes were the same in the matched tumors from both patients. In regards to SCLC, 2 specimens were of the same histologic subtype but were not characteristic of combined small-cell lung cancer (CSCLC); the characteristics of the tumor subtypes were the same in the matched tumors from this patient.
The proportions of the matched tumors with speci c tumor subtypes might differ for all patients, especially in those with ADC. Different proportions of tumor subtypes were not considered in the criteria for MPLCs because a unique clone might exhibit different proportions of histologies in matched tumors from one patient.
The results of the molecular analysis (EGFR/ALK/ROS-1 status) are shown in Table 3. Of the 24 patients tested for EGFR status, 21 patients had MFLCs with the same EGFR mutations in matched tumors (11 patients showed the same EGFR mutation; 10 patients were negative for mutations). Three patients had matched tumors with different EGFR mutations. Patient 24 exhibited metachronous bilateral nodules with an L858R mutation in the rst tumor and a T790M mutation in the second tumor. Patient 27 exhibited two synchronous nodules in the right lung, in which the upper lobe harbored an L858R mutation and the lower lobe was negative. Patient 31 had metachronous nodules in the right lung, in which the upper lobe contained an exon 19 deletion and the middle lobe was negative. In all 32 patients tested for ALK/ROS-1 by IHC, positive specimens were con rmed by FISH. Of the 32 patients, patient 21 had two synchronous nodules in the right lung (upper and lower lobes), which were ALK-positive; patient 11 had two synchronous nodules in the left lung (upper and lower lobes), which were ROS1-positive.

MiRNA analysis
First, the difference in the expression of the ve miRNAs between the primary lung tumor (T1) and the metastatic tumor in the 12 patients with lymph node involvement (T2) was compared using the ΔΔCt method (|ΔΔCt| =| Avg (Ct miR-X − Ct miR-U6 )T1 − Avg (Ct miR-X − Ct miR-U6 )T2|) and is shown in Table 4. The sum of the difference in the Ct values of the 5 miRNAs was the sum of |ΔΔCt|. The maximum sum of |ΔΔCt| was 8.15 (patient 11) and the minimum was 1.51 (patient 1). The limit of the sum of |ΔΔCt| of tumors diagnosed as identical was < 9 ( Figure 1). Therefore, if the sum of |ΔΔCt| in matched tumors is < 9, the tumors are considered identical (i.e., the primary tumor and its IPM nodule). When the sum of |ΔΔCt| in matched tumors exceeds 9, the 2 tumors are considered to be different from each other (i.e., they are MPLCs).
All 32 patients with tumors of the same histological type were evaluated by the miRNA criteria to ensure that a second primary or IPM nodule was present. The ΔΔCt method was used, and the bigger tumor (sMFLCs) or the rst tumor (mMFLCs) was designated Tumor 1 (T1), while subsequent tumors were designated Tumor 2 (T2). Patients were ranked by the sum|ΔΔCt| of the ve miRNAs ( Figure 2).The maximum sum of the differences in the Ct values of the 5 miRNAs was 24.98 (patient 32) and the minimum was 1.63 (patient 1). According to the miRNA criteria, 20 of 32 (62.5%) patients were diagnosed with newly classi ed IPM, and 12 (37.5%) patients were diagnosed with newly classi ed MPLCs.
Characterization as MPLCs or IPMs MM criteria and ACCP criteria (3rd edition) were used to evaluate the differential diagnosis of MPLCs and IPM. Assuming that components of different histologic subtypes arise in independent clones and that the components of the same subtype would be found in metastases, we found that all 44 patients with multiple resected lung lesions were evaluated according to the CHS criteria. We assume that different mutations arise in independent clones and that the same mutation would be found in metastases. All 32 patients with tumors of the same histological type were evaluated according to molecular criteria. These patients were also diagnosed according to the miRNA criteria. Twelve patients with tumors of different histological types received a de nite diagnosis of MPLCs.
According to different criteria, patients with MFLCs were determined to have MPLC or IPM (Table 5) Tumors in patients 07 and 08, for whom the time intervals were within 2 years and in whom no lymph node or systemic metastasis was detected, were classi ed as MPLCs by the MM criteria and as IPM by the ACCP criteria. Tumors in patients 06, 24, 26, and 29, for whom the time intervals were between 2 and 4 years and in whom no lymph node or systemic metastasis was detected, were classi ed as MPLCs by MM criteria and as an unde ned cluster by the ACCP criteria. Out of 32 patients, 17 patients were diagnosed with IPM and 15 patients were diagnosed with MPLCs (46.9%) by the MM criteria. Eighteen patients were diagnosed with IPM (satellite), 10 patients were diagnosed with MPLCs (31.3%) and 4 patients had tumors that were "not classi ed" by the ACCP criteria. According to the CHS criteria, 5 patients had matched tumors with different histologic subtypes, and 27 patients had matched tumors with the same histologic subtype, and patients whose tumors were of different histologic subtypes (15.6%) were diagnosed with MPLCs. According to the molecular criteria, different molecular testing results were obtained in 3 patients with matched tumors, the same result was obtained in 13 patients, negative results were obtained in 8 patients, and a molecular analysis was not performed in 8 patients. Patients with different molecular results (9.4%) were diagnosed with MPLCs.
An integrated analysis of 32 patients was performed according to the MM, ACCP, CHS and molecular criteria ( Table 5). The CHS and molecular criteria led to a de nite diagnosis of MPLCs if differences were apparent between the matched tumors (7 patients: patients 24, 25, 27, 28, 29, 31, 32). If the CHS and molecular criteria achieved the same result in the matched tumors, a conclusion was made simply by the combination of the MM and ACCP criteria. A con ict between these two criteria was resolved by an integrated analysis result of no de nite conclusion. Of the 32 patients, 7 were diagnosed with de nite MPLCs, 16 were diagnosed with IPM, and 5 were diagnosed with MPLCs; no de nite conclusion was reached in 4 patients.
With the miRNA criteria, 20 of 32 (62.5%) patients were diagnosed with newly classi ed IPM, and 12 (37.5%) patients were diagnosed with newly classi ed MPLCs (Table 5). Tumors in seven patients who were diagnosed with de nite MPLCs by integrated analysis were newly classi ed as MPLCs. Tumors in four patients who were diagnosed with "no de nite conclusion" by integrated analysis were newly classi ed with MPLCs (patient 26) or IPM (patients 06, 07, 08). Tumors in four patients who were diagnosed with MPLCs by integrated analysis were newly classi ed as IPM (patients 01, 03, 11,13). Tumors in three patients who were diagnosed with IPM by integrated analysis were newly classi ed as MPLCs (patients 21, 22, 30). The same conclusion was reached in other patients using integrated analysis and miRNA criteria: 13 with IPM, 1 with MPLCs.

Discussion
The classi cation of additional nodules according to the seventh edition[22] of the lung cancer staging system is focused on these tumor nodules in a way that may be potentially misleading. Cohorts in the IASLC database and in other databases that contain patients with additional nodules excluded those with synchronous primary cancers as well as those with systemic spread [1].This means that a large number of patients with MPLCs were not diagnosed appropriately.
On the basis of analyses of overall survival among pathologically staged cases, additional nodules in the same lobe are categorized as T3, nodules in the ipsilateral lobe are categorized as T4 and those in the Patients with a primary lung cancer and additional nodules or lesions are categorized on the basis of their clinical presentations, but it is often a challenge to manage this diagnosis clinically; clinicians may be easily confused by these lesions because the terms and de nitions are ambiguous [1,25]. The distinction of MPLCs from IPM may be easy when the matched tumors in one patient are of different histologic types, but it becomes quite di cult when the tumors are of the same histologic type. This is because the determination of whether the neoplasms are clonally related (which re ects metastatic disease) or clonally unrelated (which indicates multiple primary cancers) is impossible [1].
The MM and ACCP criteria are used to classify the matched tumors as MPLCs or IPM in a clinical setting [1,8,22]. As for patients with histologically matched tumors, diagnostic uncertainty occurs quite often due to the inherent limitations of the criteria: neither the MM nor the AACP criterion incorporates information that de nitively indicates MPLCs. These clinical criteria are faced with four problems, as follows: (1)  ADCs display mixed histologic characteristics in more than 80% of cases [26], and thus, in some instances, components of each morphologic area may help to determine whether multiple tumors are clonally related [7]. It has been suggested that ADCs can be distinguished on the basis of the components of different histologic subtypes [27][28][29]. However, CHS is associated with four limitations: (1) CHS cannot be used on specimens obtained by ne-needle biopsy. (2)Only tumors of different subtypes will be recognized as MPLCs, and out of these, the percentage is low. (3) The lineage relationships determined may be subtype-speci c, but that is not a de nite result. (4) Even when histology or histology subtypes of matched tumors are different, the possibility of metastasis cannot be excluded given the heterogeneity.
Assuming that different mutations found in separate lung tumors re ect independent clones[30], we used the molecular criteria. It has been found that EGFR/KRAS mutation testing and CHS can help to differentiate multiple primary lung ADCs from metastatic lesions [7], but that study included only seven patients. The molecular criteria have three limitations: (1) In our study, tumors were analyzed for only EGFR, Page 10/32 ALK and ROS-1 alterations. Since EGFR/ALK/ROS-1 alterations are found in only approximately 30%-50%, 3%-5%/ and 1% of resected lung tumors, respectively, there is a signi cant probability that a given tumor harbors no mutations, which means that molecular testing will be uninformative for the determination of lineage relationships. Therefore, the molecular criteria are highly speci c despite the variable sensitivity. (2) Even though all mutations and gene fusions were tested (i.e., EGFR, KRAS, HER2, MEK1, BRAF, PIK3CA, ALK fusion, ROS fusion, PDGFR amp), there may be other types of mutations, which will lead to classi cation of tumors as MPLCs, but they may not be tested. This study has some limitations. Our patient samples were limited since all patients were recruited from a single institution. Most patients with two foci were excluded from this study because they were considered metastases, and therefore, they did not undergo surgery for the second focus; this is also the primary problem in other studies and databases. The miRNA expression pro les in the plasma will be more meaningful as non-invasive test biomarkers for MPLCs, which will be demonstrated in future research if the role of miRNA expression pro les in tissues in a larger cohort of patients is con rmed.
(In addition, the cut-off value of the sum ΔΔCt was based on a small number of patients, and only ve cancer-related miRNAs were included. Patients with SMPLCs had a better overall survival than those with intrapulmonary metastases, and with the new miRNA evaluation system, the outcomes are similar. Considering the different TNM stages of patients with SMPLCs and intrapulmonary metastases in our study, and the number of cases evaluated in this study was relatively small. Therefore, further studies with larger cohorts are necessary to validate our results.)

Conclusions
The results of this study suggest that an analysis of miRNA expression pro les is helpful in the discrimination of multiple primary lung cancers from intrapulmonary metastases. There is still no gold standard for this diagnosis, which is based on the judgment of a multidisciplinary team. Declarations All participants will be informed that the result of this study will be disseminated through presentations at national and international scienti c meetings, publications in peer-reviewed journals and public events involving the local administrations of the cities where the study participants are resident. And written consent will be obtained. All data related to speci c patients will be de-identi ed.
*Sum of the differences is the value used to add up differences of miRNAs expression among 5 miRNAs.
Abbreviations: ADC, adenocarcinoma; SCC, squamous cell carcinoma; ASC, adenosquamous carcinoma; SCLC, small cell lung cancer; miR, microRNA  Figure 1 The sum of the differences in miRNA expression in patients with primary tumors and metastatic lymph nodes.