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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 7  |  Page : 1641-1647

Relationship between circulating lung-specific X protein messenger ribonucleic acid expression and micrometastasis and prognosis in patients with early-stage nonsmall cell lung cancer


1 Department of Oncology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China
2 Department of Thoracic Surgery, Xuanwu Hospital, Diagnostic and Treatment Centers of Lung Cancer, Capital Medical University, Beijing, China

Date of Submission20-Jul-2020
Date of Decision25-Aug-2020
Date of Acceptance28-Oct-2020
Date of Web Publication9-Feb-2021

Correspondence Address:
Lei Cong
Department of Oncology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_1007_20

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 > Abstract 


Aims: To investigate the diagnostic value of lung-specific X protein (LUNX) messenger ribonucleic acid (mRNA) expression in peripheral blood of patients with nonsmall cell lung cancer (NSCLC) in micrometastasis.
Materials and Methods: Peripheral blood samples of 112 patients with NSCLC were collected, and the expression of LUNX, cytokeratin 19 (CK19), and carcinoembryonic antigen (CEA) mRNA was measured by reverse transcription-polymerase chain reaction (RT-PCR).
Results: The expression of LUNX, CK19, and CEA mRNA was increased in peripheral blood of patients with NSCLC compared with that of patients with benign lung disease (P < 0.05), and the sensitivity of LUNX mRNA was higher than that of CK19 and CEA mRNA (P < 0.05). LUNX-positive expression was also associated with lymph node metastasis, tumor–node–metastasis (TNM) staging, and reduced 5-year survival rate of patients in our cohort (P < 0.05). Further, the 5-year survival improved for those LUNX-positive patients who became LUNX negative following adjuvant chemotherapy compared to those who remain LUNX positive (P < 0.05). Multivariate analysis showed that lymph node metastasis, TNM stage, and LUNX mRNA expression in peripheral blood were independent prognostic factors.
Conclusion: The detection of LUNX expression in peripheral blood of patients with NSCLC by RT-PCR is a highly specific and sensitive detection method for tumor micrometastasis that may be used for molecular diagnosis of tumor micrometastasis. LUNX mRNA expression in peripheral blood is an independent factor affecting prognosis of NSCLC and thus may reliably predict NSCLC prognosis and guide appropriate adjuvant chemotherapy treatment.

Keywords: Adjuvant chemotherapy, lung-specific X protein messenger ribonucleic acid, micrometastasis, nonsmall cell lung cancer, prognosis


How to cite this article:
Cui Y, Wang W, Yao S, Qiu Z, Cong L. Relationship between circulating lung-specific X protein messenger ribonucleic acid expression and micrometastasis and prognosis in patients with early-stage nonsmall cell lung cancer. J Can Res Ther 2020;16:1641-7

How to cite this URL:
Cui Y, Wang W, Yao S, Qiu Z, Cong L. Relationship between circulating lung-specific X protein messenger ribonucleic acid expression and micrometastasis and prognosis in patients with early-stage nonsmall cell lung cancer. J Can Res Ther [serial online] 2020 [cited 2021 Feb 28];16:1641-7. Available from: https://www.cancerjournal.net/text.asp?2020/16/7/1641/308750




 > Introduction Top


Lung cancer is one of the most common cancers and a major cause of cancer-related death worldwide. The incidence and mortality of lung cancer continue to increase yearly. The 5-year survival rate of lung cancer is as low as 18%, whereas that of patients with advanced metastatic lung cancer is 5%. In contrast, the 5-year overall survival of patients with stage I nonsmall cell lung cancer (NSCLC) is 77.9%, demonstrating that an accurate micrometastasis diagnosis enables patients to receive precise treatment with improved prognosis.[1]

Metastasis of hilar and mediastinal lymph nodes in patients with NSCLC has a direct bearing on treatment strategy determination and prognostic outcome. Immunohistochemical staining is commonly used to diagnose lymph node metastasis, but the rate of missed diagnosis is high between 10% and 30.0%. In recent years, reverse transcription polymerase chain reaction (RT-PCR) has been used to diagnose lymph node micrometastasis of lung cancer by accurately detecting highly expressed genes in cancer cells,[2] making it a powerful approach in the identification of lymph node metastasis.

To explore the diagnostic import of micrometastasis and effect of chemotherapy on expression of molecular markers, we measured expression of lung-specific X protein (LUNX), cytokeratin 19 (CK19), and carcinoembryonic antigen (CEA) messenger ribonucleic acid (mRNA) in peripheral blood of 112 patients with NSCLC and evaluated clinical significance with follow-up data.


 > Materials and Methods Top


Patients

Our cohort consisted of 112 consecutive patients diagnosed with NSCLC at our hospital from January 2013 to November 2014. All recruited patients had a definite histopathological diagnosis with no distant metastasis as determined by computer tomography (CT), emission CT, and B-scan ultrasound, followed by thoracic surgery. There were 76 males and 36 females with an average age of 61.4 ± 8.2 years. There were 50 cases of adenocarcinoma, 52 cases of squamous cell carcinoma, and 10 cases of other types. According to the American Joint Commission on Cancer/International Union Against Cancer 1997 staging system, 17 patients were at Stage I, 50 patients were at Stage II, and 45 patients were at Stage III. Our control cohort consisted of 15 patients with benign pulmonary disease and 20 healthy blood donors. Patients with Stage IB and positive for LUNX mRNA expression (LUNX-positive) as well as Stage II and III patients received four cycles of adjuvant chemotherapy.

Sample collection and ribonucleic acid extraction

Peripheral blood samples (5 mL) were aseptically taken from patients with lung cancer before surgery and from patients with benign pulmonary disease before treatment. For those patients identified as LUNX positive (i.e., detection of mRNA expression in peripheral blood), an additional 5 mL of peripheral blood was drawn the next day following the end of adjuvant chemotherapy. Nucleated cells were collected by lymphocyte separation solution and centrifugation, and the supernatant was transferred to a sterile tube. Total RNA was precipitated by adding 1000 μL Trizol reagent and appropriate volumes of chloroform and isopropanol. RNA was dissolved in 75% ethanol and sterile water, followed by RT-PCR. If RT-PCR was not immediately performed, the extracted RNA samples were cryopreserved until later use.[3] Total RNA quantity was determined by ultraviolet spectrophotometry, and RNA integrity was determined by 1%–2% agarose gel electrophoresis.

Design and synthesis of primers and polymerase chain reaction conditions

Primers for LUNX and β-actin were designed using an oligo primer design software and the National Center for Biotechnology Information gene library. Based on CK19 and CEA gene sequences, primers were designed and designated (A) for external primer and (B) internal primer [Table 1].[4] Primers were synthesized by BioSune Biotechnology (Shanghai, China).
Table 1: Primer sequences of lung-specific X protein, carcinoembryonic antigen, cytokeratin 19, and β-actin genes used in this study

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Reverse transcription was performed according to the conditions provided by the kit manufacturer using approximately 2 μL (~1 ng/μL) total RNA in a final reaction volume of 20 μL. We used a one-step PCR system for RT-PCR of LUNX and CK19 mRNA. Reactions consisted of 0.1 μL Taq (5 U/μL), 2 μL 10 × PCR buffer (containing Mg2+), 1.6 μL deoxyribonucleoside triphosphates (dNTPs) (2.5 mmol each), upstream and downstream primers (5 μm each), and 2 μL cDNA, to a final reaction volume of 20 μL. Thermocycling reaction conditions were as follows: denaturation at 95°C for 10 min, followed by 94°C for 30 s, 65°C for 30 s, and 72°C for 45 s with the annealing temperature of each cycle decreased by 1°C until it reached 55°C, at which point 20 cycles were performed, and a final 10 min extension at 72°C. CEA mRNA amplification was a two-step process. For the first step, reactions consisted of 2 μL CEA coat primer, 0.1 μL Taq (5 U/μL), 2 μL 10 × PCR buffer (containing Mg2+), 1.6 μL dNTPs (2.5 mmol each), and 2 μL cDNA, to a final reaction volume of 20 μL. Reaction conditions were as follows: denaturation at 95°C for 10 min, followed by 20 cycles of 94°C for 30 s, 65°C for 1 min, and 72°C for 1 min, and a final extension at 72°C for 5 min. For the second round of amplification, reactions were identical, but used 2 μL of the first-stage PCR reaction product as reaction template and the primers were the CEA internal primers. Thermocycling conditions were as follows: denaturation at 95°C for 10 min, followed by 20 cycles of 94°C for 30 s, 62°C for 1 min, and 72°C for 1 min, and a final extension at 72°C for 5 min.

Identification of polymerase chain reaction products

PCR products were separated by 1% agarose gel electrophoresis at 80 V for 45 min. Ethidium bromide staining was used to screen the UVP gel imaging system (Alpha Technology) and measure fluorescence intensity. Specific bands were identified for LUNX (189 base pairs [bp]), CK19 (461 bp), CEA (132 bp), and β-actin (534 bp).

Statistical analysis

Two sample rates were compared by Chi-square test and logistic regression. Survival rate was calculated by life table method and analyzed by Kaplan–Meier method and log rank test. Cox model was used for multivariate analysis. All data were statistically analyzed using IBM SPSS for Windows version 23.0 (IBM Corp., Armonk, NY, USA). P < 0.05 was considered statistically significant.


 > Results Top


Expression of lung-specific X protein, cytokeratin 19, and carcinoembryonic antigen messenger ribonucleic acid in peripheral blood

Total RNA quality based on A260/A280 ratio was between 1.74 and 1.96. After agarose gel electrophoresis, 18 S and 28 S bands were clearly visible, indicating that the extracted RNA was intact. Our results based on the expression measures of peripheral blood of 112 patients with NSCLC, 15 patients with benign pulmonary disease, and 20 healthy controls are shown in [Figure 1]. We found that the frequency of LUNX-positive patients with lung cancer was significantly higher than that of patients with benign lung disease and healthy controls [Table 2].
Figure 1: 1.1-1.3 1-5 was the reverse transcriptase polymerase chain reaction amplification of lung-specific X protein messenger ribonucleic acid, cytokeratin 19 messenger ribonucleic acid, and carcinoembryonic antigen messenger ribonucleic acid in peripheral blood of five patients. 1.1 One hundred and eighty-nine blood pressure bands were found in the 1st, 3rd, 4th, and 5th lanes, indicating lung-specific X protein messenger ribonucleic acid positive.1.2 Four hundred and sixty-one blood pressure bands were found in Lane 1, 3, and 5, indicating cytokeratin 19 messenger ribonucleic acid positive.1.3 One hundred and thirty-two blood pressure bands in Lane 1 and Lane 4, indicating carcinoembryonic antigen messenger ribonucleic acid positive. 1.4 1-5 was the expression of lung-specific X protein messenger ribonucleic acid in peripheral blood of five controls were negative. The 534 blood pressure band was the amplified product of internal reference β-actin

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Table 2: Frequency of positive lung-specific X protein, cytokeratin 19, and carcinoembryonic antigen messenger ribonucleic acid in peripheral blood

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We determined that the sensitivity of LUNX, CK19, and CEA mRNA in peripheral blood of patients with lung cancer was 62.50%, 37.50%, and 35.71%, respectively. The sensitivity of LUNX was significantly higher than that of CK19 and CEA in our patient cohort [P = 0.0002 and P = 0.0001, respectively, [Table 2]]. We found no differences in sensitivity between CK19 and CEA expression from peripheral blood in patients with NSCLC (P = 0.7815). Further, the specificity of LUNX, CK19, and CEA mRNA was 100%, 88.57%, and 85.71% in our cohort, respectively.

Relationship between lung-specific X protein messenger ribonucleic acid expression in peripheral blood and clinicopathological characteristics of patients with nonsmall cell lung cancer

Comparisons were performed to determine whether there was a relationship between LUNX mRNA expression level and age, sex, smoking history, tumor location, tumor pathological type, T stage, tumor differentiation type, lymph node metastasis, or tumor–node–metastasis (TNM) stage of our 112 patients with lung cancer. We found that the expression of LUNX mRNA in peripheral blood was not correlated with age, gender, smoking history, tumor location, tumor pathological type, T stage, or tumor differentiation type, but was associated with lymph node metastasis and TNM stage [P = 0.0002 and P < 0.0001, respectively, [Table 3]].
Table 3: Lung-specific X protein messenger ribonucleic acid expression and clinicopathological factors in patients with nonsmall cell lung cancer

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Effect of chemotherapy on lung-specific X protein messenger ribonucleic acid expression and patient survival

After chemotherapy, we found that 21 out of 70 patients who were LUNX positive became LUNX negative, indicating a conversion rate of 30%. After follow-up, we found that the 5-year survival rate was significantly higher (28.57%) for patients who became LUNX negative compared to those patients (12.24%) who remained LUNX positive following treatment [P = 0.002, [Figure 2]]. These findings indicate that the status changing from LUNX mRNA positive to negative after adjuvant chemotherapy is significantly related to patient prognosis.
Figure 2: Relationship between lung-specific X protein messenger ribonucleic acid changes and survival rate in 70 lung cancer patients with positive peripheral blood lung-specific X protein messenger ribonucleic acid after chemotherapy

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Relationship between lung-specific X protein messenger ribonucleic acid expression and patient prognosis

Our 112 patients with NSCLC were followed up for 60 months. Using Kaplan–Meier univariate survival analysis, we determined that the 5-year survival rate of patients who were LUNX positive or LUNX negative in peripheral blood was 17.14% and 35.71%, respectively. In addition, the 5-year recurrence-free survival rate of patients who were LUNX mRNA positive or negative was 14.29% and 28.57%, respectively. Both findings were statistically significant [P = 0.027 and P = 0.0368, respectively, [Figure 3]].
Figure 3: Relationship between lung-specific X protein messenger ribonucleic acid expression and survival rate in 112 cases of lung cancer

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Next, we performed Cox multifactor model analysis to identify factors that may affect patient prognosis, including gender, age, tissue type, degree of differentiation, lymph node metastasis, TNM stage, and LUNX mRNA expression status in peripheral blood. We found that lymph node metastasis, TNM stage, and peripheral blood LUNX mRNA expression status were independent factors for prognosis [Table 4].
Table 4: Cox multivariate analysis of 112 patients with nonsmall cell lung cancer

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 > Discussion Top


Lung cancer has the highest mortality of malignant tumors worldwide, including China.[5] The clinical introduction of molecular-targeted drugs and immune checkpoint inhibitors this year has led to improved survival of patients with lung cancer. However, surgery remains the only means of radical treatment, but <20%–25% of patients with NSCLC receive radical surgery. Further, the recurrence rate of these patients is high, and the 5-year survival rate is only 40%–50%.[6] In most patients with lung cancer, local recurrence and distant metastasis are the main causes of recurrence. Therefore, it is critically important to identify patients with micrometastasis after surgery and provide corresponding treatment. Imaging methods used to diagnosis lung cancer metastasis are limited, capable of identifying large metastases, but cannot be used in the diagnosis of micrometastasis in circulating blood, bone marrow, lymph nodes, and extrapulmonary organs. In recent years, with the development of molecular biology technology, RT-PCR has been used to detect the expression of a specific mRNA in solid tumor micrometastatic cells, with a sensitivity between 1 × 10−5 and 1 × 10−7.[7] However, for lung cancer, there is a lack of specific molecular markers for detection. Therefore, screening and identifying tumor-specific markers are key toward improving the specificity and reliability of micrometastasis detection in lung cancer.

LUNX is a human lung tissue-specific gene originally isolated by Iwao et al. by mRNA differential display technology.[8] It is located on chromosome 20q11.21 with a total length of 1015 bp, including an open reading frame that encodes 257 amino acids. LUNX mRNA is not expressed in normal lung tissues and lymph nodes. Therefore, the detection of LUNX mRNA in peripheral blood of patients with lung cancer may be an indicator of cancer cell metastasis.

Since the 1990s, CK19 and CEA mRNA expression has been widely used in the detection of micrometastasis in lymph node, bone marrow, and peripheral blood of epithelial malignancies such as lung cancer, exhibiting a certain sensitivity and specificity.[9],[10] However, some studies have found that as PCR amplification sensitivity increases, there is a corresponding increase in the false positive rate of CEA mRNA in lymph node and blood.[11]

In the current study, we selected LUNX, CK19, and CEA as target genes to detect micrometastasis in patients with NSCLC and used RT-PCR and measures of mRNA expression to detect micrometastasis in peripheral blood of patients with lung cancer. The advantage of molecular diagnosis of lung cancer micrometastasis lies in its sensitivity. Existing research shows that the sensitivity of a molecular diagnostic approach of lung cancer micrometastasis can reach 10−6–10−7, which is markedly better than conventional pathology-based diagnostic methods. In recent years, it was reported that the combined detection of LUNX mRNA, squamous cell carcinoma antigen, and BJ-TSA-9 can provide a detection rate up to 81.4% for tumor micrometastasis in peripheral blood of lung cancer and a rate of 71.4% in cases of adenocarcinoma.[2],[12] In fact, the positive rate of LUNX in peripheral blood alone can reach 60.0%–62.0%,[2],[12] indicating its high sensitivity. In our study, we found that the sensitivity of LUNX, CK19, and CEA mRNA in peripheral blood of 112 patients with lung cancer was 62.50%, 37.50%, and 35.71%, respectively. Further, the sensitivity of LUNX mRNA detection was significantly higher than that of CK19 and CEA mRNA in peripheral blood of patients with NSCLC. However, we also detected a number of false positives from CK19 and CEA mRNA detection, which was consistent with that found from a previous study.[11] These results suggest that the expression of LUNX mRNA in peripheral blood of patients with NSCLC has obvious advantages in sensitivity and specificity compared with detection of CK19 and CEA mRNA, thus forming a basis for the detection of micrometastasis in peripheral blood of patients with lung cancer.

At present, correlations between LUNX mRNA expression in peripheral blood and clinicopathological characteristics of patients with lung cancer are unclear. In the current study, we found that the expression of LUNX mRNA in peripheral blood of patients with NSCLC was not significantly associated with gender, age, smoking, histological type, degree of cell differentiation, size of primary tumor, and tumor location (P > 0.05), but there was evidence of an association with lymph node metastasis and staging (P < 0.05). LUNX expression increases with higher stages of P-TNM, demonstrating that molecular P-TNM staging was better than pathological P-TNM staging. Molecular diagnosis of lung cancer metastasis is of great clinical significance for surgeons to select surgical indications and the most effective postoperative treatment regimen.

Previous breast cancer studies found that a positive detection rate of micrometastasis was closely related to patient prognosis, and that it can be used for early diagnosis of tumor recurrence and distant metastasis.[13] Further, Passlick.[14] reported that micrometastases were detected in lymph node of 11 out of 72 patients with lung cancer, and that the recurrence rate of patients with micrometastasis was over four times higher than that of patients without micrometastasis. Micrometastasis of N1/2 is a risk factor for 3-year survival rate of patients with NSCLC, which affects the decision of mediastinal lymph node dissection.[15] Kaplan–Meier survival curve analysis showed that the 5-year overall survival rate and tumor-free survival rate of patients with lung cancer and LUNX positive were significantly lower than those patients who were LUNX negative (P < 0.05). Based on risk regression analysis, we found that TNM stage, lymph node metastasis, and LUNX mRNA expression in peripheral blood were significant prognostic factors in our cohort. Based on our findings and those of previous studies, we conclude that molecular diagnosis of micrometastasis in lung cancer is a relatively independent prognostic indicator.

As an important part of a comprehensive treatment regimen for lung cancer, chemotherapy has wide clinical use. We found a negative conversion rate in LUNX mRNA-positive patients after adjuvant chemotherapy, and follow-up showed that the survival rate of those patients negative for LUNX mRNA in peripheral blood was significantly higher than that of patients who were LUNX positive. From the perspective of micrometastasis, adjuvant chemotherapy can improve the survival rate of patients with lung cancer. For the high-risk patient population with early recurrence and metastasis risk, active adjuvant chemotherapy can reduce the recurrence rate and metastasis rate of patients before clinical metastasis.

There are limitations of this study. It is a single-center clinical study with a small sample size. Therefore, a future multicenter clinical study with a larger sample size is warranted to validate our findings.


 > Conclusion Top


Detection of LUNX mRNA by RT-PCR can detect micrometastasis in peripheral blood of patients with lung cancer, and its specificity and sensitivity are better than that found of CEA and CK19 mRNA detection. Furthermore, the detection of LUNX mRNA in peripheral blood may provide a theoretical and experimental basis for postoperative adjuvant chemotherapy of lung cancer, thereby guiding clinical treatment and correctly determine patient prognosis.

Financial support and sponsorship

This work was supported by the National Natural Science Foundation of China (81902350); the Shandong Provincial Natural Science Foundation, China (ZR2018BH027); the Key R and D Program of Shandong Province (2017GSF218110, 2016GSF201145); the Medical and Health Science Technology Development Program of Shandong Province (2014WS0352); and the Jinan Clinical Innovation Project (201602163).

Conflict of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
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  [Table 1], [Table 2], [Table 3], [Table 4]



 

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