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ORIGINAL ARTICLE
Year : 2015  |  Volume : 11  |  Issue : 8  |  Page : 271-274

Evaluation of melanoma antigen gene A3 expression in drug resistance of epidermal growth factor receptor-tyrosine kinase inhibitors in advanced nonsmall cell lung cancer treatment


Department of Respiration, The Second People's Hospital of Wuhu, Wuhu, Anhui, China

Date of Web Publication26-Nov-2015

Correspondence Address:
Ju Jin
Department of Respiration, The Second People's Hospital of Wuhu, Wuhu, Anhui
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.170549

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 > Abstract 

Objective: To investigate the correlation between melanoma antigen gene A3 (MAGE-A3) expression and progression-free survival (PFS) of nonsmall cell lung cancer (NSCLC) patients with epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKIs) therapy, aiming to provide a basis for research and treatment of EGFR-TKIs resistance.
Research and Methods: Retrospective analysis is conducted of PFS of 359 NSCLC patients who have been tested positive for EGFR, and experienced drug resistance during oral treatment of icotinib. MAGE-A3 expression is tested using immunology and histology chemistry methods, and T790M and c-MeT expression are tested using mutation-enriched polymerase chain reaction.
Results: (1) MAGE-A3 expression in targeted treatment of NSCLC patients shows a positive rate of 33.98%. The comparative difference between MAGE-A3 expression and T790M, c-MeT and other resistance genes was not statistically significant (P > 0.05). (2) MAGE-A3 expression was higher in patients with NSCLC targeted therapy of primary drug resistance of positive rate than acquired resistance; meanwhile the expression level differences in three modes of acquired resistance are statistically significant (P < 0.05). (3) PFS of MAGE-A3 positive expression in the targeted treatment of acquired drug resistance in patients with NSCLC is shorter than the PFS of MAGE-A3 negative expression (P = 0.01); the comparative PFS differences in the three kinds of acquired drug resistance pattern have statistical significance (P = 0.02). (4) PFS and levels of MAGE-A3 expression in NSCLC patients with the three modes of acquired resistance are negatively correlated (P < 0.01), and MAGE-A3 expression has no correlation with age, gender, pathological type or PS score (P > 0.05).
Conclusion: MAGE-A3 expression in EGFR-TKIs target therapy in NSCLC patient suggests that there might be EGFR-TKIs drug resistance, and the higher the level of expression, the shorter the time of acquired drug resistance.

Keywords: Drug resistance, lung cancer, melanoma antigen gene-A3, target therapy


How to cite this article:
Jin J, Liu BZ, Wu ZM. Evaluation of melanoma antigen gene A3 expression in drug resistance of epidermal growth factor receptor-tyrosine kinase inhibitors in advanced nonsmall cell lung cancer treatment. J Can Res Ther 2015;11, Suppl S4:271-4

How to cite this URL:
Jin J, Liu BZ, Wu ZM. Evaluation of melanoma antigen gene A3 expression in drug resistance of epidermal growth factor receptor-tyrosine kinase inhibitors in advanced nonsmall cell lung cancer treatment. J Can Res Ther [serial online] 2015 [cited 2020 Jan 17];11:271-4. Available from: http://www.cancerjournal.net/text.asp?2015/11/8/271/170549


 > Introduction Top


Currently, target therapy using epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) has become a major research area in lung cancer globally. These patients can obtain around 70% objective response rate and about 10 months' of progression-free survival (PFS) time. [1],[2] However, the example of drug resistance in EGFR target therapy has to some extent indicated the limitations of single gene target treatment strategies. The signal transduction of lung cancer is a multi-target and multi-link procedure. Multi-target lung cancer therapy research will be the new mainstream in drug resistance treatment. Melanoma antigen gene A3 (MAGE-A3) is related to chemotherapy drug resistance and poor prognosis in a variety of malignant tumors. This paper investigates the correlation between MAGE-A3 expression and drug resistance in targeted therapies of nonsmall cell lung cancer (NSCLC) patients and provides new research directions for targeted drug resistance in lung cancer treatment.


 > Research and methods Top


Research

A retrospective analysis is conducted of 359 NSCLC patients receiving icotinib drug treatment produced by Betta Pharmaceuticals in Respiration Medicine and Oncology Departments and experienced drug resistance from August 2012 to December 2014. These patients are confirmed as stage IIIB/IV either histologically or cytologically, and meet the following categories: (1) First-line patients who cannot tolerate chemotherapy or EGFR mutations, or second-line patients with ineffective chemotherapy results; (2) physically Eastern Cooperative Oncology Group score (Performance Status [PS]) between 0 and 4; (3) with a predicted survival time period of at least 12 weeks; (4) with target lesion in line with Response Evaluation Criteria in Solid Tumors (RECIST) standards; (5) with normal liver and kidney functions; and (6) consent forms signed by the patients and their family. There are 45 male patients and 314 female patients, with a median age of 56.9 (ages 40-75), of which 345 cases of lung adenocarcinoma and 14 cases of squamous cell carcinoma; all are tested EGFR positive using ARMS external examination by KingMed Diagnostics. One hundred and thirty-eight cases showed exon 19 positive, 165 showed exon 20 positive (T790M negative), and 56 showed exon 21 positive.

Methods

The method adopted is using TRIZOL lysate purchased from the US Company Invitrogen to extract RNA. RNA SERPIN, TaqDNA polymerase, and M-MLV reverse transcriptase are purchased from US company Promega; BI PRISM 7300 quantitative polymerase chain reaction (PCR) system is produced by the US company Applied Biosystems. All the reagents used for analysis are made in China. T790M and c-MeT primer are synthesized by Shanghai Invitrogen Bioengineering Company. Once all the diseases progressed the patients will go through specimen check by bronchoscope or CT-guided percutaneous transthoracic needle, using Thermo Company's kit to conduct Immunohistochemistry (Elivison 2-step method) and examine MAGE-A3 expression, and using mutation-enriched PCR method to test T790M and c-MeT expression.

Semi-quantitative predict of immunohistochemical results

The results are graded by staining intensity and the percentage of positive cells out of all cancer cells. Cytoplasms dyed light yellow and brown in the tissue slice are positive cells. The degrees of staining are graded based on the color characteristics of most cells (the lightness of the staining is in contrast with the background color): 0 point for no color, 1 for light yellow, 2 for claybank and 3 for dark brown. The positive cell percentage, for example, cells within five fields (every 40 × 10 high power field counts as 100 cells): 0 for 0-5%, 1 for 6-25%, 2 for 26-50%, 3 for 51-75% and 4 for over 75%. Five 40 × 10 fields are selected randomly from each slice, then multiply the staining degree, and positive cell percentage points: 0 is negative (−), 1-4 is weakly positive (+), 5-8 is medium positive, (++), and 9-12 is strong positive (+++).

Treatment methods

The patients take 125 mg of icotinib orally 3 times a day. The evaluation standards of RECIST solid tumors are adopted, and therapeutic evaluation is graded as complete remission, partial remission, stable disease and progressive disease (PD), with a follow-up visit every 4 weeks until December 1, 2014. Medium follow-up time is 18 months (1-26 months). PFS time is defined as the period from the beginning of the treatment till the patient shows PD or until the follow-up finish.

Therapeutic evaluation

EGFR-TKIs resistance included primary and acquired [3] resistance. Primary drug resistance refers to never obtaining therapeutic effects after the mutant application of EGFR-TKIs; acquired resistance is having clinical improvement after EGFR-TKIs therapy, but experienced tumor progression later. The three modes of acquired resistance are: [4] The first is progressing abruptly with no <3 months of disease control, a quickly growing tumor burden compared to earlier evaluation, and the symptom grade is 2; the second is progressing slowly, with no <6 months' disease control, a slightly growing tumor burden compared to earlier evaluation, and the symptom grade is ≤1; the third mode is progressing regionally, with no <3 months' of disease control and isolated extra-cranial or encephalic progression, and the symptom grade is ≤1.

Statistical analysis

Use the software SPSS 16.0 (Statistical Product and Service Solutions), adopt χ2 testing in the comparison of the drug resistance group expression, use the Kaplan-Merier method to analyze survivorship curves and log-rank test, and conduct related spearman analysis. The indication of P < 0.05 shows statistical significance.


 > Results Top


In all 359 NSCLC patients, the MAGE-A3 expression positive rate is 33.98% (122/359), T790M expression positive rate is 45.96% (165/359), the c-MeT expression positive rate is 18.11% (65/359), and other genes in Group II shows an expression positive rate of 35.93% (129/359). There are no statistical significance in the comparison between MAGE-A3 expression and the three drug-resistant genes - T790M (χ2 = 3.12, P = 0.08), c-MeT (χ2 = 1.47, P = 0.23), Group II (χ2 = 3.52, P = 0.15).

There are 141 cases of primary resistance, with 129 cases of glandular cancer and 12 cases of squamous carcinoma. There are 218 cases of acquired resistance, with 216 cases of glandular cancer and 2 of squamous carcinoma. The comparison of MAGE-A3 expression positive rates in primary resistance 44.68% (63/141) and acquired resistance 27.06% (59/218) in NSCLC patients has statistical significance (χ2 = 11.84, P = 0.01) [Table 1].
Table 1: A comparative analysis of MAGE - A3 expression in primary and acquired drug resistance

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The median PFS of the 218 NSCLC patients with acquired resistance: 59 cases of MAGE-A3 positive expression is 9.1 (8.4-9.8) months, and 159 negative expression is 10.5 (9.8-11.1) months. The survival comparative difference are statically significant (P = 0.01); the median PFS of the three modes of drug resistance in the 59 NSCLC patients with MAGE-A3 positive expression are: Explosive - 8.4 (6.7-9.0) months, slow - 10.8 (9.3-11.3) months, regional - 9.0 (7.7-10.1) months. The comparative differences have statistical significance [Chart 1 [Additional file 1]] and [Chart 2 [Additional file 2]].

Among 59 NSCLC patients with acquired resistance, the level difference of MAGE-A3 expression in three types of acquired resistance has statistical significance (χ2 = 24.92, P < 0.01), with explosive type the highest level, slow type the lowest. The coefficient of association C = 0.55 is moderate correlation [Table 2] and [Figure 1]a-c.
Figure 1: (a) Glandular cancer (+), (b) glandular cancer (++), (c) glandular cancer (+++)

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Table 2: Comparative analysis of MAGE - A3 expression level in three types of acquired drug resistance

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In the 59 cases of NSCLC patients with acquired drug resistance, MAGE-A3 expression level is negatively related to median PFS (r1= −0.76, P < 0.01), and has no correlation with age (r = −0.12, P = 0.55), gender (r = 0.19, P = 0.28), pathological pattern (r = −0.24, P = 0.18) or PS score.


 > Discussion Top


As people develop clearer understanding about biological behaviors of tumors, targeted treatment of lung cancer becomes a new research focus. Although the mechanism that underlines acquired TKI drug resistance is still not fully illustrated, different signal channels have been discovered, including T790M (exon 20), L747S (exon 19), D761Y (exon 19), T854A (exon 21 activation link), c-MeT, PIK3CA, etc., among which T790M and c-MeT mutation occupy about 70% of all drug resistant mutation. Clinically, there are still 25% of patients with an unknown molecular target of EGFR-TKI acquired resistance, therefore how to treat these patients is still an unidentified issue.

The MAGE group is widely expressed in many malignant tumor tissues, mostly melanoma, and rarely express in healthy tissues except for testicles and placenta. Research indicated that at least more than 17 subgroup members including MAGE-A, B, C, D, E, etc., are located in a person's chromosome long arm ends Xq28. MAGE-A3 in a vital member of the MAGE-A family constituted by two small and one large exons. Albumen is inside the cytoplasm. Its expression is related with tumor progression and transfer and could be used as data for independent prognosis testing. A large amount of clinical and lab researches showed that some MAGE-antigen presentation may carry drug resistance effects in tumor chemotherapy. Suzuki et al. [5] discovered in stomach cancer with advanced recurrence, the MAGE-A1 expression could predict docetaxel and paclitaxel chemotherapy drug resistance. Similar researches showed that MAGE-A expression indicates disease progression or chemotherapy resistance in medulloblastoma and increases the sensitivity of chemotherapy by eliminating miR-34a. [6] Hartmann et al. [7] used cetuximab and panitumumab target-treat oral squamous cell lines for tests and discovered that MAGE-A sub-group expression is correlated with targeted treatment failures, and can be used as new prognostic indicators. The study shows that MAGE-A3 bears no differences with the expression of other resistant gene in the EGFR-TKIs treatment of NSCLC patients and has a higher level of expression in primary resistance. In acquired resistance, the higher the expression level, the shorter the median PFS, indicating a negative correlation. MAGE-A3 could be another signal channel in the targeted drug resistance treatment mechanism of NSCLC patients. Therefore, the testing of MAGE-A3 in NSCLC patients could predict EGFR-TKIs' treatment of resistance, provide the basis for new goals of target drug resistance researches and help with the decision-making in chemotherapy.

In recent years, lung cancer immunotherapy has become the new target therapy. MAGE-A3 genes are representatives of tumor-specific antigens, when combined with human leucocyte antigen HLA1 and HLA2 molecule, will form HLA antigen - compounds recognized by toxic T cells (cytotoxic T lymphocytes [CTL]), induce proliferation of CTL and kill tumor cells with specificity. Clinical studies have been using the genes as a vaccine for immunotherapy in NSCLC patients and continuously making new progress. [8],[9]


 > Conclusion Top


EGFR-TKIs targeted NSCLC treatment has more advantages over traditional chemotherapy. However, primary and acquired drug resistance has become the bottleneck of target therapy and increased difficulties for clinical treatment of lung cancer. Nevertheless, the continuous exploring of EGFR-TKIs drug resistance mechanism could lead to new molecular markers that could predict therapeutic effects, guides the treating process, and further improves the effects of target therapy. MAGE-A3 has a relatively high expression rate, and is related to chemotherapy resistance, therefore, could be a new direction for multi-target lung cancer treatment. In the meantime, the strong immunogenicity will signature a new target for lung cancer immunotherapy.

Acknowledgments

This project is supported by Wuhu, Anhui Province (Urban Technology Project No. 2013hm27).

Financial support and sponsorship

Urban Technology Project, Wuhu, Anhui Province (Project No. 2013hm27).

Conflicts of interest

There are no conflicts of interest.

 
 > References Top

1.
Kim ES, Hirsh V, Mok T, Socinski MA, Gervais R, Wu YL, et al. Gefitinib versus docetaxel in previously treated non-small-cell lung cancer (INTEREST): A randomised phase III trial. Lancet 2008;372:1809-18.  Back to cited text no. 1
    
2.
Thatcher N, Chang A, Parikh P, Rodrigues Pereira J, Ciuleanu T, von Pawel J, et al. Gefitinib plus best supportive care in previously treated patients with refractory advanced NSCLC: Results from a randomized, placebo-controlled, multicentre study (Iressa Survival Evaluation in Lung Cancer). Lancet 2005;366:1527-37.  Back to cited text no. 2
    
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Yang JJ, Chen HJ, Yan HH, Zhang XC, Zhou Q, Su J, et al. Clinical modes of EGFR tyrosine kinase inhibitor failure and subsequent management in advanced non-small cell lung cancer. Lung Cancer 2013;79:33-9.  Back to cited text no. 3
    
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Powrózek T, Krawczyk P, Ramlau R, Sura S, Wojas-Krawczyk K, Kucharczyk T, et al. EGFR gene mutations in patients with adenosquamous lung carcinoma. Asia-Pacific Journal of Clinical Oncology 2014;10:340-45.  Back to cited text no. 4
    
5.
Suzuki T, Yoshida K, Wada Y, Hamai Y, Sentani K, Oue N, et al. Melanoma-associated antigen-A1 expression predicts resistance to docetaxel and paclitaxel in advanced and recurrent gastric cancer. Oncol Rep 2007;18:329-36.  Back to cited text no. 5
    
6.
Weeraratne SD, Amani V, Neiss A, Teider N, Scott DK, Pomeroy SL, et al. miR-34a confers chemosensitivity through modulation of MAGE-A and p53 in medulloblastoma. Neuro Oncol 2011;13:165-75.  Back to cited text no. 6
    
7.
Hartmann S, Kriegebaum U, Küchler N, Lessner G, Brands RC, Linz C, et al. Efficacy of cetuximab and panitumumab in oral squamous cell carcinoma cell lines: Prognostic value of MAGE-A subgroups for treatment success. J Craniomaxillofac Surg 2013;41:623-9.  Back to cited text no. 7
    
8.
Domingues D, Turner A, Silva MD, Marques DS, Mellidez JC, Wannesson L, et al. Immunotherapy and lung cancer: Current developments and novel targeted therapies. Immunotherapy 2014;6:1221-35.  Back to cited text no. 8
    
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Adam V, Wauters I, Vansteenkiste J. Melanoma-associated antigen-A3 vaccination in the treatment of non-small-cell lung cancer. Expert Opin Biol Ther 2014;14:365-76.  Back to cited text no. 9
    


    Figures

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    Tables

  [Table 1], [Table 2]



 

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