Int J Clin Exp Pathol. 2015 Sep 1;8(9):10523-33.

Variation in the expression levels of predictive chemotherapy biomarkers in histological subtypes of lung adenocarcinoma: an immunohistochemical study of tissue samples.

 

Fujimoto Y1, Togo S1, Tulafu M1, Shimizu K1, Hayashi T2, Uekusa T3, Honma Y1, Namba Y1, Takamochi K4, Oh S4, Suzuki K4, Takahashi K1.
  • 1Department of Respiratory Medicine, Juntendo University Faculty of Medicine & Graduate School of Medicine 2-1-1 Hongo, Bunkyo-Ku, Tokyo 113-8421, Japan ; Research Institute for Diseases of Old Ages, Juntendo University Graduate School of Medicine 2-1-1 Hongo, Bunkyo-Ku, Tokyo 113-8421, Japan.
  • 2Department of Pathology, Juntendo University School of Medicine 2-1-1 Hongo, Bunkyo-Ku, Tokyo 113-8421, Japan.
  • 3Department of Pathology, Labor Health and Welfare Organization Kanto Rosai Hospital 1-1 Kizukisumiyoshi-cho, Nakahara-Ku, Kawasaki, Kanagawa Prefecture 211-8510, Japan.
  • 4Department of General Thoracic Surgery, Juntendo University School of Medicine 2-1-1 Hongo, Bunkyo-Ku, Tokyo 113-8421, Japan.

 

Abstract

BACKGROUND: Lung adenocarcinoma is often composed of a complex and heterogeneous mixture of histological subtypes. Invasive adenocarcinomas are now classified by their predominant pattern, using the comprehensive histological subtyping of the International Association for the Study of Lung Cancer (IASLC), the American Thoracic Society (ATS), and the European Respiratory Society (ERS) classifications. This study aimed to determine whether the expression levels of predictive chemotherapy biomarkers are associated with the histological subtypes proposed by the IASLC/ATS/ERS classification.

MATERIALS AND METHODS: We reviewed data on representative tissue samples from 27 patients who received surgical resection and the expression of excision repair cross complementation group 1 (ERCC1), class III β-tubulin, thymidylate synthase (TS), ribonucleotide reductase M1 (RRM1), and c-Met were examined using immunostaining on tumor tissue slides. We assessed immunohistochemical H-scores, as calculated from the intensity and distribution of intratumor expression, according to the IASLC/ATS/ERS histological subtype.

RESULTS: The expression levels of predictive chemotherapy biomarkers varied according to histological subtype. The H-scores of TS and class III β-tubulin expression levels were higher in solid-type components than they were in lepidic-type components Tumors with solid predominant histology tended to recur earlier than non-solid predominant tumors. However, none of the H-scores in histologically predominant tissues was significantly associated with staging or overall survival.

CONCLUSIONS: Immunohistochemical H-scores of the predictive chemotherapy biomarkers were strongly associated with histological subtype. The presence of a solid subtype, which was associated with poor outcomes, might be assessed by measuring these biomarkers in mixed subtype adenocarcinomas.

KEYWORDS: Class III β-tubulin; Thymidylate synthase; c-Met; excision repair cross complementation group 1; immunohistochemistry; ribonucleotide reductase M1

PMID: 26617762

 

Supplement

Immunohistochemistry is a widely applicable method of evaluating protein expression levels of biomarkers in tumor tissue and can visually distinguish cancer cells from non-cancer cells, including stromal cells. Many experimental and clinical studies have revealed that certain molecular markers, such as TS, ERCC1, RRM1, class III β-tubulin, and c-Met, have been associated with a response to chemotherapy in non-small cell lung cancer (NSCLC) patients. However, even now, a standardized immunohistochemical scoring system to quantify the expression levels of these markers has not been developed for NSCLC, and thus, inconsistent results are obtained for predictive chemotherapy biomarker levels between different institutions. In a previous meta-analysis study that investigated the association between TS expression and efficacy of pemetrexed-based chemotherapy in advanced NSCLC, TS positivity varied from 29.6% to 72.5% in NSCLC (1). Lung adenocarcinoma is often composed of a complex and heterogeneous mixture of histologic subtypes; indeed, 90% of diagnosed invasive adenocarcinomas show a histologically mixed subtype. Thus, intratumoral histological heterogeneity may explain the inconsistent results of previous immunohistochemical scoring systems.

We analyzed the heterogeneous intensity of cancer cells according to each histological subtype in patients with mixed subtype lung adenocarcinoma. Even within a single histological subtype, intensity and distribution varied. As previous studies have shown that histologic subtyping is an important and independent prognostic variable in lung adenocarcinoma (2-4), we focused on analyzing whether the expression levels of predictive chemotherapy biomarkers according to histologic subtype may be associated with chemotherapy response. In this study, we show that immunohistochemically assessed expression levels of predictive chemotherapy biomarkers differ according to the histological subtype of adenocarcinoma; to our knowledge, no other study has focused on the expression levels of predictive chemotherapy biomarkers according to the histological subtype of lung adenocarcinoma.

We reviewed representative tissue samples from 27 patients who received surgical resection and who were pathologically diagnosed with invasive adenocarcinoma based on the IASLC/ATS/ERS classification. The H-scores of TS, ERCC1, RRM1, class III β-tubulin, c-Met, and phospho-Met (Tyr1234/1235) were examined on tumor tissue slides using our developed immunostaining scoring system. Immunohistochemical H-scores calculated from the mean value of total intensity were multiplied by the distribution of intratumoral expression in each histological subtype (Figure 1) (5).

We demonstrated that the expression levels of some predictive chemotherapy biomarkers tended to be higher in solid components than they were in non-solid components, suggesting that the different expression of predictive chemotherapy biomarkers in different histological subtypes might influence responses to chemotherapy.

Herein, we immunohistochemically examined several predictive chemotherapy biomarkers in a limited number of lung adenocarcinomas and found no direct evidence that our scoring system would further increase the predictive power of chemotherapy response. However, the present immunohistochemical scoring system should be prospectively validated for its putative predictive value of biomarker expression for response to chemotherapy to better identify patients who would benefit from biomarker analysis to predict chemotherapy response, and to develop personalized treatment algorithms.

 

 

fig1Figure 1. Quantification of H-score in mixed subtype tumor tissue. The total H-score was calculated as the sum of the intensity multiplied by the distribution in each histological subtype in 10 fields chosen randomly. All the immunostained sections were reviewed separately by 2 blinded observers under a microscope at a magnification of ×400. Finally, we calculated the mean value of each H-score according to the histological subtype in one NSCLC patient.

 

We examined the expression levels of TS, ERCC1, RRM1, class III β-tubulin, c-Met, and phospho-Met according to the histological subtype in tumor sections. TS expression levels were higher in solid areas than in papillary or lepidic areas. The H-score of TS was significantly greater in solid areas than it was in non-solid areas. The H-score of class III β-tubulin was higher in solid areas compared to that in lepidic areas (Figure 2).

 

 

fig2

Figure 2. Expression levels of predictive chemotherapy biomarkers according to histological subtype. A: TS, B: ERCC1, C: RRM1, D: class III β-tubulin. Quantitative estimation of immunohistochemical staining is summarized by an H-score. Non-solid bars indicate the combination of all subtypes except for the solid subtype. *P < 0.05 and **P < 0.01, as compared to H-scores for solid components.

 

c-Met protein expression is associated with advanced tumor stages and poor clinical outcomes, and phospho-Met (Tyr1235) overexpression is correlated with papillary or micropapillary histology of lung adenocarcinomas (6). However, the results of the present study demonstrate that both c-Met and phospho-Met expression levels tended to be higher in solid subtype than in other non-solid subtype tumors; however, these differences were not statistically significant (Figure 3).

 

 

fig3

Figure 3. c-Met expression levels according to histological subtypes. A) c-Met, B) phospho-Met (Tyr1234/1235). Quantitative estimation of immunohistochemical staining is summarized by an H-score. Non-solid bars indicate the combination of all the subtypes except the solid subtype.

 

Importance of the study:

The present study represents the first investigation of immunohistochemical H-scores of predictive chemotherapy biomarkers that are differently expressed in lung adenocarcinoma tumor tissue according to the histological subtype. We demonstrated that expression levels of some predictive chemotherapy biomarkers were higher in solid components than in non-solid components. The variation in predictive chemotherapy biomarker expression among different histological subtypes might be indicative of patient responses to chemotherapy.

 

References:

1.  Wang T, Chuan Pan C, Rui Yu J, Long Y, Hong Cai X, De Yin X, Qiong Hao L, Li Luo L. Association between TYMS expression and efficacy of pemetrexed-based chemotherapy in advanced non-small cell lung cancer: A meta-analysis. PLoS One 2013; 8: e74284.

2.  Yoshizawa A, Motoi N, Riely GJ, Sima CS, Gerald WL, Kris MG, Park BJ, Rusch VW, Travis WD. Impact of proposed IASLC/ATS/ERS classification of lung adenocarcinoma: Prognostic subgroups and implications for further revision of staging based on analysis of 514 stage I cases. Mod Pathol 2011; 24: 653-664.

3.  Russell PA, Wainer Z, Wright GM, Daniels M, Conron M, Williams RA. Does lung adenocarcinoma subtype predict patient survival: A clinicopathologic study based on the new International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society international multidisciplinary lung adenocarcinoma classification. J Thorac Oncol. 2011;6:1496–1504.

4.  Warth A, Muley T, Meister M, Stenzinger A, Thomas M, Schirmacher P, Schnabel PA, Budczies J, Hoffmann H, Weichert W. The novel histologic International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification system of lung adenocarcinoma is a stage-independent predictor of survival. J. Clin. Oncol. 2012;30:1438–1446.

5.  Vilmar A, Garcia-Foncillas J, Huarriz M, Santoni-Rugiu E, Sorensen JB. RT-PCR versus immunohistochemistry for correlation and quantification of ERCC1, BRCA1, TUBB3 and RRM1 in NSCLC. Lung Cancer 2012; 75: 306-312.

6.  Koga K, Hamasaki M, Kato F, Aoki M, Hayashi H, Iwasaki A, Kataoka H, Nabeshima K. Association of c-Met phosphorylation with micropapillary pattern and small cluster invasion in pT1-size lung adenocarcinoma. Lung Cancer 2013; 82: 413-419.

 

Acknowledgements

The authors thank Taiho Pharmaceutical Co. Ltd. for the generous gift of anti-TS polyclonal antibodies. A part of this work was supported by Leading Center for the Development and Research of Cancer Medicine, Juntendo University Graduate School of Medicine.

 

Contact

Yuichi Fujimoto, M.D. Shinsaku Togo, M.D.,Ph.D.

Division of Respiratory Medicine, Juntendo University Faculty of Medicine & Graduate School of Medicine,

3-1-3 Hongo, Bunkyo-Ku, Tokyo, Japan

yfujimo@juntendo.ac.jp, shinsaku@juntendo.ac.jp

 

 

 

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