Image analysis of immunohistochemistry is superior to visual scoring as shown for patient outcome of esophageal adenocarcinoma
Quantification of protein expression based on immunohistochemistry (IHC) is an important step in clinical diagnoses and translational tissue-based research. Manual scoring systems are used in order to evaluate protein expression based on staining intensities and distribution patterns. However, visual scoring remains an inherently subjective approach. The aim of our study was to explore whether digital image analysis proves to be an alternative or even superior tool to quantify expression of membrane-bound proteins. We analyzed five membrane-binding biomarkers (HER2, EGFR, pEGFR, β-catenin, and E-cadherin) and performed IHC on tumor tissue microarrays from 153 esophageal adenocarcinomas patients from a single center study. The tissue cores were scored visually applying an established routine scoring system as well as by using digital image analysis obtaining a continuous spectrum of average staining intensity. Subsequently, we compared both assessments by survival analysis as an end point. There were no significant correlations with patient survival using visual scoring of β-catenin, E-cadherin, pEGFR, or HER2. In contrast, the results for digital image analysis approach indicated that there were significant associations with disease-free survival for β-catenin, E-cadherin, pEGFR, and HER2 (P = 0.0125, P = 0.0014, P = 0.0299, and P = 0.0096, respectively). For EGFR, there was a greater association with patient survival when digital image analysis was used compared to when visual scoring was (visual: P = 0.0045, image analysis: P < 0.0001). The results of this study indicated that digital image analysis was superior to visual scoring. Digital image analysis is more sensitive and, therefore, better able to detect biological differences within the tissues with greater accuracy. This increased sensitivity improves the quality of quantification.
KeywordsDigital image analysis Esophageal adenocarcinoma Membrane-bound biomarker Immunohistochemistry HER2 EGFR pEGFR β-catenin
The authors gratefully acknowledge the financial support of the Ministry of Education and Research of the Federal Republic of Germany (BMBF) (SYS-Stomach: 01ZX1310B) and the Deutsche Forschungsgemeinschaft (SFB 824 TP Z02 and WA 1656/3-1). The authors would like to thank Ulrike Buchholz, Claudia-Mareike Pflüger, and Andreas Voss for excellent technical assistance.
Conflict of interest
The authors declare no conflict of interest.
- Dobson L, Conway C, Hanley A, Johnson A, Costello S, O’Grady A, Connolly Y, Magee H, O’Shea D, Jeffers M, Kay E (2010) Image analysis as an adjunct to manual HER-2 immunohistochemical review: a diagnostic tool to standardize interpretation. Histopathology 57(1):27–38. doi: 10.1111/j.1365-2559.2010.03577.x PubMedCentralPubMedCrossRefGoogle Scholar
- Gudlaugsson E, Skaland I, Janssen EA, Smaaland R, Shao Z, Malpica A, Voorhorst F, Baak JP (2012) Comparison of the effect of different techniques for measurement of Ki67 proliferation on reproducibility and prognosis prediction accuracy in breast cancer. Histopathology 61(6):1134–1144. doi: 10.1111/j.1365-2559.2012.04329.x PubMedCrossRefGoogle Scholar
- Harigopal M, Barlow WE, Tedeschi G, Porter PL, Yeh IT, Haskell C, Livingston R, Hortobagyi GN, Sledge G, Shapiro C, Ingle JN, Rimm DL, Hayes DF (2010) Multiplexed assessment of the Southwest Oncology Group-directed Intergroup Breast Cancer Trial S9313 by AQUA shows that both high and low levels of HER2 are associated with poor outcome. Am J Pathol 176(4):1639–1647. doi: 10.2353/ajpath.2010.090711 PubMedCentralPubMedCrossRefGoogle Scholar
- Messersmith W, Oppenheimer D, Peralba J, Sebastiani V, Amador M, Jimeno A, Embuscado E, Hidalgo M, Iacobuzio-Donahue C (2005) Assessment of Epidermal Growth Factor Receptor (EGFR) signaling in paired colorectal cancer and normal colon tissue samples using computer-aided immunohistochemical analysis. Cancer Biol Ther 4(12):1381–1386PubMedCrossRefGoogle Scholar
- Minot DM, Voss J, Rademacher S, Lwin T, Orsulak J, Caron B, Ketterling R, Nassar A, Chen B, Clayton A (2012) Image analysis of HER2 immunohistochemical staining. Reproducibility and concordance with fluorescence in situ hybridization of a laboratory-validated scoring technique. Am J Clin Pathol 137(2):270–276. doi: 10.1309/AJCP9MKNLHQNK2ZX PubMedCrossRefGoogle Scholar
- Mohammed ZM, Going JJ, McMillan DC, Orange C, Mallon E, Doughty JC, Edwards J (2012b) Comparison of visual and automated assessment of HER2 status and their impact on outcome in primary operable invasive ductal breast cancer. Histopathology 61(4):675–684. doi: 10.1111/j.1365-2559.2012.04280.x PubMedGoogle Scholar
- Mohammed ZM, McMillan DC, Elsberger B, Going JJ, Orange C, Mallon E, Doughty JC, Edwards J (2012c) Comparison of visual and automated assessment of Ki-67 proliferative activity and their impact on outcome in primary operable invasive ductal breast cancer. Br J Cancer 106(2):383–388. doi: 10.1038/bjc.2011.569 PubMedCentralPubMedCrossRefGoogle Scholar
- Nassar A, Cohen C, Agersborg SS, Zhou W, Lynch KA, Albitar M, Barker EA, Vanderbilt BL, Thompson J, Heyman ER, Lange H, Olson A, Siddiqui MT (2011) Trainable immunohistochemical HER2/neu image analysis: a multisite performance study using 260 breast tissue specimens. Arch Pathol Lab Med 135(7):896–902. doi: 10.1043/2010-0418-OAR1.1 PubMedGoogle Scholar
- Ong CW, Kim LG, Kong HH, Low LY, Wang TT, Supriya S, Kathiresan M, Soong R, Salto-Tellez M (2010) Computer-assisted pathological immunohistochemistry scoring is more time-effective than conventional scoring, but provides no analytical advantage. Histopathology 56(4):523–529. doi: 10.1111/j.1365-2559.2010.03496.x PubMedCrossRefGoogle Scholar
- Rauser S, Weis R, Braselmann H, Feith M, Stein HJ, Langer R, Hutzler P, Hausmann M, Lassmann S, Siewert JR, Hofler H, Werner M, Walch A (2007) Significance of HER2 low-level copy gain in Barrett’s cancer: implications for fluorescence in situ hybridization testing in tissues. Clin Cancer Res : Off J Am Assoc Cancer Res 13(17):5115–5123. doi: 10.1158/1078-0432.CCR-07-0465 CrossRefGoogle Scholar
- Rizzardi AE, Johnson AT, Vogel RI, Pambuccian SE, Henriksen J, Skubitz AP, Metzger GJ, Schmechel SC (2012) Quantitative comparison of immunohistochemical staining measured by digital image analysis versus pathologist visual scoring. Diagn Pathol 7:42. doi: 10.1186/1746-1596-7-42 PubMedCentralPubMedCrossRefGoogle Scholar
- Ruschoff J, Dietel M, Baretton G, Arbogast S, Walch A, Monges G, Chenard MP, Penault-Llorca F, Nagelmeier I, Schlake W, Hofler H, Kreipe HH (2010) HER2 diagnostics in gastric cancer-guideline validation and development of standardized immunohistochemical testing. Virchows Arch 457(3):299–307. doi: 10.1007/s00428-010-0952-2 PubMedCentralPubMedCrossRefGoogle Scholar
- Skaland I, Ovestad I, Janssen EA, Klos J, Kjellevold KH, Helliesen T, Baak JP (2008a) Comparing subjective and digital image analysis HER2/neu expression scores with conventional and modified FISH scores in breast cancer. J Clin Pathol 61(1):68–71. doi: 10.1136/jcp.2007.046763 PubMedCrossRefGoogle Scholar
- Sobin L, Gospodarowicz ML, Wittekind C (2010) TNM classification of malignant tumors. Wiley, New YorkGoogle Scholar
- Turashvili G, Leung S, Turbin D, Montgomery K, Gilks B, West R, Carrier M, Huntsman D, Aparicio S (2009) Inter-observer reproducibility of HER2 immunohistochemical assessment and concordance with fluorescent in situ hybridization (FISH): pathologist assessment compared to quantitative image analysis. BMC Cancer 9:165. doi: 10.1186/1471-2407-9-165 PubMedCentralPubMedCrossRefGoogle Scholar
- Welsh AW, Moeder CB, Kumar S, Gershkovich P, Alarid ET, Harigopal M, Haffty BG, Rimm DL (2011) Standardization of estrogen receptor measurement in breast cancer suggests false-negative results are a function of threshold intensity rather than percentage of positive cells. J Clin Oncol 29(22):2978–2984. doi: 10.1200/JCO.2010.32.9706 PubMedCentralPubMedCrossRefGoogle Scholar
- Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane LM, Allison KH, Allred DC, Bartlett JM, Bilous M, Fitzgibbons P, Hanna W, Jenkins RB, Mangu PB, Paik S, Perez EA, Press MF, Spears PA, Vance GH, Viale G, Hayes DF, American Society of Clinical O, College of American P (2013) Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol 31(31):3997–4013. doi: 10.1200/JCO.2013.50.9984 PubMedCrossRefGoogle Scholar