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Prognostic assessment of hypoxia and metabolic markers in cervical cancer using automated digital image analysis of immunohistochemistry.

Kim BW, Cho H, Chung JY, Conway C, Ylaya K, Kim JH, Hewitt SM - J Transl Med (2013)

Bottom Line: Here, we have examined the primary players in the hypoxia signaling pathway, by immunohistochemistry, but confirming their interactions, as well as defining which proteins are associated with outcome.High expression of HIF-1α and c-Met showed worse 5-year overall survival rate (P = 0.047 and P = 0.005, respectively) than low expression group, but CA9 and GLUT1 did not show significant survival difference.After adjusting the prognostic covariates, c-Met was found to be an independent risk factor (HR=3.27; 95% CI, 1.05-10.23, P = 0.041) for overall survival in cervical cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tissue Array Research Program & Applied Molecular Pathology Lab, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT

Background: Hypoxia inducible factor-1 alpha (HIF-1α), induced by tumor hypoxia, regulates tumor cell metabolism and metastasis by up-regulation of c-Met, carbonic anhydrase 9 (CA9) and glucose transporter 1 (GLUT1). The prognostic significance of hypoxia and metabolic markers is not clearly defined in cervical cancer. Here, we have examined the primary players in the hypoxia signaling pathway, by immunohistochemistry, but confirming their interactions, as well as defining which proteins are associated with outcome.

Methods: The study subjects were comprised of cervical intraepithelial neoplasia (CIN, n = 209), carcinoma in situ (CIS, n = 74), cervical cancer (n = 179), and matched nonadjacent normal tissues (n = 357). Immunohistochemistry (IHC) was performed to identify HIF-1α, c-Met, CA9, and GLUT1. IHC scoring was performed using automated digital image analysis and the association of hypoxic markers with prognostic outcome was evaluated.

Results: HIF-1α, c-Met, CA9 and GLUT1 expression were higher in cervical cancer than in CIN and normal cervix (all P < 0.001). Among these markers, expression of HIF-1α and c-Met were significantly different in FIGO stage (P < 0.001 and P = 0.019, respectively) and patients with lymph node metastasis (P < 0.001 and P = 0.010, respectively). HIF-1α expression was correlated with c-Met expression in cervical cancer (P < 0.001). High expression of HIF-1α and c-Met showed worse 5-year overall survival rate (P = 0.047 and P = 0.005, respectively) than low expression group, but CA9 and GLUT1 did not show significant survival difference. After adjusting the prognostic covariates, c-Met was found to be an independent risk factor (HR=3.27; 95% CI, 1.05-10.23, P = 0.041) for overall survival in cervical cancer.

Conclusions: We demonstrate that c-Met correlates with HIF-1α and is a poor prognostic factor in survival in cervical cancer.

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Digital image analysis of cytoplasmic and membranous staining. Cytoplasmic HIF-1α staining is shown (A) and automated image analysis utilizing TissueIA recognizes cytoplasmic HIF-1α staining highlighted in green color (B). CA9 is shown in membranous staining (C) and automated image analysis determines membranous CA9 staining highlighted in green color (D). The output from the algorithm returns a number of quantitative measurements for intensity and percentage of positive staining present. Scale bar: 100 μm.
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Figure 2: Digital image analysis of cytoplasmic and membranous staining. Cytoplasmic HIF-1α staining is shown (A) and automated image analysis utilizing TissueIA recognizes cytoplasmic HIF-1α staining highlighted in green color (B). CA9 is shown in membranous staining (C) and automated image analysis determines membranous CA9 staining highlighted in green color (D). The output from the algorithm returns a number of quantitative measurements for intensity and percentage of positive staining present. Scale bar: 100 μm.

Mentions: We examined expression of HIF-1α, c-Met, CA9, and GLUT1 in cervical neoplasias and cancer specimens by IHC. Subsequently, we performed analysis of these markers using automated digital image software. Representative immunohistochemical expression of HIF-1α, c-Met, CA9 and GLUT1 are presented in Figure 1. As shown Figure 1, c-Met (Figure 1D-1F), CA9 (Figure 1G and 1H) and GLUT1 (Figure 1I) expression was observed in the tumor cell membrane, while HIF-1α expression (Figure 1A-1C) was mainly observed in the cytoplasm, with some cases also demonstrating weak nucleus staining. The examples of IHC and digital image analysis output images are presented in Figure 2. The green color represents what is classified as positively stained by the algorithms.


Prognostic assessment of hypoxia and metabolic markers in cervical cancer using automated digital image analysis of immunohistochemistry.

Kim BW, Cho H, Chung JY, Conway C, Ylaya K, Kim JH, Hewitt SM - J Transl Med (2013)

Digital image analysis of cytoplasmic and membranous staining. Cytoplasmic HIF-1α staining is shown (A) and automated image analysis utilizing TissueIA recognizes cytoplasmic HIF-1α staining highlighted in green color (B). CA9 is shown in membranous staining (C) and automated image analysis determines membranous CA9 staining highlighted in green color (D). The output from the algorithm returns a number of quantitative measurements for intensity and percentage of positive staining present. Scale bar: 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750663&req=5

Figure 2: Digital image analysis of cytoplasmic and membranous staining. Cytoplasmic HIF-1α staining is shown (A) and automated image analysis utilizing TissueIA recognizes cytoplasmic HIF-1α staining highlighted in green color (B). CA9 is shown in membranous staining (C) and automated image analysis determines membranous CA9 staining highlighted in green color (D). The output from the algorithm returns a number of quantitative measurements for intensity and percentage of positive staining present. Scale bar: 100 μm.
Mentions: We examined expression of HIF-1α, c-Met, CA9, and GLUT1 in cervical neoplasias and cancer specimens by IHC. Subsequently, we performed analysis of these markers using automated digital image software. Representative immunohistochemical expression of HIF-1α, c-Met, CA9 and GLUT1 are presented in Figure 1. As shown Figure 1, c-Met (Figure 1D-1F), CA9 (Figure 1G and 1H) and GLUT1 (Figure 1I) expression was observed in the tumor cell membrane, while HIF-1α expression (Figure 1A-1C) was mainly observed in the cytoplasm, with some cases also demonstrating weak nucleus staining. The examples of IHC and digital image analysis output images are presented in Figure 2. The green color represents what is classified as positively stained by the algorithms.

Bottom Line: Here, we have examined the primary players in the hypoxia signaling pathway, by immunohistochemistry, but confirming their interactions, as well as defining which proteins are associated with outcome.High expression of HIF-1α and c-Met showed worse 5-year overall survival rate (P = 0.047 and P = 0.005, respectively) than low expression group, but CA9 and GLUT1 did not show significant survival difference.After adjusting the prognostic covariates, c-Met was found to be an independent risk factor (HR=3.27; 95% CI, 1.05-10.23, P = 0.041) for overall survival in cervical cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tissue Array Research Program & Applied Molecular Pathology Lab, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT

Background: Hypoxia inducible factor-1 alpha (HIF-1α), induced by tumor hypoxia, regulates tumor cell metabolism and metastasis by up-regulation of c-Met, carbonic anhydrase 9 (CA9) and glucose transporter 1 (GLUT1). The prognostic significance of hypoxia and metabolic markers is not clearly defined in cervical cancer. Here, we have examined the primary players in the hypoxia signaling pathway, by immunohistochemistry, but confirming their interactions, as well as defining which proteins are associated with outcome.

Methods: The study subjects were comprised of cervical intraepithelial neoplasia (CIN, n = 209), carcinoma in situ (CIS, n = 74), cervical cancer (n = 179), and matched nonadjacent normal tissues (n = 357). Immunohistochemistry (IHC) was performed to identify HIF-1α, c-Met, CA9, and GLUT1. IHC scoring was performed using automated digital image analysis and the association of hypoxic markers with prognostic outcome was evaluated.

Results: HIF-1α, c-Met, CA9 and GLUT1 expression were higher in cervical cancer than in CIN and normal cervix (all P < 0.001). Among these markers, expression of HIF-1α and c-Met were significantly different in FIGO stage (P < 0.001 and P = 0.019, respectively) and patients with lymph node metastasis (P < 0.001 and P = 0.010, respectively). HIF-1α expression was correlated with c-Met expression in cervical cancer (P < 0.001). High expression of HIF-1α and c-Met showed worse 5-year overall survival rate (P = 0.047 and P = 0.005, respectively) than low expression group, but CA9 and GLUT1 did not show significant survival difference. After adjusting the prognostic covariates, c-Met was found to be an independent risk factor (HR=3.27; 95% CI, 1.05-10.23, P = 0.041) for overall survival in cervical cancer.

Conclusions: We demonstrate that c-Met correlates with HIF-1α and is a poor prognostic factor in survival in cervical cancer.

Show MeSH
Related in: MedlinePlus