Original contribution Up-regulation of the hypoxia-inducible factor–1 transcriptional pathway in colorectal carcinomas ☆ Daniela Furlan BSc a , Nora Sahnane BSc a , Ileana Carnevali BSc a , Roberta Cerutti BSc a , Francesco Bertoni MD b , Ivo Kwee PhD b , Silvia Uccella MD, PhD a , Valentina Bertolini MD a , Anna Maria Chiaravalli MSc a , Carlo Capella MD a a Department of Human Morphology, Anatomic Pathology Unit, University of Insubria, Varese, Italy b Laboratory of Experimental Oncology, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland Received 13 November 2007; revised 11 February 2008; accepted 22 February 2008 Keywords: Colorectal carcinomas; HIF-1, hypoxia, invasive growth, quantitative real-time PCR Summary The aim of the study was to identify the impact on prognosis of hypoxia-inducible factor–1 genetic program in colorectal carcinomas and to develop an experimental procedure that would allow a reliable quantitative gene expression analysis in formalin-fixed and paraffin-embedded tissue. The expression of hypoxia-inducible factor–1α and 13 hypoxia-inducible factor–1 target genes (AMF , CAIX, VEGF , VEGFR1, VEGFR2, HGF, MET , TGFα, EGFR, IGF2, MMP2, PLAUR, NIX) was quantified by real-time polymerase chain reaction in 18 colorectal, poorly differentiated neuroendocrine carcinomas and in 60 invasive colorectal carcinomas. Moreover, hypoxia-inducible factor–1α protein expression was evaluated by immunohistochemistry. High levels of hypoxia-inducible factor–1α were positively associated with poorly differentiated neuroendocrine carcinoma histology (P b .005), poor differentiation (P b .025), presence of necrosis, and presence of microsatellite instability (P b .05). AMF , TGFα, IGF2, NIX, VEGF , and VEGFR2 transcripts were significantly higher in the very aggressive poorly differentiated neuroendocrine carcinomas than in exocrine colorectal carcinomas and TGFα expression was significantly associated with presence of lymph nodal metastases (P b .05). High levels of TGFα and NIX were significantly associated with decreased overall survival (P b .001; P b .01). The multivariate analysis showed that advanced stage, presence of lymph node metastases, and high TGFα expression had an independent effect on survival (P b .006; P b .01; P b .0006). Our study suggests an up-regulation of the hypoxia-inducible factor–1 transcriptional pathway in colorectal carcinomas. hypoxia-inducible factor–1α overexpression alone, has no impact on the prognosis of colorectal carcinomas likely because the consequences of hypoxia-inducible factor–1α expression/stabilization strongly depend on the genetic background of the transformed cells. Mechanisms leading to increased synthesis of hypoxia-inducible factor–1α mRNA via autocrine growth factor loops may play a crucial role in invasive growth in this site. © 2008 Elsevier Inc. All rights reserved. 1. Introduction Hypoxia is a key factor of the tumor microenvironment that has been closely associated with resistance to radiation and chemotherapy [1] and, more recently, with malignant ☆ This study has been in part supported by a grant from the University of Insubria, Varese, Italy. ⁎ Corresponding author. Department of Human Morphology, Anatomic Pathology Unit, University of Insubria, Via O. Rossi, 9, 21100 Varese, Italy. E-mail address: daniela.furlan@uninsubria.it (D. Furlan). www.elsevier.com/locate/humpath 0046-8177/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2008.02.013 Human Pathology (2008) 39, 1483–1494