Diffusion limited hypoxia estimated by vascular image analysis: comparison with pimonidazole staining in human tumors.
OBJECTIVE
To assess diffusion limited hypoxia in human tumors using image analysis of vasculature and to compare it with the bioreductive marker pimonidazole as an independent method.


METHODS
To set up the method, nine rectal adenocarcinomas and ten squamous cell carcinomas were analyzed. To validate the method, ten squamous cell carcinomas of the cervix were analyzed from patients who were injected with pimonidazole and biopsied approximately 24 h later. Sections of the rectal and esophageal tumors were stained for vasculature, while cervix tumor sections were double stained for vasculature and pimonidazole. Tumor areas were delineated on digitized images, and the proportion of tumor tissue greater than a fixed distance from the nearest blood vessel (called diffusion limited fraction, DLF) was then calculated. The proportion of tumor area stained for pimonidazole was also measured.


RESULTS
There was a wide variation between tumors in both the vascular-derived DLF and in the pimonidazole-stained fraction. Average DLFs varied between 1.5 and 92% for different tumors, with significant differences between them. The area stained by pimonidazole was significantly smaller than DLF for all tumors. The correlation between pimonidazole area and DLF was significant in three of seven tumors containing > or = 3 images. When images from all tumors (n=123) were analyzed together, the correlation was highly significant (r=0.47, P<0.0001).


CONCLUSIONS
The vascular derived DLF correlates significantly with pimonidazole staining, but there was large scatter. Both methods may underestimate perfusion limited hypoxia.
Haustermans
K
K
Division of Experimental Therapy, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, The, Amsterdam, Netherlands.
Hofland
I
I
Van de Pavert
L
L
Geboes
K
K
Varia
M
M
Raleigh
J
J
Begg
A C
AC
D000230:Adenocarcinoma / Q000098:blood supply / Q000378:metabolism; D001808:Blood Vessels / Q000378:metabolism / Q000473:pathology*; D002294:Carcinoma, Squamous Cell / Q000098:blood supply / Q000378:metabolism; D015687:Cell Hypoxia / Q000502:physiology; D004058:Diffusion; D004938:Esophageal Neoplasms / Q000098:blood supply / Q000378:metabolism*; D005260:Female; D006801:Humans; D007091:Image Processing, Computer-Assisted; D009593:Nitroimidazoles; D015588:Observer Variation; D011838:Radiation-Sensitizing Agents; D012004:Rectal Neoplasms / Q000098:blood supply / Q000378:metabolism*; D015203:Reproducibility of Results; D002583:Uterine Cervical Neoplasms / Q000098:blood supply / Q000378:metabolism*
D009593:Nitroimidazoles; D011838:Radiation-Sensitizing Agents; C033815:pimonidazole