Tissue-based assay for ornithine decarboxylase to identify patients likely to respond to difluoromethylornithine

Abstract

In a previous publication, we showed that a clinical trial of DL-alpha-difluoromethyl ornithine (DFMO), in combination with PCV (procarbazine, CCNU, vincristine) increased survival of patients with anaplastic gliomas (WHO III) but not glioblastoma multiforme (WHO IV). We believe that treatment outcome (survival) is inversely related to tumor ornithine decarboxylase (ODC) levels. To prove this, we needed to develop an assay to quantify ODC levels in formalin-fixed tumor tissues, which would enable a retrospective study of tumor biopsy specimens from the landmark clinical trial. We developed an assay using a specific polyclonal antibody coupled to an Alexa fluorescent dye. Transgenic MHC-ODC mice with differing levels of ODC in heart muscle were used to establish the relationship between mean gray-scale intensity and enzymatic ODC activity. We found a direct relationship between mean gray-scale intensity of the ODC antibody coupled to Alexa 647 dye and enzymatic activity. Preliminary analysis of a human glioma tissue array shows that tumor-specific variations in levels of ODC can be semiquantitated. We show that mean gray-scale intensity of astrocytoma:glioblastoma is 1:6 and of anaplastic astrocytoma:glioblastoma is 1:4. We also compared the intensity of antibody to Ki67 coupled with phycoerythrin simultaneously in cells but failed to see a relationship that crossed histologies. We conclude that we can measure levels of ODC in formalin-fixed tumor tissue using an antibody to ODC coupled to Alexa 647 dye, and this will enable us to conduct a future study to correlate survival of patients with gliomas of different histologies treated with DFMO to tumor ODC levels.

Figure 1

Photomicrographs taken of formalin-fixed transgenic mouse hearts sacrificed at 2, 7, 14, and 28 days of life. The tissues are part of the array stained with ODC-Ab-Alexa 647 for 48 hr. The 12-bit gray-scale images are pseudo-colored using a red-to-blue format to depict fluorescent intensity levels after 100-msec excitation by a mercury vapor light source; red represents low and blue high optical intensity. ODC activity in units of nmol/30min/μg protein for A = 3.66, B = 17.4, C = 22.2, and D = 28.0.

Figure 2

Linear least-squares plot of transgenic heart activity (nmol/30 min/μg protein) versus ODC-Ab-Alexa 647 intensity. The plot represents 22 heart samples. The integrated and bit-mapped intensity for each of three ×40 magnified fields/heart sample (1700 total measurements/heart) were used to compute a mean ± SEM. Dashed line represents the 95% confidence interval for the regression line that was fitted to the equation Y = 564.8 (±59.8) + 22.4 (±3.2) × ODC activity, with R ² = 0.71.

Figure 3

Digitalized images (×40 magnification) of (A) normal brain after Ab-ODC, secondary Ab-peroxidase, and hematoxylin stain and (B), GBM under similar conditions. Greater peroxidase positivity is seen in the nuclei of tumor cells than in normal cells, with occasional staining of cytosol. Arrows point to occasional peroxidase-obvious cells in normal brain (A) and considerably more in the GBM samples (B).

Figure 4

Photomicrographs of formalin-fixed graded human gliomas. The tissues are part of the array stained with ODC-Ab-Alexa 647 for 48 hr. The 12-bit grayscale images are pseudo-colored using a red-to-blue format to depict fluorescent intensity levels after 1000 msec excitation by a mercury vapor light source; red represents low and blue high optical intensity.

Figure 5

ODC-Ab-Alexa 647 intensity for glioblastoma (GBM), anaplastic astrocytoma (AA), anaplastic oligodendroglioma (AO), oligodendroglioma (OLIG), and astocytoma (LGA). Each point represents the mean ± SD.

Figure 6

Scatterplot of ODC-Ab-Alexa 647 intensity versus Ab-Ki67-PE intensity for three, anaplastic astrocytoma (AA), oligodendroglioma (OLIG), anaplastic oligodendroglioma (AO), and glioblastoma (GBM) tumors from the tissue array. Because there were no statistical intergroup differences within a histology, the results from the tumors were pooled and plotted as a single histologic group. The dotted lines bracket intensities that represent fluorescent saturation.