We studied the metabolic, proliferative, and angiogenic features of non-enhancing Grades 2 and 3 astrocytic gliomas to (1) confirm that the relative PC and GPC levels were consistent with previous studies of choline metabolism in gliomas, and (2) to determine whether there was any association between the metabolic and histologic markers. We compared the levels of 10 different metabolites and paid particular attention to the contributions of PC and GPC to the tCho levels in each group to determine whether histologic grade was the primary determinant of the relative PC to GPC concentrations. We also looked for associations between the levels of choline compounds, Ki-67 labeling, and VEGF staining. This was important for understanding the time course of alterations in choline metabolism with respect to changes in cellular proliferation and initiation of angiogenesis within astrocytomas.
Of the 10 metabolic measures that were compared, only PC was significantly higher in AS3 than AS2. This finding is consistent with a previous report by Sabatier (9
), who reported higher levels of PC in high grade glioma as compared with low grade glioma. There were also trends toward higher tCho and Tau in the AS3 although they did not reach statistical significance. In vivo
MRS studies have shown that high grade – in particular Grade 3 – gliomas typically have higher resonance peaks at 3.2 ppm corresponding to choline compounds (8
). The broad resonance peaks of in vivo
MRS, however, often contain resonances from multiple metabolites, such as the GPC (3.22 ppm), PC (3.21 ppm), and fCho (3.20 ppm) peaks that we summed as tCho. It is possible that the Tau triplet at 3.24 ppm may also contribute to the in vivo
choline-compound peak but this postulate needs to be confirmed. The fact that Tau had one of the highest Cramer Rao errors in our study, however, lessens the impact of the trend toward higher levels in the high grade tumors.
Despite the higher PC levels in AS3, GPC was the predominant choline compound in all 41 tissue biopsies that we studied with HRMAS. In fact, there was no difference in the PC:GPC ratio nor the ratios of each choline compound to tCho between the AS2 and AS3 samples. The GPC predominance is consistent with a recent study by Wright et al (27
) which reported mean PC = 0.45 and GPC = 0.77 mM in a cohort of five Grade 3 gliomas. However, these findings are in contrast to reports by Righi and Sabatier (5
) that stated the predominant choline compound in low grade glioma is GPC, while PC dominates in high grade glioma. One possible reason for the different findings may be the difference in the histopathological classification of the tumors in the “high grade” cohorts of the three studies. Righi et al (5
) included eight GBM in their cohort of 14 high grade astroctyomas, which are distinguished from AS2 and AS3 by the presence of microvascular hyperplasia and pseudopalisading necrosis that contains regions of hypoxia and expresses high levels of the hypoxic marker hypoxia-inducible factor-1 alpha (HIF-1alpha) (12
). Sabatier et al (9
) included oligodendroglial tumors in their high grade cohort which also have been shown to express HIF-1alpha (16
). Of the five astrocytoma (two Grade 2 and three Grade 3) studied by Sabatier et al, GPC was the predominant peak in three of them (9
). Thus, the dominance of PC that has been observed in cohorts of high grade glioma that include GBM and/or oligodendroglioma may not be a feature of non-enhancing Grade 3 astrocytoma.
The three choline compounds that were measured – PC, GPC, and fCho – all play a role in membrane phospholipid metabolism that accompanies the cell cycle (28
). Cancer-associated alterations in choline metabolism have been best characterized in breast cancer (29
) where PC has been shown to increase in a stepwise fashion during malignant transformation (31
) via increased phosphorylation by choline kinase (32
). Concomitant decreases in GPC have also been shown to accompany malignant transformation of breast cancer cells (33
). Moreover, GPC is the predominant choline compound in normal mammary epithelial cells, and a “GPC to PC switch” to a PC-dominant metabolic phenotype occurs when the cells were immortalized (31
). Interestingly, despite the role that phospholipid metabolism plays in generating sufficient membrane for new cells created during mitosis, no established correlation has been observed between any of the choline compounds and cell proliferation in breast cancer. However, a recent study showed that hypoxia increases choline kinase expression in prostate cancer cells (10
). Such a mechanism may also play a role in glioma and may explain why PC did not predominate in our AS3 tumors which do not harbor the high hypoxic fractions seen in GBM.
We demonstrated a positive correlation between GPC and Ki-67 proliferation index across all biopsies and among biopsies within the same tumor. TCho,fCho, and PC also increased with proliferation across all biopsies; however, the association with PC did not reach statistical significance. The lack of a clear association between PC and proliferation is further evidence that choline kinase expression and/or activity may not be directly associated with proliferation in gliomas. Indeed, we found that the higher PC in AS3 tumors was not due to the higher proliferation in those tumors. Cell density, on the hand, was positively associated with both PC and tCho, while fCho and GPC exhibited similar trends with cell density that did not reach statistical significance. Thus, when comparing different regions of the same tumor, the density of tumor cells within a sub-region of non-enhancing astrocytoma appears to influence the concentration of all of the choline compounds, while the proliferation in a given sub-region primarily impacts GPC concentrations.
Although none of the tumors in our study cohort exhibited frank contrast-enhancement on MRI, we questioned whether they were at different stages of angiogenesis and whether the expression of the angiogenic marker, vascular endothelial growth factor (VEGF), was associated with the choline metabolism within the tissue samples. Contrast-enhancement reflects the leakage of contrast material across the blood-brain barrier and into the interstitium, and is therefore thought to be less pronounced in tumors that do not have angiogenic neovasulature which is characteristically leaky. However, studies have shown a range of expression levels of VEGF within cohorts of non-enhancing (34
) and low grade astrocytoma (35
). Further, the VEGF levels were shown to be associated with astrocytoma histologic grade within the non-enhancing cohort (34
) and future malignant transformation and survival within the low grade cohort (35
). In the current study, none of the AS2 tumors expressed VEGF, while approximately half of the AS3 were VEGF-positive. When we grouped the tumors according to their VEGF status, there was no difference in the levels of any of the choline compounds. The only metabolite that differed between the VEGF-positive and VEGF-negative tumors was mI. VEGF-positive tumors exhibited higher mI even when only AS3 tumors were considered. These findings contradicted in vivo
studies by Castillo et al (36
) that reported a negative association between mI:Cre ratios and glioma grade in their study of previously-treated patients. In the current study, we found no difference in the mI or mI:Cre (data not shown) in the AS2 and AS3, suggesting that treatment effects may have influenced either the mI or Cre levels in the study by Castillo et al. Indeed, a study by Hattingen et al suggested that the mI levels in gliomas were associated with the presence of astrogliosis and reported no difference between the mI levels in low and high grade glioma (37
). To our knowledge, this is the first report of a possible association between VEGF and mI and we can only speculate as to the biological basis of the association. MI is a precursor to inositol phosphates that are regulated by VEGF (38
), however, more studies are needed to determine whether such a signaling pathway underlies the association between mI and VEGF observed in the astrocytomas in this study.
A limitation of this study was the limited amount of tissue for immunohistochemical analysis which prevented a direct measurement of hypoxia in the biopsy specimens using a marker such as HIF-1alpha. The exclusion of contrast-enhancing Grade 3 astrocytoma from this study also prevented us from explicitly determining whether our results are unique to non-enhancing tumors or extend to the overall population of Grade 3 astrocytomas.
In conclusion, our results showed that GPC was the predominant peak in non-enhancing astrocytic glioma, irrespective of histologic grade, which is in contrast to the predominant PC levels that have been observed in GBM and high grade breast cancer. GPC was associated with proliferation within tumors, PC and tCho were associated with cell density within tumors and no choline compound was associated with VEGF immunopositivity. Taken together, these data suggest that choline kinase activity, which is the primary source of the increased PC seen in aggressive breast cancer and hypoxic glial tumors, does not appear to be tightly linked to cell proliferation or angiogenesis in non-enhancing astrocytomas. These results add to the growing body of information regarding the role of phospholipid metabolism in the malignant progression of glial tumors.