The general ability to map genomic and proteomic expressions to in-vivo imaging information opens the potential for new insights into disease characterization and a better understanding of disease mechanisms. In turn, such new insights seem well suited for diseases like prostate cancer that show heterogeneous clinical expression and that demand more specific information upon which to base management decisions for an afflicted individual’s personalized management. MRI and MRS offer a variety of ways to probe the in-vivo features of biologic processes, including cancer.
One of the almost universal findings in MRS of tumors is the elevation of the composite resonance assigned to the trimethyl group of choline containing compounds (for a review see [17
]). The spectral patterns observed on MRS of the prostate and their interpretations have recently been reviewed by Rajesh et al. [30
] . The relative level of the choline peak has been shown to correlate with the Gleason grade of the tumor [31
]. In spite of the importance of this peak, it is still unclear as to what alterations in genetic expression leads to choline elevation in prostate cancer. The lifetime of choline in the blood and its distribution in the body, as well as its transport into the cells, are critical steps in the metabolic fate of choline. The transport is the rate-limiting step for the synthesis of acetylcholine [33
] and of PCho [34
]. The transporters of choline, as well as choline kinase isozymes, were recently characterized by Eliyahu et al. [35
] in normal and malignant mammary cells. In addition to two specific transporters, (CHT1 and OCT2), choline kinase a was shown to be up-regulated in the malignant transformation of breast tissue. Choline kinase a has been shown to be up-regulated in prostate cancer [36
], and the implications of this up-regulation for potential targeted therapies has been discussed by Glunde and Bhujwalla [37
]. In this light, it is interesting to point out that both F-18 [38
] and C-11 [40
] labeled choline have been used in PET studies to visualize primary prostate tumors. Since choline kinase a is one of the major enzymes that converts choline to phosphocholine, it is reasonable to hypothesize that these radiotracers may be probing its over-expression. The methods shown here provide a means for spatially correlating the levels of over-expression of choline kinase a with the level of the choline resonance observed on in vivo
The induction of new blood vessels is a key event in the progression of a solid tumor as it grows beyond 1–2 mm in diameter and can no longer depend on simple passive diffusion to provide nutrients and clear toxic wastes (for a review see Folkman [41
]). Both the outgrowth of new blood vessels from established vessels (angiogenesis) and the formation of new microvessels from circulating endothelial cells (vasculogenesis) can be co-opted by a cancer [42
]. Tumor induced blood vessel formation has proven to be a complex pathological event, and the early hopes for anti-angiogenesis therapies have been tempered by the realization that cancers utilize multiple molecular pathways to induce and remodel a vascular network. Model systems can be used to selectively manipulate the formation, regression and maturation of tumor blood vessels in order to identify the genetic pathways involved in these processes. However, it is unclear how faithfully these models replicate the pathologies of human tumors. Assessments of angiogenic phenotypes using a variety of biomarkers are only beginning to be included into the clinical assessments of a patient’s malignancy. There is great interest in developing, validating and utilizing, non-invasive imaging technologies to replace these more invasive tests.
Vascular endothelial growth factor (VEGF) is one of the most intensively studied angiogenic factors in human cancers, and VEGF has generally been reported to be over-expressed in prostate cancer relative to benign prostate tissue. Thus, it is reasonable to expect that most, if not all, of the tumors that exhibit abnormal DCEMRI patterns would show over-expression of VEGF. However, it has been reported that the levels of VEGF over-expression in primary human prostate cancers are highly variable, both at the RNA and protein levels [43
]. The tissue print mapping techniques described in this paper have allowed us to undertake a detailed analyses of gene expression patterns in low-VEGF, DCE-MRI positive prostate cancers to determine which pathways were involved in the angiogenesis of these tumors. Interestingly, one of the genes most highly over-expressed in the low-VEGF cancer shown in is neuropeptide Y (NPY), which showed a 49-fold ratio tumor/benign on Gene Array. This high level of NPY over-expression was confirmed by rt-PCR and immunohistochemistry. In our ACIS IHC analysis of tissue microarrays that contain cores from progressively higher grades of radical prostatectomy cancers, we observed NPY overexpression early in development of this malignancy, with the most intense staining in a subset of cases that include approximately 15% of the Gleason 7 tumors. Rasiah et al (47
) saw a similar pattern of expression in their patient population, and noted that NPY overexpression could also be detected in high grade PIN.
Although other groups have recongized NPY as one of many neuroendocrine markers that are over-expressed in a subset of prostate cancers, the ramifications of its over-expression has not yet been established [47
]. In a number of experimental systems, NPY has been shown to be a potent factor for stimulating angiogenesis in benign tissues [49
]. Moreover, NPY can act as a proangiogenesis factor in experimental models of two different neuroendocrine cancers: neuroblastoma and Ewing’s sarcoma [54
]. In addition to its direct proangiogenic activity, it has been suggested that NPY can also modify the angiogenic response to VEGF [55
]. However, very few published studies have attempted a direct evaluation of NPY as an angiogenic factor in naturally occurring human cancers. The significance of NPY over-expression is that in model systems NPY produces micro vessels that appear to be more mature and less “leaky” than those induced by VEGF [49
]. In principle, DCEMRI should be able to distinguish tumors with these more mature vessels from tumors with more leaky vessels. For example, in the models commonly employed in the pharmacokinetic analyses of DCEMRI results, e.g. the so called Tofts Model [56
], the values of the parameter, Ktrans
, should be higher in tumors with leaky vessels. If this hypothesis were correct, there would be a non-invasive way to identify high VEGF expressing tumors from high NPY expressing tumors. DCEMRI could be used to identify patients who would respond to anti-angiogenic therapy based on anti-VEGF approaches and also monitor changes in response to therapy.
In summary, the examples shown here indicate that it may be possible to map expression profiles of proteins and enzymes that are upregulated in tumors with MRI/MRS related parameters on a pixel-by-pixel basis. MR imaging data can be employed to direct tissue sampling for genetic micro-array analysis to identify candidate genes for further immunohistochemical studies. The advantage of the immunohistochemical studies using fluorescence readouts is that they can provide quantitative spatial maps of protein expression that can be directly related to the MRI/MRS results. This approach can be exploited to determine whether the overexpression of a particular protein or enzyme results in measurable change in a MRI/MRS parameter. For example, it is possible to determine whether the level of total choline seen in the prostate cancer by MRS is correlated with the level of overexpression of choline kinase a or the levels of choline transporters, or both. Using this kind of approach, it should be possible to determine which, if any, of the many MRI/MRS parameters that are currently available are true biomarkers for characterization of the disease processes being investigated.