Prostate specific membrane antigen (PSMA) expression in primary gliomas and breast cancer brain metastases

Primary and secondary brain cancers are difficult to treat and are a major cause of cancer-related death [1‐3]. Gliomas are the most common primary brain tumors, with grades I, II, and III usually progressing to a poor outcome over 2 to 10 years, while the aggressive grade IV (glioblastoma; GBM) advances very rapidly within 2–3 years, causing approximately 13,000 deaths per year [1]. Secondary brain cancers occur in an estimated 200,000 persons annually in the U.S., and account for at least 20% of all cancer deaths yearly [4, 5]. A major source of brain metastases is adenocarcinoma of the breast [4, 5]. Approximately 200,000 women develop breast cancer annually, and 2–3 years after diagnosis nearly 30% develop brain metastases [6, 7]. There are few treatment options for this relatively late and lethal complication, which is more frequent in younger patients [8].

A potentially effective therapeutic strategy may derive from the finding that the transmembrane prostate specific membrane antigen (PSMA) is robustly expressed by the tumor vascular endothelium in a variety of solid cancers, including GBM and primary adenocarcinoma of the breast, but is not evident in normal vascular endothelium, and to only very low levels in normal prostate [9‐13]. Patient autopsy evidence and preclinical models reveal that progressive gliomas and breast tumor metastases either develop new blood vessels or co-opt existing vessels [14‐17]. PSMA has not been detected elsewhere in the body, except on the luminal side of the intestinal epithelium, which is not accessible via the vasculature, and on the apical surface of renal proximal tubular cells, although PSMA targeted antibodies are too large to filter through the kidney glomerulus [18]. Hence, it may be feasible to deliver PSMA targeted agents to the tumor and its microvasculature to (1) selectively destroy the vessels perfusing the tumor tissue, (2) achieve high regional doses of drugs to overcome tumor resistance [19], and (3) spare normal tissues, which typically lack PSMA expression [11]. The vascular endothelium of malignant brain tumors is potentially accessible to a PSMA targeted agent which is important since studies show that the tumor mass may be at least partly protected from therapeutic agents by the blood–brain barrier (BBB) [18, 20]. Normal brain parenchyma which expresses low levels of PSMA in a heterogeneous, sparse pattern [21], would be expected to be protected from anti-PSMA targeted antibody agents by an intact BBB [22]. Treatment with cytotoxin-conjugated antibody or nanoparticles coated with a PSMA ligand conjugated to cytotoxins is being actively explored [23, 24], and anti-PSMA based agents have targeted and suppressed prostate tumors in preclinical models. Such agents are presently in multiple Phase II clinical trials for prostate tumors and other cancers (http://​clinicaltrials.​gov/​ct2/​show/​NCT01056029; http://​clinicaltrials.​gov/​show/​NCT01695044) [25, 26].

A potentially significant reported finding is that breast cancer metastases, while treatment resistant, do not infiltrate surrounding normal brain tissue beyond approximately 1 mm, rather they tend to increase in number and size [27]. This suggests ablation of such metastatic foci and a 1 mm surrounding zone may hold the promise of substantially reducing or eradicating the intracranial tumor burden [27]. PSMA is expressed by the blood vessels of primary breast tumors [9] and importantly, there is an initial report of PSMA expression in breast cancer brain metastases, using manual, general methods of tissue scanning and scoring [28].

For the present study we initially sought to determine, using human tissue samples and fully automated optical scanning and quantification of stained tissue sections, PSMA expression in glioma grades I through IV. We also investigated the intensity and uniformity of PSMA expression by breast cancer brain metastases across patients [9]. The overarching goals of this early feasibility effort was to extend the limited volume of current data using precise automated slide scanning and quantification technology, and to help guide subsequent large scale studies correlating tumor subtypes with patterns of PSMA expression. In addition, given the known important role of PSMA in tumor angiogenesis, further describing its expression may provide a basis for research aimed at understanding; (1) how lower grade gliomas evolve to the highly angiogenic glioblastomas, and (2) how PSMA may contribute to angiogenesis and vasculogenic mimicry in glioblastomas.

The present study measured the expression of PSMA on human primary and secondary brain cancers to explore the feasibility of PSMA targeting for tumor selective delivery of therapeutic agents. The data indicated that the PSMA is expressed on the brain tumor cells and especially the tumor blood vessels in human gliomas of grades I and IV (Figures 2A & 3). We determined that blood vessels in breast cancer brain metastases exhibit PSMA, although we also found that expression intensity varied considerably between patients (Figure 5A and B). Normal brain tissue from tumor and non-tumor bearing patients had a small number of PSMA positive cells while the microvasculature had no staining (Figure 1). This agrees with a previous report describing cellular PSMA expression according to brain anatomical region [21]. Our results for grade IV gliomas (GBM) align with those of Wernicke et a l. [18], and we extend the available data to include grade I to III gliomas. Wernicke et al. also reported that PSMA staining was uniform for 14 breast cancer brain metastases and at the same intensity as the primary tumors [28]. We also found PSMA on such metastases, although with considerable variation and in general the staining intensity on metastases was somewhat below that of the matched primary breast tumor. Using automated, complete scanning and quantification to yield a continuous, fine scale, we required relatively few samples to elaborate on Wernicke’s findings.

Despite the fact that primary and secondary brain tumors express high levels of VEGF and are densely vascularized, making anti-angiogenic therapy attractive, VEGF inhibitors have so far been of limited benefit in brain cancer patients but may be potentially be effectively combined with PSMA targeting [17, 18, 30‐33]. The basis for this is that in addition to GBM induction of angiogenesis via VEGF, (1) at least some of the brain tumor vasculature derives from the transdifferentiation of tumor cells and does not depend on VEGF [34‐36] and (2) PSMA induces VEGF-independent angiogenesis in pathological conditions [37]. It is also relevant that the stem-like cells driving brain cancers and tumor-derived angiogenesis reside in close proximity to newly formed microvessels, and the expansion of glioma stem like cells in animal models requires a microvascular supply [38, 39].

Our observed variability of PSMA staining between tumors of the same type, and the differential staining of grade IV gliomas relative to lower grades needs to be comprehensively investigated in terms of informing and personalizing treatment regimens. All the glioma samples were acquired before the patients were treated, and one explanation for differential staining may be related to previous reports indicating that grade IV gliomas are highly angiogenic compared with lower grades [40], and that PSMA is only expressed on angiogenic vessels [41, 42]. This is not entirely unexpected as for prostate cancer evidence suggests that PSMA expression is greatest in high-grade, metastatic, and hormone-insensitive tumors [43]. Another possible basis for differential staining may relate to the observation that the FOLH1 (PSMA) gene generates several splice variants, including PSMA, PSME, PSM’, PSMA-C, PSMA-D, PSMAΔ6, and PSMAΔ18 [44, 45]. Grade 1 through IV gliomas which exhibit different behavior and gene signatures, may also have differential expression of FOLH1 splice variants [46]. Moreover, PSMA influences integrin activation, various signaling pathways, and impacts survival and invasion in prostate cancer cells [42]. Since PSMA is expressed by some GBM tumor cells, it would be of potential interest to explore whether different splice variants play a role in the transdifferentiation of GBM cells into vascular elements, i.e., vasculogenic micmicry [47]. The possibility that the expression of FOLH1 splice variants changes according to glioma grade and influences vascular expansion and tumor cell invasion should be investigated in follow-up studies.

The variability in PSMA staining observed with all glioma grades aligns with the phenotypic heterogeneity of gliomas and suggests that; (1) the extent and variability of PSMA staining within glioma grades needs to be further explored in larger scale studies, and (2) tumor PSMA levels should be characterized to better identify patients that would potentially benefit from PSMA targeted therapy. The same concept may apply to breast cancer brain metastases given the heterogeneity that we observed [48].

The present study has expanded on previous data to include all grades of glioma (I through IV). This is important because lower grade gliomas often progress despite treatment and are associated with transformation to more aggressive higher grades [49]. Elaborating on PSMA data for is significant because breast cancer brain metastases represents an emerging, large clinical problem. Even patients with Her-2-positive breast tumors responding well to trastuzumab (Herceptin®) suffer from brain metastasis [8]. It should be noted that the present initial study was constrained by the difficulty in acquiring tissue specimens. A follow-up project will involve other centers to acquire a larger number of patient samples to more accurately quantify the variability of PSMA staining, and importantly, to correlate PSMA levels with different grade IV glioma (GBM) subtypes, viz., Proneural, Neural, Classical and Mesenchymal. In addition, PSMA expression will be related to breast cancer subtype.

This report evaluated PSMA staining in grades I to IV gliomas and breast cancer brain metastases. Average microvessel PSMA levels in grade IV gliomas and breast cancer brain metastases were significantly greater than for normal brain, with variability between samples taken from different patients. These data provide a foundation for further more comprehensive investigations of PSMA in primary and secondary brain tumors; (1) as a target for the selective ablation of tumor microvasculature, (2) as a means of preferentially concentrating therapeutic agents at tumor tissue, and (3) potentially to help understand the progression of low grade gliomas to glioblastoma. Based on this and other data, a prospective clinical trial in glioblastoma is in process (http://​clinicaltrials.​gov/​ct2/​show/​NCT02067156).

SK is a MD, PhD neuro-oncologist with a background in molecular biology. SK’s efforts are divided 50% to treating brain cancer patients and 50% to research. SK leads a multidisciplinary laboratory focused on understanding the developmental and molecular aberrations resulting in malignant gliomas and using this information to develop novel molecular therapeutics to improve patient survival. The laboratory is collaborative, with post-docs, graduate students and project scientists experienced in glioma stem cell biology, signal transduction, brain tumor imaging, genomic and proteomic biomarker discovery, and translational science based on clinical samples from patients at UCSD.