Using prostate specific membrane antigen (PSMA) expression in clear cell renal cell carcinoma for imaging advanced disease

Abstract

Prostate specific membrane antigen (PSMA) is a 750 amino acid, type II transmembrane glycoprotein that has been shown to be over-expressed in both prostate cancer cells and the vasculature of solid tumours.1Grauer L.S. Lawler K.D. Marignac J.L. et al.Identification, purification, and subcellular localization of prostate-specific membrane antigen PSM protein in the LNCaP prostatic carcinoma cell line.Cancer Res. 1998; 58: 4787PubMed Google Scholar Also known as folate dehydrolase, glutamate carboxypeptidase II or N-acetyl-L-aspartyl-L-glutamate peptidase I,2Chang S.S. Reuter V.E. Heston W.D. et al.Five different anti-prostate-specific membrane antigen (PSMA) antibodies confirm PSMA expression in tumor-associated neovasculature.Cancer Res. 1999; 59: 3192PubMed Google Scholar PSMA has numerous metabolic roles and high levels of expression have been associated with tumour aggressiveness and malignant potential of prostate cancer cells.3Xiao Z. Adam B.L. Cazares L.H. et al.Quantitation of serum prostate-specific membrane antigen by a novel protein biochip immunoassay discriminates benign from malignant prostate disease.Cancer Res. 2001; 61: 6029PubMed Google Scholar Using PSMA as a target, positron emission tomography (PET) has demonstrated promising clinical utility in prostate cancer. In renal cell carcinoma (RCC), PSMA has also been shown to be highly expressed in the neovasculature of clear cell RCC (ccRCC). In a histopathological study by Baccala et al., PSMA was highly expressed in the proximal tubules of normal kidney tissues, and specifically in the neovasculature of ccRCC (75%), chromophobe (31%), oncocytoma (53%) and transitional cell carcinoma (21%).4Baccala A. Sercia L. Li J. et al.Expression of prostate-specific membrane antigen in tumor-associated neovasculature of renal neoplasms.Urology. 2007; 70: 385Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar Recently, a few reports have demonstrated improvements in staging metastatic RCC using PSMA PET, in comparison to conventional imaging.5Sawicki L.M. Buchbender C. Boos J. et al.Diagnostic potential of PET/CT using a Ga-labelled prostate-specific membrane antigen ligand in whole-body staging of renal cell carcinoma: initial experience.Eur J Nucl Med Mol Imaging. 2016; Mar 21; (Epub ahead of print)PubMed Google Scholar, 6Rowe S.P. Gorin M.A. Hammers H.J. et al.Imaging of metastatic clear cell renal cell carcinoma with PSMA-targeted F-DCFPyL PET/CT.Ann Nucl Med. 2015; 29: 877-882Crossref PubMed Scopus (111) Google Scholar In this report, we identified a young 35-year-old patient with bilateral renal malignancies with left renal vein tumour thrombus and left adrenal metastasis. There was no history of hereditary renal cell cancer syndrome. Pre-operatively, the patient underwent PSMA PET, triple phase computer tomography (CT) of chest and abdomen, and duplex ultrasound of inferior vena cava (IVC). The patient proceeded to left radical nephrectomy, adrenalectomy, caval thrombectomy, and right partial nephrectomy. Small PSMA binding ligand, 68Ga-HBED-CC (HBED-CC, ABX AG, Germany), was manufactured at the Specialized PET Services Queensland Radiopharmaceutical Laboratory at Royal Brisbane and Women’s Hospital as per Eder et al.7Eder M. Löhr T. Bauder-Wüst U. et al.Pharmacokinetic properties of peptidic radiopharmaceuticals: reduced uptake of (EH)3-conjugates in important organs.J Nucl Med. 2013; 54: 1327Crossref PubMed Scopus (21) Google Scholar PET images were acquired 90 min after administration of 150 MBq of 68Ga-HBED-CC on a Biograph mCT FLOW PET/CT scanner (Siemens, Germany), which is the current local protocol for prostate cancer staging. A low dose CT scan was performed concurrently with the PET scan for anatomical and attenuation correction. Using PSMA PET, all tumour deposits including primary tumours, renal vein thrombus and adrenal metastasis were found to be highly PET avid. The anatomical position of tumour thrombus identified by CT scan was detected at the same level with PET, and confirmed intraoperatively. All lesions identified with CT were also detected with PSMA PET (Fig. 1). There were no additional lesions recognised by the PSMA PET. The tissues were formalin fixed, paraffin embedded, prepared as a tissue array and sections were cut onto Superfrost Plus slides (Thermo Scientific, USA). Standard haematoxylin and eosin staining was performed for pathological analysis. PSMA immunohistochemistry (antibody, Clone 3E6; Dako, Agilent Technologies, USA) using Autostainer Link instrument (Dako, Agilent Technologies) was performed to correlate with in vivo PSMA PET imaging (Fig. 2). Negative controls were from normal renal tissue array with primary antibody only. As expected, the neovasculature of tumours demonstrated intense PSMA activity whilst the tumour cells did not. Proximal tubules of normal renal cortical tissue and prostate cancer cells exhibited intense staining (positive controls). In order to assess the levels of PSMA expression in pure RCC cells, 5 mL of blood was collected from the patient in lithium heparin tube for circulating tumour cells (CTCs). The cells were enriched (RosetteSep CD45 negative selection kit; StemCell, USA) and cultured in customised stem cell solution as described by Gao et al.8Gao D. Vela I. Sboner A. et al.Organoid cultures derived from patients with advanced prostate cancer.Cell. 2014; 159: 176Abstract Full Text Full Text PDF PubMed Scopus (924) Google Scholar CTCs were identified by immunofluorescence imaging [negative for CD45 (Cy5), positive for pan-cytokeratin (cy3) and 4',6-diamidino-2-phenylindole (DAPI)]. CTCs were then harvested for RNA using Norgen micro DNA/RNA extraction kit (Norgen Biotek, Canada) as per manufacturer’s protocol. Extracted RNA was amplified, using Single Cell RNA Amplification kit (Repli-G WTA Single Cell kit; Qiagen, Germany). cDNA was made with Superscript reverse transcriptase (Life Technologies, USA) according to the manufacturer’s protocol. RT-PCR results were compared with PSMA positive and negative prostate cancer cells in different dilutions. Housekeeping (RPL32) and androgen receptor (AR) genes were used to compare the results. Diluted samples (10 and 100 cells/sample) of DU145 (RPL32+/PSMA–/AR–), PC3/PIP (RPL32+/PSMA+/AR–), and LNCAP (RPL32+/PSMA+/AR+) cells were used to validate the results. As expected, PSMA was expressed by PC3/PIP and LNCaP cells (positive controls) but not by DU145 (negative control) and RCC CTCs (Table 1). All cells expressed similar levels of RPL32 (housekeeping gene).Table 1Reverse transcription polymerase chain reaction (RT-PCR) of PSMA expression of renal cell carcinoma circulating tumour cells (RCC CTCs).CellsPSMA expressionAR expressionRelative PSMA expression to RPL32Relative AR expression to RPL32DU145(n=10 cells)––Not detectableNot detectableDU145(n=100 cells)––Not detectableNot detectablePC3/PIP(n=10 cells)+–4.197Not detectablePC3/PIP(n=100 cells)+–4.393Not detectableLNCaP(n=10 cells)++0.9390.693LNCaP(n=100 cells)++0.6180.405RCC CTC(n=7 cells)–+Not detectable0.012CTCs, DU145, LNCAP and PC3/PIP cells were harvested and extracted. RNA was amplified using Single Cell RNA Amplification kit (Repli-G WTA Single Cell kit). RT-PCR results were compared with PSMA positive and negative prostate cancer cells in different dilutions. Housekeeping gene (RPL32) and androgen receptor (AR) genes were used to compare the results. Diluted samples (10 and 100 cells/sample) of DU145 (RPL32+/PSMA–/AR–), PC3/PIP (RPL32+/PSMA+/AR–), and LNCaP (RPL32+/PSMA+/AR+) cells were used to validate the results. Open table in a new tab CTCs, DU145, LNCAP and PC3/PIP cells were harvested and extracted. RNA was amplified using Single Cell RNA Amplification kit (Repli-G WTA Single Cell kit). RT-PCR results were compared with PSMA positive and negative prostate cancer cells in different dilutions. Housekeeping gene (RPL32) and androgen receptor (AR) genes were used to compare the results. Diluted samples (10 and 100 cells/sample) of DU145 (RPL32+/PSMA–/AR–), PC3/PIP (RPL32+/PSMA+/AR–), and LNCaP (RPL32+/PSMA+/AR+) cells were used to validate the results. There are numerous types of RCC, with ccRCC being the most common (70–90%) and more aggressive than other common histological types such as papillary and chromophobe RCC.9Ljungberg B. Cowan N.C. Hanbury D.C. et al.EAU guidelines on renal cell carcinoma: the 2010 update.Eur Urol. 2010; 58: 398Abstract Full Text Full Text PDF PubMed Scopus (1171) Google Scholar ccRCC is mostly thought to arise from proximal renal tubular epithelium as the result of one of many von Hippel–Lindau gene mutations. Resulting angiogenesis from the mutations increases the expression of hypoxia-inducible factor (HIF)-2-alpha and in turn, vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF).10Cohen D. Zhou M. Molecular genetics of familial renal cell carcinoma syndromes.Clin Lab Med. 2005; 25: 259Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar The pathophysiological cascade that results in angiogenesis has been successfully targeted clinically by tyrosin kinase inhibtors, mTOR inhibitors and monoclonal antibodies against VEGF receptors. In this report, we have demonstrated that increased angiogenesis by ccRCC can be utilised to potentially improve staging using PSMA linked imaging technologies. We show that PSMA is highly expressed in the neovasculature of tumours, which is in contrast to normal cortical tissue where PSMA expression is shown in the epithelial cells of proximal tubule without activity within the adjacent vasculature. We also demonstrate that ccRCC cells, which originate from PSMA expressing proximal tubules, lose PSMA expression. PSMA PET CT imaging is becoming widely utilised in prostate cancer imaging. This increases the availability for PSMA PET to be utilised for staging, surgical planning and clinical decision-making in ccRCC patients. Conventional imaging will remain important, however PSMA PET may improve staging by identifying small metastatic lesions not obvious on routine imaging. The physiological distribution of PSMA such as in the salivary gland, liver, kidney, spleen, and bowel will limit its use for detecting rare metastatic lesions in these organs, underpinning the importance of multimodality imaging. Further, it would be important to recognise that PSMA PET may detect dual-pathology such as concurrent metastatic RCC and prostate cancer.