Aberrantly glycosylated PSMA in urine as a potential marker for prostate cancer.

PD29-01 THE VALUE OF 99MTC-PSMA SPECT/CT GUIDED SURGERY TO IDENTIFY AND LOCALIZE LYMPH NODE METASTASIS FOR PROSTATE CANCER PATIENTS

Prostate Specific Antigen and Human Glandular Kallikrein 2 in Early Detection of Prostate Cancer

Prostate-specific membrane antigen (PSMA) is an integral cell-surface membrane glycoprotein that is overexpressed in prostate carcinomas rendering it an appropriate target for antibody-based therapeutic strategies. The biosynthesis of PSMA in transfected COS-1 cells reveals a slow conversion of mannose-rich to complex glycosylated PSMA compatible with slow transport kinetics from the endoplasmic reticulum to the Golgi. Importantly, mannose-rich PSMA persists as a trypsin-sensitive protein throughout its entire life cycle, and only Golgi-located PSMA glycoforms acquire trypsin resistance. This resistance, used here as a tool to examine correct folding, does not depend on the type of glycosylation, because different PSMA glycoforms generated in the presence of inhibitors of carbohydrate processing in the Golgi are also trypsin resistant. The conformational transition of PSMA to a correctly folded molecule is likely to occur in the Golgi and does not implicate ER molecular chaperones, such as BiP. We show here that PSMA is not only heavily N-but also O-glycosylated. The question arising is whether glycans, which do not play a role in folding of PSMA, are implicated in its transport to the cell surface. Neither the cell-surface expression of PSMA nor its efficient apical sorting in polarized Madin-Darby canine kidney cells are influenced by modulators of N- and O-glycosylation. The acquisition of folding determinants in the Golgi, therefore, is an essential prerequisite for protein trafficking and sorting of PSMA and suggests that altered or aberrant glycosylation often occurring during tumorigenesis has no regulatory effect on the cell-surface expression of PSMA.

Objective The objective of this study was to evaluate the clinical utility of gallium-68 [ 68 Ga] prostate-specific membrane antigen (PSMA) positron emission tomography/computed tomography (PET/CT) with rising prostate-specific antigen (PSA) levels in prostate cancer diagnosis. Methods This is a retrospective, single-center, observational cross-sectional study, which is provided after ethics committee clearance, from May 2, 2022 to June 25, 2022. Study includes sample size of 50 patients with prostate adenocarcinoma with varying PSA levels and Gleason score of 6 to 9 who underwent [ 68 Ga] PSMA PET/CT scan. The patients included in this study underwent PET/CT scan on uMI550 (United Imaging Healthcare, Shanghai, China). Results All patients were divided into three groups based on PSA levels in ng/mL as: PSA ≤ 0.2 (8%), 0.2 < PSA ≤ 1 (10%), 1 < PSA ≤ 3 (8%), 3 < PSA ≤ 10 (18%), and PSA > 10 (56%). Among 50 scans, at least one PSMA avid lesion was visualized in 41 scans (78.9%). These scans were considered positive and included in this study, rest of the scans had insignificant PSMA uptake and were considered negative. [ 68 Ga] PSMA PET/CT detection rates were 75.0, 20.0, 50.0, 88.90, and 89.3% in patients with PSA ≤ 0.2, 0.2 < PSA ≤ 1, 1 < PSA ≤ 3, 3 < PSA ≤ 10, and PSA > 10, respectively. In addition to prostate bed, lesions were also visualized in lymph nodes (32%), liver (2%), skeleton (28%), and thorax (6%). Considering lesions in the prostate bed a significant direct correlation was detected between maximal standardized uptake value (SUVmax) and PSA value ( p = 0.03). Discussion PSMA PET/CT has been demonstrated to be an effective method for identifying both low-grade Gleason score tumors and low PSA levels. The study provides support for the use of [ 68 Ga] PSMA PET/CT in conjunction with PSA levels for the evaluation of prostate cancer, including local recurrence and distant metastases. Conclusion The findings of this study indicate that PSMA PET/CT is an effective method for diagnosing prostate cancer, as it allows for the detection of high SUVmax values in pathological tissues. Furthermore, high sensitivity and detection rates are noted with PSMA PET/CT scan even in cases where PSA levels were low. Therefore, this study demonstrates that [ 68 Ga] PSMA PET/CT is beneficial for the early detection of prostate cancer and the prediction of treatment outcomes.

Extracellular nucleic acids could serve as molecular markers in the early detection of cancer and in the prediction of disease outcome. In this study we examined six molecular markers, such as: variations in the quantity of DNA in plasma, glutathione-S-transferase P1 (GSTP1) gene methylation status in plasma, carcinoembryonic antigen (CEA) and prostate-specific membrane antigen (PSMA) mRNA in peripheral blood mononuclear cells (PBMC), and plasma samples from prostate cancer patients in different stages. The combination of DNA load and GSTP1 promoter methylation status identified 83% (10/12) of the prostate cancer patients before therapy. This study shows that free circulating DNA can be detected in patients with prostate cancer compared with disease-free individuals, and suggests a new, noninvasive approach for early detection of prostate cancer.

At present, increased early detection of prostate cancer appears to be the most feasible way to reduce cancer-related mortality. As a result significant efforts have been made to identify more men with curable cancer. This article reviews the role of serum prostate-specific antigen in an early detection or screening strategy and describes efforts to enhance the specificity of prostate-specific antigen testing.

321 Analysis of Combinatorial Effect of Multikinase Inhibitor (Sorafenib, Sunitinib) or Cetuximab with MEK or mTOR Inhibitors in Human Head and Neck Cancer Cells

320 Comparative Pharmacokinetics of Trastuzumab-US and Trastuzumab-EU and the Potential Biosimilar Trastuzumab-Pfizer in Male CD-1 Mice

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

In vivo simultaneous visualization of multiple biomarkers is critical to accurately diagnose disease and decipher fundamental processes at a certain pathological evolution, which however is rarely exploited. Herein, a multimodal activatable imaging probe (P-125 I) is reported with activatable fluoro-photoacoustic and radioactive signal for in vivo imaging of biomarkers (i.e., hepsin and prostate-specific membrane antigen (PSMA)) associated with prostate cancer diagnosis and prognosis. P-125 I contains a near-infrared (NIR) dye that is caged with a hepsin-cleavable peptide sequence and linked with a radiolabeled PSMA-targeted ligand (PSMAL). After systemic administration, P-125 I actively targets the tumor site via specific recognition between PSMA and PSMAL moiety and in-situ generates of activated fluoro-photoacoustic signal after reacting with hepsin to release the free dye (uncaged state). P-125 I achieves precisely early detection of prostate cancer and renal clearance to alleviate toxicity issues. In addition, the accumulated radioactive and activated photoacoustic signal of probe correlates well with the respective expression level of PSMA and hepsin, which provides valuable foreseeability for cancer progression and prognosis. Thus, this study presents a multimodal activatable probe for early detection and in-depth deciphering of prostate cancer.

BackgroundExosomes or extracellular vesicles have the potential as a diagnostic marker for various diseases including cancer. In order to identify novel exosomal markers for prostate cancer (PC), we performed proteomic analysis of exosomes isolated from PC cell lines and examined the usefulness of the marker in patients.MethodsExosomes isolated by differential centrifugation from the culture medium of androgen-dependent LNCaP prostate cancer cell line and its sublines of partially androgen-independent C4, androgen-independent C4–2 and bone metastatic C4–2B were subjected to iTRAQ-based proteomic analysis. Exosomes were also isolated by immunocapture and separated by size exclusion chromatography and density gradient centrifugation. Protein expression was determined by Western blot analysis. GGT activity was measured using a fluorescent probe, γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG). Immunohistochemical analysis of tissues was performed using anti-GGT1 antibody.ResultsAmong proteins upregulated in C4–2 and C4–2B cells than in LNCaP cells, we focused on gamma-glutamyltransferase 1 (GGT1), a cell-surface enzyme that regulates the catabolism of extracellular glutathione. The levels of both GGT1 large and small subunits were elevated in exosomes isolated from C4–2 and C4–2B cells by differential centrifugation and by immunocapture with anti-CD9 or -prostate-specific membrane antigen (PSMA) antibody. In cell lysates and exosomes, GGT1 expression correlated with GGT activity. Size exclusion chromatography of human serum demonstrated the presence of GGT activity and GGT1 subunits in fractions positive for CD9. Density gradient centrifugation revealed the co-presence of GGT1 subunits with CD9 in exosomes isolated by differential centrifugation from human serum. Since GGT activity correlated with GGT1 expression in serum exosomes isolated by differential centrifugation, we measured serum exosomal GGT activity in patients. Unexpectedly, we found that serum exosomal GGT activity was significantly higher in PC patients than in benign prostatic hyperplasia (BPH) patients. In support of this finding, immunohistochemical analysis showed increased GGT1 expression in PC tissues compared with BPH tissues.ConclusionsOur results suggest that serum exosomal GGT activity could be a useful biomarker for PC.

The present study was carried out to investigate whether PET imaging can be used as a potential substitute for immunohistochemical analysis of tumor samples in prostate cancer (PC) patients. Correlation between imaging signals of 2 PET tracers and the corresponding target structures was assessed. The first tracer was [68Ga]Ga-PSMA (prostate-specific membrane antigen)-HBED-CC (N,N'-bis [2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid) [68Ga]Ga-PSMAHBED-CC ([68Ga]PSMA), which is already implemented in clinical routines. The second tracer was 16β-[18F]fluoro-5α-dihydrotestosterone (16β-[18F]FDHT), which binds to the androgen receptor (AR). The AR is particularly interesting in PC, because AR expression status and its shift during therapy might directly influence patient care. Methods: This prospective, explorative clinical study included 10 newly diagnosed PC patients. Each patient underwent [68Ga]PSMA PET/MRI and [18F]FDHT PET/MRI scans before prostatectomy. Cancer SUVs were determined and related to background SUVs. After prostatectomy, tumor tissue was sampled, and AR and prostate-specific membrane antigen (PSMA) expression was determined. AR and PSMA expression was evaluated quantitatively with the open-source bioimage analysis software QuPath and with a 4-tier rating system. Correlation between imaging signals and marker expression was statistically assessed. Results: For [18F]FDHT, the SUVmax/SUVbackground ratio showed a significant, strong correlation (r = 0.72; P = 0.019) with the AR optical density of the correlating tissue sample. The correlation between PSMA optical density and the [68Ga]PSMA SUVmax/SUVbackground ratio was not significant (P = 0.061), yet a positive correlation trend could be observed (r = 0.61). SUVmax/SUVbackground ratios were higher for [68Ga]PSMA (mean ± SD, 34.9 ± 24.8) than for [18F]FDHT (4.8 ± 1.2). In line with these findings, the tumor detection rates were 90% for the [68Ga]PSMA PET scan but only 40% for the [18F]FDHT PET scan. The 4-tier rating of PSMA staining intensity yielded very homogeneous results, with values of 3+ for most subjects (90%). AR staining was rated as 1+ in 2 patients (20%), 2+ in 4 patients (40%), and 3+ in 4 patients (40%). Conclusion: [18F]FDHT PET may be useful for monitoring AR expression and alterations in AR expression during treatment of PC patients. This approach may facilitate early detection of treatment resistance and allows for adaptation of therapy to prevent cancer progression. [18F]FDHT PET is inferior to [68Ga]PSMA PET for primary PC diagnosis, but the correlation between [68Ga]PSMA SUVs and PSMA expression is weaker than that between [18F]FDHT and the AR.

Background Prostate-specific membrane antigen (PSMA) PET is useful in the early detection of oligorecurrent prostate cancer (PCa), but whether PSMA PET parameters can be used to identify patients who would benefit from metastasis-directed therapy (MDT) with radiation or surgery remains uncertain. Purpose To assess the association of PSMA PET parameters with outcomes of patients with oligorecurrent PCa after MDT. Materials and Methods In this retrospective analysis of a single-center phase II trial that enrolled patients with biochemical recurrence of PCa after maximal local therapy and with no evidence of disease at conventional imaging, patients underwent PSMA PET (between May 2017 and November 2021), and unveiled recurrences were treated with MDT. Maximum standardized uptake value (SUVmax) and mean standardized uptake value (SUVmean) and PSMA tumor volume derived using thresholds of 2.5 (SUVmean2.5) and 41% (SUVmean41%), respectively, were recorded for sites of recurrence on PSMA PET scans, and a molecular imaging PSMA score was assigned. These parameters were also corrected for smooth filter and partial volume effects, and the PSMA score was reassigned. Cox proportional hazards models were used to evaluate the relationship between PSMA PET parameters and outcomes. Results A total of 74 men (mean age, 68.3 years ± 6.6 [SD]) with biochemical recurrence of PCa were included. PSMA PET revealed 145 lesions in the entire cohort, of which 125 (86%) were metastatic lymph nodes. Application of the correction factor changed the PSMA score in 88 of 145 lesions (61%). Mean SUVmax, SUVmean2.5, and SUVmean41% were associated with lower risk of biochemical progression (hazard ratio [HR] range, 0.77-0.95; 95% CI: 0.61, 1.00; P = .03 to P = .04). For corrected parameters, mean SUVmax, mean SUVmean2.5, mean SUVmean41%, mean PSMA score, maximum SUVmean2.5, maximum SUVmean41%, and maximum PSMA score were associated with a lower risk of biochemical progression (HR, 0.61-0.98; 95% CI: 0.39, 1.00; P = .01 to P = .04). Conclusion Measured and corrected PSMA PET parameters were associated with biochemical progression in men with oligorecurrent PCa treated with MDT. Clinical trial registration no. NCT03160794 © RSNA, 2023 See also the editorial by Civelek in this issue.

Prostate specific membrane antigen (PSMA) is a folate gamma glutamyl carboxypeptidase that is oriented on the plasma membrane of normal and prostate cancer cells. A cytosolic version of PSMA, PSM', results from alternative splicing of the PSMA gene. Two additional alternatively spliced variants of PSMA, PSM-C and PSM-D, have been described recently. The ratio of PSMA to PSM' mRNA was higher in a small number of prostate cancer specimens compared to normal prostate cancer and benign prostatic hypertrophy (Su et al. Cancer Res 1995;55:1441). The intent of our study was to measure the gene expression of PSMA and the 3 PSMA splice variants in a large number of patient's tissues. A real-time, quantitative PCR assay was developed to quantify PSMA, PSM', PSM-C and PSM-D. Discrimination among the variants was achieved by designing unique primers and TaqMan probes for each gene. Amplification and detection was specific for the desired splice variant and was sensitive to one gene copy per reaction. The assay was used to quantify the gene expression in specimens of normal, benign, primary and metastatic prostate cancer from 72 patients. The mean PSMA expression (relative to 18S rRNA) was 2- to 3-fold lower in normal prostate (n = 4) compared to primary (n = 55, p = 0.31) and metastatic (n = 20, p = 0.33) prostate cancer. There was no difference in the PSMA expression between benign and cancerous prostate tissue from the same patients (n = 35). The ratio of PSMA to PSM' was lowest in the normal prostate and increased with increasing Gleason score (p < 0.001). The increased ratio in these tissues was a reflection of both increasing PSMA levels and decreasing PSM' mRNA. The expression of PSM-C did not differ in any of the tissue categories studied. The expression of PSM-D was similar in normal and primary prostate cancer but was 2-fold higher in lymph node (p < 0.005) and bone metastases (p < 0.05) compared to the primary tumors. Our results of the first detailed quantitative analysis of PSMA mRNA expression in patient's tissues demonstrate that PSMA and the 3 PSMA splice variants are expressed in normal, benign, cancerous and metastatic prostate cancer. We note increased PSMA expression in some malignant tissues, however, these increases are modest in magnitude. We also report that the expression of a novel splice variant, PSM-D, is elevated in prostate cancer metastases.

Introduction: Prostate cancer (PCa) is the most common non-skin malignancy and the second leading cause of cancer related death in men. Prostate cancer is a heterogeneous disease with diverse clinical outcomes ranging from indolent asymptomatic cases to very aggressive and potentially lethal metastatic forms of the disease. Early detection of PCa is predominantly through determination of serum PSA levels and digital rectal examinations (DRE). Subsequently, biopsy based Gleason Grade Scores and TNM staging, tumor size or mpMRI are used to assess disease severity and progression. However, due to the low specificity of PSA concentration in diagnosis and prognosis, biopsy complications, and high mpMRI costs, there is still a critical need to identify and develop novel noninvasive biomarkers that discriminate indolent from aggressive PCa upon diagnosis. Our long-term objective is to discover biomarkers for aggressive PCa, develop and to implement them into robust clinical testing minimally invasive assays for PCa risk stratification at diagnosis. The current focus is on Prostate-Specific Membrane Antigen (PSMA), a membrane bound glycoprotein, that is significantly over-expressed in aggressive and metastatic prostate carcinomas and with poor patient prognosis. The role of its glycosylation status and composition in disease progression and its glycoforms on biomarker development and therapeutics is unknown. Aberrant glycosylation of glycoproteins occurs in many cancers, and it influences disease progression. We rationalize that differential glycosylation of PSMA correlate with progression to aggressive PCa and that PSMA glycopeptides and glycoforms can be used as diagnostics and prognosticators for aggressive disease. Methods/Results: PSMA was isolated from risk Grade Group stratified post-DRE urines, seminal plasma and prostate cancer cell lines with different metastatic phenotypes cell line lysates. Intact glycopeptides were isolated, purified and analyzed by qualitative and quantitative mass spectrometry to identify site specific glycoforms. Our data demonstrate differential expression of unique PSMA glycoforms that correlate with disease risk and severity. Conclusions: Differential glycosylation of PSMA correlates with PCa disease progression to aggressive phenotypes and may have utility in disease stratification. Ongoing studies focus on determining if these qualitative and quantitative changes have diagnostic/prognostic value and if these be integrated with other PCa biomarkers to improve their prognostic power in discriminating aggressive PCa. Acknowledgements: This work was supported by the DOD, USAMRAA, through the Prostate Cancer Research Program under Award No. W81XWH18-1-0228. Opinions, interpretations, and conclusions are those of the author and are not necessarily endorsed by the USAMRAA. Citation Format: Stephen Mackay, Ian O. Oduor, Tanya Burch, Dean A. Troyer, Oliver J. Semmes, Julius O. Nyalwidhe. Prostate specific membrane antigen (PSMA) glycoforms and prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1853.