33 research outputs found
DNA methylation patterns in bladder cancer and washing cell sediments: a perspective for tumor recurrence detection
<p>Abstract</p> <p>Background</p> <p>Epigenetic alterations are a hallmark of human cancer. In this study, we aimed to investigate whether aberrant DNA methylation of cancer-associated genes is related to urinary bladder cancer recurrence.</p> <p>Methods</p> <p>A set of 4 genes, including <it>CDH1 </it>(E-cadherin), <it>SFN </it>(stratifin), <it>RARB </it>(retinoic acid receptor, beta) and <it>RASSF1A </it>(Ras association (RalGDS/AF-6) domain family 1), had their methylation patterns evaluated by MSP (Methylation-Specific Polymerase Chain Reaction) analysis in 49 fresh urinary bladder carcinoma tissues (including 14 cases paired with adjacent normal bladder epithelium, 3 squamous cell carcinomas and 2 adenocarcinomas) and 24 cell sediment samples from bladder washings of patients classified as cancer-free by cytological analysis (control group). A third set of samples included 39 archived tumor fragments and 23 matched washouts from 20 urinary bladder cancer patients in post-surgical monitoring. After genomic DNA isolation and sodium bisulfite modification, methylation patterns were determined and correlated with standard clinic-histopathological parameters.</p> <p>Results</p> <p><it>CDH1 </it>and <it>SFN </it>genes were methylated at high frequencies in bladder cancer as well as in paired normal adjacent tissue and exfoliated cells from cancer-free patients. Although no statistically significant differences were found between <it>RARB </it>and <it>RASSF1A </it>methylation and the clinical and histopathological parameters in bladder cancer, a sensitivity of 95% and a specificity of 71% were observed for <it>RARB </it>methylation (Fisher's Exact test (p < 0.0001; OR = 48.89) and, 58% and 17% (p < 0.05; OR = 0.29) for <it>RASSF1A </it>gene, respectively, in relation to the control group.</p> <p>Conclusion</p> <p>Indistinct DNA hypermethylation of <it>CDH1 </it>and <it>SFN </it>genes between tumoral and normal urinary bladder samples suggests that these epigenetic features are not suitable biomarkers for urinary bladder cancer. However, <it>RARB </it>and <it>RASSF1A </it>gene methylation appears to be an initial event in urinary bladder carcinogenesis and should be considered as defining a panel of differentially methylated genes in this neoplasia in order to maximize the diagnostic coverage of epigenetic markers, especially in studies aiming at early recurrence detection.</p
Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples
Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts
Pharmacological upregulation of prostate-specific membrane antigen (PSMA) expression in prostate cancer cells
BACKGROUND Prostate-specific membrane antigen (PSMA)-based imaging and therapy are increasingly used for prostate cancer management. However, limitations are a low PSMA expression in certain patients. Androgen receptor axis inhibition can induce PSMA expression in vitro. We hypothesized that different approved compounds upregulate PSMA expression and tested their effect in vitro. METHODS Androgen receptor (AR) expressing prostate cancer (LNCaP) and epithelial prostate cells (PNT1A) were treated for 7 days with enzalutamide, dutasteride, rapamycin, metformin, lovastatin, and acetylsalicylic acid (ASA). PSMA and AR protein expression was assessed using flow cytometry, immunocytochemistry and immunoblotting. Furthermore, uptake and internalization of Lu-PSMA-617 was performed. RESULTS Enzalutamide and dutasteride led to a significant (both P < 0.05) upregulation of PSMA surface levels in LNCaP cells. In addition, treatment with rapamycin showed a non-significant trend toward PSMA upregulation. No changes were detected after treatment with vehicle, metformin, lovastatin, and ASA. Total PSMA protein expression was significantly enhanced after treatment with enzalutamide and rapamycin (both P < 0.05), whereas dutasteride led to a non-significant upregulation. Uptake of Lu-PSMA-617 was significantly increased after treatment of LNCaP with enzalutamide, dutasteride, and rapamycin (P < 0.05). In addition, internalization was significantly increased by enzalutamide and rapamycin (P < 0.05), and non-significantly increased by dutasteride. CONCLUSION In conclusion, our data provide new insights into the effect of different approved pharmacological compounds that can markedly upregulate PSMA expression and radioligand uptake in vitro. Pharmacologically induced PSMA expression may prove useful to improve prostate cancer detection and to enhance anticancer effects in PSMA-based therapy
Preclinical Development of Novel PSMA-Targeting Radioligands: Modulation of Albumin-Binding Properties To Improve Prostate Cancer Therapy
The treatment of metastatic castration-resistant
prostate cancer
(mCRPC) remains challenging with current treatment options. The development
of more effective therapies is, therefore, urgently needed. Targeted
radionuclide therapy with prostate-specific membrane antigen (PSMA)-targeting
ligands has revealed promising clinical results. In an effort to optimize
this concept, it was the aim of this study to design and investigate
PSMA ligands comprising different types of albumin binders. PSMA-ALB-53
and PSMA-ALB-56 were designed by combining the glutamate-urea-based
PSMA-binding entity, a DOTA chelator and an albumin binder based on
the 4-(<i>p</i>-iodophenyl)-moiety or <i>p</i>-(tolyl)-moiety. The compounds were labeled with <sup>177</sup>Lu
(50 MBq/nmol) resulting in radioligands of high radiochemical purity
(≥98%). Both radioligands were stable (≥98%) over 24
h in the presence of l-ascorbic acid. The uptake into PSMA-positive
PC-3 PIP tumor cells in vitro was in the same range (54–58%)
for both radioligands; however, <sup>177</sup>Lu-PSMA-ALB-53 showed
a 15-fold enhanced binding to human plasma proteins. Biodistribution
studies performed in PC-3 PIP/flu tumor-bearing mice revealed high
tumor uptake of <sup>177</sup>Lu-PSMA-ALB-53 and <sup>177</sup>Lu-PSMA-ALB-56,
respectively, demonstrated by equal areas under the curves (AUCs)
for both radioligands. The increased retention of <sup>177</sup>Lu-PSMA-ALB-53
in the blood resulted in almost 5-fold lower tumor-to-blood AUC ratios
when compared to <sup>177</sup>Lu-PSMA-ALB-56. Kidney clearance of <sup>177</sup>Lu-PSMA-ALB-56 was faster, and hence, the tumor-to-kidney
AUC ratio was 3-fold higher than in the case of <sup>177</sup>Lu-PSMA-ALB-53.
Due to the more favorable tissue distribution profile, <sup>177</sup>Lu-PSMA-ALB-56 was selected for a preclinical therapy study in PC-3
PIP tumor-bearing mice. The tumor growth delay after application of <sup>177</sup>Lu-PSMA-ALB-56 and <sup>177</sup>Lu-PSMA-617 applied at
the same activities (2 or 5 MBq per mouse) revealed better antitumor
effects in the case of <sup>177</sup>Lu-PSMA-ALB-56. As a consequence,
the survival of mice treated with <sup>177</sup>Lu-PSMA-ALB-56 was
prolonged when compared to the mice, which received the same activity
of <sup>177</sup>Lu-PSMA-617. Our results demonstrated the superiority
of <sup>177</sup>Lu-PSMA-ALB-56 over <sup>177</sup>Lu-PSMA-ALB-53
indicating that the <i>p</i>-(tolyl)-moiety was more suited
as an albumin binder to optimize the tissue distribution profile. <sup>177</sup>Lu-PSMA-ALB-56 was more effective to treat tumors than <sup>177</sup>Lu-PSMA-617 resulting in complete tumor remission in four
out of six mice. This promising results warrant further investigations
to assess the potential for clinical application of <sup>177</sup>Lu-PSMA-ALB-56
Albumin-Binding PSMA Ligands: Optimization of the Tissue Distribution Profile
The prostate-specific membrane antigen
(PSMA) has emerged as an attractive prostate cancer associated target
for radiotheragnostic application using PSMA-specific radioligands.
The aim of this study was to design new PSMA ligands modified with
an albumin-binding moiety in order to optimize their tissue distribution
profile. The compounds were prepared by conjugation of a urea-based
PSMA-binding entity, a DOTA chelator, and 4-(<i>p</i>-iodophenyl)butyric acid using multistep solid phase synthesis. The three ligands (PSMA-ALB-02,
PSMA-ALB-05, and PSMA-ALB-07) were designed with varying linker entities.
Radiolabeling with <sup>177</sup>Lu was performed at a specific activity
of up to 50 MBq/nmol resulting in radioligands of >98% radiochemical
purity and high stability. In vitro investigations revealed high binding
of all three PSMA radioligands to mouse (>64%) and human plasma
proteins (>94%). Uptake and internalization into PSMA-positive
PC-3 PIP tumor cells was equally high for all radioligands. Negligible
accumulation was found in PSMA-negative PC-3 flu cells, indicating
PSMA-specific binding of all radioligands. Biodistribution and imaging
studies performed in PC-3 PIP/flu tumor-bearing mice showed enhanced
blood circulation of the new radioligands when compared to the clinically
employed <sup>177</sup>Lu-PSMA-617. The PC-3 PIP tumor uptake of all
three radioligands was very high (76.4 ± 2.5% IA/g, 79.4 ±
11.1% IA/g, and 84.6 ± 14.2% IA/g, respectively) at 24 h post
injection (p.i.) resulting in tumor-to-blood ratios of ∼176,
∼48, and ∼107, respectively, whereas uptake into PC-3
flu tumors was negligible. Kidney uptake at 24 h p.i. was lowest for <sup>177</sup>Lu-PSMA-ALB-02 (10.7 ± 0.92% IA/g), while <sup>177</sup>Lu-PSMA-ALB-05 and <sup>177</sup>Lu-PSMA-ALB-07 showed higher renal
retention (23.9 ± 4.02% IA/g and 51.9 ± 6.34% IA/g, respectively). Tumor-to-background ratios calculated from values of the
area under the curve (AUC) of time-dependent biodistribution data
were in favor of <sup>177</sup>Lu-PSMA-ALB-02 (tumor-to-blood, 46;
tumor-to-kidney, 5.9) when compared to <sup>177</sup>Lu-PSMA-ALB-05
(17 and 3.7, respectively) and <sup>177</sup>Lu-PSMA-ALB-07 (39 and
2.1, respectively). The high accumulation of the radioligands in PC-3
PIP tumors was visualized on SPECT/CT images demonstrating increasing
tumor-to-kidney ratios over time. Taking all of the characteristics
into account, <sup>177</sup>Lu-PSMA-ALB-02 emerged as the most promising
candidate. The applied concept may be attractive for future clinical
translation potentially enabling more potent and convenient prostate
cancer radionuclide therapy
Terbium-161 for PSMA-targeted radionuclide therapy of prostate cancer
PURPOSE: The prostate-specific membrane antigen (PSMA) has emerged as an interesting target for radionuclide therapy of metastasized castration-resistant prostate cancer (mCRPC). The aim of this study was to investigate 161Tb (T1/2 = 6.89 days; Eβ͞av = 154 keV) in combination with PSMA-617 as a potentially more effective therapeutic alternative to 177Lu-PSMA-617, due to the abundant co-emission of conversion and Auger electrons, resulting in an improved absorbed dose profile.
METHODS: 161Tb was used for the radiolabeling of PSMA-617 at high specific activities up to 100 MBq/nmol. 161Tb-PSMA-617 was tested in vitro and in tumor-bearing mice to confirm equal properties, as previously determined for 177Lu-PSMA-617. The effects of 161Tb-PSMA-617 and 177Lu-PSMA-617 on cell viability (MTT assay) and survival (clonogenic assay) were compared in vitro using PSMA-positive PC-3 PIP tumor cells. 161Tb-PSMA-617 was further investigated in therapy studies using PC-3 PIP tumor-bearing mice.
RESULTS: 161Tb-PSMA-617 and 177Lu-PSMA-617 displayed equal in-vitro properties and tissue distribution profiles in tumor-bearing mice. The viability and survival of PC-3 PIP tumor cells were more reduced when exposed to 161Tb-PSMA-617 as compared to the effect obtained with the same activities of 177Lu-PSMA-617 over the whole investigated concentration range. Treatment of mice with 161Tb-PSMA-617 (5.0 MBq/mouse and 10 MBq/mouse, respectively) resulted in an activity-dependent increase of the median survival (36 vs 65 days) compared to untreated control animals (19 days). Therapy studies to compare the effects of 161Tb-PSMA-617 and 177Lu-PSMA-617 indicated the anticipated superiority of 161Tb over 177Lu.
CONCLUSION: 161Tb-PSMA-617 showed superior in-vitro and in-vivo results as compared to 177Lu-PSMA-617, confirming theoretical dose calculations that indicate an additive therapeutic effect of conversion and Auger electrons in the case of 161Tb. These data warrant more preclinical research for in-depth investigations of the proposed concept, and present a basis for future clinical translation of 161Tb-PSMA-617 for the treatment of mCRPC
Development of a new class of PSMA radioligands comprising ibuprofen as an albumin-binding entity
Prostate-specific membrane antigen (PSMA)-targeted radioligands have been used for the treatment of metastatic castration-resistant prostate cancer (mCRPC). Recently, albumin-binding PSMA radioligands with enhanced blood circulation were developed to increase the tumor accumulation of activity. The present study aimed at the design, synthesis and preclinical evaluation of a novel class of PSMA-targeting radioligands equipped with ibuprofen as a weak albumin-binding entity in order to improve the pharmacokinetic properties.
Methods: Four novel glutamate-urea-based PSMA ligands were synthesized with ibuprofen, conjugated via variable amino acid-based linker entities. The albumin-binding properties of the 177Lu-labeled PSMA ligands were tested in vitro using mouse and human plasma. Affinity of the radioligands to PSMA and cellular uptake and internalization was investigated using PSMA-positive PC-3 PIP and PSMA-negative PC-3 flu tumor cells. The tissue distribution profile of the radioligands was assessed in biodistribution and imaging studies using PC-3 PIP/flu tumor-bearing nude mice.
Results: The PSMA ligands were obtained in moderate yields at high purity (>99%). 177Lu-labeling of the ligands was achieved at up to 100 MBq/nmol with >96% radiochemical purity. In vitro assays confirmed high binding of all radioligands to mouse and human plasma proteins and specific uptake and internalization into PSMA-positive PC-3 PIP tumor cells. Biodistribution studies and SPECT/CT scans revealed high accumulation in PC-3 PIP tumors but negligible uptake in PC-3 flu tumor xenografts as well as rapid clearance of activity from background organs and tissues. 177Lu-Ibu-DAB-PSMA, in which ibuprofen was conjugated via a positively-charged diaminobutyric acid (DAB) entity, showed distinguished tumor uptake and the most favorable tumor-to-blood and tumor-to-kidney ratios.
Conclusion: The high accumulation of activity in the tumor and fast clearance from background organs was a common favorable characteristic of PSMA radioligands modified with ibuprofen as albumin-binding entity. 177Lu-Ibu-DAB-PSMA emerged as the most promising candidate; hence, more detailed preclinical investigations with this radioligand are warranted in view of a clinical translation.ISSN:1838-764
Concentration-dependent effects of dutasteride on prostate-specific membrane antigen (PSMA) expression and uptake of Lu-PSMA-617 in LNCaP cells
BACKGROUND
Prostate-specific membrane antigen (PSMA)-based imaging and therapy are increasingly used in the management of prostate cancer. However, low PSMA surface expression in certain patients is a limitation for PSMA-based technologies. We have previously shown that high doses of dutasteride, a 5α-reductase inhibitor generally used for the treatment of benign prostatic enlargement, increase the PSMA expression in vitro. We now further analyzed the concentration- and time-dependent effects of dutasteride in LNCaP cells.
METHODS
Androgen receptor (AR) expressing prostate cancer cells (LNCaP) were treated for 7 to 14 days with vehicle control (0.1% dimethyl sulfoxide) or different concentrations of dutasteride (0.25 , 0.5 , 1 , and 5 μM). In addition to cell proliferation, PSMA surface expression was assessed using flow cytometry (FACS) and immunocytochemistry. Total PSMA and AR expression was analyzed by capillary western immunoassay (WES). In addition, tumor cell uptake and internalization assays of Lu-PSMA-617 were performed.
RESULTS
Dutasteride treatment resulted in a significant upregulation of PSMA surface expression compared to vehicle control after 7 days in all tested concentrations. After 14 days a further, concentration-dependent increase of PSMA surface expression was detectable. Total PSMA protein expression significantly increased after treatment of cells with high concentrations of dutasteride using 5 μM for 7 or 14 days. However, when lower concentrations were used total PSMA expression was not significantly altered compared to vehicle control. Further testing revealed a dose-dependent increase in uptake and internalization of -PSMA-617 after 7 and 14 days. Though, a significantly increased uptake was only observed using a 5 μM dutasteride concentration for 7 days as well as 1 and 5 μM for 14 days.
CONCLUSION
Our investigations revealed a concentration- and time-dependent effect of dutasteride on PSMA expression and uptake of -PSMA-617 in LNCaP cells. A short-term treatment of patients with high doses of dutasteride might increase the detection rate of PSMA-based imaging and increase the effect of -PSMA-617 therapy via upregulation of PSMA expression
44Sc-PSMA-617 for radiotheragnostics in tandem with 177Lu-PSMA-617—preclinical investigations in comparison with 68Ga-PSMA-11 and 68Ga-PSMA-617
Background
The targeting of the prostate-specific membrane antigen (PSMA) is of particular interest for radiotheragnostic purposes of prostate cancer. Radiolabeled PSMA-617, a 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA)-functionalized PSMA ligand, revealed favorable kinetics with high tumor uptake, enabling its successful application for PET imaging (68Ga) and radionuclide therapy (177Lu) in the clinics. In this study, PSMA-617 was labeled with cyclotron-produced 44Sc (T 1/2 = 4.04 h) and investigated preclinically for its use as a diagnostic match to 177Lu-PSMA-617.
Results
44Sc was produced at the research cyclotron at PSI by irradiation of enriched 44Ca targets, followed by chromatographic separation. 44Sc-PSMA-617 was prepared under standard labeling conditions at elevated temperature resulting in a radiochemical purity of >97% at a specific activity of up to 10 MBq/nmol. 44Sc-PSMA-617 was evaluated in vitro and compared to the 177Lu- and 68Ga-labeled match, as well as 68Ga-PSMA-11 using PSMA-positive PC-3 PIP and PSMA-negative PC-3 flu prostate cancer cells. In these experiments it revealed similar in vitro properties to that of 177Lu- and 68Ga-labeled PSMA-617. Moreover, 44Sc-PSMA-617 bound specifically to PSMA-expressing PC-3 PIP tumor cells, while unspecific binding to PC-3 flu cells was not observed. The radioligands were investigated with regard to their in vivo properties in PC-3 PIP/flu tumor-bearing mice. 44Sc-PSMA-617 showed high tumor uptake and a fast renal excretion. The overall tissue distribution of 44Sc-PSMA-617 resembled that of 177Lu-PSMA-617 most closely, while the 68Ga-labeled ligands, in particular 68Ga-PSMA-11, showed different distribution kinetics. 44Sc-PSMA-617 enabled distinct visualization of PC-3 PIP tumor xenografts shortly after injection, with increasing tumor-to-background contrast over time while unspecific uptake in the PC-3 flu tumors was not observed.
Conclusions
The in vitro characteristics and in vivo kinetics of 44Sc-PSMA-617 were more similar to 177Lu-PSMA-617 than to 68Ga-PSMA-617 and 68Ga-PSMA-11. Due to the almost four-fold longer half-life of 44Sc as compared to 68Ga, a centralized production of 44Sc-PSMA-617 and transport to satellite PET centers would be feasible. These features make 44Sc-PSMA-617 particularly appealing for clinical application.ISSN:2191-219
Alpha-PET for Prostate Cancer: Preclinical investigation using 149Tb-PSMA-617
In this study, it was aimed to investigateTb-PSMA-617 for targeted α-therapy (TAT) using a mouse model of prostate-specific membrane antigen (PSMA)-expressing prostate cancer.Tb-PSMA-617 was prepared with >98% radiochemical purity (6 MBq/nmol) for the treatment of mice with PSMA-positive PC-3 PIP tumors.Tb-PSMA-617 was applied at 1 × 6 MBq (Day 0) or 2 × 3 MBq (Day 0 & Day 1 or Day 0 & Day 3) and the mice were monitored over time until they had reached a pre-defined endpoint which required euthanasia. The tumor growth was significantly delayed in mice of the treated groups as compared to untreated controls (p < 0.05). TAT was most effective in mice injected with 2 × 3 MBq (Day 0 & 1) resulting in a median lifetime of 36 days, whereas in untreated mice, the median lifetime was only 20 days. Due to the β-emission ofTb, tumor localization was feasible using PET/CT after injection ofTb-PSMA-617 (5 MBq). The PET images confirmed the selective accumulation ofTb-PSMA-617 in PC-3 PIP tumor xenografts. The unique characteristics ofTb for TAT make this radionuclide of particular interest for future clinical translation, thereby, potentially enabling PET-based imaging to monitor the radioligand’s tissue distribution