Constantinos Zamboglou

Clostridial glucosylating toxins enter cells via clathrin-mediated endocytosis.

Clostridium difficile toxin A (TcdA) and toxin B (TcdB), C. sordellii lethal toxin (TcsL) and C. novyi α-toxin (TcnA) are important pathogenicity factors, which represent the family of the clostridial glucosylating toxins (CGTs). Toxin A and B are associated with antibiotic-associated diarrhea and pseudomembraneous colitis. Lethal toxin is involved in toxic shock syndrome after abortion and α-toxin in gas gangrene development. CGTs enter cells via receptor-mediated endocytosis and require an acidified endosome for translocation of the catalytic domain into the cytosol. Here we studied the endocytic processes that mediate cell internalization of the CGTs. Intoxication of cells was monitored by analyzing cell morphology, status of Rac glucosylation in cell lysates and transepithelial resistance of cell monolayers. We found that the intoxication of cultured cells by CGTs was strongly delayed when cells were preincubated with dynasore, a cell-permeable inhibitor of dynamin, or chlorpromazine, an inhibitor of the clathrin-dependent endocytic pathway. Additional evidence about the role of clathrin in the uptake of the prototypical CGT family member toxin B was achieved by expression of a dominant-negative inhibitor of the clathrin-mediated endocytosis (Eps15 DN) or by siRNA against the clathrin heavy chain. Accordingly, cells that expressed dominant-negative caveolin-1 were not protected from toxin B-induced cell rounding. In addition, lipid rafts impairment by exogenous depletion of sphingomyelin did not decelerate intoxication of HeLa cells by CGTs. Taken together, our data indicate that the endocytic uptake of the CGTs involves a dynamin-dependent process that is mainly governed by clathrin.

Comparison of (68)Ga-HBED-CC PSMA-PET/CT and multiparametric MRI for gross tumour volume detection in patients with primary prostate cancer based on slice by slice comparison with histopathology.

Purpose: The exact detection and delineation of the intraprostatic tumour burden is crucial for treatment planning in primary prostate cancer (PCa). We compared 68Ga-HBED-CC-PSMA PET/CT with multiparametric MRI (mpMRI) for diagnosis and tumour delineation in patients with primary PCa based on slice by slice correlation with histopathological reference material. Methodology: Seven patients with histopathologically proven primary PCa underwent 68Ga-HBED-CC-PSMA PET/CT and MRI followed by radical prostatectomy. Resected prostates were scanned by ex-vivo CT in a special localizer and prepared for histopathology. Invasive PCa was delineated on a HE stained histologic tissue slide and matched to ex-vivo CT to obtain gross tumor volume (GTV-)histo. Ex-vivo CT including GTV-histo and MRI data were matched to in-vivo CT(PET). Consensus contours based on MRI (GTV-MRI), PSMA PET (GTV-PET) or the combination of both (GTV-union/-intersection) were created. In each in-vivo CT slice the prostate was separated into 4 equal segments and sensitivity and specificity for PSMA PET and mpMRI were assessed by comparison with histological reference material. Furthermore, the spatial overlap between GTV-histo and GTV-PET/-MRI and the Sørensen-Dice coefficient (DSC) were calculated. In the case of multifocal PCa (4/7 patients), SUV values (PSMA PET) and ADC-values (diffusion weighted MRI) were obtained for each lesion. Results: PSMA PET and mpMRI detected PCa in all patients. GTV-histo was detected in 225 of 340 segments (66.2%). Sensitivity and specificity for GTV-PET, GTV-MRI, GTV-union and GTV-intersection were 75% and 87%, 70% and 82%, 82% and 67%, 55% and 99%, respectively. GTV-histo had on average the highest overlap with GTV-union (57±22%), which was significantly higher than overlap with GTV-MRI (p=0.016) and GTV-PET (p=0.016), respectively. The mean DSC for GTV-union, GTV-PET and GTV-MRI was 0.51 (±0.18), 0.45 (±0.17) and 0.48 (±0.19), respectively. In every patient with multifocal PCa there was one lesion which had both the highest SUV and the lowest ADC-value (mean and max). Conclusion: In a slice by slice analysis with histopathology, 68Ga-HBED-CC-PSMA PET/CT and mpMRI showed high sensitivity and specificity in detection of primary PCa. A combination of both methods performed even better in terms of sensitivity (GTV-union) and specificity (GTV-intersection). A moderate to good spatial overlap with GTV-histo was observed for PSMA PET/CT and mpMRI alone which was significantly improved by GTV-union. Further studies are warranted to analyse the impact of these preliminary findings for diagnostic (multimodal guided TRUS biopsy) and therapeutic (focal therapy) strategies in primary PCa.

68Ga-HBED-CC-PSMA PET/CT Versus Histopathology in Primary Localized Prostate Cancer: A Voxel-Wise Comparison

Purpose: We performed a voxel-wise comparison of 68Ga-HBED-CC-PSMA PET/CT with prostate histopathology to evaluate the performance of 68Ga-HBED-CC-PSMA for the detection and delineation of primary prostate cancer (PCa). Methodology: Nine patients with histopathological proven primary PCa underwent 68Ga-HBED-CC-PSMA PET/CT followed by radical prostatectomy. Resected prostates were scanned by ex-vivo CT in a special localizer and histopathologically prepared. Histopathological information was matched to ex-vivo CT. PCa volume (PCa-histo) and non-PCa tissue in the prostate (NPCa-histo) were processed to obtain a PCa-model, which was adjusted to PET-resolution (histo-PET). Each histo-PET was coregistered to in-vivo PSMA-PET/CT data. Results: Analysis of spatial overlap between histo-PET and PSMA PET revealed highly significant correlations (p < 10-5) in nine patients and moderate to high coefficients of determination (R²) from 42 to 82 % with an average of 60 ± 14 % in eight patients (in one patient R2 = 7 %). Mean SUVmean in PCa-histo and NPCa-histo was 5.6 ± 6.1 and 3.3 ± 2.5 (p = 0.012). Voxel-wise receiver-operating characteristic (ROC) analyses comparing the prediction by PSMA-PET with the non-smoothed tumor distribution from histopathology yielded an average area under the curve of 0.83 ± 0.12. Absolute and relative SUV (normalized to SUVmax) thresholds for achieving at least 90 % sensitivity were 3.19 ± 3.35 and 0.28 ± 0.09, respectively. Conclusions: Voxel-wise analyses revealed good correlations of 68Ga-HBED-CC-PSMA PET/CT and histopathology in eight out of nine patients. Thus, PSMA-PET allows a reliable detection and delineation of PCa as basis for PET-guided focal therapies.

Evaluation of intensity modulated radiation therapy dose painting for localized prostate cancer using 68Ga-HBED-CC PSMA-PET/CT: A planning study based on histopathology reference

PURPOSE To demonstrate the feasibility and to evaluate the tumour control probability (TCP) and normal tissue complication probability (NTCP) of IMRT dose painting using 68Ga-HBED-CC PSMA PET/CT for target delineation in prostate cancer (PCa). METHODS AND MATERIALS 10 patients had PSMA PET/CT scans prior to prostatectomy. GTV-PET was generated on the basis of an intraprostatic SUVmax of 30%. Two IMRT plans were generated for each patient: Plan77 which consisted of whole-prostate IMRT to 77Gy, and Plan95 which consisted of whole-prostate IMRT to 77Gy and a simultaneous integrated boost to the GTV-PET up to 95Gy (35 fractions). The feasibility of these plans was judged by their ability to adhere to the FLAME trial protocol. TCP-histo/-PET were calculated on co-registered histology (GTV-histo) and GTV-PET, respectively. NTCPs for rectum and bladder were calculated. RESULTS All plans reached prescription doses whilst adhering to dose constraints. In Plan77 and Plan95 mean doses in GTV-histo were 75.8±0.3Gy and 96.9±1Gy, respectively. Average TCP-histo values for Plan77 and Plan95 were 70% (range: 15-97%), and 96% (range: 78-100%, p<0.0001). Average TCP-PET values for Plan77 and Plan95 were 55% (range: 27-82%), and 100% (range: 99-100%, p<0.0001). There was no significant difference between TCP-PET and TCP-histo in Plan95 (p=0.25). There were no significant differences in rectal (p=0.563) and bladder (p=0.3) NTCPs. CONCLUSIONS IMRT dose painting using PSMA PET/CT was technically feasible and resulted in significantly higher TCPs without higher NTCPs.

Single fraction multimodal image guided focal salvage high-dose-rate brachytherapy for recurrent prostate cancer.

Purpose We present a novel method for treatment of locally recurrent prostate cancer (PCa) following radiation therapy: focal, multimodal image guided high-dose-rate (HDR) brachytherapy. Material and methods We treated two patients with recurrent PCa after primary (#1) or adjuvant (#2) external beam radiation therapy. Multiparametric magnetic resonance imaging (mpMRI), choline, positron emission tomography combined with computed tomography (PET/CT), or prostate-specific membrane antigen (PSMA)-PET combined with CT identified a single intraprostatic lesion. Positron emission tomography or magnetic resonance imaging – transrectal ultrasound (MRI-TRUS) fusion guided transperineal biopsy confirmed PCa within each target lesion. We defined a PET and mpMRI based gross tumor volume (GTV). A 5 mm isotropic margin was applied additionally to each lesion to generate a planning target volume (PTV), which accounts for technical fusion inaccuracies. A D90 of 18 Gy was intended in one fraction to each PTV using ultrasound guided HDR brachytherapy. Results Six month follow-up showed adequate prostate specific antygen (PSA) decline in both patients (ΔPSA 83% in patient 1 and ΔPSA 59.3% in patient 2). Follow-up 3-tesla MRI revealed regressive disease in both patients and PSMA-PET/CT showed no evidence of active disease in patient #1. No acute or late toxicities occurred. Conclusions Single fraction, focal, multimodal image guided salvage HDR brachytherapy for recurrent prostate cancer is a feasible therapy for selected patients with single lesions. This approach has to be evaluated in larger clinical trials.

Outcome after PSMA PET/CT based salvage radiotherapy in patients with biochemical recurrence after radical prostatectomy: a bi-institutional retrospective analysis

Prostate-specific membrane antigen (PSMA) PET/CT detects prostate cancer recurrence at low levels of prostate-specific antigen (PSA). Radiotherapy with dose escalation to the former prostate bed has been associated with improved biochemical recurrence-free survival (BRFS). Thus, we hypothesized that PSMA PET/CT-guided salvage radiotherapy leads to improved BRFS. Methods: In total, 204 consecutive patients were referred for salvage radiotherapy after radical prostatectomy. PSMA PET/CT scans were performed, and patients with PSA persistence (109 patients) or evidence of distant metastases (5 patients) were excluded from this analysis. Thus, the following analysis is based on a total of 90 patients who underwent PSMA PET/CT before radiotherapy due to biochemical recurrence and received salvage radiotherapy. In cases of PET-positive findings, antiandrogen therapy was commenced before initiation of radiotherapy. BRFS (PSA ≤ 0.2 ng/mL) was defined as the study endpoint. Results: PET-positive lesions were detected in 42 of 90 (47%) patients, 24 of 42 (27%) being fossa recurrence only, 12 of 42 (13%) pelvic lymph node only, and 6 of 42 (7%) both fossa and pelvic lymph node. The median PSA before radiotherapy was 0.44 ng/mL (range, 0.11–6.24 ng/mL). Cumulatively, a total dose of 70.0 Gy (range, 67.2–72 Gy) was delivered to local macroscopic tumor, 66 Gy (range, 59.4–70.2 Gy) to the prostatic fossa, 60.8 Gy (range, 54–66 Gy) to PET-positive lymph nodes, and 50.4 Gy (range, 45–50.4 Gy) to the lymphatic pathways. After a median follow-up of 23 mo, BRFS was 78%. Antiandrogen therapy was ongoing in 4 patients at the last follow-up. No significant difference in BRFS between PET-positive patients (74%) and PET-negative patients (82%; P > 0.05) was observed at the last follow-up. Two patients had late genitourinary toxicity, grade 3, and no patient had gastrointestinal toxicity of grade 3 or higher (National Cancer Institute common terminology criteria for adverse events, version 4.03). Conclusion: PSMA PET/CT-guided salvage radiotherapy is an effective and safe local treatment option. No difference in BRFS between PET-positive and PET-negative patients was observed, indicating effective targeting of PET-positive lesions. PSMA PET/CT when readily available should be offered to patients with PSA recurrence for treatment individualization.

Kombination aus perkutaner Strahlentherapie, Brachytherapie und Androgendeprivation gleichwertige Alternative zur radikalen Prostatektomie beim Prostatakarzinom

Patienten und Methode In die multizentrische, retrospektive Analyse wurden 487 Patienten mit bioptisch gesichertem PCa mit einem Gleason-Score von 9 und 10 eingeschlossen. Eine RP erhielten 170 Patienten, eine EBRT±Androgendeprivation (ADT) 230 Patienten und eine EBRT+BT±ADT 87 Patienten. Von den 87 Patienten in der BT-Gruppe erhielten 84 eine High-doserate(HDR)-BT mit 24Gy (192Ir) in 6 Fraktionen und 3 Patienten eine Low-dose-rate(LDR)-BT mit 108Gy (125I). In der EBRT-Gruppe wurde eine mediane Äquivalenzdosis (EQD2α/β = 1,5 Gy) von 76,4Gy (Spanne 65–80Gy) appliziert, in der EBRT+BT-Gruppe eine solche (EQD2α/β = 1,5 Gy) von 88,7Gy (Spanne 81,9–98,9Gy). Mithilfe von KaplanMeier-Analysen (nicht adjustiert) und multivariaten CoxRegressionsanalysen (adjustiert auf klinische Parameter) wurden nach 5 und 10 Jahren die Raten an biochemischen Rezidiven (BR), fernmetastasenfreiem Überleben (FFÜ), PCa-spezifischem Überleben (PSÜ) und Gesamtüberleben (OS) bestimmt und verglichen.

Combined high dose rate brachytherapy and external beam radiotherapy for clinically localised prostate cancer

PURPOSE To report the clinical outcomes and treatment-related toxicities after combined high-dose-rate (HDR) brachytherapy (BRT) with external beam radiotherapy (EBRT) for patients with clinically localised high-risk prostate cancer. MATERIAL AND METHODS Between 2008 and 2012, three hundred and three consecutive patients with organ-confined high-risk prostate cancer were treated with definitive radiotherapy consisting of HDR-BRT followed by supplemental EBRT. The transrectal 3D-ultrasound-based HDR-BRT boost consisted of two single-fraction implants of 10.5 Gy, prescribed to the 90% of the gland (D90), for a total physical dose of 21.0 Gy delivered to the prostatic gland. EBRT was delivered with conventional fractionation, prescribing 45.0 Gy to the prostatic gland and seminal vesicles. Biochemical failure was defined according to the Phoenix Consensus Criteria, genitourinary (GU)/gastrointestinal (GI) toxicity was evaluated using the Common Toxicity Criteria for Adverse Events (version 3.0). RESULTS The median follow-up was 71.6 months. The 7-year overall survival, biochemical control and metastasis-free-survival rates for the entire cohort were 85.7%, 88.3% and 93.8%, respectively. Androgen deprivation therapy was initiated prior to treatment for 92.7% of patients with a median duration of 12 months. Toxicity was scored per event with late Grade 2, 3 and 4 GU adverse events and was found to be 15.3%, 2.2% and 0.3%, respectively. Late Grade 2 GI toxicity accounted for 0.3% with no instances of Grade 3 or higher late adverse events. CONCLUSION HDR-BRT with supplemental EBRT results in low biochemical relapse-free survival rates associated with a very low incidence of higher-grade late adverse events.

PSMA-PET based radiotherapy: a review of initial experiences, survey on current practice and future perspectives

Abstract68Gallium prostate specific membrane antigen (PSMA) ligand positron emission tomography (PET) is an increasingly used imaging modality in prostate cancer, especially in cases of tumor recurrence after curative intended therapy. Owed to the novelty of the PSMA-targeting tracers, clinical evidence on the value of PSMA-PET is moderate but rapidly increasing. State of the art imaging is pivotal for radiotherapy treatment planning as it may affect dose prescription, target delineation and use of concomitant therapy.This review summarizes the evidence on PSMA-PET imaging from a radiation oncologist’s point of view. Additionally a short survey containing twelve examples of patients and 6 additional questions was performed in seven mayor academic centers with experience in PSMA ligand imaging and the findings are reported here.

Biological imaging for individualized therapy in radiation oncology: part I physical and technical aspects

Recently, there has been an increase in the imaging modalities available for radiotherapy planning and radiotherapy prognostic outcome: dual energy computed tomography (CT), dynamic contrast enhanced CT, dynamic contrast enhanced magnetic resonance imaging (MRI), diffusion-weighted MRI, positron emission tomography-CT, dynamic contrast enhanced ultrasound, MR spectroscopy and positron emission tomography-MR. These techniques enable more precise gross tumor volume definition than CT alone and moreover allow subvolumes within the gross tumor volume to be defined which may be given a boost dose or an individual voxelized dose prescription may be derived. With increased plan complexity care must be taken to immobilize the patient in an accurate and reproducible manner. Moreover the physical and technical limitations of the entire treatment planning chain need to be well characterized and understood, interdisciplinary collaboration ameliorated (physicians and physicists within nuclear medicine, radiology and radiotherapy) and image protocols standardized.