Proteomic distinction of renal oncocytomas and chromophobe renal cell carcinomas.

BACKGROUND: Renal oncocytomas (ROs) are benign epithelial tumors of the kidney whereas chromophobe renal cell carcinoma (chRCCs) are malignant renal tumors. The latter constitute 5-7% of renal neoplasias. ROs and chRCCs show pronounced molecular and histological similarities, which renders their differentiation demanding. We aimed for the differential proteome profiling of ROs and early-stage chRCCs in order to better understand distinguishing protein patterns. METHODS: We employed formalin-fixed, paraffin-embedded samples (six RO cases, six chRCC cases) together with isotopic triplex dimethylation and a pooled reference standard to enable cohort-wide quantitative comparison. For lysosomal-associated membrane protein 1 (LAMP1) and integrin alpha-V (ITGAV) we performed corroborative immunohistochemistry (IHC) in an extended cohort of 42 RO cases and 31 chRCC cases. RESULTS: At 1% false discovery rate, we identified > 3900 proteins, of which > 2400 proteins were consistently quantified in at least four RO and four chRCC cases. The proteomic expression profiling discriminated ROs and chRCCs and highlighted established features such as accumulation of mitochondrial proteins in ROs together with emphasizing the accumulation of endo-lysosomal proteins in chRCCs. In line with the proteomic data, IHC showed enrichment of LAMP1 in chRCC and of ITGAV in RO. CONCLUSION: We present one of the first differential proteome profiling studies on ROs and chRCCs and highlight differential abundance of LAMP1 and ITGAV in these renal tumors.

Performance of 111In-labelled PSMA ligand in patients with nodal metastatic prostate cancer: correlation between tracer uptake and histopathology from lymphadenectomy.

PURPOSE: Intraoperative identification of lymph node (LN) metastases (LNM) detected on preoperative PSMA PET/CT may be facilitated by PSMA radioguided surgery with the use of a gamma probe. We evaluated the uptake of 111In-labelled PSMA ligand DKFZ-617 (referred to as 111In-PSMA-617) in unaffected LN and LNM at the level of single LN. METHODS: Six patients with prostate cancer (PCa) with suspicion of LNM on preoperative PSMA PET/CT underwent 111In-PSMA-617-guided lymphadenectomy (LA; four salvage LA and two primary LA). 111In-PSMA-617 (109 ± 5 MBq). was injected Intravenously 48 h prior to surgery Template LAs were performed in small subregions: common, external, obturator and internal iliac vessels, and presacral and retroperitoneal subregions (n = 4). Samples from each subregion were isolated aiming at the level of single LN. Uptake was measured ex situ using a germanium detector. Receiver operating characteristic (ROC) analysis was performed based on 111In-PSMA-617 uptake expressed as standardized uptake values normalized to lean body mass (SUL). RESULTS: Overall 310 LN (mean 52 ± 19.7) were removed from 74 subregions (mean 12 ± 3.7). Of the 310 LN, 35 turned out to be LNM on histopathology. Separation of the samples from all subregions resulted in 318 single specimens: 182 PCa-negative LN samples with 275 LN, 35 single LNM samples, 3 non-nodal PCa tissue samples and 98 fibrofatty tissue samples. The median SULs of nonaffected LN (0.16) and affected LN (13.2) were significantly different (p < 0.0001). Based on 38 tumour-containing and 182 tumour-free specimens, ROC analysis revealed an area under the curve of 0.976 (95% CI 0.95-1.00, p < 0.0001). Using a SUL cut-off value of 1.136, sensitivity, specificity, positive predictive value, negative predictive value and accuracy in discriminating affected from nonaffected LN were 92.1% (35/38), 98.9% (180/182), 94.6% (35/37), 98.4% (180/183) and 97.7% (215/220), respectively. CONCLUSION: Ex situ analysis at the level of single LN showed that 111In-PSMA-617 had excellent ability to discriminate between affected and nonaffected LN in our patients with PCa. This tracer characteristic is a prerequisite for in vivo real-time measurements during surgery.

IgG4-related disease in autoimmune lymphoproliferative syndrome.

A patient with autoimmune lymphoproliferative disorder (ALPS) developed IgG4-related disease. In retrospect, he had high levels of serum IgG4 for several years prior to presenting with IgG4-related pancreatitis. These high IgG4 levels were masked by hypergammaglobulinemia, a common feature of ALPS. We next screened 18 ALPS patients; four of them displayed increased levels of IgG4. Hence, IgG4-related disease should be considered in ALPS patients, especially in those manifesting lymphocytic organ infiltration or excessive hypergammaglobulinaemia. Screening of IgG4-related disease patients for ALPS-associated mutations would provide further information on whether this disease could be a late-onset atypical presentation of ALPS.

Diagnosis of recurrent prostate cancer with PET/CT imaging using the gastrin-releasing peptide receptor antagonist 68Ga-RM2: Preliminary results in patients with negative or inconclusive [18F]Fluoroethylcholine-PET/CT.

PURPOSE/BACKGROUND: [18F]fluoroethylcholine (18FECH) has been shown to be a valuable PET-tracer in recurrent prostate cancer (PCa), but still has limited accuracy. RM2 is a gastrin-releasing peptide receptor (GRPr) antagonist that binds to GRPr on PCa cells. Recent studies suggest that GRPr imaging with PET/CT is a promising technique for staging and restaging of PCa. We explore the value of GRPr-PET using the 68Ga-labeled GRPr antagonist RM2 in a selected population of patients with biochemically recurrent PCa and a negative/inconclusive 18FECH-PET/CT. MATERIAL AND METHODS: In this retrospective study 16 men with biochemical PCa relapse and negative (n = 14) or inconclusive (n = 2) 18FECH-PET/CT underwent whole-body 68Ga-RM2-PET/CT. Mean time from 18FECH-PET/CT to 68Ga-RM2-PET/CT was 6.1 ± 6.8 months. Primary therapies in these patients were radical prostatectomy (n = 13; 81.3%) or radiotherapy (n = 3; 18.7%). 14/16 patients (87.5%) had already undergone salvage therapies because of biochemical relapse prior to 68Ga-RM2-PET/CT imaging. Mean ± SD PSA at 68Ga-RM2-PET/CT was 19.4 ± 53.5 ng/ml (range 1.06-226.4 ng/ml). RESULTS: 68Ga-RM2-PET/CT showed at least one region with focal pathological uptake in 10/16 patients (62.5%), being suggestive of local relapse (n = 4), lymph node metastases (LNM; n = 4), bone metastases (n = 1) and lung metastasis with hilar LNM (n = 1). Seven of ten positive 68Ga-RM2 scans were positively confirmed by surgical resection and histology of the lesions (n = 2), by response to site-directed therapies (n = 2) or by further imaging (n = 3). Patients with a positive 68Ga-RM2-scan showed a significantly higher median PSA (6.8 ng/ml, IQR 10.2 ng/ml) value than those with a negative scan (1.5 ng/ml, IQR 3.1 ng/ml; p = 0.016). Gleason scores or concomitant antihormonal therapy had no apparent impact on the detection of recurrent disease. CONCLUSION: Even in this highly selected population of patients with known biochemical recurrence but negative or inconclusive 18FECH-PET/CT, a 68Ga-RM2-PET/CT was helpful to localize PCa recurrence in the majority of the cases. Thus, 68Ga-RM2-PET/CT deserves further investigation as a promising imaging modality for imaging PCa recurrence.

Diabetes and beta-adrenergic blockage are risk factors for metastatic prostate cancer.

BACKGROUND: We evaluated the influence of comorbidity inferred risks for lymph node metastasis (pN1) and positive surgical margins (R1) after radical prostatectomy in order to optimize pretherapeutic risk classification. We analyzed 454 patients after radical prostatectomy (RP) between 2009 and 2014. Comorbidities were defined by patients' medication from our electronic patient chart and stratified according to the ATC WHO code. Endpoints were lymph node metastasis (pN1) and positive surgical margins (R1). RESULTS: Rates for pN1 and R1 were 21.4% (97/454) and 29.3% (133/454), respectively. In addition to CAPRA and Gleason score, we identified diabetes as a significant medication inferred risk factor for pN1 (OR 2.9, p = 0.004/OR 3.2, p = 0.001/OR 3.5, p = 0.001) and beta-blockers for R1 (OR 1.9, p = 0.020/OR 2.9, p = 0.004). Patients with diabetes showed no statistically significant difference in Gleason score, CAPRA Score, PSA, and age compared to non-diabetic patients. CONCLUSIONS: We identified diabetes and beta1 adrenergic blockage as significant risk factors for lymph node metastasis and positive surgical margins in prostate cancer (PCa). Patients at risk will need intensive pretherapeutic staging for optimal therapeutic stratification.

Adjuvant radiotherapy after salvage lymph node dissection because of nodal relapse of prostate cancer versus salvage lymph node dissection only.

BACKGROUND: Nodal pelvic/retroperitoneal recurrent prostate cancer (PCa) after primary therapy can be treated with salvage lymph node dissection (salvage-LND) in order to delay disease progression and offer cure for a subset of patients. Whether adjuvant radiotherapy (ART) in affected regions improves the outcome by elimination of residual tumour burden remains unclear. METHODS: A total of 93 patients with exclusively nodal PCa relapse underwent choline-positron-emission tomography-computed-tomography-directed pelvic/retroperitoneal salvage-LND; 46 patients had surgery only and 47 patients received ART in regions with proven lymph node metastases. In case of subsequent prostate specific antigen (PSA) progression, different imaging modalities were performed to confirm next relapse within or outside the treated region (TR). Mean follow-up was 3.2 years. RESULTS: Lymphatic tumour burden was balanced between the two groups. Additional ART resulted in delayed relapse within TR (5-year relapse-free rate 70.7 %) versus surgery only (5-year relapse-free rate 26.3 %, p < 0.0001). In both treatment arms, time to next relapse outside the TR was almost equal (median 27 months versus 29.6 months, p = 0.359). With respect to the detection of the first new lesion, regardless if present within or outside the TR, 5 years after the treatment 34.3 % of patients in the group with additional ART were free of relapse, versus 15.4 % in the surgery only group (p = 0.0122). ART had no influence on the extent of PSA reduction at latest follow-up compared to treatment with surgery only. CONCLUSION: ART after salvage-LND provides stable local control in TR and results in overall significant improved next-relapse-free survival, compared to patients who received surgery only in case of nodal PCa-relapse.

Detection of lymph node metastasis in patients with nodal prostate cancer relapse using (18)F/(11)C-choline positron emission tomography/computerized tomography.

PURPOSE: We evaluated the diagnostic accuracy of choline positron emission tomography/computerized tomography for nodal relapse of prostate cancer according to topographical site and tumor infiltration size in lymph nodes. MATERIALS AND METHODS: A total of 72 patients with nodal prostate cancer relapse after primary therapy underwent pelvic and/or retroperitoneal salvage lymph node dissection. Salvage was done after whole body positron emission tomography/computerized tomography with (11)C-choline or (18)F-fluoroethylcholine showed positron emission tomography positive lymph nodes but no other detectable metastasis. Diagnostic accuracy was evaluated in 160 dissected lymph node regions (pelvic left/right and retroperitoneal), 498 subregions (common, external and internal iliac, obturator, presacral, aortic bifurcation, aortal, vena caval and interaortocaval) and 2,122 lymph nodes. RESULTS: Lymph node metastasis was present in 32% of resected lymph nodes (681 of 2,122), resulting in 238 positive subregions and 111 positive regions. Positron emission tomography/computerized tomography was positive for 110 regions and 209 subregions. Sensitivity, specificity, positive and negative predictive values, and accuracy were 91.9%, 83.7%, 92.7%, 82.0% and 89.4% (region based), 80.7%, 93.5%, 91.9%, 84.1% and 87.3% (subregion based), and 57.0%, 98.4%, 94.5%, 82.6% and 84.9% (lesion based), respectively. Of 393 positive lymph node metastases detected by this method 278 (70.7%) were in lymph nodes with a less than 10 mm short axis diameter. Imaging sensitivity was 13.3%, 57.4% and 82.8% for a tumor infiltration depth of 2 or greater to less than 3 mm, 5 or greater to less than 6 mm and 10 or greater to less than 11 mm, respectively. Lymph node metastasis site and the radiotracer ((11)C-choline/(18)F-fluoroethylcholine) had no substantial impact on diagnostic accuracy. CONCLUSIONS: Choline positron emission tomography/computerized tomography detects affected lymph node regions (pelvic left/right and retroperitoneal) in patients with prostate cancer relapse with high accuracy and it seems helpful for guiding salvage lymph node dissection. Sensitivity decreases with the size of metastatic infiltration in lymph nodes. This technique detects metastasis in a significant fraction of lymph nodes that are not pathologically enlarged on computerized tomography.

Autosomal dominant polycystic kidney disease: prevalence of renal neoplasias in surgical kidney specimens.

BACKGROUND: The role of autosomal dominant polycystic kidney disease (ADPKD) as a risk factor for renal cell carcinoma (RCC) is still under discussion. Data on prevalence of RCC in ADPKD are limited, especially on a large population scale. The aim of this study was to analyze the prevalence of RCC in ADPKD kidneys and characterize the clinical features of this coincidence. METHODS: Based on our histopathological registry for ADPKD and the Else Kröner-Fresenius Registry, we retrospectively reviewed malignant and benign renal lesions in patients with ADPKD who had undergone renal surgery from 1988 to 2011. RESULTS: 240 ADPKD patients underwent 301 renal surgeries. Mean age at surgery was 54 years. Overall, 16 malignant and 11 benign lesions were analyzed in 301 kidneys (5.3%; 3.7%), meaning that 12/240 (5%; 1:20) patients presented with malignant renal lesions. 66.7% (8/12) of these patients had undergone dialysis prior to surgery. We found 10/16 (63%) papillary RCC, 5/16 (31%) clear cell RCC, and 1/16 (6%) papillary noninvasive urothelial cancer. Regarding all renal lesions, 6/17 (35.3%) patients had more than one histological finding in their kidneys. In 2 cases, metachronous metastases were removed. Mean follow-up was 66.7 months. CONCLUSION: Kidney-related prevalence of RCC in ADPKD kidneys was surprisingly high. Whether or not this is due to chronic dialysis or due to the underlying disease is still speculative. Like other cystic renal diseases with an increased risk for RCC, the attending physician should be aware of the malignant potential of ADPKD, especially with concomitant dialysis.

Breast cancer plasticity is restricted by a LATS1-NCOR1 repressive axis.

Breast cancer, the most frequent cancer in women, is generally classified into several distinct histological and molecular subtypes. However, single-cell technologies have revealed remarkable cellular and functional heterogeneity across subtypes and even within individual breast tumors. Much of this heterogeneity is attributable to dynamic alterations in the epigenetic landscape of the cancer cells, which promote phenotypic plasticity. Such plasticity, including transition from luminal to basal-like cell identity, can promote disease aggressiveness. We now report that the tumor suppressor LATS1, whose expression is often downregulated in human breast cancer, helps maintain luminal breast cancer cell identity by reducing the chromatin accessibility of genes that are characteristic of a "basal-like" state, preventing their spurious activation. This is achieved via interaction of LATS1 with the NCOR1 nuclear corepressor and recruitment of HDAC1, driving histone H3K27 deacetylation near NCOR1-repressed "basal-like" genes. Consequently, decreased expression of LATS1 elevates the expression of such genes and facilitates slippage towards a more basal-like phenotypic identity. We propose that by enforcing rigorous silencing of repressed genes, the LATS1-NCOR1 axis maintains luminal cell identity and restricts breast cancer progression.

Voxel-based comparison of [68Ga]Ga-RM2-PET/CT and [68Ga]Ga-PSMA-11-PET/CT with histopathology for diagnosis of primary prostate cancer.

BACKGROUND: Focal therapies or focally escalated therapies of primary prostate cancer are becoming more and more important. This increases the need to identify the exact extension of the intraprostatic tumor and possible dominant intraprostatic lesions by imaging techniques. While the prostate-specific membrane antigen (PSMA) is already a well-established target for imaging of prostate cancer cells, the gastrin-releasing peptide receptor (GRPR) seems to provide interesting additional information. Histopathology was used to examine the extent to which the single and combined image information of PET scans targeting GRPR and PSMA might lead to better tumor delineation. METHODS: Eight patients with histologically proven primary prostate cancer underwent two positron emission tomography with computer tomography scans, [68Ga]Ga-RM2-PET/CT (RM2-PET) and [68Ga]Ga-PSMA-11-PET/CT (PSMA-PET), prior to radical prostatectomy. RM2-PET data were correlated voxel-wise to a voxel-based model of the histopathologic tumor volume information. The results were compared to, correlated to, and combined with the correlation of PSMA-PET data analyzed analogously. RESULTS: In 4/8 patients, RM2-PET showed a higher signal in histologically proven tumor regions compared to PSMA. There were also tumor regions where PSMA-PET showed a higher signal than GRPR in 4/8 patients. A voxel-wise correlation of RM2-PET against histopathology yielded similar results compared to the correlation of PSMA-PET against histopathology, while PSMA-PET is the slightly better performing imaging technique. The combined information of both tracers yielded the best overall result, although this effect was not statistically significant compared to RM2-PET alone. CONCLUSIONS: Qualitative and quantitative findings in this preliminary study with 8 patients indicate that RM2-PET and PSMA-PET partially show not only the same, but also distinct regions of prostate cancer. Patients with pPCa might profit from information given by tracers targeting GRPR and PSMA simultaneously, in terms of a better delineation of the gross tumor volume.

Results from extended lymphadenectomies with [111In]PSMA-617 for intraoperative detection of PSMA-PET/CT-positive nodal metastatic prostate cancer.

PURPOSE: Identification of suspicious PSMA-PET/CT-positive lymph node (LN) metastases (LNM) from prostate cancer (PCa) during lymphadenectomy (LA) is challenging. We evaluated an 111In-labelled PSMA ligand (DKFZ-617, referred to as [111In]PSMA-617) as a γ-emitting tracer for intraoperative γ-probe application for resected tissue samples in PCa patients. Forty-eight hours prior to LA, [111In]PSMA-617 was administered intravenously in 23 patients with suspected LNM on PSMA-PET/CT (n = 21 with biochemical relapse, n = 2 at primary therapy). Resected tissue samples (LN, LNM and fibrofatty tissue) were measured ex situ by a γ-probe expressed as counts per second (CPSnorm). [111In]PSMA-617 tissue sample uptake was measured by a germanium detector for verification and calculated as %IAlbm (percent injected activity per kilogram lean body mass at time of surgery). Based on a clinical requirement for a specificity > 95%, thresholds for both ex situ measurements were chosen accordingly. Correlation of the results from PET/CT, γ-probe and germanium detector with histopathology was done. RESULTS: Eight hundred sixty-four LNs (197 LNM) were removed from 275 subregions in 23 patients, on average 8.6 ± 14.9 LNM per patient. One hundred four of 275 tissue samples showed cancer. Median γ-probe and germanium detector results were significantly different between tumour-affected (33.5 CPSnorm, 0.71 %IAlbm) and tumour-free subregions (3.0 CPSnorm, 0.03 %IAlbm) (each p value < 0.0001). For the chosen γ-probe cut-off (CPSnorm > 23) and germanium detector cut-off (%IAlbm > 0.27), 64 and 74 true-positive and 158 true-negative samples for both measurements were identified. Thirty-nine and 30 false-negative and 6 and 5 false-positive tissue samples were identified by γ-probe and germanium detector measurements. CONCLUSION: [111In]PSMA-617 application for LA is feasible in terms of an intraoperative real-time measurement with a γ-probe for detection of tumour-affected tissue samples. γ-probe results can be confirmed by precise germanium detector measurements and were significantly different between tumour-affected and tumour-free samples.

Accuracy of [68Ga]Ga-RM2-PET/CT for diagnosis of primary prostate cancer compared to histopathology.

INTRODUCTION: Prostate cancer (PCa) often shows an overexpression of the gastrin-releasing peptide receptor (GRPr). Therefore, GRPr is a possible theragnostic target. An interesting antagonist GRPr-ligand is RM2 or BAY86-7548. This study examines the accuracy of positron emission tomography (PET) with [68Ga]Ga-RM2 for diagnostic imaging of primary PCa (pPCa) compared to histopathology in patients undergoing radical prostatectomy (RP). METHODS: [68Ga]Ga-RM2-PET examinations were performed in 15 patients before RP. All prostate specimens were histopathologically examined based on predefined spatial octants. Each prostate volume on PET was subdivided into octants, which were correlated to histopathology and evaluated according to presence of tumor by two experienced examiners. Additionally, PET data was evaluated by volume of interest (VOI) analyses in terms of maximum standardized uptake value (SUVmax) and normalized SUVmax relative to background activity (rSUVmax). Receiver operating characteristic (ROC) curves for SUVmax and rSUVmax were calculated. RESULTS: At least one focus of increased [68Ga]Ga-RM2 uptake corresponding to a tumor manifestation on histology was found in 14 of 15 patients (93%). Spatial concordance of visual PET readings with histopathology was very variable. Intraindividual agreement reached from ≤2 octants in three, 3-5 octants in six to ≥6 octants in six patients, resulting in a relatively low correlation of visual PET readings with histopathology (accuracy = 0.63; p = 0.0018). Lesion-based analysis found a sensitivity of 69% and a positive predictive value of 73%. Concordantly, the octant-based ROC curves for SUVmax and rSUVmax indicated a relatively low diagnostic performance (area under the curve of 0.59 and 0.61, respectively). CONCLUSIONS: [68Ga]Ga-RM2-PET shows only a relatively low diagnostic accuracy for pPCa compared to histopathology on an octant basis, which may be explained to some extent by methodological weaknesses. Further studies need to explore, whether the observed high interindividual variability of agreement between [68Ga]Ga-RM2-PET and histopathology can be explained by different tumor biologies or other coincident prostatic pathologies.

Detection Rate of 18F-Choline PET/CT and 68Ga-PSMA-HBED-CC PET/CT for Prostate Cancer Lymph Node Metastases with Direct Link from PET to Histopathology: Dependence on the Size of Tumor Deposits in Lymph Nodes.

Accurate detection of prostate cancer lymph node metastases (LNM) through PET/CT before lymphadenectomy is crucial for successful therapy. PET/CT with choline derivatives used to be the standard tool for imaging metastases, whereas 68Ga-PSMA (prostate-specific membrane antigen) PET/CT was introduced recently. Both PET techniques were investigated with respect to what extent the detection rate of LNM depends on the size of tumor deposits (TDs) within LNM. Methods: Documenting the switch from the use of 18F-choline to 68Ga-PSMA in 2014, we used 2 patient cohorts undergoing a template lymphadenectomy because of a PET/CT indicating LNM. Forty-four and 40 patients underwent PET/CT with 18F-choline or 68Ga-PSMA ligand, respectively. In total, 226 LNM (125 18F-choline, 101 68Ga-PSMA) originated from 73 salvage lymphadenectomies at biochemical recurrence and from 11 primary lymphadenectomies at radical prostatectomy. LNM eligible for direct correlation of PET/CT to histopathology were identified from lymphadenectomies conducted in small anatomic subregions, with 1 LNM (condition 1) or 1-2 LNM (condition 2). Longitudinal and short diameters of TD within LNM were determined by histopathology, allowing linking of the size of TD in LNM to the detection threshold of PET/CT. Diameters associated with a detection rate of 50% and 90% (d50%, d90%) were calculated on the basis of logistic growth curve models fitted. Results: Gleason score, number of removed LNs, and subregions for lymphadenectomy per patient did not differ significantly between the 18F-choline and 68Ga-PSMA groups. The median prostate-specific antigen level at imaging and number of LNM per patient were significantly higher in the 18F-choline group (3.4 ng/mL, n = 34) than in the 68Ga-PSMA group (2.2 ng/mL, n = 28; both P < 0.05). Longitudinal and short diameters of TD in LNM to reach d90% were 11.2 and 7.4 mm, respectively, for 18F-choline PET/CT and 6.3 and 4.9 mm, respectively, for 68Ga-PSMA PET/CT. Corresponding diameters to reach d50% were 5.5 and 3.3 mm, respectively, for 18F-choline PET/CT and 3.7 and 2.3 mm, respectively, for 68Ga-PSMA PET/CT. Detection rates were significantly higher under 68Ga-PSMA (P = 0.005 and 0.04 for longitudinal and short diameter). Conclusion: 68Ga-PSMA PET/CT is superior to 18F-choline PET/CT in the detection of LNM. Whether those results will lead to an improved patient outcome after 68Ga-PSMA PET-guided therapy needs to be investigated by further studies.

Proteomic Characterization of Prostate Cancer to Distinguish Nonmetastasizing and Metastasizing Primary Tumors and Lymph Node Metastases.

Patients with metastatic prostate cancer (PCa) have a poorer prognosis than patients with organ-confined tumors. We strove to uncover the proteome signature of primary PCa and associated lymph node metastases (LNMs) in order to identify proteins that may indicate or potentially promote metastases formation. We performed a proteomic comparative profiling of PCa tissue from radical prostatectomy (RPE) of patients without nodal metastases or relapse at the time of surgical resection (n=5) to PCa tissue from RPE of patients who suffered from nodal relapse (n=5). For the latter group, we also included patient-matched tissue of the nodal metastases. All samples were formalin fixed and paraffin embedded. We identified and quantified more than 1200 proteins by liquid chromatography tandem mass spectrometry with subsequent label-free quantification. An increase of ribosomal or proteasomal proteins in LNM (compared to corresponding PCa) became apparent, while extracellular matrix components rather decreased. Immunohistochemistry (IHC) corroborated accumulation of poly-(ADP-ribose)-polymerase 1 and N-myc-downstream-regulated-gene 3, alpha/beta hydrolase domain-containing protein 11, and protein phosphatase slingshot homolog 3 in LNM. These findings strengthen the present interest in examining PARP inhibitors for the treatment of aggressive PCa. IHC also corroborated increased abundance of retinol dehydrogenase 11 in metastasized primary PCa compared to organ-confined PCa. Generally, metastasizing primary tumors were characterized by an enrichment of proteins involved in cellular lipid metabolic processes with concomitant decrease of cell adhesion proteins. This study highlights the usefulness of a combined proteomic-IHC approach to explore novel aspects in tumor biology. Our initial results open novel opportunities for follow-up studies.

Focal dose escalation for prostate cancer using 68Ga-HBED-CC PSMA PET/CT and MRI: a planning study based on histology reference.

BACKGROUND: Focal radiation therapy has gained of interest in treatment of patients with primary prostate cancer (PCa). The question of how to define the intraprostatic boost volume is still open. Previous studies showed that multiparametric MRI (mpMRI) or PSMA PET alone could be used for boost volume definition. However, other studies proposed that the combined usage of both has the highest sensitivity in detection of intraprostatic lesions. The aim of this study was to demonstrate the feasibility and to evaluate the tumour control probability (TCP) and normal tissue complication probability (NTCP) of radiation therapy dose painting using 68Ga-HBED-CC PSMA PET/CT, mpMRI or the combination of both in primary PCa. METHODS: Ten patients underwent PSMA PET/CT and mpMRI followed by prostatectomy. Three gross tumour volumes (GTVs) were created based on PET (GTV-PET), mpMRI (GTV-MRI) and the union of both (GTV-union). Two plans were generated for each GTV. Plan95 consisted of whole-prostate IMRT to 77 Gy in 35 fractions and a simultaneous boost to 95 Gy (Plan95PET/Plan95MRI/Plan95union). Plan80 consisted of whole-prostate IMRT to 76 Gy in 38 fractions and a simultaneous boost to 80 Gy (Plan80PET/Plan80MRI/Plan80union). TCPs were calculated for GTV-histo (TCP-histo), which was delineated based on PCa distribution in co-registered histology slices. NTCPs were assessed for bladder and rectum. RESULTS: Dose constraints of published protocols were reached in every treatment plan. Mean TCP-histo were 99.7% (range: 97%-100%) and 75.5% (range: 33%-95%) for Plan95union and Plan80union, respectively. Plan95union had significantly higher TCP-histo values than Plan95MRI (p = 0.008) and Plan95PET (p = 0.008). Plan80union had significantly higher TCP-histo values than Plan80MRI (p = 0.012), but not than Plan80PET (p = 0.472). Plan95MRI had significantly lower NTCP-rectum than Plan95union (p = 0.012). No significant differences in NTCP-rectum and NTCP-bladder were observed for all other plans (p > 0.05). CONCLUSIONS: IMRT dose escalation on GTVs based on mpMRI, PSMA PET/CT and the combination of both was feasible. Boosting GTV-union resulted in significantly higher TCP-histo with no or minimal increase of NTCPs compared to the other plans.

Diagnostic Accuracy of Ga-68-HBED-CC-PSMA-Ligand-PET/CT before Salvage Lymph Node Dissection for Recurrent Prostate Cancer.

Background: By targeting the prostate-specific membrane antigen (PSMA) on prostate cancer (PCa) cells PSMA-PET/CT shows great potential in locating the site of biochemical recurrence even at low PSA (Prostate-specific antigen)-levels. Accurate imaging of PCa recurrent lymph node metastases (LNM) is crucial for metastases directed therapies such as salvage-lymph node dissection (salvage-LND). Objective: To evaluate the diagnostic accuracy of PSMA-PET/CT for detection of affected lymph-node regions at salvage-LND for nodal recurrence of PCa. Design, setting and participants: 30 patients with the suspicion of exclusively nodal PCa-relapse after primary therapy underwent a template pelvic and/or retroperitoneal salvage-LND after whole body 68-Ga-PSMA-PET/CT. The diagnostic accuracy of PET/CT was evaluated in comparison to the histopathology of 965 resected lymph nodes (LN) dissected from 68 main regions (pelvic left/right, retroperitoneal) and 289 subregions (common iliac, external iliac, obturator, internal iliac, presacral, aortic-bifurcation, aortal, caval). LNM and tumor deposits in LNM were measured bidimensionally in the histopathology. PSMA-expression was analyzed by immunohistochemistry in LNM. Results: LNM were present in 11.4% of the resected LN (110/965) resulting in 45 positive main regions and 85 positive subregions. PET/CT was true positive in 41 main regions and 69 subregions. Three PET-negative main regions and 16 PET-negative subregions finally contained LNM, the majority of these false negative subregions (13/16) were in neighboring regions of true-positive subregions. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy were: main region-based 93.2%, 100%, 100%, 88.9% and 95.6%, subregion-based 81.2%, 99.5%, 98.6%, 92.7 and 94.1%. Median short diameters of tumor deposits in LNM resected from false-negative subregions (1.3 mm) were significantly smaller than in LNM removed from true-positive subregions (5.5 mm, p<0.0001). Based on anatomical subregions containing just one LNM, the necessary short diameter of tumor deposits in LNM required to reach a detection rate of 50% and 90% was estimated to be ≥ 2.3 mm and ≥ 4.5 mm, respectively. Conclusion: In men with biochemical PCa-relapse and positive PSMA-PET/CT, PET/CT detects metastatic affected anatomical regions with high accuracy at a main region and at a subregion-level. If the decision for salvage-LND is prompted by a positive PSMA-PET/CT, the size of metastases is crucial for accurate detection of affected regions. All LNM showed a clear PSMA-expression in the immunohistochemistry. Further studies need to investigate how to translate the high anatomical correlation observed between PET/CT and surgical findings into optimal approaches for target salvage-LND.

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.

Proteome profiling of clear cell renal cell carcinoma in von Hippel-Lindau patients highlights upregulation of Xaa-Pro aminopeptidase-1, an anti-proliferative and anti-migratory exoprotease.

Patients of the von Hippel-Lindau (VHL) disease frequently develop clear cell renal cell carcinoma (ccRCC). Using archived, formalin-fixed, paraffin-embedded (FFPE) samples, we sought to determine global proteome alterations that distinguish ccRCC tissue from adjacent, non-malignant kidney tissue in VHL-patients. Our quantitative proteomic analysis clearly discriminated tumor and non-malignant tissue. Significantly dysregulated proteins were distinguished using the linear models for microarray data algorithm. In the ccRCC tissue, we noticed a predominant under-representation of proteins involved in the tricarboxylic acid cycle and an increase in proteins involved in glycolysis. This profile possibly represents a proteomic fingerprint of the "Warburg effect", which is a molecular hallmark of ccRCC. Furthermore, we observed an increase in proteins involved in extracellular matrix organization. We also noticed differential expression of many exoproteases in the ccRCC tissue. Of particular note were opposing alterations of Xaa-Pro Aminopeptidases-1 and -2 (XPNPEP-1 and -2): a strong decrease of XPNPEP-2 in ccRCC was accompanied by abundant presence of the related protease XPNPEP-1. In both cases, we corroborated the proteomic results by immunohistochemical analysis of ccRCC and adjacent, non-malignant kidney tissue of VHL patients. To functionally investigate the role of XPNPEP-1 in ccRCC, we performed small-hairpin RNA mediated XPNPEP-1 expression silencing in 786-O ccRCC cells harboring a mutated VHL gene. We found that XPNPEP-1 expression dampens cellular proliferation and migration. These results suggest that XPNPEP-1 is likely an anti-target in ccRCC. Methodologically, our work further validates the robustness of using FFPE material for quantitative proteomics.

Comparison of 68Ga-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.