Identification of the Biosynthetic Gene Cluster for Pyracrimycin A, an Antibiotic Produced by Streptomyces sp.

Actinomycetes make a wealth of complex, structurally diverse natural products, and a key challenge is to link them to their biosynthetic gene clusters and delineate the reactions catalyzed by each of the enzymes. Here, we report the biosynthetic gene cluster for pyracrimycin A, a set of nine genes that includes a core nonribosomal peptide synthase (pymB) that utilizes serine and proline as precursors and a monooxygenase (pymC) that catalyzes Baeyer-Villiger oxidation. The cluster is similar to the one for brabantamide A; however, pyracrimycin A biosynthesis differs in that feeding experiments with isotope-labeled serine and proline suggest that a ring opening reaction takes place and a carbon is lost from serine downstream of the oxidation reaction. Based on these data, we propose a full biosynthesis pathway for pyracrimycin A.

Characterization and engineering of Streptomyces griseofuscus DSM 40191 as a potential host for heterologous expression of biosynthetic gene clusters.

Streptomyces griseofuscus DSM 40191 is a fast growing Streptomyces strain that remains largely underexplored as a heterologous host. Here, we report the genome mining of S. griseofuscus, followed by the detailed exploration of its phenotype, including the production of native secondary metabolites and ability to utilise carbon, nitrogen, sulphur and phosphorus sources. Furthermore, several routes for genetic engineering of S. griseofuscus were explored, including use of GusA-based vectors, CRISPR-Cas9 and CRISPR-cBEST-mediated knockouts. Two out of the three native plasmids were cured using CRISPR-Cas9 technology, leading to the generation of strain S. griseofuscus DEL1. DEL1 was further modified by the full deletion of a pentamycin BGC and an unknown NRPS BGC, leading to the generation of strain DEL2, lacking approx. 500 kbp of the genome, which corresponds to a 5.19% genome reduction. DEL2 can be characterized by faster growth and inability to produce three main native metabolites: lankacidin, lankamycin, pentamycin and their derivatives. To test the ability of DEL2 to heterologously produce secondary metabolites, the actinorhodin BGC was used. We were able to observe a formation of a blue halo, indicating a potential production of actinorhodin by both DEL2 and a wild type.

68Ga-PSMA PET/CT compared with MRI/CT and diffusion-weighted MRI for primary lymph node staging prior to definitive radiotherapy in prostate cancer: a prospective diagnostic test accuracy study.

BACKGROUND: The aim was to compare the diagnostic accuracy of 68Ga-PSMA PET/CT with conventional cross-sectional imaging and diffusion-weighted MRI (DW-MRI) for detecting lymph node metastasis (LNM) to stage prostate cancer patients. Twenty consecutive, newly- diagnosed prostate cancer patients were prospectively enrolled and underwent 68Ga-PSMA-11 PET/CT, anatomical MRI or contrast-enhanced CT, and DW-MRI prior to laparoscopic, template-based, extended lymph node dissection. Histopathological findings served as the reference test. RESULTS: Histopathology showed LNM in 13 of 20 patients (19 high-risk, 1 intermediate risk). Five patients had metastasis-suspected lymph nodes on 68Ga-PSMA PET/CT. Patient-based analysis showed that the sensitivity and specificity for detecting LNM were 39% and 100% with 68Ga-PSMA PET/CT, 8% and 100% with MRI/CT, and 36% and 83% with DW-MRI, respectively. The positive and negative predictive values were 100% and 49% with 68Ga-PSMA PET/C, 100% and 37% with MRI/CT, and 80% and 42% with DW-MRI. Of 573 dissected lymph nodes, 33 were LNM from 26 regions. True-positive LNM on 68Ga-PSMA PET/CT was 9-11 mm in diameter, whereas false-negative LNM had a median diameter of 4 mm, with only 3 of 30 lymph nodes being larger than 10 mm. LNM were positive for PSMA by immunostaining. CONCLUSIONS: The sensitivity of 68Ga-PSMA PET/CT was notably better than that of MRI/CT and comparable to that of DW-MRI. Some false positive findings with DW-MRI reduced its specificity and positive predictive value compared with those of 68Ga-PSMA PET/CT and MRI/CT.

Observer Agreement and Accuracy of 18F-Sodium Fluoride PET/CT in the Diagnosis of Bone Metastases in Prostate Cancer.

Our aim was to evaluate the interobserver agreement in 18F-sodium fluoride (NaF) PET/CT for the detection of bone metastases in patients with prostate cancer (PCa). Methods:18F-NaF PET/CT scans were retrieved from all patients who participated in 4 recent prospective trials. Two experienced observers independently evaluated the 18F-NaF PET/CT scans on a patient level using a 3-category scale (no bone metastases [M0], equivocal for bone metastases, and bone metastases present [M1]) and on a dichotomous scale (M0/M1). In patients with no more than 10 lesions, the location and number of lesions were recorded. On a patient level, the diagnostic performance was calculated using a sensitivity analysis, in which equivocal lesions were handled as M0 as well as M1. Results:18F-NaF PET/CT scans from 219 patients with PCa were included, of whom 129 patients were scanned for primary staging, 67 for biochemical recurrence, and 23 for metastatic castration-resistant PCa. Agreement between the observers was almost perfect on a patient level (3-category unweighted κ = 0.83 ± 0.05, linear weighted κ = 0.90 ± 0.06, and dichotomous κ = 0.91 ± 0.07). On a lesion level (dichotomous scale), the observers agreed on the number and location of bone metastases in 205 (93.6%) patients. In the remaining 14 patients, the readers disagreed on the number of lesions in 13 patients and the location of bone metastases in 1 patient. A final diagnosis of bone metastases was made for 211 of 219 patients. The sensitivity ranged from 0.86 to 0.92, specificity from 0.83 to 0.97, positive predictive value from 0.70 to 0.93, and negative predictive value from 0.94 to 0.96. Conclusion: The interobserver agreement on 18F-NaF PET/CT for the detection of bone metastases in patients with PCa was very high among trained observers, both on a patient level and on a lesion level. Moreover, the diagnostic performance of 18F-NaF PET/CT was satisfactory, rendering 18F-NaF PET/CT a robust tool in the diagnostic armamentarium.

Prospective comparison of 68Ga-PSMA PET/CT, 18F-sodium fluoride PET/CT and diffusion weighted-MRI at for the detection of bone metastases in biochemically recurrent prostate cancer.

PURPOSE: To prospectively compare diagnostic accuracies for detection of bone metastases by 68Ga-PSMA PET/CT, 18F-NaF PET/CT and diffusion-weighted MRI (DW600-MRI) in prostate cancer (PCa) patients with biochemical recurrence (BCR). METHODS: Sixty-eight PCa patients with BCR participated in this prospective study. The patients underwent 68Ga-PSMA PET/CT, a 18F-NaF PET/CT and a DW600-MRI (performed in accordance with European Society of Urogenital Radiology guidelines, with b values of 0 and 600 s/mm2). Bone lesions were categorized using a three-point scale (benign, malignant or equivocal for metastases) and a dichotomous scale (benign or metastatic) for each imaging modality by at least two experienced observers. A best valuable comparator was defined for each patient based on study-specific imaging, at least 12 months of clinical follow-up and any imaging prior to the study and during follow-up. Diagnostic performance was assessed using a sensitivity analysis where equivocal lesions were handled as non-metastatic and then as metastatic. RESULTS: Ten of the 68 patients were diagnosed with bone metastases. On a patient level, sensitivity, specificity and the area under the curve (AUC) by receiver operating characteristic analysis were, respectively, 0.80, 0.98-1.00 and 0.89-0.90 for 68Ga-PSMA PET/CT (n = 68 patients); 0.90, 0.90-0.98 and 0.90-0.94 for 18NaF PET/CT (n = 67 patients); and 0.25-0.38, 0.87-0.92 and 0.59-0.62 for DW600-MRI (n = 60 patients). The diagnostic performance of DW600-MRI was significantly lower than that of 68Ga-PSMA PET/CT and 18NaF PET/CT for diagnosing bone metastases (p < 0.01), and no significant difference in the AUC was seen between 68Ga-PSMA PET/CT and 18NaF PET/CT (p = 0.65). CONCLUSION: 68Ga-PSMA PET/CT and 18F-NaF PET/CT showed comparable and high diagnostic accuracies for detecting bone metastases in PCa patients with BCR. Both methods performed significantly better than DW600-MRI, which was inadequate for diagnosing bone metastases when conducted in accordance with European Society of Urogenital Radiology guidelines.

68Ga-PSMA PET/CT in Patients With Biochemical Recurrence of Prostate Cancer: A Prospective, 2-Center Study.

PURPOSE OF THE REPORT: The aim of this study was to prospectively investigate the detection rate of Ga-PSMA PET/CT in biochemical recurrence (BCR) of prostate cancer and its impact on patient management. MATERIALS AND METHODS: Patients with BCR after curatively intended treatment of prostate cancer were included. Each patient underwent a Ga-PSMA PET/CT. Changes in patient management based on the results of Ga-PSMA PET/CT were assessed. RESULTS: Seventy patients were included. Sixty-four patients (91%) had radical prostatectomy, of whom 17 patients (24%) received salvage radiation therapy due to first biochemical relapse. Six patients (9%) underwent radiation therapy as the primary treatment. Ga-PSMA PET/CT detected recurrent disease in 37 patients (53%). The detection rate was 22% for prostate-specific antigen (PSA) levels up to 0.5 ng/mL compared with 83% for PSA levels greater than 0.5 ng/mL. Pathological uptake of Ga-PSMA was observed in 4 (16%) of 21, 4 (44%) of 9, 0 of 1, 7 (70%) of 10, and 22 (88%) of 25 patients with PSA levels from 0.2 to 0.3 ng/mL, 0.31 to 0.4 ng/mL, 0.41 to 0.5 ng/mL, 0.51 to 1 ng/mL, and greater than 1 ng/mL, respectively. Prostate-specific antigen was significantly higher in PSMA-positive patients than in PSMA-negative patients. In 15 (22%) of 69 patients, the results caused a definite change in patient management, and in another 15 (22%) of 69 patients, Ga-PSMA PET/CT guided the choice of treatment. CONCLUSIONS: Ga-PSMA PET/CT detects lesions in a large proportion of patients with BCR. Detection rates at low PSA levels (<0.5 ng/mL) were notably below the values reported in previous retrospective studies; however, detection rates improved with increasing PSA levels.

68 Ga-PSMA PET/CT for the detection of bone metastases in prostate cancer: a systematic review of the published literature.

BACKGROUND: 68 Ga-labelled prostate-specific membrane antigen (PSMA) is a promising PET ligand for the detection of prostate cancer. Little attention has been given to the ability of 68 Ga-PSMA PET/CT to detect malignant bone lesions and whether this approach is superior to existing bone imaging modalities. AIM: To review the existing data of 68 Ga-PSMA PET/CT for the diagnosis of bone metastases in prostate cancer. METHODS: Systematic review of the peer-reviewed literature. RESULTS: Among 1858 papers in the original search, 37 papers were included in the analysis (six case reports and 31 case series). The vast majority of the studies were low-level evidence studies. Most studies presented data on detection rates without a reference standard. All but two studies were of a retrospective nature. Several cohort studies showed bone metastasis in 5-60% of patients with prostate cancer, including in patients with very low-PSA values. For primary staging, 68 Ga-PSMA PET/CT outperformed bone scans, while the superiority of 68 Ga-PSMA PET/CT compared with bone scans with respect to biochemical recurrence and metastatic castration-resistant prostate cancer (mCRPC) remains to be demonstrated. CONCLUSION: 68 Ga-PSMA PET/CT has shown to be a promising technique for use in prostate cancer. 68 Ga-PSMA PET/CT shows more lesions than bone scans, but data on diagnostic performance are very limited and indicate improved diagnostic performance in primary staging but not in mCRPC. Properly designed studies are needed to clarify the diagnostic performance of 68 Ga-PSMA PET/CT as well as its superiority over existing methods before 68 Ga-PSMA PET/CT can be routinely used for bone imaging.

68Ga-PSMA PET/CT for the detection of bone metastasis in recurrent prostate cancer and a PSA level <2 ng/ml: Two case reports and a literature review.

Localization of prostate cancer recurrence, particularly in the bones, is a major challenge with standard of care imaging in patients with biochemical recurrence following curatively intended treatment. Gallium-68-labeled prostate specific membrane antigen positron emission tomography/computed tomography (68Ga-PSMA PET/CT) is a novel and promising method for imaging in prostate cancer. The present study reports two cases of patients with prostate cancer with biochemical recurrence, with evidence of bone metastases on 68Ga-PSMA PET/CT images and low prostate specific antigen PSA levels (<2 ng/ml) and PSA doubling time >6 months. The bone metastases were verified by supplementary imaging with 18F-sodium fluoride PET/CT and magnetic resonance imaging as well as biochemical responses to androgen deprivation therapy. Therefore, 68Ga-PSMA PET/CT is promising for the restaging of patients with prostate cancer with biochemical recurrence, including patients with low PSA levels and low PSA kinetics.

A Comprehensive Safety Evaluation of 68Ga-Labeled Ligand Prostate-Specific Membrane Antigen 11 PET/CT in Prostate Cancer: The Results of 2 Prospective, Multicenter Trials.

PURPOSE: The aim of this study was to evaluate the clinical safety profile of the Ga-PSMA-11 ligand for PET/CT imaging in prospective clinical trials. METHODS: Eighty-eight patients with newly diagnosed or recurrent prostate cancer participated in 2 prospective trials. Safety reporting was identical in the 2 trials. The Ga-PSMA-11 ligand was administered as 2 MBq/kg body weight (mean, 9.2 µg, 9.7 nmol). The reporting of clinical adverse events (AEs) and the measurement of blood pressure (BP) and heart rate (HR) were performed prior to injection (baseline); immediately after injection of Ga-PSMA-11 (postinjection); at 1, 10, and 60 minutes after injection; and after acquisition of the PET/CT scan (postscan). All hemodynamic assessments were performed in the supine position, except for the postscan measurement (sitting). The patients were interviewed regarding any AEs at baseline, postinjection, or postscan. In addition, the patients were instructed to report any AEs during the investigation and to contact the investigator if AEs occurred during the rest of the day. Adverse events were classified as mild, moderate, or severe by the patients and categorized by the investigator using the CTCAE (Common Terminology Criteria for Adverse Events) version 4.0. RESULTS: There were no reported clinical AEs. There were significant decreases in systolic BP (P < 0.001) and HR (P < 0.001) over time. In comparison, the diastolic BP increased significantly (P < 0.001). After removal of the last observation (supine position), there was no time-dependent change in systolic or diastolic BP, but the significant change in HR remained. The mean changes over the entire observation period were minimal (systolic BP, -6 to 5 mm Hg; diastolic BP, -2 to 3 mm Hg; HR, decrease of 5 beats/min). No patients developed hypotension. Fifty-five patients presented with hypertension at baseline, which increased by 1 CTCAE grade in 15 patients and by 2 grades in 2 patients. A large number of cases of asymptomatic (grade 1) bradycardia were observed, primarily in patients with preexisting bradycardia. One patient developed transient grade 1 tachycardia. No patients required medical intervention for cardiovascular perturbations. CONCLUSIONS: Ga-PSMA-11 PET/CT was very well tolerated. We consider Ga-PSMA-11 to be safe for human application.

68Ga-PSMA PET/CT Uptake in Intramuscular Myxoma Imitates Prostate Cancer Metastasis.

Ga-PSMA PET/CT is becoming the most promising imaging modality for detecting recurrent prostate cancer. The modality has the advantage of being able to detect recurrent disease, even at very low prostate-specific antigen levels. However, several studies report Ga-PSMA uptake in tissue unrelated to prostate cancer. We present a 74-year-old man who underwent Ga-PSMA PET/CT for recurrent prostate cancer 5 years after radical prostatectomy. The Ga-PSMA PET/CT showed an intramuscular lesion with increased PSMA uptake in the left vastus medialis muscle. The lesion was surgically removed, and histopathology found it to be an intramuscular myxoma that showed immunohistochemical PSMA expression.

Incidental Detection of Thyroid Metastases From Renal Cell Carcinoma Using 68Ga-PSMA PET/CT to Assess Prostate Cancer Recurrence.

Ga-PSMA PET/CT is increasingly used to assess prostate cancer. Avid Ga-PSMA uptake by thyroid cancer and renal cell carcinoma (RCC) has been reported in few cases. A 75-year-old man who received a diagnosis of RCC in 2006 and prostate cancer in 2009 presented with elevated prostate-specific antigen levels (0.7 ng/mL) following prostatectomy. Ga-PSMA PET/CT showed avid Ga-PSMA uptake in 1 pelvic and 1 retroperitoneal lymph node and focal Ga-PSMA accumulation in the thyroid. Excised retroperitoneal lymph node and thyroid tissues showed metastases from RCC, whereas the pelvic lymph node exhibited metastasis from prostate cancer.