Targeted DNA sequencing of high-grade serous ovarian carcinoma reveals association of TP53 mutations with platinum resistance when combined with gene expression.

High-grade serous ovarian carcinoma (HGSC) is the most common subtype of ovarian cancer and is among the most fatal gynecological malignancies worldwide, due to late diagnosis at advanced stages and frequent therapy resistance. In 47 HGSC patients, we assessed somatic and germline genetic variability of a custom panel of 144 known or suspected HGSC-related genes by high-coverage targeted DNA sequencing to identify the genetic determinants associated with resistance to platinum-based therapy. In the germline, the most mutated genes were DNAH14 (17%), RAD51B (17%), CFTR (13%), BRCA1 (11%), and RAD51 (11%). Somatically, the most mutated gene was TP53 (98%), followed by CSMD1/2/3 (19/19/36%), and CFTR (23%). Results were compared with those from whole exome sequencing of a similar set of 35 HGSC patients. Somatic variants in TP53 were also validated using GENIE data of 1287 HGSC samples. Our approach showed increased prevalence of high impact somatic and germline mutations, especially those affecting splice sites of TP53, compared to validation datasets. Furthermore, nonsense TP53 somatic mutations were negatively associated with patient survival. Elevated TP53 transcript levels were associated with platinum resistance and presence of TP53 missense mutations, while decreased TP53 levels were found in tumors carrying mutations with predicted high impact, which was confirmed in The Cancer Genome Atlas data (n = 260). Targeted DNA sequencing of TP53 combined with transcript quantification may contribute to the concept of precision oncology of HGSC. Future studies should explore targeting the p53 pathway based on specific mutation types and co-analyze the expression and mutational profiles of other key cancer genes.

Inhibition of homologous recombination repair by Mirin in ovarian cancer ameliorates carboplatin therapy response in vitro.

Chemoresistance poses one of the most significant challenges of cancer therapy. Carboplatin (CbPt) is one of the most used chemotherapeutics in ovarian cancer (OVC) treatment. MRE11 constitutes a part of homologous recombination (HR), which is responsible for the repair of CbPt-induced DNA damage, particularly DNA crosslinks. The study's main aim was to address the role of HR in CbPt chemoresistance in OVC and to evaluate the possibility of overcoming CbPt chemoresistance by Mirin-mediated MRE11 inhibition in an OVC cell line. Lower expression of MRE11 was associated with better overall survival in a cohort of OVC patients treated with platinum drugs (TCGA dataset, p < 0.05). Using in vitro analyses, we showed that the high expression of HR genes drives the CbPt chemoresistance in our CbPt-resistant cell line model. Moreover, the HR inhibition by Mirin not only increased sensitivity to carboplatin (p < 0.05) but also rescued the sensitivity in the CbPt-resistant model (p < 0.05). Our results suggest that MRE11 inhibition with Mirin may represent a promising way to overcome OVC resistance. More therapy options will ultimately lead to better personalized cancer therapy and improvement of patients' survival.

Prognostic potential of whole exome sequencing in the clinical management of metachronous colorectal cancer liver metastases.

BACKGROUND: Colorectal cancer is a highly prevalent and deadly. The most common metastatic site is the liver. We performed a whole exome sequencing analysis of a series of metachronous colorectal cancer liver metastases (mCLM) and matched non-malignant liver tissues to investigate the genomic profile of mCLM and explore associations with the patients' prognosis and therapeutic modalities. METHODS: DNA samples from mCLM and non-malignant liver tissue pairs (n = 41) were sequenced using whole exome target enrichment and their germline and somatic genetic variability, copy number variations, and mutational signatures were assessed for associations with relapse-free (RFS) and overall survival (OS). RESULTS: Our genetic analysis could stratify all patients into existing targeted therapeutic regimens. The most commonly mutated genes in mCLM were TP53, APC, and KRAS together with PIK3CA and several passenger genes like ABCA13, FAT4, PCLO, and UNC80. Patients with somatic alterations in genes from homologous recombination repair, Notch, and Hedgehog pathways had significantly prolonged RFS, while those with altered MYC pathway genes had poor RFS. Additionally, alterations in the JAK-STAT pathway were prognostic of longer OS. Patients bearing somatic variants in VIPR2 had significantly shorter OS and those with alterations in MUC16 prolonged OS. Carriage of the KRAS-12D variant was associated with shortened survival in our and external datasets. On the other hand, tumor mutation burden, mismatch repair deficiency, microsatellite instability, mutational signatures, or copy number variation in mCLM had no prognostic value. CONCLUSIONS: The results encourage further molecular profiling for personalized treatment of colorectal cancer liver metastases discerning metachronous from synchronous scenarios.

The potential of exome sequencing of paired colorectal tumors and synchronous liver metastases to improve treatment.

Tweetable abstract Sequencing exomes of synchronous and metachronous liver metastases of colorectal cancer has potential to enhance treatment, since they can have molecular profiles distinct from primary tumors.

Integrative analysis of mRNA and miRNA expression profiles and somatic variants in oxysterol signaling in early-stage luminal breast cancer.

Oxysterols, oxidized derivatives of cholesterol, act in breast cancer (BC) as selective estrogen receptor modulators and affect cholesterol homeostasis, drug transport, nuclear and cell receptors, and other signaling proteins. Using data from three highly overlapping sets of patients (N = 162 in total) with early-stage estrogen-receptor-positive luminal BC-high-coverage targeted DNA sequencing (113 genes), mRNA sequencing, and full micro-RNA (miRNA) transcriptome microarrays-we describe complex oxysterol-related interaction (correlation) networks, with validation in public datasets (n = 538) and 11 databases. The ESR1-CH25H-INSIG1-ABCA9 axis was the most prominent, interconnected through miR-125b-5p, miR-99a-5p, miR-100-5p, miR-143-3p, miR-199b-5p, miR-376a-3p, and miR-376c-3p. Mutations in SC5D, CYP46A1, and its functionally linked gene set were associated with multiple differentially expressed oxysterol-related genes. STARD5 was upregulated in patients with positive lymph node status. High expression of hsa-miR-19b-3p was weakly associated with poor survival. This is the first study of oxysterol-related genes in BC that combines DNA, mRNA, and miRNA multiomics with detailed clinical data. Future studies should provide links between intratumoral oxysterol signaling depicted here, circulating oxysterol levels, and therapy outcomes, enabling eventual clinical exploitation of present findings.

Exome Sequencing of Paired Colorectal Carcinomas and Synchronous Liver Metastases for Prognosis and Therapy Prediction.

PURPOSE: Analysis of somatic variant profiles in retrospectively collected pairs of primary tumors and synchronous liver metastases from surgically treated patients with colorectal carcinomas. Mutational profiles were compared between groups of patients stratified by response to chemotherapy and survival. PATIENTS AND METHODS: The study used whole-exome sequencing of tumor sample pairs from 20 patients diagnosed and treated at a single center. The Cancer Genome Atlas COAD-READ data set (n = 380) was used for validation in silico, where possible. RESULTS: The most frequently altered oncodrivers were APC (55% in primaries and 60% in metastases), TP53 (50/45), TRIP11 (30/5), FAT4 (20/25), and KRAS (15/25). Harboring variants with a high or moderate predicted functional effect in KRAS in primary tumors was significantly associated with poor relapse-free survival in both our sample set and the validation data set. We found a number of additional prognostic associations, including mutational load, alterations in individual genes, oncodriver pathways, and single base substitution (SBS) signatures in primary tissues, which were not confirmed by validation. Altered ATM, DNAH11, and MUC5AC, or a higher share of SBS24 signature in metastases seemed to represent poor prognostic factors, but because of a lack of suitable validation data sets, these results must be treated with extreme caution. No gene or profile was significantly associated with response to chemotherapy. CONCLUSION: Taken together, we report subtle differences in exome mutational profiles between paired primary tumors and synchronous liver metastases and a distinct prognostic relevance of KRAS in primary tumors. Although the general scarcity of primary tumor-synchronous metastasis sample pairs with high-quality clinical data makes robust validation difficult, this study provides potentially valuable data for utilization in precision oncology and could serve as a springboard for larger studies.

Complex molecular profile of DNA repair genes in epithelial ovarian carcinoma patients with different sensitivity to platinum-based therapy.

Epithelial ovarian carcinoma (EOC) is known for high mortality due to diagnosis at advanced stages and frequent therapy resistance. Previous findings suggested that the DNA repair system is involved in the therapeutic response of cancer patients and DNA repair genes are promising targets for novel therapies. This study aimed to address complex inter-relations among gene expression levels, methylation profiles, and somatic mutations in DNA repair genes and EOC prognosis and therapy resistance status. We found significant associations of DUT expression with the presence of peritoneal metastases in EOC patients. The high-grade serous EOC subtype was enriched with TP53 mutations compared to other subtypes. Furthermore, somatic mutations in XPC and PRKDC were significantly associated with worse overall survival of EOC patients, and higher FAAP20 expression in platinum-resistant than platinum-sensitive patients was observed. We found higher methylation of RAD50 in platinum-resistant than in platinum-sensitive patients. Somatic mutations in BRCA1 and RAD9A were significantly associated with higher RBBP8 methylation in platinum-sensitive compared to platinum-resistant EOC patients. In conclusion, we discovered associations of several candidate genes from the DNA repair pathway with the prognosis and platinum resistance status of EOC patients, which deserve further validation as potential predictive biomarkers.

Anticancer regimens containing third generation taxanes SB-T-121605 and SB-T-121606 are highly effective in resistant ovarian carcinoma model.

Taxanes are widely used in the treatment of ovarian carcinomas. One of the main problems with conventional taxanes is the risk of development of multidrug resistance. New-generation synthetic experimental taxoids (Stony Brook Taxanes; SB-T) have shown promising effects against various resistant tumor models. The aim of our study was to compare the in vitro efficacy, intracellular content, and in vivo antitumor effect of clinically used paclitaxel (PTX) and SB-Ts from the previously tested second (SB-T-1214, SB-T-1216) and the newly synthesized third (SB-T-121402, SB-T-121605, and SB-T-121606) generation in PTX resistant ovarian carcinoma cells NCI/ADR-RES. The efficacy of the new SB-Ts was up to 50-times higher compared to PTX in NCI/ADR-RES cells in vitro. SB-T-121605 and SB-T-121606 induced cell cycle arrest in the G2/M phase much more effectively and their intracellular content was 10-15-times higher, when compared to PTX. Incorporation of SB-T-121605 and SB-T-121606 into therapeutic regimens containing PTX were effective in suppressing tumor growth in vivo in NCI/ADR-RES based mice xenografts at small doses (≤3 mg/kg), where their adverse effects were eliminated. In conclusion, new SB-T-121605 and SB-T-121606 analogs are promising candidates for the next phase of preclinical testing of their combination therapy with conventional taxanes in resistant ovarian carcinomas.

Whole-exome sequencing of epithelial ovarian carcinomas differing in resistance to platinum therapy.

Epithelial ovarian carcinoma (EOC) is highly fatal because of the risk of resistance to therapy and recurrence. We performed whole-exome sequencing of blood and tumor tissue pairs of 50 patients with surgically resected EOC. Compared with sensitive patients, platinum-resistant patients had a significantly higher somatic mutational rate in <i>TP53</i> and lower in several genes from the Hippo pathway. We confirmed the pivotal role of somatic mutations in homologous recombination repair genes in platinum sensitivity and favorable prognosis of EOC patients. Implementing the germline homologous recombination repair profile significantly improved the prediction. In addition, distinct mutational signatures, for example, SBS6, and overall mutational load, somatic mutations in <i>PABPC1</i>, <i>PABPC3</i>, and <i>TFAM</i> co-segregated with the resistance status, high-grade serous carcinoma subtype, or overall survival of patients. We generated germline and somatic genetic landscapes of prognostically different subgroups of EOC patients for further follow-up studies focused on utilizing the observed associations in precision oncology.

Germline and somatic genetic variability of oxysterol-related genes in breast cancer patients with early disease of the luminal subtype.

Oxysterols, oxidized derivatives of cholesterol, have been implicated in multiple pathologies, including cancer. In breast cancer, the link is especially strong due to interactions between oxysterols and estrogen receptor activity. Here, we provide the first dedicated study of 113 oxysterol-related genes in breast cancer patients of the luminal subtype, in terms of both their somatic and germline variability, using targeted high-throughput DNA sequencing of 100 normal-tumor pairs with very high coverage. In the full cohort, or subsets of patients stratified by therapy, we found 12 germline variants in ABCA1, ABCA8, ABCC1, GPR183, LDLR, MBTPS1, NR1I2, OSBPL2, OSBPL3, and OSBPL5 to associate with poor survival of patients and variants in ABCA8, ABCG2, and HSD3B7 (three in total) associated with better survival. However, no associations remained significant after correction for multiple tests. Analysis of somatic variants revealed significantly (after FDR correction) poorer survival in patients mutated in CYP46A1 and 9 interacting (according to STRING analysis) genes, as well as in OSBPL3 and a set of 20 genes that collectively associated with the progesterone receptor status of patients. We propose further exploration of these genes in an integrative manner together with gene expression and epigenomic data.

The Role of TRIP6, ABCC3 and CPS1 Expression in Resistance of Ovarian Cancer to Taxanes.

The main problem precluding successful therapy with conventional taxanes is de novo or acquired resistance to taxanes. Therefore, novel experimental taxane derivatives (Stony Brook taxanes; SB-Ts) are synthesized and tested as potential drugs against resistant solid tumors. Recently, we reported alterations in ABCC3, CPS1, and TRIP6 gene expression in a breast cancer cell line resistant to paclitaxel. The present study aimed to investigate gene expression changes of these three candidate molecules in the highly resistant ovarian carcinoma cells in vitro and corresponding in vivo models treated with paclitaxel and new experimental Stony Brook taxanes of the third generation (SB-T-121605 and SB-T-121606). We also addressed their prognostic meaning in ovarian carcinoma patients treated with taxanes. We estimated and observed changes in mRNA and protein profiles of ABCC3, CPS1, and TRIP6 in resistant and sensitive ovarian cancer cells and after the treatment of resistant ovarian cancer models with paclitaxel and Stony Brook taxanes in vitro and in vivo. Combining Stony Brook taxanes with paclitaxel caused downregulation of CPS1 in the paclitaxel-resistant mouse xenograft tumor model in vivo. Moreover, CPS1 overexpression seems to play a role of a prognostic biomarker of epithelial ovarian carcinoma patients' poor survival. ABCC3 was overexpressed in EOC tumors, but after the treatment with taxanes, its up-regulation disappeared. Based on our results, we can suggest ABCC3 and CPS1 for further investigations as potential therapeutic targets in human cancers.

Oxysterols in cancer management: From therapy to biomarkers.

Oxysterols are oxidized derivatives of cholesterol, both endogenous and exogenous. They have been implicated in numerous pathologies, including cancer. In addition to their roles in carcinogenesis, proliferation, migration, apoptosis, and multiple signalling pathways, they have been shown to modulate cancer therapy. They are known to affect therapy of hormonally positive breast cancer through modulating oestrogen receptor activity. Oxysterols have also been shown in various in vitro models to influence efficacy of chemotherapeutics, such as doxorubicin, vincristine, cisplatin, 5-fluorouracil, and others. Their effects on the immune system should also be considered in immunotherapy. Selective anti-cancer cytotoxic properties of some oxysterols make them candidates for new therapeutic molecules. Finally, differences in oxysterol levels in blood of cancer patients in different stages or versus healthy controls, and in tumour versus non-tumour tissues, show potential of oxysterols as biomarkers for cancer management and patient stratification for optimization of therapy. LINKED ARTICLES: This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc.

Pharmacogenomics to Predict Tumor Therapy Response: A Focus on ATP-Binding Cassette Transporters and Cytochromes P450.

Pharmacogenomics is an evolving tool of precision medicine. Recently, due to the introduction of next-generation sequencing and projects generating "Big Data", a plethora of new genetic variants in pharmacogenes have been discovered. Cancer resistance is a major complication often preventing successful anticancer treatments. Pharmacogenomics of both somatic mutations in tumor cells and germline variants may help optimize targeted treatments and improve the response to conventional oncological therapy. In addition, integrative approaches combining copy number variations and long noncoding RNA profiling with germline and somatic variations seem to be a promising approach as well. In pharmacology, expression and enzyme activity are traditionally the more studied aspects of ATP-binding cassette transporters and cytochromes P450. In this review, we briefly introduce the field of pharmacogenomics and the advancements driven by next-generation sequencing and outline the possible roles of genetic variation in the two large pharmacogene superfamilies. Although the evidence needs further substantiation, somatic and copy number variants as well as rare variants and common polymorphisms in these genes could all affect response to cancer therapy. Regulation by long noncoding RNAs has also been shown to play a role. However, in all these areas, more comprehensive studies on larger sets of patients are needed.

Quantum dissipation driven by electron transfer within a single molecule investigated with atomic force microscopy.

Intramolecular charge transfer processes play an important role in many biological, chemical and physical processes including photosynthesis, redox chemical reactions and electron transfer in molecular electronics. These charge transfer processes are frequently influenced by the dynamics of their molecular or atomic environments, and they are accompanied with energy dissipation into this environment. The detailed understanding of such processes is fundamental for their control and possible exploitation in future technological applications. Most of the experimental studies of the intramolecular charge transfer processes so far have been carried out using time-resolved optical spectroscopies on large molecular ensembles. This hampers detailed understanding of the charge transfer on the single molecular level. Here we build upon the recent progress in scanning probe microscopy, and demonstrate the control of mixed valence state. We report observation of single electron transfer between two ferrocene redox centers within a single molecule and the detection of energy dissipation associated with the single electron transfer.

First experience in the Czech Republic with perirectal hydrogel injection before radiotherapy for prostate cancer.

PURPOSE: SpaceOAR® is a Food and Drug Administration approved hydrogel injection used to create space between the prostate and rectum before prostate radiotherapy (RT). This bio-degradable hydrogel was not available in the Czech Republic until 2019. We present our first experience as a new established SpaceOAR® centre. We namely focused on technical difficulties with a new procedure and possible medical complications. METHODS: We injected SpaceOAR® to 58 patients indicated for prostate RT due to prostate cancer. Prospectively and retrospectively, we monitored the learning curve and complication rate and we assessed the feasibility as an out-patient procedure in the Czech medical environment. RESULTS: The procedure is technically feasible as an out-patient procedure in a urological office. The learning curve with reasonable ultrasound experience and adequate equipment is acceptably short. The number of complications which might be associated with hydrogel injection was very low, with one exception in our centre - ulceration of the rectum. CONCLUSION: SpaceOAR® injection is a minimally invasive out-patient procedure with expected minimum complications and it is easy to learn.

Chirality-Controlled Self-Assembly of Amphiphilic Dibenzo[6]helicenes into Langmuir-Blodgett Thin Films.

Racemic and highly enantioenriched 3-methoxycarbonyl, 3-carboxy, and 3-hydroxymethyl derivatives of dibenzo[6]helicene were prepared. The Langmuir layers of these helicenes were formed at the air-water interface and transferred onto solid substrates to afford Langmuir-Blodgett films, which were then studied by ambient atomic force microscopy and (chir)optical spectroscopy. Significant differences were found in the behaviour of the Langmuir layers as well as in the morphology, UV/Vis, electronic circular dichroism (ECD), and fluorescence spectra of the Langmuir-Blodgett thin films depending on the molecular chirality and nature of the polar group. The experimental results were supported by molecular dynamics simulations.

Brain activity on fMRI associated with urinary bladder filling in patients with a complete spinal cord injury.

OBJECTIVE: Patients with complete spinal cord injury (SCI) may maintain some perception of bladder fullness. The aim of the study was to evaluate brain activation arising from anticipated extraspinal sensory pathways. METHODS: Fourteen patients ages 24-54 years were enrolled, all having experienced a complete SCI (ASIA A) at C7 to T5 an average of 17 months before study entry. Urodynamic equipment was used for repeated bladder filling and detrusor activity evaluation. All functional magnetic resonance imaging measurements were performed using a Siemens Trio 3T scanner with the GRE-EPI sequence (field of view = 192 × 192 mm, voxel 3 × 3 × 3 mm, TR/TE = 3000/30 ms, 45 slices). Nine hundred dynamic scans were acquired over 45 min. Statistical analysis was done in SPM8 using a general linear model. Statistics using t-tests were thresholded at P = 0.001. RESULTS: We excluded results from two patients because of activation artifacts. In 8 of 12 patients, significant brain activity was observed during urinary bladder filling. We found significant activation clusters at the nucleus of the solitary tract (NTS) (3/8), parabrachial nucleus (PBN) (4/8), hypothalamus (4/8), thalamus (6/8), amygdala (7/8), insular lobe (5/8), anterior cingulate gyrus (5/8), and prefrontal cortex (8/8). Activations in nuclei involved in afferents likely from the vagal nerve (NTS and PBN) correlated significantly with reported bladder sensations. CONCLUSIONS: These data suggest that extraspinal sensory pathways may develop following SCI and that vagal nerve may play a role in re-innervation of the urinary bladder. Neurourol. Urodynam. 36:155-159, 2017. © 2015 Wiley Periodicals, Inc.

Brain activity during bladder filling and pelvic floor muscle contractions: a study using functional magnetic resonance imaging and synchronous urodynamics.

OBJECTIVES: To map the brain activity during bladder filling by functional magnetic resonance imaging using a refined scanning protocol including synchronous urodynamics and pelvic floor muscle contractions. METHODS: A total of 23 healthy female volunteers (age 20-68 years) were enrolled. Participants were asked to contract their pelvic floor muscles. This was followed by a urodynamic examination consisting of repeated filling cycles. Brain activity was measured by functional magnetic resonance imaging using a 3T magnetic resonance system. Measurements of brain activity consisted of 120 functional scans during pelvic floor contractions and 210 scans during bladder filling. Each functional magnetic resonance imaging scan covered the brain with 35 slices. Statistical analyses used the general linear model and independent component analysis. Areas of activation were visualized using group statistics. RESULTS: The following main clusters of activation were observed during pelvic floor muscle contractions: medial surface of the frontal lobe (primary motor area), bilaterally; supplementary motor area, bilaterally; and left gyrus precentralis. During bladder filling, activation was detected in the inferior frontal lobe bordering the frontal cingulum, left gyrus parietalis superior, left central area, right insula, brainstem and thalamus with subcortical gray matter nuclei. CONCLUSIONS: Our work extends an existing functional magnetic resonance imaging protocol for researching the neural control of the lower urinary tract. The present results are consistent with the available literature and agree with the present hypothetical functional model of lower urinary tract neural control.

Electrospray ionization mass spectrometric investigations of the complexation behavior of macrocyclic thiacrown ethers with bivalent transitional metals (Cu, Co, Ni and Zn).

RATIONALE: Heavy metals are both a problem for the environment and an important resource for industry. Their selective extraction by means of organic ligands therefore is an attractive topic. The coordination of three thiacrown ethers to late 3d-metal ions was investigated by a combination of electrospray ionization mass spectrometry (ESI-MS) and electron paramagnetic resonance (EPR). METHODS: The mass spectrometric experiments were carried out in an ion trap mass spectrometer with an ESI source. Absolute binding constants were estimated by comparison with data for 18-crown-6/Na(+). EPR spectroscopy was used as a complementary method for investigating the Cu(I) /Cu(II) redox couple. RESULTS: The study found that thiacrown ethers preferentially bind traces of copper even at an excess of other metal ions (Co(II), Ni(II), and Zn(II)). The absolute association constants of the Cu(I) complexes were about 10(8) M(-1), and about two orders of magnitude lower for the other 3d-metal cations. The EPR spectra demonstrated that the reduction from Cu(II) to Cu(I) upon formation of the [(thiacrown)Cu](+) species takes place in solution. CONCLUSIONS: ESI-MS demonstrated that the three thiacrown ligands examined had high binding constants as well as good selectivities for copper(I) at low concentrations, and in the presence of other metal ions. By a combination of ESI-MS and EPR spectrometry it was shown that the reduction from Cu(II) to Cu(I) occurred in solution.

A preliminary report on the use of functional magnetic resonance imaging with simultaneous urodynamics to record brain activity during micturition.

PURPOSE: We mapped brain activity during micturition using functional magnetic resonance imaging with simultaneous recording of urodynamic properties during slow bladder filling and micturition. MATERIALS AND METHODS: We evaluated 12 healthy female volunteers 20 to 68 years old. Eight subjects could urinate while supine. Meaningful data were obtained on 6 of these subjects. Brain activity was recorded continuously during bladder filling and micturition. Functional magnetic resonance imaging measurements made during the micturition phase were used for the final analysis. RESULTS: Using group statistics we identified clusters of brain activity in the parahippocampal gyrus, anterior cingulate gyrus, inferior temporal gyrus and inferior frontal gyrus during micturition. At the individual level we also observed activation in the upper pontine region, thalamus and posterior cingulum. In subjects unable to void brain activation was documented in the frontal lobe and posterior cingulate gyrus but not in the pons, thalamus or anterior cingulate gyrus. In 5 subjects we identified a relevant pattern of brain activity during the terminal portion of the filling phase when the patient reported a strong desire to urinate. CONCLUSIONS: This new protocol allows for the localization of brain structures that are active during micturition. Data suggest that additional validation studies are needed. Future studies will test modifications that include more detailed monitoring of bladder sensation, stratifying subjects based on age and gender, and increasing the number of data points by adding subjects and the number of micturitions recorded in a single subject.