Use of a Modified Continent Ileovesicostomy in Adults with Prior Enterocystoplasty.

BACKGROUND/AIMS: To describe our initial experience with a novel modification of the Mitrofanoff conduit technique utilizing the Yang-Monti ileovesicostomy and the serosa lined extramural tunnel of the T-pouch to create a continent catheterizable stoma in patients with a prior enterocystoplasty. METHODS: A 14 cm segment of bowel was harvested, and the distal 4 cm was divided and reconfigured utilizing the Yang-Monti technique. The remaining segment was folded into a U and secured with a serosal basting stitch. Half of the Yang-Monti tube is laid in the trough of the U-shaped segment and secured. Next, the U-shaped segment was incised along the anti-mesenteric border for the length of the tube. The newly created flaps adjacent to the tube was then laid over the tube and sutured together completing the serosa lined tunnel. The entire patch was anastomosed to a cystostomy through the previous enterocystoplasty. Finally, the proximal end of the tube was brought through the umbilicus and matured as a stoma. RESULTS: Two patients with prior enterocystoplasties underwent the procedure described above. At follow-up of 18 and 24 months, both patients reported excellent continence. To date, there have been no revisions or significant complications. CONCLUSION: The construction of continent catheterizable stoma in adults with prior history of enterocystoplasty presents many technical challenges. The combination of the Yang-Monti ileovesicostomy and the extramural tunnel of the T-pouch provides an effective option for creating a continent catheterizable stoma in adults with prior history of enterocystoplasty.

Teaching mid-urethral sling surgery to residents: Impact on operative time and postoperative outcomes.

AIMS: The purpose of this study was to determine the impact of resident teaching on outcomes of mid-urethral sling surgery. METHODS: A retrospective review of female patients who underwent an outpatient transobturator (TOT) synthetic mid-urethral sling procedure with and without concomitant prolapse repair by two surgeons (JA, KE) in a tertiary female pelvic medicine practice was performed. Total procedure time (TPT = time from incision to closure including sling placement and any prolapse procedure), estimated blood loss (EBL), and postoperative complications including urinary retention, mesh exposure, reoperation, vaginal bleeding, and leg pain were compared between cases with and without the presence of a resident. RESULTS: One hundred thirty-four women underwent an outpatient transobturator sling procedure. Fifty-seven patients (43%) had a concomitant prolapse procedure. A resident was present at 57% (76/134) of cases. The average observed TPT (±SEM) was 60.6 ± 3.1 min when a resident was present and 46.6 ± 2.5 min when a resident was not present (P = 0.001). However, residents were more likely to be present when concomitant procedures were performed (P = 0.003). After adjusting for this, the presence of a resident increased TPT by an estimated 7.9 ± 2.5 min (P = 0.002). There was no statistical difference in EBL or postoperative complications. CONCLUSIONS: Resident participation in transobturator sling procedures resulted in a statistically significant, although clinically small, increase in operative time and had no significant impact on EBL or postoperative complications.

Prospective Pilot Trial to Evaluate a High Resolution Diffusion-Weighted MRI in Prostate Cancer Patients.

OBJECTIVES: High-resolution prostate imaging may allow for detection of subtle changes in tumor size, decrease the reliance on biopsies, and help define tumor boundaries during ablation. This pilot clinical trial evaluates a novel high-resolution prostate MRI for detection of small, biopsy-proven prostate tumors. METHODS: Our team developed a software that can be loaded on any modern MRI to generate high resolution diffusion-weighted imaging sequences (HR-DWI), which were compared to standard diffusion-weighted imaging sequence (S-DWI) in a prospective pilot trial in active surveillance patients. HR-DWI captures the entire volume of the prostate rather than sections, reducing streaking artifacts and geometric distortions. Multiple shots, rather than single shots, are used to differentiate signal and noise, enhancing resolution. All images were read by two radiologists. The primary outcome was the percent of biopsy-proven zones seen in 17 patients. The trial was powered to detect discordant proportions of 0.04 and 0.40 at one-sided alpha=0.05. RESULTS: The resolution was defined using standard phantoms. HR-DWI produced a 5-fold improvement in spatial resolution when compared to S-DWI. Multiparametric (MP)-MRI incorporating S-DWI was useful for predicting biopsy results (AUC 0.72, Fisher's exact p<0.001); however, using HR-DWI allowed MP-MRI to be more highly predictive of biopsy results (AUC 0.88, Fisher's exact p<0.001). AUC for MP-MRI incorporating HR-DWI was significantly larger than MP-MRI incorporating S-DWI (p=0.002). MP-MRI with HR-DWI had a sensitivity of 95.7% and identified tumor in 22 of 23 zones proven to have cancer on biopsy. In contrast, MP-MRI with S-DWI had a sensitivity of 60.9% and only identified 14 of 23 biopsy-positive zones (p=0.004). CONCLUSION: We developed a novel DWI and evaluated its improved resolution in a clinical setting. This technology has many potential applications and should be evaluated in future clinical trials as a patient management tool.

3D high-resolution diffusion-weighted MRI at 3T: Preliminary application in prostate cancer patients undergoing active surveillance protocol for low-risk prostate cancer.

PURPOSE: To improve spatial resolution and image quality of diffusion-weighted (DW) MRI in detecting low-risk prostate cancer (lrPC) in patients undergoing active surveillance protocol (AS-PC), we propose the application of a diffusion-prepared balanced steady-state free precession (bSSFP) technique capable of multishot acquisition. METHODS: Diffusion-prepared bSSFP was compared with single-shot DW echo planar imaging (SS-DW-EPI) at two prescribed resolutions (2.1 × 2.1 × 3.5mm(3) , 0.9 × 0.9 × 3.5 mm(3) ) in nine healthy subjects and nine AS-PC patients. Geometric distortion and susceptibility artifacts were quantitatively assessed in all subjects. In AS-PC patients, lesion detection via blinded multiparametric MRI including T1-weighted, T2-weighted, dynamic contrast-enhanced imaging, and along with either of two DW methods were evaluated against 12-point biopsy. RESULTS: Geometric distortion and susceptibility artifacts were significantly less for diffusion-prepared bSSFP at both prescribed spatial resolutions than SS-DW-EPI. Apparent diffusion coefficients of healthy prostate tissue were concordant between the two DW methods at both spatial resolutions. In AS-PC patients, multiparametric MRI with diffusion-prepared bSSFP had greater sensitivity (94%, 63%), accuracy (76%, 67%), positive-predictive value (54%, 48%), negative-predictive value (97%, 82%), and area under the curve (0.80, 0.67) than with SS-DW-EPI. CONCLUSIONS: The proposed diffusion-prepared technique with higher spatial resolution and improved image quality over SS-DW-EPI resulted in better multiparametric MRI detection of lrPC in AS-PC patients.

Multiparametric MRI Improves Accuracy of Clinical Nomograms for Predicting Extracapsular Extension of Prostate Cancer.

OBJECTIVE: To compare the accuracy of multiparametric magnetic resonance imaging (MP-MRI) with the Partin tables and Memorial Sloan-Kettering (MSK) nomogram for predicting extracapsular extension (ECE) in prostate cancer and to create a tool for clinicians to estimate pathologic ECE risk. METHODS: A retrospective review of 112 patients who underwent 3T MP-MRI of the prostate and radical prostatectomy was performed. Regression analyses were carried out to identify predictors of ECE. Predictive accuracy of models based on nomogram and MP-MRI were compared. RESULTS: A total of 33 of patients (29%) had ECE on MP-MRI whereas 26 patients (23%) had ECE on final pathology. Mean age was 62.8 years and mean prostate-specific antigen was 8.2 ng/dL. MRI was a significant predictor of ECE that was independent of age, prostate-specific antigen, Gleason score, clinical stage, and percent positive cores on biopsy. Sensitivity, specificity, positive predictive value, and negative predictive value of MP-MRI for ECE were 84.6%, 87.2%, 66.7%, and 94.9%, respectively. Areas under the curve for Partin and MSK nomograms for predicting ECE were 0.85 and 0.86, respectively. Area under the curve increased to 0.92 and 0.94, respectively, when MP-MRI was added to each nomogram. We provide an online tool that integrates Partin or MSK nomogram results with ECE status determined from MRI to predict pathologic ECE. Within the typical range of risks for ECE provided by the clinical nomograms (ie, 15%-40%), MRI was useful for predicting pathologic ECE. CONCLUSION: MP-MRI may be a useful adjunct for clinically staging prostate cancer. MP-MRI improved accuracy of existing clinical nomograms for prediction of pathologic ECE.

Impact of post prostate biopsy hemorrhage on multiparametric magnetic resonance imaging.

INTRODUCTION: Hemorrhage induced by prostate biopsy can interfere with the interpretation of prostate magnetic resonance imaging (MRI). MATERIALS AND METHODS: We reviewed 101 patients who had prostate multiparametric MRI (MP-MRI) and radical prostatectomy. RESULTS: On MRI obtained within 4 weeks following the biopsy, hemorrhage was seen in 26/36 (72.2%) patients. Patients having a MRI between 4-6 weeks of the biopsy had hemorrhage in 8/14 (57.1%) cases. After 6 weeks, hemorrhage was less common but still present in 24/46 (52%) patients. There were five patients who had prostate MRI prior to biopsy and served as a control group. There was no significant correlation between the length of time beyond 6 weeks and the likelihood of having prostate hemorrhage on MRI. The overall sensitivity and specificity of MRI for predicting extracapsular extension (ECE) were 78.6% and 89%, respectively. However, if the analysis was limited to patients with MRI within 6 weeks from the time of biopsy, the sensitivity and specificity were similar: 80% and 90%, respectively. For patients with MRI obtained after 6 weeks, the sensitivity and specificity were 76.9% and 87.9%. CONCLUSIONS: Prostate hemorrhage is seen in the majority of cases within 6 weeks of biopsy and can be seen in nearly half the patients even beyond 6 weeks. However, hemorrhage within 6 weeks of a biopsy does not interfere with assessment for ECE.

Multiparametric magnetic resonance imaging localizes established extracapsular extension of prostate cancer.

OBJECTIVE: To define the accuracy of multiparametric magnetic resonance imaging (MP-MRI) for identifying focal and established extracapsular extension (ECE) in various zones of the prostate. METHODS: Between 2010 and 2013, 342 patients underwent MP-MRI of the prostate (3T, no endorectal coil with axial perfusion and diffusion images). The findings of the images were reported as negative, suspicious, or positive for ECE by a single expert radiologist. Radical prostatectomy specimens were reviewed to confirm the size and the location of ECE and further defined as focal or established ECE. Established ECE included extension that was multifocal or involving more than 5 glands. The accuracy of MRI in localizing focal and established ECE to each zone of the prostate was determined. Regression analyses were performed to identify predictors of ECE. RESULTS: We identified 112 patients who underwent prostate MP-MRI and radical prostatectomy. MRI findings considered suspicious or definite for ECE accurately predicted pathologic ECE (P<0.001). MP-MRI identified established ECE but not focal ECE. Sensitivity, specificity, positive predictive value, and negative predictive value of MP-MRI for established ECE were 70.7%, 90.6%, 57.1%, and 95.1%, respectively. MRI identified ECE to the left vs. right side as well as each zone of the prostate; however, sensitivity was lowest at the apex. On multivariate analysis, MRI was a significant predictor of ECE that was independent of prostate-specific antigen level, Gleason score, and clinical stage. CONCLUSION: MP-MRI is useful for identifying established but not focal ECE in all zones of the prostate. MRI was a significant independent predictor of established ECE and may be a useful adjunct in staging prostate cancer.

Urothelial transdifferentiation to prostate epithelia is mediated by paracrine TGF-beta signaling.

The embryonic urogenital sinus mesenchyme (UGM) induces prostate epithelial morphogenesis in development. The molecular signals that drive UGM-mediated prostatic induction have not been defined. We hypothesized that the TGF-beta signaling directed the prostatic induction. UGM from TGF-beta type II receptor stromal conditional knockout mice (Tgfbr2(fspKO)) or control mice (Tgfbr2(floxE2/floxE2)) was recombined with wild-type adult mice bladder urothelial cells. The resulting urothelium associated with Tgfbr2(floxE2/floxE2) UGM was instructively differentiated into prostatic epithelium, as expected. In contrast, the urothelium associated with Tgfbr2(fspKO) UGM permissively maintained the phenotype of bladder epithelial cells. Microarray analysis of UGM tissues suggested the down-regulation of multiple Wnt ligands and the up-regulation of the Wnt antagonist, Wif 1, by the Tgfbr2(fspKO) UGM compared with Tgfbr2(floxE2/floxE2) UGM. The overexpression of Wif-1 by wild-type UGM resulted in the inhibition of prostatic induction. These data suggest that the stromal TGF-beta activity mediated by paracrine Wnt is necessary for the induction of prostatic differentiation. As Wnt ligands mediate differentiation and maintain the stem cell phenotype, the contribution of mouse stem cells and somatic cells to prostatic epithelium in the tissue recombination models was tested. The directed differentiation of mouse embryonic stem cells by UGM is suggested by a threshold number of mouse stem cells required in prostatic differentiation. To determine the contribution of somatic cells, the adult bladder epithelial compartment was labeled with green-fluorescent vital dye (CMFDA) and the stem-like cells marked by bromodeoxyuridine (BrdU) label-retention. The resulting prostatic epithelia of the tissue recombinants maintained the CMFDA dye, suggesting minimal cell division. Thus, the UGM can induce endoderm-derived epithelia and stem cells to form prostate through a transdifferentiation mechanism that requires stromal TGF-beta signaling to mediate epithelial Wnt activity.

Temporal-spatial protein expression in bladder tissue derived from embryonic stem cells.

PURPOSE: Identifying developmental proteins could lead to markers of bladder progenitor cells, which could be used to investigate bladder diseases. We recently reported a novel embryonic stem cell model in which to study differential protein expression patterns during bladder development. Differential and temporal expressions of the endodermal proteins known as forkhead box (Foxa1 and Foxa2) were observed. In the current study we further delineated these protein expression patterns. MATERIALS AND METHODS: Epithelium was removed from the underlying mesenchyma from embryonic day 18 rat bladders. Heterospecific recombinant xenografts were created by combining embryonic stem cells plus embryonic bladder mesenchyma and placed beneath the renal capsule of mouse hosts. Grafts were harvested at 16, 18, 21, 28, 35 and 42 days, and evaluated with hematoxylin and eosin, trichrome staining, and immunohistochemistry for uroplakin, smooth muscle alpha-actin, p63, Foxa1, Foxa2 and androgen receptor. RESULTS: At 16 days uroplakin was detectable and it seemed to correlate with the loss of Foxa2, while Foxa1 remained at all time points. Androgen receptor was first noted in stroma at day 16. It localized to urothelial nuclei at day 21 and was undetectable at 42 days. Adjacent to the urothelium alpha-smooth muscle actin was seen on day 16 and it was localized in bundles to the periphery of the graft at later time points. Staining for basilar urothelium with p63 confirmed basilar orientation at all time points. CONCLUSIONS: We report the temporal spatial expression of various genes in early bladder development. This suggests that some proteins may be potential markers of bladder progenitor cells. Characterizing these markers may potentially identify bladder progenitor cells that have been directed toward a lineage path destined to become urothelial cells. Ultimately these multipotential progenitor cells could be isolated and used to study and treat diseases that affect the bladder.

Stromal transforming growth factor-beta signaling mediates prostatic response to androgen ablation by paracrine Wnt activity.

Mechanisms of androgen dependence of the prostate are critical to understanding prostate cancer progression to androgen independence associated with disease mortality. Transient elevation of transforming growth factor-beta (TGF-beta) occurs after androgen ablation. To determine the role of TGF-beta on prostate response to androgen ablation, conditional TGF-beta type II receptor knockout mouse models of the epithelia (Tgfbr2(NKX3.1KO)) and stromal fibroblasts (Tgfbr2(fspKO)) were used. After castration, the prostates of Tgfbr2(NKX3.1KO) mice had apoptosis levels similar to those expected for control Tgfbr2(floxE2/floxE2) mice. Prostates of Tgfbr2(fspKO) mice, however, had reduced regression and high levels of proliferation associated with canonical Wnt activity throughout the glandular epithelia regardless of androgen status. In contrast, Tgfbr2(floxE2/floxE2) prostates had epithelial canonical Wnt activity only in the surviving proximal ducts after castration. In vitro studies showed that androgen antagonist, bicalutamide, transiently elevated both Tgfbr2(floxE2/floxE2) and Tgfbr2(fspKO) stromal expression of Wnt-2, Wnt-3a, and Wnt-5a. The neutralization of Wnt signaling by the expression of secreted frizzled related protein-2 (SFRP-2) resulted in decreased LNCaP prostate epithelial cell proliferation in stromal conditioned media transfer experiments. In vivo tissue recombination studies using Tgfbr2(fspKO) prostatic stromal cells in combination with wild-type or SV40 large T antigen expressing epithelia resulted in prostates that were refractile to androgen ablation. The expression of SFRP-2 restored the Tgfbr2(fspKO)-associated prostate responsiveness to androgen ablation. These studies reveal a novel TGF-beta, androgen, and Wnt paracrine signaling axis that enables prostatic regression of the distal ducts after androgen ablation while supporting proximal duct survival.

Role of nicotinic and estrogen signaling during experimental acute and chronic bladder inflammation.

Inflammation is a physiological process that characterizes many bladder diseases. We hypothesized that nicotinic and estrogen signaling could down-regulate bladder inflammation. Cyclophosphamide was used to induce acute and chronic bladder inflammation. Changes in bladder inflammation were measured histologically and by inflammatory gene expression. Antagonizing nicotinic signaling with mecamylamine further aggravated acute and chronic inflammatory changes resulting from cyclophosphamide treatment. Estrogen and nicotinic signaling independently attenuated acute bladder inflammation by decreasing neutrophil recruitment and down-regulating elevated lipocalin-2 and cathepsin D expression. However, the combined signaling by the estrogen and nicotinic pathways, as measured by macrophage infiltration and up-regulation of interleukin-6 expression in the bladder, synergistically reduced chronic bladder inflammation. The elevated expression of p65 nuclear localization in bladders treated with cyclophosphamide or cyclophosphamide with mecamylamine suggested nuclear factor-kappa B activation in the chronic inflammatory process. The complementary treatment of 17 beta-estradiol and the nicotinic agonist anabasine resulted in the translocation of p65 to the cytoplasm, again greater than either alone. Activation of nuclear factor-kappaB can result in macrophage activation and/or elevation in epithelial proliferation. These data suggest that 17 beta-estradiol and anabasine reduce chronic bladder inflammation through reduction of nuclear translocation of p65 to suppress cytokine expression.

Urothelial inhibition of transforming growth factor-beta in a bladder tissue recombination model.

PURPOSE: We examined the role of transforming growth factor-beta in urothelial and bladder development. Transforming growth factor-beta signaling was attenuated in the urothelial compartment and the subsequent effects were examined in a tissue recombination model. MATERIALS AND METHODS: Urothelium was cultured from adult rat bladders and transfected with control vector C7Delta or mutant DNIIR (dominant negative transforming growth factor-beta receptor II). Grafts were created by recombining transfected urothelium plus embryonic day 18 bladder mesenchyma and placed beneath the renal capsule of athymic mouse hosts. Grafts were harvested at 21 and 42 days. Final tissues were evaluated with staining and immunohistochemistry using hematoxylin and eosin, Gomori's trichrome strain, broad-spectrum uroplakin, smooth muscle actin-alpha, phosphorylated SMAD2 and Ki67 antigen. Bladder structures were defined as having smooth muscle, suburothelial connective tissue and mature urothelium expressing uroplakin. Urothelial compartment diameters were measured and subcategorized as small--0.10 to 0.40, medium--0.41 to 1.0 and large--greater than 1.1 mm. RESULTS: At 21 days 14 C7Delta control and 15 DNIIR grafts were evaluated. No bladder tissue was seen in the C7Delta grafts vs 49 in DNIIR tissue, including 30 small, 9 medium and 10 large tissues. At 42 days 14 C7Delta and 12 DNIIR grafts were evaluated. Six bladder structures (5 small and 1 medium) were seen in the C7Delta cohort vs 27 (14 small, 7 medium and 6 large) in the DNIIR group. Immunohistochemical detection of phosphorylated-SMAD2 was significantly attenuated in DNIIR tissue. In addition, Ki67 proliferative indexes were 4.0-fold higher in the DNIIR cohort compared to those in C7Delta tissues. CONCLUSIONS: We successfully observed that primary urothelium cultures can be genetically manipulated and recombined with undifferentiated mesenchyma to grow bladder tissue. By attenuating transforming growth factor-beta signaling in the urothelium superior bladder tissue growth occurred, suggesting that transforming growth factor-beta is a growth inhibitor in this organ system.

Directed differentiation of embryonic stem cells into bladder tissue.

Manipulatable models of bladder development which interrogate specific pathways are badly needed. Such models will allow a systematic investigation of the multitude of pathologies which result from developmental defects of the urinary bladder. In the present communication, we describe a model in which mouse embryonic stem (ES) cells are directed to differentiate to form bladder tissue by specific interactions with fetal bladder mesenchyme. This model allows us to visualize the various stages in the differentiation of urothelium from ES cells, including the commitment to an endodermal cell lineage, with the temporal profile characterized by examining the induction of specific endodermal transcription factors (Foxa1 and Foxa2). In addition, final functional urothelial differentiation was characterized by examining uroplakin expression. It is well established that ES cells will spontaneously develop teratomas when grown within immunocompromised mouse hosts. We determined the specific mesenchymal to ES cell ratios necessary to dictate organ-specific differentiation while completely suppressing teratomatous growth. Embryonic mesenchyme is well established as an inductive tissue which dictates organ-specific programming of epithelial tissues. The present study demonstrates that embryonic bladder mesenchyme can also steer ES cells towards developing specific endodermal derived urothelium. These approaches allow us to capture specific stages of stem cell differentiation and to better define stem cell hierarchies.

Stromal hyperplasia in male bladders upon loss of transforming growth factor-beta signaling in fibroblasts.

PURPOSE: Rapid bladder growth associated, partial urethral obstruction and embryonic bladder development entail stromal-epithelial interactions involving signaling by the cytokine transforming growth factor-beta (TGF-beta). However, to our knowledge the role of TGF-beta in bladder stromal hyperplasia and hypertrophy is not understood. MATERIALS AND METHODS: In an effort to understand the specific role of TGF-beta signaling in bladder stroma a fibroblast specific conditional knockout mouse of the type II TGF-beta receptor gene, Tgfbr2(/spko), was generated using Cre-lox methodology. Bladders from 18, 7 to 8-week-old mice were harvested for histological and immunohistochemical analysis. RESULTS: Bladders from homozygous Tgfbr2(/spko), male mice showed marked hypertrophy in the lamina propria and smooth muscle layers in the absence of visible or functional bladder obstruction by age 8 weeks. However, age matched female mice of the same genotype maintained bladder architecture similar to that in wild-type littermate male and female controls. Immunohistochemistry for the phosphorylated form of Smad2 indicated a general loss in TGF-beta signaling in the lamina propria of bladders of male and female Tgfbr2(/spko), mice, and yet pronounced alpha-smooth muscle actin expression was noted in male Tgfbr2(/spko), bladders, which is a marker for myofibroblasts. CONCLUSIONS: A sex disparity was observed in the Tgfbr2(/spko), mouse model lacking TGF-beta signaling in fibroblasts. Deletion of TGF-beta in males leads to a hypertrophied lamina propria and muscularis externa with myofibroblast differentiation and proliferation. Female homozygous Tgfbr2(/spko), bladders appeared the same as those of wild-type male and female controls. This model suggests a role for stromal TGF-beta signaling with estrogens and androgens in bladder fibrosis.

Effect of conditional knockout of the type II TGF-beta receptor gene in mammary epithelia on mammary gland development and polyomavirus middle T antigen induced tumor formation and metastasis.

Transforming growth factor-beta (TGF-beta) isoforms are growth factors that function physiologically to regulate development, cellular proliferation, and immune responses. The role of TGF-beta signaling in mammary tumorigenesis is complex, as TGF-beta has been reported to function as both a tumor suppressor and tumor promoter. To elucidate the role of TGF-beta signaling in mammary gland development, tumorigenesis, and metastasis, the gene encoding type II TGF-beta receptor, Tgfbr2, was conditionally deleted in the mammary epithelium (Tgfbr2MGKO). Loss of Tgfbr2 in the mammary epithelium results in lobular-alveolar hyperplasia in the developing mammary gland and increased apoptosis. Tgfbr2MGKO mice were mated to the mouse mammary tumor virus-polyomavirus middle T antigen (PyVmT) transgenic mouse model of metastatic breast cancer. Loss of Tgfbr2 in the context of PyVmT expression results in a shortened median tumor latency and an increased formation of pulmonary metastases. Thus, our studies support a tumor-suppressive role for epithelial TGF-beta signaling in mammary gland tumorigenesis and show that pulmonary metastases can occur and are even enhanced in the absence of TGF-beta signaling in the carcinoma cells.