Renin synthesis and secretion by principal cells of the collecting duct (CD) is enhanced in angiotensin (Ang) II-dependent hypertension. The presence of renin/(pro)renin and its receptor, the (pro)renin receptor [(P)RR], in the CD may provide a pathway for Ang I generation with further conversion to Ang II. To assess if (P)RR activation occurs during Ang II-dependent hypertension, we examined renal (P)RR levels and soluble (P)RR (s(P)RR) excretion in the urine of chronic Ang II-infused rats (80 ng/min; for 2-weeks, n=10) and sham-operated rats (n=10). Systolic blood pressure and Ang II levels in the plasma and kidney were increased while plasma renin activity was suppressed in Ang II-infused rats. Renal (P)RR transcripts were upregulated in the cortex and medulla of Ang II-infused rats. (P)RR immunoreactivity in CD cells and the protein levels of the full-length form (37 kDa band) were significantly decreased in the medulla of Ang II-infused rats. The soluble (P)RR (28 kDa band) was detected in the renal medulla and urine samples of Ang II-infused rats which also showed increases in urinary renin content. To determine if the s(P)RR could stimulate Ang I formation, urine samples were incubated with recombinant human (pro)renin. Urine samples of Ang II-infused rats exhibited increased Ang I formation compared to sham-operated rats. Thus, in chronic Ang II-infused rats the catalytic activity of the augmented renin produced in the CD may be enhanced by the intraluminal s(P)RR and cell-surface located (P)RR, thus contributing to enhanced intratubular Ang II formation.

During renin-angiotensin system activation, cyclooxygenase-2 (COX-2)-derived prostaglandins attenuate the pressor and antinatriuretic effects of angiotensin II (AngII) in the renal medulla. The (pro)renin receptor (PRR) is abundantly expressed in the collecting ducts (CD) and its expression is augmented by AngII. PRR overexpression upregulates COX-2 via mitogen-activated kinases (MAPK/ERK1/2) in renal tissues; however, it is not clear if this effect occurs independently or in concert with AngII type 1 receptor (AT1R) activation. We hypothesized that PRR activation stimulates COX-2 expression independently of AT1R in primary cultures of rat renal inner medullary (IM) cells. The use of different cell-specific immunomarkers (aquaporin-2 for principal cells, anion exchanger type-1 for intercalated type-A cells, and tenascin C for interstitial cells) and co-staining for AT1R, COX-2 and PRR revealed that PRR and COX-2 were colocalized in intercalated and interstitial cells while principal cells did not express PRR or COX-2. In normal rat kidney sections, PRR and COX-2 were colocalized in intercalated and interstitial cells. In rat renal IM cultured cells, treatment with AngII (100 nmol/L) increased COX-2 expression via AT1R. In addition, AngII and rat recombinant prorenin (rrPR; 100 nmol/L) treatments increased ERK1/2 phosphorylation, independently. Importantly, rrPR upregulated COX-2 expression in the presence of AT1R blockade. Inhibition of MAPK/ERK1/2 suppressed COX-2 upregulation mediated by either AngII or rrPR. Furthermore, PRR knockdown using PRR-short hairpin RNA blunted the rrPR-mediated upregulation of COX-2. These results indicate that COX-2 expression is upregulated by activation of either PRR or AT1R via MAPK/ERK1/2 in rat renal IM cells.

The (pro)renin receptor [(P)RR] upregulates cyclooxygenase-2 (COX-2) in inner medullary collecting duct (IMCD) cells through ERK1/2. Intrarenal COX-2 and (P)RR are upregulated during chronic ANG II infusion. However, the duration of COX-2 and (P)RR upregulation has not been determined. We hypothesized that during the early phase of ANG II-dependent hypertension, membrane-bound (P)RR and COX-2 are augmented in the renal medulla, serving to buffer the hypertensinogenic and vasoconstricting effects of ANG II. In Sprague-Dawley rats infused with ANG II (0.4 μg·min(-1)·kg(-1)), systolic blood pressure (BP) increased by day 7 (162 ± 5 vs. 114 ± 10 mmHg) and continued to increase by day 14 (198 ± 15 vs. 115 ± 13 mmHg). Membrane-bound (P)RR was augmented at day 3 coincident with phospho-ERK1/2 levels, COX-2 expression, and PGE2 in the renal medulla. In contrast, membrane-bound (P)RR was reduced and COX-2 protein levels were not different from controls by day 14. In cultured IMCD cells, ANG II increased secretion of the soluble (P)RR. In anesthetized rats, COX-2 inhibition decreased the glomerular filtration rate (GFR) and renal blood flow (RBF) during the early phase of ANG II infusion without altering BP. However, at 14 days of ANG II infusions, COX-2 inhibition decreased mean arterial BP (MABP), RBF, and GFR. Thus, during the early phase of ANG II-dependent hypertension, the increased (P)RR and COX-2 expression in the renal medulla may contribute to attenuate the vasoconstrictor effects of ANG II on renal hemodynamics. In contrast, at 14 days the reductions in RBF and GFR caused by COX-2 inhibition paralleled the reduced MABP, suggesting that vasoconstrictor COX-2 metabolites contribute to ANG II hypertension.

Background: Manual qualitative and quantitative measures of terminal duct lobular unit (TDLU) involution were previously reported to be inversely associated with breast cancer risk. We developed and applied a deep learning method to yield quantitative measures of TDLU involution in normal breast tissue. We assessed the associations of these automated measures with breast cancer risk factors and risk. Methods: We obtained eight quantitative measures from whole slide images from a benign breast disease (BBD) nested case–control study within the Nurses' Health Studies (287 breast cancer cases and 1,083 controls). Qualitative assessments of TDLU involution were available for 177 cases and 857 controls. The associations between risk factors and quantitative measures among controls were assessed using analysis of covariance adjusting for age. The relationship between each measure and risk was evaluated using unconditional logistic regression, adjusting for the matching factors, BBD subtypes, parity, and menopausal status. Qualitative measures and breast cancer risk were evaluated accounting for matching factors and BBD subtypes. Results: Menopausal status and parity were significantly associated with all eight measures; select TDLU measures were associated with BBD histologic subtype, body mass index, and birth index (P < 0.05). No measure was correlated with body size at ages 5–10 years, age at menarche, age at first birth, or breastfeeding history (P > 0.05). Neither quantitative nor qualitative measures were associated with breast cancer risk. Conclusions: Among Nurses' Health Studies women diagnosed with BBD, TDLU involution is not a biomarker of subsequent breast cancer. Impact: TDLU involution may not impact breast cancer risk as previously thought.

Terminal ductal lobular unit (TDLU) involution is the regression of milk-producing structures in the breast. Women with less TDLU involution are more likely to develop breast cancer. A major bottleneck in studying TDLU involution in large cohort studies is the need for laborintensive manual assessment of TDLUs. We developed a computational pathology solution to automatically capture TDLU involution measures.Whole slide images (WSIs) of benign breast biopsies were obtained from the Nurses' Health Study (NHS). A first set of 92 WSIs was annotated for TDLUs, acini and adipose tissue to train deep convolutional neural network (CNN) models for detection of acini, and segmentation of TDLUs and adipose tissue. These networks were integrated into a single computational method to capture TDLU involution measures including number of TDLUs per tissue area (mm 2 ), median TDLU span (µm) and median number of acini per TDLU. We validated our method on 40 additional WSIs by comparing with manually acquired measures.Our CNN models detected acini with an F1 score of 0.73±0.09, and segmented TDLUs and adipose tissue with Dice scores of 0.86±0.11 and 0.86±0.04, respectively. The inter-observer ICC scores for manual assessments on 40 WSIs of number of TDLUs per tissue area, median TDLU span, and median acini count per TDLU were 0.71, 95% CI [0.51, 0.83], 0.81, 95% CI [0.67, 0.90], and 0.73, 95% CI [0.54, 0.85], respectively. Intra-observer reliability was evaluated on 10/40 WSIs with ICC scores of >0.8. Inter-observer ICC scores between automated results and the mean of the two observers were: 0.80, 95% CI [0.63, 0.90] for number of TDLUs per tissue area, 0.57, 95% CI [0.19, 0.77] for median TDLU span, and 0.80, 95% CI [0.62, 0.89] for median acini count per TDLU. TDLU involution measures evaluated by manual and automated assessment were inversely associated with age and menopausal status.We have developed a computational pathology method to measure TDLU involution. This technology eliminates the labor-intensiveness and subjectivity of manual TDLU assessment, and can be applied to future breast cancer risk studies. Wetstein et al. 3

Background The soluble prorenin receptor (sPRR), a member of the renin-angiotensin system (RAS), is elevated in plasma of patients with preeclampsia, hypertension, chronic kidney disease (CKD), and type 2 diabetes. Our goal was to examine the relationship between sPRR and RAS activation to define whether sexual dimorphisms in sPRR might explain sex disparities in renal outcomes in patients with type 2 diabetes. Methods Two hundred sixty-nine participants were included in the study (mean age, 48 ± 16 years; 42% men, 58% women), including 173 controls and 96 subjects with type 2 diabetes. In plasma and urine, we measured sPRR, plasma renin activity (PRA), and prorenin. In the urine, we also measured angiotensinogen along with other biomarkers of renal dysfunction. Results Plasma sPRR and PRA were significantly higher in women with type 2 diabetes compared to men. In these women, plasma sPRR was positively correlated with PRA, age, and body mass index (BMI). In contrast, in men the sPRR in urine but not in plasma positively correlated with eGFR in urine, but negatively correlated with urine renin activity, plasma glucose, age, and BMI. Conclusions In patients with type 2 diabetes, sPRR contributes to RAS stimulation in a sex-dependent fashion. In diabetic women, increased plasma sPRR parallels the activation of systemic RAS; while in diabetic men, decreased sPRR in urine matches intrarenal RAS stimulation. sPRR might be a potential indicator of intrarenal RAS activation and renal dysfunction in men and women with type 2 diabetes.

PURPOSE Metastatic prostate cancer is initially sensitive to androgen receptor inhibition, but eventually becomes castration-resistant prostate cancer (mCRPC). Early use of more intensive therapies targeting androgen receptor and other oncogenic drivers in treatment-naïve primary prostate cancer (PC) may be more effective than that in advanced mCRPC. However, analysis of primary tumors may not reveal targetable metastatic drivers that are subclonal in the primary tumor or acquired at metastatic sites. METHODS PC samples spanning one patient's clinical course: diagnostic biopsies, pre- or post-enzalutamide metastatic biopsies, and rapid autopsy samples including a patient-derived xenograft (PDX) were analyzed by targeted exome sequencing followed by phylogenetic analysis. RESULTS Left- and right-lobe primary PC tumors appeared to diverge, with the right acquiring additional shared mutations and striking differences in copy number alterations that later appeared in metastatic samples during the treatment course and at autopsy, whereas the left base tumor maintained a quiet copy number alteration landscape and partitioned into a dead-end node. RB1 loss, a common finding in advanced castration-resistant disease, was identified throughout mCRPC samples, but not in the primary tumor. Significantly, a truncal EGFR-activating mutation (R108K) was identified in the primary tumor and was also found to be maintained in the mCRPC samples and in a PDX model. Furthermore, the PDX model remained sensitive to the EGFR inhibitor erlotinib, despite the presence of both RB1 and BRCA2 losses. CONCLUSION These findings indicate that truncal alterations identified in primary PC can drive advanced mCRPC, even in the presence of additional strong oncogenic drivers (ie, RB1 and BRCA2 loss), and suggest that earlier detection and targeting of these truncal alterations may be effective at halting disease progression.

and case study poster sessions will be conducted during the 2019 College of American Pathologists Annual Meeting (CAP19), which is scheduled for September 21 to 25, 2019. The meeting will take place at the Gaylord Palms Resort & Convention Center, Kissimmee, Florida. The poster sessions will occur in the CAP19 Exhibit Hall. Specific dates and times for each poster session are listed below; “poster focus” times are dedicated poster-viewing periods. Also shown before each poster session are the subject areas that will be presented.