Targeted Mass Spectrometry Assays for Specific Quantification of Urinary proPSA Isoforms.

Prostate cancer (PCa) is the second leading cause of male cancer-related deaths in the United States. The pre-mature forms of prostate-specific antigen (PSA), proPSA, were shown to be associated with PCa. However, there is a technical challenge in the development of antibody-based immunoassays for specific recognition of each individual proPSA isoform. Herein, we report the development of highly specific, antibody-free, targeted mass spectrometry assays for simultaneous quantification of [-2], [-4], [-5], and [-7] proPSA isoforms in voided urine. The newly developed proPSA assays capitalize on Lys-C digestion to generate surrogate peptides with appropriate length (9-16 amino acids) along with long-gradient liquid chromatography separation. The assay utility of these isoform markers was evaluated in a cohort of 30 well-established clinical urine samples for distinguishing PCa patients from healthy controls. Under the 95% confidence interval, the combination of [-2] and [-4] proPSA isoforms yields the area under curve (AUC) of 0.86, and the AUC value for the combined all four isoforms was calculated to be 0.85. We have further verified [-2]proPSA, the dominant isoform, in an independent cohort of 34 clinical urine samples. Validation of proPSA isoforms in large-scale cohorts is needed to demonstrate their potential clinical utility.

The opposing action of stromal cell proenkephalin and stem cell transcription factors in prostate cancer differentiation.

BACKGROUND: Loss of prostate cancer differentiation or de-differentiation leads to an untreatable disease. Patient survival would benefit if this can be prevented or reversed. Cancer de-differentiation transforms luminal-like (differentiated) adenocarcinoma into less luminal-like and more stem-like (undifferentiated) small cell carcinoma through a sequential activation of stem cell transcription factors (scTF) POU5F1, LIN28A, SOX2 and NANOG. Like stem cells, prostate small cell carcinoma express this quartet of scTF as well as a 10-fold lower level of ß2-microglobulin (B2M) than that of differentiated cell types. In organ development, prostate stromal mesenchyme cells mediate epithelial differentiation in part by secreted factors. METHODS: The identified prostate stromal-specific factor proenkephalin (PENK) was cloned, and transfected into scTF+B2Mlo stem-like small cell carcinoma LuCaP 145.1, reprogrammed luminal-like scTF-B2Mhi LNCaP, and luminal-like scTF-B2Mhi adenocarcinoma LuCaP 70CR. The expression of scTF, B2M and anterior gradient 2 (AGR2) was analyzed in the transfected cells. RESULTS: PENK caused down-regulation of scTF and up-regulation of B2M to indicate differentiation. When transfected into reprogrammed LNCaP, PENK reversed the reprogramming by down-regulation of scTF with attendant changes in cell appearance and colony morphology. When transfected into LuCaP 70CR, PENK up-regulated the expression of adenocarcinoma antigen AGR2, a marker associated with cancer cell differentiation. CONCLUSIONS: Prostate cancer cells appear to retain their responsiveness to stromal PENK signaling. PENK can induce differentiation to counter de-differentiation caused by scTF activation. The many mutations and aneuploidy characteristic of cancer cells appear not to hinder these two processes. Loss of prostate cancer differentiation is like reprogramming from luminal-like to stem-like.

Identification of functionally important residues and structural features in a bacterial lignostilbene dioxygenase.

Lignostilbene-α,ß-dioxygenase A (LsdA) from the bacterium Sphingomonas paucimobilis TMY1009 is a nonheme iron oxygenase that catalyzes the cleavage of lignostilbene, a compound arising in lignin transformation, to two vanillin molecules. To examine LsdA's substrate specificity, we heterologously produced the dimeric enzyme with the help of chaperones. When tested on several substituted stilbenes, LsdA exhibited the greatest specificity for lignostilbene (kcatapp = 1.00 ± 0.04 × 106 m-1 s-1). These experiments further indicated that the substrate's 4-hydroxy moiety is required for catalysis and that this moiety cannot be replaced with a methoxy group. Phenylazophenol inhibited the LsdA-catalyzed cleavage of lignostilbene in a reversible, mixed fashion (Kic = 6 ± 1 µm, Kiu = 24 ± 4 µm). An X-ray crystal structure of LsdA at 2.3 Å resolution revealed a seven-bladed ß-propeller fold with an iron cofactor coordinated by four histidines, in agreement with previous observations on related carotenoid cleavage oxygenases. We noted that residues at the dimer interface are also present in LsdB, another lignostilbene dioxygenase in S. paucimobilis TMY1009, rationalizing LsdA and LsdB homo- and heterodimerization in vivo A structure of an LsdA·phenylazophenol complex identified Phe59, Tyr101, and Lys134 as contacting the 4-hydroxyphenyl moiety of the inhibitor. Phe59 and Tyr101 substitutions with His and Phe, respectively, reduced LsdA activity (kcatapp) ∼15- and 10-fold. The K134M variant did not detectably cleave lignostilbene, indicating that Lys134 plays a key catalytic role. This study expands our mechanistic understanding of LsdA and related stilbene-cleaving dioxygenases.

A Systematic Review of Mental Health-Improving Interventions in Veterinary Students.

Literature over the past 5 years has demonstrated that veterinary students globally are experiencing poor mental health. This has detrimental consequences for their emotional well-being and physical health, as well as implications for their future careers. Considering this issue, a systematic review was devised to investigate what interventions were being used, and what effect they had, in veterinary students. The review process involved a search of five databases, from which 161 records were retrieved. Following this, the screening process revealed seven articles eligible for appraisal. These studies investigated seven different interventions, six being cohort-level workshops/courses and one being a collation of several individual strategies. All seven studies reported that the interventions were effective to some degree in improving the mental health of their participants. However, the lack of repeat interventions and control groups limited the external validity of each intervention. A comparison to the research in medical students is briefly discussed. Three of the appraised articles were recommended for further investigation.

Lineage relationship between prostate adenocarcinoma and small cell carcinoma.

BACKGROUND: Prostate cancer displays different morphologies which, in turn, affect patient outcome. This fact prompted questions about the lineage relationship between differentiated, more treatable prostate adenocarcinoma and poorly differentiated, less treatable non-adenocarcinoma including small cell carcinoma, and the molecular mechanism underlying prostate cancer differentiation. METHODS: Newly available non-adenocarcinoma/small cell carcinoma PDX LuCaP lines were analyzed for expression of stem cell transcription factors (scTF) LIN28A, NANOG, POU5F1, SOX2, which are responsible for reprogramming or de-differentiation. cDNA of these genes were cloned from small cell carcinoma LuCaP 145.1 into expression vectors to determine if they could function in reprogramming. RESULTS: Expression of scTF was detected in small cell carcinoma LuCaP 93, 145.1, 145.2, and non-adenocarcinoma LuCaP 173.1, 173.2A. Transfection of scTF from LuCaP 145.1 altered the gene expression of prostate non-small cell carcinoma cells, as well as fibroblasts. The resultant cells grew in stem-like colonies. Of note was a 10-fold lower expression of B2M in the transfected cells. Low B2M was also characteristic of LuCaP 145.1. Conversely, B2M was increased when stem cells were induced to differentiate. CONCLUSIONS: This work suggested a pathway in the emergence of non-adenocarcinoma/small cell carcinoma from adenocarcinoma through activation of scTF genes that produced cancer de-differentiation.

Conversion of Prostate Adenocarcinoma to Small Cell Carcinoma-Like by Reprogramming.

The lineage relationship between prostate adenocarcinoma and small cell carcinoma was studied by using the LuCaP family of xenografts established from primary neoplasm to metastasis. Expression of four stem cell transcription factor (TF) genes, LIN28A, NANOG, POU5F1, SOX2, were analyzed in the LuCaP lines. These genes, when force expressed in differentiated cells, can reprogram the recipients into stem-like induced pluripotent stem (iPS) cells. Most LuCaP lines expressed POU5F1, while LuCaP 145.1, representative of small cell carcinoma, expressed all four. Through transcriptome database query, many small cell carcinoma genes were also found in stem cells. To test the hypothesis that prostate cancer progression from "differentiated" adenocarcinoma to "undifferentiated" small cell carcinoma could involve re-expression of stem cell genes, the four TF genes were transduced via lentiviral vectors into five adenocarcinoma LuCaP lines-70CR, 73CR, 86.2, 92, 105CR-as done in iPS cell reprogramming. The resultant cells from these five transductions displayed a morphology of small size and dark appearing unlike the parentals. Transcriptome analysis of LuCaP 70CR* ("*" to denote transfected progeny) revealed a unique gene expression close to that of LuCaP 145.1. In a prostate principal components analysis space based on cell-type transcriptomes, the different LuCaP transcriptome datapoints were aligned to suggest a possible ordered sequence of expression changes from the differentiated luminal-like adenocarcinoma cell types to the less differentiated, more stem-like small cell carcinoma types, and LuCaP 70CR*. Prostate cancer progression can thus be molecularly characterized by loss of differentiation with re-expression of stem cell genes. J. Cell. Physiol. 231: 2040-2047, 2016. © 2016 Wiley Periodicals, Inc.

Processing of voided urine for prostate cancer RNA biomarker analysis.

BACKGROUND: Voided urine samples have been shown to contain cells released from prostate tumors. Could good quality RNA from cells in urine be obtained from every donor for multimarker analysis? In addition, could urine donation be as simple as possible, a practical consideration for a lab test, without involving a prostate massage (as indicated for PCA3 testing), which precludes frequent collection; needing it done at a specific time of day (e.g., first or second urine); and requiring prompt processing of samples in clinics with limited molecular biology capability? METHODS: Collected urine samples were pelleted, and the RNA isolated was processed for cDNA synthesis and in vitro transcription to generate amplified sense aRNA. The resultant aRNA was rigorously analyzed for possible introduced changes. DMSO was used as a cell preservative for frozen storage of urine samples. RESULTS: Good quality aRNA was obtained for over 100 samples collected at two different institutions. The process of RNA amplification removed co-isolated DNA in some samples, which did not affect RNA amplification. Amplification did not amplify genes that were absent and produce other expression alterations. The sense aRNA could be used to generate urinary transcriptomes specific to individual patients. No chaotropic agents for RNA preservation were added to the urine samples so that the supernatant could be used for analysis of secreted protein biomarkers. The time of donation was not important since patients were seen during the entire day. DMSO was an effective cell preservative for freezing urine. CONCLUSIONS: Urinary RNA can be readily isolated and amplified for prostate cancer biomarker analysis. Individual patients had unique set of transcripts derived from their tumor.

High expression of AGR2 in lung cancer is predictive of poor survival.

BACKGROUND: Anterior gradient 2 (AGR2) is a protein disulfide isomerase-like protein widely expressed in many normal tissues as well as cancers. In our study, non-neoplastic bronchial epithelial cells as well as non-small cell lung cancer (NSCLC) cells express AGR2 protein. METHODS: AGR2 expression was analyzed on lung tissue microarrays. Tumor staining was correlated with clinical outcomes. RESULTS: On a lung cancer tissue microarray using immunohistochemistry, expression levels in cancer showed generally decreasing intensities in order from adenocarcinomas with mucinous components, other adenocarcinomas, squamous carcinomas, to large cell carcinomas. The study cohort was comprised of 400 cases. As a group, there was a slight trend of lower expression with increasing tumor grade. AGR2 expression level was a significant predictor of overall survival in younger patients only. Patients under 65 with lower levels showed a significantly better survival for both men and women. Patients over 65, in contrast, showed no such trend. CONCLUSIONS: Nearly all NSCLC tumors show AGR2 expression. Lung cancer expression of AGR2 has prognostic value for younger patients.

Reprogramming of prostate cancer cells--technical challenges.

Prostate cancer progression is characterized by tumor dedifferentiation. Cancer cells of less differentiated tumors have a gene expression/transcriptome more similar to that of stem cells. In dedifferentiation, cancer cells may follow a specific program of gene expression changes to a stem-like state. In order to treat cancer effectively, the stem-like cancer cells and cancer differentiation pathway need to be identified and studied. Due to the very low abundance of stem-like cancer cells, their isolation from fresh human tumors is technically challenging. Induced pluripotent stem cell technology can reprogram differentiated cells into stem-like, and this may be a tool to generate sufficient stem-like cancer cells.

A highly sensitive targeted mass spectrometric assay for quantification of AGR2 protein in human urine and serum.

Anterior gradient 2 (AGR2) is a secreted, cancer-associated protein in many types of epithelial cancer cells. We developed a highly sensitive targeted mass spectrometric assay for quantification of AGR2 in urine and serum. Digested peptides from clinical samples were processed by PRISM (high pressure and high resolution separations coupled with intelligent selection and multiplexing), which incorporates high pH reversed-phase liquid chromatography (LC) separations to fractionate and select target fractions for follow-on LC-selected reaction monitoring (LC-SRM) analyses. The PRISM-SRM assay for AGR2 showed a reproducibility of <10% CV and limit of quantification (LOQ) values of ∼130 pg/mL in serum and ∼10 pg per 100 µg of total protein mass in urine, respectively. A good correlation (R(2) = 0.91) was observed for the measurable AGR2 concentrations in urine between SRM and enzyme-linked immunosorbent assay (ELISA). On the basis of an initial cohort of 37 subjects, urinary AGR2/PSA concentration ratios showed a significant difference (P = 0.026) between noncancer and cancer. Large clinical cohort studies are needed for the validation of AGR2 as a useful diagnostic biomarker for prostate cancer. Our work validated the approach of identifying candidate secreted protein biomarkers through genomics and measurement by targeted proteomics, especially for proteins where no immunoassays are available.

Prostate cancer research: tools, cell types, and molecular targets.

Multiple cancer cell types are found in prostate tumors. They are either luminal-like adenocarcinoma or less luminal-like and more stem-like non-adenocarcinoma and small cell carcinoma. These types are lineage related through differentiation. Loss of cancer differentiation from luminal-like to stem-like is mediated by the activation of stem cell transcription factors (scTF) such as LIN28A, NANOG, POU5F1 and SOX2. scTF expression leads to down-regulation of ß2-microglobulin (B2M). Thus, cancer cells can change from the scTF˜B2Mhi phenotype of differentiated to that of scTF˙B2Mlo of dedifferentiated in the disease course. In development, epithelial cell differentiation is induced by stromal signaling and cell contact. One of the stromal factors specific to prostate encodes proenkephalin (PENK). PENK can down-regulate scTF and up-regulate B2M in stem-like small cell carcinoma LuCaP 145.1 cells indicative of exit from the stem state and differentiation. In fact, prostate cancer cells can be made to undergo dedifferentiation or reprogramming by scTF transfection and then to differentiate by PENK transfection. Therapies need to be designed for treating the different cancer cell types. Extracellular anterior gradient 2 (eAGR2) is an adenocarcinoma antigen associated with cancer differentiation that can be targeted by antibodies to lyse tumor cells with immune system components. eAGR2 is specific to cancer as normal cells express only the intracellular form (iAGR2). For AGR2-negative stem-like cancer cells, factors like PENK that can target scTF could be effective in differentiation therapy.

Targeted quantification of low ng/mL level proteins in human serum without immunoaffinity depletion.

We recently reported an antibody-free targeted protein quantification strategy, termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM), for achieving significantly enhanced sensitivity using selected reaction monitoring (SRM) mass spectrometry. Integrating PRISM with front-end IgY14 immunoaffinity depletion, sensitive detection of targeted proteins at 50-100 pg/mL levels in human blood plasma/serum was demonstrated. However, immunoaffinity depletion is often associated with undesired losses of target proteins of interest. Herein we report further evaluation of PRISM-SRM quantification of low-abundance serum proteins without immunoaffinity depletion. Limits of quantification (LOQ) at low ng/mL levels with a median coefficient of variation (CV) of ∼12% were achieved for proteins spiked into human female serum. PRISM-SRM provided >100-fold improvement in the LOQ when compared to conventional LC-SRM measurements. PRISM-SRM was then applied to measure several low-abundance endogenous serum proteins, including prostate-specific antigen (PSA), in clinical prostate cancer patient sera. PRISM-SRM enabled confident detection of all target endogenous serum proteins except the low pg/mL-level cardiac troponin T. A correlation coefficient >0.99 was observed for PSA between the results from PRISM-SRM and immunoassays. Our results demonstrate that PRISM-SRM can successfully quantify low ng/mL proteins in human plasma or serum without depletion. We anticipate broad applications for PRISM-SRM quantification of low-abundance proteins in candidate biomarker verification and systems biology studies.

Long-gradient separations coupled with selected reaction monitoring for highly sensitive, large scale targeted protein quantification in a single analysis.

Long-gradient separations coupled to tandem mass spectrometry (MS) were recently demonstrated to provide a deep proteome coverage for global proteomics; however, such long-gradient separations have not been explored for targeted proteomics. Herein, we investigate the potential performance of the long-gradient separations coupled with selected reaction monitoring (LG-SRM) for targeted protein quantification. Direct comparison of LG-SRM (5 h gradient) and conventional liquid chromatography (LC)-SRM (45 min gradient) showed that the long-gradient separations significantly reduced background interference levels and provided an 8- to 100-fold improvement in limit of quantification (LOQ) for target proteins in human female serum. On the basis of at least one surrogate peptide per protein, an LOQ of 10 ng/mL was achieved for the two spiked proteins in nondepleted human serum. The LG-SRM detection of seven out of eight endogenous plasma proteins expressed at ng/mL or subng/mL levels in clinical patient sera was also demonstrated. A correlation coefficient of >0.99 was observed for the results of LG-SRM and enzyme-linked immunosorbent assay (ELISA) measurements for prostate-specific antigen (PSA) in selected patient sera. Further enhancement of LG-SRM sensitivity was achieved by applying front-end IgY14 immunoaffinity depletion. Besides improved sensitivity, LG-SRM potentially offers much higher multiplexing capacity than conventional LC-SRM due to an increase in average peak widths (~3-fold) for a 300 min gradient compared to a 45 min gradient. Therefore, LG-SRM holds great potential for bridging the gap between global and targeted proteomics due to its advantages in both sensitivity and multiplexing capacity.

Prostate cancer cell phenotypes based on AGR2 and CD10 expression.

The combination of expression patterns of AGR2 (anterior gradient 2) and CD10 by prostate cancer provided four phenotypes that correlated with clinical outcome. Based on immunophenotyping, CD10(low)AGR2(high), CD10(high)AGR2(high), CD10(low)AGR2(low), and CD10(high)AGR2(low) were distinguished. AGR2(+) tumors were associated with longer recurrence-free survival and CD10(+) tumors with shorter recurrence-free survival. In high-stage cases, the CD10(low)AGR2(high) phenotype was associated with a ninefold higher recurrence-free survival than the CD10(high)AGR2(low) phenotype. The CD10(high)AGR2(high) and CD10(low)AGR2(low) phenotypes were intermediate. The CD10(high)AGR2(low) phenotype was most frequent in high-grade primary tumors. Conversely, bone and other soft tissue metastases, and derivative xenografts, expressed more AGR2 and less CD10. AGR2 protein was readily detected in tumor metastases. The CD10(high)AGR2(low) phenotype in primary tumors is predictive of poor outcome; however, the CD10(low)AGR2(high) phenotype is more common in metastases. It appears that AGR2 has a protective function in primary tumors but may have a role in the distal spread of tumor cells.

Palliative radiotherapy in pancreatic cancer: a retrospective study of 100 cases and regional patterns of practice.

BACKGROUND: Pancreatic cancer is often incurable and can be associated with significant pain and abdominal symptoms. This study aims to characterize the symptomatic burden of patients with pancreatic cancer receiving palliative radiotherapy and the corresponding symptomatic and radiographic responses. METHODS: Patients with pancreatic adenocarcinoma referred to BC Cancer for palliative radiation from 2006 to 2013 were retrospectively reviewed. Logistic regression and Cox proportional hazards model were used to evaluate variables predictive of symptomatic response and survival respectively. RESULTS: One hundred patients were identified. The majority had good performance status (Eastern Cooperative Oncology Group score 0-1, 82%), received only one line of chemotherapy (91%), and presented with pain (84%). The most common radiotherapy prescription was 30 Gy in 10 fractions (22%). Pain improved in 69%, early satiety and bloating improved in 59%, and hemostasis was achieved in 73% of cases. Treatment toxicity occurred in 47% of cases and were predominantly grade 1-2 with 1 case of grade 3 toxicity. Median survival was 5.1 months. Tumor size, radiotherapy dose, and concurrent chemotherapy were not predictive of symptomatic response nor prolonged survival. CONCLUSIONS: In the largest cohort to date evaluating palliative radiotherapy in pancreatic adenocarcinoma, radiation was efficacious in improving pain and gastrointestinal bleeding and was generally well-tolerated. Additional studies on the efficacy and optimal prescriptions for palliative radiotherapy are necessary in this population.

Tandem mass tag-based thermal proteome profiling for the discovery of drug-protein interactions in cancer cells.

Identification of effector targets is imperative to the characterization of the mechanisms of action of novel small molecules. Here, we describe steps to identify effector drug-protein interactions in lysates derived from cancer cell lines using a thermal proteome profiling (TPP) protocol. Building on existing TTP approaches, we detail the use of an in-solution trypsin digestion technique to streamline sample preparation, a nonparametric analysis to rank proteins for prioritization, and a follow-up strategy for identifying effector interactors. For complete details on the use and execution of this protocol, please refer to Johnson et al. (2022).1.

Development of an ELISA to detect the secreted prostate cancer biomarker AGR2 in voided urine.

BACKGROUND: Comparative transcriptomics between sorted cells identified AGR2 as one of the highest up-regulated genes in cancer. Overexpression in primary tumors was verified by tissue microarray analysis. AGR2 encodes a 19-kDa secreted protein that might be found in urine. METHODS: Monoclonal antibodies were generated against AGR2. One antibody pair, P1G4 (IgG1) to capture and P3A5 (IgG2a) to detect, showed good performance characteristics in a sandwich ELISA. This assay could detect AGR2 at sub ng/ml quantities. RESULTS: AGR2 was detected in tissue digestion media of tumor specimens and culture media of AGR2-secreting prostate cancer cell lines. Additional testings involved frozen section immunohistochemistry, immunoprecipitation, and Western blot analysis. Voided urine samples were collected from pre-operative cancer patients, and urinary protein was desalted and concentrated by filtration. The amount of AGR2 detected was scored as pg/100 µg total protein, and then converted to pg/ml urine. The developed ELISA detected AGR2 protein, ranging from 3.6 to 181 pg/ml, in an initial cohort of samples. AGR2 was not detected in the urine of non-cancer and a bladder cancer patient. CONCLUSIONS: For prostate cancer, an AGR2 urine test could be used for diagnosis. The data, although derived from a small number of samples assayed, showed that developing such a test for clinical application is viable because AGR2 is specific to cancer cells, and apparently secreted into urine.

Reprogramming of prostate cancer-associated stromal cells to embryonic stem-like.

BACKGROUND: CD90(+) prostate cancer-associated (CP) stromal cells represent a diseased cell type found only in tumor tissue. They differ from their normal counterpart in gene expression and inductive signaling. Genetic reprogramming by induced pluripotent stem (iPS) cell technology can effectively change adult cells into stem-like cells through wholesale alteration of the gene expression program. This technology might be used to 'erase' the abnormal gene expression of diseased cells. The resultant iPS cells would no longer express the disease phenotype, and behave like stem cells. METHODS: CP stromal cells, isolated from tumor tissue of a surgically resected prostate by anti-CD90-mediated sorting and cultured in vitro, were transfected with in vitro packaged lentiviral expression vectors containing stem cell transcription factor genes POU5F1, LIN28, NANOG, and SOX2. RESULTS: Alkaline phosphatase-positive iPS cells were obtained in about 3 weeks post-transfection at a frequency of 10(-4) . Their colony morphology was indistinguishable from that of human embryonic stem (ES) cells. Transcriptome analysis showed a virtually complete match in gene expression between the iPS and ES cells. CONCLUSIONS: Genes of CP stromal cells could be fully inactivated by genetic reprogramming. As a consequence, the disease phenotype was 'cured'.

Bladder expression of CD cell surface antigens and cell-type-specific transcriptomes.

Many cell types have no known functional attributes. In the bladder and prostate, basal epithelial and stromal cells appear similar in cytomorphology and share several cell surface markers. Their total gene expression (transcriptome) should provide a clear measure of the extent to which they are alike functionally. Since urologic stromal cells are known to mediate organ-specific tissue formation, these cells in cancers might exhibit aberrant gene expression affecting their function. For transcriptomes, cluster designation (CD) antigens have been identified for cell sorting. The sorted cell populations can be analyzed by DNA microarrays. Various bladder cell types have unique complements of CD molecules. CD9(+) urothelial, CD104(+) basal and CD13(+) stromal cells of the lamina propria were therefore analyzed, as were CD9(+) cancer and CD13(+) cancer-associated stromal cells. The transcriptome datasets were compared by principal components analysis for relatedness between cell types; those with similarity in gene expression indicated similar function. Although bladder and prostate basal cells shared CD markers such as CD104, CD44 and CD49f, they differed in overall gene expression. Basal cells also lacked stem cell gene expression. The bladder luminal and stromal transcriptomes were distinct from their prostate counterparts. In bladder cancer, not only the urothelial but also the stromal cells showed gene expression alteration. The cancer process in both might thus involve defective stromal signaling. These cell-type transcriptomes provide a means to monitor in vitro models in which various CD-isolated cell types can be combined to study bladder differentiation and bladder tumor development based on cell-cell interaction.

Insurance payment for artificial intelligence technology: Methods used by a stroke artificial intelligence system and strategies to qualify for the new technology add-on payment.

The first ever insurance reimbursement for an artificial intelligence (AI) system, which expedites triage of acute stroke, occurred in 2020 when the Centers for Medicare and Medicaid Services (CMS) granted approval for a New Technology Add-on Payment (NTAP). Key aspects of the AI system that led to its approval by the CMS included its unique mechanism of action, use of robotic process automation, and clear linkage of the system's output to clinical outcomes. The specific strategies employed encompass a first-case scenario of proving reimbursable value for improved stroke outcomes using AI. Given the rapid change in utilization of AI technology in stroke care, we describe the economic drivers of stroke AI systems in healthcare, focusing on concepts of reimbursement for value added by AI to the stroke care system. This report reviews (1) the successful approach used by the first NTAP-approved AI system, (2) economic variables in insurance reimbursement for AI, and (3) resultant strategies that may be utilized to facilitate qualification for NTAP reimbursement, which may be adopted by other AI systems used in stroke care.