Analytical Validation of the PreciseDx Digital Prognostic Breast Cancer Test in Early-Stage Breast Cancer.

BACKGROUND: PreciseDx Breast (PDxBr) is a digital test that predicts early-stage breast cancer recurrence within 6-years of diagnosis. MATERIALS AND METHODS: Using hematoxylin and eosin-stained whole slide images of invasive breast cancer (IBC) and artificial intelligence-enabled morphology feature array, microanatomic features are generated. Morphometric attributes in combination with patient's age, tumor size, stage, and lymph node status predict disease free survival using a proprietary algorithm. Here, analytical validation of the automated annotation process and extracted histologic digital features of the PDxBr test, including impact of methodologic variability on the composite risk score is presented. Studies of precision, repeatability, reproducibility and interference were performed on morphology feature array-derived features. The final risk score was assessed over 20-days with 2-operators, 2-runs/day, and 2-replicates across 8-patients, allowing for calculation of within-run repeatability, between-run and within-laboratory reproducibility. RESULTS: Analytical validation of features derived from whole slide images demonstrated a high degree of precision for tumor segmentation (0.98, 0.98), lymphocyte detection (0.91, 0.93), and mitotic figures (0.85, 0.84). Correlation of variation of the assay risk score for both reproducibility and repeatability were less than 2%, and interference from variation in hematoxylin and eosin staining or tumor thickness was not observed demonstrating assay robustness across standard histopathology preparations. CONCLUSION: In summary, the analytical validation of the digital IBC risk assessment test demonstrated a strong performance across all features in the model and complimented the clinical validation of the assay previously shown to accurately predict recurrence within 6-years in early-stage invasive breast cancer patients.

MicroRNA-21 deficiency suppresses prostate cancer progression through downregulation of the IRS1-SREBP-1 signaling pathway.

Sterol regulatory element-binding protein 1 (SREBP-1), a master transcription factor in lipogenesis and lipid metabolism, is critical for disease progression and associated with poor outcomes in prostate cancer (PCa) patients. However, the mechanism of SREBP-1 regulation in PCa remains elusive. Here, we report that SREBP-1 is transcriptionally regulated by microRNA-21 (miR-21) in vitro in cultured cells and in vivo in mouse models. We observed aberrant upregulation of SREBP-1, fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC) in Pten/Trp53 double-null mouse embryonic fibroblasts (MEFs) and Pten/Trp53 double-null mutant mice. Strikingly, miR-21 loss significantly reduced cell proliferation and suppressed the prostate tumorigenesis of Pten/Trp53 mutant mice. Mechanistically, miR-21 inactivation decreased the levels of SREBP-1, FASN, and ACC in human PCa cells through downregulation of insulin receptor substrate 1 (IRS1)-mediated transcription and induction of cellular senescence. Conversely, miR-21 overexpression increased cell proliferation and migration; as well as the levels of IRS1, SREBP-1, FASN, and ACC in human PCa cells. Our findings reveal that miR-21 promotes PCa progression by activating the IRS1/SREBP-1 axis, and targeting miR-21/SREBP-1 signaling pathway can be a novel strategy for controlling PCa malignancy.

Transformed bone marrow cells generate neoplasms of distinct histogenesis. a murine model of cancer transplantation.

The last several years have witnessed renewed interest regarding the contribution of cancer stem cells in tumorigenesis and neoplastic heterogeneity. It has been reported that patients who undergo bone marrow transplantation are more prone to develop a malignancy during their life time; usually hematological tumors, but solid neoplasms may also develop, which in certain instances are donor-derived. It has also been well documented that multipotent bone marrow derived cells can migrate to diverse organs, differentiating into various histological lineages. The present study reports an experimental syngeneic transplantation model, using fluorescently tagged bone marrow cells from p53 null male mice into female wild-type counterparts. We found that transplanted non-neoplastic mutant bone marrow cells can generate tumors of distinct histogenesis, including thymic lymphomas, sarcomas, and carcinomas after carcinogen induction, providing evidence that multipotent cancer-prone stem cells can reside in the bone marrow and are transplantable.

Development and validation of a novel automated Gleason grade and molecular profile that define a highly predictive prostate cancer progression algorithm-based test.

BACKGROUND: Postoperative risk assessment remains an important variable in the effective treatment of prostate cancer. There is an unmet clinical need for a test with the potential to enhance the Gleason grading system with novel features that more accurately reflect a personalized prediction of clinical failure. METHODS: A prospectively designed retrospective study utilizing 892 patients, post radical prostatectomy, followed for a median of 8 years. In training, using digital image analysis to combine microscopic pattern analysis/machine learning with biomarkers, we evaluated Precise Post-op model results to predict clinical failure in 446 patients. The derived prognostic score was validated in 446 patients. Eligible subjects required complete clinical-pathologic variables and were excluded if they had received neoadjuvant treatment including androgen deprivation, radiation or chemotherapy prior to surgery. No patients were enrolled with metastatic disease prior to surgery. Evaluate the assay using time to event concordance index (C-index), Kaplan-Meier, and hazards ratio. RESULTS: In the training cohort (n = 306), the Precise Post-op test predicted significant clinical failure with a C-index of 0.82, [95% CI: 0.76-0.86], HR:6.7, [95% CI: 3.59-12.45], p < 0.00001. Results were confirmed in validation (n = 284) with a C-index 0.77 [95% CI: 0.72-0.81], HR = 5.4, [95% CI: 2.74-10.52], p < 0.00001. By comparison, a clinical feature base model had a C-index of 0.70 with a HR = 3.7. The Post-Op test also re-classified 58% of CAPRA-S intermediate risk patients as low risk for clinical failure. CONCLUSIONS: Precise Post-op tissue-based test discriminates low from intermediate high risk prostate cancer disease progression in the postoperative setting. Guided by machine learning, the test enhances traditional Gleason grading with novel features that accurately reflect the biology of personalized risk assignment.

Ornithine Decarboxylase Is Sufficient for Prostate Tumorigenesis via Androgen Receptor Signaling.

Increased polyamine synthesis is known to play an important role in prostate cancer. We aimed to explore its functional significance in prostate tumor initiation and its link to androgen receptor (AR) signaling. For this purpose, we generated a new cell line derived from normal epithelial prostate cells (RWPE-1) with overexpression of ornithine decarboxylase (ODC) and used it for in vitro and in vivo experiments. We then comprehensively analyzed the expression of the main metabolic enzymes of the polyamine pathway and spermine abundance in 120 well-characterized cases of human prostate cancer and high-grade prostate intraepithelial neoplasia (HGPIN). Herein, we show that the ODC-overexpressing prostate cells underwent malignant transformation, revealing that ODC is sufficient for de novo tumor initiation in 94% of injected mice. This oncogenic capacity was acquired through alteration of critical signaling networks, including AR, EIF2, and mTOR/MAPK. RNA silencing experiments revealed the link between AR signaling and polyamine metabolism. Human prostate cancers consistently demonstrated up-regulation of the main polyamine enzymes analyzed (ODC, polyamine oxidase, and spermine synthase) and reduction of spermine. This phenotype was also dominant in HGPIN, rendering it a new biomarker of malignant transformation. In summary, we report that ODC plays a key role in prostate tumorigenesis and that the polyamine pathway is altered as early as HGPIN.

H-RAS mutation is a key molecular feature of pediatric urothelial bladder cancer. A detailed report of three cases.

INTRODUCTION: Urothelial carcinoma (UC) of the bladder is a rare entity in the pediatric population, with an incidence of less than 0.4% in patients younger than 20 years. These patients overwhelmingly present with non-muscle-invasive low-grade disease and an indolent behavior. OBJECTIVE: The aim was to determine the source of the different natural history between pediatric population and adults; we hypothesized that pediatric bladder cancer may stem from different molecular pathways. Our objective with this descriptive case series was to study the main genes involved in pediatric urothelial bladder carcinoma using immunohistochemical (IHC) and mutational analysis. By studying the genetic alterations and immunophenotype of the most commonly altered genes in bladder urothelial cancer in three pediatric tumors we could gain better understanding of the molecular pathogenesis in this rare disease. STUDY DESIGN: Formalin-fixed paraffin-embedded (FFPE) tissue slides of urothelial bladder tumors from three pediatric patients were retrospectively identified at Columbia University pathology archives (1990-2011) and re-evaluated. FGFR3, H-RAS, and PI3K hotspots mutational analyses were conducted by polymerase chain reaction amplification and Sanger sequencing from the FFPE tissue blocks. IHC analysis was conducted using antibodies against p53, PTEN, RB, EGFR, and HER2. Proliferative rate was assessed by Ki-67 expression. RESULTS: Two patients had low-grade Ta disease, whereas the other tumor was classified as a papillary urothelial neoplasm of low malignant potential. None of the lesions recurred. Notably, all specimens showed H-RAS G12V mutation, whereas they were characterized by wild-type FGFR3 and PI3K. Nuclear p53 was not detected, whereas PTEN and RB expression were maintained. EGFR was expressed in the three cases and HER2 was negative. The proliferation rate was very low in all cases. DISCUSSION: It is difficult to draw strong conclusions from the study of three tumors treated at the same institution and from the same referral population, and a multicentric study should be performed to confirm these preliminary results. However, we propose that H-RAS mutation analysis could be performed on urothelial bladder tumors of pediatric patients. The knowledge in the molecular basis of urothelial bladder tumors in children opens a promising field which could lead us to establish different guidelines for surveillance and follow-up of pediatric urothelial bladder cancer patients. CONCLUSION: Pediatric tumors are characterized by a consistent H-RAS mutation status, whereas FGFR3 and p53 pathways are not involved in this tumor initiation. These results may explain the few recurrences seen in this population.

Suppression of acquired docetaxel resistance in prostate cancer through depletion of notch- and hedgehog-dependent tumor-initiating cells.

Acquired resistance to Docetaxel precedes fatality in hormone-refractory prostate cancer (HRPC). However, strategies that target Docetaxel resistant cells remain elusive. Using in vitro and in vivo models, we identified a subpopulation of cells that survive Docetaxel exposure. This subpopulation lacks differentiation markers and HLA class I (HLAI) antigens, while overexpressing the Notch and Hedgehog signaling pathways. These cells were found in prostate cancer tissues and were related to tumor aggressiveness and poor patient prognosis. Notably, targeting Notch and Hedgehog signaling depleted this population through inhibition of the survival molecules AKT and Bcl-2, suggesting a therapeutic strategy for abrogating Docetaxel resistance in HRPC. Finally, these cells exhibited potent tumor-initiating capacity, establishing a link between chemotherapy resistance and tumor progression.

A BAC-based transgenic mouse specifically expresses an inducible Cre in the urothelium.

Cre-loxp mediated conditional knockout strategy has played critical roles for revealing functions of many genes essential for development, as well as the causal relationships between gene mutations and diseases in the postnatal adult mice. One key factor of this strategy is the availability of mice with tissue- or cell type-specific Cre expression. However, the success of the traditional molecular cloning approach to generate mice with tissue specific Cre expression often depends on luck. Here we provide a better alternative by using bacterial artificial chromosome (BAC)-based recombineering to insert iCreERT2 cDNA at the ATG start of the Upk2 gene. The BAC-based transgenic mice express the inducible Cre specifically in the urothelium as demonstrated by mRNA expression and staining for LacZ expression after crossing with a Rosa26 reporter mouse. Taking into consideration the size of the gene of interest and neighboring genes included in a BAC, this method should be widely applicable for generation of mice with tissue specific gene expression or deletions in a more specific manner than previously reported.

Distinct expression profiles of p63 variants during urothelial development and bladder cancer progression.

The TP63 gene, a member of the TP53 tumor suppressor gene family, can be expressed as at least six isoforms due to alternative promoter use and alternative splicing. The lack of p63 isoform-specific antibodies has limited the analysis of the biological significance of p63. We report a novel set of well-defined antibodies to examine p63 isoforms in mouse and human urothelium during embryogenesis and tumor progression, respectively. We provide evidence that basal and intermediate urothelial cells express p63 isoforms, with the TAp63 variant the first to be detected during development, whereas umbrella cells are characterized by a p63-negative phenotype. Notably, we report that p63-null mice develop a bladder with an abnormal urothelium, constituted by a single layer of cells that express uroplakin II and low molecular weight cytokeratins, consistent with an umbrella cell phenotype. Finally, analysis of 202 human bladder carcinomas revealed a new categorization of invasive tumors into basal-like (positive for ΔNp63 and high molecular weight cytokeratins and negative for low molecular weight cytokeratins) versus luminal-like (negative for ΔNp63 and high molecular weight cytokeratins and positive for low molecular weight cytokeratins) phenotypes, with ΔNp63 expression associated with an aggressive clinical course and poor prognosis. This study highlights the relevance of p63 isoforms in both urothelial development and bladder carcinoma progression, with ΔNp63 acting as an oncogene in certain invasive bladder tumors.