Probes for INS

The presence and extent of cribriform pattern of prostate cancer portend recurrence and cancer death. Therelative expressions within this morphology of the prognostically adverse loss of PTEN, and the downstream inactivation of cell cycle inhibitor p27/Kip1 had been uncertain. In this study, we examined 52 cases of cribriform cancer by immunohistochemistry (IHC) for PTEN, p27, and CD44 variant (v)7/8, and a subset of 17 casesby chromogenic in situ hybridization (ISH) using probe for PTEN or CDKN1B (gene for p27). The fractions of epithelial pixels positive by IHC and ISH were digitally assessed for benign acini, high grade prostatic intraepithelial neoplasia (PIN), and 8 morphological patterns of cancer. Immunostaining results demonstrated that: 1. PTEN loss was significant for fused small acini, cribriform-central cells, small cribriform acini, and Gleason grade 5 cells in comparison with other acini. 2. p27 loss was significant only for cribriform-peripheral cells; and borderline-significant for fused small acini in comparison with benign acini. 3. CD44v7/8 showed expression loss in cribriform-peripheral cells; other comparisons were not significant. ISH showed thatcribriform cancer had significant PTEN loss normalized to benign acini (P < .02), while Gleason 3 cancer or fused small acini did not. With CDKN1B, the degree of signal loss among various cancer morphologies was insignificant. In conclusion, molecular disparities emerged between the fused small acini and cribriform patterns of Gleason 4 cancer. PTEN or p27 loss as prognostic factors demand distinct assessment in the varieties of Gleason 4 cancer, and in the biphenotypic peripheral versus central populations in cribriform structures.

Activated Wnt signaling is critical in the pathogenesis of renal fibrosis, a final common pathway for most forms of chronic kidney disease. Therapeutic intervention by inhibition of individual Wnts or downstream Wnt/β-catenin signaling has been proposed, but these approaches do not interrupt the functions of all Wnts nor block non-canonical Wnt signaling pathways. Alternatively, an orally bioavailable small molecule, Wnt-C59, blocks the catalytic activity of the Wnt-acyl transferase porcupine, and thereby prevents secretion of all Wnt isoforms. We found that inhibiting porcupine dramatically attenuates kidney fibrosis in the murine unilateral ureteral obstruction model. Wnt-C59 treatment similarly blunts collagen mRNA expression in the obstructed kidney. Consistent with its actions to broadly arrest Wnt signaling, porcupine inhibition reduces expression of Wnt target genes and bolsters nuclear exclusion of β-catenin in the kidney following ureteral obstruction. Importantly, prevention of Wnt secretion by Wnt-C59 blunts expression of inflammatory cytokines in the obstructed kidney that otherwise provoke a positive feedback loop of Wnt expression in collagen-producing fibroblasts and epithelial cells. Thus, therapeutic targeting of porcupine abrogates kidney fibrosis not only by overcoming the redundancy of individual Wnt isoforms but also by preventing upstream cytokine-induced Wnt generation. These findings reveal a novel therapeutic maneuver to protect the kidney from fibrosis by interrupting a pathogenic crosstalk loop between locally generated inflammatory cytokines and the Wnt/β-catenin signaling pathway.

Abstract

Objectives

Recent commercialization of methods for in situ hybridization using Z-pair probe/branched DNA amplification has led to increasing adoption of this technology for interrogating RNA expression in formalin-fixed, paraffin-embedded (FFPE) tissues. Current practice for FFPE block storage is to maintain them at room temperature, often for many years.

Methods

To examine the effects of block storage time on FFPE tissues using a number of RNA in situ probes with the Advanced Cellular Diagnostic’s RNAscope assay.

Results

We report marked reductions in signals after 5 years and significant reductions often after 1 year. Furthermore, storing unstained slides cut from recent cases (<1 year old) at –20°C can preserve hybridization signals significantly better than storing the blocks at room temperature and cutting the slides fresh when needed.

Conclusions

We submit that the standard practice of storing FFPE tissue blocks at room temperature should be reevaluated to better preserve RNA for in situ hybridization.

How stem cells maintain their identity and potency as tissues change during growth is not well understood. In mammalian hair, it is unclear how hair follicle stem cells can enter an extended period of quiescence during the resting phase but retain stem cell potential and be subsequently activated for growth. Here, we use lineage tracing and gene expression mapping to show that the Wnt target gene Axin2 is constantly expressed throughout the hair cycle quiescent phase in outer bulge stem cells that produce their own Wnt signals. Ablating Wnt signaling in the bulge cells causes them to lose their stem cell potency to contribute to hair growth and undergo premature differentiation instead. Bulge cells express secreted Wnt inhibitors, including Dickkopf (Dkk) and secreted frizzled-related protein 1 (Sfrp1). However, the Dickkopf 3 (Dkk3) protein becomes localized to the Wnt-inactive inner bulge that contains differentiated cells. We find that Axin2 expression remains confined to the outer bulge, whereas Dkk3 continues to be localized to the inner bulge during the hair cycle growth phase. Our data suggest that autocrine Wnt signaling in the outer bulge maintains stem cell potency throughout hair cycle quiescence and growth, whereas paracrine Wnt inhibition of inner bulge cells reinforces differentiation.

Immunohistochemical staining for Phosphatase and Tensin Homolog (PTEN) does not have either an acceptable standard protocol or concordance of scoring between pathologists. Evaluation of PTEN mRNA with a unique and verified sequence probe may offer a realistic alternative providing a robust and reproducible protocol. In this study we have evaluated an in situ hybridization (ISH) protocol for PTEN mRNA using RNAScope technology and compared it with a standard protocol for PTEN immunohistochemistry (IHC). PTEN mRNA expression by ISH was consistently more sensitive than PTEN IHC with 56% of samples on a mixed tumour tissue microarray (TMA) showing high expressionby ISH compared to 42% by IHC. On a prostate TMA 49% of cases showed high expression by ISH compared to 43% by IHC. Variations in PTEN mRNA expression within malignant epithelium were quantifiable using image analysis on the prostate TMAs. Within tumours clear over expression of PTEN mRNA on malignant epithelium compared to benign epithelium was frequently observed and quantified. The use of Spot Studio software in the mixed tumour TMA allowed for clear demonstration of varying levels of PTEN mRNA between tumour samples by the mRNA methodology. This was evident by the quantifiable differences between distinct oropharyngeal tumours (upto 3 fold increase in average number of spots per cell between 2 cases). mRNA detection of PTEN or other biomarkers, for which optimal or standardized immunohistochemical techniques are not available, represents a means by which heterogeneity of expression within focal regions of tumour can be explored with more confidence.