Hippie Daisy Flowers Phone Case

Hippie Daisy Flowers Phone Case

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Hippie Daisy Flowers Phone Case

Fig. 5: Intratumoral heterogeneity within full-tumor section 15-096M1.

a Hematoxylin and eosin (H&E) staining of 15-096M1 lymph node metastases. Distinct areas of morphology are demonstrated, cribriform well differentiated prostatic adenocarcinoma (lower left) and undifferentiated high-grade carcinoma (upper right). N = 1 tissue section for H&E staining. B Fluorescent labeling of 15-096M1 lymph node metastases. High PanCK staining is present in the lower left and low PanCK staining is present in the upper right. N = 1 tissue section for fluorescent labeling. C Individual tumor region of interest (ROIs) (200–500 µm) with varying levels of PanCK intensity and differential tumor morphology. N = 1 tissue section for fluorescent labeling and ROI selection. Expression plots of genes known to be associated with AR+/NE− (d), AR−/NE− (e), and AR−/NE+ (f) phenotypes of ROIs 7–12 from 15-096M1. Counts were Q3 normalized and scaled (Z-score) to enable plotting of all genes on the same axes. G–k Comparison of transcript levels of specific genes in ROIs 1–6 (N = 6) from the CK+ tumor region and ROIs 10–12 (N = 3) distant from the CK+ region. Counts were log2 Q3 normalized. Significance was determined by two-sided Wilcoxon-rank tests (g–k: p = 0.024). Boxes represent the median and interquartile range (IQR) and the upper and lower whiskers extending to the values that are within 1.5 × IQR; data beyond the end of the whiskers are outliers and plotted as points.

AR-V7 expression varies within and across PC metastases Hippie Daisy Flowers Phone Case

Alternative splicing of gene transcripts occurs commonly in carcinomas. Notably, in PC, several splice variants of the AR have been identified, particularly in the context of resistance to ADT, and specific splice variants, such as AR-V7, may promote resistance to second generation AR pathway inhibitors (ARSi)37,38. We determined the presence of AR-V7 in each tumor by transcript reads spanning canonical and cryptic exons determined by bulk RNAseq and found that for most patients (25/26; 96%), both tumors evaluated were either concordantly AR-V7+ or AR-V7− (Fig. 6a). For two patients, 16–052 and 17–081, tumors were discordant with respect to AR-V7 status. We next used DSP to further evaluate the intratumoral heterogeneity of AR-V7. We designed a series of barcoded oligonucleotide probes with specificity for each of the exons comprising the full-length AR gene and cryptic exon 3 (CE3) that comprises the AR-V7 transcript (Fig. 6b). Notably, when evaluating AR-V7 expression in the individual ROIs by DSP, we observed more substantial heterogeneity: regions from the same tumor expressed high AR-V7, while other regions lacked detectable AR-V7 transcript (Fig. 6a). Further, for six tumors there was discordance between AR-V7 status measured by DSP and bulk RNAseq (Fig. 6a–d). To further assess AR-V7 expression, we evaluated AR-V7 protein by immunohistochemistry of the TMA cores. Overall, there was a significant positive correlation between AR-V7 IHC and transcript levels measured by DSP (r = 0.54, p < 0.0001, Fig. S3g) and most tumors demonstrated homogenous absence or presence of AR-V7 nuclear staining. However, there were several tumors where AR-V7 expression was heterogenous by IHC (Fig. S3f), a finding concordant with the high and low AR-V7 quantitation by DSP that varied by ROI within a tumor. We note that a degree of divergence can be expected across these measurements as the bulk RNAseq was obtained from a different portion of the metastatic tumor compared to the selection of FFPE embedded tumor cored for TMA construction. Further, the AR-V7 IHC was performed on a section of the TMA approximately ten sections, equating to ~50 µM, from the section used for DSP analyses.

Fig. 6: Alternative splicing of AR isoforms is present across metastases, as determined by DSP.

a Heatmap of 141 individual tumor ROIs grouped by 26 patients comparing bulk RNAseq AR-V7 expression to DSP. Results are expressed as gene signature Z-scores and log2 negative-normalized (NN) gene expression and presented according to color scales. B DSP–RNA probe design of AR full length and AR-V7 variant isoform. C Fluorescent labeling and ROI selection of three cores from two sites of metastasis from patients 14-043 that demonstrated divergent AR-V7 expression determined by RNAseq and DSP. N = 1 TMA section for fluorescent labeling. D Heatmap demonstrating discordance in AR-V7 expression DSP AR-V7 and AR-V7 measured by bulk RNAseq and AR-V7 immunohistochemistry (IHC) in six tumor ROIs from two sites (CC3 and H3) within patient 14-043. Results are presented according to color scales in a.

 

 

 

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