Many gene expression normalization algorithms exist for Affymetrix GeneChip microarrays. The most popular of these is RMA, primarily due to the precision and low noise produced during the process. A ...significant strength of this and similar approaches is the use of the entire set of arrays during both normalization and model-based estimation of signal. However, this leads to differing estimates of expression based on the starting set of arrays, and estimates can change when a single, additional chip is added to the set. Additionally, outlier chips can impact the signals of other arrays, and can themselves be skewed by the majority of the population.
We developed an approach, termed IRON, which uses the best-performing techniques from each of several popular processing methods while retaining the ability to incrementally renormalize data without altering previously normalized expression. This combination of approaches results in a method that performs comparably to existing approaches on artificial benchmark datasets (i.e. spike-in) and demonstrates promising improvements in segregating true signals within biologically complex experiments.
By combining approaches from existing normalization techniques, the IRON method offers several advantages. First, IRON normalization occurs pair-wise, thereby avoiding the need for all chips to be normalized together, which can be important for large data analyses. Secondly, the technique does not require similarity in signal distribution across chips for normalization, which can be important for maintaining biologically relevant differences in a heterogeneous background. Lastly, IRON introduces fewer post-processing artifacts, particularly in data whose behavior violates common assumptions. Thus, the IRON method provides a practical solution to common needs of expression analysis. A software implementation of IRON is available at http://gene.moffitt.org/libaffy/.
Clear cell renal cell carcinomas (ccRCC) are characterized by arm-wide chromosomal alterations. Loss at 14q is associated with disease aggressiveness in ccRCC, which responds poorly to ...chemotherapeutics. The 14q locus contains one of the largest miRNA clusters in the human genome; however, little is known about the contribution of these miRNAs to ccRCC pathogenesis. In this regard, we investigated the expression pattern of selected miRNAs at the 14q32 locus in TCGA kidney tumors and in ccRCC cell lines. We demonstrated that the miRNA cluster is downregulated in ccRCC (and cell lines) as well as in papillary kidney tumors relative to normal kidney tissues (and primary renal proximal tubule epithelial (RPTEC) cells). We demonstrated that agents modulating expression of DNMT1 (e.g., 5-Aza-deoxycytidine) could modulate 14q32 miRNA expression in ccRCC cell lines. Lysophosphatidic acid (LPA, a lysophospholipid mediator elevated in ccRCC) not only increased labile iron content but also modulated expression of a 14q32 miRNA. Through an overexpression approach targeting a subset of 14q32 miRNAs (specifically at subcluster A: miR-431-5p, miR-432-5p, miR-127-3p, and miR-433-3p) in 769-P cells, we uncovered changes in cellular viability and claudin-1, a tight junction marker. A global proteomic approach was implemented using these miRNA overexpressing cell lines which uncovered ATXN2 as a highly downregulated target. Collectively, these findings support a contribution of miRNAs at 14q32 in ccRCC pathogenesis.
We have interrogated a 12-chemokine gene expression signature (GES) on genomic arrays of 14,492 distinct solid tumors and show broad distribution across different histologies. We hypothesized that ...this 12-chemokine GES might accurately predict a unique intratumoral immune reaction in stage IV (non-locoregional) melanoma metastases. The 12-chemokine GES predicted the presence of unique, lymph node-like structures, containing CD20⁺ B cell follicles with prominent areas of CD3⁺ T cells (both CD4⁺ and CD8⁺ subsets). CD86⁺, but not FoxP3⁺, cells were present within these unique structures as well. The direct correlation between the 12-chemokine GES score and the presence of unique, lymph nodal structures was also associated with better overall survival of the subset of melanoma patients. The use of this novel 12-chemokine GES may reveal basic information on in situ mechanisms of the anti-tumor immune response, potentially leading to improvements in the identification and selection of melanoma patients most suitable for immunotherapy.
Most prostate cancer in African American men lacks the ETS (E26 transforming specific) family fusion event (ETS-). We aimed to establish clinically relevant biomarkers in African American men by ...studying ETS dependent gene expression patterns to identified race specific genes predictive of outcomes.
Two multicenter cohorts of a total of 1,427 men were used for the discovery and validation (635 and 792 men, respectively) of race specific predictive biomarkers. We used false discovery rate adjusted q values to identify race and ETS dependent genes which were differentially expressed in African American men who experienced biochemical recurrence within 5 years. Principal component modeling along with survival analysis was done to assess the accuracy of the gene panel in predicting recurrence.
We identified 3,047 genes which were differentially expressed based on ETS status. Of these genes 362 were differentially expressed in a race specific manner (false discovery rate 0.025 or less). A total of 81 genes were race specific and over expressed in African American men who experienced biochemical recurrence. The final gene panel included APOD, BCL6, EMP1, MYADM, SRGN and TIMP3. These genes were associated with 5-year biochemical recurrence (HR 1.97, 95% CI 1.27-3.06, p = 0.002) and they improved the predictive accuracy of clinicopathological variables only in African American men (60-month time dependent AUC 0.72).
In an effort to elucidate biological features associated with prostate cancer aggressiveness in African American men we identified ETS dependent biomarkers predicting early onset biochemical recurrence only in African American men. Thus, these ETS dependent biomarkers representing ideal candidates for biomarkers of aggressive disease in this patient population.
Tertiary lymphoid structures (TLSs) associate with better prognosis in certain cancer types, but their underlying formation and immunological benefit remain to be determined. We established a mouse ...model of TLSs to study their contribution to antitumor immunity. Because the stroma in lymph nodes (sLN) participates in architectural support, lymphogenesis, and lymphocyte recruitment, we hypothesized that TLSs can be created by sLN. We selected a sLN line with fibroblast morphology that expressed sLN surface markers and lymphoid chemokines. The subcutaneous injection of the sLN line successfully induced TLSs that attracted infiltration of host immune cell subsets. Injection of MC38 tumor lysate-pulsed dendritic cells activated TLS-residing lymphocytes to demonstrate specific cytotoxicity. The presence of TLSs suppressed MC38 tumor growth
by improving antitumor activity of tumor-infiltrating lymphocytes with downregulated immune checkpoint proteins (PD-1 and Tim-3). Future engineering of sLN lines may allow for further enhancements of TLS functions and immune cell compositions.
MYC oncoproteins regulate transcription of genes directing cell proliferation, metabolism and tumorigenesis. A variety of alterations drive
expression in acute myeloid leukemia (AML) and enforced MYC ...expression in hematopoietic progenitors is sufficient to induce AML. Here we report that AML and myeloid progenitor cell growth and survival rely on MYC-directed suppression of Transcription Factor EB (TFEB), a master regulator of the autophagy-lysosome pathway. Notably, although originally identified as an oncogene, TFEB functions as a tumor suppressor in AML, where it provokes AML cell differentiation and death. These responses reflect TFEB control of myeloid epigenetic programs, by inducing expression of isocitrate dehydrogenase-1 (IDH1) and IDH2, resulting in global hydroxylation of 5-methycytosine. Finally, activating the TFEB-IDH1/IDH2-TET2 axis is revealed as a targetable vulnerability in AML. Thus, epigenetic control by a MYC-TFEB circuit dictates myeloid cell fate and is essential for maintenance of AML.
Commercial gene expression signatures of prostate cancer prognosis were developed and validated in cohorts of predominantly European American men (EAM). Limited research exists on the value of such ...signatures in African American men (AAM), who have poor prostate cancer outcomes. We explored differences in gene expression between EAM and AAM for three commercially available panels recommended by the National Comprehensive Cancer Network for prostate cancer prognosis.
A total of 232 EAM and 95 AAM patients provided radical prostatectomy specimens. Gene expression was quantified using NanoString for 60 genes spanning the Oncotype DX Prostate, Prolaris, and Decipher panels. A continuous expression-based risk score was approximated for each. Differential expression, intrapanel coexpression, and risk by race were assessed.
Clinical and pathologic features were similar between AAM and EAM. Differential expression by race was observed for 48% of genes measured, although the magnitudes of expression differences were small. Coexpression patterns were more strongly preserved by race group for Oncotype DX and Decipher than Prolaris. Poorer prognosis was estimated in EAM versus AAM for Oncotype DX (
< 0.001), whereas negligible prognostic differences were predicted between AAM and EAM using Prolaris or Decipher (
> 0.05).
Because of observed racial differences across three commercial gene expression panels for prostate cancer prognosis, caution is warranted when applying these panels in clinical decision-making in AAM.
Differences in gene expression by race for three commercial panels for prostate cancer prognosis indicate that further study of their effectiveness in AAM with long-term follow-up is warranted.
Glioblastoma (GBM) is the most common yet uniformly fatal adult brain cancer. Intra-tumoral molecular and cellular heterogeneities are major contributory factors to therapeutic refractoriness and ...futility in GBM. Molecular heterogeneity is represented through molecular subtype clusters whereby the proneural (PN) subtype is associated with significantly increased long-term survival compared to the highly resistant mesenchymal (MES) subtype. Furthermore, it is universally recognized that a small subset of GBM cells known as GBM stem cells (GSCs) serve as reservoirs for tumor recurrence and progression. The clonal evolution of GSC molecular subtypes in response to therapy drives intra-tumoral heterogeneity and remains a critical determinant of GBM outcomes. In particular, the intra-tumoral MES reprogramming of GSCs using current GBM therapies has emerged as a leading hypothesis for therapeutic refractoriness. Preventing the intra-tumoral divergent evolution of GBM toward the MES subtype via new treatments would dramatically improve long-term survival for GBM patients and have a significant impact on GBM outcomes. In this review, we examine the challenges of the role of MES reprogramming in the malignant clonal evolution of glioblastoma and provide future perspectives for addressing the unmet therapeutic need to overcome resistance in GBM.
It is poorly understood how the tumor immune microenvironment influences disease recurrence in localized clear-cell renal cell carcinoma (ccRCC). Here we performed whole-transcriptomic profiling of ...236 tumors from patients assigned to the placebo-only arm of a randomized, adjuvant clinical trial for high-risk localized ccRCC. Unbiased pathway analysis identified myeloid-derived IL6 as a key mediator. Furthermore, a novel myeloid gene signature strongly correlated with disease recurrence and overall survival on uni- and multivariate analyses and is linked to TP53 inactivation across multiple data sets. Strikingly, effector T-cell gene signatures, infiltration patterns, and exhaustion markers were not associated with disease recurrence. Targeting immunosuppressive myeloid inflammation with an adenosine A2A receptor antagonist in a novel, immunocompetent, Tp53-inactivated mouse model significantly reduced metastatic development. Our findings suggest that myeloid inflammation promotes disease recurrence in ccRCC and is targetable as well as provide a potential biomarker-based framework for the design of future immuno-oncology trials in ccRCC.
Improved understanding of factors that influence metastatic development in localized ccRCC is greatly needed to aid accurate prediction of disease recurrence, clinical decision-making, and future adjuvant clinical trial design. Our analysis implicates intratumoral myeloid inflammation as a key driver of metastasis in patients and a novel immunocompetent mouse model. This article is highlighted in the In This Issue feature, p. 2221.
Prostate cancer (PCa) is one of the most common cancers to affect men worldwide. Androgen receptor (AR) signaling is central to PCa and PCa therapy. MicroRNAs (miRNAs) play crucial roles in the ...regulation of prostate cancer through modulation of signaling pathways. In the present study, we illustrate the functional significance and therapeutic benefit of miR-299-3p, an AR targeting microRNA, in PCa progression. We noted loss of expression of miR-299-3p in prostate tumors compared to noncancerous prostate tissues. Replenishment of miR-299-3p in C4-2B, 22Rv-1 and PC-3 cells contributed to cell cycle arrest, reduced proliferation, migration and increased expression of apoptotic markers. Additionally, overexpression of miR-299-3p induced a reduction of AR, PSA and VEGFA expression. AGO-RNA pulldown experiment showed enrichment of AR, VEGFA and miR-299-3p in C4-2B cells overexpressing miR-299-3p. miR-299-3p overexpression also inhibited epithelial mesenchymal transition, expression of Slug, TGF-β3, phospho-AKT and phospho-PRAS40, but increased expression of E-cadherin. Furthermore, miR-299 overexpression resulted in reduced tumor growth in xenograft models and increased drug sensitivity. Overall, this study has identified novel mechanisms of antitumor and antimigration function of miR-299-3p through modulation of AR and VEGFA signaling pathways which lead to improved drug sensitivity of PCa.
PDF
