miR Expression Signatures Research Articles

Combination of a six microRNA expression profile with four clinicopathological factors for response prediction of systemic treatment in patients with advanced colorectal cancer.

BackgroundFirst line chemotherapy is effective in 75 to 80% of patients with metastatic colorectal cancer (mCRC). We studied whether microRNA (miR) expression profiles can predict treatment outcome for first line fluoropyrimidine containing systemic therapy in patients with mCRC.MethodsMiR expression levels were determined by next generation sequencing from snap frozen tumor samples of 88 patients with mCRC. Predictive miRs were selected with penalized logistic regression and posterior forward selection. The prediction co-efficients of the miRs were re-estimated and validated by real-time quantitative PCR in an independent cohort of 81 patients with mCRC.ResultsExpression levels of miR-17-5p, miR-20a-5p, miR-30a-5p, miR-92a-3p, miR-92b-3p and miR-98-5p in combination with age, tumor differentiation, adjuvant therapy and type of systemic treatment, were predictive for clinical benefit in the training cohort with an AUC of 0.78. In the validation cohort the addition of the six miR signature to the four clinicopathological factors demonstrated a significant increased AUC for predicting treatment response versus those with stable disease (SD) from 0.79 to 0.90. The increase for predicting treatment response versus progressive disease (PD) and for patients with SD versus those with PD was not significant. in the validation cohort. MiR-17-5p, miR-20a-5p and miR-92a-3p were significantly upregulated in patients with treatment response in both the training and validation cohorts.ConclusionA six miR expression signature was identified that predicted treatment response to fluoropyrimidine containing first line systemic treatment in patients with mCRC when combined with four clinicopathological factors. Independent validation demonstrated added predictive value of this miR-signature for predicting treatment response versus SD. However, added predicted value for separating patients with PD could not be validated. The clinical relevance of the identified miRs for predicting treatment response has to be further explored.

Abstract 504: MicroRNA Signatures in Murine Aorta and Carotid Arteries

Atherosclerosis, a major cause of mortality, affects arteries diffusely. However, some vascular beds are preferably involved. In the last decade microRNAs (miRs) have emerged as key regulators of gene expression in physiological or pathophysiological processes. Here, we investigated whether different arteries, commonly affected by atherosclerosis, present specific miR expression signatures. For this purpose, aorta (Ao) and carotid (Ca) arteries of four healthy male Wistar rats were dissected, total RNA was extracted with Trizol, and enriched for small RNA fraction. MicroRNAs were then sequenced using massive parallel sequencing (RNA-Seq) on Ion Torrent PGM platform. Data were analyzed using CLC Genomics Workbench software. We identified 266 mature miRs in Ao and 421 in Ca, 260 were common between arteries. Differential expression analysis (EDGE) showed increased expression (IE) of 16 miRs in Ao and reduced expression (RE) of 54 miRs in Ao, relative to Ca. The lists of differently expressed miRs were subjected to in silico functional analyzes: (1) computational target prediction, using miRWalk tool, identified 1,094 target genes for IE list and 3,574 for RE list; (2) gene-set enrichment analysis in the lists of putative target genes, using EnrichR tool, showed differences in enriched biological processes, such as response to shear stress, regulation of endothelial proliferation, smooth muscle metabolism, and response to FGF signaling (which were enriched in RE list); (3) construction of regulatory networks for differently expressed miRs, using miRnet, identified metabolic pathway components differently regulated between arteries, such as inhibition of SIRT1 and increased expression of HIF1-alpha in Ao. Taken together, our results show differences in miR signature between arteries, suggesting that specific expression profiles of miR may play a role in the vascular behavior as well as in atherosclerosis pathophysiology.

Network analysis of microRNAs, genes and their regulation in diffuse and follicular B-cell lymphomas

MicroRNAs (miRs) are short non-coding regulatory RNAs that control gene expression at the post-transcriptional level and play an important role in cancer development and progression, acting either as oncogenes or as tumor suppressors. Identification of aberrantly expressed miRs in patients with hematological malignancies as compared to healthy individuals has suggested that these molecules may serve as novel clinical diagnostic and prognostic biomarkers.We conducted a systematic literature review of articles published between 2007 and 2017 and re-analyzed experimentally-validated human miR expression signatures in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) from various biological sources (tumor tissue, peripheral blood, bone marrow and cell lines). A unique miR expression pattern was observed for each disease. Compared to healthy individuals, 61 miRs were aberrantly expressed in DLBCL and 85 in FL; 20-30% of aberrantly expressed miRs overlapped between the two lymphoma subtypes.Analysis of integrative positive and negative miRNA-mRNA relationships using the Ingenuity Pathway Analysis (IPA) system revealed 970 miR-mRNA pairs for DLBCL and 90 for FL. Through gene ontology analysis, we found potential regulatory pathways that are deregulated in DLBCL and FL due to improper expression of miR target genes. By comparing the expression level of the aberrantly expressed miRs in DLBCL to their expression levels in other malignancies, we identified seven miRs that are aberrantly expressed in DLBCL tumor tissues (miR-15a, miR-16, miR-17, miR-106, miR-21, miR-155 and miR-34a-5p). This specific expression pattern may be a potential diagnostic tool for DLBCL.

Abstract 5756: Effects of Epstein-Barr virus latent membrane protein 1 (LMP1) on cell invasiveness and expression of endogenous microRNAs in human cells in vitro

Abstract Infection by the Epstein-Barr virus (EBV) is associated with a variety of human cancers, notably Burkitt lymphoma and nasopharyngeal carcinoma (NPC). Most malignant cells within EBV-associated cancers are latently infected, and some viral products expressed during the latent phase of the viral cycle have well-described carcinogenicity, including the EBV latent membrane protein 1 (LMP1). Distinct viral genotypes described so far include the B95.8, the most studied viral strain originally found in an elderly patient with transfusion-induced infectious mononucleosis, and the M81 strain, isolated from a NPC case from a Chinese patient. Compared to B95.8, the EBV M81 strain infects epithelial cells more readily, and it is more likely to induce spontaneous viral lytic reactivation. However, whether these EBV strains have distinct biological features relevant for the progression of EBV-associated cancers remains to be elucidated. Thus, this study aimed to evaluate whether the expression of B95.1 and M81 variants of EBV LMP1 have different impact on cell migration and invasiveness of human cells lines in vitro, as well as on the expression of selected endogenous microRNAs (miRs) that were previously reported to have a role on cancer progression. Expression of EBV LMP1 was achieved in 293 and NP69 cells transfected with vectors encoding its B95.8 or M81 variants. In vitro migration and invasion rates were assessed by the scratch wound healing assay and the Boyden chamber assay, respectively. The expression of 91 selected miRs were evaluated by a customized qPCR-array approach. Cells expressing EBV LMP1 showed increased migration and invasion rates compared with mock-transfected cells. However, the differences observed between B95.8 and M81 variants did not achieve statistical significance. Conversely, 293 cells expressing the M81 variant of LMP1 showed hyperexpression of human miRs 497-5p, 17-3p, and 34a-5p, compared to B95.8. The in silico target prediction for these miRs showed enrichment of gene targets involved in the MAPK, NF-κB, and PI3K/AKT intracellular signaling pathways. This would reflect in negative regulation of cell proliferation and cell motility/adhesion, as well as higher levels of apoptosis. Based on these data, it is plausible to suggest that EBV LMP1 from M81 viral strain is somewhat less effective in terms of carcinogenicity, and this might be a reflect of the improved ability of EBV M81 to induce lytic viral infection compared to the B95.8 strain, as previously reported. In conclusion, the results indicate that cells expressing LMP1 from EBV isolates B95.8 and M81 showed differences in terms of miR expression signatures, which ultimately might impact the pathogenesis and progression of EBV-associated cancers. Citation Format: Bárbara Grasiele Müller-Coan, Ethel Cesarman, Deilson Elgui De Oliveira. Effects of Epstein-Barr virus latent membrane protein 1 (LMP1) on cell invasiveness and expression of endogenous microRNAs in human cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5756. doi:10.1158/1538-7445.AM2017-5756

Abstract 1512: Functional validation of microRNA activity inferred by ActMiR in bladder cancer

Abstract Background: MicroRNAs (miRs) play a key role in cancer, both in tumorigenesis and tumor progression. In the past years, miR expression signatures have been reported as prognostic biomarkers in different tumor types including bladder cancer (BC). However, miR's expression does not always correlate with activity. We recently developed a novel computational method, named ActMiR, for explicitly inferring the activity of miRs based on the changes in expression levels of target genes. The main objective of this study was to validate the inferred miRs’ activity using BC as a model. Design: We applied ActMiR in 405 BC cases from The Cancer Genome Atlas (TCGA) database for which information from both mRNA and miR expression was available. Different BC cell lines (5637 and HT1376 for basal BC, SW780 and HT1197 for p53-like BC, and RT4 and RT112 for luminal BC) as well as the immortalized urothelial cell line [Human Urothelium cell (HUC)] were used to perform the in vitro functional validation. RNA was extracted from these cells basally and after inhibition of miR106-5p with specific anti-miR inhibitor (Taqman, Life Technologies); miR and target genes’ expression was assessed by qRT-PCR. Results: ActMir analyses revealed that a subset of 306 out of 1044 miRNAs were differentially expressed between tumor and normal samples at p-value<10-4. More importantly, at p-value<10-4, 155 out of 556 miRNAs were functionally active. From these, only four (miR106b, miR532, mir556, and mir134) were significantly differentially expressed, functionally active and showed prognostic significance. We chose to further analyze mir106b because it showed the biggest and most significant difference in activity between normal and cancer cells (p-value<2*10-11). As inferred by ActMir, mir106-5p was significantly overexpressed in all cancer cells when compared to HUC. Basal cells showed the highest fold increase (mean of 39; range: 31-47), followed by luminal (mean of 19; range: 5-32) and p53-like cells (mean of 8; range: 3-13). Inhibition of mir106b-5p was performed to assess whether the predicted target genes were consequently upregulated. Conclusion: Our results underscore the value of ActMir for inferring miR activity in BC from a systems biology perspective. It endorses ActMir as a promising tool for studying casual effects of miR activity on target genes, and ultimately for determining survival outcomes of BC patients. Citation Format: Mireia Castillo-Martin, Ana Collazo Lorduy, EunJee Li, Jun Zhu. Functional validation of microRNA activity inferred by ActMiR in bladder cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1512.

MicroRNA fingerprints in juvenile myelomonocytic leukemia (JMML) identified miR-150-5p as a tumor suppressor and potential target for treatment.

Juvenile myelomonocytic leukemia (JMML) is an aggressive leukemia of early childhood characterized by aberrant proliferation of myelomonocytic cells and hypersensitivity to GM-CSF stimulation. Mutually exclusive mutations in the RAS/ERK pathway genes such as PTPN11, NRAS, KRAS, CBL, or NF1 are found in ~90% of the cases. These mutations give rise to disease at least in part by activating STAT5 through phosphorylation and by promoting cell growth. MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression, which are often deregulated in leukemia. However, little is known about their role in JMML. Here, we report distinctive miR expression signatures associated with the molecular subgroups of JMML. Among the downregulated miRs in JMML, miR-150-5p was found to target STAT5b, a gene which is often over-activated in JMML, and contributes to the characteristic aberrant signaling of this disorder. Moreover, loss of miR-150-5p and upregulation of STAT5b expression were also identified in a murine model of JMML. Ectopic overexpression of miR-150-5p in mononuclear cells from three JMML patients significantly decreased cell proliferation. Altogether, our data indicate that miR expression is deregulated in JMML and may play a role in the pathogenesis of this disorder by modulating key effectors of cytokine receptor pathways.

Function of microRNA activity by ActMiR in bladder cancer.

4531Background: MicroRNAs (miRs) play a key role in cancer, in tumorigenesis and tumor progression. In the past years, miR expression signatures have been reported as prognostic biomarkers in bladd...

A stem cell-like gene expression signature associates with inferior outcomes and a distinct microRNA expression profile in adults with primary cytogenetically normal acute myeloid leukemia

Acute myeloid leukemia (AML) is hypothesized to be sustained by self-renewing leukemia stem cells (LSCs). Recently, gene expression signatures (GES) from functionally defined AML LSC populations were reported, and expression of a 'core enriched' (CE) GES, representing 44 genes activated in LCSs, conferred shorter survival in cytogenetically normal (CN) AML. The prognostic impact of the CE GES in the context of other molecular markers, including gene mutations and microRNA (miR) expression alterations, is unknown and its clinical utility is unclear. We studied associations of the CE GES with known molecular prognosticators, miR expression profiles, and outcomes in 364 well-characterized CN-AML patients. A high CE score (CE(high)) associated with FLT3-internal tandem duplication, WT1 and RUNX1 mutations, wild-type CEBPA and TET2, and high ERG, BAALC and miR-155 expression. CE(high) patients had a lower complete remission (CR) rate (P=0.003) and shorter disease-free (DFS, P<0.001) and overall survival (OS, P<0.001) than CE(low) patients. These associations persisted in multivariable analyses adjusting for other prognosticators (CR, P=0.02; DFS, P<0.001; and OS, P<0.001). CE(high) status was accompanied by a characteristic miR expression signature. Fifteen miRs were upregulated in both younger and older CE(high) patients, including miRs relevant for stem cell function. Our results support the clinical relevance of LSCs and improve risk stratification in AML.

MicroRNA as a Diagnostic Tool in Fine-Needle Aspiration Biopsy of Thyroid Nodules

Thyroid cancer accounts for 1.5% of all malignancies in the U.S., and it is the most common endocrine malignancy. Detection of thyroid cancer mostly relies on evaluation of thyroid nodules, which are very common but only 5%-7% harbor malignancy. Fine-needle aspiration biopsy (FNAB) is currently the most important tool for the evaluation of thyroid nodules; however, it is limited in that it provides only a cytology assessment of the aspirated cells, and indeterminate diagnoses are present in up to 30% of FNAB results. This limitation can be overcome by the molecular analysis of FNAB, and more specifically with the use of microRNAs (miRs). miRs constitute a class of endogenous small noncoding RNA fragments that regulate gene expression, and in vitro studies have shown that miRs play a significant role in cancer and regulate major processes, such as proliferation, differentiation, and cell death. Several studies have investigated the miR expression signature in different thyroid cancers, and data support its use as a diagnostic tool that is highly accurate for thyroid nodules. The purpose of this study is to review the accumulated data on miR dysregulation in the different thyroid cancer types, critically assess its diagnostic utility, and conclude with future study strategies.

Poor Outcome of RUNX1-Mutated (RUNX1-mut) Patients (Pts) with Primary, Cytogenetically Normal Acute Myeloid Leukemia (CN-AML) and Associated Gene- and MicroRNA (miR) Expression Signatures,

Abstract 3454RUNX1 encodes the α subunit of core binding factor, a heterodimeric transcription factor required for normal hematopoiesis. Acquired RUNX1 mutations (muts) have been associated with poor clinical outcome in AML; however, prior studies analyzed pts heterogeneous for cytogenetics, age, AML type (primary or secondary), and treatment received [including allogeneic stem cell transplant (alloSCT) in 1st complete remission (CR1)] and contained limited data regarding the potential molecular drivers of the worse outcome. We report a relatively large study testing the prognostic impact of RUNX1 muts in primary CN-AML pts (n=392) treated similarly with intensive cytarabine/anthracycline-based 1st-line therapy and without alloSCT in CR1. This cohort comprised both younger [<60 years (y); n=173] and older (≥60 y; n=219) pts. Pretreatment marrow (n=303) and blood (n=89) were analyzed centrally for RUNX1 muts by PCR and direct sequencing, and for FLT3-ITD, FLT3-TKD, MLL-PTD and NPM1, CEBPA, WT1, IDH1, IDH2 and TET2 muts. Gene and miR expression profiles were derived using microarrays. RUNX1 muts were found in 12.5% of pts (8% younger, 16% older), and were associated with lower hemoglobin (P=.01), white blood cells (WBC; P=.04), and blood blasts (P=.006). RUNX1-mut pts harbored NPM1 (P<.001) and CEBPA muts (P=.06) less frequently than RUNX1-wild-type (RUNX1-wt) pts. RUNX1-mut pts had lower CR rates (P=.005 in younger; P=.006 in older), and shorter disease-free (DFS; P=.058 in younger; P<.001 in older), overall (OS; P=.003 in younger; P<.001 in older) and event-free (EFS; P<.001 for younger and older; Figures 1 and 2) survival than RUNX1-wt pts. In multivariable models, RUNX1 muts remained associated with lower CR rate (P<.001) and shorter DFS (P<.001), OS (P<.001), and EFS (P<.001; Table) after adjustment for clinical and molecular variables. [Display omitted] [Display omitted] Table 1Multivariable analysis for EFS according to RUNX1-mut status in all CN-AML ptsHREFSPRUNX1, mut v wt2.271.65–3.12<.001FLT3-ITD, ITD v no ITD1.571.27–1.95<.001WT1, mut v wt1.441.02–2.01.04WBC, continuous 50 unit increase1.131.04–1.23.006Age group, ≥60y v <60y1.801.46–2.22<.001Note: A hazard ratio (HR) >1 corresponds to a higher risk for higher values of continuous variables and the 1st level listed of a dichotomous variable.To gain biological insight, RUNX1 mut-associated gene and miR expression signatures were derived in CN-AML for the first time. Older, NPM1-wt pts were analyzed since RUNX1 muts are more common in this age group and are nearly exclusive from NPM1 muts, which have their own characteristic gene-expression signature. This yielded 484 probe sets representing 278 named genes differentially expressed between RUNX1-mut (n=31) and RUNX1-wt (n=45) pts (P<.001). Genes normally expressed in hematopoietic stem (HSC) and early progenitor cells, including DNTT, BAALC, MN-1, CD109, P2RY14, FOXO1 and FLT-3 were upregulated in RUNX1-mut pts, as were components of the Wnt-signaling pathway, LRP6 and TCF4, that promote self-renewal and proliferation of HSCs. Genes upregulated (SETBP1, RBPMS, and SLC37A3) and downregulated (CCNA1 and RNASE3) in AML stem cells relative to AML progenitors were similarly deregulated in the RUNX1-mut signature. B cell lineage genes BLNK, IGHM, IRF8 and several class II MHC molecules were upregulated in RUNX1-mut pts while CEBPA, a key promoter of granulopoiesis, was downregulated. Genes implicated in chemoresistance, GAS6, PRKCE, and PTK2, were upregulated and MYCN, a promoter of both proliferation and apoptosis of myeloid cells, was downregulated in RUNX1-mut pts. Seven miRs were differentially expressed between RUNX1-mut and RUNX1-wt pts. Two members of the let-7 tumor suppressor family, which represses self-renewal and promotes differentiation of stem cells, were downregulated, as was miR-223, a positive regulator of granulopoiesis. MiRs -99a and -100 were also downregulated and miRs -211 and -595 upregulated in association with RUNX1 muts.In summary, RUNX1 muts are twice as common in older CN-AML pts than younger. They negatively impact on outcome in both younger and older pts not receiving alloSCT in CR1. RUNX1-mut blasts have molecular features of normal/malignant stem cells and B cells, which may explain their chemoresistance and guide novel therapeutic approaches. Disclosures:No relevant conflicts of interest to declare.

A microRNA expression signature of osteoclastogenesis

AbstractMicroRNAs (miRs) are small noncoding RNAs that principally function in the spatiotemporal regulation of protein translation in animal cells. Although emerging evidence suggests that some miRs play important roles in osteoblastogenesis and skeletal homeostasis, much less is known in osteoclastogenesis. Here, we show that receptor activator of nuclear factor κB ligand (RANKL)–induced osteoclastogenesis is mediated by miR-21. MiR-21 was identified as an miR expression signature of RANKL-induced osteoclastogenesis that down-regulates programmed cell death 4 (PDCD4) protein levels. Diminished PDCD4 removes a repression from c-Fos, a critical transcription factor for osteoclastogenesis and osteoclast-specific downstream target genes. In addition, RANKL-induced c-Fos up-regulates miR-21 gene expression. Bone marrow–derived monocyte/macrophage precursors deficient of DiGeorge syndrome critical region gene 8, an RNA binding protein associated with miR biogenesis, and Dicer, an endoribonuclease in the RNaseIII family associated with miR biogenesis, possessed significantly decreased miR-21 levels and increased PDCD4 protein levels so that RANKL-induced osteoclastogenesis was impaired in those cells. However, forced expression of miR-21 rescued osteoclast development because of down-regulation of PDCD4 protein expression levels. Thus, our studies provide a new molecular mechanism, including a positive feedback loop of c-Fos/miR-21/PDCD4, regulating osteoclastogenesis.

Mutations In the Tet Oncogene Family Member 2 (TET2) Gene Refine the New European LeukemiaNet Risk Classification of Primary, Cytogenetically Normal Acute Myeloid Leukemia (CN-AML) In Adults: A Cancer and Leukemia Group B (CALGB) Study

AbstractAbstract 98Mutations in the TET2 gene were recently identified in a variety of myeloid neoplasms including AML. However, the frequency and clinical relevance of TET2 mutations in CN-AML, the largest cytogenetic subgroup of adult AML, have not been well defined. We report here the frequency and spectrum of TET2 mutations, and their associations with clinical and molecular characteristics, treatment outcomes and genome-wide gene- and microRNA (miR)-expression signatures in a relatively large cohort of 427 patients (pts) with primary CN-AML. The pts, aged 18–83 years, were intensively treated on CALGB frontline protocols, and were analyzed centrally for TET2 mutations by PCR and direct sequencing, and for other prognostic gene mutations (FLT3 internal tandem duplications [ITD] and tyrosine kinase domain mutations, MLL partial tandem duplications, and mutations in NPM1, CEBPA, WT1 and IDH1&IDH2). If available, buccal swabs or remission marrow samples were used to determine TET2 germline status. Gene- and miR-expression profiles were derived using microarrays (Affymetrix HG-U133 plus 2.0 and OSUCCC custom miR array v4.0). At least 1 sequence variation in TET2 was found in 104 pts. Frameshift (n=59) and nonsense (n=34) variations were distributed throughout all coding exons, while missense changes (n=37) clustered mainly (28/37) in 2 evolutionarily conserved domains of TET2. The remaining missense variations in 9 pts were located outside the conserved domains, and analysis of available buccal swabs or remission samples showed that these sequence changes were present in the germline. Since it is unclear whether they represent innocent polymorphisms or disease-relevant mutations, these 9 pts were excluded from further analyses. TET2-mutated (TET2-mut) pts were older (P<.001), had higher white blood counts (P=.04), a lower frequency of IDH1 and IDH2 mutations (P<.001), and showed a trend towards higher frequency of CEBPA mutations (P=.07) compared with TET2 wild-type (TET2-wt) pts. The European LeukemiaNet (ELN) recently proposed a standardized reporting system for AML, in which CN-AML pts are assigned to Favorable-risk (Fav; pts with mutated CEBPA and/or mutated NPM1 without FLT3-ITD) or Intermediate-I-risk (Int-I; all remaining CN-AML pts) categories. We assessed the prognostic relevance of TET2 mutations in the context of the Fav (n=199 pts) and Int-I (n=219) ELN categories. TET2 mutations tended to be more frequent in Fav than in Int-I CN-AML pts (27% v 19%, P=.08), even though types and location of mutations were similar in both groups. Within the Fav category, TET2-mut pts had shorter event-free survival (EFS; P<.001), a lower complete remission (CR) rate (P=.007) and shorter disease-free survival (DFS; P=.003; Fig 1), and shorter overall survival (OS; P=.001; Fig 2) compared with TET2-wt pts. In contrast, in the Int-I category, no difference in EFS (P=.45), CR rates (P=1.0), DFS (P=.36; Fig 1) or OS (P=.72; Fig 2) was found between TET2-mut and TET2-wt pts. In multivariable models, TET2 mutations associated with shorter EFS (P=.004; hazard ratio [HR], 1.71), lower CR rate (P=.03; odds ratio, 0.62) and shorter DFS (P=.049; HR, 1.54) only among Fav, but not among Int-I, CN-AML pts. A TET2 mutation-associated gene-expression signature consisting of 213 probe sets (136 named genes) was identified in ELN Fav CN-AML pts and included genes previously implicated in AML pathogenesis, e.g., upregulated CEBPA, APP, NCAM1 and IDH1, and downregulated MLL. In contrast, no signature of differentially expressed genes was identified in Int-I pts. miR profiling revealed distinct TET2 mutation-associated miR-expression signatures in the ELN Fav and Int-I risk groups. Among miRs upregulated in ELN Fav/TET2-mut pts were miR-148a (targeting DNA methyltransferases, highly expressed in refractory chronic lymphocytic leukemia) and miR-24 (stimulating myeloid cell proliferation, blocking granulocytic and erythroid differentiation). In Int-I/TET2-mut pts, one of the upregulated miRs was miR-204 (targeting HOXA10 and MEIS1, downregulated in NPM1-mut AML). We conclude that TET2 mutations are associated with lower remission rates and inferior survival in the ELN Fav category of CN-AML, and may be useful to refine the ELN molecular classification. TET2 mutation-associated gene- and miR-expression signatures, first identified here, may contribute to our understanding of the biology of TET2-mutated CN-AML. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

MicroRNA dynamics in the stages of tumorigenesis correlate with hallmark capabilities of cancer

While altered expression of microRNAs (miRs) in tumors has been well documented, it remains unclear how the miR transcriptome intersects neoplastic progression. By profiling the miR transcriptome we identified miR expression signatures associated with steps in tumorigenesis and the acquisition of hallmark capabilities in a prototypical mouse model of cancer. Metastases and a rare subset of primary tumors shared a distinct miR signature, implicating a discrete lineage for metastatic tumors. The miR-200 family is strongly down-regulated in metastases and met-like primary tumors, thereby relieving repression of the mesenchymal transcription factor Zeb1, which in turn suppresses E-cadherin. Treatment with a clinically approved angiogenesis inhibitor normalized angiogenic signature miRs in primary tumors, while altering expression of metastatic signature miRs similarly to liver metastases, suggesting their involvement in adaptive resistance to anti-angiogenic therapy via enhanced metastasis. Many of the miR changes associated with specific stages and hallmark capabilities in the mouse model are similarly altered in human tumors, including cognate pancreatic neuroendocrine tumors, implying a generality.

Assessment of Microrna Expression in Mantle Cell Lymphoma Using High Throughput Techniques

Mantle cell lymphoma (MCL) accounts for 3–10% of all lymphomas and demonstrates a poor clinical response to current therapeutic approaches, with a median survival of 3–5 years. The natural history of MCL is heterogeneous and not well-defined by standard clinical markers as patients may die within months of diagnosis or experience long-term survival. There remains a need for reliable biomarkers of MCL prognosis. To date, global gene expression signatures have not been determined for formalin-fixed, paraffin-embedded (FFPE) MCL samples due to difficulties isolating full-length mRNA transcripts from FFPE tissues. Examining microRNA expression in FFPE samples may circumvent this problem, as this population of RNAs remains intact during processing of FFPE samples. MicroRNAs (miRs) are small, non-coding RNAs, which regulate gene expression by inhibiting mRNA translation. Although miR expression signatures have been derived for other hematological malignancies, assessment of miR expression in patients with MCL has yet to be undertaken. We hypothesize that different pathological subtypes of MCL have unique miR expression signatures, distinct from miR expression profiles of other B-cell non-Hodgkin lymphomas. Our objectives were two-fold:To determine and validate miR expression in different pathological subtypes of MCL; and,To compare miR expression in MCL to other B-cell non-Hodgkin lymphomas.Total RNA was extracted from FFPE samples [17 conventional MCL, 11 blastoid MCL, 4 follicular lymphomas (Grades 1, 2, 3a, and 3b), 1 nodal marginal zone lymphoma, 1 small lymphocytic lymphoma (SLL/CLL) and 3 benign, reactive lymph nodes (normal controls)] using the RecoverAll kit for FFPE tissues (Ambion). RNA was subjected to quantitative real-time PCR (qRT-PCR) for 365 miRs and 3 endogenous control small nucleolar RNAs to obtain miR expression profiles using the TaqMan Low Density Array (TLDA) v1.0 platform (MicroFluidic card, Applied Biosystems). TLDAs were run on the ABI7900 HT analyzer with TLDA upgrade and analysed with RQ Manager software provided by Applied Biosystems. Expression profiles were correlated to pathological subtype, and hierarchical clustering, principal component analysis (PCA), and ANOVA were performed using Partek software [Partek Genomics Suite for Gene Expression Data]. Results indicate that miR expression profiles differ between B-cell, non-Hodgkin lymphoma and MCL samples. Clustering analysis and PCA both demonstrated different profiles between MCL and B-cell, non-Hodgkin lymphomas. Although PCA did not demonstrate significant differences between the conventional and blastoid MCL samples, a set of fifteen miRs may be able to distinguish these two groups of MCL, since these 15 miRs are relatively upregulated in blastoid MCL in comparison to conventional MCL. In addition, PCA revealed five (3 conventional MCL samples and 2 blastoid samples), which did not cluster with their respective groups. These five samples were from patients known to have progressive disease, indicating that such patients may have different miR expression profiles compared to patients with non-progressive disease. We conclude that high-throughput miR expression profiles can be generated from FFPE samples in B-cell non-Hodgkin lymphomas and that miR expression profiles for MCL samples differ from those of other B-cell non-Hodgkin lymphomas. Blastoid and conventional MCL samples may not have significantly differing profiles, however a set of 15 miRs appears to be able to distinguish between these two groups. Of note, samples from patients with known progressive disease have significantly different profiles from those with non-progressive disease.

A common microRNA signature in mouse models of retinal degeneration

Perturbed microRNA (miR) expression is a feature of, and may play a fundamental role in, certain disease states such as different forms of cancer. Retinitis pigmentosa (RP) a group of inherited retinal degenerations is characterised by a progressive loss of photoreceptor cells and consequent visual handicap. We have previously reported an altered pan-retinal expression of miR-96, -183, -1 and -133 in a P347S-Rhodopsin transgenic mouse model of RP. As many different mutations in Rhodopsin and other genes such as RDS/Peripherin can lead to RP, it was of interest to explore whether the characterized retinal miR expression signature was observed in three other mouse models of RP linked to rhodopsin and rds/peripherin. Therefore, pan-retinal expression of miR-96, -182, -183, -1, -133 and -142 was analysed using quantitative real-time RT-PCR. A common signature of altered miR expression was found; expression of miR-96, -182 and -183 decreased by 14.1-53.2%, while expression of miR-1, -133 and -142 was up-regulated by 186.1-538.5%. Significantly, the detected pan-retinal miR signature was mirrored by similar miR expression profiles in FACS-isolated rod photoreceptors from these mice. In an attempt to understand the function of these miRs, corresponding target genes were predicted using computational means. Many ‘enriched’ targets (with binding sites for at least two of the above miRs) were found to be regulatory molecules and members of intracellular signalling circuits. However, further studies are required to highlight which of the large number of in silico predicted targets are actually controlled by these miRs.

MicroRNA (miR) expression signatures in molecular subsets of cytogenetically normal (CN) acute myeloid leukemia (AML): A Cancer and Leukemia Group B (CALGB) study

7010 Background: Aberrantly expressed miRs contribute to malignant transformation by hybridizing to specific mRNA targets and suppressing their translation into proteins. In CN AML, the largest cytogenetic AML subset, several molecular markers are associated with favorable [e.g., NPM1 mutation (mutNPM1)] or unfavorable [e.g., FLT3 internal tandem duplication (ITD) or high ERG expression (↑ERG)] outcome. We explored if differentially expressed miRs are associated with the aforementioned markers. If so, this might indicate a role for miRs in leukemogenesis and prognosis in CN AML. Methods: Diagnostic samples from 100 adults &lt;60 years, treated on CALGB 19808 (Blood 2005; 106:122a), were analyzed on miR microarrays. Among 305 miR probes, we identified differentially expressed miRs (P&lt;0.005) when comparing patients (pts) with or without mutNPM1, FLT3 ITD or ↑ERG. Results: Nine miRs were differentially expressed in FLT3 ITD compared with FLT3 wild-type pts. miR 155, previously reported in aggressive cancers, was upregulated in FLT3 ITD pts. Strong signatures were associated with mutNPM1 (42 miR probes) and ↑ERG groups (54 miR probes). Consistent with their better prognosis, pts with mutNPM1 had upregulated miRs 10 and 196 targeting HOX genes, miRs 15 and 16 targeting BCL2 and let-7 targeting RAS. Consistent with a more aggressive phenotype, ↑ERG pts had downregulation of let-7 and miR 15 and 16 but also miRs 30, 103, and 24 which target, respectively, CDK6, FBXW11 and MAPK14, all involved in cell cycle regulation and proliferation. Four miRs were associated with event free survival (EFS); all were also associated with ↑ERG. High expression of miRs 124, 129, and 194 was significantly associated with worse EFS and ↑ERG. Low expression of miR 181 was associated with worse EFS and ↑ERG. Notably, downregulation of mir 181 has also been reported in pts with aggressive B-cell chronic lymphocytic leukemia. Conclusions: We report the first miR expression signatures within molecular subsets of CN AML. Further study is warranted to elucidate mechanisms by which miRs contribute to leukemogenesis and impact prognosis, and to determine whether miRs can be therapeutically targeted. No significant financial relationships to disclose.