Univariate and multivariate analysis of miR-148a expression and DFS in stage II/III CRC patients.

a<p>Proximal colon, located above splenic flexture; distal colon, located in splenic flexture or below.</p>b<p>p<0.05.</p

The Clinical Significance of MiR-148a as a Predictive Biomarker in Patients with Advanced Colorectal Cancer

<div><h3>Aim</h3><p>Development of robust prognostic and/or predictive biomarkers in patients with colorectal cancer (CRC) is imperative for advancing treatment strategies for this disease. We aimed to determine whether expression status of certain miRNAs might have prognostic/predictive value in CRC patients treated with conventional cytotoxic chemotherapies.</p> <h3>Methods</h3><p>We studied a cohort of 273 CRC specimens from stage II/III patients treated with 5-fluorouracil-based adjuvant chemotherapy and stage IV patients subjected to 5-fluorouracil and oxaliplatin-based chemotherapy. In a screening set (n = 44), 13 of 21 candidate miRNAs were successfully quantified by multiplex quantitative RT-PCR. In the validation set comprising of the entire patient cohort, miR-148a expression status was assessed by quantitative RT-PCR, and its promoter methylation was quantified by bisulfite pyrosequencing. Lastly, we analyzed the associations between miR-148a expression and patient survival.</p> <h3>Results</h3><p>Among the candidate miRNAs studied, miR-148a expression was most significantly down-regulated in advanced CRC tissues. In stage III and IV CRC, low miR-148a expression was associated with significantly shorter disease free-survival (DFS), a worse therapeutic response, and poor overall survival (OS). Furthermore, miR-148a methylation status correlated inversely with its expression, and was associated with worse survival in stage IV CRC. In multivariate analysis, miR-148a expression was an independent prognostic/predictive biomarker for advanced CRC patients (DFS in stage III, low vs. high expression, HR 2.11; OS in stage IV, HR 1.93).</p> <h3>Discussion</h3><p>MiR-148a status has a prognostic/predictive value in advanced CRC patients treated with conventional chemotherapy, which has important clinical implications in improving therapeutic strategies and personalized management of this malignancy.</p> </div

DataSheet_1_Epigenetic therapy reprograms M2-type tumor-associated macrophages into an M1-like phenotype by upregulating miR-7083-5p.docx

Reprogramming M2-type, pro-tumoral tumor-associated macrophages (TAMs) into M1-type, anti-tumoral macrophages is a key strategy in cancer therapy. In this study, we exploited epigenetic therapy using the DNA methylation inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) and the histone deacetylation inhibitor trichostatin A (TSA), to reprogram M2-type macrophages into an M1-like phenotype. Treatment of M2-type macrophages with the combination of 5-aza-dC and TSA decreased the levels of M2 macrophage cytokines while increasing those of M1 macrophage cytokines, as compared to the use of either therapy alone. Conditioned medium of M2 macrophages treated with the combination of 5-aza-dC and TSA sensitized the tumor cells to paclitaxel. Moreover, treatment with the combination inhibited tumor growth and improved anti-tumor immunity in the tumor microenvironment. Depletion of macrophages reduced the anti-tumor growth activity of the combination therapy. Profiling of miRNAs revealed that the expression of miR-7083-5p was remarkably upregulated in M2 macrophages, following treatment with 5-aza-dC and TSA. Transfection of miR-7083-5p reprogrammed the M2-type macrophages towards an M1-like phenotype, and adoptive transfer of M2 macrophages pre-treated with miR-7083-5p into mice inhibited tumor growth. miR-7083-5p inhibited the expression of colony-stimulating factor 2 receptor alpha and CD43 as candidate targets. These results show that epigenetic therapy upon treatment with the combination of 5-aza-dC and TSA skews M2-type TAMs towards the M1-like phenotype by upregulating miR-7083-5p, which contributes to the inhibition of tumor growth.</p

Associations between miR-148a status and therapeutic response or survival in stage IV CRC patients treated with 5-FU and oxaliplatin.

<p><b>A</b>. Therapeutic response according to miR-148a expression. Complete response, CR; partial response, PR; stable disease, SD; progressive disease; PD. <b>B</b>. Kaplan-Meyer curves for progression-free survival (PFS, left panel) and OS (right panel) in stage IV patients according to miR-148a expression. <b>C</b>. Kaplan-Meyer curves for PFS (left panel) and OS (right panel) in stage IV patients according to miR-148a methylation.</p

MiR-148 expression status and clinicopathologic characteristics of CRC patients.

a<p>The difference was analyzed by Mann-Whitney U test.</p>b<p>The difference was analyzed by Fisher's exact test.</p>c<p>Proximal colon, located above splenic flexure; distal colon, located in splenic flexure or below.</p>d<p>The difference was analyzed by the chi-square test.</p

Survival analysis in stage II/III patients treated with 5-FU-based chemotherapy.

<p>Kaplan-Meyer curves for <b>A</b>, Disease-free survival (DFS) and <b>B</b>, overall survival (OS) in stage II/III patients according to miR-148a expression. Kaplan-Meyer curves for <b>C</b>, DFS in stage II and <b>D</b>, stage III DFS depending upon miR-148a expression.</p

Univariate and multivariate analysis of miR-148a expression, methylation and overall survival in stage IV MSS CRC patients.

a<p>p<0.05.</p

MiR-148a expression and methylation in colonic mucosa from healthy individuals and in CRC tissues from patients.

<p><b>A</b>. miR-148a expression in colonic mucosa from healthy controls (NC), and in stage II, III and IV CRCs; the number of patients (N) and median expression (Median) are listed below the graph. <b>B</b>. <i>In situ</i> hybridization for miR-148a in CRC tumors and normal mucosa, in which the chromogen stains red, and the counterstain blue. Representative photomicrographs are shown from a normal colonic mucosa (top panels), a tumor with low miR-148a expression (middle panels), and a tumor with high miR-148a expression (lower panels) at indicated magnifications. A photomicrograph is shown from a tumor with high miR-148a expression using a scramble probe as a negative control (bottom, left panel). <b>C</b>. miR-148a methylation levels in stage IV tumors. The putative promoter region of miR-148a, and the position of pyrosequencing primers are illustrated in the top panel. The scatter plot of miR-148a expression and methylation levels are shown in the bottom panel. <b>D</b>. miR-148a expression levels are shown for methylated and non-methylated CRCs in the top panel. miR-148a methylation levels are shown for tumors with high and low miR-148a expression in the bottom panel. One outlier value (the methylation level; 48%) is excluded from the methylated group in the bottom graph.</p

LINE-1 methylation results in different clinical subgroups.

<p>CRC, colorectal cancer; SD, standard deviation.</p><p>Mann Whitney test was used to compare the LINE-1 values.</p>1<p>Values for comparison between normal colonic mucosa and other groups of CRC.</p>2<p>Values for comparison between early onset CRC (n = 185) and other groups of CRC.</p

Clinical, pathological and molecular features of patients with mismatch repair deficiency.

1<p>MSI-H and/or loss of expression of MMR proteins by immunohistochemistry.</p>2<p>MSS and normal expression of MMR proteins by immunohistochemistry.</p>3<p>Including first and second degree relatives; Lynch syndrome-associated neoplasia includes: endometrium, stomach, ovaries, urinary tract, small intestine, pancreas, bile ducts, brain or sebaceous glands.</p>4<p>Signet ring cells and/or Crohn’s-like lymphocytic reaction and/or tumor infiltrating lymphocytes and/or medullary growth pattern and/or anaplastic tumor.</p