2382 resistance to gemcitabine has led to identiﬁcation of several candidate predictive markers. These include genes related to gemcitabine metabolism and transport, such as deoxy- cytidine kinase, ribonucleotide reductase, and human equilibrative nucleoside transporter-1.11,12 However, the usefulness of such markers in a clinical setting remains unclear because of difﬁculties in evaluating their protein or mRNA levels. Even when well-established methods are used, immunohistochemical evaluation and its interpreta- tion are different between laboratories.13 Also, accurate quantitative analyses of mRNA from clinical samples are often difﬁcult as a result of degradation. Therefore, we need more reliable methods-based biomarkers to predict responses to gemcitabine. MicroRNAs (miRNAs) are small noncoding RNA gene products of approximately 22 nt that are found in a variety of organisms. They play key roles in regulating the translation and degradation of mRNAs through base pairing to partially complementary sites, predominantly in the 30-untranslated regions of mRNAs.14,15 Because microRNAs are well pre- served, even in formalin-ﬁxed, parafﬁn-embedded (FFPE) samples, the quantitative analysis of miRNA in many types of clinical sample is accurate and reliable.16,17 Therefore, miRNAs are promising molecular markers. There is increasing evidence that miRNAs are mutated or differentially expressed in many types of cancers, and thus are also potential diagnostic markers.18–20 The expression levels of several miRNAs, such as miR-21, miR-196a-2, miR-155, and miR-210 in pancreatic cancer, are known to correlate with a poor prognosis.19,21,22 Thus, there is a possibility that speciﬁc miRNAs that can be used as predictive markers for a gemcitabine response exist. Here, to identify predictive markers, we established two gemcitabine-resistant pancreatic cancer cell lines and per- formed global miRNA expression analyses. We then further investigated 11 selected miRNAs as potential pre- dictive markers using macrodissected FFPE samples derived from 90 patients with resected pancreatic cancer. MATERIALS AND METHODS Cell Lines and Establishment of Gemcitabine-Resistant Cells Two human pancreatic cancer cell lines, SUIT-2 and CAPAN-1 (the gift of Dr. H. Iguchi, National Shikoku Cancer Center, Matsuyama, Japan), were used in this study. Gemcitabine-resistant cells were generated by exposing these cell lines to gradually increasing concentrations of gemcitabine. The initial concentration of gemcitabine was 1 nM, which did not seem to affect the proliferation of either the SUIT-2 or CAPAN-1 cells. When the cells had K. Ohuchida et al. adapted to the drug, the concentration of gemcitabine was gradually increased by 10–100 nM per week to a ﬁnal concentration of 200 nM (SUIT-2) and 1 lM (CAPAN-1). Gemcitabine was dissolved in phosphate-buffered saline and added to the cell culture media. Propidium Iodide Assay Cell proliferation was evaluated by measuring the ﬂuorescence intensity of propidium iodide as described previously.23 The ﬂuorescence intensity corresponding to the total cells was measured with a CytoFluor multiwell plate reader (PerSeptive Biosystems, Framingham, MA, USA). The results were converted to percentage survival rates by comparing treated cells with untreated cells. miRNA Expression Proﬁling Expression proﬁles for the 365 mature miRNAs were obtained by quantitative polymerase chain reaction (qPCR) using the TaqMan Array Human MiRNA (TLDA) or TaqMan MiRNA Assays (Applied Biosystems; ABI) on an ABI Prism 7900HT according to the manufacturer’s instructions. Mature miRNAs were normalized to RNU44 and are expressed as fold changes relative to controls. Patients and Pancreatic Tissues From 1992 through 2008, a total of 104 patients underwent pancreatic resection for pancreatic cancer at the Department of Surgery and Oncology, Kyushu University Hospital (Fukuoka, Japan). Survival was measured from the time of pancreatic resection, and death was the end point. The follow-up data for 103 cases were available. Thirteen of the 103 patients were excluded from the present study because they had received combination therapy with gemcitabine and other chemotherapeutic agents such as S1(tegafur/gimestat/potassium oxonate). Our ﬁnal study series consisted of 90 patients with resected pancreatic cancer with available follow-up data. The patients (57 men and 32 women) had a median age of 65 years (range, 36–86 years). The median observation time for overall survival was 14.7 months, ranging 0.5–108 months. Sixty-three patients died during follow- up; the other patients were alive and censored. All resected specimens were ﬁxed in formalin and embedded in parafﬁn for pathological diagnosis. All tissues adjacent to the specimens were evaluated histologically according to the criteria of the World Health Organization.24 Diagnoses were conﬁrmed independently by two patholo- gists with regard to the pathological features of all cases. Tumor stage was assessed according to the International Union Against Cancer classiﬁcation.25 Clinicopathological