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  • br Disclosure statement br Acknowledgment This


    Disclosure statement
    Acknowledgment This work was supported by the National Natural Science Foundation of China (81270704, 81330018).
    Background Over-expression of cyclooxygenase-2 (COX-2) is common in many malignancies including non-small cell lung cancer (NSCLC) and is associated with poor prognosis [1], [2], [3]. COX-2 inhibition has been clinically tested in NSCLC with mixed results [4], [5]. COX-2 catalyzes the transformation of arachidonate to prostaglandin E2 (PGE2), thereby promoting cancer progression through many mechanisms including tumor cell proliferation, survival, invasion, metastasis, angiogenesis and immune suppression [6]. However, inhibitors of COX-2 (COXibs) have the potential for cardiovascular toxicities; COXibs reduce levels of prostacyclin which plays a key role in platelet homeostasis, antiproliferation, and antiangiogenesis [7]. By targeting the COX-2 pathway downstream of COX-2 inhibition, there is the potential to maintain prostacyclin levels and decrease the risk of toxicities while also improving efficacy. Multiple studies have shown that, via prostaglandin E (EP) receptor EP4, PGE2 induces proliferation and metastatic spread and immunosuppression in many cancers including prostate, breast and colon [8], [9], [10], [11], [12], [13]. To date, no study evaluating EP4 Pemetrexed disodium hemipenta hydrate in lung cancer has been performed. Based upon the above data, we hypothesized that EP4 expression would correlate with outcome and patterns of relapse after treatment of stage IIIA locally advanced NSCLC (LANSCLC).
    Methods We identified 41 patients with stage IIIA (AJCC 7th edition) LANSCLC with at least N2 disease who were treated with curative intent at our institution for whom tissue specimens were available. A tissue microarray (TMA) was created from formalin-fixed, paraffin embedded specimens. Representative areas of tumor were identified by a pathologist, and 1.0mm tissue cores were obtained in duplicate and embedded in a TMA, along with 6 normal lung control specimens and one normal liver control specimen. Sections cut from the TMA were deparaffinized in xylene and rehydrated in a graded series of ethanols. Sections were pretreated with heat-induced epitope retrieval using a pressure cooker and Target Retrieval Solution pH 6.1 (Dako TRS, S1699/1700), followed by endogenous peroxidase blocking for 5min with 0.3% hydrogen peroxide. The sections were then incubated 1h with Prostaglandin E Receptor EP4 (PTGER4) Rabbit anti-human Polyclonal antibody (Lifespan Biosciences #LS-A3898) at 1:800 dilution at room temperature. The sections were incubated with secondary antibodies (EnVision+ dual link system, Dako) for 30min at room temperature. All slides were developed for 5min using diaminobenzidine as the chromagen (Dako, Carpinteria, CA) and were counterstained with hematoxylin. Staining was performed on a DAKO automatic stainer using EnVision+ (DAKO), a biotin-free detection system that consists of a secondary antibody covalently linked to peroxidase coated dextrose polymers. A negative control was performed using identical methods but with omission of the antibody. Tumor expression of EP4 was evaluated by a pathologist blinded to the clinical data. Nuclear and cytoplasmic staining were separately scored using a semiquantitative score: 0 (no identifiable staining), 1+ (faint), 2+ (weak), 3+ (moderate), or 4+ (strong). The percentage of positive cells for each compartment (nuclear or cytoplasmic) was also scored; however, all tissue cores showed a consistent degree of staining across nearly all (>75%) of the tumor cells. Each tissue core was evaluated independently. There was a one-step (e.g. 0 versus 1+) scoring discrepancy between duplicate cores from a single tumor for 6 of 82 observations (2/41 for nuclear staining; 4/41 for cytoplasmic staining), and no two-step scoring discrepancies. For that reason, survival evaluation was dichotomized as 0–1 staining vs. 2+. In cases of scoring discrepancies, both cores were re-reviewed by the pathologist and a single overall score was assigned for the tumor. In 6 cases, no tumor was present on one of the cores, and the score was based on the single evaluable core. The normal lung control cores displayed no nuclear staining in any case, with cytoplasmic staining of 2+ in 5/6 and 1+ in 1/6.