No abstract

Barrett's esophagus (BE)/Barrett's metaplasia (BM) is a recognized precursor of esophageal adenocarcinoma (EA) with an intermediary stage of dysplasia. The low yield and high cost of endoscopic screening of patients with BE underscores the need for novel biomarkers, such as microRNA (miRNA), which have emerged as important players in neoplastic progression for risk assessment of developing dysplasia/adenocarcinoma. Recently, we reported highly elevated levels of miRNA-196a (miR-196a) in EA and demonstrated its growth-promoting and anti-apoptotic functions. Here, we evaluated miR-196a as a marker of BE progression to low-grade dysplasia, high-grade dysplasia, and EA using microdissected paraffin-embedded tissues from 11 patients. Higher levels of miR196a were observed in EA, BE, and dysplastic lesions compared with normal squamous mucosa, and in high-grade dysplasia compared with BE and lowgrade dysplasia. Using frozen tumor tissues from 10 additional patients who had advanced EA, we evaluated the correlation of miR-196a with its in silicopredicted targets, keratin 5 (KRT5), small prolinerich protein 2C (SPRR2C), and S100 calcium-binding protein A9 (S100A9), which are down-regulated during BE progression. MiR-196a levels inversely correlated with the predicted target mRNA levels in EA. We confirmed that miR-196a specifically targets KRT5 , SPRR2C , and S100A9 3 UTRs using miR-196a-mimic and luciferase reporter-based assays. In conclusion , this study identified miR-196a as a potential marker of progression of BE and KRT5, SPRR2C, and S100A9 as its targets.

Imatinib (Gleevec) resistance (IR) in CML is a multifactorial phenomenon related to ineffective blockage of the bcr-abl kinase through point mutation and/or gene amplification, or alternatively kinase bypass through clonal evolution or presently unknown mechanisms. The newer generation of bcr-abl small molecule inhibitors (dasatinib, nilotinib) may be useful in overcoming ineffective bcr-abl blockage in IR-CML but may not be effective in other mechanisms of resistance. As a tool in classifying the pattern of disease resistance, we profiled kinase levels in IR-CML following switch to new kinase inhibitors or with imatinib dose escalation. In Ficoll-purified blood and bone marrow samples, we examined levels of BCR-ABL autophosphorylation (Tyr245), and phosphorylation of two bcr-abl targets STAT5 (Tyr694/699) and CRKL (Tyr207) by Western blot. Kinase profiling was compared to hematologic responses, cytogenetic analysis, FISH for the bcr-abl locus using dual fusion probes and quantitative(q)PCR for bcr-abl transcript. We profiled 26 patients with IR-CML undergoing therapy shifts as well as serial samples from 10 patients receiving frontline dasatinib therapy for newly diagnosed CML. In all but 1 of 68 analyzed samples, detection of phospho(p)-CRKL in unsorted white blood cells was associated with bcr-abl/abl qPCR ratios above 1%. Among all 68 samples, pSTAT5 and pBCR-ABL levels declined in parallel to pCRKL but were absent in more samples than pCRKL. Persistence of detectable pCRKL despite switch to nilotinib, dasatinib or high dose imatinib was seen in 5 patients with bcr-abl kinase domain (KD) mutations including 2 with G250E and 1 each with L248V, F311L and T315I, 2 patients with bcr-abl gene amplification and 3 patients without KD mutations including 2 with cytogenetic clonal evolution. Among these 10 patients with IR-CML and persistent pCRKL, complete hematologic responses were noted in 4 and complete cytogenetic response in 3. Response to therapy switch and absence of pCRKL was seen in tumors with E355G, L387M and E459K KD mutations and in 12 patients with no mutation or genomic amplification. Among the patients receiving frontline therapy with dasatinib, effective hematologic responses correlated well with loss of detectable pCRKL and pSTAT5. However, detectable pCRKL did not disappear until at least 8 weeks following initiation of therapy, and lagged hematologic response. The 2 patients treated with frontline dasatinib therapy who had persistent pCRKL after 20 weeks of treatment also showed amplification of the bcr-abl locus in tumor cells by FISH. Kinase profiling, particularly for pCRKL, is useful for monitoring molecular response in the therapy switch period in IR-CML and correlates well with the predicted pattern of response in tumors with KD mutations. The value of monitoring kinase levels frequently following initiation of frontline kinase inhibitor therapy in newly diagnosed CML remains to be established.