Patients with ALK gene rearrangements often manifest dramatic responses to crizotinib, an ALK inhibitor. Accurate identification of patients with ALK-positive non-small cell lung cancer (NSCLC) is essential for the clinical application of ALK-targeted therapy. However, assessing EML4-ALK rearrangement in NSCLC remains challenging in routine pathology practice. The aim of this study was to compare the diagnostic accuracy of FISH, immunohistochemistry (IHC), and real-time quantitative RT-PCR (QPCR) methodologies for detection of EML4-ALK rearrangement in NSCLC and to appraise immunohistochemistry as a pre-screening tool. In this study, a total of 473 paraffin-embedded NSCLC samples from surgical resections and biopsies were analyzed by IHC with ALK antibody. ALK rearrangement was further confirmed by FISH and QPCR. ALK protein expression was detected in twenty patients (20/473, 4.2%). Of the 20 ALK-positive cases by IHC, 15 cases were further confirmed as ALK rearrangement by FISH, and 5 cases were not interpretable. Also, we evaluated 13 out of the 20 IHC-positive tissues by QPCR in additional to FISH, and found that 9 cases were positive and 2 cases were equivocal, whereas 2 cases were negative although they were positive by both IHC and FISH. The ALK status was concordant in 5 out of 8 cases that were interpretable by three methods. Additionally, none of the 110 IHC-negative cases with adenocarcinoma histology showed ALK rearrangements by FISH. Histologically, almost all the ALK-rearranged cases were adenocarcinoma, except that one case was sarcomatoid carcinoma. A solid signet-ring cell pattern or mucinous cribriform pattern was presented at least focally in all ALK-positive tumors. In conclusion, our findings suggested that ALK rearrangement was associated with ALK protein expression. The conventional IHC assay is a valuable tool for the pre-screening of patients with ALK rearrangement in clinical practice and a combination of FISH and QPCR is required for further confirmation.

Autophagy can be tumor suppressive as well as promotive in regulation of tumorigenesis and disease progression. Accordingly, the prognostic significance of autophagy key regulator Beclin 1 was varied among different tumors. Here, we detected the clinicopathological and prognostic effect of Beclin 1 in the subtypes of intrahepatic cholangiocarcinoma (ICC) and extrahepatic cholangiocarcinoma (ECC). Beclin 1 expression level was detected by immunohistochemistry staining in 106 ICC and 74 ECC patients. We found that Beclin 1 was lowly expressed in 126 (70%) cholangiocarcinoma patients, consist of 72 ICC and 54 ECC. Moreover, the cholangiocarcinoma patients with lymph node metastasis (N1) had a lower Beclin 1 level than that of N0 subgroup (P=0.012). However, we did not detect any correlations between Beclin 1 and other clinicopathological features, including tumor subtypes, vascular invasion, HBV infection, liver cirrhosis, cholecystolithiasis and TNM stage. Survival analysis showed that, compared with the high expression subset, Beclin 1 low expression was correlated with a poorer 3-year progression-free survival (PFS, 69.1% VS 46.8%, P=041) for cholangiocarcinoma. Importantly, our stratified univariate and multivariate analysis confirmed that Beclin 1 lowly expressed ICC had an inferior PFS as well as overall survival than ECC, particularly than that of Beclin 1 highly expressed ECC patients. Thus, our study demonstrated that Beclin 1low expression, correlated with lymph node metastasis, and might be a negative prognostic biomarker for cholangiocarcinoma. Combined Beclin 1 level with the anatomical location might lead to refined prognosis for the subtypes of ICC and ECC.

We and others had proven that hypoxia-induced autophagy was essential to regulate cancer cell destiny under anticancer therapeutic stress. Here, we addressed the clinicopathologic effect of HIF-1α and autophagic Beclin 1 in primary duodenal adenocarcinoma (PDA). HIF-1α and Beclin 1 expression level were semi-quantitatively evaluated using tissue microarrays and immunohistochemistry (IHC) staining in 141 PDA patients. Among these patients, 77 acted as training set to select HIF-1α and Beclin 1 IHC cutoff score for patient outcome, and 64 cases were used as testing set to evaluate their prognostic effect. We found that Beclin 1 was cytoplasmic overexpressed, defined by training set fixed cutoff point, in 49.6 % PDA tissue, compared to 46.8 % patients had HIF-1α high expression. In testing set, Beclin 1 overexpression predicted a superior 5-year overall survival (OS) in both univariate (P = 0.010) and multivariate (P = 0.017) analyses. However, we did not detect any correlation between HIF-1α level and patient prognosis (P = 0.989). Significantly, among Beclin 1 overexpressed patients, radical surgery plus adjuvant chemotherapy had a 23.1-month OS improvement than given radical surgery alone (59.2 vs 36.1 months; P = 0.01). For Beclin 1 lowly expressed patients, radical surgery plus adjuvant chemotherapy and given radical surgery alone had the similar OS (P = 0.283). Contrary to previous studies, we failed to detect any correlation between Beclin 1 and HIF-1α levels in PDA (correlation coefficient 0.217, P = 0.099). In conclusions, our results confirmed that Beclin 1 was a favorable prognostic biomarker for PDA, and might be used to identify particular patients for more selective therapy.