Tissue array and immunohistochemistry

Sixty de-identified lung cancer tissues with paired adjacent noncancerous tissues were collected from the hospital between January 2007 and March 2008 and developed into tissue arrays by Shanghai OUTDO Biotech (Shanghai, China). Twenty adenocarcinomas, 10 squamous carcinomas, 10 adenosquamous carcinomas, 10 large cell carcinomas and 10 small cell carcinomas were included. The study protocols were approved by the SJTUSM (Shanghai Jiao Tong University School of Medicine) Ethics Committee. All procedures adhere to the BRISQ Guidelines for reporting research on human biospecimens.

Immunohistochemical detection of Rab1A was performed using a streptavidin-biotin complex method as described previously [8]. The primary antibody against Rab1A (Proteintech Group) was used at a concentration of 1:100. To score a tumor cell as positive, cytoplasmic or nuclear staining was performed. For quantitative analysis, a Histo (H) score was calculated based on the staining intensity and percentage of stained cells using Aperio ScanScope systems (Vista, CA, USA). The intensity score was defined as follows: 0, no appreciable staining in cells; 1, weak staining in cells comparable with stromal cells; 2, intermediate staining; 3, strong staining. The fraction of positive cells was scored as 0%-100%. The H score was calculated as previously described, by multiplying the intensity score and the fraction score [8], producing a total range of 0-300. Based on the distribution of the data, a cutoff of 67.5 was used for Rab1A positivity. Tissue sections were examined and scored separately by two independent investigators blinded to the clinicopathologic data.