The proliferation marker Ki67 has prognostic and predictive values in breast cancer, and the cutoff of the Ki67 label index (LI) is a key index for chemotherapy. However, poor interobserver consistency in Ki67 assessment has limited the clinical use of Ki67, especially in luminal cancers. Here, we reported a modified Ki67 assessment method, size-set semiautomatic counting (SSSAC) and investigated its interobserver reproducibility. One hundred invasive breast cancer tissues were set immunostained for Ki67 in one laboratory, scanned as digital slides, and sent to 41 pathologists at the laboratories of 16 hospitals for Ki67 LI assessment using size-set semiautomatic counting (SSSAC), size-set visual assessment (SSVA) and size-set digital image analysis (SSDIA) with a specific image viewing software (Aperio Image Scope, Leica, Germany). The intraclass correlation coefficient (ICC) and Bland-Altman plot were used to evaluate interobserver reproducibility. The Wilcoxon signed-rank test was used to analyze the difference in the Ki67 values assessed by SSSAC and SSDIA. SSSAC demonstrated better interobserver reproducibility (ICC = 0.942) than SSVA (ICC = 0.802). The interobserver reproducibility was better in Ki67 homogeneously stained slides and centralized hot-spot slides than in scattered hot-spot slides. The Ki67 value assessed with SSSAC was obviously higher than that assessed with SSDIA (negative ranks (SSDIA < SSSAC): N = 80, sum of ranks = 4274.50; positive ranks (SSDIA > SSSAC): N = 17, sum of ranks = 478.50; Z = -6.837; P < 0.001). SSSAC shows satisfactory interobserver reproducibility in the Ki67 assessment of breast cancer and may be a candidate standard method for Ki67 LI assessment in breast cancer and other malignancies.

The ras genes play an important role in the development and progression of human tumours. Neutralizing Ras proteins in the cytoplasm could be an effective approach to blocking ras signalling. In this study, we prepared anti-p21Ras single chain fragment variable antibody (scFv) and investigated its immunoreactivity with human tumours. The coding sequences of H-ras, K-ras, and N-ras were separately ligated into the vector pET-28a(+). Then, recombinant expressing plasmids were induced by IPTG for p21Ras expression in E. coli. Hybridoma cell lines producing anti-p21Ras monoclonal antibodies were isolated using wildtype p21Ras proteins as immunogens. Anti-p21Ras scFv antibody was prepared from the hybridoma by the phage scFv display method. The immunoreactivity of the anti-p21Ras monoclonal antibody and the scFv antibody was identified by ELISA and immunocytochemistry. We prokaryotically expressed wildtype H-p21Ras, K-p21Ras and N-p21Ras and generated the hybridoma cell line KGH-R1, producing anti-p21Ras monoclonal antibodies. It was demonstrated that KGH-R1 monoclonal antibody could recognize wildtype and mutated H-p21Ras, K-p21Ras and N-p21Ras in human tumour cell lines. In all 14 types of primary human cancer tissues tested, the monoclonal antibody presented strong immunoreactivity but showed weak or negative immunoreactivity in the corresponding normal tissues. Subsequently, we prepared anti-p21Ras scFv from hybridoma KGH-R1, which showed the same immunoreactivity as the original monoclonal antibody. Sequence analysis demonstrated that the nucleotides and amino acids of the scFv exhibited an approximately 50 % difference from the anti-p21Ras scFv reported previously. This study presents a novel anti-p21Ras scFv antibody. Our data suggest that the scFv may be useful for ras signalling blockage and may be a potential therapeutic antibody for ras-derived tumours.

Colorectal cancer (CRC) is the most common type of gastrointestinal cancer. CRC gene therapy mediated by adenovirus holds great promise for the treatment of malignancies. However, intravenous delivery of adenovirus exhibits limited anti-tumor activity in vivo when used alone. In this study, the antitumor activity of the recombinant adenovirus KGHV500 was assessed with the MTT, TUNEL, Matrigel invasion and cell migration assays. To enhance the intravenous delivery of KGHV500 in vivo, cytokine-induced killer (CIK) cells were used as a second vector to carry KGHV500. We explored whether CIK cells could carry the recombinant adenovirus KGHV500 containing the anti-p21Ras single chain fragment variable antibody (scFv) gene into tumors and enhance antitumor potency. Our results showed that KGHV500 exhibited significant antitumor activity in vitro. In the nude mouse SW480 tumor xenograft model, the combination of CIK cells with KGHV500 could induce higher antitumor activity against colorectal cancer in vivo than that induced by either CIK or KGHV500 alone. After seven days of treatment, adenovirus and scFv were detected in tumor tissue but were not detected in normal tissues by immunohistochemistry. Therefore, KGHV500 replicates in tumors and successfully expresses anti-p21Ras scFv in a colorectal cancer xenograft model. Our study provides a novel strategy for the treatment of colorectal cancer by combining CIK cells with the recombinant adenovirus KGHV500 which carried anti-p21 Ras scFv.

Colorectal cancer (CRC) is the most common gastrointestinal type of cancer. The overexpression of Ras proteins, particularly p21Ras, are involved in the development of CRC. However, the subtypes of the p21Ras proteins that are overexpressed and the mutation status remain unknown restricting the development of therapeutic antibodies targeting p21Ras proteins. The present study aimed to investigate the mutation status of ras genes associated with Ras proteins that are overexpressed in CRC and explore whether or not wild-type p21Ras could be a target for CRC therapy. p21Ras expression was examined immunohistochemically in normal colorectal epithelium, benign lesions and malignant colorectal tumor tissues by monoclonal antibody (Mab) KGH-R1 which is able to react with three types of p21Ras proteins: H-p21Ras, N-p21Ras and K-p21Ras. Then, the expression levels of p21Ras subtypes were determined in CRC by a specific Mab for each p21Ras subtype. Mutation status of ras genes in p21Ras-overexpressing CRC was detected by DNA sequencing. There was rare p21Ras expression in normal colorectal epithelium but a high level of p21Ras expression in CRC, with a significant increase from normal colorectal epithelium to inflammatory polyps, low-grade intraepithelial neoplasia, high-grade intraepithelial neoplasia and invasive colorectal adenocarcinoma, respectively. Overexpression of K-p21Ras was found in all CRC tissues tested, overexpression of N-p21Ras was found in 85.7% of the CRC tissues, while H-p21Ras expression was not found in any CRC tissue. DNA sequencing showed that there were no K-ras mutations in 60% of the K-p21Ras-overexpressing CRC, while 40% of the CRC tissues harbored K-ras mutations. N-ras mutations were not found in any N-p21Ras-overexpressing CRC. Our findings indicate that overexpression of wild-type p21Ras may play a prominent role in the development of CRC in addition to ras mutations and could be a promising target for CRC therapy.