Optimisation and validation of immunohistochemistry protocols for cancer research.

BACKGROUND: Immunohistochemistry (IHC) has become a valuable laboratory technique for diagnosing, evaluating metastasis and informing treatment selection in several cancers. Standardization however remains a limiting factor in IHC. The main aim of this research study was to optimise, validate and standardize antibodies and IHC protocols for cancer research. METHODS: Seven monoclonal mouse and rabbit antibodies were optimised using formalin-fixed paraffin embedded (FFPE) human tissue blocks. 4um sections of FFPE block were stained using the Roche Ventana XT or Ventana ULTRA IHC automated analysers. This study modified manufacturer recommended protocols by using a unique antigen retrieval method, adding an amplification step, varying primary antibody incubation times, as well as using the Roche Ventana Ultraview detection system. RESULTS: Optimum antibody localisation was observed in modified IHC protocols in comparison with manufacturer recommended protocols for anti-CEACAM-1, anti-CD31, anti-COX-2, anti-HER-2/neu, anti-S100P, anti-thrombomodulin and anti-VEGFR-3. Majority of antibodies required more than one modification of the initial protocol. For anti-VEGFR-3 optimum staining was observed following 4 protocol modifications. CONCLUSIONS: This study has optimised and standardized several tissue-based biomarkers that may be, in the future, used to screen, diagnose and monitor patients with certain cancer, such as bladder cancer. Accurate data on optimised protocols reduce time and resources wasted on experimental protocols, and ultimately help identify biomarkers or biomarker panels, which may be used to select treatment regimens for various cancers.

The role of antibody expression and their association with bladder cancer recurrence: a single-centre prospective clinical-pilot study in 35 patients.

BACKGROUND: Bladder cancer (BC) is the 10th most common cancer in the UK, with about 10,000 new cases annually. About 75-85% of BC are non-muscle invasive (NMIBC), which is associated with high recurrence and progression rates (50-60% within 7-10 years). There are no routine biomarkers currently available for identifying BC patients at increased risk of developing recurrence. The focus of this research study was to evaluate antibody expression in BC patients and their association with cancer recurrence. METHODS: 35 patients scheduled for TURBT were recruited after written informed consent. Ethical approval for the project was granted via IRAS (REC4: 14/WA/0033). Following surgical procedure, tissues were preserved in 10% buffered formalin and processed within 24 h in FFPE blocks. 7 sections (4 µm each) were cut from each block and stained for CD31, Human epidermal growth factor receptor-2 (HER-2), S100P, Cyclooxygenase-2 (COX-2), VEGFR-3 thrombomodulin and CEACAM-1 using immunohistochemistry. Clinical outcome measures (obtained via cystoscopy) were monitored for up to 6 months following surgical procedure. RESULTS: There was significantly increased expression of CD31 (p < 0.001), HER-2 (p = 0.032), S100P (p < 0.001), COX-2 (p < 0.001), VEGFR-3 (p < 0.001) and decreased expression of thrombomodulin (p = 0.010) and CEACAM-1 (p < 0.001) in bladder tumours compared to normal bladder tissues. HER-2 expression was also significantly associated with cancer grade (p = 0.003), especially between grade 1 and grade 2 (p = 0.002) and between grade 1 and grade 3 (p = 0.004). There was also a significant association between cancer stage and HER-2 expression (p < 0.001). Although recurrence was significantly associated with cancer grade, there was no association with antibody expression. CONCLUSION: Findings from the present study may indicate an alternative approach in the monitoring and management of patients with BC. It is proposed that by allowing urological surgeons access to laboratory markers such as HER-2, Thrombomodulin and CD31 (biomarker profile), potentially, in the future, these biomarkers may be used in addition to, or in combination with, currently used scoring systems to predict cancer recurrence. However, verification and validation of these biomarkers are needed using larger cohorts.