PURPOSE To identify novel genetic markers predictive of clinical benefit from epidermal growth factor receptor-directed antibody therapy in patients with metastatic colorectal cancer (mCRC). PATIENTS AND METHODS Seventy-six consecutive patients who received cetuximab or panitumumab, either alone or in combination with chemotherapy, with available tumor tissue were included. Tumor tissue was tested for mutations at known hotspots in the KRAS, BRAF, PIK3CA, PIK3R1, AKT1, and PTEN genes by pyrosequencing. PTEN promoter methylation status was analyzed by methylation-specific PCR, and expression determined by immunohistochemistry (IHC). Forty-four patients had ≥ 4 weeks of therapy and were considered for clinical correlates. RESULTS Consistent with previous studies, KRAS gene mutations were associated with a shorter progression free (PFS) and overall survival (OS). Among the KRAS wild type patients, preservation of PTEN expression and PIK3CA WT status was associated with improved OS (median OS, 80.4 vs 32.5 weeks, HR: 0.33, p=0.0008) and a trend towards improved PFS (median PFS, 24.8 vs 15.2 weeks, HR: 0.51, p=0.06), compared to PTEN negative or PIK3CA mutant tumors. PTEN methylation was more common in the metastases than the primary (p=0.02). Simultaneous presence of methylation and mutation in the PTEN gene was associated with IHC negativity (p=0.026). CONCLUSION In addition to KRAS mutation, loss of PTEN expression (by IHC) and PIK3CA mutation is likely to be predictive of lack of benefit to anti-EGFR therapy in mCRC. PTEN promoter methylation and mutation status was predictive of PTEN expression, and may be utilized as an alternative means of predicting response to EGFR-targeted therapy.

The regenerative potential of mesenchymal stromal cells (MSC) holds great promise in using them for treatment of a wide range of debilitating diseases. Several types of culture media and systems have been used for large-scale expansion of MSCs in vitro; however, the majority of them rely heavily on using foetal bovine serum (FBS)-supplement for optimal cell proliferation. FBS-based cultures pose the potential threat of spread of transmissible spongiform encephalopathy and bovine spongiform encephalopathy to MSCs and then to their recipients. A recent trend in cell culture is to change from serum-use to serum-free media (SFM). In this context, the current review focuses specifically on employment of various SFM for MSCs and discusses existences of various options with which to substitute FBS. In addition, we analyse MSC population growth kinetic patterns using various SFM for large-scale production of MSCs.

Critical limb ischemia (CLI) due to Buerger’s disease is a major unmet medical need with a high incidence of morbidity. This phase II, prospective, nonrandomized, open‐label, multicentric, dose‐ranging study was conducted to assess the efficacy and safety of i.m. injection of adult human bone marrow‐derived, cultured, pooled, allogeneic mesenchymal stromal cells (BMMSC) in CLI due to Buerger’s disease. Patients were allocated to three groups: 1 and 2 million cells/kg body weight (36 patients each) and standard of care (SOC) (18 patients). BMMSCs were administered as 40–60 injections in the calf muscle and locally, around the ulcer. Most patients were young (age range, 38–42 years) and ex‐smokers, and all patients had at least one ulcer. Both the primary endpoints—reduction in rest pain (0.3 units per month [SE, 0.13]) and healing of ulcers (11% decrease in size per month [SE, 0.05])—were significantly better in the group receiving 2 million cells/kg body weight than in the SOC arm. Improvement in secondary endpoints, such as ankle brachial pressure index (0.03 [SE, 0.01] unit increase per month) and total walking distance (1.03 [SE, 0.02] times higher per month), were also significant in the group receiving 2 million cells/kg as compared with the SOC arm. Adverse events reported were remotely related or unrelated to BMMSCs. In conclusion, i.m. administration of BMMSC at a dose of 2 million cells/kg showed clinical benefit and may be the best regimen in patients with CLI due to Buerger’s disease. However, further randomized controlled trials are required to confirm the most appropriate dose. Stem Cells Translational Medicine 2017;6:689–699

Reovirus is a double stranded RNA virus, with an intrinsic preference for replication in KRAS mutant cells. As 45% of human colorectal cancers (CRC) harbor KRAS mutations, we sought to investigate its efficacy in KRAS mutant CRC cells, and examine its impact in combination with the topoisimerase-1 inhibitor, irinotecan. Reovirus efficacy was examined in the KRAS mutant HCT116, and the isogenic KRAS WT Hke3 cell line, and in the non-malignant rat intestinal epithelial cell line. Apoptosis was determined by flow cytometry and TUNEL staining. Combination treatment with reovirus and irintoecan was investigated in 15 CRC cell lines, including the HCT116 p21 isogenic cell lines. Reovirus preferentially induced apoptosis in KRAS mutant HCT116 cells compared to its isogenic KRAS WT derivative, and in KRAS mutant IEC cells. Reovirus showed a greater degree of caspase 3 activation with PARP 1 cleavage, and preferential inhibition of p21 protein expression in KRAS mutant cells. Reovirus synergistically induced growth inhibition when combined with irinotecan. This synergy was lost upon p21 gene knock out. Reovirus preferentially induces apoptosis in KRAS mutant colon cancer cells. Reovirus and irinotecan combination therapy is synergistic, p21 mediated, and represents a novel potential treatment for patients with CRC.