[New Classification for Advanced Colorectal Cancer Using CancerPlex®Genomic Tests].

Recently, targeted drugs have been developed for the treatment of colorectal cancer(CRC). Among targets, it is well known that KRAS mutations are associated with resistance to epidermal growth factor receptor(EGFR)monoclonal antibodies. However, response rates using anti-EGFR monotherapy for CRC were less than 20-30% in previous clinical studies. Thus, because the RAS/MAP2K/MAPK and PI3K/AKT pathways are associated with CRC resistance to chemotherapy, we analyzed gene mutations in Stage IV CRC patients using a genomic test(CancerPlex®). Medical records were reviewed for 112 patients who received treatment for CRC between 2007 and 2015 in Niigata University Medical and Dental Hospital or Niigata Cancer Center Hospital. There were 66 male and 46 female patients, and their median age was 62.5(range, 30-86) years. Cluster analyses were performed in 110 non-hypermutated Japanese CRC patients using Euclidean distance and Ward's clustering method, and 6 typical groups were identified. Among these, patients with all wild-type actionable genes benefited from anti-EGFR therapies. The expense of targeted drugs warrants consideration of cost-effectiveness during treatment decision-making for advanced CRC patients. To this end, based on the genetic information on CRC, it is possible to develop precision medicine using CancerPlex®.

Trastuzumab-induced CCL20 and interleukin-8 mRNA in human whole blood ex vivo.

Heparinized human whole blood from 16 adult volunteers was stimulated with achievable blood concentrations of trastuzumab and rituximab at 37 degrees C for 4 h, then CCL20, IL8, and beta-actin mRNA were quantified. The fold increase of beta-actin was all less than 1.5, and heat aggregated IgG induced both IL8 and CCL20 mRNA in all cases, suggesting that the assay was performed appropriately. Rituximab reduced the levels of CCL20 mRNA in approximately 1/3 of subjects, whereas 50 µg/ml trastuzumab induced IL8 and CCL20 mRNA in more than half of subjects. Although the results do not directly indicate the toxicity of antibody medicines, the individual variation found under physiological ex vivo condition will be an interesting clinical research model for drug safety analysis.

Utility of comprehensive genomic sequencing for detecting HER2-positive colorectal cancer.

HER2-targeted therapy is considered effective for KRAS codon 12/13 wild-type, HER2-positive metastatic colorectal cancer (CRC). In general, HER2 status is determined by the use of immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Comprehensive genomic sequencing (CGS) enables the detection of gene mutations and copy number alterations including KRAS mutation and HER2 amplification; however, little is known about the utility of CGS for detecting HER2-positive CRC. To assess its utility, we retrospectively investigated 201 patients with stage I-IV CRC. The HER2 status of the primary site was assessed using IHC and FISH, and HER2 amplification of the primary site was also assessed using CGS, and the findings of these approaches were compared in each patient. CGS successfully detected alterations in 415 genes including KRAS codon 12/13 mutation and HER2 amplification. Fifty-nine (29%) patients had a KRAS codon 12/13 mutation. Ten (5%) patients were diagnosed as HER2 positive because of HER2 IHC 3+, and the same 10 (5%) patients had HER2 amplification evaluated using CGS. The results of HER2 status and HER2 amplification were completely identical in all 201 patients (P < .001). Nine of the 10 HER2-positive patients were KRAS 12/13 wild-type and were considered possible candidates for HER2-targeted therapy. CGS has the same utility as IHC and FISH for detecting HER2-positive patients who are candidates for HER2-targeted therapy, and facilitates precision medicine and tailor-made treatment.

Genomic landscape of colorectal cancer in Japan: clinical implications of comprehensive genomic sequencing for precision medicine.

BACKGROUND: Comprehensive genomic sequencing (CGS) has the potential to revolutionize precision medicine for cancer patients across the globe. However, to date large-scale genomic sequencing of cancer patients has been limited to Western populations. In order to understand possible ethnic and geographic differences and to explore the broader application of CGS to other populations, we sequenced a panel of 415 important cancer genes to characterize clinically actionable genomic driver events in 201 Japanese patients with colorectal cancer (CRC). METHODS: Using next-generation sequencing methods, we examined all exons of 415 known cancer genes in Japanese CRC patients (n = 201) and evaluated for concordance among independent data obtained from US patients with CRC (n = 108) and from The Cancer Genome Atlas-CRC whole exome sequencing (WES) database (n = 224). Mutation data from non-hypermutated Japanese CRC patients were extracted and clustered by gene mutation patterns. Two different sets of genes from the 415-gene panel were used for clustering: 61 genes with frequent alteration in CRC and 26 genes that are clinically actionable in CRC. RESULTS: The 415-gene panel is able to identify all of the critical mutations in tumor samples as well as WES, including identifying hypermutated tumors. Although the overall mutation spectrum of the Japanese patients is similar to that of the Western population, we found significant differences in the frequencies of mutations in ERBB2 and BRAF. We show that the 415-gene panel identifies a number of clinically actionable mutations in KRAS, NRAS, and BRAF that are not detected by hot-spot testing. We also discovered that 26% of cases have mutations in genes involved in DNA double-strand break repair pathway. Unsupervised clustering revealed that a panel of 26 genes can be used to classify the patients into eight different categories, each of which can optimally be treated with a particular combination therapy. CONCLUSIONS: Use of a panel of 415 genes can reliably identify all of the critical mutations in CRC patients and this information of CGS can be used to determine the most optimal treatment for patients of all ethnicities.

Ex vivo simulation of the action of antileukemia drugs by measuring apoptosis-related mRNA in blood.

BACKGROUND: In conventional bioassays, isolated cells are suspended in culture media, incubated in vitro for several days, and then characterized with respect to any cellular changes. In developing new molecular tests under physiological ex vivo conditions, we quantified the production of mRNAs for p21 and PUMA (p53 up-regulated modulator of apoptosis), which are involved in cell cycle arrest and apoptosis, respectively. METHODS: We stimulated human whole blood with a chemotherapeutic drug (cytarabine, daunorubicin, mitoxantrone, aclarubicin, etoposide, or idarubicin) for 4 h and then quantified mRNA by assessing mRNA recovery and cDNA-synthesis efficiency in each sample. We also used immunoassay and flow cytometry to investigate nucleosome and annexin V, respectively, as apoptosis markers. RESULTS: Ex vivo mRNA analysis yielded more positive results than nucleosome and annexin V analyses. The concentrations of cytarabine- and daunorubicin-induced p21 and PUMA mRNAs were significantly lower in acute myelogenous leukemia (AML) patients than in healthy controls (P <0.0001), whereas idarubicin induced significantly greater responses in AML patients than in controls (P = 0.01). The patients had different mRNA-response patterns, which were largely classifiable into 4 groups. Prednisone enhanced cytarabine or mitoxantrone induction of p21 and PUMA mRNAs in 3 (2.6%) of 114 reactions. All 15 patients who achieved complete remission had received at least one drug that produced positive mRNA responses, whereas we observed a lack of mRNA response to the clinically used drugs in all 3 cases in which the therapy failed to induce any hematologic improvement. CONCLUSION: This study introduced ex vivo mRNA analysis as a candidate platform for drug-sensitivity tests in leukemia.

Ex vivo induction of mRNA in human whole blood as a new platform of drug and dietary supplement development.

PURPOSE: We introduced a new concept of ex vivo gene expression analysis (Mitsuhashi, Clin Chem 53:148-149, 2007), where drug action was simulated under physiological conditions. This model system was applied to study various fields of drug development. MATERIALS AND METHODS: Heparinized human whole blood was incubated with drugs for less than 4h. The changes of specific mRNA were then quantified using the method we developed (Mitsuhashi, Tomozawa, Endo, and Shinagawa, Clin Chem 52:634-642, 2006). RESULTS: The mRNA quantitation method was used as a model system to study the following areas: (1) identification of respondents and non-respondents, (2) ex vivo compound screening, (3) determination of individually optimized doses, (4) drug-to-drug comparison, (5) assessment of leukocyte toxicity, (6) discovery of molecular targets, (7) assessment of the action of dietary supplements, and (8) characterization of respondents and non-respondents for various dietary supplements. CONCLUSION: Since ex vivo assays are safe, a large number of healthy donors and disease patients can be recruited to identify individual-to-individual variations, which is not available from current preclinical study models. Although each system should be validated using a large number of samples, the ex vivo analysis will be a new tool for the development of drugs and dietary supplements in future.