RAS mutation is an established predictive biomarker of resistance to anti-epidermal growth factor receptor(EGFR)therapy in metastatic colorectal cancer. In addition, previous studies identified mutations in ERBB2, FGFR1, PDGFRA, BRAF, MAP2K1, PTEN, and PIK3CA as potential mechanisms of resistance to anti-EGFR therapy. Testing for these mutations might be necessary to determine eligibility for anti-EGFR therapy in patients with metastatic colorectal cancer. CancerPlex®is a nextgeneration sequencer for 413 cancer genes. An analysis panel includes genes that may be associated with resistance to anti- EGFR therapy. A 65-year-old man with unresectable rectal cancer, multiple lung metastases, and a bulky liver metastasis was evaluated for expression of genes associated with resistance to anti-EGFR. The analysis found that all genes indicating resistance were wild-type genes. Cetuximab monotherapy was administered after rectal resection, with dramatic shrinkage of the metastatic tumors. A more accurate selection of patients according to tumor genetic status using CancerPlex®might improve the risk-benefit profile of anti-EGFR therapy.

Mismatch repair(MMR)protein deficiency in colorectal cancer is well correlated with high-level microsatellite instability (MSI-H). There are little data on mismatch repair deficiency(dMMR)colorectal cancers in Japan. In addition, we have no available data on the therapeutic efficacy of oxaliplatin(oxa)-based chemotherapy, one of the standard treatment regimens for metastatic colorectal cancer, for patients with dMMR colorectal cancer. The subjects were 254 patients with Stage IV colorectal cancer whose tumors were immunohistochemically stained for MMR proteins, MLH1, MSH2, MSH6, and PMS2. Patients who underwent R0 resection were excluded. Clinicopathologic factors and the efficacy of oxa-based chemotherapy were compared between patients with dMMR colorectal cancer and those with mismatch repair proficient(pMMR)colorectal cancer. There were 7(2.8%)patients with dMMR. Four patients demonstrated both MLH1 and PMS2 loss, while 3 patients demonstrated both MSH2 and MSH6 loss. Though the dMMR had a higher frequency in female patients(p=0.02) and a lower frequency in those with liver metastasis(p<0.01), the other clinicopathologic factors evaluated did not significantly differ between the dMMR group and the pMMR group. One hundred and fifty patients with unresectable disease or R1/2 resection received first-line oxa-based chemotherapy. The median overall survival was 23.2 months and 16.2 months in patients with dMMR(n=4)and those with pMMR, respectively(n=146)(p=0.33). The frequency of dMMR amongStag e IV colorectal cancers was lower than those(4-11%)reported in Western countries. Therefore, the clinical significance of universal screeningfor dMMR in all colorectal cancer samples may not be valid. Concerningsurvival benefit, oxa-based chemotherapy seems to be an effective alternative in clinical practice for metastatic colorectal cancer patients with dMMR.

BRAF V600E mutation plays an important role in the serrated neoplasia pathway of colorectal tumorigenesis and is a negative predictive factor for chemotherapy response as well as a prognostic factor in patients with colorectal cancer. To evaluate BRAF V600E mutations, a conventional polymerase chain reaction(PCR)is performed but recently immunohistochemistry (IHC)with a BRAF antibody has been used. Although similarities between the PCR and IHC methods have been reported, some investigators have doubts about the usefulness of IHC for BRAF mutation analysis. The subjects were 38 colorectal cancer patients with tumors demonstrating loss of both MLH1 and PMS2, and high-level microsatellite instability. Of the original 39 patients, 1 was excluded due to Lynch syndrome, which was identified using germline mutation testing. The mutation rate of BRAF V600E was 57.9% using both methods, but the concordance rate was 68.4%, with a kappa-value of 0.33. We should consider the usefulness of the IHC method in the evaluation of BRAF mutations in colorectal cancer patients.

Information on the cancer genome has accumulated rapidly, since approximately 2009, with the use of second-generation sequencing technology. Malignant lymphoma is no exception. In mature B-cell lymphomas, which constitute the vast majority of non-Hodgkin lymphomas, frequent mutations are identified in genes involved in signaling pathways, histone-modifying molecules, the DNA damage-response pathway, etc. The signaling pathways in which multiple genes are mutated include immune cell-specific pathways such as the B-cell receptor, the Toll-like receptor, and their downstream NF-κB signaling pathways, as well as the NOTCH signaling pathway. In mature T/NK-cell lymphomas, mutations accumulate in genes involved in the T-cell receptor pathway and its downstream NF-κB signaling pathway, regulators of DNA methylation, the JAK-STAT pathway, etc. Many of these mutations are found in multiple types of lymphomas but the frequencies of each gene mutation differ among diseases, demonstrating characteristic profiles. This cumulative and growing fund of knowledge provides an important basis for the development of new molecular-targeted drugs.

Acute myeloid leukemia (AML) is a heterogeneous disease, the onset of which involves a variety of chromosomal abnormalities and gene mutations. In recent years, the use of next-generation sequencers has facilitated intensive exploration of gene mutations resulting in the discovery of many AML-related gene mutations, and in clarifying the clonal evolution process of relapse. Epigenetic regulatory gene mutations have occurred in pre-leukemic cells with normal differentiation potential, and the acquisition of numerous genetic mutations was found to be strongly associated with AML onset, with further co-mutations contributing to clonal diversity and leading to the generation of treatment-resistant clones. As a result of these fruitful findings, the gene mutations of AML are becoming useful as not only prognostic factors but also the targets of molecular medicines such as FLT3 and IDH inhibitors. Most notably, several guidelines have proposed a prognostic classification that groups FLT3ITD, NPM1 mutation, and CEBPA mutation together under conventional chromosomal aberrations. This review outlines recent findings pertaining to the gene mutations in AML.

This review outlines recent advances in the understanding of gene alterations and the genetic background associated with myeloproliferative neoplasms (MPNs), as well as describing the roles of these genetic factors in the development of MPNs. JAK2, CALR, and MPL mutations that are specifically found in patients with MPNs have been shown to constitutively activate cytokine receptors. Other mutations that are commonly found in hematopoietic malignancies have been demonstrated to synergize with disease-specific mutations and to accelerate the development of MPN, or to define the disease subtype. However, some of these mutations are found in healthy elderly persons, such that the mechanism of MPN development remains elusive. Further analyses including those for genetic factors associated with the occurrence of MPN will lead to a complete understanding of MPN development.

The advent and long-term use of tyrosine kinase inhibitors in molecular target therapy for chronic myeloid leukemia have resulted in a marked improvement of treatment outcomes. This has changed'the algorithm of the laboratory process for the diagnosis and therapeutic monitoring of chronic myeloid leukemia. It includes defining the molecular typing of BCR-ABL1 to establish the diagnosis, and a quantitative and/or high- sensitivity assay for minimal residual disease to evaluate the treatment response. Along with improved long-term survival outcomes, new issues have arisen regarding the best index to use for the improved clinical outcomes, such as treatment-free remission. To this end, clinical and laboratory monitoring of CML patients has been investigated. Novel methodologies and technologies have been applied to improve decision-making on patient care, tailoring the treatment to the individual characteristics of each patient, including an early index for the treatment response and deeper molecular response to realize treatment-free survival, and BCR-ABLI kinase domain mutation screening for refractory disease. In response to the advancement and applications of these emerging technologies, proper laboratory practice with the quality assurance of testing is expected. [Review].

FMS-like tyrosine kinase 3 (FLT3), a class III tyrosine kinase receptor, plays an important role in the pro- liferation, survival, and differentiation of hematopoietic stem/progenitor cells. Approximately 30% of pa- tients with cytogenetically normal acute myeloid leukemia (CN-AML) harbor FLT3 mutations. The most frequent FLT3 mutations are internal tandem duplications (ITDs) in the juxtamembrane domain. FLT3-ITD mutations cause ligand-independent dimerization of FLT3 and the constitutive activation of its downstream signaling pathways, such as PI3K/AKT, A4APK/ERK, and STAT5, leading to dysregulated cellular prolifera- tion. The relapse risk of CN-AML patients with FLT3-ITD is higher and the overall survival (OS) of such patients is shorter than those of patients with wild-type FLT3. Recently genome-wide studies with next-generation sequencing have suggested that mutational combinations of genes related to signal transduction, transcription, splicing, cancer suppressors, and epigenetics contribute to the pathogenesis of AML. These mutations including FLT3-ITD may be prognostic factors facilitating the risk stratification for CN-AML. The point mutations D835/I836 in the tyrosine kinase domain (TKD) are detected in 5-7% of AML patients. The clinical relevance of these FLT3-TKD mutations remains unclear. FLT3-TKD mutations are detected even in patients treated with FLT3 inhibitors as secondary mutations, suggesting that the mutations are as- sociated with the resistance. Therefore, the detection of these mutations might provide us with the opportunity to consider appropriate treatment for patients. The molecular abnormalities in AML patients give us insights into the pathology of AML and clinically significant information required for the diagnosis, prognosis, and treatment decisions. [Review].

The majority of patients with acute promyelocytic leukemia (APL) harbor the t (15;17) (q22;q12) transloca- tion, which results in the expression of PML-RARA mRNA. All-trans retinoic acid (ATRA) is a representa- tive molecular-targeted drug and is directed against PML-RARA. Therefore, the detection of PML-RARA mRNA has become indispensable for the diagnosis of APL and the decision regarding the treatment policy. Once the diagnosis is confirmed by genetic testing, ATRA-based induction therapy can be initiated. This is also applicable in atypical cases such as the M3 variant. Furthermore, after ATRA-based induction therapy, the curative effect is evaluated by quantitative PCR analysis. Thus, genetic testing is important in the follow-up of patients with APL. [Review].

A 67-year-old woman was detected stage III B adenocarcinoma of the left lung, with epidermal growth factor receptor(EGFR) -mutation. Since chest computed tomography showed enlarged lymph nodes in stations 7 and 1R-4R, radiochemotherapy was recommended. However, she declined radiochemotherapy and the chemotherapy after tumor reduction surgery was chosen. She underwent resection of the left lower lobe and lingular segment, and mediastinal lymph node dissection through a posterolateral thoracotomy by extended bronchoplasty(type C) due to the extranodal invasion of lymph nodes to the lingular segmental bronchus. At postoperative day 21, she underwent right superior mediastinal lymph node dissection through a median sternotomy. Postoperatively, she received 6 cycles of chemotherapy with carboplatin plus paclitaxel followed by gefitinib. 1.5 years later, gamma knife surgery was done for multiple cerebral metastases, followed by 4 cycles of chemotherapy with cisplatin plus pemetrexed and then erlotinib. Six years after surgery, she is surviving without any evidence of disease recurrence. Tumor reduction surgery, when followed by chemotherapy, for EGFR-mutant lung adenocarcinoma is likely to lead to long-term survival.

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®.

Leukemia is derived from hematopoietic stem/progenitor cells that have acquired genetic abnormalities, leading to malignant transformation. The basis of therapyfor leukemia is a combination of anti-cancer drugs based on risk stratification. The overall 5-year survival rate in leukemia patients of all ages is still 40%, although it has improved in pediatric patients. Leuke- mia itself is a heterogeneous disease that includes various entities/subtypes with different pathogenic gene aberrations. Selection of the treatment strategylargelydepends on risk stratification, and this in turn is mainlybased on specific recurrent chromosome aberrations. However, in acute myeloid leukemia(AML), a significant proportion of patients present with a normal karyotype according to conventional cytogenetic analysis and are classified into an intermediate-risk group, which actuallyconsists of various subtypes with different prognoses. In addition, leukemic cells usuallyharbor one or more driver mutations among their various genetic aberrations, and these driver mutations could affect prognosis. The discoveryof additional mutations in genes such as NPM1, CEBPA and FLT3, which are frequent in AML patients with a normal karyotype, have improved the precision of risk stratification in AML. In this regard, array-based gene expression analysis and whole exome/ transcriptome sequencing could be useful tools for identifying the whole spectrum of genetic aberrations, or for compiling a complete list of mutated genes within leukemic cells. Genetic profiling information obtained using these newlydeveloped methods could provide more accurate information for molecular subtyping and risk stratification in leukemia.

Multigene assays that simultaneously measure the expression of various breast cancer genes have been developed to guide the use of adjuvant chemotherapy in early breast cancer. The efficacy of adjuvant therapies depends on the recurrence risk for an individual patient. As a result, accurate prediction of the recurrence risk is vital for precise adjuvant chemotherapy in individual breast cancer patients. The recurrence risk as typically assessed by conventional examination of histological data of immuno-histological biomarkers(ER, PR, HER2, and Ki-67)is not sufficient to select subsets of patients. Therefore, validated gene expression signatures are useful, in addition to well-established clinico-pathological factors. Available gene expression assay systems, such as MammaPrint®, Oncotype DX®, PAM50 ROR, GGI, EndoPredict®(EP), Breast Cancer IndexSM(BCI), and Curebest®95GC Breast, are recommended. While MammaPrint®and Oncotype DX®are the most predictive of the recurrence risk within the first 5 years of diagnosis, BCI and EPclin have demonstrated utility in predicting late recurrence. In addition, PAM50 provides further biological insights by classifying breast cancers into intrinsic molecular subtypes. These gene expression signatures have become important tools in clinical practice for the identification of low-risk endocrine-positive patients in whom chemotherapy could be avoided. However, there is much work left in the development of a molecular classification considering the biology and novel therapeutic targets in high-risk recurrent disease because chemotherapy has only modest benefits in this population. The recognition of genomic mutations and their relationship to a patient's responsiveness to various therapies will provide important breakthroughs toward precision medicine.

Pancreatic cancer has one of the highest rates of mortality among malignancies and the development of promising future therapies is strongly required. Recently, the utility of gene aberrations as biomarkers for determining therapeutic strategies has been demonstrated in several types of cancer. The detection of druggable mutations that aid in the selection of effective molecular targeting drugs is feasible in clinical settings for certain cancers. On the other hand, personalized therapy for pancreatic cancer guided by genomic biomarkers has not yet been realized and suitable molecular targets for the disease have been unclear until now. KRAS, CDKN2A, TP53, and SMAD4 have been recognized as major driver genes in pancreatic carcinogenesis. However, it is considered difficult to develop treatment strategies to target genetic aberrations of these four genes. In recent years, genome sequencing has progressively revealed the molecular biological characteristics of pancreatic cancer, including the discovery of novel potential therapeutic targets and low-frequency druggable genetic aberrations. Gene profilebased novel treatment strategies and subsequent attempts to realize precision medicine for pancreatic cancer are steadily ongoing in an effort to achieve improved treatment outcomes.

Personalized lung cancer therapy has progressed by targeting several oncogenic aberrations that drive lung carcinogenesis. Recent advances in gene analysis technologies, including next-generation sequencing that yields large amounts of genomic data, have greatly contributed to this progress. In addition, immune checkpoint blockade therapy has become available in Japan, and extensive searches for biomarkers predictive of therapeutic response have been carried out. "Clinical sequencing" which analyzes aberrations in a set of therapy-related genes in patient cancer specimens, has been actively conducted in Japan and other countries. This will help to establish more efficient and effective precision cancer medicine based on gene information. Herein, we summarize the recent progress in personalized lung cancer therapy research, including clinical sequencing.

Genome-wide analyses have recently been reported for ovarian cancer. High-grade serous ovarian carcinoma(HGSOC) almost exclusively harbor TP53 mutations and prominent copy number aberrations. Approximately 20% of HGSOCs harbor BRCA mutations, in which case PARP inhibitors may be effective. HGSOCs are classified into 4 molecular subtypes with distinct histopathological features by transcriptional profiling. These subtypes differ in prognosis and drug sensitivity. Additionally, a whole-genome analysis for HGSOC has revealed various factors that can induce resistance to chemotherapy. On the other hand, ovarian clear cell carcinoma(OCCC), a chemoresistant subtype, develops through oxidative stress conditions in an endometriotic cyst. OCCC specific genes include HNF1B and its downstream genes and genes related to oxidative stress. HNF1B mediates resistance to oxidative stress and platinum in OCCC cells. In the future, development of new therapeutic strategies based on these OCCC specific features is expected.