Direct molecular evidence for both multicentric and monoclonal carcinogenesis followed by transdifferentiation from hepatocellular carcinoma to cholangiocarcinoma in a case of metachronous liver cancer.

Frequent recurrence is a major issue in liver cancer and histological heterogeneity frequently occurs in this cancer type. However, it has remained elusive whether such cancers are multicentric or monoclonal. To elucidate the clonal evolution of hepatocellular carcinoma (HCC) recurrence and combined hepatocellular-cholangiocarcinoma (cHCC-CCA) development, the somatic mutation frequency and signatures in a patient with triple occurrence of liver cancer every three years were examined, with samples designated as #1HCC, #2HCC and #3cHCC-CCA, respectively. A total of four tumor regions, including HCC (#3HCC) and intrahepatic CCA (#3iCCA) components of #3cHCC-CCA, and three nontumor regions (#1N, #2N and #3N) were precisely dissected from formalin-fixed paraffin-embedded tissues of each surgical specimen. DNA was extracted and subjected to tumor-specific somatic mutation determination. Of note, five nonsynonymous single-nucleotide variants (SNVs), namely those of KMT2D, TP53, DNMT3A, PKHD1 and TLR4, were identified in #3cHCC-CCA. All five SNVs were detected in both #3HCC and #3iCCA and #2HCC but not in #1HCC. The telomerase reverse transcriptase (TERT) promoter mutation C228T, but not C250T, was observed in all tumors. Digital PCR of C228T also indicated the presence of the TERT promoter mutation C228T in nontumorous liver tissues (#1N, #2N and #3N) at a frequency of 0.11-0.83% compared with normal liver and blood samples. These results suggest the following phylogenetic evolution of three metachronous liver cancers: #1HCC was not related to #2HCC, #3HCC and #3iCCA; both #3HCC and #3iCCA arose from #2HCC. From the above, three novel findings were deduced: i) Both multicentric occurrence and intrahepatic metastasis may be involved in liver cancer in a three-year interval; ii) transdifferentiation from HCC to iCCA is a possible pathogenic mechanism of cHCC-CCA; and iii) a nontumorous, noncirrhotic liver may contain a preneoplastic region with a cancer driver mutation in the TERT promoter.

Identification of a novel frameshift c.930delG MEN1 germline mutation (p.Glu273LysfsTer7) in a sporadic case of multiple endocrine neoplasia type 1: A case report.

Multiple endocrine neoplasia type 1 (MEN1) is a rare genetic disorder that is inherited in an autosomal dominant manner. The characteristics of the disease are the combined occurrence of tumors in glands of the endocrine system, such as the parathyroid glands, pituitary gland and endocrine pancreas. Germline mutations in the MEN1 gene are associated with the occurrence of MEN1 and genetic testing for this gene is generally used as a basis for diagnosis. In this paper, a case of MEN1 in a middle­aged Japanese woman is reported. Direct sequencing analysis of the patient's DNA was performed and it revealed a MEN1 gene heterozygous germline (NM_130799.2:c.930delG) mutation in exon 5. This deletion/frameshift mutation produced a stop codon in the downstream sequence (NP_570711.1:p.Glu273LysfsTer7). To the best of our knowledge, this is the first report describing the NM_130799.2:c.930delG mutation as the basis for MEN1.

Correction: Importance of gastric cancer for the diagnosis and surveillance of Japanese Lynch syndrome patients.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Identification of a Novel ACVRL1 Gene Mutation (c.100T>A, p.Cys34Ser) in a Japanese Patient with Possible Hereditary Hemorrhagic Telangiectasia (Osler-Weber-Rendu Disease).

Hereditary hemorrhagic telangiectasia (HHT; also known as Osler-Weber-Rendu disease) is an autosomal dominant genetic disorder that causes frequent epistaxis, mucocutaneous telangiectasia, and visceral arteriovenous malformations. Four genes (ENG, ACVRL1, SMAD4, and GDF2) have been identified as pathogenic in HHT. We describe the case of a 50-year-old Japanese man highly suspected of having HHT due to recurrent epistaxis, mucocutaneous telangiectasia, and a family history. Genomic analysis revealed a novel missense mutation of c.100T>A, p.Cys34Ser in the patient's ACVRL1 gene. We used 6 freeware programs to perform an in silico analysis of this mutation. The results demonstrated the mutation's high pathogenicity.

Importance of gastric cancer for the diagnosis and surveillance of Japanese Lynch syndrome patients.

Lynch syndrome (LS) is an autosomal dominantly inherited disease predisposed to not only colorectal cancer but also other LS-related tumors. Although the clinical and genetic characteristics of LS in Western countries have been well characterized, the information of Japanese LS is limited. As a collaborative study of Japanese Society for Cancer of the Colon and Rectum (JSCCR), we registered colorectal cancer (CRC) patients who fulfilled the modified Amsterdam II criteria including gastric cancer as an LS-related tumor. Among 4030 CRC patients initially registered in this project, 85 patients (2.1%) fulfilled the modified criteria. An additional 26 patients who met the same criteria were enrolled in the analysis. We analyzed three major responsible genes, MLH1, MSH2, and MSH6 by direct sequencing, and further performed multiplex ligation-dependent probe amplification for MLH1 and MSH2. Consequently, we identified pathogenic variants in 64 of the 111 patients comprising of 34 patients in MLH1, 28 in MSH2, and 2 in MSH6. It is of note that large structural alterations were found in 17 patients. Among the 64 patients, 11 patients would not have been enrolled in the analysis if gastric cancer were not included in the modified criteria. In addition, 10 of the 64 variant carriers (15.6%) had medical history of gastric cancer. Furthermore, the standardized incidence ratio of gastric cancer in the LS patients to the Japanese population is estimated to be as high as 20.2. These data underscore the importance of gastric cancer in the diagnosis and healthcare of Japanese LS patients.

A novel fusion gene involving PDGFRB and GCC2 in a chronic eosinophilic leukemia patient harboring t(2;5)(q37;q31).

BACKGROUND: Platelet-derived growth factor receptor beta (PDGFRB) rearrangement has been reported in a number of patients with chronic eosinophilic leukemia (CEL), B-acute lymphoblastic leukemia, myeloproliferative neoplasms, and juvenile myelomonocytic leukemia. Here, we report a case of CEL carrying a novel fusion gene involving PDGFRB and GRIP and coiled-coil domain containing 2 (GCC2). PATIENT AND METHODS: A 54-year-old man presenting with a cough and dyspnea was diagnosed with acute eosinophilic pneumonia. Cytogenetic analysis of the bone marrow revealed the presence of t(2;5)(q37;q31). Fluorescence in situ hybridization analysis in the peripheral blood leukocytes revealed the presence of a split signal at PDGFRB gene. Imatinib treatment was effective, and disappearance of t(2;5)(q37;q31) in the bone marrow was confirmed after three months of imatinib therapy. Whole-genome sequencing was performed in peripheral blood leukocytes collected before imatinib therapy. RESULTS: A novel fusion gene between exon 22 of GCC2 and exon 12 of PDGFRB was detected and the presence of GCC2-PDGFRB was confirmed by PCR. CONCLUSION: This is the first case report demonstrating the GCC2 gene as a partner of PDGFRB in the pathogenesis of CEL.

Late-onset Cerebrotendinous Xanthomatosis with a Novel Mutation in the CYP27A1 Gene.

Cerebrotendinous xanthomatosis (CTX) is a rare, autosomal recessive, inborn disruption in bile acid synthesis characterized by severe systemic xanthomas, cataracts and neurological injuries occurring before adolescence without elevation of the serum cholesterol or triglyceride levels. CTX is caused by a deficiency of the mitochondrial enzyme sterol 27-hydroxylase, which is encoded by the CYP27A1 gene. We herein report a 50-year-old Japanese woman with late-onset CTX who had no relevant symptoms before the development of bilateral Achilles tendon xanthomas in middle age. A genetic analysis revealed a compound heterozygous mutation in the CYP27A1 gene with a previously known missense mutation (NM_000784.3:c.1421 G>A) and a novel frame shift mutation of NM_000784.3:c.1342_1343insCACC.

Progress and issues of the genome-wide association study for hypertension.

Over the past few years, use of the genome-wide association study (GWAS) has made it possible to identify the primary genetic mechanisms of essential hypertension. GWAS results have helped identify many loci in or near genes that generally were not expected to be associated with blood pressure or essential hypertension. However, considering the great expectations of improving clinical outcomes and the billions of dollars that have been spent on various GWASs, the progress made so far has been slow. There are several factors that could be responsible for the relative lack of success of GWASs. First, it is possible that the number of people enrolled in the various GWASs was not enough, thereby limiting the power to detect additional markers. Second, although the alleles that are associated with a modest increase in risk are constantly being found, their discriminatory ability and use as predictive markers has been quite low. Difficulties with control group selection along with unrepeatability have also been problematic when using GWASs. The current paper summarizes the recent progress attained when using a GWAS of hypertension to identify the many loci associated with essential hypertension. In this review, we discuss the progress and issues of a GWAS for hypertension.

Determination of splice-site mutations in Lynch syndrome (hereditary non-polyposis colorectal cancer) patients using functional splicing assay.

Lynch syndrome (hereditary non-polyposis colorectal cancer) is an inherited disease caused by germ-line mutation in mismatch repair genes such as MLH1, MSH2, and MSH6. The mutations include missense and nonsense mutations, small insertions and deletions, and gross genetic alterations including large deletions and duplications. In addition to these genetic changes, mutations in introns are also involved in the pathogenesis. However, it is sometimes difficult to interpret correctly the pathogenicity of variants in exons as well as introns. To evaluate the effect of splice-site mutations in two Lynch syndrome patients, we carried out a functional splicing assay using minigenes. Consequently, this assay showed that the mutation of c.1731+5G>A in MLH1 led to exon15 skipping, and that the mutation of c.211+1G>C in MSH2 created an activated cryptic splice-site 17-nucleotides upstream in exon1. These aberrant splicing patterns were not observed when wild type sequence was used for the assay. We also obtained concordant results by RT-PCR experiments with transcripts from the patients. Furthermore, additional functional splicing assays using two different intronic mutations described in earlier studies revealed splicing alterations that were in complete agreement with the reports. Therefore, functional splicing assay is helpful for evaluating the effects of genetic variants on splicing.

Validation of the revised 2008 WHO diagnostic criteria in 75 suspected cases of myeloproliferative neoplasm.

The objective of this study was to validate the recently revised 2008 WHO diagnostic criteria of myeloproliferative neoplasms (MPN) together with the analysis of correlation of JAK2 (Janus kinase 2)-V617F mutant allele burden with clinical/laboratory findings on each patient. We made a diagnosis of 75 suspected MPN patients based on both diagnostic criteria of the 2001 WHO classification and the revised 2008 WHO classification, and found that both criteria show a quite similar diagnostic power except for two patients (idiopathic erythrocytosis (IE) and thrombocytosis) who were diagnosed as essential thrombocythemia by the 2008 WHO criteria. From JAK2-V617F analysis, hemoglobin and hematocrit values were significantly higher and platelet count was lower in JAK2-V617F high allele burden group than JAK2-V617F middle allele burden group. Mutant allele burden of polycythemia vera (PV) group was higher than that of essential thrombocythemia group. Therefore, the amount of mutant allele seemed to define the disease phenotypes. We further found a PV case presenting a rare type of JAK2-exon12 mutation. In contrast, IE presented a good prognosis unlike MPN. Hereafter, the 2008 WHO criteria with JAK2 gene analysis are useful for precise diagnosis of MPN and the patients with erythrocytosis.

Effects of peripheral cannabinoid receptor ligands on motility and polarization in neutrophil-like HL60 cells and human neutrophils.

The possible role of the peripheral cannabinoid receptor (CB2) in neutrophil migration was investigated by using human promyelocytic HL60 cells differentiated into neutrophil-like cells and human neutrophils isolated from whole blood. Cell surface expression of CB2 on HL60 cells, on neutrophil-like HL60 cells, and on human neutrophils was confirmed by flow cytometry. Upon stimulation with either of the CB2 ligands JWH015 and 2-arachidonoylglycerol (2-AG), neutrophil-like HL60 cells rapidly extended and retracted one or more pseudopods containing F-actin in different directions instead of developing front/rear polarity typically exhibited by migrating leukocytes. Activity of the Rho-GTPase RhoA decreased in response to CB2 stimulation, whereas Rac1, Rac2, and Cdc42 activity increased. Moreover, treatment of cells with RhoA-dependent protein kinase (p160-ROCK) inhibitor Y27632 yielded cytoskeletal organization similar to that of CB2-stimulated cells. In human neutrophils, neither JWH015 nor 2-AG induced motility or morphologic alterations. However, pretreatment of neutrophils with these ligands disrupted N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced front/rear polarization and migration and also substantially suppressed fMLP-induced RhoA activity. These results suggest that CB2 might play a role in regulating excessive inflammatory response by controlling RhoA activation, thereby suppressing neutrophil migration.