Kouki Ohtsuka

Abnormalities of epidermal growth factor receptor in lung squamous-cell carcinomas, adenosquamous carcinomas, and large-cell carcinomas: tyrosine kinase domain mutations are not rare in tumors with an adenocarcinoma component.

Tyrosine kinase domain (TKD) gene mutations of the epidermal growth factor receptor gene (EGFR) have proven to be clinically significant in nonsmall‐cell lung cancer (NSCLC), particularly in adenocarcinoma. However, TKD mutations together with deletion mutations in the extracellular domain of EGFR (EGFRvIII) have not been fully investigated in NSCLC except for adenocarcinoma. The present study sought to gain further insight into the significance of EGFR mutations in NSCLC by focusing on nonadenocarcinoma NSCLC.

Clinico-Pathological and Biological Significance of Tyrosine Kinase Domain Gene Mutations and Overexpression of Epidermal Growth Factor Receptor for Lung Adenocarcinoma

Introduction: Mutations in the tyrosine kinase domain (TKD) of the epidermal growth factor receptor (EGFR) gene have proven to be clinically significant in non-small cell lung cancer. However, relationships between these mutations and EGFR expression or deletion mutations in the extracellular domain of EGFR (EGFRvIII) remain unclear. The purpose of this study was to gain further insight into the clinical significance of these molecular abnormalities in lung adenocarcinoma. Methods: We investigated EGFR TKD mutations using direct sequencing, EGFR protein expression using Western blotting, and EGFRvIII using reverse transcriptase–polymerase chain reaction in samples from 48 adenocarcinoma patients. Correlations with various clinico-pathological features were analyzed. Results: EGFR TKD mutations were detected in 25 of 48 adenocarcinomas (52.1%), and overexpression of EGFR protein was identified in 19 patients (39.6%). Presence of EGFR TKD mutations was significantly correlated with EGFR overexpression (p = 0.021). EGFR TKD mutations were significantly correlated with never-smoker status (p = 0.043), absence of emphysematous or fibrotic appearance on computed tomography (p = 0.001), papillary subtype (p = 0.041), and bronchioloalveolar carcinoma features (p = 0.045). EGFRvIII was not detected in any adenocarcinomas. Retrospective analysis revealed that patients with EGFR TKD mutations displayed better postoperative prognosis than patients with wild-type EGFR (p = 0.033). Conclusions: These results suggest that EGFR TKD mutation is associated with EGFR overexpression, representing an important factor for consideration when investigating the clinical significance, including susceptibility to chemotherapy, of EGFR TKD mutations in adenocarcinoma. EGFRvIII does not seem to play a major role in the development of lung adenocarcinoma.

A simple and sensitive method for detecting major mutations within the tyrosine kinase domain of the epidermal growth factor receptor gene in non-small-cell lung carcinoma.

The detection of mutations in the epidermal growth factor receptor (EGFR) gene impacts therapeutic decision-making for non–small-cell lung carcinoma (NSCLC). Although direct sequencing has been most frequently used to detect EGFR mutations, this method displays several disadvantages. We set up simple mutation-specific polymerase chain reaction (PCR) for common delE746-A750 and L858R mutations of EGFR using primers specific to these mutations. Both mutation-specific PCR and direct sequencing methods were used to investigate 62 samples of NSCLC, and the results were compared. To evaluate the sensitivity of mutation-specific PCR, DNA mixtures containing various proportions of mutant alleles were analyzed. Mutation-specific PCR revealed delE746-A750 in 8 samples and L858R in 14 samples. All samples with either delE746-A750 or L858R mutation revealed by direct sequencing also displayed positive results for mutation-specific PCR. Conversely, mutations in 3 samples revealed by L858R-specific PCR were barely detectable by direct sequencing. In DNA mixture analysis, DNA mixtures containing 2.5% of delE746-A750 allele or 0.25% of L858R allele yielded positive results with mutation-specific PCR. Our mutation-specific PCR showed satisfactory sensitivity and reliability for detecting major EGFR mutations in clinical NSCLC samples. Given the practical availability, this method could be widely applicable to the treatment of lung cancers.

Visceral fat thickness in overweight men correlates with alterations in serum fatty acid composition.

BACKGROUND We examined relationships between visceral fat amount and alterations in serum fatty acid composition, both of which represent critical factors in the development of metabolic syndrome. METHODS Correlations were analyzed between visceral fat thickness as measured by ultrasonography and proportions of individual fatty acids in 21 normal-weight and 24 overweight Japanese men. RESULTS Significant associations were identified in overweight subjects. Visceral fat thickness displayed positive correlations to levels of palmitic acid and saturated fatty acids (r=0.475, P<0.05 and r=0.545, P<0.01, respectively); and negative correlations to levels of linoleic acid and polyunsaturated fatty acids (r=-0.513, P<0.05 and r=-0.428, P<0.05, respectively). Visceral fat thickness was also correlated with estimated desaturase activities, with positive correlations to Delta9- and Delta6-desaturase activities and negative correlations to Delta5-desaturase activity (r=0.580, P<0.01, r=0.669, P<0.01 and r=-0.559, P<0.01, respectively). No significant associations were identified in normal-weight subjects. CONCLUSIONS Significant associations between visceral fat amount and alterations in serum fatty acid composition were identified, but only in overweight individuals.

Overexpression of MET is a new predictive marker for anti-EGFR therapy in metastatic colorectal cancer with wild-type KRAS.

AbstractPurposeSince the KRAS mutation is not responsible for all metastatic colorectal cancer (mCRC) patients with resistance to anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MoAb) therapy, new predictive and prognostic factors are actively being sought. MethodsWe retrospectively evaluated the efficacy of anti-EGFR MoAb-based therapies in 91 patients with mCRC according to KRAS, BRAF, and PIK3CA mutational status as well as PTEN and MET expression.ResultsIn the patient group with wild-type KRAS, the presence of BRAF mutation or PIK3CA mutations was associated with lower disease control rate (DCR), shorter progression-free survival (PFS), and shorter overall survival. Patients with MET overexpression also showed lower DCR and shorter PFS when compared with patients with normal MET expression. In a separate analysis, 44 patients harboring wild-type KRAS tumors were sorted into subgroups of 25 patients without abnormality in three molecules (BRAF, PIK3CA and MET) and 19 patients with abnormality in at least one of these three molecules. The former group showed significantly higher DCR and longer PFS following anti-EGFR therapy than the latter group.ConclusionsOur data point to the usefulness of MET overexpression, in addition to BRAF and PIK3CA mutations, as a new predictive marker for responsiveness to anti-EGFR MoAbs in mCRC patients with wild-type KRAS. This study also suggests that application of multiple biomarkers is more effective than the use of a single marker in selecting patients who might benefit from anti-EGFR therapy.

ATP-binding cassette transporters in primary central nervous system lymphoma: Decreased expression of MDR1 P-glycoprotein and breast cancer resistance protein in tumor capillary endothelial cells

Most tumors in the central nervous system are drug resistant partly because of the presence of the blood-brain barrier (BBB) between circulating blood and tumor tissues. Primary central nervous system lymphoma (PCNSL) is one of the exceptions, as it is highly sensitive to high-dose methotrexate (MTX)-based chemotherapy. The aim of this study was to evaluate the BBB function of tumor capillary endothelial cells in PCNSL. Expression of three major drug efflux transporters that belong to the ATP-binding cassette (ABC) superfamily, P-glycoprotein encoded by the human multidrug resistance gene (MDR1 Pgp; ABCB1), breast cancer resistance protein (BCRP; ABCG2), and multidrug resistance-associated protein 1 (MRP1; ABCC1), was evaluated. Immunohistochemistry was performed in capillary endothelial cells of 30 tumor areas from 22 PCNSL cases and compared with that of 30 gliomas. The microenvironment around tumor capillaries was assessed by examining the distribution of astrocytes and by counting the number of macrophages and T-cells, the principal cytokine producers. In PCNSL, expression of MDR1 Pgp and BCRP in tumor capillary endothelial cells was decreased in 63 and 93% of tumor areas examined, respectively, and these reduction levels differed significantly from those of gliomas (P<0.05). When the PCNSLs were further segregated by way of infiltration of tumor cells into three patterns (dense, perivascular and sparse), decreased MDR1 Pgp and BCRP in tumor capillary endothelial cells were much more prominent in dense and perivascular patterns. In all tumors and non-tumor areas of the brain, MRP1 was undetected on capillary endothelial cells. Assessment of the microenvironment around the tumor capillaries suggested that dissociation from astrocytes and infiltration of macrophages and T-cells was involved in the down-regulation of MDR1 Pgp and BCRP in capillary endothelial cells. In conclusion, we report that expression of the major ABC transporters of the BBB, MDR1 Pgp and BCRP, decreases in tumor capillary endothelial cells of PCNSL. Thus, decreased expression may permit delivery of chemotherapeutic agents to the tumor tissues.

Low concentrations of serum n-3 polyunsaturated fatty acids in non-alcoholic fatty liver disease patients with liver injury

Tomonori Kishino*, Hiroaki Ohnishi, Kouki Ohtsuka, Satsuki Matsushima, Tsuyoshi Urata, Keiko Watanebe, Yukihisa Honda, Yoshitake Mine, Motoko Matsumoto, Kaori Nishikawa, Hideaki Mori, Shin’ichi Takahashi, Hitoshi Ishida and Takashi Watanabe 1 Department of Laboratory Medicine, Kyorin University School of Medicine, Tokyo, Japan 2 Department of Clinical Laboratory, Kyorin University Hospital, Tokyo, Japan 3 The Third Department of Internal Medicine, Kyorin University School of Medicine, Tokyo, Japan

First isolation of Mycobacterium kyorinense from clinical specimens in Brazil.

ABSTRACT In this article, the first isolation of Mycobacterium kyorinense specimens in Brazil is described. M. kyorinense is a recently identified species, with a few strains reported only in Japan. The Brazilian isolates were initially identified as Mycobacterium celatum by PCR restriction enzyme pattern analysis (PRA) with hsp65. However, biochemical tests indicated the same profile of M. kyorinense and distinguished them from M. celatum and Mycobacterium branderi. The sequencing of the hsp65, rpoB, and 16S rRNA genes allowed the accurate identification of isolates as M. kyorinense.

Effect of Carryover of Clot Activators on Coagulation Tests During Phlebotomy

We investigated the effect of clot activators carried over from the serum tube on major coagulation tests during phlebotomy. First, blood specimens from 30 normal subjects were mixed with small amounts of fluid containing clot activators, and their effects on various coagulation tests were determined. Only the value of fibrin monomer complex displayed a remarkable change when thrombin-containing fluid was added to the blood specimens. Subsequently, 100 paired blood specimens (taken from 75 healthy volunteers and 25 patients taking warfarin) were collected in coagulation tubes before and after the serum tube using standard phlebotomy procedures. Various coagulation tests were performed to determine the effect of contamination of thrombin-containing blood on coagulation parameters. Differences between the 2 tubes were minimal but significant for some of the coagulation tests. Therefore, we conclude that the effect of clot activators in the serum tube on coagulation tests is minimal when standard phlebotomy procedures are used.

Impact of genetic profiles on the efficacy of anti-EGFR antibodies in metastatic colorectal cancer with KRAS mutation.

Reports indicate that, even in KRAS-mutated colon cancer, there are subsets of patients who benefit from anti-EGFR monoclonal antibody (MoAb) treatment. The aim of the present study was to identify genetic profiles that contribute to the responsiveness of metastatic colorectal cancer (mCRC) to anti-EGFR MoAb. We retrospectively evaluated the efficacy of anti-EGFR MoAb in mCRC patients with KRAS mutations according to KRAS mutational subtypes, BRAF and PIK3CA mutational status and PTEN and MET expression. Among 21 patients with KRAS-mutant tumors, 8 (38%) harbored p.G13D, 7 (33%) harbored p.G12V, 5 (24%) harbored p.G12D, and 1 (5%) harbored p.G12C mutation. Patients with the p.G13D mutation exhibited a significantly higher disease control rate than patients with other KRAS mutations (P=0.042), and tended to show a longer progression-free survival (PFS) than patients with other KRAS mutations with marginal significance (P=0.074). Patients with loss of PTEN had significantly shorter PFS than those with normal PTEN expression in patients with KRAS mutations (P=0.044). MET overexpression was significantly associated with shorter PFS compared to normal MET expression in patients with KRAS mutations (P=0.016). Our data demonstrated the potential utility of alterations in PTEN and MET expression as predictive markers for response to anti-EGFR MoAbs in mCRC patients with KRAS mutations. In addition, we confirmed the predictive value of the KRAS p.G13D mutation for better response to anti-EGFR therapies in comparison with other KRAS mutations.