Hiroaki Ohnishi

Tandem duplication of the FLT3 gene is found in acute lymphoblastic leukaemia as well as acute myeloid leukaemia but not in myelodysplastic syndrome or juvenile chronic myelogenous leukaemia in children

We examined mRNA expression and internal tandem duplication of the Fms‐like tyrosine kinase 3 (FLT3) gene in haematological malignancies by reverse transcriptase‐polymerase chain reaction (RT‐PCR) and genomic PCR followed by sequencing. By RT‐PCR, expression of FLT3 was detected in 45/74 (61%) leukaemia cell lines and the frequency of expression of FLT3 was significantly higher in undifferentiated type (B‐precursor acute lymphoblastic leukaemia; ALL) than in differentiated type cell lines (B‐ALL) (P = 0.0076). Using the genomic PCR method, 194 fresh samples including 87 acute myeloid leukaemias, 60 ALLs, 32 myelodysplastic syndromes (MDSs) and 15 juvenile chronic myelogenous leukaemias (JCMLs) were examined. Tandem duplication was found in 12 (13.8%) AMLs and two (3.3%) ALLs. Sequence analyses of the 14 samples with the duplication revealed that eight showed a simple tandem duplication and six a tandem duplication with insertion. Most of these tandem duplications occurred within exon 11, and two duplications occurred from exon 11 to intron 11 and exon 12. No tandem duplications of FLT3 gene were detected in MDS or JCML. The frequency of tandem duplication of FLT3 gene in childhood AML was lower than that in adult AML so far reported. All of the 12 AML patients with the duplication died within 47 months after diagnosis, whereas two ALL patients with the duplication have survived 44 and 72 months, respectively. These two ALL patients expressed both lymphoid and myeloid antigens and were considered to have biphenotypic leukaemia. These results suggest that tandem duplication is involved in ALL in addition to AML, but not in childhood MDS or JCML, and that childhood AML patients with the tandem duplication have a poor prognosis.

Alterations of the p53, p21, p16, p15 and RAS genes in childhood T-cell acute lymphoblastic leukemia

We investigated the alterations of the p53, p21, p16, p15 and RAS genes in childhood T-cell acute lymphoblastic leukemia (T-ALL) and T-ALL cell lines by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing. Mutations of the p53 gene were found in three of 57 (5%) patients at diagnosis, one of 14 (7%) patients at relapse and in 12 of 18 (67%) cell lines. In these 12 cell lines, four had more than two mutations of the p53 gene. The p53 mutations were found in four of five cell lines whose original fresh leukemic cells were simultaneously examined original fresh leukemic cells. However, only one of the four fresh leukemic cells had the same mutation. All patients with p53 mutations in the course of disease died. Mutations of the p21 gene were not identified in 71 fresh samples and in 18 cell lines. N-RAS mutations were found in two of 57 (4%) fresh T-ALL patients at diagnosis, and four of 18 cell lines (22%), whereas no mutations were detected in any samples at relapse. Alterations of the p16 gene were found in 18 of 47 (38%) patients at diagnosis and in seven of 14 (50%) at relapse. These differences were not statistically significant. There were no differences in the frequency of alteration of the p16 and p15 genes between event-free patients and the remaining patients. Furthermore, we found the methylation of p16 gene in three of seven patients lacking homozygous deletions, suggesting higher frequency of p16 inactivation than previous reports in T-ALL. Interestingly, we found that one allele is inactivated by methylation and another allele had nonsense mutation in one cell line (KOPT-KI), resulting in loss of protein expression of p16. This type of p16 inactivation has not been so far reported in leukemia. We conclude that, (1) p53 mutations are infrequent at diagnosis but tend to be associated with poor clinical outcome; (2) RAS and p21 mutations may not be involved in the pathogenesis of T-ALL; (3) not only frequent alterations of p16 and p15 genes but also methylation of p16 gene are involved in initiating the leukemogenesis of T-ALLs, and (4) these 5 genes are independently involved in T-ALL.

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.

Constitutive activation of PI3K is involved in the spontaneous proliferation of primary acute myeloid leukemia cells: direct evidence of PI3K activation.

Constitutive activation of PI3K is involved in the spontaneous proliferation of primary acute myeloid leukemia cells: direct evidence of PI3K activation

Consistent detection of CALM-AF10 chimaeric transcripts in haematological malignancies with t(10;11)(p13;q14) and identification of novel transcripts

The t(10;11)(p13‐14;q14‐21) is a rare but recurring translocation associated with acute lymphoblastic leukaemia (ALL) and acute myeloid leukaemia (AML). Recently the CALM gene was cloned from the t(10;11) breakpoint of U937 and fused to AF10, a putative transcription factor, which had been identified as one of the fusion partners of the MLL gene. In order to define the involvement of these genes in primary leukaemias and cell lines with t(10;11), we analysed the expression of fusion transcripts by reverse transcriptase‐polymerase chain reaction (RT‐PCR) in five patient samples including ALL, AML and lymphoblastic lymphoma, and three monocytic cell lines (P31/Fujioka, KP‐Mo‐TS and U937). The CALM‐AF10 fusion transcript was detected in all samples; however, the AF10‐CALM fusion was not detected in two patient samples and one cell line. In RT‐PCR analysis there were six isoforms of the CALM‐AF10 fusion transcripts and five of AF10‐CALM fusion transcripts. We also detected novel transcripts in U937. Sequence analysis revealed that all these isoforms had in‐frame junctions and that some of them resulted from alternative splicing at different exons of CALM and others from different breakpoints at CALM and/or AF10. There were at least two different breakpoints of CALM and three of AF10 gene. Our results suggest that the CALM‐AF10 fusion gene is a constant feature and is involved in the pathogenesis of haematological malignancies with t(10;11)(p13‐14;q14‐21), showing various and often multilineage phenotypes. Thus, t(10;11) needs to be investigated by RT‐PCR for identification of the genes involved.

Impact of fresh-frozen plasma from male-only donors versus mixed-sex donors on postoperative respiratory function in surgical patients: a prospective case-controlled study

BACKGROUND: To reduce the risk of transfusion‐related acute lung injury (TRALI), plasma products are mainly made from male donors in some countries because of the lower possibility of alloimmunization; other countries are considering this policy. The advantage of male‐only fresh‐frozen plasma (FFP) should be examined in a prospective case‐control study.

Chronic eosinophilic leukaemia with FIP1L1–PDGFRA fusion and T674I mutation that evolved from Langerhans cell histiocytosis with eosinophilia after chemotherapy

MUC1 was expressed strongly but transiently in early erythroid differentiation, however, it was absent in the circulating erythrocytes (Rughetti et al, 2003). Moreover, increased serum CA 15-3 has also been reported among patients with homozygous b-thalassaemia and sickle cellthalassaemia (Symeonidis et al, 2006). These observations may suggest that in SCD, the erythroid hyperplasia might express MUC1 glycoform similar to those expressed by carcinoma cells. Our data indicates that serum CA 15-3 levels may be elevated in individuals with SCD. The serum concentration of CA 15-3 should thus be considered when malignant disease is suspected.

Mutations of the RAS genes in childhood acute myeloid leukemia, myelodysplastic syndrome and juvenile chronic myelocytic leukemia

Using the polymerase chain reaction-single strand conformation polymorphism method and direct sequencing, 12 acute myeloid leukemia (AML) cell lines and 108 fresh childhood myeloid tumor specimens, including 67 AML, 29 myelodysplastic syndrome (MDS), and 12 juvenile chronic myelocytic leukemia (JCML) were examined for mutation in H-, K-, and N-RAS genes. The mutation was found in eight of the 120 samples (6.7%), which consisted of four cell lines (33.3%) and four fresh myeloid tumors (3.7%). The frequency of the mutation in the cell lines was apparently higher than that in fresh myeloid tumors. K-RAS gene mutations were found in two of the 67 fresh AML specimens (3%). Interestingly, these two patients had 11q23 translocations. The N-RAS gene mutation was found in one of the 29 specimens (3.4%) of MDS and in one of the 12 specimens (8.3%) of JCML. All mutations were found in codon 12, 13 or 61 of the N-RAS and K-RAS genes. Frequency of mutation of RAS genes in fresh myeloid malignancies was very low. These findings suggest that mutation of RAS genes does not play an important role in the development of childhood myeloid malignancies.

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.