Yasuko Fujita

Biological characteristics of the leukemia-associated transcriptional factor AML1 disclosed by hematopoietic rescue of AML1-deficient embryonic stem cells by using a knock-in strategy.

ABSTRACT AML1 is one of the most frequently mutated genes associated with human acute leukemia and encodes the DNA-binding subunit of the heterodimering transcriptional factor complex, core-binding factor (CBF) (or polyoma enhancer binding protein 2 [PEBP2]). A null mutation in either AML1 or its dimerizing partner, CBFβ, results in embryonic lethality secondary to a complete block in fetal liver hematopoiesis, indicating an essential role of this transcription complex in the development of definitive hematopoiesis. The hematopoietic phenotype that results from the loss of AML1 can be replicated in vitro with a two-step culture system of murine embryonic stem (ES) cells. Using this experimental system, we now demonstrate that this hematopoietic defect can be rescued by expressing thePEBP2αB1 (AML1b) isoform under the endogenousAML1-regulatory sequences through a knock-in (targeted insertion) approach. Moreover, we demonstrate that the rescuedAML1 −/− ES cell clones contribute to lymphohematopoiesis within the context of chimeric animals. Rescue requires the transcription activation domain of AML1 but does not require the C-terminal VWRPY motif, which is conserved in all AML1 family members and has been shown to interact with the transcriptional corepressor, Groucho/transducin-like Enhancer of split. Taken together, these data provide compelling evidence that the phenotype seen inAML1-deficient mice is due solely to the loss of transcriptionally active AML1.

Successful treatment with Erwinia L-asparaginase for recurrent natural killer/T cell lymphoma.

We describe a patient with natural killer (NK)/T cell lymphoma who relapsed after autologous peripheral blood stem cell transplantation (auto-PBSCT) and was successfully treated with Escherichia coli (E. coli ) and Erwinia l -asparaginase. A 38-year-old male patient with ulcerated tumor at the left thigh was diagnosed as having nasal type NK/T cell lymphoma on the basis of histopathological and flowcytometric findings of tumor, revealing diffuse infiltration of atypical lymphoid cells into blood vessels and expression of CD7 and CD56 antigens, but not CD3. He had tumor infiltration in the bone marrow and at the right lower lung field. After five cycles of CHOP (cyclophosphamide, doxorubicin, vincristine and prednisolone) therapy, the patient achieved complete remission and received high-dose chemotherapy with auto-PBSCT, although the tumor recurred in the right leg 10 months later. Despite salvage chemotherapy, followed by local irradiation and surgical amputation, a tumor recurred at the left upper gingiva 10 days after. Using E. coli l -asparaginase (6000 U/m 2 /day), the tumor regressed, fever was alleviated and the serum lactate dehydrogenase decreased to normal range after several days. The asparagine synthetase expression in tumor cells was immunohistochemically negative on paraffin-embedded tissues. Because of the anaphylactoid reaction developing after E. coli l -asparaginase, alternative Erwinia l -asparaginase (6000 U/m 2 /day) was administered, resulting in regression of tumor and fever lysis. l -asparaginase is a promising agent for the treatment of NK/T cell lymphoma.

Novel loss-of-function mutations of the haematopoiesis-related transcription factor, acute myeloid leukaemia 1/runt-related transcription factor 1, detected in acute myeloblastic leukaemia and myelodysplastic syndrome.

AML1/RUNX1, which encodes a transcription factor essential for definitive haematopoiesis, is a frequent target of leukaemia‐associated chromosome translocations. Point mutations of this gene have also recently been associated with leukaemia and myelodysplastic syndrome (MDS). To further define the frequency and biological characteristics of AML1 mutations, we have examined 170 cases of such diseases. Mutations within the runt‐domain were identified in five cases: one of de novo acute myeloid leukaemia (AML) and four of MDS. Where multiple time point samples were available, mutations were detected in the earliest samples, which persisted throughout the disease course. Of the five mutations, one was a silent mutation, two were apparent loss‐of‐function mutations caused by N‐terminal truncation, and two were insertions, I150ins and K168ins, which preserved most of the AML1 DNA‐binding domain. Both AML1 molecules with insertion mutations were non‐functional in that they were unable to rescue haematological defects in AML1‐deficient mouse embryonic stem cells. In addition, activating mutations of N‐ras, deletion of chromosome 12p, or inactivation of TP53 accompanied some of the AML1 mutations. Together, these observations strongly suggest that one‐allele inactivation of AML1 serves as an initial or early event that plays an important role in the eventual development of overt diseases with additional genetic alterations.

Differentiation of follicular from mucosa-associated lymphoid tissue lymphoma by detection of t(14;18) on single-cell preparations and paraffin-embedded sections.

A 57‐year‐old woman was referred to the Kyoto Prefectural University of Medicine because of multiple polypoid lesions in the duodenum. On the basis of the histopathologic findings, mucosa‐associated lymphoid tissue lymphoma had been diagnosed. The polypoid lesions did not show any improvement in spite of antimicrobial therapy for elimination of Helicobacter pylori. Because the disease remained stable during the clinical course, no other specific treatment was administered. We performed fluorescence in situ hybridization (FISH) analysis on a single‐cell preparation obtained from the duodenal lesions, to assess the specific chromosome translocation. We identified BCL2/IGH fusion at a frequency of 83%. Two‐color FISH was also performed on paraffin‐embedded tissue sections, which demonstrated BCL2/IGH fusion–positive cells in neoplastic follicles. These findings, together with the CD10+ immunophenotyping of tumor cells, led to a diagnosis of primary follicular lymphoma of the duodenum. Interphase FISH with the IGH gene and oncogene probes is a rapid and powerful tool for assessing genomic changes in gastrointestinal lymphoma on single‐cell preparations and tissue sections. This technique is particularly useful in view of the increasingly small core biopsy samples and needle aspirations obtained for diagnostic purposes.

Molecular-cytogenetic Characterization of Non-Hodgkin's Lymphoma with Double and Cryptic Translocations of the Immunoglobulin Heavy Chain Gene

The present study aimed to characterize the clinical and molecular-cytogenetic features of non-Hodgkins lymphoma (NHL) with double translocation of the immunoglobulin heavy chain (IGH) gene. G-banding analysis, fluorescence in situ hybridization (FISH) with the IGH (Cgamma and VH) and oncogene (c-MYC, BCL1, BCL2, and BCL6) probes, and long-distance polymerase chain reaction (LD-PCR) were performed on 6 patients with B-cell lymphoma, one with angioimmunoblastic T-cell lymphoma, and one with acute lymphoblastic leukemia (ALL) with B-cell phenotype. G-banding analysis detected two different 14q32 translocations, t(14,18) and add (14)(q32) in a patient with ALL. Two distinct partners of double IGH translocation identified by FISH were as follows: c-MYC + BCL2 in 3 patients, c-MYC + BCL1 in 2, c-MYC + BCL6 in one, BCL2 + 9q22 in one, and 1q21 + 6q27 in one. Colocalization of BCL1 and c-MYC probes was demonstrated in a patient with mantle cell lymphoma. LD-PCR detected c-MYC/Cmu, c-MYC/Calpha and BCL6/Cmu, and c-MYC/Calpha fusion in each one patient. Seven of 8 patients showed high serum LDH. Central nervous system and leukemic involvement was observed in 5 and 6 patients, respectively. Median survival time of patients with c-MYC/IGH translocation was 9 months. The results defined a clinical subset of B-cell lymphoma/leukemia showing extremely poor prognosis. C-MYC/IGH translocation is possibly an evolutionary alteration following the primary IGH translocation with BCL1, BCL2, or BCL6. Furthermore, FISH identified one novel (9q22) and one cryptic chromosomal breakpoints (6q27) involved in IGH translocation.

Linked color imaging improves the visibility of colorectal polyps: a video study

Background/study aim  Linked color imaging (LCI) by a laser endoscope (Fujifilm Co, Tokyo, Japan) is a novel narrow band light observation. In this study, we aimed to investigate whether LCI could improve the visibility of colorectal polyps using endoscopic videos. Patients and methods  We prospectively recorded videos of consecutive polyps 2 – 20 mm in size diagnosed as neoplastic polyps. Three videos, white light (WL), blue laser imaging (BLI)-bright, and LCI, were recorded for each polyp by one expert. After excluding inappropriate videos, all videos were evaluated in random order by two experts and two non-experts according to a published polyp visibility score from four (excellent visibility) to one (poor visibility). Additionally, the relationship between polyp visibility scores in LCI and various clinical characteristics including location, size, histology, morphology, and preparation were analyzed compared to WL and BLI-bright. Results  We analyzed 101 colorectal polyps (94 neoplastic) in 66 patients (303 videos). The mean polyp size was 9.0 ± 8.1 mm and 54 polyps were non-polypoid. The mean polyp visibility scores for LCI (2.86 ± 1.08) were significantly higher than for WL and BLI-bright (2.53 ± 1.15, P  < 0.001; 2.73 ± 1.47, P  < 0.041). The ratio of poor visibility (score 1 and 2) was significantly lower in LCI for experts and non-experts (35.6 %, 33.6 %) compared with WL (49.6 %, P  = 0.015, 50.5 %, P  = 0.046). The polyp visibility scores for LCI were significantly higher than those for WL for all of the factors. With respect to the comparison between BLI-bright and WL, the polyp visibility scores for BLI-bright were not higher than WL for right-sided location, < 10 mm size, sessile serrated adenoma and polyp histology, and poor preparation. For those characteristics, LCI improved the lesions with right-sided location, SSA/P histology, and poor preparation significantly better than BLI. Conclusions  LCI improved polyp visibility compared to WL for both expert and non-expert endoscopists. It is useful for improving polyp visibility in any location, any size, any morphology, any histology, and any preparation level.

Histopathological analysis of cold snare polypectomy and its indication for colorectal polyps 10–14 mm in diameter

Cold snare polypectomy (CSP) is commonly used for treating colorectal polyps <10 mm in diameter. We evaluated the analysis and safety of CSP for larger polyps.

Multiple bone lesions after allogeneic bone marrow transplantation in a patient with relapsed adult acute lymphoblastic leukemia: minimal residual disease analysis may predict extramedullary relapse.

We describe a patient with acute lymphoblastic leukemia (ALL, L2) who relapsed with multiple bone lesions after allogeneic bone marrow transplantation (allo-BMT). Allo-BMT was performed from an HLA-identical sibling during the first hematological complete remission (CR). Minimal residual disease (MRD) assessed by polymerase chain reaction (PCR) with primers for T cell receptor δ (TCRδ) gene became positive in the bone marrow sample on day 46 after allo-BMT. On day 113, the patient complained of a painful tumor at the right clavicle. The examination of biopsy specimen revealed infiltration of leukemic cells. After partial response was achieved by local radiotherapy, disseminated bone lesions were demonstrated by 99mTC scintigraphy scan, followed by bone marrow relapse on day 137. The patient died of cardiac tamponade on day 236 after Allo-BMT. MRD assessed by PCR assay for TCRδ gene in the bone marrow is useful for the prediction of extramedullary as well as medullary relapse after BMT.

Magnifying Endoscopy with Blue Laser Imaging Improves the Microstructure Visualization in Early Gastric Cancer: Comparison of Magnifying Endoscopy with Narrow-Band Imaging

Backgrounds Magnifying endoscopy with blue laser imaging (ME-BLI) for diagnosis of early gastric cancer (EGC) is as effective as magnifying endoscopy with narrow-band imaging (ME-NBI). However, there are different EGCs in microstructure visualization between ME-BLI and ME-NBI. This study aimed to clarify the pathological features of the EGCs, in which microstructure visualization was different between ME-NBI and ME-BLI. Methods EGCs were classified into groups A (irregular microsurface pattern (MSP) in ME-BLI and absent MSP in ME-NBI), B (irregular MSP in two modalities), or C (absent MSP in two modalities), according to the vessel plus surface classification. We compared the pathological features of EGCs between the three groups. Results 17, four, and five lesions could be evaluated in detail in groups A, B and C, respectively. Well-differentiated adenocarcinomas with shallow crypts were more frequent in group A than in group B (58.8 and 0%, resp.). The mean crypt depth of group A was significantly shallower than that of group B (56 ± 20, 265 ± 64 μm, resp., P = 0.0002). Conclusions ME-BLI could better visualize the microstructures of the EGCs with shallow crypts compared with ME-NBI. Therefore, ME-BLI could enable a more accurate diagnosis of EGC with shallow crypts.

Molecular profiling and genome-wide analysis based on somatic copy number alterations in advanced colorectal cancers

To characterize somatic alterations in colorectal cancer (CRC), we conducted a genome‐scale analysis of 106 CRC specimens. We assessed comprehensive somatic copy number alterations (SCNAs) in these CRC specimens. In addition, we examined microsatellite instability (MSI; low and high), genetic mutations (KRAS, BRAF, TP53, and PIK3CA), and DNA methylation status (classified into low, intermediate, and high type). We stratified molecular alterations in the CRCs using a hierarchical cluster analysis. The examined CRCs could be categorized into three subgroups using hierarchical cluster analysis. Tumors in subgroup 1 were characterized by a low frequency of SCNAs and a high frequency of MSI‐high status, whereas tumors in subgroups 2 and 3 were closely associated with a high frequency of SCNAs. Tumors in subgroup 1 were preferentially present in the right‐sided colon and showed frequent MSI‐high status. Subgroup 3 was distinguished by specific alterations, including gains at 1q23‐44, 1p11‐36, 10q11‐26, 10p11‐13, 12q24‐24, and 13q33‐33. In contrast, tumors in subgroup 2 were characterized by copy‐neutral LOH at 12p12‐13, 1q24‐25, and 10q22. In addition, KRAS mutations were more frequently found in subgroup 3 than in subgroup 1. TP53 mutations and intermediate levels of DNA methylation were common alterations in the three subgroups. SCNAs contributed to sporadic CRC, and there were three subgroups based on SCNAs that played a different role in driving the development of this disease.