Activation of RHOA-VAV1 signaling in angioimmunoblastic T-cell lymphoma.

Somatic G17V RHOA mutations were found in 50-70% of angioimmunoblastic T-cell lymphoma (AITL). The mutant RHOA lacks GTP binding capacity, suggesting defects in the classical RHOA signaling. Here, we discovered the novel function of the G17V RHOA: VAV1 was identified as a G17V RHOA-specific binding partner via high-throughput screening. We found that binding of G17V RHOA to VAV1 augmented its adaptor function through phosphorylation of 174Tyr, resulting in acceleration of T-cell receptor (TCR) signaling. Enrichment of cytokine and chemokine-related pathways was also evident by the expression of G17V RHOA. We further identified VAV1 mutations and a new translocation, VAV1-STAP2, in seven of the 85 RHOA mutation-negative samples (8.2%), whereas none of the 41 RHOA mutation-positive samples exhibited VAV1 mutations. Augmentation of 174Tyr phosphorylation was also demonstrated in VAV1-STAP2. Dasatinib, a multikinase inhibitor, efficiently blocked the accelerated VAV1 phosphorylation and the associating TCR signaling by both G17V RHOA and VAV1-STAP2 expression. Phospho-VAV1 staining was demonstrated in the clinical specimens harboring G17V RHOA and VAV1 mutations at a higher frequency than those without. Our findings indicate that the G17V RHOA-VAV1 axis may provide a new therapeutic target in AITL.

Prognostic value of sequencing-based minimal residual disease detection in patients with multiple myeloma who underwent autologous stem-cell transplantation.

BACKGROUND: Most patients with multiple myeloma (MM) are considered to be incurable, and relapse owing to minimal residual disease (MRD) is the main cause of death among these patients. Therefore, new technologies to assess deeper response are required. PATIENTS AND METHODS: We retrospectively analyzed 125 patients with MM who underwent high-dose melphalan plus autologous stem-cell transplantation (ASCT) to detect MRD in autograft/bone marrow (BM) cells using a next-generation sequencing (NGS)-based method and allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR). RESULTS: NGS-based method was applicable to 90% and this method had at least one to two logs greater sensitivity compared to ASO-PCR. MRD negative by NGS [MRDNGS(-)] (defined as <10-6) in post-ASCT BM cases (n = 26) showed a significantly better progression-free survival (PFS) (96% at 4 years, P < 0.001) and overall survival (OS) (100% at 4 years, P =0.04) than MRDNGS(+) in post-ASCT BM cases (n = 25). When restricting the analysis to the 39 complete response cases, patients who were MRDNGS(-) (n = 24) showed a significantly better PFS than those that were MRDNGS(+) (n = 15) (P =0.02). Moreover, MRDNGS(-) in post-ASCT BM cases (n = 12) showed significantly a better PFS than MRDNGS(+) cases (n = 7) where MRD was not detected by ASO-PCR (P = 0.001). Patients whose autografts were negative by NGS-based MRD assessment (<10-7) (n = 19) had 92% PFS and 100% OS at 4 years post-ASCT. Conversely, the NGS-based MRD positive patients who received post-ASCT treatment using novel agents (n = 49) had a significantly better PFS (P = 0.001) and tended to have a better OS (P= 0.214) than those that were untreated (n = 33). CONCLUSIONS: Low level MRD detected by NGS-based platform but not ASO-PCR has significant prognostic value when assessing either the autograft product or BM cells post-ASCT.

Whole-body computed tomography versus conventional skeletal survey in patients with multiple myeloma: a study of the International Myeloma Working Group.

For decades, conventional skeletal survey (CSS) has been the standard imaging technique for multiple myeloma (MM). However, recently whole-body computed tomography (WBCT) has been implemented into the diagnostic criteria of MM. This analysis compares sensitivity and prognostic significance of WBCT and CSS in patients with smoldering MM (SMM) and MM. Fifty-four of 212 patients (25.5%) had a negative CSS and a positive WBCT for osteolytic lesions (P<0.0001). Of 66 patients with SMM based on CSS, 12 (22.2%) had osteolytic lesions on WBCT. In comparison, WBCT failed to detect some bone destructions in the appendicular skeleton possibly due to limitations of the field of view. Presence of lytic bone lesions in WBCT was of borderline prognostic significance (P=0.051) for SMM patients, with a median time to progression of 38 versus 82 months for those without bone destructions. In conclusion, WBCT identifies significantly more sites of bone destruction than CSS. More than 20% of patients with SMM according to CSS have in fact active MM detectable with WBCT. On the basis of this and other studies, WBCT (either computed tomography (CT) alone or as part of a positron emission tomography-CT protocol) should be considered the current standard for the detection of osteolytic lesions in MM.

The novel BMI-1 inhibitor PTC596 downregulates MCL-1 and induces p53-independent mitochondrial apoptosis in acute myeloid leukemia progenitor cells.

Disease recurrence is the major problem in the treatment of acute myeloid leukemia (AML). Relapse is driven by leukemia stem cells, a chemoresistant subpopulation capable of re-establishing disease. Patients with p53 mutant AML are at an extremely high risk of relapse. B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is required for the self-renewal and maintenance of AML stem cells. Here we studied the effects of a novel small molecule inhibitor of BMI-1, PTC596, in AML cells. Treatment with PTC596 reduced MCL-1 expression and triggered several molecular events consistent with induction of mitochondrial apoptosis: loss of mitochondrial membrane potential, BAX conformational change, caspase-3 cleavage and phosphatidylserine externalization. PTC596 induced apoptosis in a p53-independent manner. PTC596 induced apoptosis along with the reduction of MCL-1 and phosphorylated AKT in patient-derived CD34+CD38low/- stem/progenitor cells. Mouse xenograft models demonstrated in vivo anti-leukemia activity of PTC596, which inhibited leukemia cell growth in vivo while sparing normal hematopoietic cells. Our results indicate that PTC596 deserves further evaluation in clinical trials for refractory or relapsed AML patients, especially for those with unfavorable complex karyotype or therapy-related AML that are frequently associated with p53 mutations.

Clinical and prognostic significance of bone marrow abnormalities in the appendicular skeleton detected by low-dose whole-body multidetector computed tomography in patients with multiple myeloma.

Clinical significance of medullary abnormalities in the appendicular skeleton (AS) detected by low-dose whole-body multidetector computed tomography (MDCT) in patients with multiple myeloma (MM) was investigated. A total of 172 patients with monoclonal gammopathy of undetermined significance (MGUS) (n=17), smoldering MM (n=47) and symptomatic MM (n=108) underwent low-dose MDCT. CT values (CTv) of medullary density of AS⩾0 Hounsfield unit (HU) was considered as abnormal. Percentage of medullary abnormalities and the mean CTv of AS in patients with MGUS, smoldering MM and symptomatic MM were 18, 55 and 62% and -44.5 , -20.3 and 11.2 HU, respectively (P<0.001 and P<0.001). Disease progression of MM was independently associated with high CTv on multivariate analysis. In symptomatic MM, the presence of abnormal medullary lesions was associated with increased incidence of high-risk cytogenetic abnormalities (34.4% vs 7.7%; P=0.002) and extramedullary disease (10.4% vs 0%; P=0.032). It was also an independent poor prognostic predictor (hazard ratio 3.546, P=0.04). This study showed that CTv of AS by MDCT is correlated with disease progression of MM, and the presence of abnormal medullary lesions is a predictor for poor survival.

Percentage of urinary albumin excretion and serum-free light-chain reduction are important determinants of renal response in myeloma patients with moderate to severe renal impairment.

Reversal of renal dysfunction significantly affects the prognosis of multiple myeloma (MM) with renal impairment (RI). There is no reliable test for predicting reversibility of RI in MM patients. We postulated that MM with high albuminuria may reflect glomerular disease that is difficult to reverse. Here, we examined the impact of urinary albumin excretion. We retrospectively analyzed 279 patients admitted to our hospital from April 2000 to December 2013. Clinical variables and laboratory data that may affect myeloma treatment response were extracted. The results were examined for relationship to renal response by univariate and multivariate analysis. RI (estimated glomerular filtration rate ≦50 ml/min per 1.73 m(2)) was observed in 116 patients (46%) and renal responses of renal complete response, renal partial response, renal minor response and no response were obtained in 46 (40%), 15 (13%), 13 (11%) and 42 (36%) patients, respectively. Although renal recovery was significantly associated with Durie-Salmon 1 or 2 (P=0.02), myeloma response better than very good partial response (P=0.03), involved free light-chain (iFLC) reduction from baseline 80% at day 12 (P=0.005), ≧95% at day 21 (P<0.001) and urinary albumin ≦25% on admission (P<0.001) on univariate analysis, only reduction of iFLC 95% at day 21 (P=0.015) and urinary albumin ≦25% (P=0.007) remained significant for any renal response. Our observation indicates that increased urinary albumin excretion >25% and reduction of iFLC ≦95% on day 21 were associated with favorable renal recovery in MM patients with RI, and were considered as negative predictors for renal response.

Geometric frustration of icosahedron in metallic glasses.

Icosahedral order has been suggested as the prevalent atomic motif of supercooled liquids and metallic glasses for more than half a century, because the icosahedron is highly close-packed but is difficult to grow, owing to structure frustration and the lack of translational periodicity. By means of angstrom-beam electron diffraction of single icosahedra, we report experimental observation of local icosahedral order in metallic glasses. All the detected icosahedra were found to be distorted with partially face-centered cubic symmetry, presenting compelling evidence on geometric frustration of local icosahedral order in metallic glasses.

Objective determination of fatigue development following sun exposure using Advanced Trail Making Test.

Sun exposure during leisure activity evokes fatigue. We employed the Advanced Trail Making Test (ATMT), a recently developed objective method of evaluating brain function performance used to measure mental fatigue, for objective determination of fatigue development caused by solar exposure to the human body. First, a survey of consumer awareness was performed, and fatigue development from solar exposure was generally recognized in both summer and spring. In the field test, 15 males (26-41 years old) received sun exposure equivalent to 100 kJ m(-2) of ultraviolet radiation three to four times each day for 3 days, during which the subjects wore a short sleeve shirt and a short pant, and covered their head with a towel. A significant increase in scores for subjective sense of fatigue was observed in the evening of all 3 days following sun exposure and on the fourth day, which had no exposure, as well as in the morning of the third and fourth days, as compared with those periods during the control week, which did not have experimental solar exposure. ATMT showed a significant increase in average value in the evening of the first and second days following sun exposure, as well as in the morning of the third and fourth days. In addition, increases in body temperature and heart rate were observed during the exposure periods. The results of multiple regression analysis of subjective feelings showed that fatigue caused by solar exposure was qualitatively different from that in the control week. These results suggest that brain function performance declined following solar exposure as did fatigue development. ATMT results may be useful for quantitative and objective evaluation of mental fatigue caused by sun exposure, along with development of sun care products for the prevention of solar-caused fatigue.

Eradication of residual bcr-abl-positive clones by inducing graft-versus-host disease after allogeneic stem cell transplantation in patients with Philadelphia chromosome-positive acute lymphoblastic leukemia.

Persistence of bcr-abl transcripts after marrow grafting is thought to convey a high risk for relapse in patients with Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL). Donor leukocyte infusion (DLI) is closely associated with development of graft-versus-host disease (GVHD) and has well-defined activity against relapsed chronic myelogenous leukemia (CML) but not ALL. We report two patients with Ph-positive ALL who remained bcr-abl positive by reverse transcriptase polymerase chain reaction (RT-PCR) after marrow grafting. Residual bcr-abl transcripts in both patients were eliminated following acute GVHD, which was induced by either DLI or rapid reduction of immunosuppression. Both patients have continued in complete molecular remission for 18 months and 8 months following transplantation, respectively. Our observation suggests that induction of GVHD may eliminate minimal residual disease, thereby preventing leukemia relapse in patients transplanted for Ph-positive ALL.

Immunosuppressive properties of CD95L-transduced "killer" hybrids created by fusing donor- and recipient-derived dendritic cells.

Allogeneic immune responses, which are initiated by dendritic cells (DCs) of both donor and host origins, remain a major obstacle in organ transplantation. Presentation of intact major histocompatibility complex (MHC) molecules by allogeneic DCs and allogeneic peptides by syngeneic DCs leads to complex allogeneic immune responses. This study reports a novel strategy designed to suppress both pathways. A stable DC line XS106 (A/J mouse origin) was transfected with CD95L cDNA and fused with splenic DCs purified from allogeneic BALB/c mice. The resulting "killer" DC-DC hybrids: (1) expressed CD95L and MHC class I and class II molecules of both A/J and BALB/c origins, while maintaining otherwise characteristic surface phenotypes of mature DCs; (2) inhibited MHC class I- and class II-restricted mixed leukocyte reactions between the parental strains by triggering apoptosis of alloreactive T cells; and (3) abolished delayed-type hypersensitivity responses of A/J (and BALB/c) mice to BALB/c-associated (and A/J-associated) alloantigens when injected intravenously into A/J (and BALB/c) mice. The onset of graft-versus-host disease in (BALB/c x A/J) F1 hosts receiving A/J-derived hematopoietic cell transplantation was suppressed significantly (P <.001) by killer DC-DC hybrid treatment. These results form both technical and conceptual frameworks for clinical applications of CD95L-transduced killer hybrids created between donor DCs and recipient DCs in the prevention of allogeneic immune responses following organ transplantation.

Dominant negative isoform of the Ikaros gene in patients with adult B-cell acute lymphoblastic leukemia.

Gene targeting studies in mice have shown that the transcription factor Ikaros plays an essential role in lymphoid development and as a tumor suppressor in T cells, whereas the related gene Aiolos functions as a tumor suppressor in B cells. We analyzed the expression levels of the Ikaros gene family, Ikaros and Aiolos, in human bone marrow samples from patients with adult acute lymphoblastic leukemia [ALL (n = 46; B-cell ALL = 41; T-cell ALL = 5)]. Overexpression of the dominant negative isoform of Ikaros gene Ik-6 was observed in 14 of 41 B-cell ALL patients by reverse transcription-PCR, and the results were confirmed by sequencing analysis and immunoblotting. None of the other dominant negative isoforms of the Ikaros gene were detected by reverse transcription-PCR analysis. Southern blotting analysis with PstI digestion revealed that those patients with the dominant negative isoform Ik-6 might have small mutations in the Ikaros locus. We did not detect any overexpression of dominant negative isoforms of Aiolos in adult ALL patients. These results suggest that Ikaros plays a key role in human B-cell malignancies through the dominant negative isoform Ik-6.

Successful treatment of disseminated nasal NK/T-cell lymphoma using double autologous peripheral blood stem cell transplantation.

Nasal natural killer (NK)/T-cell lymphoma is a rare disease with an aggressive clinical course. Prognosis is generally poor and the disease is invariably fatal after systemic dissemination. We report a patient with aggressive nasal NK/T-cell lymphoma who was resistant to therapy and developed systemic dissemination involving the intestine, skin, and stomach. Epstein-Barr virus (EBV) was detected by Southern blotting with EBV-terminal repeat probe and by in situ EBV-encoded small nuclear early region-1 hybridization. The patient was treated using double high-dose chemotherapy and autologous peripheral blood stem cell transplantation (auto-PBSCT). Pretransplant conditioning for the first auto-PBSCT was MCVC (high-dose ranimustine, carboplatin, etoposide [VP16], and cyclophosphamide), and for the second auto-PBSCT, modified ICE (high-dose ifosfamide, VP16, and carboplatin). The patient obtained a complete remission and has been free of disease for 3.0 years since the second PBSCT. These observations suggest that double high-dose chemotherapy with PBSCT support may be effective in resistant nasal NK/T-cell lymphoma.

Induction of antigen-specific immunosuppression by CD95L cDNA-transfected 'killer' dendritic cells.

Dendritic cells (DCs) are special subsets of antigen-presenting cells characterized by their highly potent capacity to activate immunologically naive T cells. Here we report that DCs that are transfected with CD95 ligand (CD95L) cDNA, called 'killer' DCs, deliver death signals, instead of activation signals, to T cells after antigen-specific interaction. Injection of antigen-pulsed killer DCs into mice before sensitization induced antigen-specific immunosuppression. When administered after sensitization, killer DCs suppressed immune responses almost completely after subsequent challenge. Thus, killer DCs represent an entirely new immunomodulatory protocol, which may become directly applicable in preventing and even treating T cell-mediated inflammatory diseases.

[Development of cerebellar astrocytoma in a patient with acute myeloid leukemia 8 years after his second bone marrow transplant].

We report a case of cerebellar astrocytoma occurring 8 years after the second bone marrow transplantation (BMT) in 32-year-old man. The patient was admitted to our hospital in December 1997 because of dysarthria and gait disturbance. He had been treated earlier for acute myeloid leukemia (AML M2) with chemotherapy and cranial irradiation followed by allogeneic BMT from a sibling in december 1988. Three months after the first BMT, testicular relapse was observed and followed by systemic relapse. The patient received reinduction therapy and a second successful BMT. He had been well until about 1 month before admission to our hospital. Neurological examination revealed left cerebellar ataxia, and brain magnetic resonance imaging disclosed a left cerebellar tumor. The tumor was surgically resected and a histological diagnosis of cerebellar astrocytoma was made. The patient was further treated by irradiation for residual tumor and discharged without progression of the disease.

Monitoring of human herpesviruses after allogeneic peripheral blood stem cell transplantation and bone marrow transplantation.

Herpesviruses frequently cause serious complications after allogeneic bone marrow transplantation (allo-BMT). Recent studies have shown more rapid immune reconstitution after allogeneic peripheral blood stem cell transplantation (allo-PBSCT) compared with allo-BMT. However, it has not been clarified whether the improved immune reconstitution after allo-PBSCT is associated with a lower incidence of herpesvirus infections. We monitored the emergence of Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV-6) and HHV-7 DNA by a nested-double polymerase chain reaction in peripheral blood leucocytes from 22 allo-BMT and 16 allo-PBSCT patients. Each virus had an unique temporal profile of detection. HHV-6 DNA was detected most frequently at 3 weeks after transplantation, whereas CMV and EBV DNA were detected later (2-3 months). Detection rates of HHV-6 DNA at 3 and 4 weeks after allo-BMT were significantly higher than those after allo-PBSCT (9/16 v 2/13 at 3 weeks, P < 0.01; 10/21 v 1/15 at 4 weeks, P < 0.01). Detection rates of the other three herpesviruses after the two types of allogeneic transplantation were not significantly different throughout observation period. Furthermore, detection of HHV-6 DNA within the first 4 weeks was associated with delayed platelet engraftment after both allo-BMT and allo-PBSCT (P < 0.01). These results suggest an advantage for allo-PBSCT over allo-BMT in terms of suppression of HHV-6 reactivation and prevention of subsequent complications.