Bortezomib-based strategy with autologous stem cell transplantation for newly diagnosed multiple myeloma: a phase II study by the Japan Study Group for Cell Therapy and Transplantation (JSCT-MM12).

BACKGROUND: The Japan Study Group for Cell Therapy and Transplantation (JSCT) organized a phase II study to evaluate the efficacy and safety of a treatment protocol (JSCT-MM12) for multiple myeloma (MM) patients who were previously untreated and transplantation-eligible. Since bortezomib-based therapy is known to be effective for MM, the protocol is intensified more than the previous protocol (JSCT-MM10) and comprised the subsequent treatments: bortezomib + cyclophosphamide + dexamethasone (VCD) induction; bortezomib + high-dose-melphalan (B-HDM) conditioning with autologous stem cell transplantation (ASCT); bortezomib + thalidomide + dexamethasone (VTD) consolidation; and lenalidomide (LEN) maintenance. METHODS: Sixty-four symptomatic patients aged between 20 and 65 years were enrolled for treatment and received three cycles of VCD, followed by cyclophosphamide administration for autologous stem cell harvest and B-HDM/ASCT, and subsequently two cycles of VTD, after that LEN for 1 year. RESULTS: Complete response (CR)/stringent CR (sCR) rates for induction, ASCT, consolidation, and maintenance therapies were 20, 39, 52, and 56%, respectively. The grade 3/4 toxicities (≥ 10%) with VCD treatment included neutropenia (27%), anemia (19%), and thrombocytopenia (11%). There was no treatment-related mortality. After median follow-up of 41 months, estimated 3-year progression-free survival (PFS) and overall survival (OS) rates were 64% and 88%, respectively. The high-risk group revealed lower CR/sCR, PFS, and OS than the standard-risk group. CONCLUSIONS: The study revealed that the treatment protocol consisting of VCD induction, B-HDM/ASCT followed by VTD consolidation, and LEN maintenance could produce highly beneficial responses and favorable tolerability in newly diagnosed MM. However, future study is required for improving treatment in the high-risk group.

[Systemic mastocytosis with refractory ascites successfully treated with interferon-α and a leukotriene receptor antagonist].

A 67-year-old male was referred to our hospital because of anemia, thrombocytopenia, and massive ascites. A diagnosis of systemic mastocytosis was made based on the observation of many mast cells in his bone marrow, elevated serum tryptase levels, and the presence of c-kit point mutation Asp816Val. Dasatinib and cladribine were ineffective, and a large volume of ascites was removed approximately every 3 days. Then, following an asthma attack, the patient was treated with pranlukast, a leukotriene receptor antagonist (LTRA). After LTRA treatment initiation, the frequency of ascites drainage decreased, and no puncture was necessary from the 10th day after the start of LTRA. Interferon α (IFN-α) was administered from the 15th day after the start of LTRA. Thereafter, his anemia and thrombocytopenia gradually improved, the ascites disappeared, the mast cells in his bone marrow were significantly reduced, and the Asp816Val mutation disappeared. Because persistent monocytosis was evident, he was suspected of chronic myelomonocytic leukemia but has not been diagnosed and is undergoing watchful waiting. This was considered to be a rare case of refractory ascites in which IFN-α was effective and LTRA might have been beneficial.

Phase 2 study of arsenic trioxide followed by autologous hematopoietic cell transplantation for relapsed acute promyelocytic leukemia.

The optimal treatments for relapsed acute promyelocytic leukemia (APL) remain equivocal. We conducted a phase 2 study to evaluate the efficacy and feasibility of a sequential treatment consisting of induction and consolidation with arsenic trioxide (ATO), peripheral blood stem cell (PBSC) harvest after high-dose cytarabine chemotherapy, and autologous hematopoietic cell transplantation (HCT). Between 2005 and 2009, 35 patients (26 with hematologic and 9 with molecular relapse) were enrolled. Induction therapy resulted in complete remission in 81% of those with hematologic relapse, and most patients became negative for PML-RARα after the first ATO consolidation course, but 4 remained positive. Administration of the second ATO consolidation course further decreased the transcript levels in 3 patients. In total, 25 patients proceeded to PBSC harvest, all of whom successfully achieved the target CD34+ cell doses, and 23 underwent autologous HCT with PML-RARα-negative PBSC graft. Posttransplant relapse occurred in 3 patients, and there was no transplant-related mortality. With a median follow-up of 4.9 years, the 5-year event-free and overall survival rates were 65% and 77%, respectively. These findings demonstrate the outstanding efficacy and feasibility of the sequential treatment featuring ATO and autologous HCT for relapsed APL. This study was registered at http://www.umin.ac.jp/ctr/ as #C000000302.

Real-world data of AML in Japan: results of JALSG clinical observational study-11 (JALSG-CS-11).

This report covers acute myeloid leukemia (AML) results from a multicenter, prospective observational study of AML, myelodysplastic syndromes, and chronic myelomonocytic leukemia in Japan. From August 2011 to January 2016, 3728 AML patients were registered. Among them, 42% were younger than 65, and the male-to-female ratio was 1.57:1. With a median follow-up time of 1807 days (95% confidence interval [CI]: 1732-1844 days), the estimated 5-year overall survival (OS) rate in AML patients (n = 3707) was 31.1% (95% CI: 29.5-32.8%). Trial-enrolled patients had a 1.7-fold higher OS rate than non-enrolled patients (5-year OS, 58.9% [95% CI: 54.5-63.1%] vs 35.5% [33.3-37.8%], p < 0.0001). Women had a higher OS rate than men (5-year OS, 34% [95% CI; 31.4-36.7%] vs 27.7% [25.7-29.7%], p < 0.0001). The OS rate was lower in patients aged 40 and older than those under 40, and even lower in those over 65 (5-year OS for ages < 40, 40-64, 65-74, ≥ 75: 74.5% [95% CI; 69.3-79.0%] vs 47.5% [44.4-50.6%] vs 19.3% [16.8-22.0%] vs 7.3% [5.5-9.4%], respectively). This is the first paper to present large-scale data on survival and clinical characteristics in Japanese AML patients.

Real-world data of MDS and CMML in Japan: results of JALSG clinical observational study-11 (JALSG-CS-11).

We conducted a multicenter, prospective observational study of acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and chronic myelomonocytic leukemia (CMML) in Japan. From August 2011 to January 2016, we enrolled 6568 patients. Herein, we report the results for MDS (n = 2747) and CMML (n = 182). The percentage of patients aged 65 years or older was 79.5% for MDS and 79.7% for CMML. The estimated overall survival (OS) rate and cumulative incidence of AML evolution at 5 years were 32.3% (95% confidence interval: 30.2-34.5%) and 25.7% (23.9-27.6%) for MDS, and 15.0% (8.9-22.7%) and 39.4% (31.1-47.6%) for CMML. Both diseases were more common in men. The most common treatment for MDS was azacitidine, which was used in 45.4% of higher-risk and 12.7% of lower-risk MDS patients. The 5-year OS rate after treatment with azacitidine was 12.1% (9.5-15.1%) for of higher-risk MDS patients and 33.9% (25.6-42.4%) for lower-risk patients. The second most common treatment was erythropoiesis-stimulating agents, given to just 20% of lower-risk patients. This is the first paper presenting large-scale, Japanese data on survival and clinical characteristics in patients with MDS and CMML.

Structure changes upon deprotonation of the proton release group in the bacteriorhodopsin photocycle.

In the photocycle of bacteriorhodopsin at pH 7, a proton is ejected to the extracellular medium during the protonation of Asp-85 upon formation of the M intermediate. The group that releases the ejected proton does not become reprotonated until the prephotolysis state is restored from the N and O intermediates. In contrast, at acidic pH, this proton release group remains protonated to the end of the cycle. Time-resolved Fourier transform infrared measurements obtained at pH 5 and 7 were fitted to obtain spectra of kinetic intermediates, from which the spectra of M and N/O versus unphotolyzed state were calculated. Vibrational features that appear in both M and N/O spectra at pH 7, but not at pH 5, are attributable to deprotonation from the proton release group and resulting structural alterations. Our results agree with the earlier conclusion that this group is a protonated internal water cluster, and provide a stronger experimental basis for this assignment. A decrease in local polarity at the N-C bond of the side chain of Lys-216 resulting from deprotonation of this water cluster may be responsible for the increase in the proton affinity of Asp-85 through M and N/O, which is crucial for maintaining the directionality of proton pumping.

Dehydration of main-chain amides in the final folding step of single-chain monellin revealed by time-resolved infrared spectroscopy.

Kinetic IR spectroscopy was used to reveal beta-sheet formation and water expulsion in the folding of single-chain monellin (SMN) composed of a five-stranded beta-sheet and an alpha-helix. The time-resolved IR spectra between 100 mus and 10 s were analyzed based on two consecutive intermediates, I(1) and I(2), appearing within 100 mus and with a time constant of approximately 100 ms, respectively. The initial unfolded state showed broad amide I' corresponded to a fluctuating conformation. In contrast, I(1) possessed a feature at 1,636 cm(-1) for solvated helix and weak features assignable to turns, demonstrating the rapid formation of helix and turns. I(2) possessed a line for solvated helix at 1,637 cm(-1) and major and minor lines for beta-sheet at 1,625 and 1,680 cm(-1), respectively. The splitting of the major and minor lines is smaller than that of the native state, implying an incomplete formation of the beta-sheet. Furthermore, both major and minor lines demonstrated a low-frequency shift compared to those of the native state, which was interpreted to be caused by hydration of the C O group in the beta-sheet. Together with the identification of solvated helix, the core domain of I(2) was interpreted as being hydrated. Finally, slow conversion of the water-penetrated core of I(2) to the dehydrated core of the native state was observed. We propose that both the expulsion of water, hydrogen-bonded to main-chain amides, and the completion of the secondary structure formation contribute to the energetic barrier of the rate-limiting step in SMN folding.

Coordinating the structural rearrangements associated with unidirectional proton transfer in the bacteriorhodopsin photocycle induced by deprotonation of the proton-release group: a time-resolved difference FTIR spectroscopic study.

In the photocycle of bacteriorhodopsin at pH 7, proton release from the proton releasing group (PRG) to the extracellular medium occurs during formation of the M intermediate. This proton release is inhibited at acidic pH, below the pK(a) of the PRG, approximately 6 in M, and instead occurs later in the cycle as the initial state is restored from the O intermediate. Here, structural changes related to deprotonation of the PRG have been investigated by time-resolved FTIR spectroscopy at 25 degrees C. The vibrational features at 2100-1790, 1730-1685, 1661, and 1130-1045 cm(-1) have greater negative intensity in the pure M-minus-BR spectrum and even in the M-minus-BR spectrum, that is present earlier together with the L-minus-BR spectrum, at pH 7, than in the corresponding M-minus-BR spectra at pH 5 or 4. The D212N mutation abolishes the decreases in the intensities of the broad feature between 1730 and 1685 cm(-1) and the band at 1661 cm(-1). The 1730-1685 cm(-1) feature may arise from transition dipole coupling of the backbone carbonyl groups of Glu204, Phe208, Asp212, and Lys216 interacting with Tyr57 and C(15)-H of the chromophore. The 1661 cm(-1) band, which is insensitive to D(2)O substitution, may arise by interaction of the backbone carbonyl of Asp212 with C(15)-H. The 2100-1790 cm(-1) feature with a trough at 1885 cm(-1) could be due to a water cluster. Depletion of these bands upon deprotonation of the PRG is attributable to disruption of a coordinated structure, held in place by interactions of Asp212. Deprotonation of the PRG is also accompanied by disruption of the interaction of the water molecule near Arg82. The liberated Asp212 may stabilize the protonated state of Asp85 and thus confer unidirectionality to the transport.

Structural changes due to the deprotonation of the proton release group in the M-photointermediate of bacteriorhodopsin as revealed by time-resolved FTIR spectroscopy.

One of the steps in the proton pumping cycle of bacteriorhodopsin (BR) is the release of a proton from the proton-release group (PRG) on the extracellular side of the Schiff base. This proton release takes place shortly after deprotonation of the Schiff base (L-to-M transition) and results in an increase in the pKa of Asp85, which is a crucial mechanistic step for one-way proton transfer for the entire photocycle. Deprotonation of the PRG can also be brought about without photoactivation, by raising the pH of the enzyme (pKa of PRG; approximately 9). Thus, comparison of the FTIR difference spectrum for formation of the M intermediate (M minus initial unphotolyzed BR state) at pH 7 to the corresponding spectrum generated at pH 10 may reveal structural changes specifically associated with deprotonation of the PRG. Vibrational bands of BR that change upon M formation are distributed across a broad region between 2120 and 1685 cm(-1). This broad band is made up of two parts. The band above 1780 cm(-1), which is insensitive to C15-deuteration of the retinal, may be due to a proton delocalized in the PRG. The band between 1725 and 1685 cm(-1), on the lower frequency side of the broad band, is sensitive to C15-deuteration. This band may arise from transition dipole coupling of the vibrations of backbone carbonyl groups in helix G with the side chain of Tyr57 and with the C15H of the Schiff base. In M, these broad bands are abolished, and the 3657 cm(-1) band, which is due to the disruption of the hydrogen bonding of a water molecule, probably with Arg82, appears. Loss of the interaction of the backbone carbonyl groups in helix G with Tyr57 and the Schiff base, and separation of Tyr57 from Arg82, may be causes of these spectral changes, leading to the stabilization of the protonated Asp85 in M.

A role for internal water molecules in proton affinity changes in the Schiff base and Asp85 for one-way proton transfer in bacteriorhodopsin.

Light-induced proton pumping in bacteriorhodospin is carried out through five proton transfer steps. We propose that the proton transfer to Asp85 from the Schiff base in the L-to-M transition is accompanied by the relocation of a water cluster on the cytoplasmic side of the Schiff base from a site close to the Schiff base in L to the Phe219-Thr46 region in M. The water cluster present in L, formed at 170 K, is more rigid than that at room temperature. This may be responsible for blocking the conversion of L to M at 170 K. In the photocycle at room temperature, this water cluster returns to the site close to the Schiff base in N, with a rigid structure similar to that of L at 170 K. The increase in the proton affinity of Asp85, which is a prerequisite for the one-way proton transfer in the M-to-N transition, is suggested to be facilitated by a structural change which disrupts interactions between Asp212 and the Schiff base, and between Asp212 and Arg82. We propose that this liberation of Asp212 is accompanied by a rearrangement of the structure of water molecules between Asp85 and Asp212, stabilizing the protonated Asp85 in M.

Water as a cofactor in the unidirectional light-driven proton transfer steps in bacteriorhodopsin.

Recent evidence for involvement of internal water molecules in the mechanism of bacteriorhodopsin is reviewed. Water O-H stretching vibration bands in the Fourier transform IR difference spectra of the L, M and N intermediates of bacteriorhodopsin were analyzed by photoreactions at cryogenic temperatures. A broad vibrational band in L was shown to be due to formation of a structure of water molecules connecting the Schiff base to the Thr46-Asp96 region. This structure disappears in the M intermediate, suggesting that it is involved in transient stabilization of the L intermediate prior to proton transfer from the Schiff base to Asp85. The interaction of the Schiff base with a water molecule is restored in the N intermediate. We propose that water is a critical mobile component of bacteriorhodopsin, forming organized structures in the transient intermediates during the photocycle and, to a large extent, determining the chemical behavior of these transient states.

CD56-positive diffuse large B-cell lymphoma: possible association with extranodal involvement and bcl-6 expression.

Among B-cell non-Hodgkin's lymphomas, neural cell adhesion molecule/CD56 expression is exceptional. In this study, seven cases of CD56-positive diffuse large B-cell lymphoma (DLBCL) are described. The frequency of CD56-positive DLBCL was 7% in our hospital. Four of seven (57.1%) cases expressed both CD10 and bcl-6 suggestive of a germinal center B-cell phenotype. Six of seven (85.7%) cases expressed bcl-6. Two cases expressed aberrant T cell-associated antigens, one each of CD7 and CD8. However, none of these seven cases showed CD5 expression. No significant difference was observed between CD56-positive and CD56-negative DLBCL in terms of the five international prognostic index risk factors. However, all seven cases had at least one extranodal involvement and showed a good response to initial treatment. The predominance of extranodal involvement in our series may be associated with the adhesion-related function of CD56. A high frequency of bcl-6 expression may be associated with a more favorable clinical course and prognosis.

Development of a technique for the investigation of folding dynamics of single proteins for extended time periods.

A technique was developed for the detection of fluorescence signals from free single molecules for extended time periods and was applied to the characterization of the unfolded states of iso-1-cytochrome c (cyt c). Protein molecules labeled with fluorescent dye were slowly injected into a capillary at concentrations that allow for the observation of one molecule at a time. A laser was introduced into the capillary coaxially, and the fluorescence was imaged as traces by using a lens with a large focal depth and wide field of view. Thus, the traces reflect the time-dependent changes in the fluorescence signals from single proteins. Cyt c was labeled with Alexa Fluor 532 at the C-terminal cysteine (cyt c-Alexa). In bulk experiments, cyt c-Alexa was shown to possess different fluorescence intensity for the native state, the unfolded state (U), and the intermediate state. Single-molecule traces of cyt c-Alexa were recorded by using the device. Intensity histograms of the traces revealed two distributions with broad and narrow widths, which were interpreted to correspond to the U and intermediate state, respectively, observed in the bulk measurements. The broad width of the U suggested the existence of a relatively slow conformational dynamics, which might be consistent with the correlation time ( approximately 15 ms) estimated from the traces assignable to the U. The technique was expected to reveal dynamics of proteins along the folding processes without artifacts caused by immobilization.

Water structural changes in the L and M photocycle intermediates of bacteriorhodopsin as revealed by time-resolved step-scan Fourier transform infrared (FTIR) spectroscopy.

In previous Fourier transform infrared (FTIR) studies of the photocycle intermediates of bacteriorhodopsin at cryogenic temperatures, water molecules were observed in the L intermediate, in the region surrounded by protein residues between the Schiff base and Asp96. In the M intermediate, the water molecules had moved away toward the Phe219-Thr46 region. To evaluate the relevance of this scheme at room temperature, time-resolved FTIR difference spectra of bacteriorhodopsin, including the water O-H stretching vibration frequency regions, were recorded in the micro- and millisecond time ranges. Vibrational changes of weakly hydrogen-bonded water molecules were observed in L, M, and N. In each of these intermediates, the depletion of a water O-H stretching vibration at 3645 cm-1, originating from the initial unphotolyzed bacteriorhodopsin, was observed as a trough in the difference spectrum. This vibration is due to the dangling O-H group of a water molecule, which interacts with Asp85, and its absence in each of these intermediates indicates that there is perturbation of this O-H group. The formation of M is accompanied by the appearance of water O-H stretching vibrations at 3670 and 3657 cm-1, the latter of which persists to N. The 3670 cm-1 band of M is due to water molecules present in the region surrounded by Thr46, Asp96, and Phe219. The formation of L at 298 K is accompanied by the perturbations of Asp96 and the Schiff base, although in different ways from what is observed at 170 K. Changes in a broad water vibrational feature, centered around 3610 cm-1, are kinetically correlated with the L-M transition. These results imply that, even at room temperature, water molecules interact with Asp96 and the Schiff base in L, although with a less rigid structure than at cryogenic temperatures.

t(14;18)(q32;q21)-bearing pleural MALT lymphoma with IgM paraproteinemia: value of detection of specific cytogenetic abnormalities in the differential diagnosis of MALT lymphoma and lymphoplasmacytic lymphoma.

A 67-year-old woman presented with a pleural effusion and a tumor in the right pleural wall. Histological examination of thoracoscopic tumor and pleural biopsy specimens showed infiltration by medium sized cells, some of which showed plasmacytoid differentiation. In view of the presence of IgM paraproteinemia and bone marrow involvement by lymphoma cells, the patient was diagnosed tentatively as having lymphoplasmacytic lymphoma (LPL). However, chromosomal analysis of the cells in the pleural fluid detected t(14;18)(q32;q21), while fluorescence in situ hybridization was positive for 11% of the MALT1 split signal. Because of the presence of characteristic genetic abnormalities and notable extranodal involvement, the patient was diagnosed as having MALT lymphoma. She was treated with three courses of cladribine and rituximab, and achieved complete regression of the tumor. In this case the detection of t(14;18)(q32;q21) involving IGH and MALT1 was useful for the differential diagnosis of LPL and MALT lymphoma.

Ca2+ -independent phospholipase A2-dependent sustained Rho-kinase activation exhibits all-or-none response.

Sustained contraction of cells depends on sustained Rho-associated kinase (Rho-kinase) activation. We developed a computational model of the Rho-kinase pathway to understand the systems characteristics. Thrombin-dependent in vivo transient responses of Rho activation and Ca2+ increase could be reproduced in silico. Low and high thrombin stimulation induced transient and sustained phosphorylation, respectively, of myosin light chain (MLC) and myosin phosphatase targeting subunit 1 (MYPT1) in vivo. The transient phosphorylation of MLC and MYPT1 could be reproduced in silico, but their sustained phosphorylation could not. This discrepancy between in vivo and in silico in the sustained responses downstream of Rho-kinase indicates that a missing pathway(s) may be responsible for the sustained Rho-kinase activation. We found, experimentally, that the sustained phosphorylation of MLC and MYPT1 exhibit all-or-none responses. Bromoenol lactone, a specific inhibitor of Ca2+ -independent phospholipase A2 (iPLA2), inhibited sustained phosphorylation of MLC and MYPT1, which indicates that sustained Rho-kinase activation requires iPLA2 activity. Thus, the systems analysis of the Rho-kinase pathway identified a novel iPLA2-dependent mechanism of the sustained Rho-kinase activation, which exhibits an all-or-none response.

Relocation of water molecules between the Schiff base and the Thr46-Asp96 region during light-driven unidirectional proton transport by bacteriorhodopsin: an FTIR study of the N intermediate.

A key event in light-driven proton pumping by bacteriorhodopsin is the formation of the L intermediate, whose transition to M is accompanied by the first proton transfer step, from the Schiff base to Asp85 on the extracellular side. Subsequent reprotonation of the Schiff base from the other side of the membrane to form the N intermediate is crucial for unidirectional proton transport. Previous FTIR studies have suggested that the intense water O-D stretching vibration bands which appear in L at 2589, 2605, and 2621 cm(-)(1) are due to a cluster of polarized water molecules connecting the Schiff base to the Thr46-Asp96 region closer to the cytoplasmic surface. In the present study the difference spectrum was obtained of the N intermediate with its photoproduct N', formed after irradiating N at 80 K. The water O-D stretching vibrations of N appear as a broad feature in a similar frequency region with a similar intensity to those of L. This feature is also affected by T46V like in L. However, the intensities of these water vibrations of N nearly returned to the initial unphotolyzed state upon formation of N', unlike those of L which are preserved in L'. An exception was V49A, which preserved the intense water vibrations of N in N'. The results suggest that both L and N have a water cluster extending from the Schiff base to Thr46. The surrounding protein moiety stabilizes the water cluster in L, but in N it is stabilized mostly by interaction with the Schiff base.