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Background: We conducted a phase II study to evaluate the efficacy and safety of dasatinib in patients with newly diagnosed chronic-phase chronic myeloid leukemia (CML-CP) in Japan (IMIDAS PART2 study; UMIN000006358). Several groups reported that some of CML patients who achieved stable deep molecular response (DMR) level or deeper could stop Tyrosine Kinase Inhibitor (TKI) and approximately 40% of these patients could keep therapy free survival by cessation of TKI. Discontinuation of TKI has emerged as a new goal of treatment in CML. Achievement of DMR is necessary for discontinuation of TKI. The aim of the present study was to analyze the prognostic significance of (i) BCR-ABL transcript International Scale (BCR-ABL IS) levels, (ii) the halving time and (iii) velocity of BCR-ABL transcript elimination using an optimized cutoff according to receiver operating characteristic (ROC) analysis. Methods: Eighty newly diagnosed CML-CP patients were included in this study. Patients received dasatinib 100mg once daily. Treatment has continued until disease progression or unacceptable toxicity. Clinical efficacy and safety was partially reported in 55th ASH Meeting. We sought to investigate the impacts of above 3 parameters within the initial 1 or 3 months of therapy. Halving time was calculated by the method, described by Branford et al. Velocity of BCR-ABL transcript elimination at 1 or 3 months (V-BCR-ABL1m or 3m respectively) was calculated as BCR-ABL IS at 1 or 3 months (BCR-ABL IS1m or 3m respectively) divided by that at diagnosis. Results: One patient was withdrawal before administration of dasatinib. Seventy-nine patients administered dasatinib 100 mg once daily. The estimated MMR and DMR rates were 92.1 % (95%CI, 76.8-97.3 %) and 60.9% (95%CI; 42.3-73.4 %) by 12 months respectively. The patients who had already achieved DMR at 3 months were excluded from landmark analysis. The cut off values for prediction of DMR at 12 months were obtained by ROC analysis. Those of BCR-ABL IS1m and BCR-ABL IS3m were 11.7% and 0.284% respectively. Those of halving times on 0-1 month and 0-3months (halving time1m and 3m) were 17.8 and 13.6 days respectively. Those of V-BCR-ABL1m and V-BCR-ABL3m were 0.321 and 0.018 respectively. The estimated DMR at 12 months, 95% CI and probability (P), obtained by Kaplan-Myer analysis, were shown in Figure 1. Odd' ratio, obtained by Chi-square test, was shown in Table 1. The patients with less than 0.321 at V-BCR-ABL1m showed the highest DMR at 12 months (80%), the least probability (P=0.009) and the least odd' ratio (0.175). At 3 months, there were similar data in these parameters among BCR-ABL IS3m, halving time3m and V-BCR-ABL3m. Figure 1 showed the cumulative DMR rate according to the cutoff values in V-BCR-ABL1m and V-BCR-ABL3m. V-BCR-ABL1m 0.321 and V-BCR-ABL3m 0.018 separated best. Conclusion: These data strongly suggested that V-BCR-ABL1m,3m would be a significant landmark to predict DMR at 12 months as well as BCR-ABL IS1m,3m, halving time1m,3m. Among them, less than 0.321 in V-BCR-ABL1m was identified as an optimized predictive cutoff value of DMR at 12 months. Disclosures Ishida: Bristol-Myers Squibb: Honoraria.

Background: Most patients with multiple myeloma (MM) are considered to be incurable, and relapse, due to minimal residual disease (MRD), is the main cause of death among these patients. Even though allele-specific oligonucleotide real-time quantitative PCR (ASO-qPCR) of immunoglobulin heavy chain gene rearrangement has been used to assess the MRD in MM due to its excellent sensitivity and specificity, ASO-qPCR has a major limitation in its relative quantification method because it needs a reference standard curve, which is usually made with dilutions of diagnostic myeloma DNA or from plasmids containing the target IgH rearrangement gene. Recently, droplet digital PCR (ddPCR) was developed to perform reliable absolute quantification of target genes. Based on the principle of single target gene detection, the sensitivity can be increased when the larger amount of DNA is analyzed. Here we assessed the prognostic value of MRD assessment in autografts from MM patients in the autologous stem cell transplantation (ASCT) setting using ASO-qPCR, ddPCR and next-generation sequencing (NGS) approaches. Methods: Twenty-three Japanese patients with newly diagnosed MM who received various induction regimens prior to ASCT without any post-ASCT therapy were retrospectively analyzed. Median age 57 (range 39-67); males 11, females 12; ISS 1 (n=7), 2 (n=12), 3 (n=3), not assessed (n=1). 11 patients were analyzed by G-banding and FISH (t(4;14), del17p, t(14;16)) and 4 patients showed high-risk chromosomal abnormalities (t(4;14) (n=2), t(14;16) (n=1), -13 by G-banding (n=1)). All patients had achieved a very good partial response (VGPR) or better after ASCT. Analyzed samples included: (1) BM slides from 20 MM patients at diagnosis, (2) fresh/frozen BM cells from 3 MM patients at diagnosis, and (3) obtained autografts. IGH-based ASO-qPCR was performed as described previously (Methods Mol Biol 2009). ddPCR was performed by the QX200 Droplet Digital PCR system (Bio-RAD Inc.) with a total 6000 ng of genomic DNA combined with the same ASO-primers and TaqMan-probes used in the ASO-qPCR. Droplets were generated by the QX200 droplet generator. End-point PCR (40 cycles) was performed on a C1000 Touch Thermal cycler (Bio-RAD Inc). The PCR product was loaded in the QX200 droplet reader and analyzed by QuantaSoft 1.7.4 (Bio-Rad Inc). NGS-based MRD assessment was performed using the immunosequencing platform (Adaptive Biotechnologies, South San Francisco, CA) (Martinez-Lopez et al Blood 2014). Results: Nineteen patients could be analyzed by ASO-qPCR and ddPCR, while all 23 patients could be analyzed by NGS. We compared MRD results in autografts between ddPCR and NGS. We observed a high correlation between ddPCR and NGS results of MRD (r=0.82, P Conclusions: In this study, we showed the prognostic value of ddPCR and NGS-based MRD assessment in autografts of patients with MM. Although the ddPCR had improved sensitivity in detecting MRD in autografts and demonstrated higher prognostic value compared with ASO-qPCR, the NGS platform showed the highest sensitivity and prognostic value among these methods. Disclosures Zheng: Adaptive Biotechnologies Corp: Employment, Equity Ownership. Moorhead:Adaptive Biotechnologies Corp: Employment, Equity Ownership. Faham:Adaptive Biotechnologies Corp.: Employment, Other: Stockholder.

PO-272 Survivin suppressant YM155 induces cell death via proteasomal degradation of c-Myc in multiple myeloma cells M. Asahi, S. Ito, Y. Ishida Hematology & Oncology, Department of Internal Medicine, Department of Medical Oncology, Iwate Medical University School of

e242 [U266 (p53 mutant), OPM-2 (p53 mutant), KMS-18 (p53 WT/-), JJN3(p53 WT/-), MM.1S (p53 WT)]. Effects on downstream substrates were analysed by Western blotting. Elevated HUWE1 expression was confirmed in MM cell lines and primary CD138+ MM cells compared to healthy CD138+ cells at both the gene and protein level ( 2-fold). shRNA knockdown of HUWE1 significantly decreased (p 0.01) the proliferation of all MM cell lines, regardless of p53 status; this effect was maintained when cells were cultured in the presence of bone marrow stromal cells. Conversely, we show that MM cell lines that carry mutant p53 were largely resistant to the MDM2 inhibitor Nutlin-3. Finally, we have demonstrated that HUWE1 depletion is associated with a significant increase of cells in G2/M, an accumulation of p53 and MIZ1 and a decrease in MYC protein levels (p 0.05). The proteasome is an established target in MM and there are opportunities for more specific targeting of enzymes within the UPS. Here we demonstrate that the E3 ligase HUWE1 is important in the growth and survival of MM cells and offers a more valuable therapeutic target than MDM2. Future work will investigate the effect of small molecule inhibitors of HUWE1 in MM.

e214 Conclusions: Our results demonstrate the potent, rapid activity of filanesib which induces a cell cycle blockade through the inhibition of KSP, leading to apoptosis in MM. Furthermore, this agent showed significant synergy with pomalidomide and dexamethasone, what supports the clinical ““Pomdefil”” trial, recently activated in the context of the Spanish Myeloma Group (GEM).

We analyzed cytosolic high-Km 5'-nucleotidase (cN-II) and deoxycytidine kinase (dCK) mRNA expression in bone marrow mononuclear cells (BMMNC) of patients with high-risk myelodysplastic syndrome (MDS) using quantitative real-time polymerase chain reaction (rt-PCR). At diagnosis, the cN-II mRNA expression of patients was higher than that of healthy volunteers, but the dCK mRNA expression showed no significant difference. Patients with ara-C-containing chemotherapies whose BMMNC showed a high level of cN-II expression (greater than the median value) had shorter median overall survival (15 months versus 22 months, p0.01) and shorter median post-chemotherapy survival (10 months versus 16 months, p=0.012). These data suggest that the expression level of cN-II mRNA might be a prognostic factor of high-risk MDS.

The surface property of commercially pure titanium (cpTi) was improved by forming a thin hydroxyapatite (HA) layer by anodic oxidation and hydrothermal treatment (HA/cpTi). We hypothesize that the adhesion of macrophages to HA/cpTi surfaces is important to the process of osseointegration. This study investigates the effect of adhesion of macrophages to HA/cpTi surfaces on the expression of bone morphogenetic protein-2 (BMP-2). The murine macrophage cell line J774A.1 was cultured on HA/cpTi and polished cpTi (S/cpTi). Macrophage cell adhesion was examined by SEM, 0-72 h following plating onto HA/cpTi and S/cpTi. BMP-2 gene expression was examined by RT-PCR analysis. The level of BMP-2 secreted into the supernatant was measured using an ELISA assay. The extent of macrophage adhesion increased with time on both the HA/cpTi and S/cpTi surfaces, with a" higher degree of spreading observed on HA/cpTi than onS/cpTi surfaces after 24 or 72 h. The ratio of BMP-2 mRNA was higher on HA/cpTi than on S/cpTi after 24 h (0.348 vs. 0, p < 0.05) and 72 h (0.584 vs. 0.189, p < 0.05). After 24 h, secretion of BMP-2 was detected in cultures grown on HA/cpTi, but not on S/cpTi. After 72 h, secretion of BMP-2 was detected in cultures grown on S/cpTi, but the levels were higher in cultures grown on HA/cpTi. These findings show that macrophages have the capacity to adhere to HA/cpTi endosseous implants and provide a source of osteoinductive cytokines that may play a key role in the process of osseointegration.

Minimally invasive direct coronary artery bypass grafting (MIDCAB) using an H graft was performed on a 74-year-old man with advanced prostatic cancer who needed coronary revascularization. Through a left anterior small thoracotomy, the left internal thoracic artery (LITA) and the left anterior descending artery (LAD) were cleared, and a short radial artery (RA) was placed in an end-to-side fashion between the LITA and LAD. The distal LITA was ligated to avoid potential steal phenomenon. A flow pattern through the RA graft evaluated by transit time flow measurements demonstrated good diastolic flow with a mean value of 37 mL/min. The total surgical duration was 80 min, and no blood products were required. A postoperative angiogram showed a widely patent H graft. The patient was relieved of chest pain and was discharged. The H graft procedure is a useful alternative technique to minimize the surgical trauma in limited situations such as a high-risk case.

High frequency electromagnetic fields in the 120 kHz band emitted from card readers for access control systems in radiation control areas cause abnormally high and erroneous indicated dose readings on semiconductor-type electronic personal dosimeters (SEPDs). All SEPDs malfunctioned but recovered their normal performance by resetting after the exposure ceased. The minimum distances required to prevent electromagnetic interference varied from 5.0 to 38.0 cm. The electric and magnetic immunity levels ranged from 35.1 to 267.6 V m(-1) and from 1.0 to 16.6 A m(-1), respectively. Electromagnetic immunity levels of SEPDs should be strengthened from the standpoint of radiation protection.

Background Previous clinical studies have documented successful neck pain relief in whiplash patients using nerve block and radiofrequency ablation of facet joint afferents, including capsular ligament nerves. No previous study has documented injuries to the neck ligaments as determined by altered dynamic mechanical properties due to whiplash. The goal of the present study was to determine the dynamic mechanical properties of whiplash-exposed human cervical spine ligaments. Additionally, the present data were compared to previously reported control data. The ligaments included the anterior and posterior longitudinal, capsular, and interspinous and supraspinous ligaments, middle-third disc, and ligamentum flavum. Methods A total of 98 bone-ligament-bone specimens (C2–C3 to C7-T1) were prepared from six cervical spines following 3.5, 5, 6.5, and 8 g rear impacts and pre- and post-impact flexibility testing. The specimens were elongated to failure at a peak rate of 725 (SD 95) mm/s. Failure force, elongation, and energy absorbed, as well as stiffness were determined. The mechanical properties were statistically compared among ligaments, and to the control data (significance level: P < 0.05; trend: P < 0.1). The average physiological ligament elongation was determined using a mathematical model. Results For all whiplash-exposed ligaments, the average failure elongation exceeded the average physiological elongation. The highest average failure force of 204.6 N was observed in the ligamentum flavum, significantly greater than in middle-third disc and interspinous and supraspinous ligaments. The highest average failure elongation of 4.9 mm was observed in the interspinous and supraspinous ligaments, significantly greater than in the anterior longitudinal ligament, middle-third disc, and ligamentum flavum. The average energy absorbed ranged from 0.04 J by the middle-third disc to 0.44 J by the capsular ligament. The ligamentum flavum was the stiffest ligament, while the interspinous and supraspinous ligaments were most flexible. The whiplash-exposed ligaments had significantly lower (P = 0.036) failure force, 149.4 vs. 186.0 N, and a trend (P = 0.078) towards less energy absorption capacity, 308.6 vs. 397.0 J, as compared to the control data. Conclusion The present decreases in neck ligament strength due to whiplash provide support for the ligament-injury hypothesis of whiplash syndrome.

Study Design. A biomechanical study of intervertebral foraminal narrowing during simulated automotive rear impacts. Objectives. To quantify foraminal width, height, and area narrowing during simulated rear impact, and evaluate the potential for nerve root and ganglion impingement in individuals with and without foraminal spondylosis. Summary of Background Data. Muscle weakness and paresthesias, documented in whiplash patients, have been associated with neural compression within the cervical intervertebral foramen. To our knowledge, no studies have comprehensively examined dynamic changes in foramen dimensions. Methods. There were 6 whole cervical spine specimens (average age 70.8 years) with muscle force replication and surrogate head that underwent simulated rear impact at 3.5, 5, 6.5, and 8 g, following noninjurious baseline 2 g acceleration. Peak dynamic narrowing of foraminal width, height, and area were determined during each impact and statistically compared to baseline narrowing. Results. Significant increases (P 0.05) in average peak foraminal width narrowing above baseline were observed at C5‐C6 beginning with 3.5 g impact. No significant increases in average peak foraminal height narrowing were observed, while average peak foraminal areas were significantly narrower than baseline at C4‐C5 at 3.5, 5, and 6.5 g. Conclusions. Extrapolation of the present results indicated that the highest potential for ganglia compression injury was at the lower cervical spine, C5‐C6 and C6‐C7. Acute ganglia compression may produce a sensitized neural response to repeat compression, leading to chronic radiculopathy following rear impact.

Whole cervical spine model with muscle force replication was subjected to simulated frontal impacts of increasing severity, and resulting injuries were evaluated via flexibility testing.To identify and quantify cervical spine soft tissue injury and the injury threshold acceleration due to frontal impact.Cervical spine instability may result from automotive collisions. No previous studies have quantified soft tissue injuries due to frontal impact.Six human cervical specimens (occiput-T1) with muscle force replication were subjected to frontal impacts of 4, 6, 8, and 10 g. Before frontal impact, baseline flexibility data were collected following a 2 g simulation. Flexibility parameters of total (flexion plus extension) neutral zone (NZ), flexion NZ, total range of motion (ROM), and flexion ROM were obtained following each impact and compared with baseline flexibility. Injury was a significant increase (P0.05) in intervertebral flexibility due to frontal impact over baseline. Injury threshold was the lowest T1 peak acceleration that caused injury.The injury threshold acceleration was 8 g, as determined by significant increases of 12.6 to 51.4% over the baseline flexibility, in the C4-C5 total NZ, and the C6-C7 total NZ, flexion NZ, total ROM, and flexion ROM. Following 10 g, significant increases in flexibility parameters were observed at C2-C3, C3-C4, C4-C5, C6-C7, and C7-T1.Middle (C2-C3 to C4-C5) and lower (C6-C7 and C7-T1) cervical spine were at risk for injury during frontal impacts, for the experimental conditions studied.

The Intervertebral Neck Injury Criterion (IV-NIC) is based on the hypothesis that dynamic intervertebral motion beyond physiological limits may injure soft tissues. In contrast, the Neck Injury Criterion (NIC) hypothesizes that sudden change in spinal fluid pressure may cause neural injuries. The goals of this study, using the biofidelic whole human cervical spine model with muscle force replication, were to determine the IV-NIC injury threshold due to frontal impact at each intervertebral level, and to compare the IV-NIC and NIC in determining injury.Using a bench-top apparatus, frontal impacts were simulated at 4, 6, 8, and 10 g horizontal accelerations of the T1 vertebra. Pre- and post-trauma flexibility testing measured the soft tissue injury; that is, a significant increase (p0.05) in neutral zone or range of motion at any intervertebral level, above the corresponding physiological limit.Results indicated that the soft tissue injury occurred due to flexion mode of injury and its threshold was 8 g. The average IV-NIC injury threshold (95% confidence interval) was 2.0 (1.2-2.8) at C4-C5 and 2.3 (1.6-3.0) at C6-C7, while the average NIC injury threshold was 18.4 (17.9-19.0) m(2)/s(2). The NIC injury threshold was reached significantly earlier than all the IV-NIC injury thresholds, demonstrating that the NIC may be unable to predict facet and soft tissue injury caused by non-physiologic inververtebral rotation.Present results suggest that IV-NIC is an effective tool for determining soft tissue neck injuries by identifying the intervertebral level, mode, time, and severity of injury.

A 54-year-old woman, who had undergone pancreatoduodenectomy with resection of the portal vein and intraoperative radiation therapy for cancer of the lower bile duct 16 months before, visited our institution complaining of melena. To identify the cause of bleeding and severe anemia, we performed gastrointestinal endoscopy but could detect no obvious source. The portal phase of the superior mesenteric arteriography and percutaneous transhepatic portography revealed severe stenosis of the extrahepatic portal vein, which corresponded to the end-to-end anastomosis of the portal vein, and hepatofugal collaterals. Extravasations into the afferent loop of the jejunum were detected only with portography. These findings suggested that portal hypertension due to extrahepatic portal obstruction led to bleeding varices. Subsequent to percutaneous transhepatic portography, we dilated the stenosis of the extrahepatic portal vein using a balloon catheter and placed an expandable metallic stent there. Portography after the treatment revealed the disappearance of the hepatofugal flow to collaterals and extravasations, and the patient has had no further episodes of gastrointestinal bleeding since. In conclusion, for patients with bleeding varices due to extrahepatic portal obstruction, especially after abdominal surgery, percutaneous transhepatic angioplasty is considered to be the treatment of choice because of its efficiency and minimal invasiveness.

Background Aortic surgery is an invasive, high-risk noncardiac procedure and the patients who require it have a high prevalence of coronary artery disease. Therefore, preoperative risk stratification for this subset is essential. Methods and Results To assess the perioperative risk for aortic surgery, pharmacologic stress single-photon emission computed tomography (SPECT) was performed in 302 patients: aortic dissection in 56, thoracic aortic aneurysm in 124, and abdominal aortic aneurysm in 122. Not only was the presence or absence of perfusion defects analyzed, but also the 20-segment model. Pharmacologic thallium SPECT revealed negative findings in 210 patients and positives in 92. Perioperative cardiac events occurred in 9 patients: 7 occurred in patients with positive SPECT, and in only 2 of those with negative SPECT (2/210 vs 7/92; p

Background Native flow competition is a significant factor affecting bypass graft patency. The objective of this study was to compare the effect of competitive flow on conduit flow dynamics in the gastroepiploic artery (GEA) and the saphenous vein graft (SVG). Methods In 51 patents, 23 GEAs (in-situ grafts) and 28 SVGs (aortocoronary grafts) were examined using a Doppler-tipped guidewire during coronary angiography after coronary artery bypass. Graft flow volume at rest and maximum graft flow volume during hyperemia were calculated from graft diameter and average peak velocity at rest and maximum average peak velocity induced by papaverine hydrochloride injection. Grafts were classified according to the grade of native coronary artery stenosis; group S (14 GEAs and 16 SVGs) displayed over 75% stenosis and group M (9 GEAs and 12 SVGs) exhibited over 50% up to 75% stenosis. Results In group S, no difference in flow volume was apparent between the GEA and the SVG at rest (36± 17 vs 42 ± 16) and during hyperemia (78 ± 30 vs 88 ± 28). In group M, flow volume of the GEA was significantly lower than that of the SVG at rest (17 ± 11 vs 38 ± 12; p = 0.029) and during hyperemia (32 ± 19 vs 94 ± 46; p = 0.001). Conclusions These data suggest that in intermediate coronary stenosis, GEA flow is compromised by native flow competition, whereas the SVG flow dynamics is maintained. However, the GEA can provide comparable flow capacity to the SVG and will achieve good surgical results when target coronary artery selection is appropriate.

The biological significance of CD56 antigen expression in patients with acute promyelocytic leukemia (APL) has been under investigation. We investigated the clinical and biologic features of CD56 + APL. In our series, CD56 antigen was positive in 4 of 28 (14%) APL patients. No differences were found regarding age, gender, performance status (PS), initial leukocyte and platelet counts, lactate dehydrogenase (LDH) and fibrinogen (Fbg) levels according to CD56 expression. CD34 antigen was co-expressed in 3 of the 4 patients with CD56 ' APL, in contrast to 2 of the 24 patients with CD56 - APL (P = .01). Extramedullary relapse occurred in 3 of the 4 patients with CD56 + APL, in contrast to none of the 24 patients with CD56 - APL (P = .001). Median remission duration was 4 months in CD56 + APL and was not reached in CD56 APL. The CD56 + population had a shorter remission duration (P

Study Design. A biofidelic whole cervical spine model with muscle force replication was used to evaluate spinal canal pinch diameter (CPD) narrowing during simulated whiplash. Objectives. To quantify CPD narrowing during simulated whiplash and to determine if whiplash resulted in a narrower post-whiplash CPD. Summary of Background Data. Spinal cord injuries are uncommon in whiplash patients, although such injuries have been reported in those with narrow canals. It has been hypothesized mat increased cerebral spinal fluid pressure during whiplash could injure neural tissues. Methods. The biofidelic model and a bench-top whiplash apparatus were used to simulate whiplash at 3.5, 5, 6.5, and 8 g accelarations, of the T1 vertebra. The CPD was measured in the intact specimen in the nautral pasture (neutral posture CPD) and under a 1.5 Nm static extension load (pre-whiplash CPD), during simulated whlplasn (dynamic whipiash CPD), and again under a 1.5 Nm extension load following each whiplash simulation (post-whiplash CPD). Results. The average dynamic whiplash CPDs were significantly narrower (P < 0.05) than the corresponding pre-whiplash CPDs at accelerations of 3.5 g and above. The narrowest CPD was observed at C5-C6 during the 8.5 g simulation and was 3.5 mm narrower than the neutral posture CPD. in general, the average post-whiplash CPDs were not significantly narrower than the corresponding pre-whipfash CPDs. Conclusions. Spinal cord injury during whiplash is unlikely in patients with average normal canal diameters. Cord compression following whiplash due to physiologic extension loading is not likely. Previous clinical studies have found that whiplash patients with narrow canals may be at risk of injury, and our results do not disprove it.

STUDY DESIGN A newly developed biofidelic whole cervical spine (WCS) model with muscle force replication (MFR) was subjected to whiplash simulations of varying intensity, and the resulting injuries were evaluated through changes in the intervertebral flexibility. OBJECTIVES To identify the soft tissue injury threshold based on the peak T1 horizontal acceleration and the association between acceleration magnitude and injury severity resulting from simulated whiplash using the WCS + MFR model. SUMMARY OF BACKGROUND DATA Whiplash has been simulated using mathematical models, whole cadavers, volunteers, and WCSs. The measurement of injury (difference between prewhiplash and postwhiplash flexibilities) is possible only using the WCS model. METHODS Six WCS + MFR specimens (C0-T1) were incrementally rear-impacted at nominal T1 horizontal maximum accelerations of 3.5, 5, 6.5, and 8 g, and the changes in the intervertebral flexibility parameters of neutral zone and range of motion were determined. The injury threshold acceleration was the lowest T1 horizontal peak acceleration that caused a significant increase in the intervertebral flexibility. RESULTS The first significant increase (P

The Intervertebral Neck Injury Criterion (IV-NIC) is based on the hypothesis that intervertebral motion beyond the physiological limit may injure spinal soft tissues during whiplash, while the Neck Injury Criterion (NIC) hypothesizes that sudden changes in spinal fluid pressure may cause neural injury. Goals of the present study, using a biofidelic whole cervical spine model with muscle force replication, were to correlate IV-NIC with soft-tissue injury, determine the IV-NIC injury threshold, and compare IV-NIC and NIC. Using a bench-top apparatus, rear-impacts were simulated at 3.5, 5, 6.5, and 8 g horizontal accelerations of the T1 vertebra. Pre- and post-whiplash flexibility tests measured the soft tissue injury threshold, i.e. significant increases in the intervertebral neutral zone (NZ) or range of motion (ROM) above corresponding baseline values. Extension IV-NIC peaks correlated well with NZ and ROM increases at C0–C1 and at C3–C4 through C7–T1 (r=0.64 and 0.62 respectively, p

Objectives: Graft failure has been reported when the arterial conduit, such as the internal thoracic artery (ITA) or the right gastroepiploic artery (GEA), is grafted to a lower grade coronary artery stenosis. The shear stress as a significant factor affecting graft patency was compared between the arterial conduit and the saphenous vein graft (SVG) after surgery. Methods: In 101 patients, 40 ITAs, 27 GEAs and 34 SVGs were examined using a Doppler-tipped guide wire during postoperative angiography. The graft flow volume and shear stress were calculated from velocity and diameter data. The study grafts were classified according to the grade of native coronary artery stenosis: group L had more than 50 up to 75% stenosis, and group H had more than 75% stenosis. Group H consisted of 25 ITAs, 17 GEAs and 21 SVGs, while group L consisted of 15 ITAs, 10 GEAs and 13 SVGs. Results: In group H, graft flow volume did not significantly differ among the ITA (34 ^ 11 ml/min), GEA (36 ^ 16 ml/min) and SVG (41 ^ 15 ml/min), and graft shear stress significantly (ITA vs. GEA P , 0:0001; GEA vs. SVG P , 0:01) differed among the ITA (16.0 ^ 4.8 dyn/cm 2 ), GEA (9.1 ^ 3.2 dyn/cm 2 ) and SVG (4.8 ^ 1.6 dyn/cm 2 ). In group L, flow volume was lower ðP , 0:001Þ in the ITA (18 ^ 6 ml/min) and GEA (13 ^ 8 ml/min) than in the SVG (35 ^ 16 ml/min), and shear stress was significantly ðP , 0:001Þ greater in the ITA (13.7 ^ 4.9 dyn/cm 2 ) than the GEA (5.6 ^ 2.0 dyn/cm 2 ) or SVG (4.6 ^ 2.0 dyn/cm 2 ). Conclusions: These data suggest that shear stress of the ITA is superior and maintained despite the flow volume being reduced by flow competition. Lower shear stress of the GEA for intermediate stenosis may be associated with the development of conduit failure. q 2003 Elsevier B.V. All rights reserved.

Cervical disc injury due to frontal impact has been observed in both clinical and biomechanical investigations; however, there is a lack of data that elucidate the mechanisms of disc injury during these collisions. The goals of the current study were to determine the peak dynamic disc annular tissue strain and disc shear strain during simulated frontal impact of the whole human cervical spine model with muscle force replication at 4 g, 6 g, 8 g and 10 g horizontal accelerations of the T1 vertebra. These data were compared with those obtained during physiological loading, and with previously reported rear impact data. Peak disc shear strain and peak annular tissue strain during frontal impact exceeded (p

Whiplash has been simulated using volunteers, whole cadavers, mathematical models, anthropometric test dummies, and whole cervical spines. Many previous in vitro whiplash models lack dynamic biofidelity. The goals of this study were to (1) develop a new dynamic whole cervical spine whiplash model that will incorporate anterior, lateral and posterior muscle force replication, (2) evaluate its performance experimentally and (3) compare the results with in vivo data. To evaluate the new model, rear-impact whiplash simulations were performed using the incremental trauma approach at maximum measured T1 horizontal accelerations of 3.6 g, 4.7 g, 6.6 g, and 7.9 g. The kinematic response of the new model, e.g., peak head–T1 extension and peak intervertebral rotations, were compared with the corresponding in vivo data. The average peak head–T1 extension was within the in vivo corridor during the 3.6 g whiplash simulation (9.1 kph delta V). The peak in vivo intervertebral rotations obtained during a 4.6 g whiplash simulation of a young volunteer were within, or only marginally in excess of, the 95% confidence limits of the average peak intervertebral rotations measured during the 4.7 g whiplash simulation of the present study. Thus, the new whole cervical spine model with muscle force replication produced biofidelic dynamic responses to simulated whiplash. The new model is capable of generating important biomechanical data that may help improve our understanding of whiplash injuries and injury mechanisms.

We describe unusual cytogenetic findings in a 33-year-old male with blastic phase of Philadelphia chromosome (Ph)-positive chronic myeloid leukemia. In addition to the t(9;22)(q34;q11), which was detected in all metaphases, a t(11;19)(q23;p13.3) was also identified as an evolutional change in all 20 metaphases. Fluorescence in situ hybridization (FISH) analysis showed that fusion signals of the ABL/BCR probes were found in 95% of blastic cells. Southern blotting and FISH analysis also revealed involvement of the MLL gene on 11q23. Clinical course was aggressive and the patient responded poorly to therapy. These findings suggest an association between Ph and 11q23 with poor prognosis, and that t(11;19)(q23;p13.3) was the essential pathogenic factor in our case.

We isolated bacterial strains tolerant to high concentrations of oleic acid from soil samples, and studied biotransformation of fatty acids with the bacteria. We found that a strain BL0–3 identified as Micrococcus luteus metabolized oleic acid to 10-hydroxystearic acid, 10-oxostearic acid, and 4-oxolauric acid. Palmitoleic acid and myristoleic acid also served as substrates in the biotransformation, and 10-oxopalmitic acid and 10-oxomyristic acid were produced, respectively. The formation of 4-oxolauric acid from oleic acid was inhibited by compounds which inhibit β-oxidation of fatty acids.

Anterior longitudinal ligament (ALL) injuries following whiplash have been documented both in vivo and in vitro; however, ALL strains during the whiplash trauma remain unknown. A new in vitro whiplash model and a bench-top trauma sled were used in an incremental trauma protocol to simulate whiplash at 3.5, 5, 6.5 and 8 g accelerations, and peak ALL strains were determined for each trauma. Following the final trauma, the ALLs were inspected and classified as uninjured, partially injured or completely injured. Peak strain, peak intervertebral extension and increases in flexibility parameters were compared among the three injury classification groups. Peak ALL strains were largest in the lower cervical spine, and increased with impact acceleration, reaching a maximum of 29.3% at C6-C7 at 8 g. Significant increases (P

Aim: To investigate the diagnostic accuracy of power Doppler ultrasonography (US) in assessing the vascular invasion by pancreatic cancer. Methods: A prospective study of 40 consecutive patients with pancreatic cancer (head 35, body 5) was performed. All patients underwent surgery. The relationships between tumor and each vessel were classified into four types according to the closest circumferential contact of the tumor with the vessel. A type 0 indicated no contact; type 1 indicated less than one third contact; type 2 indicated one third to 99% contact, and type 3 indicated encasement. Vascular invasion was diagnosed in types 2 and 3. The diagnostic accuracy was evaluated in the portal vein and in the splanchnic arteries (celiac artery, common hepatic artery, and superior mesenteric artery). The power Doppler US findings were confirmed by the operative findings. The results of power Doppler US were compared with those of CT scan and angiography. Results: Portal vein invasion was confirmed in resected specimens in 23 cases and by operative findings in 5 cases. For the diagnosis of portal vein invasion, sensitivity, specificity, and overall accuracy of power Doppler US were, respectively, 79.3, 90.9, and 82.5%. The respective values were 79.3, 100, and 85% for CT and 72.4, 81.8, and 75% for angiography. For the diagnosis of arterial invasion, sensitivity, specificity, and overall accuracy of power Doppler US were 80, 92, and 90%, respectively. The corresponding values were 47, 88, and 73% for CT and 47, 100, and 80% for angiography. Conclusion: Power Doppler US proved to be useful for the diagnosis of vascular invasion by pancreatic cancer.

Purpose The aim of this study was to evaluate the accuracy of sonography for measuring the volume of the left lateral segment of the liver. Methods The volume of the left lateral segment of the liver was measured with sonography in 101 consecutive patients who were hospitalized between December 1998 and January 2000 for hepatic, biliary, or pancreatic disease or who were donors for a living related liver transplantation. The results were compared with those obtained using CT. Results The mean calculated volume of the left lateral segment of the liver ± standard deviation was 261 ± 118 cm3 by sonography compared with 274 ± 123 cm3 by CT. The relationship between the results of both imaging modalities was linear and statistically significant (r = 0.93; p < 0.0001). Conclusions Our study demonstrated that sonography has an acceptable level of accuracy and reliability for routine measurement of the volume of the left lateral segment of the liver. © 2002 Wiley Periodicals, Inc. J Clin Ultrasound 30:117–122, 2002; DOI 10.1002/jcu.10050

BACKGROUND: Febrile neutropenia (FN) is often observed in cases of hematological malignancy such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and myelodysplastic syndrome (MDS). Carbapenem antibiotics having a potent and broad antimicrobial activity against both gram-positive and gram-negative bacteria including Pseudomonas aeruginosa, are good candidates as the first-line therapy drug in high-risk FN patients. In the IDSA guideline 2010 on the use of antimicrobial agents in FN, mono-therapy with an anti-pseudomonal beta-lactam, such as piperacillin-tazobactam, cefepime or carbapenem [only meropenem (MEPM) and imipenem-cilastatin], is recommended as an empirical therapy for high-risk FN patients. Doripenem (DRPM) is a newer carbapenem with few data available as for the efficacy and safety in the setting of FN patients. Therefore, we conducted a randomized, cooperative group, open-label trial comparing DRPM (1.0 g every 8 hours) with MEPM (1.0 g every 8 hours) as the first-line empirical antibacterial therapy for high-risk FN patients with hematological malignancy including AML, ALL and high-risk MDS. PATIENTS and METHODS: One hundred and forty-six hospitalized high-risk FN patients with hematological malignancy (AML 76, ALL 43, APL 13, MDS-AML 9, MDS-RAEB 5 cases) during or after chemotherapy were randomized to each drug group (DRPM: n=73, MEPM: n=73). The study drug was started to administer as a mono-therapy and continued at least for 5 days without drug toxicity, and the efficacy and safety were evaluated. RESULTS: The overall response rate at 7 days in DRPM and MEPM group were not significantly different (DRPM: 67.6%, MEPM: 52.9%, respectively, P=0.098). Both the resolution of fever by mono-therapy at day 3 to 5 (DRPM: 56.9%, MEPM: 47.0%, respectively, p=0.26), and survival at day 30 (DRPM: 98.4%, MEPM: 98.5%, respectively, p=0.312) were not significantly different in the two groups. The frequency of cases needed for anti-MRSA agent and antifungal agent were a little less often in DRPM rather than in MEPM group [DRPM: 36.9%, MEPM: 50.0% (p=0.185), DRPM: 26.1%, MEPM: 32.3% (p=0.535), respectively]. Only grade 1-2 adverse events were observed in both groups (liver dysfunction, renal dysfunction, diarrhea and rash), and they were less often in DRPM group significantly (DRPM: 29.8%, MEPM: 40.8%, respectively, p=0.046). These adverse events were clinically acceptable in the two groups, and most of patients could continue the treatment by both study drugs. CONCLUSIONS: Our clinical study suggested that DRPM had the non-inferiority of efficacy in comparison with MEPM as the first-line empirical therapy in high-risk FN patients with hematological malignancy, and both drugs could be generally well tolerated. Disclosures No relevant conflicts of interest to declare.

Regulatory T (Treg) cells are a subset of CD4+ T cells that play key roles in the maintenance of immunologic self-tolerance and negative control of immune responses. Treg cells are defined based on expression of CD4, CD25, and transcription factor forkhead box P3 (FoxP3). FoxP3 controls the development and function of Treg cells and can be used as a reliable marker of Treg cells, however, its intracellular localization prohibits its use for the isolation of viable Treg cells. Most FoxP3+ Treg cells express low levels of CD127 in adult peripheral blood, however, the expression of CD127 in cord blood (CB) Treg cells of preterm and term infants at each gestational age remains unknown. Being that CD127 is a cell surface marker, it enables the isolation of viable Treg cells by flow cytometric sorting for further analysis. We analyzed CD4+CD25+FoxP3+ and CD4+CD25+CD127− T cells in preterm and term CB at various gestational ages. CB samples were obtained at Iwate Medical University hospital from preterm and term neonates who had no hereditary disorders, hematologic abnormalities, or immunodeficiency diseases. Of 103 total samples, 36 were obtained from preterm (24–36 gestational weeks) neonates and 67 were obtained from term (37–41 gestational weeks) neonates. Out of 36 preterm infants, 9 were diagnosed with chorioamnionitis histologically. None of the mothers involved in the study had any specific underlying diseases. CB mononuclear cells were isolated and analyzed by FACSCalibur flow cytometer and CellQuestPro software (BD Biosciences). No differences were observed in the proportions of CD25+FoxP3+ (median 4.5%) and CD25+CD127− (median 5.0%) cells in the CD4+ T-cell population. A strong correlation was found between the proportions of CD25+FoxP3+ and CD25+CD127− cells in the CD4+ T-cell population (r=0.96). The proportion of CD25+FoxP3+ cells in the CD4+ T-cell population in the CB of neonates at 24-26, 27-29, and 30-32 gestational weeks was significantly higher than in the CB of neonates at 37-38 and 39-41 gestational weeks. The proportion of CD25+CD127− cells in the CD4+ T-cell population in the CB of neonates at 24-26, 27-29, and 30-32 gestational weeks was also higher than in the CB of neonates at 37-38 and 39-41 gestational weeks. The absolute number of both CD4+CD25+FoxP3+ and CD4+CD25+CD127− T cells did not differ between the CB of preterm and term neonates. Absolute lymphocyte counts in preterm CB were significantly lower than term infants (P We showed that CD127 is preferable to FoxP3 as a marker for identifying Treg cells in CB of various gestational ages and would be useful for separating Treg cells by flow cytometry for therapeutic applications. The proportions of Treg cells in the CB of preterm infants were significantly higher than in the CB of term infants. The high Treg proportion during early gestation suggests that fetal naive T cells have a high propensity to differentiate into Treg cells in response to antigenic stimulation, such as by maternal alloantigens. The functional implications of the high proportion of Treg cells in preterm CB are unknown but may be important for the suppression of undesirable alloimmune reactions to maternal derivatives in utero and the maintenance of tolerance during pregnancy. Disclosures No relevant conflicts of interest to declare.

Background: Immune thrombocytopenia (ITP) is an autoimmune disease in which anti-platelet antibody (APA) is produced. APA-coated platelets are captured and phagocytized by macrophages in the spleen. Recent study revealed that spleen tyrosine kinase (Syk) inhibitor is effective in the treatment of ITP, because Syk phosphorylation is the key step of the phagocytosis by macrophages. Activated Fc receptor signal transduction is initiated by phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) tyrosine residues by SRC family kinases. Recruitment of Syk to dually phosphorylated ITAMs triggers the activation of Syk. To prove the hypothesis that the inhibition of SRC family kinase induces the decreased phosphorylation of Syk, resulting in decreased phagocytosis by macrophages, a SRC family kinase inhibitor, dasatinib, was used in the experiments. Methods, Results and Discussion: In vitro study; Murine macrophage cell line, RAW, was incubated with APA coated murine platelets for 30 minutes. Phagocytosis by RAW was significantly decreased with dasatinib (100nM, p In vivo study; (1) Three hours before APA intra-peritoneal injection, dasatinib (2.5mg/kg) was oral-administrated. Six hours after APA injection, platelet counts were measured. The platelet counts were 366 ± 164 x109/L with dasatinib (n=4, mean ± SD) and 114 ± 51 x109/L without dasatinib (n=4)(P=0.026)(Figure 2). (2) Osmotic pump, filled with APA, were inserted in murine intra-peritoneal cavity and dasatinib (2.5mg/kg) was oral-administered once daily for 7 days. The platelet counts were 499 ± 98 x109/L with dasatinib (n=4, mean ± SD) and 82 ± 131 x109/L without dasatinib (n=4) at day 7 (p Conclusion: Dasatinib inhibits phosphorylation of Syk, inducing decreased phagocytosis of APA-coated platelets via decreased SRC family kinase activity. These findings reveal that SRC family kinase controls the efficiency of phagocytosis in part through the regulation of Syk function. Dasatinib might be effective in the treatment of ITP. Disclosures No relevant conflicts of interest to declare.

Introduction: The immunophenotypic analysis of plasma cells using flow cytometry (FCM) is useful for the diagnosis for multiple myeloma (MM) and the detection of MM cells after treatment. CD138 is a well-known surface marker identifying plasma cells, although decreased CD138 expression on plasma cells is frequently observed in MM patients with relapsed or progressive disease. Thus, more stable MM antigens are needed. The SLAM family molecule CD229 has recently been reported to be specifically overexpressed on MM cells including their clonogenic precursors, suggesting that it may be a good marker to identify MM cells. Furthermore, some surface antigens, i.e., costimulatory molecule CD86 (B7-2), its receptor CD28, IL-6 receptor (CD126), insulin-like growth factor-1 (CD221), and coinhibitory molecule B7-H1 (PD-L1, CD274), were reported to be associated with poor prognosis. This study aimed to validate the potential of CD229 as a new MM cell marker and the efficacy of immunophenotypes on MM cells as prognostic markers in a multicenter study. Patients & Methods: Two-hundred thirteen patients comprising 144 newly diagnosed (18 asymptomatic and 126 symptomatic) MM patients, 25 refractory/relapsed MM patients, and 44 monoclonal gammopathy of undetermined significance (MGUS) patients were enrolled. Immunophenotyping of plasma cells in bone marrow was performed with standard 3-color FCM, in which plasma cells were gated by CD38-highly positive cells and 9 parameters, i.e., expression of MM antigens (CD138, CD229) and prognosis-related antigens previously reported (CD28, CD45, CD56, CD86, CD126, CD221, CD274) on MM cells, were analyzed. Expression levels of prognosis-related antigens were compared between patients in high-risk categories, i.e., International Scoring System (ISS) stage II/III, high-risk chromosomal abnormalities [t(4:14), t(14:16), del17p] by FISH, high-risk group stratified by the International Myeloma Working Group (IMWG) [ISS stage II/II and t(4:14) and/or del17p], high serum lactase dehydrogenase (LDH) level, or revised ISS stage III, and other patients. The revised ISS is a powerful new tool to predict outcome in MM patients treated with novel agents based on 3 factors: serum LDH level, ISS stage, and high-risk chromosomal abnormalities [Oliva S, et al. EHA2014, Abstract #S1289]. Differences between continuous variables were evaluated using the Mann-Whitney U-test. Results: 1) CD229 was expressed on almost all MM cells, even on low CD138-expressing MM cells. MM cells from ISS stage II/III patients expressed significantly lower levels of CD138 than those from ISS stage I patients (P = 0.0004). Furthermore, although CD138 expression levels in symptomatic MM patients were lower than those in patients with asymptomatic MM and MGUS and the expression was decreased in refractory/relapsed MM patients, CD229 expression levels on plasma cells were similar in MGUS and MM patients. 2) Newly diagnosed MM patients with high-risk chromosomal abnormalities or in the IMWG high-risk group had higher levels of CD86 and CD126 compared with other patients (P = 0.0056 and 0.0248 in high-risk chromosomal abnormalities, P = 0.0221 and 0.0396 in IMWG high-risk group, respectively). Furthermore, patients in revised ISS stage III had higher levels of CD126 compared with others (P = 0.0646). 3) Although the serum IL-6 level was significantly higher in ISS stage II/III patients, in patients with high LDH levels, and in revised ISS stage III patients compared with other groups, there was no correlation between serum IL-6 levels and expression levels of the IL-6 receptor CD126 on MM cells. Conclusion: CD229 has the potential to become a new marker for the identification of MM cells and target for immunotherapy. High expression levels of CD86 and CD126 were associated with high-risk patients, and CD126 may be associated with survival in patients treated with novel agents. FCM analysis may be useful for predicting prognosis as well as detecting malignant clones. Further studies are in progress to clarify the significance of those molecules on MM cells. Disclosures No relevant conflicts of interest to declare.

Introduction: B7-H1 (also known as PD-L1 or CD274), a co-inhibitory molecule of the B7 family, is detected on various tumor cells and associated with tumor evasion from cytotoxic T lymphocyte (CTL)-mediated immune surveillance. Our previous study showed that B7-H1 expression levels on plasma cells from multiple myeloma (MM) patients were significantly upregulated compared with those cells from monoclonal gammopathy of undetermined significance patients and healthy volunteers. B7-H1-expressing MM cells had a proliferative advantage and were resistant to antimyeloma agents (Tamura et al. Leukemia 2013). However, it remains unknown whether cellular responses in B7-H1-expressing MM cells are affected by the interaction of B7-H1 molecules with their receptors, i.e., PD-1 and CD80. We thus investigated the reverse signal derived from B7-H1 binding to their receptors on MM cells and examined the clinical characteristics of B7-H1-highly positive MM patients in a multicenter study. Methods: 1) We established B7-H1-expressing MM cell lines called MOSTI expressing high levels of CD38 and CD138 from bone marrow mononuclear cells of myeloma patients and B7-H1-positive MM cells (B7-H1.KMS28PE) stably transfected with the B7-H1 gene. 2) B7-H1 knockdown MOSTI cell lines were obtained using B7-H1-specific short-hairpin RNA expressing a lentiviral vector. 3) The proliferative potential was examined by BrdU incorporation using flow cytometry (FCM) and the MTT assay. 4) Drug-induced apoptotic cells were stained with annexin V and propidium iodide (PI) and detected in FCM. 5) Magnetic Dynabeads were coupled with PD-1-Ig or CD80-Ig fusion proteins. B7-H1-expressing MM cells were co-cultured with the beads, and the binding capacity of the beads to B7-H1+ MM cells, drug sensitivity, and cell proliferation of B7-H1+ MM cells were analyzed. 6) We classified 105 cases with newly diagnosed MM into two groups according to B7-H1 expression levels on plasma cells and compared the clinical characteristics associated with prognosis between B7-H1-highly-expressing MM patients (n=43) and other patients (n=62). Results: 1) Knockdown of B7-H1 expression in MOSTI cells significantly suppressed cell proliferation and increased melphalan-induced apoptosis. These results demonstrated that B7-H1 expression is directly associated with aggressive myeloma behavior including cell growth and drug resistance. 2) B7-H1 molecules on MOSTI and B7-H1.KMS28PE cells bound more strongly to PD-1-Fc than to CD80-Fc. The binding of PD-1-Fc to MOSTI cells was inhibited by anti-B7-H1 antibody in a concentration-dependent manner. In MOSTI cells treated with PD-1-Fc beads, apoptosis induced by both melphalan and bortezomib was markedly inhibited in comparison with the cells treated with control Ig. PD-1-Fc bead-treated B7-H1.KMS28PE cells were also resistant to melphalan-induced apoptosis. However, CD80-Fc bead-treated cells did not show drug resistance. Resistance to antimyeloma agents via the reverse signal from PD-1 to MM cells was inhibited by the PI3K/AKT inhibitor LY294002. In Western blot analysis, phospho-AKT expression was significantly upregulated in PD-1-Fc-treated MM cells. However, the cell growth of PD-1-Fc-treated MOSTI cells was the same as that of control Ig-treated cells. These data indicate that the reverse signal delivered from B7-H1 expressed on MM cells bound to PD-1 induced the drug resistance of MM cells thorough the Akt signaling pathway. 3) Patients with B7-H1 highly-expressing MM cells tended to have the poor-risk cytogenetic abnormality t(4;14) (P=0.0703). Furthermore, fibroblast growth factor receptor 3 was significantly upregulated on MM cells in those B7-H1-highly positive patients (P=0.0141). Expression levels of CD56, CD45, and CD221, which were reported to be poor prognostic markers in MM, were siginificantly higher in B7-H1-highly positive patients compared with others. Conclusion: Our study revealed a new mechanism via which the interaction between B7-H1 on MM cells and PD-1 molecules not only inhibits tumor-specific CTLs but also induces the drug resistance of MM cells through the Akt signaling pathway. Furthermore, B7-H1 expression on MM cells may be associated with t(4;14) translocation and poor prognostic MM antigens. Thus, B7-H1 may be a reasonable target for immunotherapy. Disclosures No relevant conflicts of interest to declare.

BACKGROUND: Dasatinib is a second-generation BCR-ABL inhibitor that has a 325-fold higher potency than imatinib and a 16-fold higher potency than nilotinib in vitro. Recent reports suggested that absolute lymphocytosis in response to dasatinib for chronic phase (CP) –chronic myeloid leukemia (CML) might be associated with favorable response. However, as the fluctuation of lymphocytosis was often observed, it is sometimes difficult to define lymphocytosis. To evaluate how much increase and how long continuation of lymphocytosis affects the better clinical efficacy, we developed an index (DL index; Dasatinib induced Lymphocytosis index) that combines duration and increase of lymphocytosis. The aim of this study was to test the DL index as a predictor of Major Molecular Response (MMR) at 12 month. PATIENTS and METHODS: The DL area was based on a graph plotting the absolute lymphocyte counts and was the area under the lymphocyte curve using trapezoidal method. DL index was DL area divided by weeks. For example, DL index12W was defined as (DL area from 4 to 12 weeks) divided by (12-4) weeks. We tested the DL index12W in 30 patients who developed MMR at 12 months (MMR group) and 9 patients without MMR (non-MMR group). RESULTS and DISCUSSION: DL score12w in MMR group and non-MMR group were 2714.0 (95% CI; 2286.0-3142.2, n=30) and 1559.9 (95% CI; 1006.9- 2113.0, n=9) respectively. There was a significant difference between MMR group and non-MMR group in DL index12W by t-test(Figure 1, p=0.0016). A significant linear relationship was obtained between DL index12w and the peaked lymphocyte counts within 12 weeks by Spearman's correlation coefficient rank test (Figure 2, p=1.97E-07, correlation coefficient=0.91). The DL index12w, 2,113, which was the upper limit of non-MMR group 95% CI, is equivalent to 2,909/µl in lymphocyte count, and the DL index12w, 2,286, which is the lower limit of MMR group 95% CI, equivalent to 3,123/µl in lymphocyte count. Therefore, we divided all patients (n=39) into two groups, equal or more than 3,123/µl and less than 3,123/µl in the peaked lymphocyte counts. The patients with the peaked lymphocyte counts ≥ 3,123/µl reached MMR at 12 months (19/20=95.0%) to compare those less than 3,123/µl (11/19=57.9%) (p=0.0075). CONCLUSION: The peaked lymphocyte number, induced by the DL index12w, which calculation was extremely easy, was associated with molecular response. These data suggests that this tool can be useful in interpreting the results of molecular response at 12 months. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Background: Although molecular complete remission (mCR) in multiple myeloma (MM) can be assessed by allele-specific oligonucleotide (ASO)-PCR, this technique requires preparation of clonotype-specific primers for each individual, which is laborious and time-consuming. We utilized the LymphoSIGHTTM platform, which employs consensus primers and next-generation sequencing (NGS) to amplify and sequence all rearranged immunoglobulin gene segments present in a myeloma clone, to assess mCR. This technique has been shown to have 1-2 logs greater sensitivity compared to ASO-PCR and flow cytometry, respectively (Faham et al, Blood 2012). Usage of the sequencing method for minimal residual disease (MRD) detection in MM may provide increased sensitivity and specificity, while overcoming the challenges associated with ASO-PCR. We compared the LymphoSIGHTTM method with ASO-qPCR for MRD detection in autografts and bone marrow (BM) in the autologous peripheral blood stem cell (PBSC) transplantation (ASCT) setting. Methods: One hundred and nine Japanese patients with newly diagnosed MM who received various induction regimens prior to ASCT were retrospectively analyzed. All patients had achieved a partial response (PR) or better after ASCT. BM slides from 84 MM patients and fresh/frozen BM cells from 25 MM patients at diagnosis, as well as autografts/post-ASCT BM cells from each patient, were obtained for DNA extraction. IGH-based ASO-qPCR was performed as described previously (Methods Mol Biol 2009). Using universal primer sets, we amplified IGH variable (V), diversity (D), and joining (J) gene segments, IGH-DJ, and IGK from genomic DNA. Amplified products were subjected to deep sequencing using NGS. Reads were analyzed using standardized algorithms for clonotype determination. Myeloma-specific clonotypes were identified for each patient based on their high frequency in BM samples. Results : Myeloma clonotypes could be identified in autografts/post-ASCT BM cells in 98 of 109 patients (90%) and by ASO-qPCR in 63 of 101 patients (62%). MRD by NGS was assessed in autografts of 89 patients. 70 of 89 patients (79%) were positive by NGS; 28 of 62 patients (45%) were positive by ASO-qPCR. Although we observed a high correlation between NGS and PCR MRD results at MRD levels of 10-5 or higher, the sensitivity of ASO-PCR was 10-4-10-5, whereas that of NGS was 10-6 or lower when a sufficient amount of DNA was available for analysis. Eight cases where MRD was not detected in the autograft by NGS (MRDNGS(-)) and 38 MRDNGS(+) cases received post-ASCT therapy using novel agents such as bortezomib/lenalidomide/thalidomide, while 11 MRDNGS(-) cases and 32 MRDNGS(+) cases were followed without post-ASCT therapy. The MRDNGS(-) cases without post-ASCT therapy showed significantly better progression-free survival (PFS) than the MRDNGS(+) cases without post-ASCT therapy (P = 0.012) (Figure 1A) although overall survival rates were comparable between these groups. To investigate the value of sensitive detection by NGS, we compared PFS in 11 MRDNGS(-) cases (Group 1) with the 12 MRDNGS(+) cases where MRD was not detected by ASO-qPCR (MRDASO(-)) (Group 2). The patients in both groups did not receive any post-ASCT therapy. Group 1 showed significantly better PFS than Group 2 (P = 0.027) (Figure 1B). Furthermore, 9 MRDNGS(-) in post-ASCT BM cases tended to show a better PFS than 18 MRDNGS(+) in post-ASCT BM cases (P = 0.075) (Figure 1C). In a multivariate analysis, post-ASCT therapy using novel agents (P Conclusions: In this study, we showed the prognostic value of MRD detection using the NGS-based LymphoSIGHT platform in autografts of patients with MM who received and in those who did not receive post-ASCT therapy with novel agents. The NGS platform has improved sensitivity compared with ASO-qPCR in detecting MRD in autografts. Patients with low level MRD detected by NGS but not by ASO-qPCR have worse prognosis compared to patients who are MRD negative by sequencing, which underscores the need for sensitive detection. Figure 1 Figure 1. Disclosures Zheng: Sequenta, Inc.: Employment. Moorhead:Sequenta, Inc.: Employment. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Bendamustine is an alkylator containing a benzimidazole ring; it only has partial cross-resistance with the other alkylating agents. Several studies have shown that bendamustine is effective as a single agent or as combined treatment with rituximab for low-grade B-cell lymphoma. Although bendamustine has a favorable safety profile, the occurrence of myelosuppression and lymphocytopenia is relatively frequent. In lymphocytopenia, CD4-positive (CD4+) T-cell recovery is specifically impaired. If this occurs over a prolonged period of time, patients are at risk of opportunistic infections, such as cytomegalovirus (CMV) reactivation. This is a risk for bendamustine-treated patients, although the actual frequency of CMV reactivation in these patients is not clear. In this study, we prospectively evaluated the occurrence of CMV reactivation in relapsed or refractory low-grade B-cell lymphoma patients who were treated with bendamustine alone or in combination with rituximab. We simultaneously investigated the immune state of the patients. We analyzed CMV pp65 antigen in leukocytes, immunoglobulin (Ig) G, IgM, and IgA levels, and the CD4+:CD8+ lymphocyte ratio prior to treatment and after the third and sixth courses of treatment. There were 29 patients enrolled in the study. The median age was 68 years old (range, 28–83 years). Patients were treated a median of 2 times prior to bendamustine treatment; these treatments included chemotherapy and radiotherapy. There were 15 patients (51%) who completed 6 courses of chemotherapy. The immune status of the patients is shown in Figure 1. The median CD4+ lymphocyte count prior to treatment was 218/µL, which decreased to 75/µL by the end of treatment. The median of IgG, IgM, and IgA concentrations before treatment were 909, 52, and 147 mg/dL, respectively; by the end of treatment, the IgG, IgM, IgA concentrations had decreased to 832, 30, and 110 mg/dL, respectively. The extent of CD4+ T-cell depression during treatment appeared to be more severe than the Ig depression. CMV pp65 antigen was detected in 3 patients after the third course of treatment, and in 2 patients after the sixth course of treatment. None of the patients had any symptoms and CMV pp65 antigen count returned to negative without any treatment. The median CD4+ lymphocytes count in patients with or without CMV reactivation is shown in Table 1. There is a trend that the depression of CD4+ cells after the 6th course of bendamustine increases the risk of CMV reactivation. This is the first study to prospectively analyze CMV reactivation in low-grade B-cell lymphoma patients treated with bendamustine. In this study, we found that CMV reactivation by bendamustine occurred in approximately 15% of patients. On the other hand, the risk of symptomatic CMV disease, which would require medical intervention, does not appear to be high in bendamustine-treated patients. Most patients with relapsed or refractory low-grade B-cell lymphoma who are administered bendamustine therapy are in an immunosuppressed condition at the beginning of treatment. This is due to heavy chemotherapy pretreatment, which is often performed using rituximab. In addition to this, the decrease in CD4+ lymphocytes observed during bendamustine treatment, is likely to increase the risk of opportunistic infections, such as CMV reactivation. In future studies, we aim to investigate the risk factors for symptomatic cytomegalovirus infection in patients treated with bendamustine, especially in combination with rituximab. We hope that our study, which evaluates the risk of opportunistic infections in bendamustine treated patients, will be beneficial in determining the optimal timing for a preemptive approach for CMV infection. This will be helpful in establishing safe bendamustine treatment regimens in relapsed or refractory low-grade B-cell lymphoma patients. Figure 1 Figure 1. Table 1. The median number of lymphocytes, CD4+ lymphocytes and CD4/8 ratio in patients with or without cytomegalovirus reactivation Parameter All patients CMV reactivation p value + - Before treatment (n=24) Lymph (/µl) 996 1518 (n=5) 987 (n=19) 0.24 CD4+ cells (/µl) 218 261 218 0.96 CD4/8 ratio 0.69 0.60 0.69 0.54 After the 3rd course (n=23) Lymph (/µl) 980 1330 (n=5) 944 (n=18) 0.39 CD4+ cells (/µl) 130 118 130 0.52 CD4/8 ratio 0.20 0.12 0.20 0.16 After the 6th course (n=15) Lymph (/µl) 628 579 (n=5) 902 (n=10) 0.27 CD4+ cells (/µl) 75 36 118 0.035 * CD4/8 ratio 0.20 0.17 0.21 0.35 *; significant Disclosures No relevant conflicts of interest to declare.

The European Myeloma Network (EMN) has recently proposed an algorithm for the optimal starting dose and dose modification of antimyeloma drugs for elderly or unfit patients with multiple myeloma. However, the feasibility of this algorithm remains unclear. Therefore, we retrospectively assessed the feasibility of lenalidomide therapy for 20 patients with relapsed or refractory multiple myeloma (RRMM). We divided the patients into two groups, the recommended dose (RD) group (n = 12) in which the administered dose corresponded to the recommended dose according to the EMN algorithm, and the non-recommended dose (NRD) group (n = 8) in which the administered dose did not correspond to the recommended dose and we compared the efficacy and toxicity of lenalidomide therapy between the two groups. The overall response rate was 67% and 63% in the RD and NRD groups, respectively. There was no significant difference in time to progression between the two groups (9 vs. 14 months P = 0.75). Grade 3/4 neutropenia and grade 2/3 fatigue were less frequent in the RD group than in the NRD group (50% vs. 88% and 8% vs. 38%, respectively). The discontinuation rate due to severe adverse events was much lower in the RD group than in the NRD group (17% vs. 50%). These results indicated that the dose adjustment strategy of lenalidomide according to the algorithm may maintain efficacy and improve the safety profile in patients with RRMM. From our results, the algorithm seemed to be feasible for elderly or unfit myeloma patients in daily practice. A larger prospective study is needed to confirm the feasibility of this algorithm.

Background: Febrile neutropenia (FN) are often observed in hematological malignancy (HEM) such as acute leukemia (AL). Carbapenem antibiotics having a potent and broad antibacterial activity against gram-positive and gram-negative bacteria including Pseudomonas aeruginosa, are recommended to use early and sufficiently as empiric first-line therapy for FN. Several beta-lactam antibiotics including imipenem/cilastatin (IPM/CS), meropenem (MEPM), ceftazidime (CAZ) or cefepime (CFPM) have been studied as empirical therapy for FN. However, limited data are available concerning the efficacy of doripenem (DRPM) in FN patients with HEM. Methods: We conducted a randomized, cooperative group, open-label trial comparing DRPM (1.0g every 8 hours) with MEPM (1.0g every 8 hours) as first-line empirical antibacterial treatment for FN patients. 146 hospitalized FN patients with AL during or after chemotherapy were randomized to each drug group (DRPM, 73; MEPM, 73). These study drugs were administered as mono-therapy at least for 5 days without drug toxicity. The efficacy and safety were evaluated. Results: The overall response rate within seven days of DRPM and MEPM were not significantly different (DRPM: 67.3%, MEPM: 52.9%, P = 0.098). Both the resolution of fever by mono-therapy at day 3 to 5 (DRPM: 57.8%, MEPM: 47.4% (p = 0.26)), or survival at day 30 (DRPM: 98.2%, MEPM: 100% (p = 0.312)) was not significantly different in the two groups. Only grade 1-2 adverse events: liver dysfunction, renal dysfunction, diarrhea and rash were observed in some cases, and a little less often in DRPM group (DRPM: 29.8%, MEPM: 40.8% (p = 0.046)). These adverse events were clinically minimal in the two groups, and most of patients could continue the treatment by both study drugs. Conclusions: We could prove the non-inferiority of DRPM in comparison with MEPM for the initial treatment in AL patients with FN. Both drugs were well tolerated as first line therapy for FN.

Objectives: Patients with haematological malignancy who experience persistent fever and neutropenia often receive empirical antifungal therapy for the prevention and early treatment of invasive fungal infections. Micafungin (MCFG), a member of the echinocandin class of compounds, may be an effective alternative that is better tolerated than voriconazole (VRCZ). Methods: We conducted a randomised, cooperative group, open-label trial, comparing the efficacy and safety of micafungin with those of voriconazole as empirical antifungal therapy in febrile neutropenic patients with haematological malignancy. A successful outcome was defined as the fulfilment of all components of a 5-part composite endpoint. Results: Efficacy was evaluated in 100 patients (MCFG: 50, VRCZ: 50). Overall, favourable responses were observed in 52.0% of MCFG and 34.0% of VRCZ recipients (p = 0.069). There were no significant differences between the 2 groups in the rates of successful treatment of baseline fungal infection, absence of breakthrough fungal infection, survival for ≥7 days after treatment completion, or resolution of fever in the setting of neutropenia. Premature study discontinuation occurred less often in the MCFG group than in the VRCZ group (p = 0.001). Fewer patients who received MCFG experienced drug-related adverse events (p = 0.002). Conclusions: MCFG is as effective as and is generally better tolerated than VRCZ when given as empirical antifungal therapy in patients with persistent fever and neutropenia.

Background: The high-dose melphalan followed by autologous blood stem cell transplantation (ASCT) is a standard treatment for younger patients with multiple myeloma (MM). Recent reports have shown that achieving molecular complete response is associated with longer progression-free survival. However, the impact of minimal residual disease (MRD) in peripheral blood cell autografts on outcome remains unknown. We report the clinical and molecular follow-up of 6 MM patients who underwent ASCT. Methods: Six patients in whom the treatment was performed at Iwate Medical University Hospital and MRD in autografts was evaluable were included in the study. All patients had achieved at least a partial response after ASCT. Fresh bone marrow cells at diagnosis as well as autografts were obtained for DNA extraction. The CDR III coding region of the IgH gene was studied by direct sequencing of PCR product from bone marrow samples at diagnosis, and then MRD in each autograft was measured by ASO-PCR. Results: Four of 6 patients are alive: 2 who had no MRD in their autografts maintain a stringent complete response (CR) for 40 and 8 months after ASCT, while 2 who had MRD in them have serum M protein assessable by immunofixation for 20 and 10 months after ASCT. Two patients died from disease progression, both of which had MRD in their autografts and had have serum M protein after ASCT. Conclusion: These observations suggest that no MRD in peripheral blood cell autografts may be associated with a durable CR after ASCT. In addition, the post-ASCT treatment such as consolidation or maintenance therapy should be considered for MM patients who have MRD in autografts. Larger study and serial follow-up should be recommended to establish the clinical impact of MRD measurement in autografts in MM patients. As of writing, follow-up MRD analyses for total 13 MM patients are ongoing. Updated results will be presented.

Transportation of samples is essential for large-scale biobank projects. However, RNA degradation during pre-analytical operations prior to transportation can cause systematic bias in transcriptome data, which may prevent subsequent biomarker identification. Therefore, to collect high-quality biobank samples for expression analysis, specimens must be transported under stable conditions. In this study, we examined the effectiveness of RNA-stabilizing reagents to prevent RNA degradation during pre-analytical operations with an emphasis on RNA from peripheral blood mononuclear cells (PBMCs) to establish a protocol for reducing systematic bias. To this end, we obtained PBMCs from 11 healthy volunteers and analyzed the purity, yield, and integrity of extracted RNA after performing pre-analytical operations for freezing PBMCs at -80°C. We randomly chose 7 samples from 11 samples individually, and systematic bias in expression levels was examined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), RNA sequencing (RNA-Seq) experiments and data analysis. Our data demonstrated that omission of stabilizing reagents significantly lowered RNA integrity, suggesting substantial degradation of RNA molecules due to pre-analytical freezing. qRT-PCR experiments for 19 selected transcripts revealed systematic bias in the expression levels of five transcripts. RNA-Seq for 25,223 transcripts also suggested that about 40% of transcripts were systematically biased. These results indicated that appropriate reduction in systematic bias is essential in protocols for collection of RNA from PBMCs for large-scale biobank projects. Among the seven commercially available stabilizing reagents examined in this study, qRT-PCR and RNA-Seq experiments consistently suggested that RNALock, RNA/DNA Stabilization Reagent for Blood and Bone Marrow, and 1-Thioglycerol/Homogenization solution could reduce systematic bias. On the basis of the results of this study, we established a protocol to reduce systematic bias in the expression levels of RNA transcripts isolated from PBMCs. We believe that these data provide a novel methodology for collection of high-quality RNA from PBMCs for biobank researchers.

To evaluate the availability of quantification of blasts with aberrant antigen expression (AAE) using CD45 gating for minimal residual disease (MRD), 15 patients with acute leukemia (AL) and myelodysplastic syndrome (MDS) were studied. In patients with complete remission (CR), the frequency of blasts with AAE (%AAE) by CD45/side scatter (SSC) gating was significantly higher than that by the traditional forward scatter (FSC)/SSC combination (median, 4.1 vs. 0.3%, P

BACKGROUND When skeletal metastasis is the presenting problem and the primary site is occult, there is a need to identify the primary site as soon as possible. However, the search for the primary tumor is often time-consuming and difficult. The purpose of this study was to analyze the efficacy of particular diagnostic approaches and to devise an efficient and optimal diagnostic strategy. METHODS Among 213 patients with skeletal metastasis treated between 1990 and 1996 were 64 in whom skeletal lesions were the first manifestation of malignancy. The authors retrospectively analyzed both the final diagnosis and the process by which it was made in these 64 cases. RESULTS The primary cancer was identified antemortem in 56 (88%) of the 64 patients by examination and in 3 patients at autopsy. Lung carcinoma, the most frequently observed primary lesion, was identified in 23 patients. Other primary lesions were prostate carcinoma in 11 patients, breast carcinoma in 5, and hepatocellular carcinoma in 5. The primary malignancy was not determined in 5 patients. Thoracic and abdominal computed tomography (CT) scans were useful, especially in the diagnosis of patients with lung, hepatocellular, renal cell, and pancreatic carcinomas. Tumor markers were abnormally elevated in 73% of patients with carcinomas. CONCLUSIONS Although thoracic and abdominal CT scans were useful, examination of the gastrointestinal tract and pelvic CT scan seldom revealed the primary lesion and therefore should not be performed as an initial routine study in the absence of abdominal symptoms. Tumor markers are useful in differentiating carcinoma from hematologic malignancy and primary bone tumor. Cancer 1999;86:533–7. © 1999 American Cancer Society.

A 62-year-old man with left main coronary artery disease had coronary artery bypass grafting. Angiography disclosed total occlusion of the left common iliac artery. The left internal thoracic artery and the left inferior epigastric artery were well developed as collateral pathways to the left external iliac artery. With concomitant femoro-femoral crossover bypass, these two large arterial conduits were harvested and grafted to the coronary artery.

The PAS-Port system allows for the rapid deployment of a clampless proximal anastomosis between a saphenous vein graft and the aorta. We have developed a simple technique of establishing traction of the ascending aorta with the Starfish heart positioner during proximal saphenous vein anastomosis using the PAS-Port system in off-pump coronary artery bypass grafting.