Negative B-ALL Research Articles

Inotuzumab ozogamicin is effective in relapsed/refractory extramedullary B acute lymphoblastic leukemia

BackgroundExtramedullary involvement of B-cell Acute Lymphoblastic Leukemia (EM-ALL) is a rare occurrence, characterized by dismal outcome and the absence of a defined and shared therapeutic approach. In the landscape of innovative compounds, inotuzumab ozogamicin (IO) is a promising drug, whose mechanism of action relies on the killing of CD22 positive leukemic cells, through the delivery, after cell binding, of a molecule of calicheamicin.Case presentationWe report two cases of CD22 positive relapsed EM-ALL treated with IO, obtained as compassionate use. Case 1, a 66 years old woman, affected by Philadelphia (Ph) negative B-ALL, relapsed with extramedullary involvement after 6 standard chemotherapy courses, who reached a complete metabolic response with IO treatment. Case 2, a 67 years old man with Ph positive B-ALL, initially treated with ponatinib, a third generation tyrosine-kinase inhibitor (TKI), obtaining a prolonged deep molecular remission. Nevertheless, for skin relapse during TKI treatment, the patient received local radiotherapy and, shortly after, standard chemotherapy, as multiple abdominal sites of relapse were detected too, with no response. The patient then received IO, obtained as compassionate use, with a good metabolic response.ConclusionsThese two cases suggest a possible key role of IO in the setting of advanced CD22 positive ALL, and underline its potential activity also in patients with EM involvement, relapsed after or refractory to conventional chemotherapy. Despite the well known hepatotoxic effect of the compound (Sinusoid Occlusive Syndrome), neither of them had such adverse event, moreover the second patient safely underwent allogeneic bone marrow transplantation.

Application of disposable sealed spiral negative pressure drainage ball after breast surgery

Application of disposable sealed spiral negative pressure drainage ball after breast surgery

Application research of presacral space drainage tube combined with subcutaneous vacuum pressure suction in the laparoscopic-assisted abdominoperineal resection

To study the management for the perineal incision after laparoscopic-assisted abdominoperineal resection for rectal cancer. Clinical data of 87 patients undergoing laparoscopic Miles operation for lower rectal cancer from June 2009 to February 2014 were collected and studied. Presacral space drainage group: presacral space drainage tube was applied in 42 patients. Combined drainage group: presacral space drainage tube combined with subcutaneous vacuum pressure suction was applied in 45 cases. In combined drainage group, except the presacral drainage tube, another drainage tube was placed subcutaneously and connected to a negative pressure ball, which was fixed on the lateral anterior of perineal wound by the further incision and drainage. After subcutaneous tube was placed for 2 weeks, as drainage fluid was limpid and <15 ml/d for 3 days, meanwhile no obvious pelvic fluid was detected by ultrasound, and the wound healed quite well without redness and edema, then the subcutaneous tube with the negative pressure ball could be removed. There were 51 males and 36 females with the mean age of 26-78(56.9±10.8) years old. The laparoscopic Miles operation was successfully completed in all the cases without death and complications. The drainage tube was placed for 4-13(8.0±2.5) days in presacral space drainage group, and for 4-14(6.7±2.4) days in combined drainage group. The subcutaneous tube was placed for 14-24(15.8±3.0) days. The primary healing rate of perineal wound in presacral space drainage group and combined drainage group was 66.7%(28/42) and 91.1%(41/45) respectively, while the perineal wound infection rate was 21.4%(9/42) and 4.4%(2/45) respectively, whose differences between two groups were both significant (χ2=7.911, P=0.005 and χ2=5.674, P=0.017). Presacral space drainage tube combined with subcutaneous vacuum pressure suction in laparoscopic-assisted abdominoperineal resection for rectal cancer has better efficacy and lower infection rate for perineal incision, which is worth wide application.

A Retrospective Study of Ph Negative Adolescent and Young Adults with Acute B Lymphoblastic Leukemia in Two Centers

To analyze the therapeutic efficacy of different consolidation therapies after induction remission on Ph negative adolescent and young adults with acute B lymphoblastic leukemia, and to explore the effect of different risk factors on prognosis. The treatment and efficacy of 80 Ph negative B-ALL in patients of 16-39 years old in the Hematology Department of 301(65 cases) and 309(15 cases) hospital from 1999 to 2016 are retrospectively analyzed. The patients received combined induction chemotherapy of 4 or 5 chemotherapeutic drugs (VDCLP/ VDLP/ DOLP/ IOLP). After remission patients received consolidation protocols of 3-5 cycls, and then received allo-HSCT or haploidentical HSCT. The median follow-up time was 29 (6-153) months. HSCT was carried out after CR1. The 5-year OS and EFS of allo-HSCT group(n=29) was (73±16)% and (67±17)%, respectively, while those of haploidentical-HSCT group(n=20) were (53±22)% and (53±22)%, respectively, and those of pediatric-inspired protocols(n=31) was (63±17)% and (50±18)%, respectively. The difference between OS and EFS in 3 group was not statistically significant(P>0.05). The re-remission rate of recurrent patients was (50±23)%. On the one side, the cumulative incidence of TRM of pediatric-inspired protocol was better than that of HSCT (P<0.05). On the other side, the cummulative incidence of relapse (CIR) of pediatric-inspired protocol was poorer than that of HSCT, yet without significant difference (P>0.05). The median remission time of CR2 in patients was 14(2-36) months. Univariate and multivariate analysis were performed in 65 patients, and showed an abnormal result of CD13 or CD33 positive, CD22 negative, indicating a poor prognosis(P<0.05). In the adolescent and young adult patients with Ph- B-ALL treated by pediatric-inspired protocols, the survival time is similar with that in allo-HSCT group. However, more prospective clinical studies of random control test(RCT) should be carried out.

Quantification of Acute Lymphoblastic Leukemia Clonotypes in Leukapheresed Peripheral Blood Progenitor Cells Predicts Relapse Risk after Autologous Hematopoietic Stem Cell Transplantation

Since the incorporation of tyrosine kinase inhibitors into the treatment of Philadelphia chromosome–positive (Ph+) acute lymphoblastic leukemia (ALL), the notion that all patients with “high-risk” ALL uniformly require allogeneic (allo) hematopoietic cell transplantation (HCT) has received increasing scrutiny. Although multiple studies have shown superiority of alloHCT over autologous (auto) hematopoietic cell transplantation for high-risk patients, these findings may be explained, in part, by contamination of the peripheral blood progenitor cell (PBPC) leukapheresis product by residual leukemic cells in patients undergoing autoHCT. We retrospectively evaluated minimal residual disease (MRD) using next-generation sequencing (NGS) in the PBPC leukapheresis product of 32 ALL patients who underwent autoHCT. Twenty-eight patients (88%) had diagnostic samples with quantifiable immunoreceptor rearrangements to follow for MRD. Twelve (38%) patients had Ph+ B-ALL, 12 (38%) had Philadelphia chromosome–negative (Ph−) B-ALL, and 4 (14%) had T cell ALL. With a median follow-up of 41 months (range, 3 to 217), median relapse-free survival (RFS) and overall survival for the entire cohort were 3.2 and 4.2 years, respectively; at 5 years after transplantation, 42% of patients remain alive and relapse free. Using MRD detection at a threshold of ≥ 1 × 10−6, median RFS for patients with detectable MRD was 6.5 months and was not reached for patients without detectable disease (P = .0005). In multivariate analysis, the only factor significantly associated with relapse was the presence of MRD ≥1 × 10−6 (odds ratio, 23.8; confidence interval, 1.8 to 312.9; P = .0158). Our findings suggest that NGS for MRD detection can predict long-term RFS in patients undergoing autoHCT for high-risk ALL.

The application of negative pressure ball in patients with thoracoscopic lobectomy

Objective To observe the negative pressure ball aplication effect of thoracoscopic loectomy in patients. Methods 50 cases of patients with lung resection operation were divided into conventional group (water-sealed drainage bottle) and improved group (negative pressure ball) by random digital table method, and each group had 25 cases. The effect of nursing care and the satisfaction degree of patients were compared between the two groups. Results After different methods of nursing, the postoperative bed time in the improved group was (12.0±0.8) hours, hospitalization time was (8.0±1.5) days, which were significantly lower than (48.0±2.5) hours, (12.0±2.0) days in the conventional group. The difference was statistically significant (t=2.401, 2.302,P <0.05); The satisfaction of patients in the improvement group was 92.0% (23/25) which was significantly higher than 72.0% (18/25) of the control group, and the difference was statistically significant (χ2=4.920,P <0.05). Conclusions Negative pressure ball in thoracoscopic lobectomy patients after application can significantly improve the effect of clinical nursing and patient satisfaction, worthy of promotion and application in clinical work. Key words: Thoracic cavity; Negative pressure ball; Lobectomy

Evidence for Heterogeneous Genetic Associations with Acute Lymphoblastic Leukemia (ALL) By Cytogenetics and Sex in High-Risk Patients Treated with Matched Unrelated Donor Allogeneic Blood or Marrow Transplant (URD-BMT)

Inherited genetic polymorphisms in ARID5B, GATA3, and IKZF1 have been reported to be associated with ALL and vary by cytogenetic subgroups. For example, ARID5B variants are associated with hyperdiploid B-ALL in pediatric patients, which occurs in approximately 30% of ALL patients and is a marker of good prognosis. In contrast, a variant in GATA3 (rs3824662) has been consistently associated with ALL regardless of cytogenetics. While associations by subtype and across age have been explored in ALL patients, the effect of sex on the association of germline variants with ALL has not been tested, despite the higher incidence and worse prognosis of ALL in males than females. We tested the association of previously reported ALL variants in a high-risk ALL population of URD-BMT recipients, assessing the association of these variants with different cytogenetic subgroups and for heterogeneity of effect by sex.Genotyping was performed using the Illumina Omni-Express BeadChip containing approximately 730,000 single nucleotide polymorphisms (SNPs). After quality control, cohort 1 (C1) and cohort 2 (C2) included 364 (C1) and 82 (C2) B-ALL cases of continental European ancestry and 2,229 (C1) and 809 (C2) controls (unrelated healthy donors). The sample size of non-European cases was not adequate for genetic analyses. Cases were a subset of the genome wide association study (GWAS) named Determining the Influence of Susceptibility COnveying Variants Related to one-Year mortality after BMT (DISCOVeRY-BMT) a parent GWAS study in collaboration with the Center for International Blood and Marrow Transplant Research. We tested SNPs within GATA3, IKZF1 and ARID5B for associations with disease status using logistic regression assuming an additive model adjusted for age. Analyses were done separately for the following B-ALL subgroups: hyperdiploid negative ALL, Ph-negative ALL, Abnormal cytogenetics and Normal cytogenetics. P-values for each cohort were combined using METAL software with weights proportional to the square root of the sample size(Pmeta). Sex-stratified analyses were performed using all B-ALL cases and by subgroup. Tests for heterogeneity using Cochran's Q method and I² statistic were used to calculate the percentage of variation across sex and cytogenetic group, abnormal versus normal).The study population consisted mostly of non-hyperdiploid and Ph negative B-ALL. The GATA3 variant conferred a 78% (P =2.9x10-10) and 82% (P =0.001) increased odds of B-ALL in C1 and C2, respectively, Pmeta=1.7x10-12 (Table 1). The GATA3 SNP was significantly associated, Pmeta < 5x10-8, with all subgroups except abnormal cytogenetics ALL. The SNP in IKZF1 wasassociated (Pmeta < 5x10-8) with B-ALL overall, Ph negative and normal cytogenetics ALL, with some evidence of heterogeneity in odds ratios (ORs) between normal and abnormal cytogenetics B-ALL in both cohorts. The ARID5B variant, previously shown to be strongly associated with pediatric ALL, showed weak or no evidence of association (Table 1). IKZF1 (rs11980379) associations were stronger across all associated subgroups in males (Table 2). When comparing ORs calculated for males and females in there was evidence of significant heterogeneity (Table 2) for B-ALL overall (P =0.02), non-hyperdiploid (P =0.02) and normal cytogenetics (P =0.004).GATA3 showed consistent association, with replication, across the cytogenetic subgroups analyzed. IKZF1 variant demonstrated differences by sex, with significant associations between this SNP and B-ALL, observed primarily in males, regardless of cytogenetics. Thus, sex-specific associations were not an artifact of underlying cytogenetic distributions between males and females. The variation between males and females indicates the IKZF1 variant may impact ALL risk differently in males and females, potentially explaining both the overall greater risk of ALL in males and the risk of ALL with worse outcomes. ARID5B associations have previously been associated with hyperdiploid ALL, however the majority of our high-risk study population is non-hyperdiploid, so ARID5B may be a marker of good risk ALL which is rarely treated with URD-BMT. [Display omitted] [Display omitted] DisclosuresHahn:Novartis: Equity Ownership; NIH/NHLBI: Research Funding. McCarthy:Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; The Binding Site: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sucheston-Campbell:NIH/NHLBI: Research Funding.

Outcome of patients with adult acute lymphoblastic leukemia between 2000 and 2013: experience from single center

目的分析北京大学人民医院14年间连续收治的成人急性淋巴细胞白血病（ALL）患者2006年前后的疗效差异。方法回顾性分析北京大学人民医院2000年1月至2013年12月连续收治的成人ALL患者477例，其中Ph染色体阴性B细胞ALL（Ph−-B-ALL）276例（57.9%），T-ALL 69例（14.5%），Ph染色体阳性ALL（Ph+-ALL）132例（27.7%）；2006年之前收治111例（23.3%），2006年之后（含2006年）收治366例（76.7%）。获得完全缓解（CR）患者中，持续化疗187例（43.0%），接受异基因造血干细胞移植（allo-HSCT）患者248例（57.0%）。结果全部患者中位随访19个月，存活患者中位随访35个月，总CR率为92.0%，435例CR患者5年累积复发、无病生存（DFS）和总体生存（OS）率分别为42.5%、46.2％和47.6%。2006年之后与2006年之前相比：①Ph−-B-ALL、T-ALL患者总CR率差异无统计学意义，Ph+-ALL患者总CR率显著增高（P=0.036）；②全部CR患者5年DFS率（P=0.001）及OS率（P<0.001）显著增高，其中Ph−-B-ALL患者5年OS率显著增高（P=0.046），T-ALL（P=0.013和P=0.036）和Ph+-ALL（P=0.003和P<0.001）患者5年DFS率和OS率均显著增高，尤其是诱导期加用伊马替尼的Ph+-ALL患者（P<0.001和P<0.001）；③allo-HSCT患者5年DFS率（P=0.001）及OS率（P<0.001）显著增高，而未行移植患者差异无统计学意义。2006年之后，诱导期加用伊马替尼的Ph+-ALL患者5年DFS、OS率显著高于Ph−-B-ALL和T-ALL患者（P=0.009和P=0.001）。多因素分析显示，接受allo-HSCT和诱导期伊马替尼联合化疗是改善ALL患者预后的2个重要因素。结论在过去的14年间以2006年为界，ALL患者的治疗结果明显改善，尤以Ph+-ALL患者为著。

High Content Screening Identifies Synthetic Lethality Of Retinoid Receptor Agonists In IKZF1-Mutated BCR-ABL1 positive Acute Lymphoblastic Leukemia

Expression of BCR-ABL1 is a hallmark of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL). While CML is typically responsive to tyrosine kinase inhibitor monotherapy, BCR-ABL1-positive ALL is associated with a high frequency of treatment failure and relapse, even in the era of TKI therapy. Alterations of the lymphoid transcription factor gene IKZF1 (IKAROS) are frequent in BCR-ABL1 ALL and in CML at lymphoid blast crisis. Moreover, IKZF1 alterations are associated with poor outcome in BCR-ABL1-positive and -negative B-ALL. IKZF1 alterations are usually heterozygous and include loss-of-function deletions (∼60% of cases), focal deletion resulting in expression of a dominant negative isoform lacking the N-terminal DNA-binding zinc fingers, IK6 (∼30%) and missense mutations (∼10%). In addition, over half of BCR-ABL1-positive ALL cases harbor deletions of CDKN2A/B (INK4/ARF).We used two murine models of BCR-ABL1 leukemia to examine the role of IKZF1 alterations (deletions and IK6) and ARF loss in disease lineage and treatment responsiveness. In the first model, lineage negative Arf-/-, Ikzf1+/- or wild type (WT) bone marrow was transduced with p185 BCR-ABL1-expressing retrovirus and either an IK6-expressing retrovirus or empty vector followed by transplantation into irradiated wild type mice. p185-transduced WT marrow invariably induced a CML-like leukemia, whereas Arf loss and Ikzf1 alterations synergistically drove an aggressive pre-B ALL. In a complementary model, unmanipulated Arf-/-, Ikzf1+/-, or WT marrow was transduced with p185 BCR-ABL and transplanted immediately or after in vitro derivation of pre-B cell cultures. In this model, Arf loss and Ikzf1 haploinsuffiency or expression of IK6 reduced the latency of ALL and reduced responsiveness to dasatinib in vitro and in vivo. Strikingly, perturbation of Ikzf1 activity by haploinsufficiency, expression of IK6, zinc finger missense mutations, or shRNA mediated knockdown was associated with increased expression of the stem cell marker CD90 (Thy1) and the growth of cells in large aggregates in liquid cultures. Ikzf1 alteration was also associated with induction of a hematopoietic stem cell-like gene expression program, and increased self-renewal manifested as enhanced serial colony forming potential. This aberrant growth and self-renewal phenotype was abrogated by neutralizing anti-Thy1 antibodies. Thy1 is a known IKZF1 target, and these findings suggest that IKZF1 alterations derepress Thy1 expression resulting in enhanced stemness and treatment resistance.To identify agents that may enhance TKI responsiveness, we screened the activity of 384 FDA approved compounds in Arf-/- BCR-ABL1+ IK6+ pre-B. We assessed cell viability using CellTiter Glo, and measured the number and size of cell aggregates by high throughput microscopy to identify drugs that selectively inhibit cell aggregation. This identified the retinoid X receptor agonist bexarotene as a potent inhibitor of cell aggregation. A follow-up screen of 128 nuclear hormone receptor effector compounds identified potent anti-aggregation activity of 9-cis retinoic acid, 13-cis retinoic acid, all-trans retinoic acid and carbacyclin. In contrast, N-oleoylethanolamine, an antagonist of perioxisome proliferator-activated receptors that heterodimerize with RXRs, markedly increased cell aggregation. All retinoid receptor agonists induced cell cycle arrest and reduced proliferation without inducing apoptosis, and attenuated Thy1 expression, abrogated colony formation and enhanced the efficacy of dasatinib in vitro and in mice transplanted with Arf-/- p185 IK6 cells. Notably, Ikzf1is a predicted retinoid receptor target, and bexarotene treatment upregulated expression and nuclear localization of wild type IKZF1 in Arf-/- p185 pre-B cells, with and without IK6.Thus, IKZF1 alterations drive lymphoid lineage, and induce self-renewal and drug resistance in BCR-ABL1 ALL. IKZF1 alterations are associated with overexpression of Thy1, a target of IKZF1 transcriptional repression. This phenotype is reversed by retinoid receptor agonists that induce expression of IKZF1, attenuate Thy1 expression and increase sensitivity to TKI therapy. These data provide important new insights into the role of IKZF1 alterations in leukemogenesis, and identify a new therapeutic option in high risk B-ALL. Disclosures:No relevant conflicts of interest to declare.

Expression and clinical significance of IKZF1 gene IK6 isoform in adult acute lymphoblastic leukemia

This study was aimed to detect the expression of IKZF1 gene isoforms in bone marrow cells of patients with adult acute lymphoblastic leukemia and to investigate the clinical characteristics and prognosis of patients with IK6 isoform. The expression of IKZF1 gene isoforms were measured by nested RT-PCR in 79 newly diagnosed ALL patients. The clinical characteristics of IK6 positive patients and overall survival, disease-free survival of the IK6 positive group and IK6 negative group were compared. The results showed that IK1 and IK2/3 were the functional isoform while the IK4, IK6, IK8 and IK9 were the dominant negative isoform in adult ALL. The dominant negative isoform IK6 accounted for 34.4% in B-ALL patients and accounted for 22.2% in T-ALL patients. The BCR/ABL1 positive rate and the percentage of high risk patients in IK6 positive group was higher than that of IK6 negtive group in B-ALL patients (P = 0.027, P = 0.048). The expression of IK6 isoform did not correlate with sex, age and WBC count of B-ALL and T-ALL patients. The overall survival and disease-free survival of IK6 positive group were both lower than that of IK6 negtive group in Ph negative B-ALL patients (P = 0.009, P = 0.002). It is concluded that IK6 is a main isoform of the expression of IKZF1 gene in adult ALL patients, and can be used as a prognostic factor for guiding treatment in Ph negative B-ALL patients.

Epigenetic Inactivation of Notch-Hes Pathway in Human B-Cell Acute Lymphoblastic Leukemia

The Notch pathway can have both oncogenic and tumor suppressor roles, depending on cell context. For example, Notch signaling promotes T cell differentiation and is leukemogenic in T cells, whereas it inhibits early B cell differentiation and acts as a tumor suppressor in B cell leukemia where it induces growth arrest and apoptosis. The regulatory mechanisms that contribute to these opposing roles are not understood. Aberrant promoter DNA methylation and histone modifications are associated with silencing of tumor suppressor genes and have been implicated in leukemogenesis. Using methylated CpG island amplification (MCA)/DNA promoter microarray, we identified Notch3 and Hes5 as hypermethylated in human B cell acute lymphoblastic leukemia (ALL). We investigated the methylation status of other Notch pathway genes by bisulfite pyrosequencing. Notch3, JAG1, Hes2, Hes4 and Hes5 were frequently hypermethylated in B leukemia cell lines and primary B-ALL, in contrast to T-ALL cell lines and patient samples. Aberrant methylation of Notch3 and Hes5 in B-ALL was associated with gene silencing and was accompanied by decrease of H3K4 trimethylation and H3K9 acetylation and gain of H3K9 trimethylation and H3K27 trimethylation. 5-aza-2′-deoxycytidine treatment restored Hes5 expression and decreased promoter hypermethylation in most leukemia cell lines and primary B-ALL samples. Restoration of Hes5 expression by lentiviral transduction resulted in growth arrest and apoptosis in Hes5 negative B-ALL cells but not in Hes5 expressing T-ALL cells. These data suggest that epigenetic modifications are implicated in silencing of tumor suppressor of Notch/Hes pathway in B-ALL.

CALLA negative precursor B lymphoblastic leukemia with MLL gene translocation and an unusual FISH signal pattern

Rearrangements of the mixed lineage leukemia (MLL) gene at 11q23 commonly occur in infants with CALLA negative B lymphoblastic leukemia (B-ALL). Most often, these are detected by conventional karyotyping; however, fluorescent in-situ hybridization (FISH) with the help of a dual-color break-apart probe is used to identify cryptic translocations. When there is an MLL gene translocation, the usual FISH signal pattern is 1 red-1 green-1 yellow fusion signal pattern. We present a case of an infant with CALLA negative precursor B-ALL with a characteristic translocation t(4;11) (q21;q23), however, with an unusual MLL FISH signal pattern.

Characterization of CDR3 Structure of Vβ21 T Cell Clones In Patients with P210BCR-ABL Positive CML and B-ALL

AbstractAbstract 4455The clonally expanded T cells identified in most cancer patients which were considered to respond to tumour association antigen (TAA) have definitely specific anti-tumor cytotoxicity. P210BCR-ABL protein regards as the hallmark of CML and known to be causative of the disease, and also expresses in partial acute lymphoblastic leukaemia (ALL) can induce to yield tumor-specific cytotoxic T lymphocytes (CTL) which can kill bar-abl+ tumor cells in many trials. In our previous study, we found the oligoclonal expansion of TCR Vβ21 subfamily in the peripheral blood (PB) of 8 cases with P210BCR-ABL positive CML and 3 cases with P210BCR-ABL positive B-ALL patients and 1 case with P210BCR-ABL negative B-ALL patients using the genescan technology. To further elucidate the oligoclonal TCR Vβ21 features, we carried out sequencing analysis of CDR3 region of each oligoclonal TCR Vβ21 and determined the primary structure of the CDR3 region. The 12 TCR Vβ21 clones used different Jβ segments, Jβ2.3 segment was used in 4 clones, both Jβ2.1 and Jβ1.1 segments were used in 2 clones respectively, Jβ1.2, Jβ1.3, Jβ2.2 and Jβ2.7 segments were used in 1 clone. Only a conserved amino acid motif (SLxxV) was found within the CDR3 region from 3 patients with CML. Then we constructed the three-dimensional structures of the CDR3 sequences from all 12 clones by homology modeling methods and through pairwise comparing with the three-dimensional structures of all CDR3 sequences, the results showed that the conformation of the CDR3 region either containing the conserved amino acid motif or using the same Jβ segment displayed low similarity. Interesting, the conformation among some clones whose CDR3 region contain different amino acid motif and did not use the same Jβ segment were high resemble. In conclusions, our findings described that the high frequency of TCR Vβ21 subfamily expansion emerged in p210BCR-ABL positive CML and B-ALL patients, and according to perplexing features of CDR3 region, it could not easy to confirmed that the oligoclonal expanded TCR Vβ21 subfamily must recognize to p210BCR-ABL protein, however, primary structure characterization of CDR3 region, in combination with spatial structure features, ultimately might help to identify the tumor-associated antigen immune attack on tumor cell in p210BCR-ABL positive CML or B-ALL patients. Disclosures:Li:The study was supported by grants from National “863” project (No. 2006AA02Z114) and Key project of Natural Science Foundation of Guangdong province, China (No. 05103293, 9251063201000001).: Research Funding.

The study of perioperative nursing of laparoscopic cholecystectomy patients

Objective To evaluate perioperative nursing of laparoscopic cholecystectomy patients. Methods Laparoscopic cholecystectomy appeared hypogastric complications and special circumstances of treatment and before surgery, perioperative nursing. Results Of 138 cases in 132 cases of patients with laparoscopic cholecystectomy was successful Six cases of bleeding occurred after laparotomy transit line cholecystectomy. The other three cases of postoperative occurred biliary fistula, 2 weeks after removal of negative pressure drainage ball were recovered. Conclusions Although laparoscopic cholecystectomy less traumatic, but there are certain risks and complications. Only scientific and meticulous job in perioperative treatment and nursing is an important part of successful operation. Key words: Laparoscopic cholecystectomy; Perioperative period; Nursing

In-Vitro Efficacy of the Deoxyguanoside Analogs Forodesine (BCX- 1777) and ARA-G in Pediatric Acute Lymphoblastic Leukemia Cells.

Purine nucleotide phosphorylase (PNP) deficiency in humans is associated with elevated dGuo plasma levels. This results in the intra-cellular conversion of dGuo into dGTP, following 3 consecutive kinase steps and depletion of T-cells resulting in immune deficiency. This T-cell toxicity provided the rationale for the development of deoxyguanosine analogues as potential therapeutic compounds for T-cell malignancies. Forodesine (BCX-1777; BioCryst-Mundipharma) is an efficient blocker of PNP activity. Forodesine facilitates the conversion of dGuo into dGTP raising the intracellular dGTP pool. AraG (9-b-D-arabinofuranosyl-guanine) is a compound that is resistant to PNP-mediated degradation resulting in phosphorylation of AraG into AraGTP. AraGTP becomes incorporated in the DNA and blocks DNA synthesis resulting in apoptosis. In a phase II clinical trial, the AraG prodrug Nelarabine enforced a complete remission rate of 55% for pediatric T-ALL patients at 1st relapse. (Berg, JCO 2005). Clinical data of forodesine treatment in pediatric ALL patients are not yet available.The cytotoxic effect of Forodesine was investigated on primary leukemia cells from newly diagnosed pediatric acute lymphoblastic leukemia (ALL) patients in-vitro. Cells were incubated with a fixed concentration of Forodesine (1μM) in the presence of increasing concentrations of dGuo (0.001–50μM). The dGTP levels under conditions where PNPactivity was completely blocked was monitored.Incubation of primary leukemic cells obtained from 6 pediatric ALL patients (4 T-ALL, 2 B-ALL) with 10μM dGuo results in rapid dGuo degradation (t½<4hrs) by the PNP enzyme that is completely abolished by the addition of 1μM of Forodesine. Cells consequently accumulate dGTP levels upon Forodesine treatment to a median 7.9 (range 0.5–378 fold) that is comparable between T-ALL (n=31) and B-ALL (n=11) patient samples. This reflects equal intrinsic ability of de-novo nucleotide synthesis for both T-ALL and B-ALL cells. In accordance with T-cell selective toxicity, T-ALL cells were more sensitive to Forodesine/ dGuo treatment (median T-ALL LC50 value: 1.1μM dGuo/1μM Forodesine, n=27, p=0.001) compared to B-ALL cells, which had a median LC50 value of 8.8μM dGuo/1μM Forodesine (n=30). All patients that responded demonstrated dGTP accumulation (1.5– 222.1 fold), but the magnitude of dGTP accumulation did not relate to Forodesine/dGuo toxicity.Studying in-vitro responsiveness to AraG, T-ALL cells were more sensitive compared to B-ALL cells (p=0.0002) with a median AraG LC50 value of 20.5μM for T-ALL samples (n=24) versus 48.3μM for B-ALL samples (n=20). However, TELAML1 negative B-ALL cases were sensitive to AraG where as TELAML1 positive B-ALL cases were remarkable insensitive to AraG treatment (median LC50 value >50μM, n=9). No correlation was identified between in-vitro Forodesine/dGuo and AraG cytotoxicities. Most patient samples that displayed AraG resistance still responded to Forodesine/dGuo treatment.In contrast, AraG cytotoxicity strongly correlated with AraC cytotoxicity (r2=0.71, p<0.0001). In conclusion, T-ALL cells are sensitive to Forodesine/dGuo treatment in-vitro in contrast to B-ALL cells that have nearly 8 fold higher LC50 values. In-vitro Forodesine mediated cytotoxicity seems more potent in pediatric ALL than AraG treatment. Resistance to AraG treatment does not preclude responsiveness to Forodesine treatment and vice versa, indicating that Forodesine and AraG rely on different cellular mechanisms for cytotoxicity.

Nitric oxide synthase inhibition blocks octopus touch learning without producing sensory or motor dysfunction

We trained a set of eight octopuses to discriminate by touch between smooth and rough plastic balls. A food reward was given for taking the positive ball and a 5-6 V AC electric shock for taking the negative ball. The animals were trained until they regularly accepted positive balls and rejected negative balls. We then injected four with L-NAME, blocking nitric oxide synthase, and four with D-NAME for control. We continued the training and found no alterations in behaviour during six successive training sessions. We looked, in particular, for signs of sensory or motor dysfunction and observed none. We then reversed the paradigm, making the formerly positive ball negative. All controls readily learned the reversal; soon taking all the positive balls and rejecting the negative ones entirely. The L-NAME injected animals did not learn the reversal. However, they gradually stopped taking the newly negative smooth ball and increased their positive responses to the formerly negative rough ball, eventually taking approximately equal numbers of each. This experiment provided evidence that L-NAME impaired learning without affecting sensory or motor functions or central retrieval and/or retention mechanisms.