JAK/STAT pathway inhibition overcomes IL7-induced glucocorticoid resistance in a subset of human T-cell acute lymphoblastic leukemias.

While outcomes for children with T-cell acute lymphoblastic leukemia (T-ALL) have improved dramatically, survival rates for patients with relapsed/refractory disease remain dismal. Prior studies indicate that glucocorticoid (GC) resistance is more common than resistance to other chemotherapies at relapse. In addition, failure to clear peripheral blasts during a prednisone prophase correlates with an elevated risk of relapse in newly diagnosed patients. Here we show that intrinsic GC resistance is present at diagnosis in early thymic precursor (ETP) T-ALLs as well as in a subset of non-ETP T-ALLs. GC-resistant non-ETP T-ALLs are characterized by strong induction of JAK/STAT signaling in response to interleukin-7 (IL7) stimulation. Removing IL7 or inhibiting JAK/STAT signaling sensitizes these T-ALLs, and a subset of ETP T-ALLs, to GCs. The combination of the GC dexamethasone and the JAK1/2 inhibitor ruxolitinib altered the balance between pro- and anti-apoptotic factors in samples with IL7-dependent GC resistance, but not in samples with IL7-independent GC resistance. Together, these data suggest that the addition of ruxolitinib or other inhibitors of IL7 receptor/JAK/STAT signaling may enhance the efficacy of GCs in a biologically defined subset of T-ALL.

RasGRP1 overexpression in T-ALL increases basal nucleotide exchange on Ras rendering the Ras/PI3K/Akt pathway responsive to protumorigenic cytokines.

Ras GTPases are activated by RasGEFs and inactivated by RasGAPs, which stimulate the hydrolysis of RasGTP to inactive RasGDP. GTPase-impairing somatic mutations in RAS genes, such as KRAS(G12D), are among the most common oncogenic events in metastatic cancer. A different type of cancer Ras signal, driven by overexpression of the RasGEF RasGRP1 (Ras guanine nucleotide-releasing protein 1), was recently implicated in pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients and murine models, in which RasGRP1 T-ALLs expand in response to treatment with interleukins (ILs) 2, 7 and 9. Here, we demonstrate that IL-2/7/9 stimulation activates Erk and Akt pathways downstream of Ras in RasGRP1 T-ALL but not in normal thymocytes. In normal lymphocytes, RasGRP1 is recruited to the membrane by diacylglycerol (DAG) in a phospholipase C-γ (PLCγ)-dependent manner. Surprisingly, we find that leukemic RasGRP1-triggered Ras-Akt signals do not depend on acute activation of PLCγ to generate DAG but rely on baseline DAG levels instead. In agreement, using three distinct assays that measure different aspects of the RasGTP/GDP cycle, we established that overexpression of RasGRP1 in T-ALLs results in a constitutively high GTP-loading rate of Ras, which is constantly counterbalanced by hydrolysis of RasGTP. KRAS(G12D) T-ALLs do not show constitutive GTP loading of Ras. Thus, we reveal an entirely novel type of leukemogenic Ras signals that is based on a RasGRP1-driven increased in flux through the RasGTP/GDP cycle, which is mechanistically very different from KRAS(G12D) signals. Our studies highlight the dynamic balance between RasGEF and RasGAP in these T-ALLs and put forth a new model in which IL-2/7/9 decrease RasGAP activity.

Macrophage and NK-mediated killing of precursor-B acute lymphoblastic leukemia cells targeted with a-fucosylated anti-CD19 humanized antibodies.

This work reports the tumoricidal effects of a novel investigational humanized anti-CD19 monoclonal antibody (Medi-551). An a-fucosylated antibody with increased affinity for human FcγRIIIA, Medi-551 is shown to mediate both antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Medi-551/CD19 complexes internalize slowly (>5 h) and thus remain accessible to effector cells for prolonged periods. We evaluated in vitro ADCC and ADCP activities of primary human natural killer (NK) cells and macrophages against precursor-B (pre-B) acute lymphoblastic leukemia (ALL) cell lines and pediatric patient blasts. Fluorescent imaging studies document immunological synapses formed between anti-CD19-bound target leukemia cells and effector cells and capture the kinetics of both NK-mediated killing and macrophage phagocytosis. Genetic polymorphisms in FcγRIIIA-158F/V modulate in vitro activities of effector cells, with FcγRIIIA-158V homozygotes or heterozygotes showing the strongest activity. Medi-551 treatment of severe combined immunodeficiency (SCID) mice engrafted with human pre-B cells led to prolonged animal survival and markedly reduced disease burden in blood, liver and bone marrow. These data show that anti-CD19 antibodies effectively recruit immune cells to pre-B ALL cells and support a move forward to early phase trials in this disease.

GSI-I (Z-LLNle-CHO) inhibits γ-secretase and the proteosome to trigger cell death in precursor-B acute lymphoblastic leukemia.

Gamma secretase inhibitors (GSIs) comprise a growing class of compounds that interfere with the membrane-bound Notch signaling protein and its downstream intra-nuclear transcriptional targets. As GSI-I (Z-LLNle-CHO) is also a derivative of a widely used proteosome inhibitor MG-132, we hypothesized that this compound might be active in precursor-B acute lymphoblastic leukemia (ALL) cell lines and patient samples. We found that GSI-I treatment of precursor-B ALL blasts induced apoptotic cell death within 18-24 h. With confirmation using RNA and protein analyses, GSI-I blocked nuclear accumulation of cleaved Notch1 and Notch2, and inhibited Notch targets Hey2 and Myc. Microarray analyses of 207 children with high-risk precursor-B ALL demonstrate that Notch pathway expression is a common feature of these neoplasms. However, microarray studies also implicated additional transcriptional targets in GSI-I-dependent cell death, including genes in the unfolded protein response, nuclear factor-κB and p53 pathways. Z-LLNle-CHO blocks both γ-secretase and proteosome activity, inducing more robust cell death in precursor-B ALL cells than either proteosome-selective or γ-secretase-selective inhibitors alone. Using Z-LLNle-CHO in a nonobese diabetes/severe combined immunodeficiency (NOD/SCID) precursor-B ALL xenograft model, we found that GSI-I alone delayed or prevented engraftment of B-lymphoblasts in 50% of the animals comprising the experimental group, suggesting that this compound is worthy of additional testing.

Bone marrow stroma-supported culture of T-lineage acute lymphoblastic leukemic cells predicts treatment outcome in children: a Pediatric Oncology Group study.

Significant predictors of treatment outcome are poorly defined for patients with T-lineage acute lymphoblastic leukemia (T-ALL). A high WBC at diagnosis, which has traditionally been a predictor of poor response in T-ALL, has considerably weakened prognostic significance in the face of modern, more intensive chemotherapy. To test the hypothesis that bone marrow stroma-supported leukemic cell recovery might identify children at high risk for relapse, we measured the ex vivo recovery of T-ALL lymphoblasts from 29 newly diagnosed patients using a stromal cell co-culture assay. In all cases the T-ALL lymphoblasts showed an increase in recovery of T-ALL cells (RTC), ranging from 4 to 343%, in comparison to samples maintained without stroma. Since we were blinded to patient outcome in this case-control study, we then correlated patient outcome with RTC. The RTC for 18 patients in complete continuous remission (CCR) for greater than 4 years was stochastically larger than for the 11 patients who eventually relapsed (P = 0.011, by the two-sided Wilcoxon test). Furthermore, 100% of patients with an RTC of more than 26% had a CCR greater than 4 years while 78% of the patients with an RTC of less than 25% relapsed within 4 years. This is the first report to show that higher lymphoblast recovery may predict a more favorable outcome for children with T-ALL. A prospective study is needed to test whether stroma-supported leukemic cell recovery might serve as a basis for assigning risk-adjusted therapy.

Enhanced T-lineage acute lymphoblastic leukaemia cell survival on bone marrow stroma requires involvement of LFA-1 and ICAM-1.

The bone marrow (BM) microenvironment supports leukaemia cell survival and proliferation. The roles played by adhesive receptor interactions in the survival of T-lineage acute lymphoblastic leukaemia (T-ALL) cells on BM stromal cells are not well understood. Recently, we have developed an assay that partially recapitulates the BM microenvironment using HS-5 BM stromal cells. In this assay, the magnitude of ex vivo T-ALL lymphoblast survival predicts patient outcome. We examined the molecular basis for cell-cell adhesive events leading to T-ALL lymphoblast survival on HS-5 and on donor-derived BM stroma. Lympho cyte function-associated antigen-1 (LFA-1) on T-ALL cell lines bound intercellular adhesion molecule-1 (ICAM-1) on HS-5 monolayers, and survival was inhibited 85-98% with monoclonal antibodies directed against LFA-1 or ICAM-1. We compared these results with patient-derived T-ALL lymphoblasts co-cultured on either HS-5 BM or normal BM monolayers and found that LFA-1 and ICAM-1 were required, but not alone sufficient for ex vivo leukaemic cell survival. On normal BM stroma, but not HS-5 monolayers, two additional adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, were highly expressed and contributed to T-ALL cell survival. This is the first report to demonstrate the importance of LFA-1/ICAM-1-mediated adhesion as a critical event in a cascade of cell surface receptor-ligand interactions that regulate T-ALL survival in the BM microenvironment.

Computerized system for outcomes-based antiemetic therapy in children.

OBJECTIVE: To introduce a computerized data collection system used for an outcomes-based approach to antiemetic therapy in children, and to present data collected with this system in support of a new antiemetic dosing regimen. METHODS: A validated nausea/vomiting survey was used to collect data on nausea severity (NSEV), vomiting severity (VSEV), daily activity interference (DAI), and the number of vomiting episodes. NSEV, VSEV, and DAI were rated as 0 = none to 3 = severe. All children and/or their parents were surveyed following the completion of each highly emetogenic chemotherapy regimen. This survey was computerized and transferred to a handheld data entry unit. Time and motion studies were performed to compare the time required to collect nausea/vomiting data and transfer the data to a computerized database with the hand-held system versus traditional paper (manual) surveys. The hand-held technology was used to collect survey data for children receiving a new antiemetic regimen (daily ondansetron and dexamethasone [OD]), which was then compared with data obtained with a previously employed regimen (thrice-daily ondansetron and daily methylprednisolone [OM]). Statistical analysis and a cost-effectiveness analysis (CEA) were performed to compare the two antiemetic regimens. RESULTS: The mean time required for total data entry with the manual system was 5.2 minutes per survey versus 2.4 minutes with the hand-held technology (p = 0.0026). A total of 376 nausea/vomiting surveys in 78 children receiving the OM antiemetic regimen were compared with 153 surveys in 38 children treated with the OD regimen. The mean survey scores were as follows: NSEV (1.2 vs. 0.8), VSEV (1.0 vs. 0.7), DAI (1.0 vs. 0.7), and number of vomiting episodes (4.3 vs. 2.1) for OM and OD, respectively; all were significantly lower with the OD regimen (p < 0.05). The percentage of patients with complete control of nausea and vomiting (19.2% vs. 39.2%) and good control (55.6% vs. 65.4%) were significantly greater with the OD regimen (p < 0.05). The CEA revealed that the OD resulted in a reduction of approximately $31 per patient for good protection and a $258 reduction for complete protection from nausea and vomiting. CONCLUSIONS: A computerized outcomes-based system aided by handheld technology allowed for more prompt and efficient collection of nausea/vomiting data. The OD antiemetic regimen was shown to be a more cost-effective alternative for children receiving severely emetogenic chemotherapy.

Benign hematogone-rich lymphoid proliferations can be distinguished from B-lineage acute lymphoblastic leukemia by integration of morphology, immunophenotype, adhesion molecule expression, and architectural features.

Distinction of normal B-lymphoid proliferations including precursors known as hematogones from acute lymphoblastic leukemia (ALL) is critical for disease management. We present a multiparameter assessment of 27 bone marrow samples containing at least 25% hematogones (range, 25%-72%) by morphologic review. We used flow cytometry to evaluate B-cell differentiation antigen and adhesion molecule expression and immunohistochemistry on clot sections to evaluate architectural distribution. Flow cytometry revealed that intermediately differentiated cells (CD19+, CD10+) predominated, followed in frequency by CD20+, surface immunoglobulin-positive cells, with CD34+, terminal deoxynucleotidyl transferase (TdT)-positive cells as the smallest subset. Adhesion molecules (CD44, CD54) were expressed more heterogeneously compared with expression in acute lymphoblastic leukemia. Immunohistochemistry revealed that CD34+, TdT-positive cells were dispersed without significant clustering, while CD20+ cells exceeded CD34/TdT-positive cells in 24 of 25 cases. This multidisciplinary study demonstrates that hematogone-rich lymphoid proliferations exhibit a spectrum of B-lymphoid differentiation antigen expression with predominance of intermediate and mature B-lineage cells, heterogeneity of adhesion molecule expression, and nonclustered bone marrow architectural distribution.

Survey ranking of emetogenic control in children receiving chemotherapy.

PURPOSE: To determine the effect of standard antiemetic treatment in children receiving various combination chemotherapy regimens. METHODS: A validated nausea/vomiting survey was administered to 78 patients receiving 13 different emetogenic chemotherapy regimens. Patients received antiemetic prophylaxis with ondansetron (0.3 mg/kg/day) alone for moderately emetogenic chemotherapy regimens or ondansetron (0.45 mg/kg/day) and methylprednisolone (4 mg/kg/day) for severely emetogenic chemotherapy regimens. A total of 324 different courses of chemotherapy were surveyed. Nausea and vomiting severity, duration, number of emetic episodes, appetite, daily activity interference, and rates of both complete and good antiemetic protection were determined for each chemotherapy protocol. Differences between genders and ages were analyzed. RESULTS: Chemotherapy combinations containing platinum compounds were found to be highly emetogenic and nauseating despite antiemetic therapy with ondansetron plus a corticosteroid. In addition, complete antiemetic protection for the combination of vincristine, cyclophosphamide, and dactinomycin was poor. For most of the severely emetogenic chemotherapy protocols, patients experienced good protection from nausea and vomiting less than 60% of the time, despite the use of ondansetron plus methylprednisolone. Significant differences were found in rates of residual nausea and vomiting and failure of antiemetic protection among the severely emetogenic chemotherapy protocols despite identical antiemetic therapy. Good protection rates were higher for moderately emetogenic chemotherapy treated with ondansetron alone. CONCLUSIONS: The currently recommended prophylactic therapy for pediatric patients receiving severely emetogenic chemotherapy fails to provide protection for many patients receiving commonly administered chemotherapy regimens and for most pediatric patients receiving platinum-containing chemotherapy combinations. New and refined antiemetic strategies are needed to improve efficacy in the pediatric population.

Improved quantification of cell survival on stromal monolayers by flow cytometric analyses.

BACKGROUND: The ex vivo survival of leukemic cells maintained on bone marrow stroma is an important tool for the investigation of cell survival and leukemogenesis. Currently, ex vivo survival of leukemic cell survival is measured by coculture on stromal cell monolayers. In these assays, we postulated that two important sources of error might be introduced through either variations in flow volume or in donor stromal cells. METHODS: A previously reported coculture assay that maintains leukemic cells on bone marrow stromal cells was employed. RESULTS: We identified two means of optimizing the coculture assay. First, biologically inert beads having well-characterized fluorescent properties were added to each sample to mathematically adjust for flow-based variations in volume acquisition. The inclusion of fluorescent beads to the basic stromal cell assay showed a significantly lower coefficient of variation as compared to samples analyzed without beads or manually counted using a hemacytometer. Second, in order to minimize variability in bone marrow hematopoietic function between donors, an adherent stromal cell line known to support hematopoiesis (HS-5) was used. When normal human donor stromal cells were used, variability in the survival of leukemic cells was observed on stromal cells derived from different donors. In contrast, statistically significant variability in survival of leukemic cells was not seen on HS-5 monolayers. Finally, we demonstrate that patient-derived leukemic samples may be examined for cell survival using these modifications. CONCLUSIONS: The novel use of fluorescent beads and a hematopoietic-supportive stromal cell line together makes the quantification of stroma-supported cell survival more reproducible, accurate, and amenable to patient-derived samples. These improvements in flow cytometry-based cell quantification are an important step in establishing a role for stromal cell assays in the study of leukemia biology and therapy.

Pre-B acute lymphoblastic leukemia with b3a2 (p210) and e1a2 (p190) BCR-ABL fusion transcripts relapsing as chronic myelogenous leukemia with a less differentiated b3a2 (p210) clone.

The Philadelphia chromosome translocation t(9;22)(q34;q11) may give rise to different BCR/ABL fusion mRNAs due to different genomic breakpoints and alternative splicing. The e1a2, b2a2 or b3a2 and c3a2 fusion mRNAs encode distinct fusion proteins (p190, p210 and p230, respectively), which are associated with different forms of leukemogenesis in humans and animal models. Our patient presented with acute pre-B cell lymphoblastic leukemia (ALL) with normal cytogenetics. After 3 years of standard ALL therapy, he relapsed with t(9;22)-positive chronic myelogenous leukemia (CML). Retrospective molecular analyses of the pre-treatment pre-B cell ALL sample showed the b3a2 (p210) and e1a2 (p190) BCR/ABL fusion transcripts. Only the b3a2 (p210) transcript was detected at relapse. Southern and immunoglobulin heavy chain (IgH) analyses of the presentation and relapse samples revealed an identical BCR rearrangement in both samples. However, only the ALL sample harbored an IgH gene rearrangement. These findings show a clonal relationship between the more differentiated pre-B cell and less differentiated CML clones and that the p210 and p190 fusion mRNAs were alternatively spliced from a single genomic breakpoint. Our patient's unusual molecular findings provide circumstantial evidence that the p190 protein may promote a more differentiated phenotype in a comparatively less differentiated p210-transformed precursor cell.

Myelodysplastic syndromes in the adolescent.

Myelodysplastic syndromes (MDS) are a group of acquired blood diseases that are the result of abnormal bone marrow function. The ineffective production of red cells, platelets, and white blood cells can lead to symptomatic anemia, bruising, infections, and the likelihood of evolution into acute myelogenous leukemia. While MDS is uncommon in the adolescent patient, a surprising number of affected individuals are also affected with a predisposing constitutional syndrome. The treatment of MDS in the adolescent patient is in part determined by symptoms and also by the historical outcomes associated with each of five morphologic categories of presentation. Improved supportive care has allowed for increasingly more children with MDS to survive into the second decade of life. The management of MDS in affected adolescents presents a number of interesting and worthwhile challenges to health care professionals.

The presence of CD34+ cell clusters predicts impending relapse in children with acute lymphoblastic leukemia receiving maintenance chemotherapy.

Early detection of relapse in children with acute lymphoblastic leukemia (ALL), as well as distinction of leukemic blasts from hematogones, can be difficult by morphologic examination alone. Using CD34 and terminal deoxynucleotidyl transferase (TdT) immunoperoxidase stains, we studied specimens from 25 children with ALL in morphologic remission to determine if we could identify children at risk of relapse. We studied morphologic remission bone marrow specimens from 9 patients who experienced relapse during the subsequent 6 months and 16 children who remained in complete remission, including 10 specimens with increased numbers of hematogones. Despite morphologic remission, clusters of more than 5 CD34+ and/or TdT-positive cells were identified before overt relapse in 6 of 9 cases of relapse, but were noted in only 1 of 10 specimens from children in continuous complete remission and none of 10 specimens with increased numbers of hematogones. Clusters of CD34+ or TdT-positive cells can identify individual patients at risk for imminent relapse. Hematogones may be differentiated from lymphoblasts by this method.

Assessment of the emetogenic potential of intrathecal chemotherapy and response to prophylactic treatment with ondansetron.

The purpose of this study was to document the emetogenic potential of intrathecal chemotherapy (IC) in children and to evaluate the efficacy of ondansetron in reducing nausea and vomiting with this chemotherapy treatment. Patients less than 18 years of age with acute lymphoblastic leukemia were eligible to participate in a survey project measuring the emetogenic potential of various chemotherapy treatments. Patients surveyed for 1 or more IC treatments were included in this report. The IC consisted of methotrexate, hydrocortisone and cytarabine, dosed according to patient age. A nausea/vomiting survey instrument was completed by each patient and/or parent following IC treatment. The instrument rated nausea, vomiting and daily activity interference (DAI) on a 4-point scale of 0 = none, 1 = mild, 2 = moderate and 3 = severe, and collected data on the number of vomiting and/or retching episodes in addition to the child's appetite following the chemotherapy treatment. When ondansetron was employed, it was administered in an i.v. infusion at a dose of 0.15 mg/kg before and after chemotherapy or as an oral dose of 4 mg or 8 mg before chemotherapy. Courses of IC without antiemetics were analyzed to determine the emetogenic potential of IC. For patients receiving IC both with and without ondansetron, courses were compared with each patient used as their own control to determine the influence of ondansetron upon survey responses. Statistical analysis consisted of nonparametric Friedman 2-way ANOVA for ordinal variables and a paired t-test for continuous variables. The binomial test was employed to analyze for differences between ondansetron and no antiemetic in the number of patients with complete control of both nausea and vomiting or vomiting alone. A total of 63 children with a mean age of 7.6 +/- 4.2 years were each studied on one or more occasions. Thirty-seven children were surveyed for 87 IC treatments without antiemetics (group I), and 17 children from this group were surveyed for 48 IC courses with i.v. ondansetron (group IA). An additional 18 children were subsequently surveyed for 39 IC courses with i.v. ondansetron (group II). Fifteen patients (7 of whom were members of group I) were surveyed following 33 IC courses with oral ondansetron (group III). The survey scores for group I patients were: nausea severity 1.3 +/- 1.1, vomiting severity 1.2 +/- 1.1, DAI 1.2 +/- 1.0 and mean number of emetic episodes 4.7 +/- 8.4. The mean appetite score was 1.5 +/- 1.1. For patients in group IA, nausea severity (0.8 +/- 0.9), vomiting severity (0.5 +/- 0.8), DAI (0.7 +/- 0.8), and the number of emetic episodes (1.4 +/- 2.8) were all significantly lower than with prior IC treatments without ondansetron. For complete protection, children receiving i.v. ondansetron had greater complete protection rates from both nausea and vomiting or vomiting alone than did patients receiving no antiemetic. Survey responses were also lower for patients receiving oral ondansetron, but insufficient control data did not allow for statistical analysis. IC results in mild to moderate nausea and vomiting in children. The emetogenic potential of IC is significantly reduced by i.v. ondansetron.

Fluorescence in situ hybridization (FISH) detects clonal instability in an optic nerve relapse of acute lymphoblastic leukemia.

PURPOSE: We report a case of an isolated optic nerve relapse 3.5 years after diagnosis of hyperdiploid acute lymphoblastic leukemia (ALL) in a 6-year-old boy who had been off treatment for 3 months. The use of interphase fluorescence in situ hybridization (FISH) for clonal identification of chromosome abnormalities is described. PATIENT AND METHODS: An asymptomatic lesion over the right optic nerve head was identified on routine funduscopic exam. Fine needle aspiration of the optic nerve infiltrate provided tissue for morphologic, immunohistochemical, and FISH analyses. RESULTS: FISH showed similar but not identical chromosome makeup of the leukemic blasts at the time of relapse as compared to tissue samples obtained at the time of diagnosis. CONCLUSION: Despite antimetabolite therapy, hyperdiploid ALL can rarely recur isolated to an optic nerve. FISH is a useful adjunct for confirming relapse when low numbers of white blood cells are obtained with fine needle aspiration.

Differences among raters evaluating the success of EMLA cream in alleviating procedure-related pain in children with cancer.

We evaluated the analgesic efficacy of EMLA cream after repeated bone marrow aspirations or lumbar punctures (LPs) in children with cancer, and compared the ratings among patients, their parents, physicians, and nurses. Data from LPs were analyzed at the last procedure without EMLA (T1) and the first and last procedures with EMLA (T2 and T3). Friedman's nonparametric analysis of variance was used for statistical analysis. A total of 272 procedures in 29 children were analyzed. For 179 procedures without EMLA, physicians rated pain lower than other raters, and for the 93 with EMLA physicians rated pain less than the children. Children rated pain at T2 lower than at T1 or T3. Physicians rated pain at T2 less than at T3. Both children and physicians rated pain at T3 as not different from that at T1. No differences were noted at these time points for other raters in LP distress ratings, or in bone marrow aspiration pain or distress ratings. Thus EMLA was associated with decreased pain ratings for LPs, but this effect was not sustainable with repeated procedures. The cream alone should not be relied on to control pain of bone marrow aspiration or repeated LPs in children. Physicians underestimated pain, which may have implications for undertreatment in this patient population.