Steven M. Kornblau

MicroRNA Signatures Associated with Cytogenetics and Prognosis in Acute Myeloid Leukemia

MicroRNAs (miRNAs) are small RNAs of 19 to 25 nucleotides that are negative regulators of gene expression. To determine whether miRNAs are associated with cytogenetic abnormalities and clinical features in acute myeloid leukemia (AML), we evaluated the miRNA expression of CD34(+) cells and 122 untreated adult AML cases using a microarray platform. After background subtraction and normalization using a set of housekeeping genes, data were analyzed using Significance Analysis of Microarrays. An independent set of 60 untreated AML patients was used to validate the outcome signatures using real-time polymerase chain reaction. We identified several miRNAs differentially expressed between CD34(+) normal cells and the AML samples. miRNA expression was also closely associated with selected cytogenetic and molecular abnormalities, such as t(11q23), isolated trisomy 8, and FLT3-ITD mutations. Furthermore, patients with high expression of miR-191 and miR-199a had significantly worse overall and event-free survival than AML patients with low expression (overall survival: miR-191, P = .03; and miR-199a, P = .001, Cox regression). In conclusion, miRNA expression in AML is closely associated with cytogenetics and FLT3-ITD mutations. A small subset of miRNAs is correlated with survival.

Reverse phase protein array: validation of a novel proteomic technology and utility for analysis of primary leukemia specimens and hematopoietic stem cells

Proteomics has the potential to provide answers in cancer pathogenesis and to direct targeted therapy through the comprehensive analysis of protein expression levels and activation status. The realization of this potential requires the development of new, rapid, high-throughput technologies for performing protein arrays on patient samples, as well as novel analytic techniques to interpret them. Herein, we describe the validation and robustness of using reverse phase protein arrays (RPPA) for the analysis of primary acute myelogenous leukemia samples as well as leukemic and normal stem cells. In this report, we show that array printing, detection, amplification, and staining precision are very high, reproducible, and that they correlate with traditional Western blotting. Using replicates of the same sample on the same and/or separate arrays, or using separate protein samples prepared from the same starting sample, the intra- and interarray reproducibility was extremely high. No statistically significant difference in protein signal intensities could be detected within the array setups. The activation status (phosphorylation) was maintained in experiments testing delayed processing and preparation from multiple freeze-thawed samples. Differences in protein expression could reliably be detected in as few as three cell protein equivalents. RPPA prepared from rare populations of normal and leukemic stem cells were successfully done and showed differences from bulk populations of cells. Examples show how RPPAs are ideally suited for the large-scale analysis of target identification, validation, and drug discovery. In summary, RPPA is a highly reliable, reproducible, high-throughput system that allows for the rapid large-scale proteomic analysis of protein expression and phosphorylation state in primary acute myelogenous leukemia cells, cell lines, and in human stem cells. [Mol Cancer Ther 2006;5(10):2512–21]

Targetable Kinase-Activating Lesions in Ph-like Acute Lymphoblastic Leukemia

BACKGROUND Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is characterized by a gene-expression profile similar to that of BCR-ABL1-positive ALL, alterations of lymphoid transcription factor genes, and a poor outcome. The frequency and spectrum of genetic alterations in Ph-like ALL and its responsiveness to tyrosine kinase inhibition are undefined, especially in adolescents and adults. METHODS We performed genomic profiling of 1725 patients with precursor B-cell ALL and detailed genomic analysis of 154 patients with Ph-like ALL. We examined the functional effects of fusion proteins and the efficacy of tyrosine kinase inhibitors in mouse pre-B cells and xenografts of human Ph-like ALL. RESULTS Ph-like ALL increased in frequency from 10% among children with standard-risk ALL to 27% among young adults with ALL and was associated with a poor outcome. Kinase-activating alterations were identified in 91% of patients with Ph-like ALL; rearrangements involving ABL1, ABL2, CRLF2, CSF1R, EPOR, JAK2, NTRK3, PDGFRB, PTK2B, TSLP, or TYK2 and sequence mutations involving FLT3, IL7R, or SH2B3 were most common. Expression of ABL1, ABL2, CSF1R, JAK2, and PDGFRB fusions resulted in cytokine-independent proliferation and activation of phosphorylated STAT5. Cell lines and human leukemic cells expressing ABL1, ABL2, CSF1R, and PDGFRB fusions were sensitive in vitro to dasatinib, EPOR and JAK2 rearrangements were sensitive to ruxolitinib, and the ETV6-NTRK3 fusion was sensitive to crizotinib. CONCLUSIONS Ph-like ALL was found to be characterized by a range of genomic alterations that activate a limited number of signaling pathways, all of which may be amenable to inhibition with approved tyrosine kinase inhibitors. Trials identifying Ph-like ALL are needed to assess whether adding tyrosine kinase inhibitors to current therapy will improve the survival of patients with this type of leukemia. (Funded by the American Lebanese Syrian Associated Charities and others.).

MicroRNA 29b functions in acute myeloid leukemia.

MicroRNAs (miRNAs) are associated with cytogenetics and molecular subtypes of acute myelogeneous leukemia (AML), but their impact on AML pathogenesis is poorly understood. We have previously shown that miR-29b expression is deregulated in primary AML blasts. In this work, we investigated the functional role of miR-29b in leukemogenesis. Restoration of miR-29b in AML cell lines and primary samples induces apoptosis and dramatically reduces tumorigenicity in a xenograft leukemia model. Transcriptome analysis after ectopic transfection of synthetic miR-29b into leukemia cells indicates that miR-29b target apoptosis, cell cycle, and proliferation pathways. A significant enrichment for apoptosis genes, including MCL-1, was found among the mRNAs inversely correlated with miR-29b expression in 45 primary AML samples. Together, the data support a tumor suppressor role for miR-29 and provide a rationale for the use of synthetic miR-29b oligonucleotides as a novel strategy to improve treatment response in AML.

Treatment of adult T-cell leukemia-lymphoma with a combination of interferon alfa and zidovudine

BACKGROUND Infection with the human T-cell lymphotropic virus type I, a retrovirus, can cause a distinctive cancer, adult T-cell leukemia-lymphoma. The median survival of patients with the acute and lymphomatous forms of the disease is short, despite the use of cytotoxic chemotherapy. METHODS We treated 19 patients with acute or lymphomatous forms of adult T-cell leukemia-lymphoma with oral zidovudine (200 mg five times daily) and interferon alfa (Intron A, 5 to 10 million units subcutaneously each day). Seven of these patients had either relapsed after multiagent cytotoxic chemotherapy or failed to respond to that treatment. RESULTS Major responses were achieved in 58 percent of the patients (11 of 19), including complete remission in 26 percent (5 of 19). Four patients in whom prior cytotoxic therapy had failed had major responses, two of which were complete remissions. Six patients have survived for more than 12 months, with the longest remission since the discontinuation of treatment lasting more than 59 months. CONCLUSIONS The combination of zidovudine and interferon alfa has activity against adult T-cell leukemia-lymphoma, even in patients in whom prior cytotoxic therapy has failed. This regimen should be evaluated further for its role in the treatment of adult T-cell leukemia-lymphoma.

Use of granulocyte colony-stimulating factor before, during, and after fludarabine plus cytarabine induction therapy of newly diagnosed acute myelogenous leukemia or myelodysplastic syndromes: comparison with fludarabine plus cytarabine without granulocyte colony-stimulating factor.

PURPOSE To determine whether granulocyte colony-stimulating factor (G-CSF) administered before, during, and after fludarabine plus cytarabine (ara-C; FA) chemotherapy affected complete response (CR) rate, infection rate, blood count recovery, or survival in patients with newly diagnosed acute myelogenous leukemia (AML) or myelodysplastic syndromes (MDS). PATIENTS AND METHODS A total of 112 patients with newly diagnosed AML (n = 69) or MDS (n = 43) received G-CSF 400 micrograms/m2/d 1 day before (presenting WBC count < 50,000/microL) and/or during (all patients) fludarabine 30 mg/m2/d and ara-C 2 g/m2/d for 5 days (FLAG). G-CSF continued until a CR was achieved. Results were compared with those in 85 newly diagnosed patients (54 AML, 31 MDS) previously treated with FA without G-CSF. RESULTS Patients in both groups were relatively old (median age of all patients, 63 years), and were likely to have prognostically unfavorable cytogenetic abnormalities (36% had abnormalities of chromosomes 5 and 7 [-5/-7]). G-CSF accelerated recovery to > or = 1,000 neutrophils (P < .0001; median, 34 days for FA, 21 days for FLAG), but logistic regression provided no evidence that the CR rate was higher with FLAG than with FA (P = .50), with unadjusted CR rates of 63% and 53%, respectively. This may reflect relatively high rates of death before neutrophil recovery in both groups. Rates of infection were similar in both groups. The follow-up duration in remission is short, and much of these data remain censored. To date, survival is similar with FA and FLAG. CONCLUSION On average, G-CSF before, during, and after FA had no effect on CR or infection rates in this population, in which elderly patients and poor prognostic factors were prevalent. The use of FA and laminar airflow rooms rather than more usual therapy needs to be considered when analyzing the results.

Functional proteomic profiling of AML predicts response and survival

Because protein function regulates the phenotypic characteristics of cancer, a functional proteomic classification system could provide important information for pathogenesis and prognosis. With the goal of ultimately developing a proteomic-based classification of acute myeloid leukemia (AML), we assayed leukemia-enriched cells from 256 newly diagnosed AML patients, for 51 total and phosphoproteins from apoptosis, cell-cycle, and signal-transduction pathways, using reverse-phase protein arrays. Expression in matched blood and marrow samples were similar for 44 proteins; another 7 had small fold changes (8%-55%), suggesting that functional proteomics of leukemia-enriched cells in the marrow and periphery are similar. Protein expression patterns were independent of clinical characteristics. However, 24 proteins were significantly different between French-American-British subtypes, defining distinct signatures for each. Expression signatures for AML with cytogenetic abnormalities involving -5 or -7 were similar suggesting mechanistic commonalities. Distinct expression patterns for FMS-like tyrosine kinase 3-internal tandem duplication were also identified. Principal component analysis defined 7 protein signature groups, with prognostic information distinct from cytogenetics that correlated with remission attainment, relapse, and overall survival. In conclusion, protein expression profiling patterns in AML correlate with known morphologic features, cytogenetics, and outcome. Confirmation in independent studies may also provide pathophysiologic insights facilitating triage of patients to emerging targeted therapies.

Safety of retroviral gene marking with a truncated NGF receptor

To the editor—Random integration into the host cell genome and inappropriate transgene expression are major safety concerns for the clinical use of retroviral vectors. Li et al. recently reported a leukemic transformation of mouse bone marrow cells caused by integration of a transgene-carrying retroviral vector into the Evi1 proto-oncogene. They suggested that expression of the transgene, a truncated form of the p75 low-affinity nerve growth factor receptor (∆LNGFR) with most of the intracytoplasmic tail deleted (from residue 248), contributed to the leukemic progression. Because ∆LNGFR is used as a surface marker in gene therapy clinical trials aimed at controlling graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), a critical assessment of the potential risks associated with the use of such a molecule is essential. In a collaborative effort between 17 independent groups of investigators, we have accumulated both pre-clinical and clinical evidence supporting the safety of ∆LNGFR as a cell-marking molecule. Cumulative data obtained from >300 mice transplanted with bone marrow cells transduced with ∆LNGFR-expressing retroviral vectors showed normal engraftment, persistence and differentiation of ∆LNGFR-expressing hematopoietic stemprogenitor cells (HSCs) in primary, secondary and tertiary BMT recipients, with no adverse events (Table 1 and Supplementary Information online). Over 100 of these mice were monitored for >20 weeks after BMT; more than 70 animals, including 16 recipients of secondary or tertiary BMT, were monitored for >28 weeks. Considering that a total of >1 × 10 transduced cells were transplanted, and assuming an average of one retroviral integration per cell, we estimate the risk of oncogenic transformation after transduction with a ∆LNGFR-encoding retroviral vector to be <1 in 10 integration events. Therefore, expression of ∆LNGFR could not have increased the expected frequency of an insertional oncogenesis event, which has been previously estimated at 10 to 10 per insertion event. Expression of ∆LNGFR did not alter the function or survival of T lymphocytes derived from peripheral blood mononuclear cells transduced with a variety of vectors and studied in different animal models. In pre-clinical models of post-BMT GVHD, no difference in the ability to induce donor chimerism or to mediate GVHD was observed for ∆LNGFR-expressing T cells, as compared with control T cells, in 356 mice, 200 rats and 3 dogs (Table 1 and Supplementary Information online), again with no adverse events. Analysis of 102 independent transductions of human peripheral lymphocytes with two different vectors (SFCMM-3 and SFCM) encoding the same ∆LNGFR detected no change in the expression of markers of lineage, activation or adhesion, or in the proliferative capacity of T cells, as assayed by limiting dilution after polyclonal in vitro stimulation. All cells remained strictly dependent on interleukin-2 for growth and survival, and the Safety of retroviral gene marking with a truncated NGF receptor

The haematopoietic cell transplantation comorbidity index score is predictive of early death and survival in patients over 60 years of age receiving induction therapy for acute myeloid leukaemia

The haematopoietic cell transplantation comorbidity index (HCTCI) predicts nonrelapse mortality and overall survival (OS) post‐stem cell transplantation. HCTCI scores were assessed in 177 patients over 60 years of age receiving acute myeloid leukaemia (AML) induction therapy. HCTCI score was 0 in 22% of patients, 1–2 in 30%, and ≥3 in 48%. In patients with scores of 0, 1–2, or ≥3, early death rates were 3%, 11% and 29% (P < 0·001) respectively; median OS was 45, 31 and 19 weeks (P = 0·04) respectively. The HCTCI score is predictive of early death rates and OS in older patients receiving AML induction therapy.

Topotecan and Cytarabine Is an Active Combination Regimen in Myelodysplastic Syndromes and Chronic Myelomonocytic Leukemia

PURPOSE To evaluate the efficacy and safety of the combination of topotecan and cytarabine in patients with myelodysplastic syndromes (MDSs) and chronic myelomonocytic leukemia (CMML). PATIENTS AND METHODS Fifty-nine patients with MDSs and 27 with CMML were enrolled. They were either previously untreated (66%) or had received only biologic agents (14%) or chemotherapy with or without biologic agents (20%). Treatment consisted of topotecan 1.25 mg/m(2) by continuous intravenous infusion daily for 5 days and cytarabine 1. 0 g/m(2) by infusion over 2 hours daily for 5 days. Prophylaxis included antibacterial, antifungal, and antiviral agents. At a median follow-up of 7 months, all 86 patients were assessable for response and toxicity. RESULTS Complete remission (CR) was observed in 48 patients (56%; 61% with MDSs, 44% with CMML; P =.15). Similar CR rates were observed for patients with good-risk and poor-risk MDS (70% and 56%, respectively). The treatment effectively induced CR in patients with a poor-prognosis karyotype involving chromosomes 5 and 7 (CR, 71%) and secondary MDSs (CR, 72%). Fifty-four patients received one induction course, 25 patients received two, and the rest received more than two. The median number of continuation courses was two. The median overall duration of CR was 34 weeks (50 weeks for MDSs and 33 weeks for CMML). The median survival was 60 weeks for MDS and 44 weeks for CMML patients. CR and survival durations were longer in patients with refractory anemia with excess blasts (RAEB). Grade 3 or 4 mucositis or diarrhea was observed in three patients each. Fever was observed in 63%, and infections in 49% of patients. Six patients (7%) died during induction therapy. CONCLUSION Topotecan and cytarabine induced high CR rates in unselected patients with MDSs and CMML, particularly among patients with poor-prognosis cytogenetics and secondary MDSs. Topotecan-cytarabine is an active induction regimen in MDS and CMML patients, is well tolerated, and is associated with a low mortality rate.