Amy S. Ruppert

Adverse Prognostic Significance of KIT Mutations in Adult Acute Myeloid Leukemia With inv(16) and t(8;21): A Cancer and Leukemia Group B Study

PURPOSE To analyze the prognostic impact of mutated KIT (mutKIT) in core-binding factor acute myeloid leukemia (AML) with inv(16)(p13q22) and t(8;21)(q22;q22). PATIENTS AND METHODS Sixty-one adults with inv(16) and 49 adults with t(8;21), assigned to postremission therapy with repetitive cycles of higher dose cytarabine were analyzed for mutKIT in exon 17 (mutKIT17) and 8 (mutKIT8) by denaturing high-performance liquid chromatography and direct sequencing at diagnosis. The median follow-up was 5.3 years. RESULTS Among patients with inv(16), 29.5% had mutKIT (16% with mutKIT17 and 13% with sole mutKIT8). Among patients with t(8;21), 22% had mutKIT (18% with mutKIT17 and 4% with sole mutKIT8). Complete remission rates of patients with mutKIT and wild-type KIT (wtKIT) were similar in both cytogenetic groups. In inv(16), the cumulative incidence of relapse (CIR) was higher for patients with mutKIT (P = .05; 5-year CIR, 56% v 29%) and those with mutKIT17 (P = .002; 5-year CIR, 80% v 29%) compared with wtKIT patients. Once data were adjusted for sex, mutKIT predicted worse overall survival (OS). In t(8;21), mutKIT predicted higher CIR (P = .017; 5-year CIR, 70% v 36%), but did not influence OS. CONCLUSION We report for the first time that mutKIT, and particularly mutKIT17, confer higher relapse risk, and both mutKIT17 and mutKIT8 appear to adversely affect OS in AML with inv(16). We also confirm the adverse impact of mutKIT on relapse risk in t(8;21) AML. We suggest that patients with core-binding factor AML should be screened for mutKIT at diagnosis for both prognostic and therapeutic purposes, given that activated KIT potentially can be targeted with novel tyrosine kinase inhibitors.

Resistance Mechanisms for the Bruton's Tyrosine Kinase Inhibitor Ibrutinib

BACKGROUND Ibrutinib is an irreversible inhibitor of Brutons tyrosine kinase (BTK) and is effective in chronic lymphocytic leukemia (CLL). Resistance to irreversible kinase inhibitors and resistance associated with BTK inhibition have not been characterized. Although only a small proportion of patients have had a relapse during ibrutinib therapy, an understanding of resistance mechanisms is important. We evaluated patients with relapsed disease to identify mutations that may mediate ibrutinib resistance. METHODS We performed whole-exome sequencing at baseline and the time of relapse on samples from six patients with acquired resistance to ibrutinib therapy. We then performed functional analysis of identified mutations. In addition, we performed Ion Torrent sequencing for identified resistance mutations on samples from nine patients with prolonged lymphocytosis. RESULTS We identified a cysteine-to-serine mutation in BTK at the binding site of ibrutinib in five patients and identified three distinct mutations in PLCγ2 in two patients. Functional analysis showed that the C481S mutation of BTK results in a protein that is only reversibly inhibited by ibrutinib. The R665W and L845F mutations in PLCγ2 are both potentially gain-of-function mutations that lead to autonomous B-cell-receptor activity. These mutations were not found in any of the patients with prolonged lymphocytosis who were taking ibrutinib. CONCLUSIONS Resistance to the irreversible BTK inhibitor ibrutinib often involves mutation of a cysteine residue where ibrutinib binding occurs. This finding, combined with two additional mutations in PLCγ2 that are immediately downstream of BTK, underscores the importance of the B-cell-receptor pathway in the mechanism of action of ibrutinib in CLL. (Funded by the National Cancer Institute and others.).

MicroRNA Expression in Cytogenetically Normal Acute Myeloid Leukemia

BACKGROUND A role of microRNAs in cancer has recently been recognized. However, little is known about the role of microRNAs in acute myeloid leukemia (AML). METHODS Using microRNA expression profiling, we studied samples of leukemia cells from adults under the age of 60 years who had cytogenetically normal AML and high-risk molecular features--that is, an internal tandem duplication in the fms-related tyrosine kinase 3 gene (FLT3-ITD), a wild-type nucleophosmin (NPM1), or both. A microRNA signature that was associated with event-free survival was derived from a training group of 64 patients and tested in a validation group of 55 patients. For the latter, a microRNA compound covariate predictor (called a microRNA summary value) was computed on the basis of weighted levels of the microRNAs forming the outcome signature. RESULTS Of 305 microRNA probes, 12 (including 5 representing microRNA-181 family members) were associated with event-free survival in the training group (P<0.005). In the validation group, the microRNA summary value was inversely associated with event-free survival (P=0.03). In multivariable analysis, the microRNA summary value remained associated with event-free survival (P=0.04) after adjustment for the allelic ratio of FLT3-ITD to wild-type FLT3 and for the white-cell count. Using results of gene-expression microarray analysis, we found that expression levels of the microRNA-181 family were inversely correlated with expression levels of predicted target genes encoding proteins involved in pathways of innate immunity mediated by toll-like receptors and interleukin-1beta. CONCLUSIONS A microRNA signature in molecularly defined, high-risk, cytogenetically normal AML is associated with the clinical outcome and with target genes encoding proteins involved in specific innate-immunity pathways.

Prognostic Significance of, and Gene and MicroRNA Expression Signatures Associated With, CEBPA Mutations in Cytogenetically Normal Acute Myeloid Leukemia With High-Risk Molecular Features: A Cancer and Leukemia Group B Study

PURPOSE To evaluate the prognostic significance of CEBPA mutations in the context of established molecular markers in cytogenetically normal (CN) acute myeloid leukemia (AML) and gain biologic insights into leukemogenesis of the CN-AML molecular high-risk subset (FLT3 internal tandem duplication [ITD] positive and/or NPM1 wild type) that has a significantly higher incidence of CEBPA mutations than the molecular low-risk subset (FLT3-ITD negative and NPM1 mutated). PATIENTS AND METHODS One hundred seventy-five adults age less than 60 years with untreated primary CN-AML were screened before treatment for CEBPA, FLT3, MLL, WT1, and NPM1 mutations and BAALC and ERG expression levels. Gene and microRNA (miRNA) expression profiles were obtained for the CN-AML molecular high-risk patients. RESULTS CEBPA mutations predicted better event-free (P = .007), disease-free (P = .014), and overall survival (P < .001) independently of other molecular and clinical prognosticators. Among patients with CEBPA mutations, 91% were in the CN-AML molecular high-risk group. Within this group, CEBPA mutations predicted better event-free (P < .001), disease-free (P = .004), and overall survival (P = .009) independently of other molecular and clinical characteristics and were associated with unique gene and miRNA expression profiles. The major features of these profiles were upregulation of genes (eg, GATA1, ZFPM1, EPOR, and GFI1B) and miRNAs (ie, the miR-181 family) involved in erythroid differentiation and downregulation of homeobox genes. CONCLUSION Pretreatment testing for CEBPA mutations identifies CN-AML patients with different outcomes, particularly in the molecular high-risk group, thus improving molecular risk-based classification of this large cytogenetic subset of AML. The gene and miRNA expression profiling provided insights into leukemogenesis of the CN-AML molecular high-risk group, indicating that CEBPA mutations are associated with partial erythroid differentiation.

Prognostic Factors and Outcome of Core Binding Factor Acute Myeloid Leukemia Patients With t(8;21) Differ From Those of Patients With inv(16): A Cancer and Leukemia Group B Study

PURPOSE Because both t(8;21) and inv(16) disrupt core binding factor (CBF) in acute myeloid leukemia (AML) and confer relatively favorable prognoses, these cytogenetic groups are often treated similarly. Recent studies, however, have shown different gene profiling for the two groups, underscoring potential biologic differences. Therefore, we sought to determine whether these two cytogenetic groups should also be considered separate entities from a clinical standpoint. PATIENTS AND METHODS We analyzed 144 consecutive adults with t(8;21) and 168 with inv(16) treated on Cancer and Leukemia Group B front-line studies. We compared pretreatment features, probability of achieving complete remission (CR), overall survival (OS) and cumulative incidence of relapse (CIR) between the two groups. RESULTS With a median follow-up of 6.4 years, for CBF AML as a whole, the CR rate was 88%, 5-year OS was 50% and CIR was 53%. After adjusting for covariates, patients with t(8;21) had shorter OS (hazard ratio [HR] = 1.5; P = .045) and survival after first relapse (HR = 1.7; P = .009) than patients with inv(16). Unexpectedly, race was an important predictor for t(8;21) AML, in that nonwhites failed induction more often (odds ratio = 5.7; P = .006) and had shorter OS than whites when certain secondary cytogenetic abnormalities were present. In patients with t(8;21) younger than 60 years, type of induction also correlated with relapse risk. For inv(16) AML, secondary cytogenetic abnormalities (especially +22) and male sex predicted better outcome. CONCLUSION When the prognostic impact of race, secondary cytogenetic abnormalities, sex, and response to salvage treatment is considered, t(8;21) and inv(16) AMLs seem to be distinct clinical entities and should be stratified and reported separately.

Overexpression of the ETS-Related Gene, ERG, Predicts a Worse Outcome in Acute Myeloid Leukemia With Normal Karyotype: A Cancer and Leukemia Group B Study

PURPOSE To test the prognostic significance of ETS-related gene (ERG) expression in cytogenetically normal primary acute myeloid leukemia (AML). PATIENTS AND METHODS Pretreatment blood samples from 84 cytogenetically normal AML patients aged less than 60 years, who were characterized for BAALC expression, FLT3 internal tandem duplication (ITD), and MLL partial tandem duplication (PTD) and uniformly treated on Cancer and Leukemia Group B 9621 protocol, were analyzed for ERG expression by real-time reverse transcriptase polymerase chain reaction. Patients were divided into quartiles according to ERG levels and were compared for clinical outcome. High-density oligonucleotide arrays were used to identify genes differentially expressed between high and low ERG expressers. RESULTS With a median follow-up of 5.7 years, patients with the upper 25% of ERG expression values had a worse cumulative incidence of relapse (CIR; P < .001) and overall survival (OS; P = .011) than the remaining patients. In a multivariable analysis, high ERG expression (P < .001) and the presence of MLL PTD (P = .027) predicted worse CIR. With regard to OS, an interaction was observed between expression of ERG and BAALC (P = .013), with ERG overexpression predicting shorter survival only in low BAALC expressers (P = .002). ERG overexpression was an independent prognostic factor even when the unfavorable group of FLT3 ITD patients lacking an FLT3 wild-type allele was included. High ERG expression was associated with upregulation of 112 expressed-sequenced tags and named genes, many of which are involved in cell proliferation, differentiation, and apoptosis. CONCLUSION ERG overexpression in AML patients with normal cytogenetics predicts an adverse clinical outcome and seems to be associated with a specific molecular signature.

Phase II Study of Flavopiridol in Relapsed Chronic Lymphocytic Leukemia Demonstrating High Response Rates in Genetically High-Risk Disease

PURPOSE Patients with chronic lymphocytic leukemia (CLL) with high-risk genomic features achieve poor outcomes with traditional therapies. A phase I study of a pharmacokinetically derived schedule of flavopiridol suggested promising activity in CLL, irrespective of high-risk features. Given the relevance of these findings to treating genetically high-risk CLL, a prospective confirmatory study was initiated. PATIENTS AND METHODS Patients with relapsed CLL were treated with single-agent flavopiridol, with subsequent addition of dexamethasone to suppress cytokine release syndrome (CRS). High-risk genomic features were prospectively assessed for response to therapy. RESULTS Sixty-four patients were enrolled. Median age was 60 years, median number of prior therapies was four, and all patients had received prior purine analog therapy. If patients tolerated treatment during week 1, dose escalation occurred during week 2. Dose escalation did not occur in four patients, as a result of severe tumor lysis syndrome; three of these patients required hemodialysis. Thirty-four patients (53%) achieved response, including 30 partial responses (PRs; 47%), three nodular PRs (5%), and one complete response (1.6%). A majority of high-risk patients responded; 12 (57%) of 21 patients with del(17p13.1) and 14 (50%) of 28 patients with del(11q22.3) responded irrespective of lymph node size. Median progression-free survival among responders was 10 to 12 months across all cytogenetic risk groups. Reducing the number of weekly treatments per cycle from four to three and adding prophylactic dexamethasone, which abrogated interleukin-6 release and CRS (P < or = .01), resulted in improved tolerability and treatment delivery. CONCLUSION Flavopiridol achieves significant clinical activity in patients with relapsed CLL, including those with high-risk genomic features and bulky lymphadenopathy. Subsequent clinical trials should use the amended treatment schedule developed herein and prophylactic corticosteroids.

Prolonged lymphocytosis during ibrutinib therapy is associated with distinct molecular characteristics and does not indicate a suboptimal response to therapy

The Brutons tyrosine kinase (BTK) inhibitor ibrutinib has outstanding activity in patients with chronic lymphocytic leukemia. Most patients experience lymphocytosis, representing lymphocyte egress from nodal compartments. This resolves within 8 months in the majority of patients, but a subgroup has lymphocytosis lasting >12 months. Here we report a detailed characterization of patients with persistent lymphocytosis during ibrutinib therapy. Signaling evaluation showed that while BTK is inhibited, downstream mediators of B-cell receptor (BCR) signaling are activated in persistent lymphocytes. These cells cannot be stimulated through the BCR and do not show evidence of target gene activation. Flow cytometry for κ and λ expression, IGHV sequencing, Zap-70 methylation, and targeted gene sequencing in these patients are identical at baseline and later time points, suggesting that persistent lymphocytes do not represent clonal evolution. In vitro treatment with targeted kinase inhibitors shows that they are not addicted to a single survival pathway. Finally, progression-free survival is not inferior for patients with prolonged lymphocytosis vs those with traditional responses. Thus, prolonged lymphocytosis is common following ibrutinib treatment, likely represents the persistence of a quiescent clone, and does not predict a subgroup of patients likely to relapse early.

High BAALC expression associates with other molecular prognostic markers, poor outcome, and a distinct gene-expression signature in cytogenetically normal patients younger than 60 years with acute myeloid leukemia: A Cancer and Leukemia Group B (CALGB) study

BAALC expression is considered an independent prognostic factor in cytogenetically normal acute myeloid leukemia (CN-AML), but has yet to be investigated together with multiple other established prognostic molecular markers in CN-AML. We analyzed BAALC expression in 172 primary CN-AML patients younger than 60 years of age, treated similarly on CALGB protocols. High BAALC expression was associated with FLT3-ITD (P = .04), wild-type NPM1 (P < .001), mutated CEBPA (P = .003), MLL-PTD (P = .009), absent FLT3-TKD (P = .005), and high ERG expression (P = .05). In multivariable analysis, high BAALC expression independently predicted lower complete remission rates (P = .04) when adjusting for ERG expression and age, and shorter survival (P = .04) when adjusting for FLT3-ITD, NPM1, CEBPA, and white blood cell count. A gene-expression signature of 312 probe sets differentiating high from low BAALC expressers was identified. High BAALC expression was associated with overexpression of genes involved in drug resistance (MDR1) and stem cell markers (CD133, CD34, KIT). Global microRNA-expression analysis did not reveal significant differences between BAALC expression groups. However, an analysis of microRNAs that putatively target BAALC revealed a potentially interesting inverse association between expression of miR-148a and BAALC. We conclude that high BAALC expression is an independent adverse prognostic factor and is associated with a specific gene-expression profile.

Repetitive Cycles of High-Dose Cytarabine Benefit Patients With Acute Myeloid Leukemia and inv(16)(p13q22) or t(16;16)(p13;q22): Results from CALGB 8461

PURPOSE To study the impact of repetitive (three to four courses) versus a single course of high-dose cytarabine (HDAC) consolidation therapy on outcome of patients with acute myeloid leukemia (AML) and inv(16)(p13q22) or t(16;16)(p13;q22). PATIENTS AND METHODS We examined the cumulative incidence of relapse (CIR), relapse-free survival (RFS), and overall survival (OS) for 48 adults younger than 60 years with inv(16)/t(16;16) who had attained a complete remission on one of four consecutive clinical trials and were assigned to receive HDAC consolidation therapy. Twenty-eight patients were assigned to either three or four courses of HDAC, and 20 patients were assigned to one course of HDAC followed by alternative intensive consolidation therapy. RESULTS Pretreatment features were similar for the two groups. The CIR was significantly decreased in patients assigned to receive three to four cycles of HDAC compared with patients assigned to one course (P=.03; 5-year CIR, 43% v 70%, respectively). The difference in RFS also approached statistical significance (P=.06). In a multivariable analysis that adjusted for potential confounding covariates, only treatment assignment (three to four cycles of HDAC) predicted for superior RFS (P=.02). The OS of both groups was similar (P=.93; 5-year OS, 75% for the three to four cycles of HDAC group v 70% for the one cycle of HDAC group), reflecting a high success rate with stem-cell transplantation salvage treatment administered among patients in both treatment groups. CONCLUSION We conclude that, in AML patients with inv(16)/t(16;16), repetitive HDAC therapy decreases the likelihood of relapse compared with consolidation regimens including less HDAC.