The search for molecular markers in AML that allow prediction of outcome has recently focused on genes involved in the regulation of programmed cell death (PCD). The aim of our study was to determine whether mRNA levels of Mdm-2, Bcl-2, Bclx L , Bad, and Bax are independent prognostic parameters for outcome. Transcript levels were analyzed by real-time quantitative RT-PCR in 232 samples collected either at diagnosis or following induction chemotherapy (ICT). Multivariate COX regression analysis adjusted for chemotherapy protocol, de novo vs secondary AML, and de novo vs relapsed AML indicated: (1) At diagnosis, high expression of Bad (P ‫؍‬ 0.015) and even more so high Bax and Bad levels (P ‫؍‬ 0.018) predicted adverse outcome, regardless of the response to ICT. In patients who subsequently failed to enter complete remission (CR), high levels of Bad, Bax and Bax high/Bad high were associated with an increased relative risk (RR) to die from tumor (RR ‫؍‬ 5.0 for Bad, 3.4 for Bax and 6.14 for Bax high/Bad high). (2) Following ICT, high expression of Bax (P ‫؍‬ 0.005) and high Bcl-2/Bax ratios (P ‫؍‬ 0.004) were independent predictors of unfavorable outcome, regardless of response to ICT. We conclude that high levels of Bax and Bad correlate with poor outcome, particularly in patients who do not enter CR and may serve as prognostic markers in AML.

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Mastocytosis is a myeloproliferative neoplasm characterized by expansion of abnormal mast cells (MCs) in various tissues, including skin, bone marrow, gastrointestinal tract, liver, spleen, or lymph nodes. Subtypes include indolent systemic mastocytosis, smoldering systemic mastocytosis and advanced systemic mastocytosis (AdvSM), a term collectively used for the three most aggressive forms of the disease: aggressive systemic mastocytosis, mast cell leukemia, and systemic mastocytosis with an associated clonal hematological non-mast cell disease (SM-AHNMD). MC activation and proliferation is physiologically controlled in part through stem cell factor (SCF) binding to its cognate receptor, KIT. Gain-of-function KIT mutations that lead to ligand-independent kinase activation are found in most SM subtypes, and the overwhelming majority of AdvSM patients harbor the KIT D816V mutation. Several approved tyrosine kinase inhibitors (TKIs), such as imatinib and nilotinib, have activity against wild-type KIT but lack activity against KIT D816V . Midostaurin, a broad spectrum TKI with activity against KIT D816V , has a 60% clinical response rate, and is currently the only drug specifically approved for AdvSM. While this agent improves the prognosis of AdvSM patients and provides proof of principle for targeting KIT D816V as a driver mutation, most responses are partial and/or not sustained, indicating that more potent and/or specific inhibitors are required. Avapritinib, a KIT and PDGFRα inhibitor, was specifically designed to inhibit KIT D816V . Early results from a Phase 1 trial suggest that avapritinib has potent antineoplastic activity in AdvSM, extending to patients who failed midostaurin. Patients exhibited a rapid reduction in both symptoms as well as reductions of bone marrow MCs, serum tryptase, and KIT D816V mutant allele burden. Adverse effects include expected toxicities such as myelosuppression and periorbital edema, but also cognitive impairment in some patients. Although considerable excitement about avapritinib exists, more data are needed to assess long-term responses and adverse effects of this novel TKI.

Background: Patients with chronic phase CML who achieve a complete cytogenetic response (CCR) have a low risk of disease progression. Since patients unlikely to achieve a CCR may benefit from more aggressive therapy, it would be clinically advantageous to identify those patients prior to therapy. Based on the hypothesis that cytogenetic refractoriness may be a property of leukemic progenitor cells, we explored the potential of gene expression profiling of CD34+ cells as a tool for predicting failure to achieve CCR. Methods: Fifty-one patients with CML who either achieved a CCR within 1 year of imatinib therapy (R, n = 35), or remained at least 65% Ph+ (NR, n = 16) were included in the study. Pre-imatinib CD34+ cells were FACS isolated from cryopreserved bone marrow mononuclear cells. RNA was extracted from the CD34+ cells and samples with >=5ng of high quality total RNA were amplified and labelled with the Affymetrix two cycle cDNA synthesis and IVT labeling protocol using <20ng input RNA; 10μg of labelled target cRNA were hybridized to Affymetrix HG-U133 Plus 2.0 GeneChip® arrays. Gene-by-gene ANOVA determined differential expression between NR and R. Low-level analysis was done using PLIER (Affymetrix) and RMA. Ranked p-values of n-way ANOVA results were used to select candidate classifiers and 90 classification models were tested. Best model selection and conserved accuracy estimate were done by 1 and 2 level nested cross validation respectively. Following hierarchical clustering and Principal Component Analysis (PCA), we identified the gene onotologies and associated pathways of the classifiers that had been selected in high frequency in the tested classification models. Results: The selection procedure yielded CD34+ cells with a median purity of 95.9% and median cell number of 1.0x104. Despite relatively low numbers of input cells, successful hybridization was achieved for 36 patients (24 R and 12 NR). Partial separation of NR and R was seen following hierarchical clustering and PCA, with R vs. NR being identified as a significant source of variation. Regardless of the specific low-level analysis approach, the classifiers performed similarly (conserved estimate of accuracy: 64% PLIER, 65% RMA). Certain biological pathways were associated with those classifiers who were selected in high frequency in the tested classification models (table). The majority of transcripts associated with these pathways were common to both low-level analysis methods, while additional unique transcripts for each pathway were identified with each method. Conclusions: (i) Gene expression profiling of CD34+ cells selected from cryopreserved bone marrow is feasible. (ii) This pilot study suggests that transcript profiling may have clinically useful predictive value in identifying patients unlikely to respond to imatinib. A larger study is needed to determine the predictive accuracy and obtain a clear understanding of the clinical relevance of those values. (iii) The classifiers are dominated by transcripts associated with PI3K and MAPK signalling, cell cycle, cell adhesion and nucleic acid metabolism. Pathway RMA Unique Transcripts PLIER Unique Transcripts Shared Transcripts Cell Cycle 4 1 14 Complement & Coagulation Cascades 8 2 9 Oxidative Phosphorylation 8 3 4 Phosphatidylinositol Signaling System 8 1 15 Purine Metabolism 9 2 13 Pyrimidine Metabolism 9 2 9