Longitudinal analysis of 25 sequential sample-pairs using a custom multiple myeloma mutation sequencing panel (M(3)P).

Recent advances in genomic sequencing technologies now allow results from deep next-generation sequencing to be obtained within clinically meaningful timeframes, making this an attractive approach to better guide personalized treatment strategies. No multiple myeloma-specific gene panel has been established so far; we therefore designed a 47-gene-targeting gene panel, containing 39 genes known to be mutated in ≥3 % of multiple myeloma cases and eight genes in pathways therapeutically targeted in multiple myeloma (MM). We performed targeted sequencing on tumor/germline DNA of 25 MM patients in which we also had a sequential sample post treatment. Mutation analysis revealed KRAS as the most commonly mutated gene (36 % in each time point), followed by NRAS (20 and 16 %), TP53 (16 and 16 %), DIS3 (16 and 16 %), FAM46C (12 and 16 %), and SP140 (12 and 12 %). We successfully tracked clonal evolution and identified mutation acquisition and/or loss in FAM46C, FAT1, KRAS, NRAS, SPEN, PRDM1, NEB, and TP53 as well as two mutations in XBP1, a gene associated with bortezomib resistance. Thus, we present the first longitudinal analysis of a MM-specific targeted sequencing gene panel that can be used for individual tumor characterization and for tracking clonal evolution over time.

The bone marrow of multiple myeloma patients contains B cell populations at different stages of differentiation that are clonally related to the malignant plasma cell.

One of the distinguishing features of multiple myeloma (MM) is the proliferation of a clonal plasma cell population in the bone marrow (BM). It is of particular interest that the tumor plasma cells appear to be restricted to the microenvironment of the BM and are rarely detected in the peripheral system, yet the disease is found widely disseminated throughout the axial skeleton. Furthermore, isolation of MM tumor cell lines has proven to be quite problematic due to their slow growth rate. These observations have instigated the search for earlier cells in the B cell lineage that are clonally related to the plasma cell tumor and that may represent the growth fraction of the tumor. We used allele-specific oligonucleotides (ASO) derived from the third complementarity determining region of the rearranged tumor immunoglobulin heavy chain gene to detect isotypes clonally related to the plasma cell tumor. By reverse transcribing RNA from the BM with a panel of CH primers (mu, delta, alpha, and gamma), followed by ASO-polymerase chain reaction amplification, we demonstrate the existence of preswitch isotype species that are clonally related to the myeloma tumor. Furthermore, we show that separation of the BM cells into CD45+ and CD38+ cell populations results in a lineage-specific expression of the clonally related RNA molecules, with the C mu and C delta in the CD45+, and C gamma in the CD38+ population. Interestingly, clonally related C alpha transcripts are also derived from the CD45+ fraction. These results confirm the presence of B cell populations clonally related to the plasma cell tumor and are consistent with models that propose the existence of myeloma precursors.

Prognostic factors for hyperdiploid-myeloma: effects of chromosome 13 deletions and IgH translocations.

Chromosomal hyperdiploidy is the defining genetic signature in 40-50% of myeloma (MM) patients. We characterize hyperdiploid-MM (H-MM) in terms of its clinical and prognostic features in a cohort of 220 H-MM patients entered into clinical trials. Hyperdiploid-myeloma is associated with male sex, kappa immunoglobulin subtype, symptomatic bone disease and better survival compared to nonhyperdiploid-MM (median overall survival 48 vs 35 months, log-rank P = 0.023), despite similar response to treatment. Among 108 H-MM cases with FISH studies for common genetic abnormalities, survival is negatively affected by the existence of immunoglobulin heavy chain (IgH) translocations, especially those involving unknown partners, while the presence of chromosome 13 deletion by FISH did not significantly affect survival (median overall survival 50 vs 47 months, log-rank P = 0.47). Hyperdiploid-myeloma is therefore a unique genetic subtype of MM associated with improved outcome with distinct clinical features. The existence of IgH translocations but not chromosome 13 deletion by FISH negatively impacts survival and may allow further risk stratification of this population of MM patients.

Prognostic value of chromosome 1q21 gain by fluorescent in situ hybridization and increase CKS1B expression in myeloma.

A specific role for increased level of expression of CKS1B, as a consequence of chromosome 1q21 copy number gain, has been postulated as both pathogenic, as well as a powerful clinical prognostic factor in multiple myeloma (MM). The purpose of this study is to determine the clinical associations and prognostic impact of copy number gain at chromosome 1q21 (with a bacteria artificial chromosome clone containing CKS1B) and CKS1B gene level of expression in MM. We studied the chromosome region 1q21 for copy number change in a cohort of myeloma patients treated by high-dose therapy with stem-cell rescue (HDT) (n = 159). A separate cohort of patients, treated by HDT was studied for CKS1B messenger RNA expression by gene expression profiling (n = 67). 1q21 gain was then correlated with clinical parameters and survival. Gain of 1q21 copy number was detected in about a third of MM and was associated with more proliferative disease and poor-risk cytogenetic categories such as t(4;14), and chromosome 13 deletion. Both 1q21 gain and increase gene expression level were significantly associated with reduced survival. However, neither is an independent prognostic marker in MM on multivariate Cox proportional hazard analysis.

Ploidy status rarely changes in myeloma patients at disease progression.

Hyperdiploid and non-hyperdiploid multiple myeloma represents distinct biological entities characterized by different patterns of genetic changes. We sought to determine whether ploidy category (non-hyperdiploid versus hyperdiploid) remains stable over time from diagnosis to progression. Of the 43 patients studied (39 by flow cytometry DNA index and 4 by a FISH-based index), only five (12%) altered their ploidy status at progression. In three of these patients, the change may possibly be attributable to technical artifacts because of the low absolute change in DNA index. For those who retain their ploidy subtypes, the DNA index change minimally (3.75+/-4.87%). It would appear that the initiating genetic events underlying hyperdiploid and non-hyperdiploid MM that marks them out as distinct entities continue to dominate and persist during disease evolution and progression.

Coexistence of aneuploid subclones within a myeloma cell line that exhibits clonal immunoglobulin gene rearrangement: clinical implications.

A new human myeloma cell line, ANBL-6, was established and characterized at the genotypic and phenotypic levels. The cells exhibit a clonally rearranged immunoglobulin gene locus and resemble plasma cells morphologically. The ANBL-6 cells also exhibited an absolute dependence on exogenous interleukin 6 for growth. Of interest, DNA ploidy analysis suggested the existence of a near-diploid as well as a near-tetraploid population in this cell line. Cytogenetic studies confirmed the existence of two aneuploid karyotypes and further revealed a clonal relationship between the two karyotypes, as evidenced by numerous shared structural abnormalities. To determine whether the near-diploid cells functioned as stem cells for the near-tetraploid population, the near-diploid population was separated via flow cytometry and recultured prior to ploidy analysis. This population was observed to remain predominantly near-diploid over time, suggesting that these cells did not function as stem cells for the near-tetraploid population. However, the near-tetraploid cells did exhibit a growth advantage in vitro. Moreover, sequential ploidy analysis performed retrospectively on fresh bone marrow cells from the patient also suggested that there was an expansion of the near-tetraploid population during clinical relapse. These results suggest that both populations are self-regenerating and reflect the consequences of clonal evolution in the myeloma tumor. The coexistence of clonally related subclones with shared chromosomal abnormalities, however, suggests that the near-tetraploid subclone was derived from the near-diploid subclone at an unknown time during tumorigenesis.

Panel sequencing for clinically oriented variant screening and copy number detection in 142 untreated multiple myeloma patients.

We employed a customized Multiple Myeloma (MM)-specific Mutation Panel (M(3)P) to screen a homogenous cohort of 142 untreated MM patients for relevant mutations in a selection of disease-specific genes. M(3)Pv2.0 includes 77 genes selected for being either actionable targets, potentially related to drug-response or part of known key pathways in MM biology. We identified mutations in potentially actionable genes in 49% of patients and provided prognostic evidence of STAT3 mutations. This panel may serve as a practical alternative to more comprehensive sequencing approaches, providing genomic information in a timely and cost-effective manner, thus allowing clinically oriented variant screening in MM.

Establishment and characterization of three myeloma cell lines that demonstrate variable cytokine responses and abilities to produce autocrine interleukin-6.

A consensus regarding myeloma cell growth factor responsiveness and ability to produce autocrine interleukin (IL)-6 has not yet been obtained. In this study, we have established three new human myeloma cell lines (DP-6, KAS-6/1 and KP-6) from patients with aggressive disease. Extensive characterization of these cell lines revealed considerable heterogeneity at several levels. Growth factor responsiveness was initially addressed. Although the potent myeloma cell growth factor, IL-6, induced the proliferation and allowed for the expansion of all three cell lines, a panel of other cytokines elicited heterogeneous responses in each cell line. IL-3, IL-10, IL-11, insulin-like growth factor-I and tumor necrosis factor-alpha also stimulated DNA synthesis in all three cell lines; however, the magnitude of the response was generally lower than that observed in cultures containing IL-6. Transforming growth factor-beta, by contrast, uniformly inhibited the growth of all three cell lines. IL-1alpha and IL-1beta induced the proliferation of the DP-6 cells, but had minimal effects on the KAS-6/1 and KP-6 cells. Interferon (IFN)-alpha stimulated DNA synthesis in the KAS-6/1 cells, but inhibited the proliferation of the DP-6 and KP-6 cells. By comparison, IFN-gamma induced the growth of the KAS-6/1 and DP-6 cells, but inhibited the KP-6 cells. The gp130-associated cytokines, IL-11, leukemia inhibitory factor and oncostatin M, stimulated the growth of the KAS-6/1 cells, but had minimal effects on the DP-6 and KP-6 cells. The cell lines were also analyzed for IL-6 expression. RT-PCR analysis demonstrated that all three cell lines expressed IL-6 mRNA. However, when culture supernatants were tested using a sensitive IL-6 ELISA or IL-6 bioassay only the DP-6 and KP-6 cells were shown to be secreting biologically active IL-6. In summary, although all three of these cell lines were established from myeloma patients, the heterogeneity observed between these cell lines was considerable and may reflect, as well as provide tools to study, the heterogeneity observed in clinical disease.

Contrast in cytokine expression between patients with monoclonal gammopathy of undetermined significance or multiple myeloma.

We investigated whether differences in IL-6 and IL-1beta expression could be detected in monoclonal plasma cells from patients with MGUS or MM. Expression of IL-6 and IL-1beta in bone marrow cells was determined using cell sorting to enrich for plasma cells followed by reverse transcriptase/polymerase chain reaction (RT/PCR). Nineteen patients (six MGUS, two primary amyloid (AL), 11 MM) were studied. IL-6 mRNA expression was detectable in the sorted CD38+/CD45- plasma cell populations from 0/6 MGUS, 0/2 AL and 5/11 MM patients. All five MM patients with autocrine IL-6 expression demonstrated an elevated plasma cell labeling index. IL-1beta mRNA was detectable in the sorted CD38+/CD45- plasma cell populations from 1/6 MGUS, 0/2 AL and 10/11 MM patients. In situ hybridization (ISH) confirmed that the IL-1beta producing cells were plasma cells. In conclusion, autocrine production of IL-6 parallels a high labeling index and aberrant expression of IL-1beta correlates with the diagnosis of MM. Follow-up of IL-1beta-positive MGUS patients will determine whether aberrant expression of IL-1beta will predict those MGUS patients that will eventually progress to MM.

Marrow transplantation for stable-phase chronic granulocytic leukemia.

Ten patients with chronic granulocytic leukemia in the stable phase underwent marrow transplantation from HLA-identical siblings (nine cases) or an identical twin (one case) following preparation with cytarabine, cyclophosphamide, and total body irradiation. Marrow cytogenetics on all patients prior to transplantation revealed the Philadelphia chromosome without other evidence of aneuploidy. The immediate posttransplant course was in most cases relatively uncomplicated with only two serious infections and one death. All patients recovered with cytogentically normal marrow and leukemia has recurred only in the syngeneic transplant recipient. At present, nine patients are surviving from 358 to 961 days (median 597 days) after bone marrow transplantation. Bone marrow transplantation is capable of eliminating the abnormal clone of myeloid cells in patients with stable-phase chronic granulocytic leukemia and can be performed relatively safely in this "healthy" group of patients.

T cell depletion using anti-CD2 coated magnetic beads simplifies the detection of peripheral blood plasma cells.

The detection of peripheral blood plasma cells is clinically important, but difficult to perform by use of routine smears. To simplify the detection process, the peripheral blood T cells from ten patients with known active multiple myeloma were depleted using anti-CD2 coated magnetic beads. In all cases, there was enrichment of the immunoglobulin (Ig) positive cells after T cell depletion (mean enrichment factor, 3.4; median, 3.2; range, 1.2-5.1) with a mean pre-bead %Ig+ cells of 7.3 compared to 20.4 for the post-bead sample (p = 0.005). The monoclonal plasma cells were similarly enriched and more easily enumerated on the microscope slides prepared from the T cell depleted sample.

Plasma interleukin-6 and C-reactive protein levels in reactive versus clonal thrombocytosis.

PURPOSE: Evaluate the discriminatory value of plasma interleukin-6 or C-reactive protein levels in clonal thrombocytosis compared with those in reactive thrombocytosis. PATIENTS AND METHODS: A comparative analysis of quantitatively measured laboratory values in a prospectively studied group of consecutive patients. The setting was a tertiary referral center consisting of two hospitals and an outpatient clinic. Plasma interleukin-6 and C-reactive protein levels were measured in 91 consecutive patients with thrombocytosis (platelet count > or = 600 x 10(9)/L). The cause of thrombocytosis was determined by reviewing the medical histories and follow-up data without knowledge of the corresponding laboratory values. Sixty-four patients had reactive thrombocytosis, 20 had clonal thrombocytosis, and 7 had clonal thrombocytosis plus reactive thrombocytosis. Plasma interleukin-6 was measured by an enzyme-linked immunosorbent assay, and C-reactive protein was measured with rate immunonephelometry. RESULTS: Interleukin-6 levels were undetectable in all the patients with clonal thrombocytosis, whereas they were increased in 60% of the patients with reactive thrombocytosis or clonal thrombocytosis plus reactive thrombocytosis. There was a correlation between interleukin-6 and C-reactive protein levels (r = .6), and the median and range values of both levels differed significantly between the clonal thrombocytosis group and the other two groups (P < 0.0001). In 81% of the patients with reactive thrombocytosis, levels of either interleukin-6 or C-reactive protein were elevated. There was no correlation between interleukin-6 and C-reactive protein levels and the platelet count. CONCLUSIONS: An elevated interleukin-6 level is rare in uncomplicated clonal thrombocytosis and suggests reactive thrombocytosis. However, an isolated normal value has little discriminatory value. Measurement of C-reactive protein level may be used as a less expensive surrogate for measurement of interleukin-6. Repeatedly low levels of both interleukin-6 and C-reactive protein are most consistent with clonal thrombocytosis.

Nosocomial Legionnaires' disease. Occurrence in recipients of bone marrow transplants.

Nosocomial pneumonia caused by Legionella pneumophila serogroup 1 occurred in five patients after bone marrow transplantation for hematologic malignancies. Two patients died as a result of the infection despite treatment with erythromycin. Serologic screening revealed no other cases of Legionnaires' disease in 40 consecutive recipients of bone marrow transplants, giving a frequency of infection of 13 percent. These five cases represent 23 percent of the pneumonia occurring in this group of patients. Patients undergoing bone marrow transplantation are highly susceptible to infectious complications. Legionnaires' disease must now be added to the list of pathogens infecting this group of patients. Erythromycin is not generally a part of standard empiric antibiotic regimens in febrile neutropenic patients, but appears to be a reasonable addition when pneumonia does not respond to conventional, empiric treatment. Even with appropriate therapy, Legionnaires' disease remains a highly lethal infection in immunocompromised hosts.

Fluorescent in situ hybridization studies of lymphocytes and neutrophils in chronic granulocytic leukemia.

Using immunomagnetic cell separation and fluorescent in situ hybridization (FISH), we studied nine patients who had chronic granulocytic leukemia (CGL) for the proportion of interphase nuclei with Mbcr/abl fusion in a direct preparation of the bone marrow and also in the mononuclear cell (MNC), neutrophil, and B- and T-cell fractions of the peripheral blood. In five untreated patients, conventional cytogenetics revealed 97% to 100% Philadelphia chromosome (Ph)+ metaphases. In three of these five patients, FISH studies on bone marrow direct preparations and peripheral blood MNCs indicated that an Mbcr/abl fusion occurred in 62% to 69% of the cells. We observed 69% to 88% of nuclei with Mbcr/abl fusion within the neutrophil fractions. In contrast, the values were 12% to 39% within the T-cell fractions in the four patients we studied. B-cell fractions were studied in three patients, and only one had an abnormal value (58%). In the four patients receiving alpha-interferon therapy, the degree of conventional cytogenetic remission correlated best with the degree of FISH remission observed in the peripheral blood neutrophil fraction. Our results are in agreement with earlier studies in that both B and T lymphocytes may be involved with the clonal process in CGL. The FISH-based detection of Mbcr/abl fusion in the peripheral blood neutrophil compartment provided the best estimate for the proportion of Ph metaphases determined by conventional cytogenetics.

A novel three-color, clone-specific fluorescence in situ hybridization procedure for monoclonal gammopathies.

We have developed a three-color cytoplasmic immunoglobulin (cIg) and fluorescence in situ hybridization (FISH) technique to detect plasma cell chromosomal aneuploidy in patients with multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and amyloidosis (AL). Immunofluorescent-labeled antibodies to detect light chain expression and six directly labeled alpha-satellite chromosome specific enumeration probes (CEP) were used simultaneously to detect aneuploidy of the plasma cells. The six probes were specific for chromosomes 7, 9, 11, 15, 18, and X. The technique was tested in 12 consecutive patient samples (5 MM, 2 MGUS, 3 SMM, and 2 AL). Based on the alpha-satellite signals, we found trisomic clones for CEP 7 (4 of 12), CEP 11 (4 of 12), CEP X (1 of 12), CEP 9 (8 of 12), CEP 15 (7 of 12), and CEP 18 (5 of 12). Trisomic clones of at least one of the six chromosomes were present in 9 of 12 patients. We believe that this technique efficiently identifies monotypic plasma cells and permits simultaneous analysis of numeric chromosome anomalies by FISH in emerging neoplastic cells. We are in the process of applying this technique to a series of about 100 newly diagnosed monoclonal gammopathy patients.

Is multidrug resistance (P-glycoprotein) an intrinsic characteristic of plasma cells in patients with monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma and amyloidosis?

Multiple myeloma is not a curable disease, and most patients relapse after plateau phase. Drug resistance is a major problem in effective chemotherapy in this kind of disease. Current approaches are aimed at reversing or preventing drug resistance late in the disease. We studied a drug resistance marker, P-glycoprotein (P-gp), in a total of 43 patients with monoclonal gammopathy. This group included eight with monoclonal gammopathy of undetermined significance (MGUS), five with plasmacytoma (PCM), nineteen with multiple myeloma (MM; six newly diagnosed, seven plateau, five refractory, one relapse) and eleven amyloidosis (seven newly diagnosed, four after treatment). Using 3-color flow cytometry, a plasma cell gate was selected on the basis of CD38+/45-(dim) staining and the population was examined for the expression of P-gp using two monoclonal antibodies (MRK16 and UIC2). P-gp expression was positive on marrow plasma cells in 42/43 patients. The resistance index (RI) in these cases (range 2.0-7.07) is comparable to that in the positive cell line KG-1A (3.05-3.08). In 2 of 5 patients with refractory MM, the RI for P-gp (5.4, 6.36) was higher than in plateau phase. These data suggest that relative resistance due to the P-gp mechanism is likely to be an intrinsic property of plasma cells in monoclonal gammopathies and may provide a partial explanation as to why these diseases always relapse. The results of our study support strategies for MDR reversal earlier in the course.

Clinical significance of the translocation (11;14)(q13;q32) in multiple myeloma.

The most common chromosomal translocation in multiple myeloma (MM) is t(11;14)(q13;q32). Here, we describe the clinical characteristics of patients with MM who have this translocation. We have identified 24 patients at our institution who had t(11;14)(q13;q32) as determined by standard cytogenetic analysis (CC). Seven patients had the translocation detected at the time of original diagnosis and 17 at the time of relapse. Median survival in all patients after original diagnosis was 43 months; median survival after the translocation was detected was 11.9 months. Four patients had a clinical diagnosis of plasma cell leukemia. Most patients had an elevated beta2-microglobulin (13/20 had >4 microg/ml). The bone marrow (BM) labeling index (LI) of patients, at the time of translocation detection, was elevated in most (median 1.4%, 17/23 patients had BMLI > or = 1%). Of the 24 patients, 19 (79%) died of disease progression and 5 (21%) were alive with disease at last follow-up. Lytic lesions, bone pain, or compression fractures eventually developed in all patients. Patients with MM who have t(11;14)(q13;q32) detected by standard cytogenetics seem to have an aggressive clinical course.

Fish demonstrates treatment-related chromosome damage in myeloid but not plasma cells in primary systemic amyloidosis.

Conventional cytogenetic analysis is limited in the evaluation of plasma cell disorders because, relative to normal hematopoietic elements, plasma cells divide slowly. Moreover, it is difficult to know whether abnormal metaphases originate from malignant plasma cells or myeloid cells harboring other abnormalities. We studied a patient with primary systemic amyloidosis who had previously been treated with an alkylating agent. Bone marrow cells were analyzed by cytoplasmic-immunoglobulin fluorescent staining combined with fluorescent in situ hybridization (cIg-FISH). Both chromosome enumeration probes for chromosome 1 and 7 and loci-specific probes for the short and long arm of chromosome 7 were used. Cytogenetic analysis disclosed the following abnormality: +der(1;7)(q10;p10). On cIg-FISH, the myeloid cells had fusion signals between chromosome enumeration probes for chromosomes 1 and 7, whereas plasma cells had the normal appearance of two pairs of signals. There was a second clone of abnormal myeloid cells with monosomy of chromosome 7. The bone marrow did not show any evidence of myelodysplasia. Interphase cIg-FISH is a useful technique for assigning the lineage of chromosomal abnormalities in plasma cell disorders.

A comparison of bone marrow transplantation with maintenance chemotherapy for patients with acute nonlymphoblastic leukemia in first complete remission.

Twenty patients treated with maintenance chemotherapy for acute nonlymphoblastic leukemia after achieving complete remission were compared with 13 patients who underwent bone marrow transplantation from an HLA-identical sibling. The median age was 27 years for both maintenance chemotherapy patients (range 17-42 years) and for patients undergoing bone marrow transplantation (range 16-42 years). The 1-year survival for maintenance chemotherapy was 80% vs. 54% with bone marrow transplantation (p = NS). Complete remission durability was 70% at 1 year for maintenance chemotherapy (34% projected for 5 years) compared with no relapses in the first year with bone marrow transplantation (p = 0.01). Patients on maintenance chemotherapy were hospitalized for an average of 22 days (range 0-171 days) during the first 12 months of treatment. Patients undergoing bone marrow transplantation were hospitalized for an average of 82 days (range 41-113 days) in the same time period. Severe hematologic toxicity was seen in 13/13 bone marrow transplantation patients and 6/20 maintenance chemotherapy patients. Chronic graft-vs.-host disease occurred in 3/7 surviving bone marrow transplantation patients. Maintenance chemotherapy had an average first year cost of +3,076.00 for patients who did not relapse and +48,827.00 for patients that relapsed. The first year costs for bone marrow transplantation averaged +84,102.00. Thus, maintenance chemotherapy was associated with a better early survival, less toxicity, and lower cost than bone marrow transplantation in the first year after initiating therapy. However, fewer relapses with bone marrow transplantation suggest that it will yield a higher long-term survival rate.

BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies.

Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified antiapoptotic BCL-2 family members as potent 5-Azacytidine-sensitizing targets. In further dissecting BCL-XL, BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-XL and MCL-1, but not BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-XL, BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared with the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-XL, BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-XL increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. On the basis of these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response.