The implementation of targeted therapies for acute myeloid leukaemia (AML) has been challenging because of the complex mutational patterns within and across patients as well as a dearth of ...pharmacologic agents for most mutational events. Here we report initial findings from the Beat AML programme on a cohort of 672 tumour specimens collected from 562 patients. We assessed these specimens using whole-exome sequencing, RNA sequencing and analyses of ex vivo drug sensitivity. Our data reveal mutational events that have not previously been detected in AML. We show that the response to drugs is associated with mutational status, including instances of drug sensitivity that are specific to combinatorial mutational events. Integration with RNA sequencing also revealed gene expression signatures, which predict a role for specific gene networks in the drug response. Collectively, we have generated a dataset-accessible through the Beat AML data viewer (Vizome)-that can be leveraged to address clinical, genomic, transcriptomic and functional analyses of the biology of AML.
Acute myeloid leukemia (AML) is a cancer of myeloid-lineage cells with limited therapeutic options. We previously combined ex vivo drug sensitivity with genomic, transcriptomic, and clinical ...annotations for a large cohort of AML patients, which facilitated discovery of functional genomic correlates. Here, we present a dataset that has been harmonized with our initial report to yield a cumulative cohort of 805 patients (942 specimens). We show strong cross-cohort concordance and identify features of drug response. Further, deconvoluting transcriptomic data shows that drug sensitivity is governed broadly by AML cell differentiation state, sometimes conditionally affecting other correlates of response. Finally, modeling of clinical outcome reveals a single gene, PEAR1, to be among the strongest predictors of patient survival, especially for young patients. Collectively, this report expands a large functional genomic resource, offers avenues for mechanistic exploration and drug development, and reveals tools for predicting outcome in AML.
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•Acute myeloid leukemia patient cohort with clinical, molecular, drug response data•Validation and discovery of diverse biological features of drug response•Broad mapping of tumor cell differentiation state affecting response to drugs•Modeling reveals a strong and targetable determinant of clinical outcome
Bottomly et al. present a functional genomic resource composed of molecular, clinical, and drug response data on acute myeloid leukemia patient specimens. Through integration of all of these data, they identify genetic and cell differentiation state features that predict drug response, and they utilize modeling to identify targetable determinants of clinical outcome.
Introduction: T-cell prolymphocytic leukemia (T-PLL) is an aggressive lymphoproliferative disorder and accounts for ~2% of cases of mature lymphocytic leukemia in adults. The sparsity of patients ...with this disease poses a challenge to effectively develop targeted treatments. Recent studies suggest chromosome 14 abnormalities are most common in T-PLL (75% of patients), resulting in overexpression of the proto-oncogene TCL1 and activation of protein kinase B (Stengel, Anna, et al. 2016). Presently alemtuzumab is the only effective, available therapeutic option, with almost all patients relapsing within months after completion of therapy. With the aim of identifying new therapeutics for T-PLL, we assessed the sensitivity of primary patient samples to various targeted agents using an ex vivo functional screening platform.
Methods: We have previously screened over 1000 primary specimens from patients with various hematologic malignancies against a panel of 143 single-agent small-molecular inhibitors, including kinase inhibitors, bromodomain inhibitors, BHM mimetics, proteasome inhibitors, IDH1/2 inhibitors, and several other drug classes. Primary patient specimens were cultured in the presence of each drug in a 7-point dose series. Sensitivity was assessed by a tetrazolium-based viability assay on day 3. An IC50 value was then calculated for each inhibitor and compared with the median of the IC50 values for the full patient sample cohort screened to date. Inhibitors for which the IC50 value was < 20% of the cohort median were considered effective. T-PLL primary patient samples were assessed for sensitivities using this historical drug sensitivity data and we ranked all drugs by their IC50.
Results: Our database identified 5 adults (3 male, 2 female) with T-PLL who had analyzable data obtained within 1 month of diagnosis (Table 1). Median age was 65 years (range, 49-88 years). At diagnosis, the median white blood cell count was 56.2 K/cu mm (16-243.65 K/cu mm) and all patients had detectable 14q11 abnormalities and TCR gene rearrangements. All patients were treated with standard first-line therapy with IV alemtuzumab (30 mg 3x/week) for an average of 11.6 weeks (8.59 -14.75 weeks). Their median progression free survival (PFS) was 9.3 months (5.4-20 months) and duration of response was 5.6 months (2.9 -16.8 months). Three patients received second-line therapy after alemtuzumab failure with a PFS of 6.9 months (1.1- 9.8 months). Three patients went on to an allogeneic stem cell transplants with PFS of 6.7 months (4.5-7.7 months). The median overall survival was 19.1 months (2.07-11.6 months).
Our ex vivo assay showed that 9 (6.2%) of 143 small-molecular inhibitors were considered effective (20% of median IC50) in the majority of subjects. This included 6 FDA approved tyrosine kinase inhibitors (axitinib, dasatinib, sunitinib, sorafenib, pazopanib, and ponatinib), midostaurin, an NF-kB activation inhibitor, and CSF1R inhibitors. Sensitivity of T-PLL patients to these drugs is shown in the log plot (Figure 1). Patient 4 had no strong targets identified. Dasatinib showed the most efficacy in 4 of the 5 patients, with an average IC50 of 0.003 ± 0.0007 µm, followed by CSFIRJ inhibitor with an average IC50 of 0.02 ± 0.015 µm.
Conclusions: Our functional ex vivo screen of primary patient samples allowed us to evaluate the effectiveness of several FDA approved and investigational therapeutic agents targeting multiple oncogenic pathways in T-PLL, a rare disease with limited existing therapies. Our approach indicates that several existing inhibitors may be effective in this aggressive leukemia and can be tested in prospective clinical trials.
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Druker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy.
Despite improved understanding of molecular lesions in relapsed/refractory acute leukemia, outcomes remain dismal. Aberrantly activated tyrosine kinase signaling pathways play a critical role in the ...pathogenesis of acute leukemia. Identification of drugs that target the drivers of disease has the potential to improve treatment. We have developed an FDA regulated (IDE# G110198) in vitro kinase inhibitor assay that can identify potential therapeutic targets in primary acute leukemia samples and provide individualized treatment options in a clinically relevant time frame. We designed a Phase 2 clinical trial (NCT01620216) to validate the role of this pre-clinical kinase inhibitor screen for selecting effective individualized therapies.
After obtaining informed consent, peripheral blood or bone marrow is isolated using a ficoll-density gradient and plated with dose-escalating concentration gradients of five kinase inhibitors (nilotinib, dasatinib, sorafenib, sunitinib, and ponatinib). Cell line controls are used for each drug. Samples are deemed sensitive based on IC50 and comparison to the median of all samples tested in our laboratory (over 1000 primary samples to date). Patients who satisfy eligibility are treated with FDA approved dosing using the selected inhibitor.
Inclusion is limited to patients age ≥ 21 with relapsed/refractory acute leukemia (AML and ALL) and patients age ≥ 65 with a history of myelodysplasia who have developed AML, have failed hypomethylating agents, and are not candidates for standard induction. Only patients with samples that demonstrate in vitro sensitivity to 1 of the trial drugs are eligible. Additional eligibility includes: ECOG ≤ 2, adequate organ function, and no active GVHD. Cycles are 28-days. Marrow biopsy for response assessment and correlative studies is obtained on days 15, 28, and day 1 of subsequent cycles. The primary objective is to determine the clinical activity, defined as > 25% decrease in bone marrow blast counts. Secondary objectives include overall response (defined by the International Working Group), overall survival, and progression free survival. Correlative studies include high-throughput sequencing, expression profiling, correlation of target inhibition with clinical response, and PK analysis. The treatment approach is deemed worthy of ongoing study if clinical activity is observed in at least 4 of 24 patients.
43 patients have been screened with 12 demonstrating in vitro sensitivity. Nine have been enrolled on treatment. Median age is 64 (31-71) years with 5 males and 4 females. Two patients had ALL. The remainder had AML. Drugs utilized to date include dasatinib (n=3) and sorafenib (n=6). Of the three patients who exhibited in vitro sensitivity but were not enrolled, sensitivity to sorafenib, sunitinib, and dasatinib was seen. Clinical activity was seen in 4 subjects with AML -all treated with sorafenib- with median change in marrow blast in this group of 69% (44-95%). Median time on treatment for responding patients was 39 days (18-110 days). One subject with refractory AML who had failed allogeneic transplant had a 95% decrease in marrow blasts with prolonged disease control (Figure 1). Two patients were taken off study due to drug-related AEs (pancreatitis and diarrhea). No unanticipated AEs were seen.
Our in vitro kinase inhibitor assay identified potential therapies for the treatment of ALL and AML. Although the numbers are too small to draw any specific conclusions regarding response, the 4 responders were FLT3-ITD+ AML, a known target of sorafenib, and appear to have proliferative disease with higher baseline blast counts. The relatively small panel of available treatment drugs with overlapping target profiles may have contributed to the preferential treatment of patients with FLT3-ITD+ AML and to the significant screen fail rate. Our results using a larger panel of targeted agents have shown a high level of sensitivity to at least one drug. Accordingly, we are expanding the platform of clinically available targeted agents. The study continues to enroll patients with the goals of defining additional molecular abnormalities and optimizing assay characteristics, while prospectively testing the therapeutic potential of additional drugs. Correlative studies are ongoing. We have initiated another study using this assay to help select targeted therapies to be added to AML induction (NCT02779283).
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Spurgeon:Gilead Sciences: Research Funding; Bristol Myers Squibb: Research Funding; Acerta Pharma: Research Funding; Genentech: Research Funding; Janssen: Research Funding. Maziarz:Incyte: Membership on an entity’s Board of Directors or advisory committees; Athersys: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Tyner:Janssen Research & Development: Research Funding; Inctye: Research Funding; Genentech: Research Funding; Constellation Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Leap Oncology: Consultancy; Agios Pharmaceuticals: Research Funding; Array Biopharma: Research Funding; Aptose Biosciences: Research Funding; Seattle Genetics: Research Funding; Takeda Pharmaceuticals: Research Funding. Druker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy.
Atypical chronic myeloid leukemia (aCML) and chronic neutrophilic leukemia (CNL) are rare hematologic neoplasms characterized by leukocytosis and a hypercellular bone marrow. Although recurrent ...mutations in the colony-stimulating factor 3 receptor (CSF3R) are frequently observed in patients with (CNL), the mutational landscape in (aCML) is less well-defined. In this report, we describe an 81-year-old male who was diagnosed with aCML. He presented with leukocytosis and anemia but no significant clinical symptoms. Standard laboratory studies revealed the absence of the Philadelphia chromosome. Massively parallel sequencing demonstrated no mutations in CSF3R, but the presence of a heterozygous NRAS-G12D variant (47% allele frequency). The patient was started on treatment with trametinib, an MEK1/2 inhibitor with Food and Drug Administration approval for malignant melanoma. Therapy with trametinib resulted in exceptional improvements in his blood counts and continued disease control with 14 months of follow-up. This case highlights the need for clinical trials evaluating the safety and efficacy of MEK1/2 as a therapeutic target for the treatment of patients with NRAS-mutated aCML/CNL.
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