scholarly journals Custom High Throughput Drug Sensitivity Assay Reveals Therapeutic Options for Chronic Myeloid Leukemia Patients Resistant to or Intolerant of Tyrosine Kinase Inhibitors

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 268-268
Author(s):  
Vivian G. Oehler ◽  
Sylvia Chien ◽  
Jin Dai ◽  
Carrie L. Cummings ◽  
James Annis ◽  
...  
Keyword(s):  
High Throughput ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Kinase Inhibitors ◽  
Blast Phase ◽  
Drug Sensitivity Assay ◽  
Ic50 Values ◽  
Investigational Agents ◽  
New Diagnosis

Abstract Introduction. Tyrosine kinase inhibitors (TKIs) have revolutionized chronic phase (CP) chronic myeloid leukemia (CML) care with many patients achieving major and deeper molecular responses. However, for those who are resistant to or do not tolerate the approved TKIs, there are few alternatives. We therefore developed a custom high throughput drug screen comprised of both FDA approved and investigational agents. Methods. Fifty-six samples (50 individual patients) have undergone testing in the drug sensitivity assay, for which a large fraction exhibited resistance to approved agents. The Quellos High Throughput Core Laboratory's Cancer Drug Sensitivity has been CLIA approved for leukemia since 2015. Blood and bone marrow samples were obtained from CML patients with written informed consent. Mononuclear cells were isolated by density depletion. The myeloid population was obtained by lineage depletion of non-myeloid cells using magnetic beads and antibodies to erythroid lineage (CD235a), T (CD3) and B (CD19) lymphocytes, and NK (CD56) cells. Flow cytometry confirmed successful enrichment of the myeloid cell population. Cells were plated on extracellular matrix coated 384 well plates to test under conditions of adhesion mediated chemotherapy resistance. Initially, the assay was comprised of 32 drugs (11 patients) selected based on published activity in CML and resistant CML. The assay was then expanded to 64 drugs. Compounds are added (ranging from 5 pM to 100 μM) to patient samples using the CyBio CyBi-Well Vario and incubated at 37°C, 5% CO2 for 72 hours, then viability is assessed by CellTiterGlo. IC50s and AUCs are calculated for each drug using XLFit (IDBS) and a standard 4 parameter logistical model. Transcriptome analysis is planned for these samples. Results. Clinical characteristics are shown in Table 1. Mean and median BCR-ABL1 transcripts were 69% and 70% in diagnosis samples and 63% and 55% in resistant samples, respectively (P=0.607). ABL mutations were present in 5 patients (M244V, T315I, F359I). Additional myeloid mutations were present in 5 of 6 evaluable advanced phase samples, 4 of 17 evaluable diagnostic samples, and 3 of 10 evaluable resistant samples and included ASXL1, DNMT3A, IDH1, JAK2V617F, NRAS, RUNX1, and TET2. Figure 1 illustrates the breadth of sensitivity to agents in the assay. Figure 2 is a heat map of area under the curve (AUCs) illustrating the unique drug sensitivity patterns for all patients, with unsupervised clustering. For new diagnosis patients, the TKIs imatinib, dasatinib, nilotinib, bosutinib, and ponatinib ranked in the top 8 drugs. For primary resistant patients, the IC50 values for imatinib, nilotinib, bosutinib, and ponatinib were higher than the new diagnosis patients. For example, for ponatinib, the mean IC50 was 402.6 ± 354.7 X 10 E-9 M for primary resistant samples vs. 1.65 ± 0.45 X 10 E-9 M for diagnosis group, p=0.015 (Welch t test), or about 250-fold higher (less sensitive). In accelerated and blast phase samples drugs with IC50 values lower than 0.1 µM, a range that could correlate with in vivo drug response, were identified for all patients (range, 3-20 drugs per patient). Top candidates included proteasome and kinase inhibitors. In 2 patients harboring NRAS mutations, IC50 for trametinib was less than 0.1 µM as compared to patients without NRAS mutations, where the IC50s were higher. Clinical outcomes are available for nearly all patients. Although the study was not designed to select next-line TKI therapy in resistant patients, drug profiling was informative in many cases. Data for 7 resistant patients are shown in Table 2. For example, CML-012 had the lowest IC50 value (indicating most sensitive) for dasatinib, 4.1 X 10E-8 M and responded to dasatinib after failing imatinib (IC50 8.4 X 10E-7M). CML-003 did not respond to bosutinib (IC50 1.2 X 10E-6M) and did respond to dasatinib (IC50 1.2 X 10E-7M). CML-056 did not respond to nilotinib (IC50 1.4 X 10E-6M), dasatinib (IC50 6.9 X 10E-4M), or ponatinib (IC50 1.0 X 10E-6M). Notably, in all resistant patient samples we identified drugs with IC50 values lower than 0.1 µM. These therapeutics can be prioritized for further evaluation, either alone or in combination with TKIs, in resistant CML patients. Conclusion. In vitro drug sensitivity testing provides data for potential agents for patients with resistance or intolerance to FDA approved TKIs, or those that have entered accelerated phase or blast phase. Figure 1 Figure 1. Disclosures Oehler: Blueprint Medicines: Consultancy; Takeda: Consultancy; Pfizer: Research Funding; OncLive: Honoraria; BMS: Consultancy. Becker: Abbie: Research Funding; SecuraBio: Research Funding; Cardiff Oncology: Research Funding; Pfizer: Research Funding; BMS: Research Funding; CVS Caremark: Consultancy; Glycomimetics: Research Funding. OffLabel Disclosure: We developed a custom high throughput drug screen comprised of both FDA approved and investigational agents

scholarly journals Precision Medicine Assays Detect Novel Targetable FLT3 Fusions Amenable to Therapeutic Intervention in a Patient with Refractory Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5170-5170
Author(s):  
Bradley A Patay ◽  
Andrew Carson ◽  
Timothy J Martins ◽  
Sylvia Chien ◽  
Mary-Elizabeth M. Percival ◽  
...  
Keyword(s):  
High Throughput ◽  
Myeloid Leukemia ◽  
Drug Sensitivity ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Monosomy 7 ◽  
Drug Sensitivity Assay ◽  
Generation Sequencing ◽  
Acute Myeloid ◽  
Refractory Aml

Abstract Context: The 5-year survival rate for acute myeloid leukemia (AML) is 26.6%. The prognosis of patients with adverse events such as older age or unfavorable risk cytogenetics remains poor, even for those who undergo allogeneic hematopoietic cell transplant. AML is a heterogeneous disease and novel N-of-1 clinical trial designs may offer benefit to individuals compared to conventional clinical trials by offering improved utilization of investigational chemotherapy regimens. Precision medicine based assays that reveal a deeper understanding of the cancer biology and potential for novel therapeutics may improve survival in the future. Objective: The goal of the clinical trial on which this patient was enrolled, Individualized Treatment for Relapsed/Refractory Acute Leukemia Based on Chemosensitivity and Genomics/Gene Expression Data (ClinicalTrials.gov Identifier:NCT01872819) was to determine feasibility of a study that utilized results of comprehensive mutation analysis and an in vitrohigh throughput functional assay to choose treatment for individual patients with refractory AML. Feasibility was defined as initiating chosen treatment within 21 days. A secondary objective was to achieve a response (cytoreduction or at least partial response) greater than that expected for comparable refractory patient populations with other salvage regimens. Design, Setting, and Patient: A single center enrolled individuals who had failed at least 2 inductions at initial diagnosis or >1 salvage regimen for relapsed AML. Patients could receive any FDA approved drug or combination regimen based on molecular analysis and high throughput drug sensitivity assay. A 58 year old female was enrolled into this protocol with MECOM (EVI1) rearranged, Monosomy 7 refractory AML. Methods and Main Outcome Measures: The patient had various assays performed on her samples, including next generation sequencing and a high throughput in vitro assay that analyzed enriched blast samples for sensitivity to 150 drugs or drug combinations. MyAML™, a next generation sequencing panel, analyzed 194 genes including breakpoint hotspot loci with long paired end sequencing and high depth that optimized detection of large insertion and deletions and other structural variants found in AML at low variant allele frequency. Results: The MyAML assay detected multiple variants including: NRAS:c.38G>A; p.Gly13Asp VAF = 100%, RUNX1:c.494_495ins; p.165_R166ins VAF = 42%, WT1:c.1149_1150ins; p.E384Pfs*5 VAF=38%. Fusions were also detected: t(13;17)(q12.2;q11.2), t(8;13)(q21.13;q12.2), and t(9;12)(q32;p13.2) which involved FLT3 novel fusions. FLT3 internal tandem duplications (ITD) or tyrosine kinase domain (TKD) variants were not detected. The assay also detected the Monosomy 7 and confirmed the t(2;3) as a THADA-MECOM (EVI1) fusion to the nucleotide breakpoint. These variants involved activated signaling, myeloid transcription factor and DNA demethylation pathways. The high throughput drug sensitivity assay identified sensitivity to kinase inhibitors such as the MEK inhibitor selumetinib (IC50 8.1 X 10e-9 M), Flt3 inhibitor staurosporine (IC50 1.4 X 10e-8 M) and Abl kinase inhibitor ponatinib (IC50 2.7 X 10e-8 M). Insurance coverage for these agents was not able to be obtained as this indication would be considered off label. However, based on the FLT3 fusion we identified using MyAML, we decided to treat the patient with the tyrosine kinase inhibitor sorafenib that we were able to obtain through a patient assistance program. This novel case is the first demonstration of a FLT3 fusion detection with a drug sensitivity assay suggesting that kinase inhibitors with multiple targets might be suitable for this type of variant. Conclusion: Specialized assays designed to identify clonal and subclonal architecture of genes associated with specific diseases can reveal variants that present therapeutic options not currently utilized for high risk patients, suggesting broader use of this approach could improve current clinical outcomes. Disclosures Patay: Invivoscribe, Inc: Consultancy. Carson:Invivoscribe, Inc: Employment. Becker:GlycoMimetics: Research Funding.

Toward Individualized Therapy: Correlation of Mutation Analysis with in Vitro high Throughput Drug Sensitivity Testing in New Diagnosis and Relapsed Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3851-3851
Author(s):  
Pamela S. Becker ◽  
Michael W. Schmitt ◽  
Zhiyi Xie ◽  
Andrew R Carson ◽  
Bradley Patay ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
High Throughput ◽  
Myeloid Leukemia ◽  
Drug Sensitivity ◽  
Missense Mutations ◽  
Sensitivity Testing ◽  
New Diagnosis ◽  
Duplex Sequencing ◽  
Acute Myeloid

Abstract Introduction: Whole genome sequencing has demonstrated tremendous heterogeneity in the mutations and chromosomal translocations associated with acute myeloid leukemia (AML), and there are several correlates with prognosis, yet we remain quite limited in our ability to predict specific chemotherapy drug sensitivity based on genomics with the exception of a few selected mutations or translocations, such as FLT3 -ITD or PML-RARA. One third of new diagnosis patients and over half of relapsed patients will not respond to initial chemotherapy regimens that incur appreciable toxicity and result in prolonged hospitalization. We therefore seek to define molecular information that might better predict response to conventional or novel therapies. Methods: MyAML™ uses next generation sequencing (NGS) to analyze the 3' and 5' UTR and exonic regions of 194 genes and potential genomic breakpoints within known somatic gene fusion breakpoints known to be associated with AML. Fragmented genomic DNA (~3.4Mb) is captured with a customized probe design, and sequenced with 300bp paired end reads on an Illumina MiSeq instrument to an average depth of coverage >1000x. Using a custom bioinformatics pipeline, MyInformatics™, single nucleotide variants (SNVs), insertion/deletions (indels), inversions and translocations are identified, annotated, characterized, and allelic frequencies calculated. Commonly associated variants in dbSNP and 1000 genomes may be eliminated, as well as variants with allele frequencies less than 5%. High throughput drug sensitivity testing was performed against a panel of 160 drugs, of which 56 are FDA approved and 104 are investigational. De-identified samples from 12 patients with de novo AML and 12 patients with relapsed AML were analyzed. For 2 patient samples, Duplex Sequencing was also performed to detect sub-clonal mutations below the detection limit of conventional NGS. Pearson and Spearman correlations were performed between all possible pairs of genes containing missense or indel mutations and the in vitro cytotoxicity response across the same set of 24 patients. Results: From the 24 patient samples analyzed to date, an average of 129 missense mutations were identified in each sample with an allelic frequency >5%. Of these, an average of over 21 missense variants were observed in COSMIC and less than 3 were novel (not in dbSNP). These samples also contained an average of over 12 coding indels (~5 frameshift and 7 inframe indels per sample). In addition, MyAML™ identified 3 samples with inv(16) and 6 samples with translocations, including the cryptic NUP98-NSD1 t(5;11) that was not detected by karyotyping. For 2 of the samples, Duplex Sequencing was performed at a depth of at least 6000X, and an accuracy of 10-7, and showed concordance of some of the mutations, with each method identifying additional mutations not observed by the other, an expected finding, as each method targeted distinct regions, and Duplex Sequencing had a greater depth of coverage. Fourteen genes were observed to exhibit at least one indel with a frameshift at frequency greater than 5% in more than one patient. In order to identify significantly associated drugs and genes containing indel mutations, we computed Pearson and Spearman correlations between drugs and these 14 genes across 24 patients. The correlation analyses revealed significant associations (p= 0.006 to 0.04) between indel mutations in three genes and chemosensitivity to drugs commonly used in AML such as cladribine, clofarabine, cytarabine, daunorubicin, etoposide, fludarabine and mitoxantrone. Similarly, significant associations (p<0.05) were identified between missense mutations in 5 genes and chemosensitivity to these drugs. Conclusion: Personalized data derived from a targeted genomic assay and in vitro chemotherapy sensitivity testing of individual patient AML samples will likely lead to innovation in treatment, identification of novel targeted agents, and improved outcomes in AML. Disclosures Xie: Invivoscribe: Employment. Carson:Genection: Employment. Patay:Genection: Employment.

scholarly journals Novel drug candidates for blast phase chronic myeloid leukemia from high-throughput drug sensitivity and resistance testing

2015 ◽  
Vol 5 (5) ◽  
pp. e309-e309 ◽  
Author(s):  
P O Pietarinen ◽  
T Pemovska ◽  
M Kontro ◽  
B Yadav ◽  
J P Mpindi ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
High Throughput ◽  
Myeloid Leukemia ◽  
Drug Sensitivity ◽  
Resistance Testing ◽  
Blast Phase ◽  
Drug Candidates ◽  
Novel Drug

scholarly journals High Throughput Drug Synergy Testing in a Clinical Trial of Panobinostat, Carfilzomib, Dexamethasone to Define Response Biomarkers for Relapsed/Refractory Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1902-1902
Author(s):  
Pamela S. Becker ◽  
Kim Quach ◽  
Ted A Gooley ◽  
Edward N. Libby ◽  
Andrew J. Cowan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Growth Inhibition ◽  
Clinical Response ◽  
High Throughput ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Combination Index ◽  
Drug Sensitivity Assay ◽  
Synergy Testing

Background: The treatment of multiple myeloma (MM) is optimized by use of combination regimens consisting of agents with different mechanisms of action. Panobinostat is a pan-inhibitor of histone deacetylases types I,II, and IV. Panobinostat, bortezomib, dexamethasone was shown to be an effective regimen (San Miguel et al Lancet Hematol 2016; Richardson et al Blood 2016), leading to the FDA approval of panobinostat for patients with relapsed/refractory MM. Carfilzomib is a proteasome inhibitor that was FDA approved in relapsed/refractory MM with the advantage of minimal neuropathy. Panobinostat and carfilzomib has also been shown to be a highly active regimen in relapsed/refractory MM with an overall response rate of up to 75% (Berdeja et al, Haematologica, 2015). With the heterogeneity of MM, individual patients exhibit wide variability in responses to drug combinations. A test that could predict patient responses to specific agents might enable clinicians to optimize therapy for patients, improving outcomes. We developed an in vitro high throughput drug sensitivity assay with formal synergy testing to predict response. In this ongoing trial, Panobinostat with Carfilzomib and Dexamethasone for Relapsed/Refractory Multiple Myeloma: Correlation with In Vitro Chemosensitivity Testing (NCT03256045), we will correlate individual patient in vitro sensitivity assay results with individual clinical response to the same triple drug regimen. Study Design and Methods: This study's objective is to directly demonstrate the utility of a high throughput drug sensitivity assay in determining biomarkers (e.g. individual IC50s, AUCs and/or synergy scores) to accurately predict response to combination therapy that was given prospectively to all enrolled patients. We are enrolling patients with relapsed/refractory MM by IMWG criteria with measurable disease defined by the detection of a quantifiable monoclonal protein in the urine or serum or an abnormal serum free light chain ratio. Additionally, patients must have adequate blood counts and organ function. Patients who have had prior autologous or allogeneic transplants or CAR-T cell therapy are eligible. The regimen consists of panobinostat 20 mg orally on days 1,3,5,15,17,19; carfilzomib 20 mg/m2/dose IV on days 1,2 of cycle 1, then dose escalation up to 45 mg/m2/dose days 8,9,15,16 and all days for subsequent cycles; and dexamethasone 20 mg orally on days of carfilzomib. Dose reductions of all three drugs are permitted per patient tolerance to allow continuation on study treatment. Up to 12 cycles of treatment are permitted. Patients are monitored by serial electrocardiograms and assessments of cardiac function. Safety parameters including adverse events are recorded. CD138+ plasma cells are procured from the patient bone marrow (aspiration and biopsy) and blood (when present) by magnetic bead separation. Cells are then added to 384-well plates and incubated overnight before the drugs are added. Cells are exposed to 8 concentrations (spanning 4 logs) of panobinostat, carfilzomib, or dexamethasone as singlet, doublet and triplet combinations for 72 hours. Cell viability is determined using CellTiter-Glo and IC50 and AUC values are are calculated by fitting data using least squares method to the standard four-parameter logistic model. Curve fitting is performed using IDBS XLFit software. The combination index is calculated by the method described by Chou and Talalay, Trends Pharmacol Sci 1983;4:450-4. Concentrations of Drug1 and Drug2 (that is, panobinostat and dexamethasone or panobinostat and carfilzomib) alone or in combinations are determined that give rise to 90% growth inhibition. At 90% Growth Inhibition, the Combination Index or CI = ([D1] in the combination / [D1] alone) + ([D2] in the combination / [D2] alone). All patients are treated with panobinostat, carfilzomib, and dexamethasone and evaluated for response using the IMWG response criteria. At the completion of enrollment at 35 patients, we plan to correlate the in vitro testing data with in vivo clinical response to determine appropriate biomarkers. This will be done by correlating the IC50s and AUCs for the individual drugs for responders vs. non-responders (including degree of response VGPR vs PR vs SD), as well as correlations of the synergy scores for each of the pairs of drugs in the responders vs. non-responders. Enrollment was initiated in April 2018. Disclosures Becker: Accordant Health Services/Caremark: Consultancy; AbbVie, Amgen, Bristol-Myers Squibb, Glycomimetics, Invivoscribe, JW Pharmaceuticals, Novartis, Trovagene: Research Funding; The France Foundation: Honoraria. Libby:Abbvie: Consultancy; Pharmacyclics and Janssen: Consultancy; Akcea: Consultancy; Alnylam: Consultancy. Cowan:Juno: Research Funding; Abbvie: Research Funding; Sanofi: Consultancy; Janssen: Consultancy, Research Funding; Cellectar: Consultancy; Celgene: Consultancy, Research Funding. Hammer:Glycomimetics: Consultancy.

Rapid Reduction of Chronic Myeloid Leukemia Stem Cells After Treatment with Second-Generation BCR-ABL Kinase Inhibitors, Dasatinib and Nilotinib

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4457-4457
Author(s):  
Yosuke Minami ◽  
Akihiro Abe ◽  
Yuka Nomura ◽  
Miho Minami ◽  
Yachiyo Kuwatsuka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Second Generation ◽  
Kinase Inhibitors ◽  
Newly Diagnosed ◽  
Rapid Reduction ◽  
Population Total ◽  
Significant Difference

Abstract Abstract 4457 Chronic myeloid leukemia (CML) is effectively treated with imatinib (IM), however, several mathematical models and ex vivo-examinations suggested that IM-therapy does not eradicate BCR-ABL-positive hematopoietic stem cells (HSC). We prospectively (0, 3, 6 and 12 months after IM-therapy) investigated 16 newly diagnosed and 22 long-term followed CML-chronic phase (CP) cases using methods previously reported (Jamieson et al., N Engl J Med, 2004. and Abe et al., Int J Hematol, 2008) (Figure 1) with FACSAria™ and quantitative RT-PCR of BCR-ABL among each sorted population; total mononuclear cells, HSC/Thy-1+, HSC/Thy-1–, common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP) and megakaryocyte erythroid progenitors (MEP). In optimal responders to IM-therapy, BCR-ABL transcripts in the HSC populations (HSC/Thy-1+ and HSC/Thy-1–) tended to be more retentive than other populations while gradual reduction was observed during the first 12 months in all populations. And discrepancy of minimum residual diseases (MRD) between the HSC populations and other populations was larger in patients after longer IM-therapy. In evaluating properties of CML stem cells and other markers, we observed irrelevant distribution of side population (SP) and expressions of ABC transporters (ABCB1 and ABCG2) in comparison with CD34/38 expression. We also prospectively investigated BCR-ABL transcripts in each population of 23 IM-resistant or -intolerant CML-CP cases and one newly diagnosed CML-accelerated phase (AP) case during treatment with second-generation tyrosine kinase inhibitors (2nd TKIs), dasatinib or nilotinib. Treatment with each inhibitor induced more rapid reduction of BCR-ABL transcripts even in the HSC population (CD34+CD38–) during the first 6 months and there was no significant difference of MRD among each population in optimal responders to 2nd TKIs-therapy. In the stromal co-culturing system using primary cells and leukemic NOD/SCID/IL2rgnull (NOG) mice xenotransplanted with Ph+ leukemia cells, retention of quiescent slow-cycling (Hoechst 33342low/Pyronin Ylow) CD34+ population after IM-treatment were observed and cell death mechanisms after treatment with 2nd TKIs are also under investigation. These results imply that therapy with 2nd TKIs could be a promising approach for quick and efficient reduction of the CML stem cells and cure of disease. Figure 1 Figure 1. Disclosures: Naoe: Kyowa-Kirin: Research Funding; Novartis: Research Funding; Bristol-Myers Squibb: Research Funding.

Health Related Quality of Life of Bosutinib in Imatinib-Resistant or Imatinib-Intolerant Patients with Advanced Chronic Myeloid Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4448-4448 ◽  
Author(s):  
Jennifer Whiteley ◽  
Arlene Reisman ◽  
Virginia Kelly ◽  
Jorge E Cortes ◽  
David Cella
Keyword(s):  
Myeloid Leukemia ◽  
Research Funding ◽  
Well Being ◽  
Health Related Quality ◽  
Employment Equity ◽  
Blast Phase ◽  
Related Quality ◽  
Health Related ◽  
Equity Ownership

Abstract Abstract 4448 Purpose: Bosutinib is a dual Src/Abl tyrosine kinase inhibitor (TKI), which has demonstrated efficacy in a phase I/II study of patients with Advanced Phase Chronic Myeloid Leukemia (CML). The objective was to evaluate the effect of bosutinib on health -related quality of life (HRQoL) in patients with advanced CML after failure with imatinib. Methods: Patient reported HRQoL was an exploratory objective in the clinical trial and measured using the 44-item Functional Assessment of Cancer Therapy-Leukemia (FACT-Leu). The FACT-Leu is a modular approach to assess patient HRQoL using a core set of general cancer questions as well as a cancer site specific leukemia subscale with 5 domains: Social Well-being (SWB), Emotional Well-being (EWB), Physical Well-being (PWB), Functional Well-being (FWB) and Leukemia Subscale (LeuS); and 3 summary scales: FACT-General, FACT Trial Outcome Index (TOI) and FACT-Leu Total. The item responses for each scale are summed to provide scores; higher scores indicate better HRQoL. The FACT-Leu was completed at weeks 4, 8, 12 and every 12 weeks thereafter, as well as treatment completion. Within cohort comparisons were assessed using paired t-tests. Results: Of the 164 patients with advanced leukemia included in the trial, 76 had accelerated phase (AP) CML and 64 blast phase (BP) CML. At 24 weeks, AP patients reported statistically significant improvements in PWB (p=0.02), EWB (p<0.001), LeuS (p<0.001), FACT-G (p<0.001), FACT-Leu (p<0.001) and FACT-TOI (p<0.001) with the PWB, FACT-G, LeuS, TOI and FACT-Leu exceeding minimally important differences (MID) at 24 weeks. Blast phase patients reported significant improvements in PWB (p=0.02), EWB (p=0.02), FWB (p=0.04), LeuS (p=0.01), FACT-G (p<0.001), FACT-Leu (p<0.001) and FACT-TOI (p=0.01) at 24-weeks with all scales exceeding MID except the SWB. At 48-weeks the AP patients continued to have statistically significant improvements in PWB (p=0.05), EWB (p=0.02), and FACT-G (p=0.03) and PWB, FACT-G, TOI and FACT-Leu exceeded the MID at 48 weeks. There were no statistically significant deteriorations in HRQoL through week 48 (Figure 1) Conclusions: These data suggest that CML patients treated with bosutinib demonstrate improved HRQoL. Confirmation in a controlled study is needed. Disclosures: Whiteley: Pfizer Inc: Employment, Equity Ownership. Reisman:Pfizer Inc: Employment, Equity Ownership. Kelly:Pfizer Inc: Employment, Equity Ownership. Cortes:Novartis, Bristol Myers Squibb, Pfizer, Ariad, Chemgenex: Consultancy, Research Funding. Cella:Pfizer Inc: Honoraria, Research Funding.

Peripheral Blood Flow-Cytometry Chronic Myeloid Leukemia Stem Cells Detection and Quantification during Tyrosine Kinase Inhibitors Therapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 942-942 ◽  
Author(s):  
Monica Bocchia ◽  
Lara Aprile ◽  
Santina Sirianni ◽  
Elisabetta Abruzzese ◽  
Antonella Gozzini ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Fish Analysis ◽  
Molecular Response ◽  
Line Treatment ◽  
First Line ◽  
Qrt Pcr ◽  
First Line Treatment

Abstract Introduction: In chronic myeloid leukemia (CML), tyrosine Kinase Inhibitors (TKIs) treatment is a potentially life-time therapy for the majority of patients (pts), as few of them, only after achieving a deep and stable molecular response, may discontinue TKIs without recurrence of disease. Available data suggest that relapse after TKIs discontinuation is due to the persistence of leukemic stem cells (LSCs) intrinsically resistant to TKIs. Survival of CML LSCs may be the consequence of activation of several pathways BCR-ABL1 independent. qRT-PCR, the most sensitive assay to monitor disease status in CML pts, may be inappropriate to quantify residual quiescent CML LSCs that are transcriptionally silent. Therefore, the possibility to easily quantify LSCs during TKIs treatment is a great opportunity to better understand the behavior of residual LSCs and potentially to identify those pts candidates to safely discontinue TKIs. Recently, Valent et al described that CD34+/CD38-/Lin- CML LSCs specifically co-express dipeptidylpeptidase IV (CD26) and that CD26 is a potential biomarker for the quantification and isolation of CML LSCs, in bone marrow samples of CML patients. Furthermore, Culen et al. quantified CD26+ LSCs bone marrow compartment in 31 CML patients at diagnosis and their number appears to correlate with response to TKIs treatment. In the present study we wanted to explore the feasibility, rate and potential implication of detecting CD26+ LSCs in peripheral blood (PB) from CML pts during TKI treatment. Methods: CML pts during first line treatment with any approved TKIs, referring to several Italian Hematology Centers, entered this non interventional cross sectional study after signing a proper informed consent. During a routine follow up visit, in which pts were checked for molecular response by standard PB qRT-PCR BCR-ABL1 analysis, additional 3 mls of PB were collected in EDTA and sent within 24 hours to Siena Hematology Lab to detect CD34+/CD38-/CD26+ LSCs by multicolor flow cytometry. After red blood cells lysis, cells were incubated with anti CD45 (BD Biosciences), CD34 (581), CD38 (HIT2), CD26 (M-A261) (BD Pharmigen). After washing, acquisition and analysis were performed by FACSCanto II (BD Biosciences, NR Nannini) using DIVA 8 software (BD, Biosciences). CD45+ cells acquired for each sample ranged from 500,000 to 1,000,000. Isotype controls were included in each staining. In 5 pts a FISH analysis of PB sorted CML LSCs population was also performed. Results: to validate our assay we first performed a FISH analysis of both PB sorted CD34+/CD38-/CD26+ and CD34+/CD38-/CD26- in 5 CML patients at 3-6 months after starting treatment, confirming Ph+ cells only in the CD26+ fraction. Afterward, we checked for circulating CML LSCs a total of 202 CML pts in first line treatment with TKIs for a median of 39 months (range 1-175). Type of TKI, length of treatment, molecular response and quantification of LSCs are summarized in Table 1. PB CML LSCs were detectable in 146/202 (72.3%) pts with a median number of CD26+ of 0,0165 cells/µL (range 0,0018-0,66). Kendall rank correlation coefficient used to analyze the relation between the measurable variables showed no correlation between BCR-ABL/ABLIS ratio (median 0,004 range 0-61) and number of residual LSCs (r 0.118 p=0.097). In 56/202 (27.7%) pts CD26+ LSCs were undetectable, yet we found no correlation with the concomitant degree of molecular response. Conclusions: this study represents the first attempt to measure in a large cohort of CML patients residual circulating LSCs during TKIs treatment. In our hands PB LSCs flow-cytometry assay appeared feasible, specific and sensitive and thus suitable for routine monitoring. As expected, the majority of CML patients, even in deep molecular response, still harbor residual LSCs and the number of PB CD26+ did not correlate with the number of BCR-ABL1 copies. This evidence suggests that the molecular response refers to transcriptionally active CML progenitor cells and not to quiescent, TKIs resistant, CML LSCs. Prospective studies evaluating the behavior of PB CML LSCs during different TKIs treatment, as well as studies monitoring PB CD26+ in CML pts that discontinued TKIs treatment are ongoing. Our goal is to rule out the impact, if any, of a "stem cell response" in addition to the standard molecular response in the management of CML patients mainly to identify those pts candidates for a safe TKI discontinuation. Disclosures Bocchia: Janssen: Honoraria; Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Aprile:Novartis: Honoraria. Castagnetti:Pfizer: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; ARIAD Pharmaceuticals: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Tiribelli:Ariad Pharmaceuticals: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau. Breccia:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Ariad: Honoraria; Pfizer: Honoraria. Rosti:Roche: Honoraria, Research Funding, Speakers Bureau; Incyte: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau.

scholarly journals Allogeneic stem cell transplantation for patients harboring T315I BCR-ABL mutated leukemias

Blood ◽  
2011 ◽  
Vol 118 (20) ◽  
pp. 5697-5700 ◽  
Author(s):  
Franck Emmanuel Nicolini ◽  
Grzegorz W. Basak ◽  
Simona Soverini ◽  
Giovanni Martinelli ◽  
Michael J. Mauro ◽  
...  
Keyword(s):  
Overall Survival ◽  
Stem Cell ◽  
Chronic Myeloid Leukemia ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Philadelphia Chromosome ◽  
Blast Phase

Abstract T315I+ Philadelphia chromosome–positive leukemias are inherently resistant to all licensed tyrosine kinase inhibitors, and therapeutic options remain limited. We report the outcome of allogeneic stem cell transplantation in 64 patients with documented BCR-ABLT315I mutations. Median follow-up was 52 months from mutation detection and 26 months from transplantation. At transplantation, 51.5% of patients with chronic myeloid leukemia were in the chronic phase and 4.5% were in advanced phases. Median overall survival after transplantation was 10.3 months (range 5.7 months to not reached [ie, still alive]) for those with chronic myeloid leukemia in the blast phase and 7.4 months (range 1.4 months to not reached [ie, still alive]) for those with Philadelphia chromosome–positive acute lymphoblastic leukemia but has not yet been reached for those in the chronic and accelerated phases of chronic myeloid leukemia. The occurrence of chronic GVHD had a positive impact on overall survival (P = .047). Transplant-related mortality rates were low. Multivariate analysis identified only blast phase at transplantation (hazard ratio 3.68, P = .0011) and unrelated stem cell donor (hazard ratio 2.98, P = .011) as unfavorable factors. We conclude that allogeneic stem cell transplantation represents a valuable therapeutic tool for eligible patients with BCR-ABLT315I mutation, a tool that may or may not be replaced by third-generation tyrosine kinase inhibitors.

scholarly journals Failure to achieve a complete hematologic response at the time of a major cytogenetic response with second-generation tyrosine kinase inhibitors is associated with a poor prognosis among patients with chronic myeloid leukemia in accelerated or blast phase

Blood ◽  
2009 ◽  
Vol 113 (21) ◽  
pp. 5058-5063 ◽  
Author(s):  
Carmen Fava ◽  
Hagop M. Kantarjian ◽  
Elias Jabbour ◽  
Susan O'Brien ◽  
Nitin Jain ◽  
...  
Keyword(s):  
Survival Rate ◽  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Myeloid Leukemia ◽  
Second Generation ◽  
Kinase Inhibitors ◽  
Cytogenetic Response ◽  
Blast Phase ◽  
Complete Hematologic Response

Abstract Second-generation tyrosine kinase inhibitors are effective in Philadelphia chromosome–positive (Ph+) acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML). Occasionally, patients with Ph+ ALL, or accelerated phase (AP) or blast phase (BP) CML achieve a major cytogenetic response (MCyR) but not a complete hematologic response (CHR). We analyzed 126 patients with CML in AP or BP, or with Ph+ ALL treated with dasatinib or nilotinib after imatinib failure. Twenty patients received sequential treatment with both dasatinib and nilotinib for a total of 146 instances. CHR and MCyR rates were 54% and 37%, respectively in AP, 17% and 39% in BP, and 33% and 50% in Ph+ ALL. Failure to achieve a CHR at the time of achievement of a MCyR was associated with an inferior outcome, similar to that of patients without a MCyR (2-year survival rate, 37% and 35%, respectively). In contrast, patients with MCyR and concomitant CHR had a 77% 2-year survival rate. Twelve of 29 patients with MCyR without concomitant CHR later achieved a CHR; the 2-year survival rate for these patients was 55% compared with 22% for those who never achieved a CHR. These results suggest that achievement of a MCyR without concomitant CHR is associated with poor outcome.

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