Pamela S. Becker

Relation of Clinical Response and Minimal Residual Disease and Their Prognostic Impact on Outcome in Acute Myeloid Leukemia

PURPOSE Both presence of minimal residual disease (MRD) and achievement of complete remission (CR) with incomplete platelet recovery (CRp) rather than CR after induction therapy predict relapse in acute myeloid leukemia (AML). These results suggest a correlation between response (peripheral count recovery) and MRD at the time of morphologic remission. Here we examine this hypothesis and whether MRD and response provide independent prognostic information after accounting for other relevant covariates. PATIENTS AND METHODS We retrospectively analyzed data from 245 adults with AML who achieved CR, CRp, or CR with incomplete blood count recovery (CRi) after induction therapy. Bone marrow samples were collected on or closest to the first date of blood count recovery, and MRD was determined by 10-color multiparameter flow cytometry. RESULTS The 71.0% of patients who achieved CR had MRD less frequently and had lower levels of MRD than the 19.6% of patients achieving CRp and 9.4% achieving CRi. Although pretreatment covariates such as cytogenetics, monosomal karyotype, relapsed or refractory rather than newly diagnosed AML, and FLT3 internal tandem duplication were associated with relapse, their prognostic effect was much lower once MRD and response were taken into account, the univariable statistical effect of which was not materially affected by inclusion of pretreatment covariates. CONCLUSION Our data indicate that post-therapy parameters including MRD status and response are important independent prognostic factors for outcome in patients with AML achieving remission. MRD status and type of response (CR v CRp or CRi) should play important, and perhaps dominant, roles in planning postinduction therapy.

Romiplostim monotherapy in thrombocytopenic patients with myelodysplastic syndromes: long-term safety and efficacy

Romiplostim can improve platelet counts in about 50% of patients with low‐ or intermediate 1‐risk (lower risk) myelodysplastic syndromes (MDS) and thrombocytopenia, but its long‐term toxicity and efficacy are not known. This open‐label extension study evaluated the long‐term safety and efficacy of romiplostim in 60 patients with lower risk MDS and platelet counts ≤50 × 109/l. The primary endpoint was adverse event (AE) incidence. Secondary endpoints were efficacy parameters, including bleeding events and platelet response. Median (range) treatment time in the extension study and the median observation times thereafter were 25 (2–181) and 57 (11–209) weeks, respectively. Treatment‐related AEs and serious AEs were reported in 14/60 (23%) and 4/60 (7%) patients, respectively. Progression to acute myeloid leukaemia (AML) occurred in two patients after 44 and 46 weeks. Patients (n = 34, 57%) with a platelet response were further evaluated for length of response. Median (range) response duration was 33 (7–174) weeks; 28/34 (82%) patients had a continuous response. Five of 34 patients (15%) had grade ≥3 bleeding events; three when the platelet count was >50 × 109/l. There were no new safety concerns and the rate of progression to AML was low; response to romiplostim was maintained for most patients.

Novel lineage depletion preserves autologous blood stem cells for gene therapy of Fanconi anemia complementation group A

A hallmark of Fanconi anemia is accelerated decline in hematopoietic stem and progenitor cells (CD34 +) leading to bone marrow failure. Long-term treatment requires hematopoietic cell transplantation from an unaffected donor but is associated with potentially severe side-effects. Gene therapy to correct the genetic defect in the patient’s own CD34+ cells has been limited by low CD34+ cell numbers and viability. Here we demonstrate an altered ratio of CD34Hi to CD34Lo cells in Fanconi patients relative to healthy donors, with exclusive in vitro repopulating ability in only CD34Hi cells, underscoring a need for novel strategies to preserve limited CD34+ cells. To address this need, we developed a clinical protocol to deplete lineage+(CD3+, CD14+, CD16+ and CD19+) cells from blood and marrow products. This process depletes >90% of lineage+cells while retaining ≥60% of the initial CD34+cell fraction, reduces total nucleated cells by 1–2 logs, and maintains transduction efficiency and cell viability following gene transfer. Importantly, transduced lineage− cell products engrafted equivalently to that of purified CD34+ cells from the same donor when xenotransplanted at matched CD34+ cell doses. This novel selection strategy has been approved by the regulatory agencies in a gene therapy study for Fanconi anemia patients (NCI Clinical Trial Reporting Program Registry ID NCI-2011-00202; clinicaltrials.gov identifier: 01331018).

Tandem autologous/allogeneic hematopoietic cell transplantation with bortezomib maintenance therapy for high-risk myeloma

We evaluated tandem autologous/allogeneic hematopoietic cell transplantation followed by bortezomib maintenance therapy in a prospective phase 2 trial of treatment of high-risk multiple myeloma. The high-dose conditioning regimen for autologous hematopoietic cell transplantation consisted of melphalan 200 mg/m2. The nonmyeloablative conditioning regimen for the allogeneic transplant involved low-dose total body irradiation (2 Gy) with or without fludarabine (30 mg/m2 × 3 days). Among the 31 patients enrolled, 26 (84%) proceeded to HLA-matched allogeneic hematopoietic cell transplantation at a median of 61 (range, 41-168) days following the autologous transplant. Twenty-one patients (68%) started bortezomib (1.6 mg/m2 IV or 2.6 mg/m2 subcutaneously every 14 days for 9 months) at a median of 79 (range, 63-103) days after allogeneic transplantation. With a median follow-up of 51 (range, 16-86) months and based on intention to treat, the 2-year and 4-year progression-free survival and overall survival estimates among 24 newly diagnosed high-risk patients were 71% and 75%, and 52% and 61%, respectively. The 7 patients enrolled with relapsed or persistent disease had a 2-year and 4-year progression-free survival and overall survival rates of 14% and 43%, and 14% and 29%, respectively. These findings suggest that for patients with newly diagnosed high-risk multiple myeloma, bortezomib maintenance therapy after tandem autologous/allogeneic hematopoietic cell transplantation is safe and may prevent disease progression until full establishment of a graft-versus-myeloma effect. This benefit, however, does not extend to patients who enroll after unsuccessful prior therapy. This trial was registered at www.clinicaltrials.gov as #NCT00793572.

215 Update of open-label extension study evaluating the long-term safety and efficacy of romiplostim in thrombocytopenic patients with myelodysplastic syndromes (MDS)

Case 3: Male 78 years-old diagnosed of SRA (low IPSS, normal cytogenetic) 1 year ago. He had a transitory respond to EPO. 2 months later, he began 5-aza therapy, with good respond 3 months later. He did keep up to 15th cycle. Later, he has initiated Lenalidomide (10mg/d), he has managered the independent transfusional. Initial Hb was 6 g/dL, and now Hb is 9 g/dL, after 8 months of treatment. Case 4: Female 63 years-old was diagnosed of SRA (low IPSS, normal cytogenetic) 20 years ago. She began 5-aza therapy, with good respond 3 months later, and this was maintained 24 months later. She has initiated Lenalidomide (10mg/d). At present, she hasn’t respond yet, after 5 months. Results: 3 patients reached the independent transfusion, but we were obligated to tapering dosage and we must wait 6–10 months to research the respond. Conclusions: 1. In low risk MDS patients, non 5q−, lenalidomide is an alternative therapeutic, when 5-aza has failed; 2. Lenalidomide used dosage, is lower than recommended by toxicity; 3. The response is later than we hope.

Infusion of a non-HLA-matched ex-vivo expanded cord blood progenitor cell product after intensive acute myeloid leukaemia chemotherapy

BACKGROUND The intensive chemotherapy regimens used to treat acute myeloid leukaemia routinely result in serious infections, largely due to prolonged neutropenia. We investigated the use of non-HLA-matched ex-vivo expanded cord blood progenitor cells to accelerate haemopoietic recovery and reduce infections after chemotherapy. METHODS We enrolled patients with a diagnosis of acute myeloid leukaemia by WHO criteria and aged 18-70 years inclusive at our institution (Fred Hutchinson Cancer Research Center) into this phase 1 trial. The primary endpoint of the study was safety of infusion of non-HLA-matched expanded cord blood progenitor cells after administration of clofarabine, cytarabine, and granulocyte-colony stimulating factor priming. The protocol is closed to accrual and analysis was performed per protocol. The trial is registered with ClinicalTrials.gov, NCT01031368. FINDINGS Between June 29, 2010, and June 26, 2012, 29 patients with acute myeloid leukaemia (19 newly diagnosed, ten relapsed or refractory) were enrolled. The most common adverse events were fever (27 [93%] of 29 patients) and infections (25 [86%] of 29 patients). We observed one case of acute infusional toxicity (attributed to an allergic reaction to dimethyl sulfoxide) in the 29 patients enrolled, who received 42 infusions of expanded progenitor cells. The following additional serious but expected adverse events were observed (each in one patient): grade 4 atrial fibrillation, grade 4 febrile neutropenia, lung infection with grade 4 absolute neutrophil count, colon infection with grade 4 absolute neutrophil count, grade 4 changed mental status, and one death from liver failure. No unexpected toxicity or graft-versus-host disease was observed. There was no evidence of in-vivo persistence of the expanded progenitor cell product in any patient beyond 14 days or induced alloimmunisation. INTERPRETATION Infusion of the expanded progenitor cell product seemed safe and might provide a promising treatment method for patients with acute myeloid leukaemia. FUNDING Biomedical Advanced Research and Development Authority in the US Department of Health and Human Services and Genzyme (Sanofi).

ArticlesInfusion of a non-HLA-matched ex-vivo expanded cord blood progenitor cell product after intensive acute myeloid leukaemia chemotherapy: a phase 1 trial

BACKGROUND The intensive chemotherapy regimens used to treat acute myeloid leukaemia routinely result in serious infections, largely due to prolonged neutropenia. We investigated the use of non-HLA-matched ex-vivo expanded cord blood progenitor cells to accelerate haemopoietic recovery and reduce infections after chemotherapy. METHODS We enrolled patients with a diagnosis of acute myeloid leukaemia by WHO criteria and aged 18-70 years inclusive at our institution (Fred Hutchinson Cancer Research Center) into this phase 1 trial. The primary endpoint of the study was safety of infusion of non-HLA-matched expanded cord blood progenitor cells after administration of clofarabine, cytarabine, and granulocyte-colony stimulating factor priming. The protocol is closed to accrual and analysis was performed per protocol. The trial is registered with ClinicalTrials.gov, NCT01031368. FINDINGS Between June 29, 2010, and June 26, 2012, 29 patients with acute myeloid leukaemia (19 newly diagnosed, ten relapsed or refractory) were enrolled. The most common adverse events were fever (27 [93%] of 29 patients) and infections (25 [86%] of 29 patients). We observed one case of acute infusional toxicity (attributed to an allergic reaction to dimethyl sulfoxide) in the 29 patients enrolled, who received 42 infusions of expanded progenitor cells. The following additional serious but expected adverse events were observed (each in one patient): grade 4 atrial fibrillation, grade 4 febrile neutropenia, lung infection with grade 4 absolute neutrophil count, colon infection with grade 4 absolute neutrophil count, grade 4 changed mental status, and one death from liver failure. No unexpected toxicity or graft-versus-host disease was observed. There was no evidence of in-vivo persistence of the expanded progenitor cell product in any patient beyond 14 days or induced alloimmunisation. INTERPRETATION Infusion of the expanded progenitor cell product seemed safe and might provide a promising treatment method for patients with acute myeloid leukaemia. FUNDING Biomedical Advanced Research and Development Authority in the US Department of Health and Human Services and Genzyme (Sanofi).

Does outcome of second salvage therapy in relapsed or refractory acute myeloid leukemia depend on intensity of either first or second salvage therapy

Daisuke Araki, Megan Othus, Roland B. Walter, Pamela S. Becker, Vicky Sandhu and Elihu H. Estey Department of Medicine, University of Washington, Seattle, WA, USA; Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; Department of Medicine, Division of Hematology, University of Washington, Seattle, WA, USA; Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

Pre-transplantation novel agent induction predicts progression-free survival for patients with immunoglobulin light-chain amyloidosis undergoing high-dose melphalan and autologous stem cell transplantation.

Abstract Introduction: High-dose melphalan and autologous stem cell transplantation (HDM/SCT) is an effective treatment modality for immunoglobulin light-chain (AL) amyloidosis; however, its application remains restricted to patients with good performance status and limited organ involvement. In recent years, the paradigm for AL amyloidosis has changed with the introduction of novel agents such as immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs). We hypothesized that use of novel agent induction regimens has improved outcomes for patients with AL amyloidosis undergoing HDM/SCT at our center. Methods: All patients with AL amyloidosis, age ≥18 years who underwent HDM/SCT between 2001 and 2014 at the Fred Hutchinson Cancer Research Center and University of Washington Medical Center were included in this study. Any regimen administered within 6 months prior to HDM/SCT including an IMiD or a PI was considered a novel induction regimen. Use of induction regimen was evaluated in a Cox proportional hazard model for association with progression-free survival (PFS) and overall survival (OS). Results: Forty-five patients with AL amyloidosis underwent HDM/SCT. The median age was 57.2 years (range 39–74.4), 15 (33.3%) were women. The median number of organs involved was 2 (range 1–5), with 20 patients having only 1 (44.4%), 10 patients having 2 (22.2%), and 15 patients (33.3%) having ≥ 3 organs involved. Novel agent induction regimens were used prior to HDM/SCT in 21 patients (46.7%); these comprised PI in 13/21 (57.1%), IMiD alone in 6/21 (28.6%), PI and cyclophosphamide (CyBorD) in 3/21 (14.3%), and IMiD and PI in 3/21 (14.3%). Use of a novel agent induction regimen was associated with improved, but not OS. The 3-year PFS for patients who received a novel agent induction was 79%, while for those who did not was 53% (hazard ratio [HR] = 0.317, p = 0.048). The 3-year OS for patients who received novel agent induction regimens was 95%, while for those who did not was 71% (HR = 0.454, p = 0.247). Discussion: Our data suggest that use of a novel agent induction regimen including an IMiD or PI prior to HDM/SCT for patients with AL amyloidosis could improve outcomes, with improvement in PFS. Although these results are limited by sample size and lack of randomization, these results support possible further investigation of novel agent induction regimens in the context of a prospective clinical trial.

Abstract LB-283: Human cancers harbor extensive subclonal mutations

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Recent advances in next-generation DNA sequencing (NGS) have revealed greater than expected mutational heterogeneity, not only between tumors of similar cancer type, but also within individual tumors. This mutational heterogeneity could serve as a reservoir for the emergence of new phenotypes, including resistance to therapy. While mutational diversity has been reported in many human cancers, the high error-rate of conventional NGS limits its ability to confidently resolve mutations present in less than 1.0 to 5% of the cells that comprise a tumor. These low-level subclonal mutations likely contribute to the rapid emergence of resistance to radiation and chemotherapy and the ability of cancer cells to invade adjacent tissues and to metastasize. In order to study subclonal mutations, we have developed a highly accurate sequencing protocol, termed Duplex Sequencing, which takes advantage of the double-stranded nature of DNA to increase the accuracy of DNA sequencing by more than 10,000-fold; Duplex Sequencing has an unprecedented background error frequency of 1,000,000-fold, with up to 99.8% of resultant sequencing reads on target. Using these new methodologies, we have investigated the subclonal makeup of several cancers, including acute myeloid leukemia (AML), colon cancers (CRC), and glioblastoma (GBM). Upon targeting genes identified by NGS as drivers of clonal proliferation, we identified multiple subclonal mutations in each of these cancers, some with the potential to elicit drug resistance and others to drive tumor cell proliferation. We also investigated the five human replicative DNA polymerases. No reports have previously implicated mutations in these polymerases in either AML or GBM, nor in sporadic CRC with the exception of mutations in the exonuclease domain of DNA polymerase epsilon. However, our approach, combining targeted gene capture with Duplex Sequencing, revealed multiple subclonal mutations in the catalytic domains of all five replicative DNA polymerases in each of these tumors. The presence of subclonal mutations in the catalytic domains of replicative DNA polymerases supports the mutator phenotype hypothesis, which posits that an increased mutation rate is a driving force during early tumorigenesis. Furthermore, extrapolating the results on the number of subclonal mutations in target genes to the entire genome, our results indicate that sites harboring subclonal mutations are >100-fold more frequent than sites with clonal mutations. Thus, by the time a tumor is clinically diagnosed, every position in the genome could be mutated in at least one cell in the tumor; these subclones could be the reservoir for the emergence of new phenotypes and resistance to therapy. Citation Format: Michael W. Schmitt, Edward J. Fox, Marc J. Prindle, Kate S. Reid – Bayliss, Pamela S. Becker, Lawrence A. Loeb. Human cancers harbor extensive subclonal mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-283. doi:10.1158/1538-7445.AM2015-LB-283