Impact of Prior Inotuzumab Ozogamicin Treatment on Brexucabtagene Autoleucel outcomes in Adults with B-cell ALL.

The effect of prior inotuzumab ozogamicin (InO) treatment on brexucabtagene autoleucel (brexu-cel) outcomes remains unclear in adults with acute lymphoblastic leukemia (ALL), particularly the influence off previous InO response and the timing of administration. We conducted a retrospective multicenter analysis of 189 patients with relapsed/refractory (r/r) ALL treated with brexu-cel. Over half of the patients received InO before brexu-cel (InO-exposed). InO-exposed patients were more heavily pretreated (p= 0.02) and frequently had active marrow disease pre-apheresis (p= 0.03). Response rate and toxicity profile following brexu-cel were comparable for InO-exposed and InO-naïve; however, consolidation therapy post brexu-cel response was utilized at a higher rate in InO-naïve patients (p= 0.005). With a median follow up of 11.4 months, InO-exposed patients had inferior progression-free survival (PFS) (p=0.013) and overall survival (OS) (p=0.006) in univariate analyses; however, prior InO exposure did not influence PFS (HR 1.20, 95%CI, 0.71-2.03) in multivariate models. When InO-exposed patients were stratified according to prior InO response, InO responders had superior PFS (p=0.002) and OS (p<0.0001) relative to InO-refractory. The timing of administering InO did not affect brexu-cel outcomes, with comparable PFS (p=0.51) and OS (p=0.86) for patients receiving InO as bridging therapy or pre-apheresis. In conclusion, while InO exposure was associated with inferior survival outcomes following brexu-cel in unadjusted analyses, these associations were no longer significant in multivariate analyses, suggesting it is unlikely that InO negatively impacts brexu-cel efficacy. Our data instead imply that InO-exposed recipients of brexu-cel tend to be higher-risk patients with intrinsic adverse leukemia biology.

Impact of prior therapies and subsequent transplantation on outcomes in adult patients with relapsed or refractory B-cell acute lymphoblastic leukemia treated with brexucabtagene autoleucel in ZUMA-3.

BACKGROUND: Brexucabtagene autoleucel (brexu-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved in the USA for adults with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL) and in the European Union for patients ≥26 years with R/R B-ALL. After 2 years of follow-up in ZUMA-3, the overall complete remission (CR) rate (CR+CR with incomplete hematological recovery (CRi)) was 73%, and the median overall survival (OS) was 25.4 months in 78 Phase 1 and 2 patients with R/R B-ALL who received the pivotal dose of brexu-cel. Outcomes by prior therapies and subsequent allogeneic stem cell transplantation (alloSCT) are reported. METHODS: Eligible adults had R/R B-ALL and received one infusion of brexu-cel (1×106 CAR T cells/kg) following conditioning chemotherapy. The primary endpoint was the CR/CRi rate per central review. Post hoc subgroup analyses were exploratory with descriptive statistics provided. RESULTS: Phase 1 and 2 patients (N=78) were included with median follow-up of 29.7 months (range, 20.7-58.3). High CR/CRi rates were observed across all prior therapy subgroups examined: 1 prior line of therapy (87%, n=15) and ≥2 prior lines (70%, n=63); prior blinatumomab (63%, n=38) and no prior blinatumomab (83%, n=40); prior inotuzumab (59%, n=17) and no prior inotuzumab (77%, n=61); and prior alloSCT (76%, n=29) and no prior alloSCT (71%, n=49). The frequency of Grade ≥3 cytokine release syndrome, neurological events, and treatment-related Grade 5 adverse events were largely similar among prior therapy subgroups.Median duration of remission (DOR) in responders with (n=14) and without (n=43) subsequent alloSCT was 44.2 (95% CI, 8.1 to not estimable (NE)) and 18.6 months (95% CI, 9.4 to NE); median OS was 47.0 months (95% CI, 10.2 to NE) and not reached (95% CI, 23.2 to NE), respectively. Median DOR and OS were not reached in responders without prior or subsequent alloSCT (n=22). CONCLUSIONS: In ZUMA-3, adults with R/R B-ALL benefited from brexu-cel, regardless of prior therapies and subsequent alloSCT status, though survival appeared better in patients without certain prior therapies and in earlier lines of therapy. Additional studies are needed to determine the impact prior therapies and subsequent alloSCT have on outcomes of patients who receive brexu-cel.

Referral to and receipt of allogeneic hematopoietic stem cell transplantation in older adults with acute myeloid leukemia.

INTRODUCTION: Recent data have shown improved outcomes in selected older adults with acute myeloid leukemia (AML) following allogeneic hematopoietic stem cell transplantation (HSCT). Nonetheless, practice patterns for referring and performing HSCT vary. We aimed to evaluate referral, utilization, and reasons for not referring/proceeding to HSCT in older adults with AML. MATERIALS AND METHODS: This is a single center retrospective analysis of patients aged ≥60 years diagnosed with AML evaluating rates of HSCT referral and utilization. Fisher's exact test was used to compare rates of referral and utilization across age groups and years of diagnosis. RESULTS: Median age of the 97 patients was 70 years (range 61-95); 30% (29/97) were referred for HSCT and of these, 69% (20/29) received HSCT. Common documented reasons (can be multiple) for not referring were performance status (n = 21), advanced age (n = 16), patient refusal (n = 15), refractory disease (n = 14), and prohibitive comorbidity (n = 6). Among patients who were referred but did not receive HSCT (n = 9/29), documented reasons for not proceeding with HSCT were refractory disease (n = 5), advanced age (n = 2), and prohibitive comorbidity (n = 2). HSCT referral and utilization rates significantly decreased with age (p < 0.01) but were generally stable over time from 2014 to 2017 (p = 0.40 for referral and p = 0.56 for utilization). DISCUSSION: Despite improvements in supportive care and HSCT techniques, HSCT referral and utilization rates remained low among older adults with AML but stable over time.

Dasatinib/prednisone induction followed by blinatumomab/dasatinib in Ph+ acute lymphoblastic leukemia.

Novel treatment strategies are needed for the treatment of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) in older patients. This trial evaluated the feasibility and outcomes with the anti-CD19 bispecific T-cell-engaging antibody, blinatumomab, in combination with dasatinib and steroids. Patients 65 years of age or older with Ph+ or Ph-like ALL (with dasatinib-sensitive fusions/mutations) were eligible and could be newly diagnosed or relapsed/refractory. Induction therapy consisted of dasatinib/prednisone. Patients not achieving response by day 56 proceeded to blinatumomab reinduction therapy. Patients achieving response with induction or reinduction therapy proceeded to blinatumomab/dasatinib postremission therapy for 3 cycles followed by dasatinib/prednisone maintenance. All patients received central nervous system prophylaxis with intrathecal methotrexate for a total of 8 doses. Response was assessed at days 28, 56, and 84 and at additional time points based on response parameters. Measurable residual disease was assessed centrally by 8-color flow cytometry at day 28. A total of 24 eligible patients with newly diagnosed Ph+ ALL were enrolled with a median age of 73 years (range, 65-87 years). This combination was safe and feasible. With a median of 2.7 years of follow-up, 3-year overall survival and disease-free survival were 87% (95% confidence interval [CI], 64-96) and 77% (95% CI, 54-90), respectively. Although longer follow-up is needed, these results are encouraging, and future trials are building on this backbone regimen. This trial was registered at www.clinicaltrials.gov as #NCT02143414.

Optimal approach to T-cell ALL.

T-lineage acute lymphoblastic leukemia (T-ALL) is curable for most children and adolescent and young adult patients with contemporary frontline chemotherapy regimens. During the past decade, improved survival rates have resulted from the optimization of frontline chemotherapy regimens, the use of minimal residual disease (MRD) assessment for evaluating a patient's risk for relapse, and the intensification of treatment based on the persistence of MRD. Optimization of initial therapy is critical because relapsed T-ALL after initial intensive chemotherapy is incurable for most adult patients. Current T-ALL salvage chemotherapy regimens are minimally effective, and unlike in B-cell ALL, there are no approved antibody therapies or chimeric antigen receptor T-cell therapies for relapsed disease. Immunotherapy and small-molecule inhibitors are beginning to be tested in relapsed T-ALL and have the potential to advance the treatment. Until effective salvage strategies are discovered, however, intensive frontline therapy is required for cure. In this article I review the current frontline chemotherapy regimens for adult patients with T-ALL, summarize the novel targeted and immune therapeutics currently in early-phase clinical trials, and outline how these therapies are helping to define an optimal approach for T-ALL.

Two-year follow-up of KTE-X19 in patients with relapsed or refractory adult B-cell acute lymphoblastic leukemia in ZUMA-3 and its contextualization with SCHOLAR-3, an external historical control study.

BACKGROUND: Brexucabtagene autoleucel (KTE-X19) is an autologous anti-CD19 CAR T-cell therapy approved in the USA to treat adult patients with relapsed or refractory B-precursor acute lymphoblastic leukemia (R/R B-ALL) based on ZUMA-3 study results. We report updated ZUMA-3 outcomes with longer follow-up and an extended data set along with contextualization of outcomes to historical standard of care. METHODS: Adults with R/R B-ALL received a single infusion of KTE-X19 (1 × 106 CAR T cells/kg). Long-term post hoc subgroup assessments of ZUMA-3 were conducted. Outcomes from matched patients between historical clinical trials and ZUMA-3 patients were assessed in the retrospective historical control study SCHOLAR-3. RESULTS: After 26.8-months median follow-up, the overall complete remission (CR) rate (CR + CR with incomplete hematological recovery) among treated patients (N = 55) in phase 2 was 71% (56% CR rate); medians for duration of remission and overall survival (OS) were 14.6 and 25.4 months, respectively. Most patients responded to KTE-X19 regardless of age or baseline bone marrow blast percentage, but less so in patients with > 75% blasts. No new safety signals were observed. Similar outcomes were observed in a pooled analysis of phase 1 and 2 patients (N = 78). In SCHOLAR-3, the median OS for treated patients from ZUMA-3 (N = 49) and matched historical controls (N = 40) was 25.4 and 5.5 months, respectively. CONCLUSIONS: These data, representing the longest follow-up of CAR T-cell therapy in a multicenter study of adult R/R B-ALL, suggest that KTE-X19 provides a clinically meaningful survival benefit with manageable toxicity in this population. TRIAL REGISTRATION: NCT02614066.

SWOG 1318: A Phase II Trial of Blinatumomab Followed by POMP Maintenance in Older Patients With Newly Diagnosed Philadelphia Chromosome-Negative B-Cell Acute Lymphoblastic Leukemia.

PURPOSE: Chemotherapy outcomes in older patients with Philadelphia (Ph) chromosome-negative B-acute lymphoblastic leukemia (ALL) are very poor. Here, we evaluated blinatumomab as induction and consolidation therapy followed by prednisone, vincristine, 6-mercaptopurine, and methotrexate (POMP) maintenance chemotherapy in this patient population. PATIENTS AND METHODS: Patients were treated at National Clinical Trial Network sites. Eligibility criteria included age ≥ 65 years and newly diagnosed Ph chromosome-negative B-ALL. Patients received blinatumomab as induction for one-two cycles until attainment of response (complete remission (CR) and CR with incomplete count recovery). Patients then received three cycles of consolidation with blinatumomab followed by 18 months of POMP maintenance chemotherapy. Eight doses of intrathecal methotrexate were administered as central nervous system prophylaxis. RESULTS: Twenty-nine eligible patients were enrolled. The median age was 75 years, and the median bone marrow blast count at diagnosis was 87%. Cytogenetic risk was poor in 10 patients (34%), and five of 14 patients (36%) tested had the Ph-like ALL gene signature. Nineteen patients (66%; 95% CI, 46 to 82) achieved CR. Kaplan-Meier 3-year disease-free survival and overall survival estimates were 37% (95% CI, 17 to 57) and 37% (95% CI, 20 to 55), respectively. CONCLUSION: Blinatumomab was well tolerated and effective in the treatment of older patients with newly diagnosed Ph chromosome-negative B-ALL, including patients with poor-risk cytogenetics. The 3-year disease-free survival and overall survival results are encouraging and suggest that this approach should be further explored.

A phase II study of sequential decitabine and rapamycin in acute myelogenous leukemia.

A phase II study was conducted to ascertain whether sequential exposure to decitabine followed by rapamycin, an mTOR (mechanistic target of rapamycin) inhibitor would result in better responses than decitabine alone. Newly diagnosed acute myelogenous leukemia (AML) patients who were >65 years old and not eligible for intensive induction regimens or patients with relapsed or refractory AML received 10 days of decitabine followed by 12 days of rapamycin in cycle 1 and 5 days of decitabine followed by 17 days of rapamycin in subsequent cycles. The composite complete remission rate (CR) was 33 % (CR plus CR with incomplete count recovery). Median overall survival was 7.7 months in newly diagnosed elderly AML patients and 6.6 months in relapsed/refractory AML patients. Twenty-four evaluable patients were enrolled, and the study did not meet its primary endpoint of demonstrating a significant improvement in composite CR rate with the combination as compared to an established historical CR rate of 25 % with decitabine alone. Despite that, the survival rates in relapsed/refractory cases appear comparable to what is reported with other salvage regimens, and no significant patterns of non-hematologic toxicity were noted. 50 % of subjects in the de novo group achieved a composite CR which is significantly higher (p = 0.02) than the rate of 25 % with decitabine alone. This trial is registered at clinical trials.gov as NCT02109744.

KTE-X19 for relapsed or refractory adult B-cell acute lymphoblastic leukaemia: phase 2 results of the single-arm, open-label, multicentre ZUMA-3 study.

BACKGROUND: Despite treatment with novel therapies and allogeneic stem-cell transplant (allo-SCT) consolidation, outcomes in adult patients with relapsed or refractory B-precursor acute lymphoblastic leukaemia remain poor, underlining the need for more effective therapies. METHODS: We report the pivotal phase 2 results of ZUMA-3, an international, multicentre, single-arm, open-label study evaluating the efficacy and safety of the autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy KTE-X19 in adult patients with relapsed or refractory B-precursor acute lymphoblastic leukaemia. Patients were enrolled at 25 sites in the USA, Canada, and Europe. Eligible patients were aged 18 years or older, with Eastern Cooperative Oncology Group performance status of 0-1, and morphological disease in the bone marrow (>5% blasts). After leukapheresis and conditioning chemotherapy, patients received a single KTE-X19 infusion (1 × 106 CAR T cells per kg bodyweight). The primary endpoint was the rate of overall complete remission or complete remission with incomplete haematological recovery by central assessment. Duration of remission and relapse-free survival, overall survival, minimal residual disease (MRD) negativity rate, and allo-SCT rate were assessed as secondary endpoints. Efficacy and safety analyses were done in the treated population (all patients who received a dose of KTE-X19). This study is registered with ClinicalTrials.gov, NCT02614066. FINDINGS: Between Oct 1, 2018, and Oct 9, 2019, 71 patients were enrolled and underwent leukapheresis. KTE-X19 was successfully manufactured for 65 (92%) patients and administered to 55 (77%). The median age of treated patients was 40 years (IQR 28-52). At the median follow-up of 16·4 months (13·8-19·6), 39 patients (71%; 95% CI 57-82, p<0·0001) had complete remission or complete remission with incomplete haematological recovery, with 31 (56%) patients reaching complete remission. Median duration of remission was 12·8 months (95% CI 8·7-not estimable), median relapse-free survival was 11·6 months (2·7-15·5), and median overall survival was 18·2 months (15·9-not estimable). Among responders, the median overall survival was not reached, and 38 (97%) patients had MRD negativity. Ten (18%) patients received allo-SCT consolidation after KTE-X19 infusion. The most common adverse events of grade 3 or higher were anaemia (27 [49%] patients) and pyrexia (20 [36%] patients). 14 (25%) patients had infections of grade 3 or higher. Two grade 5 KTE-X19-related events occurred (brain herniation and septic shock). Cytokine release syndrome of grade 3 or higher occurred in 13 (24%) patients and neurological events of grade 3 or higher occurred in 14 (25%) patients. INTERPRETATION: KTE-X19 showed a high rate of complete remission or complete remission with incomplete haematological recovery in adult patients with relapsed or refractory B-precursor acute lymphoblastic leukaemia, with the median overall survival not reached in responding patients, and a manageable safety profile. These findings indicate that KTE-X19 has the potential to confer long-term clinical benefit to these patients. FUNDING: Kite, a Gilead Company.

KTE-X19 anti-CD19 CAR T-cell therapy in adult relapsed/refractory acute lymphoblastic leukemia: ZUMA-3 phase 1 results.

ZUMA-3 is a phase 1/2 study evaluating KTE-X19, an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy, in adult relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). We report the phase 1 results. After fludarabine-cyclophosphamide lymphodepletion, patients received a single infusion of KTE-X19 at 2 × 106, 1 × 106, or 0.5 × 106 cells per kg. The rate of dose-limiting toxicities (DLTs) within 28 days after KTE-X19 infusion was the primary end point. KTE-X19 was manufactured for 54 enrolled patients and administered to 45 (median age, 46 years; range, 18-77 years). No DLTs occurred in the DLT-evaluable cohort. Grade ≥3 cytokine release syndrome (CRS) and neurologic events (NEs) occurred in 31% and 38% of patients, respectively. To optimize the risk-benefit ratio, revised adverse event (AE) management for CRS and NEs (earlier steroid use for NEs and tocilizumab only for CRS) was evaluated at 1 × 106 cells per kg KTE-X19. In the 9 patients treated under revised AE management, 33% had grade 3 CRS and 11% had grade 3 NEs, with no grade 4 or 5 NEs. The overall complete remission rate correlated with CAR T-cell expansion and was 83% in patients treated with 1 × 106 cells per kg and 69% in all patients. Minimal residual disease was undetectable in all responding patients. At a median follow-up of 22.1 months (range, 7.1-36.1 months), the median duration of remission was 17.6 months (95% confidence interval [CI], 5.8-17.6 months) in patients treated with 1 × 106 cells per kg and 14.5 months (95% CI, 5.8-18.1 months) in all patients. KTE-X19 treatment provided a high response rate and tolerable safety in adults with R/R B-ALL. Phase 2 is ongoing at 1 × 106 cells per kg with revised AE management. This trial is registered at www.clinicaltrials.gov as #NCT02614066.

The challenge to further improvements in survival of patients with T-ALL: Current treatments and new insights from disease pathogenesis.

Survival rates for children and adult patients with T-cell acute lymphoblastic leukemia (T-ALL) have improved during the past decade due to optimization of frontline multiagent chemotherapy regimens. The outcome for relapsed T-ALL after initial intensive chemotherapy is frequently fatal, however, because no effective salvage regimens have been developed. Immunotherapy and small molecule inhibitors are beginning to be tested in T-ALL and have the potential to advance the treatment, especially the frontline regimen by eradicating minimal residual disease thus inducing more durable remissions. In this paper, I review the current chemotherapy regimens for adult patients with T-ALL and summarize the novel immunotherapies and small molecule inhibitors that are currently in early phase clinical trials.

Novel mHealth App to Deliver Geriatric Assessment-Driven Interventions for Older Adults With Cancer: Pilot Feasibility and Usability Study.

BACKGROUND: Older patients with cancer are at an increased risk of adverse outcomes. A geriatric assessment (GA) is a compilation of reliable and validated tools to assess domains that are predictors of morbidity and mortality, and it can be used to guide interventions. However, the implementation of GA and GA-driven interventions is low due to resource and time limitations. GA-driven interventions delivered through a mobile app may support the complex needs of older patients with cancer and their caregivers. OBJECTIVE: We aimed to evaluate the feasibility and usability of a novel app (TouchStream) and to identify barriers to its use. As an exploratory aim, we gathered preliminary data on symptom burden, health care utilization, and satisfaction. METHODS: In a single-site pilot study, we included patients aged ≥65 years undergoing treatment for systemic cancer and their caregivers. TouchStream consists of a mobile app and a Web portal. Patients underwent a GA at baseline with the study team (on paper), and the results were used to guide interventions delivered through the app. A tablet preloaded with the app was provided for use at home for 4 weeks. Feasibility metrics included usability (system usability scale of >68 is considered above average), recruitment, retention (number of subjects consented who completed postintervention assessments), and percentage of days subjects used the app. For the last 8 patients, we assessed their symptom burden (severity and interference with 17-items scored from 0-10 where a higher score indicates worse symptoms) using a clinical symptom inventory, health care utilization from the electronic medical records, and satisfaction (6 items scored on a 5-point Likert Scale for both patients and caregivers where a higher score indicates higher satisfaction) using a modified satisfaction survey. Barriers to use were elicited through interviews. RESULTS: A total of 18 patients (mean age 76.8, range 68-87) and 13 caregivers (mean age 69.8, range 38-81) completed the baseline assessment. Recruitment and retention rates were 67% and 80%, respectively. The mean SUS score was 74.0 for patients and 72.2 for caregivers. Mean percentage of days the TouchStream app was used was 78.7%. Mean symptom severity and interference scores were 1.6 and 2.8 at preintervention, and 0.9 and 1.5 at postintervention, respectively. There was a total of 27 clinic calls during the intervention period and 15 during the postintervention period (week 5-8). One patient was hospitalized during the intervention period (week 1-4) and two patients during the postintervention period (week 5-8). Mean satisfaction scores of patients and caregivers with the mobile app were 20.4 and 23.4, respectively. Barriers fell into 3 themes: general experience, design, and functionality. CONCLUSIONS: TouchStream is feasible and usable for older patients on cancer treatment and their caregivers. Future studies should evaluate the effects of the TouchStream on symptoms and health care utilization in a randomized fashion.

Can we incorporate geriatric assessment in the management of acute lymphoblastic leukemia in older adults?

Acute lymphoblastic leukemia (ALL) is an uncommon disease. Approximately 14% of new ALL cases occur in adults aged 60 and over, and the three-year overall survival in this population is poor at 12.8%. Older adults with ALL are heterogeneous in terms of their underlying health status, which can make treatment selection challenging given the disease rarity and limited inclusion of older patients in clinical trials. A comprehensive geriatric assessment (CGA) is a compilation of tools to assess multiple domains such as physical function and cognition, and may assist in guiding treatment selection and supportive care interventions. However, studies on the use of CGA are limited in older adults with ALL. In this review, we discuss the utility of CGA in patients with various hematologic malignancies. Using two patient cases of ALL, we also describe how CGA may be use to guide treatment and supportive care interventions.

Philadelphia chromosome negative B-cell acute lymphoblastic leukemia in older adults: Current treatment and novel therapies.

Older adults with Philadelphia chromosome negative (Ph-),-B-cell acute lymphoblastic leukemia (ALL) have the highest rates of treatment failure and treatment complications with current therapy, and, thus, there is no standard treatment for these patients. Approximately 16 percent of patients with newly diagnosed Ph- B-cell ALL are aged 60 years or older [1]. The five-year overall survival for this older cohort of patients is approximately 20 percent, and there has been no improvement in their survival in decades [2]. The challenge in managing older patients with ALL is achieving balance between efficacy of treatment and the toxicity of multi-agent chemotherapy. The latter approach is highly effective in younger adults, but greatly limited by toxicity in older adults. New classes of agents, bi-specific T-cell engager (BiTE) monoclonal antibody and antibody drug conjugates (ADC) have been introduced into the treatment of ALL, and these agents have achieved therapeutic responses and manageable toxicity in patients of all ages with relapsed refractory ALL. These newer immunotherapy agents may improve the treatment of older adults. This review focuses on the new approaches to treatment of Ph- B-cell ALL in older patients. Other reviews in this special edition of ALL will focus on Philadelphia chromosome positive ALL, Philadelphia-like ALL, and allogeneic stem cell transplant as related to older adults.

Chemoenzymatic synthesis and antileukemic activity of novel C9- and C14-functionalized parthenolide analogs.

Parthenolide is a naturally occurring terpene with promising anticancer properties, particularly in the context of acute myeloid leukemia (AML). Optimization of this natural product has been challenged by limited opportunities for the late-stage functionalization of this molecule without affecting the pharmacologically important α-methylene-γ-lactone moiety. Here, we report the further development and application of a chemoenzymatic strategy to afford a series of new analogs of parthenolide functionalized at the aliphatic positions C9 and C14. Several of these compounds were determined to be able to kill leukemia cells and patient-derived primary AML specimens with improved activity compared to parthenolide, exhibiting LC50 values in the low micromolar range. These studies demonstrate that different O-H functionalization chemistries can be applied to elaborate the parthenolide scaffold and that modifications at the C9 or C14 position can effectively enhance the antileukemic properties of this natural product. The C9-functionalized analogs 22a and 25b were identified as the most interesting compounds in terms of antileukemic potency and selectivity toward AML versus healthy blood cells.

Evolution of acute myelogenous leukemia stem cell properties after treatment and progression.

Most cancers evolve over time as patients initially responsive to therapy acquire resistance to the same drugs at relapse. Cancer stem cells have been postulated to represent a therapy-refractory reservoir for relapse, but formal proof of this model is lacking. We prospectively characterized leukemia stem cell populations (LSCs) from a well-defined cohort of patients with acute myelogenous leukemia (AML) at diagnosis and relapse to assess the effect of the disease course on these critical populations. Leukemic samples were collected from patients with newly diagnosed AML before therapy and after relapse, and LSC frequency was assessed by limiting dilution analyses. LSC populations were identified using fluorescent-labeled cell sorting and transplantation into immunodeficient NOD/SCID/interleukin 2 receptor γ chain null mice. The surface antigen expression profiles of pretherapy and postrelapse LSCs were determined for published LSC markers. We demonstrate a 9- to 90-fold increase in LSC frequency between diagnosis and relapse. LSC activity at relapse was identified in populations of leukemic blasts that did not demonstrate this activity before treatment and relapse. In addition, we describe genetic instability and exceptional phenotypic changes that accompany the evolution of these new LSC populations. This study is the first to characterize the evolution of LSCs in vivo after chemotherapy, identifying a dramatic change in the physiology of primitive AML cells when the disease progresses. Taken together, these findings provide a new frame of reference by which to evaluate candidate AML therapies in which both disease control and the induction of more advanced forms of disease should be considered.

When to Treat Adults Like Children: Optimizing Therapy for Lymphoblastic Lymphoma in Young Adults.

A 23-year-old man was urgently referred for evaluation of rapidly enlarging cervical lymphadenopathy, progressive dyspnea, fatigue, night sweats, and an unintentional weight loss of 25 pounds. A computed tomography scan of the neck performed 30 days before referral revealed bilateral cervical and supraclavicular lymphadenopathy (6 × 5 cm). A fine-needle aspirate of nasopharyngeal tissue demonstrated fibroadipose tissue. Tissue collected by core needle biopsy of a left internal jugular lymph node demonstrated a reactive lymph node but no malignancy. The patient was admitted to an academic medical center hospital. His physical examination was remarkable for bulky cervical and supraclavicular lymphadenopathy. A testicular examination was normal. The patient's lactate dehydrogenase concentration was 327 U/L (normal range, 118-225 U/L). A positron emission tomography scan revealed bilateral cervical and supraclavicular lymphadenopathy (6 × 5 cm with a standardized uptake value [SUV] of 14), a 1.3-cm subcutaneous nodule in the left thigh (SUV 16), and two 2.7-cm liver lesions (SUV 14). He underwent an excisional lymph node biopsy. The lymph node revealed effacement of the architecture by an interfollicular infiltrate of lymphoid cells (Fig 1). Mitotic figures were abundant (Ki-67 stain 80% to 90% positive) and there were multiple foci of tissue necrosis. The lymphoblasts were examined by flow cytometry and immunohistochemistry and expressed the T-cell markers CD2, CD3, CD4, and terminal deoxynucleotidyl transferase. A subpopulation of T cells was positive for both CD4 and CD8. Polymerase chain reaction studies revealed a clonal rearrangement of the T-cell receptor γ gene. A marrow aspirate and biopsy revealed normal trilineage hematopoiesis with no evidence of lymphoma and a normal male karyotype (46, XY). A lumbar puncture sample did not contain lymphoma cells. The patient's diagnosis was T-lymphoblastic lymphoma.

Discovery of potent parthenolide-based antileukemic agents enabled by late-stage P450-mediated C-H functionalization.

The sesquiterpene lactone parthenolide has recently attracted considerable attention owing to its promising antitumor properties, in particular in the context of stem-cell cancers including leukemia. Yet, the lack of viable synthetic routes for re-elaborating this complex natural product has represented a fundamental obstacle toward further optimization of its pharmacological properties. Here, we demonstrate how this challenge could be addressed via selective, late-stage sp(3) C-H bond functionalization mediated by P450 catalysts with tailored site-selectivity. Taking advantage of our recently introduced tools for high-throughput P450 fingerprinting and fingerprint-driven P450 reactivity prediction, we evolved P450 variants useful for carrying out the highly regioselective hydroxylation of two aliphatic sites (C9 and C14) in parthenolide carbocyclic backbone. By chemoenzymatic synthesis, a panel of novel C9- and C14-modified parthenolide analogs were generated in order to gain initial structure-activity insights on these previously inaccessible sites of the molecule. Notably, some of these compounds were found to possess significantly improved antileukemic potency against primary acute myeloid leukemia cells, while exhibiting low toxicity against normal mature and progenitor hematopoietic cells. By identifying two 'hot spots' for improving the anticancer properties of parthenolide, this study highlights the potential of P450-mediated C-H functionalization as an enabling, new strategy for the late-stage manipulation of bioactive natural product scaffolds.

Targeting aberrant glutathione metabolism to eradicate human acute myelogenous leukemia cells.

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34(+)) leukemic versus normal specimens. Our data indicate that CD34(+) AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34(+) AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34(+) cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34(+) AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34(+) cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells.

BCL-2 inhibition targets oxidative phosphorylation and selectively eradicates quiescent human leukemia stem cells.

Most forms of chemotherapy employ mechanisms involving induction of oxidative stress, a strategy that can be effective due to the elevated oxidative state commonly observed in cancer cells. However, recent studies have shown that relative redox levels in primary tumors can be heterogeneous, suggesting that regimens dependent on differential oxidative state may not be uniformly effective. To investigate this issue in hematological malignancies, we evaluated mechanisms controlling oxidative state in primary specimens derived from acute myelogenous leukemia (AML) patients. Our studies demonstrate three striking findings. First, the majority of functionally defined leukemia stem cells (LSCs) are characterized by relatively low levels of reactive oxygen species (termed "ROS-low"). Second, ROS-low LSCs aberrantly overexpress BCL-2. Third, BCL-2 inhibition reduced oxidative phosphorylation and selectively eradicated quiescent LSCs. Based on these findings, we propose a model wherein the unique physiology of ROS-low LSCs provides an opportunity for selective targeting via disruption of BCL-2-dependent oxidative phosphorylation.