Identification of cells of leukemic stem cell origin with non-canonical regenerative properties.

Despite most acute myeloid leukemia (AML) patients entering remission following chemotherapy, outcomes remain poor due to surviving leukemic cells that contribute to relapse. The nature of these enduring cells is poorly understood. Here, through temporal single-cell transcriptomic characterization of AML hierarchical regeneration in response to chemotherapy, we reveal a cell population: AML regeneration enriched cells (RECs). RECs are defined by CD74/CD68 expression, and although derived from leukemic stem cells (LSCs), are devoid of stem/progenitor capacity. Based on REC in situ proximity to CD34-expressing cells identified using spatial transcriptomics on AML patient bone marrow samples, RECs demonstrate the ability to augment or reduce leukemic regeneration in vivo based on transfusion or depletion, respectively. Furthermore, RECs are prognostic for patient survival as well as predictive of treatment failure in AML cohorts. Our study reveals RECs as a previously unknown functional catalyst of LSC-driven regeneration contributing to the non-canonical framework of AML regeneration.

Merkel cell carcinoma mimicking acute leukemia.

Bone marrow aspirate showed diffuse infiltration by a population of monomorphic cells with scant cytoplasm, markedly increased nuclear-to-cytoplasmic ratio, and numerous indistinct nucleoli. Bone marrow biopsy confirmed extensive marrow infiltration by a malignant neoplasm with strong and diffuse expression of synaptophysin by immunohistochemistry, consistent with metastases from Merkel Cell carcinoma.

Serologic Response to Vaccine for COVID-19 in Patients with Hematologic Malignancy: A Prospective Cohort Study.

BACKGROUND: Patients with hematological cancers have increased COVID-19 morbidity and mortality, and these patients show attenuated vaccine responses. This study aimed to characterize the longitudinal humoral immune responses to COVID-19 vaccination in patients with hematological malignancies. PATIENTS AND METHODS: We conducted a prospective cohort study, collecting samples from March 2021 to July 2022, from patients seen at a cancer treatment center in London, Ontario, Canada, who met the following eligibility criteria: age ≥18 years, diagnosed with a hematological malignancy, recipient of a COVID-19 vaccine during the study period, and able to provide informed consent. RESULTS: Median anti-S titers (MST) were 0.0, 64.0, and 680.5 U/mL following first (V1), second (V2), and third (V3) vaccine doses, respectively. Patients with lymphoid malignancies' response to vaccination was attenuated compared to myeloid malignancy patients after V2 and V3 (P < .001, P < .01). Active treatment was associated with lower antibody titers (MST 10) compared to treatment 12-24 months (MST 465, P = .04367) and >24 months (MST 1660.5, P = .0025) prior to vaccination. V3 significantly increased antibody titers compared to V2 for patients less than 3 months from treatment. Increasing age was associated with smaller antibody response following V2 (P < .05), but not following V3. Patients receiving anti-CD20 therapy did not demonstrate increased antibody titer levels after V3 (V2 MST 0, V3 MST 0; P > .05). CONCLUSION: We report an attenuated serologic response to COVID-19 vaccination in our study population of patients with hematological malignancy. The immune response to vaccination was affected by patient age, diagnosis, treatment, and timing of treatment exposure.

Final report of TKI discontinuation trial with dasatinib for the second attempt of treatment-free remission after failing the first attempt with imatinib: Treatment-free Remission Accomplished by Dasatinib (TRAD) study.

Multiple studies have reported a significant treatment-free remission (TFR) rate of 50%-60% in patients with chronic myeloid leukaemia (CML) who discontinue tyrosine kinase inhibitor (TKI) therapy. However, the remaining half of these patients still require re-initiation of TKI therapy for leukaemia control. It remains unclear if TKI drugs should be switched for re-therapy in patients who failed the first TFR (TFR1) attempt. Our study attempted to determine whether dasatinib therapy after TFR1 failure post-imatinib discontinuation could improve the likelihood of TFR2. Of 59 patients who lost molecular response after imatinib discontinuation for TFR1, 55 patients (93.2%) were treated with dasatinib, of whom 49 (89.1%) regained MR4.5 or deeper response, with a median time of 1.85 months to achieve MR4.5. Dasatinib was discontinued in 35 patients for TFR2 attempt, of whom 26 patients (74.28%) lost MMR and 6 (17.14%) MR4. Risk factor analysis for the TFR2 after dasatinib discontinuation suggested three significant factors: (1) doubling time of BCR::ABL1 transcript following TFR1 attempt, (2) rapid regaining of molecular response following dasatinib therapy and (3) undetectable BCR::ABL1 transcript prior to TFR2 attempt. The present study showed that dasatinib does not increase the TFR2 rate in general, but a selected group of patients could benefit from this approach.

Canadian real-world experience of asciminib treatment in heavily pre-treated chronic myeloid leukemia (CML) patients who failed multiple lines of tyrosine kinase inhibitor (TKI) therapy.

BACKGROUND: Asciminib is a novel drug specifically targeting ABL myristoyl pocket in the ABL1 protein. METHODS: Forty one patients with chronic myeloid leukemia treated with asciminib from 2018 to 2022 were reviewed and analyzed for the efficacy and tolerability of asciminib using real-world experience data. RESULTS: The median age was 60 years (range 17-90) with a past history of a cardiovascular event in 21 patients (51%). Patients were pretreated with a median of 3 previous tyrosine kinase inhibitors (range 1-5). After a median of 12 months of asciminib (range 3-41), major molecular response (MMR) rate was 39% (n = 11/28) and 42% (n = 5/12) at 6 and 12 months, respectively. Molecular response with 2 log reduction (MR2) was noted in 54% (n = 15/28) and 50% (n = 6/12) at 6 and 12 months. The cumulative incidence of MMR and MR2 was 46.3% and 66% at 12 months. Five patients discontinued asciminib due to treatment failure (n = 3) or thrombocytopenia (n = 2). There were no cardiovascular events. Out of 7 patients treated with high dose asciminib for T315I mutation, 5 patients achieved MMR or deeper response. The event-free survival was 63% at 12 months. CONCLUSION: This study confirmed clinical efficacy and tolerability of asciminib with real-world experience.

Outpatient consolidation chemotherapy with intermediate dose cytarabine has similar survival and relapses rates in acute myeloid leukemia as compared to high dose cytarabine: A single center analysis.

INTRODUCTION: The last decade has seen advances in delivering outpatient consolidation therapy for acute myeloid leukemia (AML). The standard of care involves high-dose cytarabine or intermediate-dose cytarabine, given twice daily for three alternating days. At the London Regional Cancer Program, we have transitioned the administration of outpatient cytarabine to a once-daily regimen over six consecutive days. The outcomes of a longer duration interval of high-dose cytarabine and intermediate-dose cytarabine is currently unknown. This study aims to assess the feasibility of administering a continuous 6-day protocol of high-dose (HDAC-16) and intermediate-dose cytarabine (IDAC-16) consolidation therapy in the outpatient setting. METHODS: This is a retrospective chart review to analyze AML patients treated with outpatient high-dose or intermediate-dose cytarabine consolidation therapy at the London Regional Cancer Program from January 1, 2019, through November 1, 2022. The primary objective was to determine the outcomes of the 6-day outpatient administration of once daily high-dose cytarabine or intermediate-dose cytarabine. RESULTS: Forty-five patients received 89 cycles of cytarabine as outpatients; males were 55.6% of the total population, with a median age of ~57 years. Our overall 2-year survival of HDAC-16 (57.1%) and IDAC-16 (83.3%) is consistent with the reported literature. There was no difference in delays, relapse rates, and nonrelapse mortality between both HDAC and IDAC groups. The 2-year relapse free survival was 57.1% for HDAC-16 and 66.7% for IDAC-16. CONCLUSION: Outpatient administration of intermediate-dose cytarabine once daily over six consecutive days results in similar overall survival and relapse rates as compared to high dose cytarabine consolidation chemotherapy. Moving to a once daily administration schedule can alleviate logistical and/or accessibility hurdles for outpatient oncology clinics. Prospective randomized trials are needed in this setting to validate our results.

Leukemic progenitor compartment serves as a prognostic measure of cancer stemness in patients with acute myeloid leukemia.

We systematically investigate functional and molecular measures of stemness in patients with acute myeloid leukemia (AML) using a cohort of 121 individuals. We confirm that the presence of leukemic stem cells (LSCs) detected through in vivo xenograft transplantation is associated with poor survival. However, the measurement of leukemic progenitor cells (LPCs) through in vitro colony-forming assays provides an even stronger predictor of overall and event-free survival. LPCs not only capture patient-specific mutations but also retain serial re-plating ability, demonstrating their biological relevance. Notably, LPC content represents an independent prognostic factor in multivariate analyses including clinical guidelines of risk stratification. Our findings suggest that LPCs provide a robust functional measure of AML, enabling quantitative and rapid assessment of a wide range of patients. This highlights the potential of LPCs as a valuable prognostic factor in AML management.

Propensity score matching analysis comparing the efficacy of Ruxolitinib to historical controls in second-line or beyond treatment for chronic GvHD after steroid failure.

Established first-line therapy for chronic graft-versus-host disease (cGvHD) comprises corticosteroids with/without calcineurin inhibitors, but about half of cGvHD patients are refractory to corticosteroid therapy. The present study retrospectively analyzed treatment outcomes in 426 patients and undertook a propensity-score matching (PSM) analysis between ruxolitinib (RUX) treated group and a historical group of cGvHD patients treated with best available treatment (BAT). PSM process adjusted unbalanced risk factors between the 2 groups, including GvHD severity, HCT-CI score, and treatment line, extracting 88 patients (44 in BAT/RUX groups each) for final analysis. In PSM subgroup, RUX group showed 74.7% 12 months' FFS rate vs 19.1% for BAT group (p < 0.001), whereas 12 months' OS rates were 89.2% and 77.7%, respectively. Multivariate analysis for FFS confirmed RUX superiority over BAT together with HCT-CI score 0-2 vs ≥3. For OS, RUX was superior to BAT, while age ≥60 years and severe grade cGvHD adversely impacted OS. In PSM subgroup, at months 0, 3, and 6, 4.5%, 12.2% and 22.2% more patients in RUX group could discontinue prednisone compared to BAT group, respectively. In conclusion, the current study showed that for FFS, RUX was superior to BAT as second-line therapy or beyond in cGvHD patients after therapy failure.

A Multicenter, Retrospective Study Evaluating Clinical Outcomes of Ruxolitinib Therapy In Heavily Pretreated Chronic GVHD Patients With Steroid Failure.

Although ruxolitinib is emerging as the treatment of choice for steroid-refractory or -dependent chronic graft versus host disease (cGVHD) based on randomized control trial data, there is relatively little real-world data published on ruxolitinib for this indication. We wanted to evaluate the real-world efficacy and safety of ruxolitinib in cGVHD patients who have failed any previous systemic therapy for cGVHD. We retrospectively evaluated the efficacy of ruxolitinib in 115 heavily pretreated patients with steroid-refractory or -dependent chronic GVHD across 5 transplantation centers. The majority of the study population had severe cGVHD (60%) and received ruxolitinib at the fourth treatment line or beyond (82%, n = 96). The median duration of follow-up in this study population was 13 months. The overall response rate (ORR) was 48.6%, 54.9%, and 48.5% at 3, 6, and 12 months, respectively. Clinical benefit (an outcome metric combining ORR with steroid reduction) was observed in 58.7%, 64.8%, and 60.6% of patients at 3, 6, and 12 months, respectively. Approximately one third of patients (37.9%) were able to discontinue prednisone at 12 months, and 63.8% were able to taper prednisone to a daily dose <0.1 mg/kg at 12 months. Failure-free survival at 12 months was 64.6% (54.1%-73.2%). Multivariate analysis identified that patients with severe cGVHD were at a higher risk of failure because of a therapy switch, whereas a pretransplantation hematopoietic stem cell transplantation-comorbidity index score ≥ 3 was associated with a high risk of failure because of increasing risk of non-relapse mortality. Overall, this study demonstrates the therapeutic efficacy of ruxolitinib for cGVHD in a heavily pretreated real-world population.

When donor T cells attack: The curious case of liver transplant-associated acute graft-versus-host-disease.

Graft versus host disease is a rare but deadly complication of solid organ transplant. Clinical features of graft-versus-host-disease are non-specific, which may lead to delayed diagnosis as more common conditions including infections or drug reactions are considered. We describe a 54-year-old male patient who underwent liver transplantation for alcohol use disorder-related cirrhosis and developed acute graft-versus-host disease. Initial clinical presentation included dermatitis, bone marrow failure and enteritis. Results of skin biopsy and cytogenetic studies were consistent with liver transplant-associated acute graft-versus-host disease. The importance of this case is to highlight to transplant physicians and surgeons the challenges of diagnosing graft-versus-host-disease. In our case, pre-existing partnerships among the liver and hematopoietic stem cell transplant teams, transfusion medicine specialists, critical care specialists and facilitated timely communication relevant to confirming graft-versus-host disease. We propose an algorithm to assist in the workup of suspected graft-versus-host disease. Because this condition is characterized by high mortality, a high index of suspicion is imperative for prompt diagnosis and optimal management of the donor-recipient immune interaction when patients present with classic clinical features.

Clinical Utility of Implementing a Frontline NGS-Based DNA and RNA Fusion Panel Test for Patients with Suspected Myeloid Malignancies.

BACKGROUND: The use of molecular genetic biomarkers is rapidly advancing to aid diagnosis, prognosis, and clinical management of hematological disorders. We have implemented a next-generation sequencing (NGS) assay for detection of genetic variants and fusions as a frontline test for patients suspected with myeloid malignancy. In this study, we summarize the findings and assess the clinical impact in the first 1613 patients tested. METHODS: All patients were assessed using NGS based Oncomine Myeloid Research Assay (ThermoFisher) including 40 genes (17 full genes and 23 genes with clinically relevant "hotspot" regions), along with a panel of 29 fusion driver genes (including over fusion 600 partners). RESULTS: Among 1613 patients with suspected myeloid malignancy, 43% patients harbored at least one clinically relevant variant: 91% (90/100) in acute myeloid leukemia patients, 71.7% (160/223) in myelodysplastic syndrome (MDS), 77.5% (308/397) in myeloproliferative neoplasm (MPN), 83% (34/41) in MPN/MDS, and 100% (40/40) in chronic myeloid leukemia patients. Comparison of NGS and cytogenetics results revealed a high degree of concordance in gene fusion detection. CONCLUSIONS: Our findings demonstrate clinical utility and feasibility of integrating a NGS-based gene mutation and fusion testing assay as a frontline diagnostic test in a large reported cohort of patients with suspected myeloid malignancy, in a clinical laboratory setting. Overlap with cytogenetic test results provides opportunity for testing reduction and streamlining.

Reducing cytogenetic testing in the era of next generation sequencing: Are we choosing wisely?

INTRODUCTION: In most laboratories, next generation sequencing (NGS) has been added without consideration for redundancy compared to conventional cytogenetics (CG). We tested a streamlined approach to genomic testing in patients with suspected myeloid and plasma cell neoplasms using next generation sequencing ("NGS first") as the primary testing modality and limiting cytogenetics (CG) to samples with morphologic abnormalities in the marrow aspirate. METHODS: Based on morphologic interpretation of bone marrow aspirate and flow cytometry, samples were triaged into four groups: (a) Samples with dysplasia or excess blasts had both NGS and karyotyping; (b) Samples without excess blasts or dysplasia had NGS only; (c) Repeat samples with previous NGS and/or CG studies were not retested; (d) Samples for suspected myeloma with less than 5% plasma cell had CG testing cancelled. RESULTS: Seven hundred eleven adult bone marrow (BM) samples met the study criteria. The NGS first algorithm eliminated CG testing in 229/303 (75.6%) of patients, primarily by reducing repeat testing. Potential cost avoided was approximately $124 000 per annum. Hematologists overruled the triage comment in only 11/303 (3.6%) cases requesting CG testing for a specific indication. CONCLUSIONS: Utilizing NGS as the primary genomic testing modality NGS was feasible and well accepted, reducing over three quarters of all CG requests and improving the financial case for adoption of NGS. Key factors for the success of this study were collaboration of clinical and genomic diagnostic teams in developing the algorithm, rapid turnaround time for BM interpretation for triage, and communication between laboratories.

BCR-ABL1 transcript doubling time as a predictor for treatment-free remission failure after imatinib discontinuation in chronic myeloid leukaemia in chronic phase.

The doubling time (DT) of the BCR-ABL1 quantitative polymerase chain reaction (qPCR) transcript level reflects the re-growing fraction of leukaemic cells after discontinuation of tyrosine kinase inhibitor (TKI). The present study analyzed monthly DT within six months after imatinib discontinuation in 131 patients. Monthly DT was calculated as x = ln(2)/K, where x is the DT and K is the fold BCR-ABL1 change from the previous value divided by the number of days between each measurement. The optimal DT value was determined as 12·75 days at two months using a recursive partitioning method. The patients were stratified into three groups: the high-risk group (DT<12·75 days but >0, with rapidly proliferating chronic myeloid leukaemia (CML) cells; n = 26) showed the lowest molecular relapse-free survival (mRFS) of 7·7% at 12 months, compared to 53·6% in the intermediate-risk group (DT≥12·75 days, with slowly proliferating CML cells; n = 16) or 90·0% in the low-risk group (DT≤0, i.e., without proliferating CML cells; n = 71; P < 0·001). Monthly assessment of DT helps identify high-risk patients for treatment-free remission failure with an imminent risk of molecular recurrence, and to define low-risk patients who can be spared the frequent monitoring of monthly molecular tests.

Optimal duration of imatinib treatment/deep molecular response for treatment-free remission after imatinib discontinuation from a Canadian tyrosine kinase inhibitor discontinuation trial.

Although total duration of tyrosine kinase inhibitor (TKI) therapy and of molecular response at 4 log reduction or deeper (MR4) correlates with treatment-free remission (TFR) success after TKI discontinuation, the optimal cut-off values of the duration remain unresolved. Thus, 131 patients were enrolled into the Canadian TKI discontinuation study. The molecular relapse-free survival (mRFS) was defined from imatinib discontinuation till molecular recurrence, that is, major molecular response (MMR) loss and/or MR4 loss. We evaluated mRFS at 12 months after imatinib discontinuation, analyzed it according to the imatinib treatment duration and MR4 duration, and calculated P value, positive (PPV) and negative predictive value (NPV) in the yearly cut-off period of time. The shortest cut-off was sought that met the joint criteria of a P value ≤ 0·05, PPV ≥ 60% and NPV ≥ 60%. We propose six years as the shortest imatinib duration cut-off with a P value 0·01, PPV 68% and NPV 62%: The patients treated with imatinib duration ≥ 6 years showed a superior mRFS rate (61·8%) compared to those with less treatment (36·0%). Also, 4·5 years MR4 duration as the shortest cut-off with a P value 0·003, PPV 63% and NPV 61%: those with MR4 duration ≥ 4·5 years showed a higher mRFS rate (64·2%) than those with a shorter MR4 duration (41·9%).

Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients.

The aberrant expression of dopamine receptors (DRDs) in acute myeloid leukemia (AML) cells has encouraged the repurposing of DRD antagonists such as thioridazine (TDZ) as anti-leukemic agents. Here, we access patient cells from a Phase I dose escalation trial to resolve the cellular and molecular bases of response to TDZ, and we extend these findings to an additional independent cohort of AML patient samples tested preclinically. We reveal that in DRD2+ AML patients, DRD signaling in leukemic progenitors provides leukemia-exclusive networks of sensitivity that spare healthy hematopoiesis. AML progenitor cell suppression can be increased by the isolation of the positive enantiomer from the racemic TDZ mixture (TDZ+), and this is accompanied by reduced cardiac liability. Our study indicates that the development of DRD-directed therapies provides a targeting strategy for a subset of AML patients and potentially other cancers that acquire DRD expression upon transformation from healthy tissue.

Clinical value of next-generation sequencing compared to cytogenetics in patients with suspected myelodysplastic syndrome.

Next-generation sequencing (NGS) increasingly influences diagnosis, prognosis and management of myelodysplastic syndrome (MDS). In addition to marrow morphology and flow cytometry, our institution performs cytogenetics (CG) and NGS-based testing routinely in patients with suspected MDS. We evaluated the relative value of NGS in the assessment of patients with suspected MDS. We initially compared the diagnostic and prognostic information derived from CG and NGS in 134 patients. NGS enhanced the diagnostic yield compared to CG for clonal myeloid disorders (sensitivity 77% vs. 42·2%; specificity 90·2% vs. 78%; positive predictive value 92·8% vs. 76%; and negative predictive value 70·8% vs. 45·5%). The identification of poor prognosis mutations by NGS altered risk category in 27/39 (69·2%) patients with MDS with good/intermediate risk CG. Subsequently, we prospectively evaluated 70 patients with suspected MDS using an 'NGS-first approach' with CG restricted to samples with morphological abnormalities. We rarely identified mutations or CG abnormalities in patients without dysplastic features. NGS has a superior diagnostic performance compared to CG in patients with suspected MDS. We estimate that by using an 'NGS-first approach' we could reduce karyotyping by approximately 30%.

Liberal Versus Restrictive Red Blood Cell Transfusion Thresholds in Hematopoietic Cell Transplantation: A Randomized, Open Label, Phase III, Noninferiority Trial.

PURPOSE: Evidence regarding red blood cell (RBC) transfusion practices and their impact on hematopoietic cell transplantation (HCT) outcomes are poorly understood. PATIENTS AND METHODS: We performed a noninferiority randomized controlled trial in four different centers that evaluated patients with hematologic malignancies requiring HCT who were randomly assigned to either a restrictive (hemoglobin [Hb] threshold < 70 g/L) or liberal (Hb threshold < 90 g/L) RBC transfusion strategy between day 0 and day 100. The noninferiority margin corresponds to a 12% absolute difference between groups in Functional Assessment of Cancer Therapy-Bone Marrow Transplant (FACT-BMT) score relative to baseline. The primary outcome was health-related quality of life (HRQOL) measured by FACT-BMT score at day 100. Additional end points were collected: HRQOL by FACT-BMT score at baseline and at days 7, 14, 28, 60, and 100; transplantation-related mortality; length of hospital stay; intensive care unit admissions; acute graft-versus-host disease; Bearman toxicity score; sinusoidal obstruction syndrome; serious infections; WHO Bleeding Scale; transfusion requirements; and reactions to therapy. RESULTS: A total of 300 patients were randomly assigned to either restrictive-strategy or liberal-strategy treatment groups between 2011 and 2016 at four Canadian adult HCT centers. After HCT, mean pre-transfusion Hb levels were 70.9 g/L in the restrictive-strategy group and 84.6 g/L in the liberal-strategy group (P < .0001). The number of RBC units transfused was lower in the restrictive-strategy group than in the liberal-strategy group (mean, 2.73 units [standard deviation, 4.81 units] v 5.02 units [standard deviation, 6.13 units]; P = .0004). After adjusting for transfusion type and baseline FACT-BMT score, the restrictive-strategy group had a higher FACT-BMT score at day 100 (difference of 1.6 points; 95% CI, -2.5 to 5.6 points), which was noninferior compared with that of the liberal-strategy group. There were no significant differences in clinical outcomes between the transfusion strategies. CONCLUSION: In patients undergoing HCT, the use of a restrictive RBC transfusion strategy threshold of 70 g/L was as effective as a threshold of 90 g/L and resulted in similar HRQOL and HCT outcomes with fewer transfusions.