Flow Cytometry Of Bone Marrow Aspirates Research Articles

Prospective Evaluation of Measurable Residual Disease (MRD) and Sustainability of the Response in Patients with Systemic AL Amyloidosis Treated with Daratumumab

Background: Daratumumab can provide deep hematologic responses for patients with light chain (AL) amyloidosis, both through its direct anti-clonal activity and immunomodulatory effects on other cell populations in the bone marrow (BM) microenvironment. Promising efficacy in the frontline setting when combined with CyBorD (per the ANDROMEDA trial), as well as in the relapsed/refractory setting as monotherapy, has led to daratumumab becoming the first FDA-approved therapy for AL amyloidosis. Though it is administered for up to 24 cycles, the optimal duration of treatment is unclear. Dynamic monitoring of measurable residual disease (MRD) may provide insights into changes in the aberrant plasma cell (PC) clone and sustainability of the hematologic response in relation to daratumumab therapy. Aims: We sought to evaluate the rates of MRD clearance and resurgence both during and after daratumumab therapy for AL amyloidosis. Methods: MRD status was assessed for patients followed at the Boston University Amyloidosis Center, who were treated with a daratumumab-based regimen and achieved a hematologic very good partial response (hemVGPR) or complete response (hemCR) according to consensus criteria (Palladini, 2012). MRD assessments were performed by multiparametric 10-color flow cytometry of BM aspirates (sensitivity level of ≤10 -5) and repeated at 12-month intervals during follow-up. Clonal progression was defined as growth in the aberrant PC clone size of >1-log. Results: Between 2019 and 2023, a total of 66 patients achieved ≥hemVGPR and were tested for MRD; 43 (65%) received daratumumab monotherapy in the relapsed/refractory setting and 23 (35%) in combination with CyBorD as frontline therapy. Collectively, 48 (73%) achieved a hemCR and 18 (27%) a hemVGPR. The median age at diagnosis was 60 years (range 35-78); 19 (29%) patients were female; and 11 (17%) identified as non-white race. MRD assessment was performed once, twice or ≥3 times for 33, 18 and 15 patients, respectively. The first MRD evaluation occurred while still receiving daratumumab for 33 patients (at median 12 cycles, range 6-23); the remaining patients had their first MRD evaluation after therapy completion (median one month after, range 0-25). Of the entire cohort, 28 (42%) patients achieved MRD negativity and 38 (58%) MRD positivity (median estimated aberrant PC clone size 1.5 x 10 -4, range 1.4 x 10 -5 to 1.1 x 10 -2, representing 46.9% ± 32.8% of total PCs) at first evaluation. All but one patient with a hemVGPR had detectable MRD. Of the 23 patients who received frontline daratumumab-CyBorD (18 hemCR and 5 hemVGPR), 8 (35%) achieved MRD negativity at first evaluation; whereas 20 (47%) of the 43 patients who received daratumumab as monotherapy (30 hemCR and 13 hemVGPR) achieved MRD negativity. We surveyed MRD profiles based on the number of daratumumab treatment cycles received (Table 1) and found no apparent patterns with increasing cycles. Serial MRD monitoring was performed for 33 patients (Figure 1). Of 10 patients who continued to receive daratumumab between MRD assessments (group 1), 6 had sustained MRD negativity; 2 had persistent MRD positivity (of whom one had clonal progression); and 2 had MRD clearance (conversion from positive to negative). Of 23 patients who underwent serial MRD assessments after completing daratumumab (group 2), 9 (39%) had sustained MRD negativity; 10 (43%) had persistent MRD positivity; 2 (9%) had MRD clearance; and 2 (9%) had MRD resurgence (conversion from negative to positive), which occurred 17 and 26 months after daratumumab cessation. Clonal progression was observed in 5 of the 10 patients with persistent positivity, of whom 3 experienced a hematologic relapse. Conclusions: Our findings show that patients with AL amyloidosis can attain deep responses with daratumumab-based treatments, even to the depth of undetectable MRD. Clearance of MRD was frequent not only during treatment with daratumumab but also after completion of therapy, suggesting continuing effects on the plasma cell clone. Clonal progression after daratumumab completion was often associated with hematologic relapse. Prospective studies are needed to investigate whether daratumumab may be stopped early for those with undetectable MRD, or whether those with persistent detectable disease may benefit from extending daratumumab treatment beyond 24 cycles.

Waldenstrom's macroglobulinemia with peripheral neuropathy and favorable long-term treatment responses

Abstract Rationale: Waldenstrom's Macroglobulinemia (WM), is a rare hematological disorder with an indolent course that affects about 1 in 1,000,000 people over the age of 50. The two cases presented here highlight the favorable responses to targeted therapies and that such responses may show a longer progression-free survival than the standard regimens for WM (Dexamethasone + Rituximab + Cyclophosphamide and Bendamustine + Rituximab). Patient concerns: We report 2 Filipino patients with WM presenting with symptoms of peripheral nerve involvement characterized by numbness, dysesthesia and weakness. Diagnosis: Serum immunoelectrophoresis showed distinct bands in IgM and Kappa light chain channels, indicative of IgM kappa monoclonal gammopathy. Bone marrow aspirate flow cytometry showed a B-lymphoid population, bright expression of Kappa light chain surface immunoglobulin, all consistent with a B-cell neoplasm. Interventions: The first case received Dexamethasone + Rituximab + Cyclophosphamide. The second case was successfully treated with Rituximab + Bendamustine. Outcomes: The first case achieved complete remission for 9 years. However, in the 10th year of follow-up, there was recurrence of peripheral nerve symptomatology, which prompted a repeat course Rituximab monotherapy, with favorable response. The second case was successfully treated with Rituximab + Bendamustine over a 3-year course. Lessons: To the best of our knowledge, WM presenting with peripheral neuropathy with a long progression-free survival as described in these patients have not been reported before. The two cases highlight the potentially favorable responses to targeted therapies. Though the possibility of developing recurrence cannot be discounted, favorable outcomes with the use of the standard chemotherapies could still be expected.

Phase 1 trial of anti-CD22 recombinant immunotoxin moxetumomab pasudotox combined with rituximab for relapsed/refractory hairy cell leukemia.

7036 Background: Anti-CD22 recombinant immunotoxin moxetumomab pasudotox (Moxe) is FDA-approved for hairy cell leukemia (HCL) patients who have received at least two prior systemic therapies including a purine nucleoside analog. In phase 3 testing the complete remission (CR) rate was 41%, and response was higher in patients with lower tumor burden and lower titers of antidrug antibodies (ADA). Phase 1 testing indicated that most CRs were without minimal residual disease (MRD) and eradication of MRD was associated with prolonged CR duration. Monoclonal antibody (Mab) rituximab binds to CD20 on HCL cells and induces apoptosis or immune-mediated killing, but as a single-agent achieved only 13% CRs in relapsed HCL requiring therapy. In a phase 1 trial to determine safety, rituximab was combined with Moxe, with the goal to help reduce tumor burden and to prevent or delay ADA by killing normal B-cells. Methods: To allow rituximab sufficient time to accomplish both goals, it was infused 3 days before day 1 of cycle 1 at 375 mg/m2, and Moxe was given by 30-minute infusion on days 1, 3 and 5. On repeat cycles of Moxe days 1, 3 and 5, rituximab was given on day 1. Cycles were generally spaced 4 weeks apart. Moxe was begun at a lower dose, 30 rather than the 40 mcg/kg dose used in phase 3 in case the rituximab would increase its toxicity. Bone marrow aspirate flow cytometry, which can detect 0.002% HCL cells, was the most sensitive test used for MRD detection, much more sensitive than BRAF V600E digital droplet PCR (ddPCR) or bone marrow biopsy immunohistochemistry (IHC). Patients could receive 4 cycles past MRD-free CR, but not more than 8 cycles. Results: Three patients received Moxe at 30 mcg/Kg/dose and 6 received 40 mcg/Kg/dose, all without dose limiting toxicity (DLT). There was no evidence of hemolytic uremic syndrome or capillary leak syndrome. To prevent intravascular hypovolemia due to expected third spacing, patients were encouraged to drink one cup per hour of water or other fluid from days 1 to 8 and take dexamethasone 4 mg orally if headache or nausea prevented good oral hydration. Of the 9 patients, 7 (78%) achieved CR after 2 (n = 6) or 3 (n = 1) cycles, and achieved MRD-free CR after 2 (n = 3), 4 (n = 3) or 6 (n = 1) cycles. No patients became infected with COVID-19. Conclusions: This phase 1 trial met its primary endpoint of determining whether rituximab could be safely combined with Moxe and will enroll 4 additional patients to further access clinical activity. Further testing will determine whether addition of a CD20 Mab to Moxe significantly improves clinical outcome compared to Moxe alone, particularly long-term MRD-free CR rate. Clinical trial information: NCT03805932.

Early Pharmacodynamic Changes in T-Cell Activation, Proliferation, and Cytokine Production Confirm the Mode of Action of BFCR4350A, a FcRH5/CD3 T-Cell-Engaging Bispecific Antibody, in Patients with Relapsed/Refractory Multiple Myeloma

Early Pharmacodynamic Changes in T-Cell Activation, Proliferation, and Cytokine Production Confirm the Mode of Action of BFCR4350A, a FcRH5/CD3 T-Cell-Engaging Bispecific Antibody, in Patients with Relapsed/Refractory Multiple Myeloma

Abstract CN07-03: Recombinant immunotoxins for hematologic malignancies

Abstract Recombinant immunotoxins are composed of fragments of monoclonal antibodies (Mabs) and protein toxins, enabling the toxin to bind to a target cell recognized by the antibody, and the toxin to kill the cell after internalization. Recombinant immunotoxins are similar to but distinct from growth-factor fusions toxins like denileukin diftitox and Tagraxofusp, FDA-approved in 1999 and 2018, respectively. Unlike chemical conjugates, Recombinant immunotoxins have a peptide that links the cell-binding to the toxin domains. After proteolytic cleavage, the toxin separates from the binding domain, undergoes intracellular trafficking and enters the cytosol, resulting in apoptotic cell death. Recombinant immunotoxins containing Pseudomonas exotoxin A contain an Fv or Fab fragment of a Mab replacing the native cell-binding toxin domain. The first recombinant immunotoxin we made contained an anti-CD25 single-chain Fv fused to a 38 kDa fragment of Pseudomonas exotoxin called PE38. Anti-CD25 recombinant immunotoxin LMB-2 was active in several hematologic malignancies, notably hairy cell leukemia (HCL) and adult T-cell leukemia (ATL). LMB-2 achieved a high complete remission (CR) rate in ATL when combined with chemotherapy to reduce immunogenicity and progression between cycles. Improved targeting of HCL was achieved with anti-CD22 recombinant immunotoxin Moxetumomab Pasudotox (Moxe) that was stabilized with a disulfide bond in the Fv and has a high affinity for CD22. Moxe achieved investigator-assessed CR rates of 51-64% in relapsed/refractory HCL in Phase 1 and 3 trials, leading to FDA-approval in 2018. Minimal residual disease (MRD) was negative in most CRs. On the phase I trial, which had adequate follow-up, MRD eradication by the most sensitive standard assay, flow cytometry of the bone marrow aspirate (BMA), was associated with longer CR duration. Extra or ‘consolidation’ cycles past documentation of CR was also associated with longer CR duration. To detect MRD with higher sensitivity, patient specific immunoglobulin heavy chain (IgH) rearrangements were cloned and real-time quantitative PCR (RQ-PCR) performed. Using patient-specific primers and probe, 1 HCL cell could be detected in 106 normal cells. We found that phase 1-3 patients achieving MRD-free CR by blood RQ-PCR had significantly prolonged CR duration (p<0.0001) compared to those remaining blood RQ-PCR+. Both BMA flow cytometry and RQ-PCR were associated with prolonged CR duration if negative, but blood flow cytometry was not helpful since only one patient with CR remained flow-positive. Molecular remissions assessed by blood analysis may be useful to determine the number of cycles of Moxe to administer to achieve long-term CR or possibly cure. To achieve molecular remissions with fewer cycles, Moxe is being tested with rituximab to facilitate tumor reduction and decrease anti-drug antibody formation. Citation Format: Robert Kreitman, Daniel Gorelik, Maryalice Stetler-Stevenson, Constance M Yuan, Hao-Wei Wang, Hong Zhou, Katherine Potocka, Erin Fykes, Evgeny Arons, Ira Pastan. Recombinant immunotoxins for hematologic malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr CN07-03. doi:10.1158/1535-7163.TARG-19-CN07-03

Long Term Follow-up of a Phase II Study of Cladribine with Concurrent Rituximab in Patients with Hairy Cell Leukemia Variant

Background Hairy cell leukemia (HCL) variant (HCLv) is considered a separate, more aggressive entity compared to classic HCL. HCLv responds poorly to single-agent purine analog with complete response (CR) rates below 10% and overall response rates under 50%. Rituximab combined with purine-analog can improve response rate and duration, but long-term data have not been reported for HCLv, particularly regarding minimal residual disease (MRD). We therefore update the results of a phase II trial with cladribine and concurrent rituximab in patients with HCLv, previously reported for 10 of the 20 patients enrolled. Methods Patients with HCLv with 0 to 1 prior courses of cladribine, and/or 0-1 prior courses of rituximab, received cladribine (0.15 mg/kg days 1-5), with 8 weekly doses of rituximab (375 mg/m2) beginning day 1. The primary endpoint was to determine CR rate and secondary endpoints included evaluating minimal residual disease (MRD) by blood and bone marrow aspirate flow cytometry, and bone marrow biopsy immunohistochemistry. Patients were able to receive a 2nd course of rituximab ≥ 6 months after the first, if and when MRD was detected in blood. Results Twenty patients were enrolled. Median age was 67 (range: 42-86) years. No patients had prior concurrent cladribine-rituximab. Eight were previously untreated, 1 had only splenectomy, 6 had prior cladribine, 1 had prior cladribine and splenectomy, 1 had prior rituximab, 1 had prior rituximab and splenectomy, 1 had cladribine, rituximab, and splenectomy, and 1 had combination rituximab-containing chemotherapy followed by cladribine. Out of 20 patients receiving concurrent cladribine-rituximab (CDAR), the CR rate was 95% (95% CI: 75-100%). This CR rate was superior to a historical control group of 3 of 39 HCLv patients who achieved CR to cladribine alone (p<0.0001). Sixteen (80%, 95% CI: 56-94%) of 20 patients became MRD-free at 6 months; median duration of MRD-free CR was 72.0 months, with 9 of 16 still MRD-free at 5-108 (median 29.1) months. With median potential follow up of 88 months (range: 7-123 months), 10 patients received delayed rituximab and 4 re-achieved MRD-free CR. Six patients required alternative treatment and 6 patients died, 5 with HCLv including 1 with HCLv limiting treatment for lung cancer, and 1 with Parkinson's disease but still MRD-free. Time from progression of HCLv to death was 5.9-30.0 (median 28.1) months. Achieving MRD-free CR by 6 months after CDAR (16 vs 4 patients) was important for median progression free survival [PFS, unreached vs 17.4 mo, hazard ratio (HR) 0.031, 95% CI 0.003-0.29, p<0.0001] and overall survival (OS, unreached vs 38.2 months, HR infinite since all 4 MRD+ deaths were prior to deaths of 2 patients who achieved MRD-free CR, p<0.0001). A significant relationship between prior purine analog therapy or unmutated IGHV4-34 (n=7) status and either PFS or OS has not yet been observed. Conclusion Concurrent cladribine with rituximab is highly effective in HCLv irrespective of prior purine-analog treatment or IGHV4-34 status and should replace purine analog monotherapy as treatment. Patients with long-term MRD-free CR are being followed to determine whether concurrent cladribine-rituximab as 1st-3rd line systemic therapy can permanently eradicate HCLv. Patients who progress have limited OS. This provides a rationale for the testing of higher intensity approaches up front and the identification of additional treatment options for HCLv. Disclosures Kreitman: Genentech: Research Funding. OffLabel Disclosure: Rituximab for hairy cell leukemia

Clinical and Laboratory Features Associated with Flow Cytometric CD49f Expression in Pediatric B Cell Acute Lymphoblastic Leukemia

Introduction: B cell acute lymphoblastic leukemia (B-ALL) is the most common childhood malignancy and its diagnosis depends on flow cytometry. Aberrant antigen expression is common in B-ALL and can be useful in both diagnosis and minimal residual disease (MRD) assessment. CD49f (α6 integrin) is commonly overexpressed in lymphoblasts of B-ALL relative to benign B lymphoblasts, and research suggests a role for CD49f in central nervous system (CNS) invasion in B-ALL. We aimed to characterize clinical and laboratory features associated with lymphoblast CD49f expression in pediatric B-ALL. Methods: We reviewed clinical and laboratory features, including diagnostic and day 29 (D29) MRD bone marrow aspirate flow cytometry, for patients newly diagnosed with B-ALL at our pediatric centre using current diagnostic flow cytometry panels implemented in 2013. CD49f positivity (CD49f+) was defined as greater than 20% of leukemic blasts expressing CD49f at fluorescence intensity greater than the upper limit of an internal lymphocyte population. MRD studies were considered positive with leukemic blasts ≥0.01% of mononuclear cells. Frequency of CD49f+ and CD49f median fluorescence intensity (MFI) were compared between groups defined by recurrent cytogenetic abnormalities, molecular abnormalities detected by comparative genomic hybridization, CNS involvement at diagnosis, and occurrence of relapse by Chi-square, Fisher exact, Kruskal-Wallis, and Mann-Whitney testing. Results: 158 patients were reviewed with median age at diagnosis of 56 months (range 4 to 225 months) and median 33 months follow-up (range 1 to 67 months). 82 patients had CD49f+ B-ALL (median 62.2% CD49f+ blasts, CD49f MFI 3.4) and 76 patients had CD49f negative (CD49f-) B-ALL (median 3.7% CD49f+ blasts, CD49f MFI 0.8). CD49f+ and CD49f- cases did not differ significantly in median age at diagnosis (CD49f+, 51 months; CD49f-, 64.5 months) or presenting white blood cell count (CD49f+, 8.7x109/L; CD49f-, 8.8x109/L). 24 patients had CNS involvement at diagnosis, with which CD49f expression status was not significantly associated (CD49f+, 17.1%; CD49f-, 13.4%). Median CD49f MFI (1.8 CNS negative; 1.3 CNS positive) and proportion of CD49f+ leukemic blasts (27.8% CNS negative; 15.8% CNS positive) did not significantly differ between patients presenting with or without CNS involvement. Recurrent cytogenetic abnormalities associated significantly with CD49f status (p=0.001): CD49f+ associated with higher frequency of ETV6-RUNX1 (36.6% of CD49f+ and 11.8% of CD49f-; p=0.0004) and lower frequency (without statistical significance) of KMT2A rearrangement, hypodiploidy (each 1.2% CD49f+; 6.6% CD49f-), intrachromosomal amplification of chromosome 21, and TCF3-PBX1 (neither of which occurred in patients with CD49f+). Copy number abnormalities detectable by comparative genomic hybridization in CDKN2A, CDKN2B, PAX5, IKZF1, BTG1, ERG, TP53, RB1, EBF1, and CRLF2 did not differ in frequency between patients with CD49f+ and CD49f-. D29 MRD data were available for 154 patients, 32 of whom had positive MRD results. Frequency of D29 MRD positivity was not significantly associated with CD49f expression status at diagnosis (19.7% CD49f+; 21.9% CD49f-). Relapse occurred in ten patients and its occurrence was not associated with CD49f expression status at diagnosis (4.9% CD49f+; 7.9% CD49f-). Nine patients had evaluable relapse flow cytometry data, with three demonstrating increased proportions of CD49f+ blasts (1.5- to 3.2-fold increase from diagnosis), two demonstrating similar proportions, and three demonstrating decreased proportions of CD49f+ blasts (1.9- to 25-fold decrease from diagnosis). Two had new CNS involvement at relapse (one patient with 27% CD49f+ blasts at diagnosis and 1.1% at relapse; one patient with 93.3% CD49f+ blasts at diagnosis and 99.5% at relapse) and one had CNS involvement at diagnosis without involvement at relapse (with 12.8% CD49f+ blasts at diagnosis and 12.1% at relapse). Conclusion: Using the aforementioned criteria for flow cytometry data analysis, CD49f positivity in pediatric B-ALL was not associated with increased frequency of CNS involvement at diagnosis, day 29 MRD positivity, or relapse. CD49f expression patterns changed inconsistently in patients experiencing relapse and positivity or negativity at diagnosis or relapse was not clearly associated with CNS involvement at relapse. Disclosures No relevant conflicts of interest to declare.

POEMS Syndrome: Bone Marrow, Laboratory, and Clinical Findings in 24 Korean Patients.

POEMS syndrome is a rare paraneoplastic syndrome, which includes polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes due to plasma cell (PC) neoplasm. Diagnosis of this disease is challenging because of its rarity and complex clinical manifestations. We attempted to identify the key clinical features and characteristic bone marrow (BM) findings of POEMS syndrome, by reviewing the medical records and BM analyses of 24 Korean patients. Frequent clinical manifestations included polyneuropathy (100%), monoclonal gammopathy (100%), organomegaly (92%), extravascular volume overload (79%), and endocrinopathy (63%). The BM analyses revealed mild PC hyperplasia (median PCs: 5.5%) and frequent megakaryocytic hyperplasia (88%), megakaryocyte clusters (88%), and hyperlobation (100%). Flow cytometry of BM aspirates using CD138/CD38/CD45/CD19/CD56 showed normal (67%, 4/6) or neoplastic PC immunophenotypes (33%, 2/6). A diagnosis of POEMS syndrome must be considered when a patient suspected of having PC dyscrasia shows the above clinical presentation and BM findings.

Using MALDI-TOF mass spectrometry for tracking of minimal residual disease in peripheral blood from patients with multiple myeloma.

e19525 Background: Minimal residual disease (MRD) negativity after completed therapy is associated with longer progression-free survival (PFS) in patients with multiple myeloma (MM). Current standard of care for MRD testing use flow cytometry and/or next generation sequencing (NGS)-based assays applied on bone marrow (BM) aspirate samples. To develop a strategy for MRD tracking in peripheral blood (PB), we were motivated to evaluate MALDI-TOF head-to-head with established bone marrow-based MRD assays. Methods: We used MALDI-TOF mass spectrometry to detect M-proteins in PB. Our cohort included patients who had serum samples available at 2 timepoints including during active disease and within 60 days of MRD results as determined by flow cytometry of BM aspirates. The cohort enrolled 71 patients (26 females, 45 males) with a median age of 61 years (37-78 years). Twenty-seven patients had high-risk cytogenetics at baseline. Patients were classified at diagnosis as ISS1 (n = 38), ISS2 (n = 18) or ISS3 (n = 6). The flow cytometry based MRD assay was performed using MSKCCs 10-color, single-tube method. MALDI-TOF analysis was performed as described by Mills et al. Samples taken during active disease were used to identify the mass/charge ratio of the M-protein at baseline and in follow-up samples. MALDI-TOF results were compared to flow cytometry bone marrow-based MRD results. Results: The median time between diagnosis and the MRD timepoint was 13.4 months (3.4-91 months). MALDI-TOF in PB and flow cytometry BM-based MRD results were concordant for 44/71 (62%) patients (8+/+, 36 -/- respectively) while 27 were discordant (10 +/-, 17-/+). Fifty-four of 71 patients were in complete response (CR) (45/54 in sCR) at the time of MRD. MALDI-TOF was still positive in 13 of these 54 CR patients. In this cohort, the median PFS since MRD assessment was not reached in the 2 subgroups of double negative patients (n = 31) or in patients with a positive result in at least one technique (n = 23) with a median follow-up of 11.2 months (0-34.6 months). Conclusions: In 44/71 (62%) samples, MALDI-TOF of PB results and flow cytometry BM-based MRD results were concordant. MALDI-TOF of PB may be useful for detecting measurable residual disease and for the monitoring of MM patients during maintenance therapy with the future goal to rule out early recurrent disease.

Molecular Remissions with Anti-CD22 Recombinant Immunotoxin Moxetumomab Pasudotox Are Associated with Improved Complete Remission Durations during Phase I and III Testing

BACKGROUNDHairy cell leukemia (HCL) is an indolent B-cell malignancy strongly expressing CD22, with high sensitivity to purine analog chemotherapy, but relapses frequently occur, associated with minimal residual disease (MRD). Targeted non-chemotherapeutic approaches have shown efficacy but leave MRD. Moxetumomab pasudotox is an anti-CD22 recombinant immunotoxin recently reported in relapsed/refractory HCL to achieve 57% and 51% complete remission (CR) rates in phase 1 and 3 testing, respectively. Eradication of MRD by bone marrow flow cytometry or immunohistochemistry was associated with improved CR duration. We have previously reported that Taqman real-time quantitative PCR (RQPCR), which amplifies patient- and HCL-specific B-cell immunoglobulin rearrangements (IGHV), can detect one HCL cell in 106 normal cells. In the present study we determined whether MRD eradication measured by RQPCR is associated with improved CR duration. METHODSA total of 28 patients achieving CR to moxetumomab pasudotox in phase 1 (n=18) and phase 3 (n=10) trials were studied at the National Institutes of Health by multicolor flow cytometry of the bone marrow aspirate (BMA) and blood. Patients received moxetumomab pasudotox at 40-50 mcg/Kg on days 1, 3 and 5 of 2-7 cycles (median 5) at 4-week intervals. Blood and bone marrow samples were obtained to determine CR. In addition to end of treatment, bone marrow samples for MRD were generally obtained every 6-12 months for 2.5 years, then every other year. Blood flow cytometry was obtained every 6 months for 2.5 years, then yearly. Follow-up (time since treatment initiation) was 26.9-121.8 (median 84) months. For this analysis, CR for each bone marrow time point was determined by standard criteria for all patients independent of specific data-reporting requirements and data cut-off time points. RQPCR testing for each patient required cloning the immunoglobulin heavy chain variable regions and designing sequence-specific primers and probes. RNA samples were made from PAXgene tubes of blood and sodium-heparin tubes of BMA. RESULTSWe found that BMA RQPCR was most sensitive, followed by BMA flow cytometry, blood RQPCR, and blood flow cytometry. Of the 28 patients studied, 12 (43%) achieved MRD-free CR by all 4 tests and 2 of 12 patients (17%) relapsed. Seven (25%) were MRD+ by BMA RQPCR but negative by the other 3 studies, and none relapsed. One patient RQPCR+ and flow-negative in both blood and BMA relapsed. Seven patients flow-negative but RQPCR+ in blood, flow+ in BMA, and either BMA RQPCR+ or RQPCR not done, all relapsed. One patient with BMA RQPCR not done and the other 3 studies positive relapsed. With respect to RQPCR of blood as a single test, 3 of 20 negative vs 8 of 8 positive patients relapsed (p<0.001). Median CR duration was 13.0 months in blood RQPCR+ patients, vs not reached in blood RQPCR-negative patients, 17 (85%) of whom remain in CR at 11.6-112.9 (median 53) months of follow-up. With respect to BMA status, 2 of 12 patients negative vs 9 of 16 patients positive by either flow or RQPCR relapsed (p=0.054), and the 12 BMA MRD-negative patients had longer CR duration than the other 16 patients (p=0.018). CONCLUSIONSRQPCR negativity of blood is strongly associated with prolonged CR duration after moxetumomab pasudotox and may be a useful measure of MRD when bone marrow studies cannot be done. Additional testing will be needed to determine if blood RQPCR can be used to determine the optimal number of cycles of immunotoxin to administer. BMA RQPCR, though more sensitive than blood RQPCR, was not as useful due to fewer patients being evaluable and more time needed for BMA-RQPCR+ patients to relapse. Additional testing and follow-up may determine if BMA RQPCR negativity will correlate significantly with CR duration. [Display omitted] DisclosuresYao:MedImmune: Employment. Marshall:MedImmume: Employment. Pastan:NIH: Patents & Royalties: Co-inventor on the NIH patent for Moxetumomab Pasudotox. Kreitman:NIH: Patents & Royalties: Co-inventor on the NIH patent for Moxetumomab Pasudotox.

Minimal residual hairy cell leukemia eradication with moxetumomab pasudotox: phase 1 results and long-term follow-up

AbstractAnti-CD22 moxetumomab pasudotox achieved 46% complete remissions (CRs) in previously reported phase 1 testing in relapsed/refractory hairy cell leukemia (HCL; n = 28). The importance of minimal residual disease (MRD) after CR in HCL is unknown. A 21-patient extension cohort received 50 µg/kg every other day for 3 doses in 4-week cycles. These patients plus 12 previously reported at this upper dose level received 143 cycles without dose-limiting toxicity. The combined 33-patient cohort achieved 64% CR and 88% overall response rates, with median CR duration of 42.4 months. Of 32 50-µg/kg patients evaluable for MRD by bone marrow aspirate flow cytometry (most stringent assessment), median CR duration was 13.5 (4.9-42.4) months in 9 MRD-positive CRs vs 42.1 (24.0-69.2) months in 11 MRD-negative CRs (P < .001). Among MRD-negative CRs, 10 patients had ongoing CR, 9 without MRD, at end of study. To our knowledge, moxetumomab pasudotox is the only nonchemotherapy regimen that can eliminate MRD in a significant percentage of HCL patients, to enhance CR duration. Repeated dosing, despite early neutralizing antibodies, increased active drug levels without detectable toxicity from immunogenicity. The activity and safety profiles of moxetumomab pasudotox support ongoing phase 3 testing in HCL. This trial was registered at www.clinicaltrials.gov as #NCT00586924.

P022 CROHN’S DISEASE AND ACUTE B LYMPHOBLASTIC LEUKEMIA – COINCIDENCE OR ASSOCIATION?

A 34-year-old male with Crohn’s disease (CD) (A2-L3-B3) and bad prognostic criteria at diagnosis, was treated with a “top down” strategy with infliximab and azathioprine, with clinical remission but focal endoscopic activity. After 3 years, a toxidermic reaction to infliximab led to its suspension. On azathioprine monotherapy for 6 months, a severe exacerbation occurred and adalimumab was added. In 2015 the patient presented with anorexia, asthenia, myalgia, fever and nasal congestion. Blood tests revealed bicytopenia, acute kidney injury, hyperuricemia and high LDH. The patient was hospitalized and immunosuppressive therapy was suspended. Blood smear showed blast cells, with bone marrow aspirate flow cytometry analysis confirming an acute B lymphoblastic leukemia (FISH: t(14,18)). He underwent 7 cycles of chemotherapy, and is currently awaiting autologous stem cell transplantation. Despite several infectious complications the patient is stable and CD is in clinical remission. CD is not often associated with acute leukemia, and most reported cases refer to the myeloid type. A possible association between these 2 pathologies may be multifactorial, involving therapy adverse effects, immune system disturbances and genetic predisposition. Case analysis may help to increase clinical awareness and to gain better understanding of treatment response and prognosis for this combination of diseases.

Primary systemic amyloidosis presenting as macroglossia

Primary amyloidosis is a group of monoclonal plasma cell disorders, characterized by extracellular deposition of immunoglobulin light chain fibrils in multiple organs leading to progressive multiorgan dysfunction. We report a 61-year-old female patient with an enlarged tongue, difficulty in speech, gradually changing quality of voice, and loss of weight. Diagnosis of amyloidosis and systemic nature confirmed by tongue biopsy and anterior abdominal fat biopsy using Hematoxylin and Eosin staining and Congo red staining. Urine examination positive for Bence–Jones proteins. Bone marrow aspiration cytology, bone marrow aspirate flow cytometry immunophenotyping, serum immunofixation electrophoresis, and urine immunoelectrophoresis revealed plasmacytosis, clonal plasma cell population, marked increased in lambda light chain in serum and urine, respectively. Based on these observations, a definitive diagnosis of primary systemic amyloidosis associated with plasma cell dyscrasia was made. He was treated with intravenous (IV) bortezomib and IV dexamethasone. A battery of tests is necessary to differentiate localized amyloidosis from its systemic forms for diagnosis, treatment and to assess treatment outcome.

High Response Rate of Moxetumomab Pasudotox in Relapsed/Refractory Hairy Cell Leukemia Includes Eradication of Minimal Residual Disease: Potential Importance for Outcome

Abstract Background: In patients with hairy cell leukemia (HCL), the clinical importance of minimal residual disease is unknown; particularly by bone marrow aspirate (BMA) multicolor flow cytometry. It is often assumed that agents failing to eradicate MRD may prove equally effective as those that do. Moxetumomab pasudotox is a recombinant immunotoxin containing truncated Pseudomonas exotoxin fused to an anti-CD22 Fv. After the dose-escalation phase of a phase I trial in 28 patients with relapsed/refractory HCL treated with 5-50 ug/Kg every other day for 3 doses (QOD x3), a complete remission (CR) rate of 46% and overall response rate (ORR) of 86% were reported. Two cases of reversible grade-2 hemolytic uremic syndrome (HUS) were also reported, with transient grade I platelet and creatinine changes. Methods: To determine response and toxicity at a fixed dose, Twenty-one additional patients were enrolled at the highest dose level, totaling 33 patients at 50 ug/Kg QOD x3 and 49 patients overall. Patients required at least 2 prior courses of purine analog, or 1 course each of purine analog and rituximab if response duration to the first purine analog was &lt;2 years. CR criteria included absence of HCL cells by morphologic stains of bone marrow and blood. In CRs, multicolor flow cytometry was used to detect MRD in the BMA as well as blood. Results: No dose limiting toxicity or additional HUS cases were observed in any of the 202 cycles administered to 49 patients. Of 33 patients receiving 143 cycles of 50 ug/Kg QOD x3, the overall response rate was 88% and 21 (64%) achieved CR. Of 20 CRs followed for response duration, the median duration of CR (disease-free survival, DFS) was 42 mo (range 5 - 72), with 12 (60%) of 20 remaining in CR at a median of 42 mo. In addition to the 21 CRs achieved at 50 ug/Kg, there were 2 CRs out of 4 patients treated at 40 ug/Kg, and 5 CRs out of 12 patients treated at lower dose levels. Of the 28 CRs, MRD by BMA flow cytometry was evaluated in all except in 1 patient each at 50 and 40 ug/Kg, and 2 patients at lower doses. By BMA flow cytometry, MRD-negative CR was achieved in 11 (34%) of 32 at 50 ug/Kg, 1 of 3 at 40 ug/Kg, and 0 of 10 at lower doses (Fisher's Exact comparison above vs below 40 ug/Kg, p=0.041). MRD+ CRs included 9 at 50 ug/Kg, 0 at 40 ug/Kg, and 3 at lower doses. For the 12 MRD-negative CRs at 40-50 ug/Kg, DFS has not been reached, compared to a DFS of 17 mo in the 9 remaining MRD+ CRs at 40-50 ug/Kg (p&lt;0.0001). Of the 12 MRD-negative CRs, 11 (92%) remain in CR at 16-79 (median 42) mo of follow-up. Conclusion: Moxetumomab pasudotox can eliminate HCL MRD, and MRD eradication significantly correlated with CR durability. In HCL, particularly after multiple relapses, CRs should optimally be achieved without MRD. To our knowledge, moxetumomab pasudotox is the only known agent capable of eliminating MRD in HCL in a high percentage of patients without causing myelosuppressive toxicities common to chemotherapy. Its activity and safety profile support multicenter pivotal phase III testing, which is currently ongoing. This summary contains investigator reported data. This study was sponsored by MedImmune and supported by NCI's Intramural Research Program and the Hairy Cell Leukemia Research Foundation. Disclosures Lanasa: MedImmune: Employment. FitzGerald:NIH: Patents &amp; Royalties: Coinventor on NIH Patent. Pastan:NIH: Patents &amp; Royalties: Coinventor on NIH Patent.

Altered leucocyte progenitor profile in human bone marrow from patients with major trauma during the recovery phase

Changes in human bone marrow associated with the systemic inflammatory response to injury are little understood. It was hypothesized that major trauma results in an altered bone marrow leucocyte progenitor profile, with either uniform depletion or the balance between multipotent and committed progenitors varying, depending on whether self-renewal is favoured over differentiation. Bone marrow aspirate and peripheral blood samples were obtained at definitive surgery in adults with pelvic fractures from blunt trauma (major trauma with Injury Severity Score (ISS) at least 18, or isolated fractures) and control patients undergoing iliac crest bone grafting. ISS, interval to surgery and transfusion in the first 24 h were recorded. Bone marrow aspirate flow cytometry was used to identify haemopoietic progenitor cells (CD34(+) ), multipotent cells (CD34(+) CD45(+) CD38(-) ) and oligopotent cells (CD34(+) CD45(+) CD38(lo/+) and CD34(+) CD45(+) CD38(BRIGHT(++ +)) subsets). Peripheral blood levels of inflammatory markers were measured, and the ratio of immature to mature (CD35(-) /CD35(+) ) granulocytes was determined. The median (range) interval between injury and sampling was 7 (1-21) and 5 (1-21) days in the major trauma and isolated fracture groups respectively. The CD34(+) pool was significantly depleted in the major trauma group (P = 0·017), particularly the CD34(+) CD45(+) CD38(BRIGHT(++ +)) oligopotent pool (P = 0·003). Immature CD35(-) granulocytes increased in bone marrow with increasing injury severity (P = 0·024) and massive transfusion (P = 0·019), and in peripheral blood with increasing interval to surgery (P = 0·005). Major blunt trauma resulted in changes in the bone marrow CD34(+) progenitor pool. At the point in recovery when these samples were obtained, oligopotent progenitors were lost from the bone marrow, with continued release of immature cells.

Leukemia Lineage-Specific Chimerism Analysis and Molecular Monitoring Improve Outcome of Donor Lymphocyte Infusions

A retrospective analysis was performed of 118 patients with hematologic malignancies who received donor lymphocyte infusions (DLIs) after allogeneic stem cell transplantation (ASCT). Treatment was either given because of hematologic relapse (n = 44), molecular/cytogenetic relapse (n = 52), or other causes (n = 22). Molecular relapse was in most cases based on leukemia lineage-specific chimerism analysis. Patients with acute leukemia and myelodysplastic syndrome showed a 3-year survival of 42% if DLI treatment was given because ofmolecular relapse, compared to 16% in hematologic relapse (P < .006). In multivariate analysis, there was a correlation between response to DLI and nonhematologic relapse (risk ratio [RR] 3.36, P = .001), chronic graft-versus-host disease (cGVHD) (RR = 1.51, P = .005), and late relapse (RR = 2.06, P = .017). The overall incidences of acute GVHD (aGVHD) grades I-II and grades III-IV aGVHD were 33% and 8.5%, respectively. Probability of cGVHD was 33%. The development of aGVHD or cGVHD did not significantly influence the response of DLI in patients with molecular/cytogenetic relapse. However, the development of cGVHD was significantly associated with a better response in patients with hematologic relapse because only 4 of 29 patients without cGVHD responded compared to 7 of 12 with cGVHD (P = .007). The development of cGVHD increased significantly if DLI was given >12 months after ASCT (46% versus 27%, P = .04). In contrast, time between ASCT and start of DLI treatment had no significant influence on the risk of developing aGVHD. To conclude, monitoring of leukemia lineage-specific chimerism is of utmost importance for DLI response after ASCT.