Jacob, Eapen K.

Background: Upfront autologous stem cell transplant (ASCT) is the standard-of-care for newly diagnosed multiple myeloma (MM) patients. However, relapse and the development of therapy-related myeloid neoplasms (t-MN) post ASCT results in considerable morbidity and mortality. We hypothesized that enrichment of abnormal myeloid progenitors and immune effector cells (IEC) in peripheral blood stem cells (PBSCs) of patients with MM is associated with a higher risk of relapse and development of t-MN. Methods: After institutional review board approval, we screened patients with active MM that were evaluated at Mayo Clinic, Rochester, MN between 01/01/2003 and 12/31/2020. Patients that underwent an ASCT and had cryopreserved PBSC product available for research were included. Using mass cytometry, we performed a comprehensive immunophenotyping of mobilized cryopreserved PBSCs from 54 MM patients undergoing 1 st ASCT, using validated antibody panel of 37 lymphoid- and myeloid-based markers each. Flow cytometry standard files were normalized and concatenated using the Fluidigm acquisition software. Flow cytometry standard files were uploaded to the OMIQ software from Dotmatics (www.omiq.ai, www.dotmatics.com), where transformation and cleaning (doublets, debris) were performed. To correct for between-sample technical variations (“batch effects”), we used fdaNorm within the omiq.ai platform. Clustering and visualization of normalized and concatenaded flowcytometry files were performed within the omiq.ai platform using PhenoGraph and UMAP, respectively. We performed principal component analysis (PCA) to identify major immunomic drivers of variability across patients. We explored the associations between immune subsets and the dichotomized clinical outcomes of interest: progression-free (PFS), myeloid neoplasm-free (MNFS) and overall survival (OS) from ASCT. A 2-sided false discovery rate adjusted P-value of <0.05 was considered significant when multiple comparisons were performed; otherwise, a P-value of <0.05 was considered significant. Results: The most abundant cell populations were T-cells (49% of the CD45 +) and myeloid cells (43% of the CD45 + cells). Clustering on the CD45 + cells revealed 30 unique lymphoid clusters and 27 unique myeloid clusters. Subclustering on NK-cells identified an additional 16 NK-cell clusters. On PCA, principal components-1 (PC-1) and 2 (PC-2) explained 15.2% and 12.5% of the variability in the data, respectively. A high PC-2 score was associated with inferior median PFS (20 vs. 52 months; P=0.005, Figure 1A), MNFS (52 vs. 85 months, P=0.03, Figure 1B) and OS (64 vs. 106 months, P=0.01) from ASCT. Immune subsets enriched in the high PC-2 cohort included the ‘NKT-like’ (CD56 +) T-cell subset which expressed the inhibitory CD159a receptor and TIGIT; a terminally differentiated (CD27/CD28 -, CD57/KLRG1 +) and exhausted (TIGIT/PD-1 +) T cell subset, 2 exhausted (TIGIT/PD-1 +) T cell subsets; an immunosenescent (CD27/CD28 -, CD57/KLRG1 +) T cell subset as well as a terminally exhausted (C27/C28 -, KLRG1/PD-1/TIGIT +) TCRgamma-delta subset. Immune subsets with high loadings within PC-1 (associated with improved clinical outcomes) consisted exclusively of CD4 + cells with early memory phenotypes (CD27/CD28/CD127 +). Abnormal expression of CD7 and HLA-DR expression on myeloid progenitor cell subsets was associated with an inferior PFS, MNFS, and OS. A NK-cell subcluster, characterized by an immature (CD56 high), inhibitory (CD159a +, NKG2A) phenotype, was enriched in patients developing t-MN (0.33% vs. 0.17%, P=0.006). Conclusion: We identify abnormal myeloid and immune effector cells in the PBSCs of patients with MM, even prior to ASCT. Patients with a PBSC product that is enriched in immunosuppressive T and myeloid cell subsets, and with a lower proportion of antigen presenting cells, progenitor populations, and anti-tumor macrophages have a shorter PFS and MNFS. This suggests that the predisposition to t-MN and inferior outcomes for MM are possibly determined early in the disease course. These findings need external validation and have the potential to guide sequencing of future treatment modalities.

AbstractBackgroundHigh titers of cold agglutinins jeopardize the quality of an apheresis product meant for autologous or allogeneic transplant. Management of transplant patients with cold agglutinin disease (CAD) is often experience‐based and under reported, yet decisions must be made quickly to optimize product management and patient outcomes. There remains a lack of data quantifying cell recovery and viability when using various warming methodologies.Study Design and MethodsTo expand the published experimental data on this subject, our human cellular therapy lab compared cellular recoveries and viabilities after manipulation of cryopreserved apheresis products through various warming methodologies: (1) extended warming in a water bath, (2) warming via blood warmer and infusion pump, and (3) warming in a water bath followed by infusion pump as a control to assess potential shear stress effects.ResultsThe presented studies demonstrate that all methods of product warming produce the same rates of recovery of total and viable cells across vital cell types prior to patient administration. Statistically, use of an extended water bath protocol provided a marginal benefit in recovery of total nucleated cells, though this effect is diminished when products are held for an extended period to simulate a delay in administration.DiscussionThese results can inform decisions to improve patient care and minimize product manipulation and loss. Centers are encouraged to use this information to guide proactive measures to establish a standard operating procedure to manage CAD cases.

Abstract Background: The use of immunotherapeutic agents in multiple myeloma (MM) has shown improvements in clinical outcomes, emphasizing the role the host immune system plays in disease control. In recognition of this relationship, efforts aimed at identifying biomarkers of immune surveillance in MM have identified prognostic value in the peripheral absolute lymphocyte (ALC) and absolute monocyte (AMC) counts at various stages of disease and following autologous stem cell transplant (ASCT), with lower ALC and AMC serving as a surrogate for immune dysregulation and correlating with inferior progression free (PFS) and overall survival (OS). With ASCT remaining the standard of care in the treatment of MM, we sought to examine whether CD3 content of the apheresis product during stem cell collection could be used as a biomarker for immune competence and whether it influences outcomes. Methods: A retrospective study was conducted on 1086 MM patients who underwent stem cell (CD34) collection with available CD3 data and subsequent ASCT at our institution. We recorded the total absolute CD3 and CD34 cell count upon completion of mobilization, with a higher CD3 count serving as a surrogate for immune competence. In addition to investigating the absolute CD3 count, we calculated the CD3/CD34 ratio given variation in the CD34 goals. Patients were dichotomized based on whether their absolute CD3 and CD3/CD34 ratio values were above or below medians. A Kaplan-Meier model was used to compare median PFS and OS between groups. A cox proportional hazards model was used to determine its prognostic impact, adjusting for ISS stage 3, high risk FISH, achievement of CR near day 100, and maintenance therapy received post-ASCT. Results: The median length of follow-up from date of ASCT for the entire cohort (N=1086) was 34.1 months (range: 0.26-157 months) and the median time from stem cell collection to ASCT was 9 days (range: 3-3143 days). The most common mobilizing regimen was Neupogen and plerixafor (N=425, 39%) and 694 (64%) patients received ASCT in the first line setting. The median absolute total CD3 count was 4.3 x 10^6/kg (range: 0.1-21.9) with 542 patients above and 544 patients at or below the median. The median absolute total CD34 count was 8.53 x 10^6/kg (range: 0.2-37.0) and median CD3/CD34 ratio was 0.50 (range: 0.01-15), with 536 patients above and 550 patients at or below the median ratio. The median PFS among patients with a CD3 count > 4.25 x 10^6/kg was 23.1 vs. 16.9 months among patients with CD3 count ≤ 4.25 x 10^6/kg (p<0.0001) and median OS was 65.3 months vs. 41.6 months (p<0.0001), respectively. A similar trend was observed when dividing patients based on CD3/CD34 ratio, with median PFS of 22.4 vs. 17.5 months (p=.0003) and median OS of 61.5 vs. 45.7 months (p=.0001), both favoring the cohort with a CD3/CD34 ratio > 0.5 (Figure 1). The prognostic value among patients with a CD3/CD34 ratio > 0.5 was retained after adjusting for ISS stage III, high risk FISH features, and use of maintenance therapy as follows: PFS HR: 0.73 (95% CI: 0.62-0.86; p=0.0002); OS 0.71 (95% CI: 0.59-0.88; p=0.001). Conclusions: Our study demonstrates that patients who have higher CD3 content in their apheresis product have better PFS and OS following ASCT. These findings reveal a possible role for using absolute CD3 and CD3/CD34 ratios in the apheresis product as a surrogate marker for immune competence and in predicting clinical outcomes. Figure 1 Figure 1. Disclosures Gertz: Akcea Therapeutics, Alnylam Pharmaceuticals Inc, Prothena: Consultancy; Akcea Therapeutics, Ambry Genetics, Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Karyopharm Therapeutics, Pfizer Inc (to Institution), Sanofi Genzyme: Honoraria; Aurora Biopharma: Other: Stock option; AbbVie Inc, Celgene Corporation: Other: Data Safetly & Monitoring; Ionis Pharmaceuticals: Other: Advisory Board. Dingli: GSK: Consultancy; Sanofi: Consultancy; Janssen: Consultancy; Alexion: Consultancy; Apellis: Consultancy; Novartis: Research Funding. Dispenzieri: Takeda: Research Funding; Pfizer: Research Funding; Alnylam: Research Funding; Oncopeptides: Consultancy; Sorrento Therapeutics: Consultancy; Janssen: Consultancy, Research Funding. Kapoor: Sanofi: Research Funding; Takeda: Research Funding; Ichnos Sciences: Research Funding; Glaxo SmithKline: Research Funding; Regeneron Pharmaceuticals: Research Funding; AbbVie: Research Funding; Karyopharm: Research Funding; Karyopharm: Consultancy; Cellectar: Consultancy; BeiGene: Consultancy; Pharmacyclics: Consultancy; Sanofi: Consultancy; Amgen: Research Funding. Kumar: Novartis: Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Antengene: Consultancy, Honoraria; Tenebio: Research Funding; Amgen: Consultancy, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bluebird Bio: Consultancy; BMS: Consultancy, Research Funding; Oncopeptides: Consultancy; Carsgen: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche-Genentech: Consultancy, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Beigene: Consultancy; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding.

AbstractBackgroundDaratumumab (Dara), an anti‐CD38 monoclonal antibody for hematologic malignancies, interferes with routine blood bank testing, specifically affecting the antibody screen and identification panels. In 2016, the AABB recommended performing a baseline phenotype or genotype before a patient (Pt) begins taking anti‐CD38 to avoid this interference and potential problems with transfusion. The objective of this study was to assess red blood cell (RBC) utilization and subsequent incidence of alloimmunization to the transfused RBCs in patients receiving Dara.Methods and MaterialsWe monitored 244 patients taking Dara to determine their red blood cell transfusions and incidence of clinically significant antibody formation before and following administration of Dara. Poisson generalized estimating equations with log link were used comparing the post‐Dara incidence and prevalence to those prior, with significance defined as p < .05.ResultsFrom September 1, 2015 to December 22, 2018, 244 patients on Dara were identified, of which 145 patients (59.4%) received a red blood cell transfusion. Antibody screens were performed on 97 of the 145 patients at least 2 weeks following RBC transfusion. Four of the total transfused patients (2.8% total, 4.1% patients with follow‐up antibody screen testing) formed new clinically significant alloantibodies, which was not significantly different from Asare's hematologic incidence (p = .98/p = .49).ConclusionsThis study showed our patients on Dara did not form alloantibodies following RBC transfusion at a higher incidence than similar patient populations.

Abstract Background The two major types of cold autoimmune hemolytic anemias are cold agglutinin disease (CAD) and paroxysmal cold hemoglobinuria (PCH). In CAD, the cold agglutinin is usually IgM with anti-I specificity. In PCH, the Donath-Landsteiner (DL) antibody is a cold reacting IgG with anti-P specificity. In this study, we describe the clinical and serological characteristics of patients with autoimmune hemolytic anemia positive for both cold agglutinin and DL antibodies. Methods On review of our immunohematology reference laboratory database, we identified patients over a 16-year period (January 2000- March 2016) with the following: i) age ≥18 years, ii) hemoglobin (Hb) &lt;12g/dL, iii) positive direct antiglobulin test (DAT) with hemolysis (increased lactate dehydrogenase/low serum haptoglobin/ elevated indirect bilirubin), and iv) tested for both CAD and DL antibodies. We classified the patients into 3 cohorts. Cohort 1 included patients positive for both cold agglutinin (titer ≥1:64) and DL antibodies. Cohort 2 consisted of patients with DL antibody but no cold agglutinin (titer &lt;1:64), while Cohort 3 was comprised of patients with cold agglutinin but no DL antibody. We evaluated the clinical response based on the GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) criteria (Barcellini W, et al . Blood 2014) defined as i) complete (CR): hemoglobin ≥12 g/dL with normalization of at least one previously abnormal hemolytic marker; ii) partial (PR): hemoglobin 10-12 g/dL with hemolysis; or iii) none (NR): if any of the above criteria were not met. Results Seven patients had cold autoimmune hemolytic anemia with concomitant cold agglutinin and DL antibodies (Cohort 1). The clinical and serological characteristics are described in table 1. The median age at diagnosis was 68 years (range: 59-78) and the median hemoglobin at onset was 10.3 g/dL (range: 8.2-10.6). Six had red cell agglutination in the blood smear. Two patients had a recent history of infection (1 with Mycoplasma pneumoniae and 1 with upper respiratory tract infection). The median follow-up was 4.8 months (range: 1.0-166.4). Five patients received steroid/other immunosuppressants and two were managed conservatively. The clinical response to treatment was CR in 28.6% patients, PR in 42.9%, and NR in 14.3%. In comparison, Cohort 2 had a 20% CR, 20% PR and 40% NR, while Cohort 3 had a 50% CR, 25% PR and 25% NR, respectively. Conclusion Our study is the first series describing patients with cold autoimmune hemolytic anemia with concomitant cold agglutinin and DL antibodies. In terms of clinical response, patients with negative DL antibodies and cold agglutinin titers &gt;1:64 had better response (50% CR, Cohort 3) to immunosuppressants compared to patients with positive DL antibody (20-30% CR, Cohorts 1 and 2). Large scale studies may be warranted to determine the treatment strategies among patients with concomitant DL and high cold agglutinin titers. Disclosures Kay: Agios: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Research Funding; Tolero Corporation: Research Funding; Gilead: Research Funding. Winters: Fresenius Kabi USA: Consultancy; Eliaz Therapeutics Inc: Membership on an entity's Board of Directors or advisory committees; Sanofi Inc: Other: Moderated opinion leader's forum; Wiley Blackwell: Employment; Regional Health Inc: Consultancy; Mayo Clinic: Employment; Grifols International SA: Consultancy.

Introduction Day 15 absolute lymphocyte count (ALC-15) recovery after autologous peripheral blood hematopoietic stem cell transplantation (APBHSCT) is recognized as a prognostic factor for survival. ALC-15 recovery is directly dependent on the amount of collected and infused autograft-absolute lymphocyte count (A-ALC). Our group published a double-blind phase III study which showed that patients infused with an A-ALC ≥ 0.5 x 109 cells/kg experienced superior survival post-APBHSCT. Based on the results from our phase III study, on April 1, 2017, our Bone Marrow Transplant Program changed our standard clinical practice to collect an A-ALC ≥ 0.5 x 109 cells/kg in addition to stem cells for lymphoma patients undergoing APBHSCT. To continue to assess the prognostic ability of A-ALC, we evaluated the overall survival (OS) and progression-free survival (PFS) of diffuse large B-cell lymphoma (DLBCL) patients that underwent APBHSCT after April 1, 2017 compared with a matched-control group in a 1:1:1 ratio with DLBCL patients infused with an A-ALC &lt; 0.5 x 109 cells/kg or an A-ALC ≥ 0.5 x 109 cells/kg prior to April 1, 2017. Methods Eight-five consecutive DLBCL patients after our practice changed on April 1, 2017 received an A-ALC ≥ 0.5 x 109 cells/kg. For each DLBCL patient infused with an A-ALC ≥ 0.5 x 109 cells/kg after April 1, 2017, the set of potential controls was restricted to those DLBCL patients infused with an A-ALC &lt; 0.5 x 109 cells/kg and an A-ALC ≥ 0.5 x 109 cells/kg prior to April 1 2017 . Within this group of potential controls, the subjects were chosen who best matched for the cases in terms of clinical risk variables, including gender, age, stage, lactate dehydrogenase (LDH), performance status, extra-nodal disease, International prognostic index (IPI), and disease status prior to APBHSCT (complete or partial response), using the GREEDY algorithm. Cases were defined as DLBCL patients infused with an A-ALC ≥ 0.5 x 109 cells/kg starting from April 1 2017. Control 1 was defined as DLBCL patients infused with an A-ALC &lt; 0.5 x 109 cells/kg prior to April 1 2017. Control 2 was defined as DLBCL patients infused with an A-ALC ≥ 0.5 x 109 cells/kg prior to April 1 2017. We truncated the survival follow-up to 3 years starting from the date of transplant to match follow-up time between cases and controls. Both matched cases and control groups patients received BEAM conditioning. This retrospective study was approved by the Mayo Clinic Institutional Review Board according to the regulation of the Declaration of Helsinki. Results Cases, control 1 and control 2 were balanced to the clinical risk variables; age (p = 0.8); gender (p = 0.9); LDH (p = 0.6); stage (p = 0.3); performance status (p = 0.5); extra-nodal disease (p = 0.2); IPI (p = 0.6); and clinical status prior to transplant (p = 0.2). Figure 1A showed the OS and figure 1B the PFS. Both the cases and control 2 showed superior OS and PFS in comparison to control 1. Cases and control 2 showed overall similar OS and PFS. Multivariate analysis including all patients continue to depict the A-ALC ≥ 0.5 x 109 cells/kg as an independent predictor for OS (HR = 0.382, 95%CI=0.241-0.605, p &lt; 0.0001) and PFS (HR = 0.437, 95%CI = 0.279-0.629, p &lt;0.0001). Subgroup analysis, including all patients, shows superior OS and PFS for patients infused with an A-ALC ≥ 0.5 x 109 cells/kg for each of the following clinical variables: age [&gt; 60 years: OS (p &lt;0.0001); PFS (p &lt;0.0001) or ≤ 60 years: OS (p &lt;0.01); PFS (p , 0.02)]; extra-nodal disease [&gt; 1: OS (p &lt;0.008); PFS (p &lt;0.01) or ≤1: OS (p &lt; 0.001): PFS (p &lt;0.0005)]; IPI [&gt; 2: OS (p &lt;0.01); PFS (p &lt;0.02) or ≤ 2: OS (p &lt;0.0008); PFS (p &lt;0.0006)]; LDH [abnormal: OS (p &lt;0.006); PFS (p &lt;0.005) or normal: OS (p &lt;0.002); PFS (p &lt;0.002)]; performance status [&gt; 1: OS (p &lt;0.04); PFS (p &lt;0.03) or ≤ 1: OS (p &lt;0.0001); PFS (p &lt;0.0001)]; stage [I/II: OS (p &lt;0.006); PFS (p &lt;0.006) or III/IV: OS (p &lt;0.002); PFS (p &lt;0.001)]; response prior to transplant [complete response: OS (p &lt;0.0007); PFS (p &lt;0.0001) or partial response: OS (p &lt;0.01); PFS (p &lt;0.02)]. Conclusion Our match-control study continues to support the results of our Phase III study showing that the infusion of A-ALC is a prognostic factor for survival of DLBCL patients undergoing APBHSCT. Our studies support the practice of not only collecting enough stem cells (i.e., CD34) for hematologic engraftment, but also enough immune effector cells (i.e., A-ALC) to improve clinical outcomes in DLBCL patients post-APBHSCT. Figure 1 Disclosures Ansell: Regeneron: Research Funding; Bristol Myers Squibb: Research Funding; Trillium: Research Funding; ADC Therapeutics: Research Funding; Takeda: Research Funding; AI Therapeutics: Research Funding; Affimed: Research Funding; Seattle Genetics: Research Funding.

AbstractBackgroundPrimary cold agglutinin disease (CAD) is a monoclonal antibody (M‐protein) and complement‐mediated chronic hemolytic disease process. Antibody glycosylation can play a role in both antibody half‐life and complement fixation. Recently, M‐protein light chain (LC) glycosylation has been shown to be associated with AL amyloidosis. We hypothesized that M‐protein LC glycosylation is also associated with cold agglutinin (CA) titers and CA‐mediated hemolysis.Study Design and MethodsA cross‐sectional study of patients undergoing CA titer evaluation underwent mass spectrometric analysis for M‐proteins and M‐protein LC glycosylation. A subset of serum samples also underwent evaluation for the ability to trigger cold hemolysis in vitro. M‐protein and M‐protein LC glycosylation rates were compared across CA titer groups, clinical diagnosis, direct antiglobulin testing (DAT) results, and cold in vitro hemolysis rates.ResultsBoth M‐protein and M‐protein LC glycosylation rates significantly differed across CA titer groups with the highest rates in those with elevated CA titers. M‐protein LC glycosylation occurred almost exclusively on IgM kappa M‐proteins and was significantly associated with positive DAT results and a clinical diagnosis of CAD. Cold in vitro hemolysis was demonstrated in two patients who both had a CA titer of more than 512 but there was no significant association with CA titer group or M‐protein LC glycosylation status.ConclusionM‐protein LC glycosylation is significantly associated with higher CA titer levels. Given the role that antibody glycosylation can play in antibody half‐life and complement fixation, further studies are needed to clarify the effects of LC glycosylation within the context of CAD.

AbstractBackgroundHemolytic disease of the fetus and newborn (HDFN) attributable to anti‐M is rare, although case reports implicate anti‐M in varying severities of HDFN, including fetal hydrops and intrauterine death.Case DescriptionWe describe the case of a newborn with HDFN associated with an atypical immunoglobulin (Ig) G anti‐M that reacted best at cold temperatures. The maternal antibody detected in pregnancy was not reactive at 37°C, and a direct antiglobulin test (DAT) on red blood cells (RBCs) from the newborn was negative, suggesting an anti‐M that should not have been clinically relevant. However, the infant developed hyperbilirubinemia (bilirubin level, 17.6 mg/dL), hemolytic anemia (hemoglobin nadir, 5.5 g/dL), and reticulocytopenia. Laboratory testing demonstrated the presence of an IgG anti‐M in maternal and neonatal samples reacting best at 4°C. This passively acquired IgG anti‐M provoked hemolytic anemia in the infant and likely suppressed erythropoiesis, resulting in reticulocytopenia with prolonged anemia. He was treated for IgG anti‐M HDFN with 10 intravenous Ig infusions and 10 days of oral prednisone followed by a taper. He required seven transfusions with M− RBCs. His hemoglobin level normalized at 3 months of age. Follow‐up at 2 years revealed no hematologic or neuro‐developmental concerns.ConclusionTo our knowledge, this is the second report of HDFN attributable to an IgG anti‐M reacting preferentially at cold temperature with no 37°C reactivity. Clinically relevant IgG anti‐M may elude standard testing. Early recognition and testing for cold‐reacting IgG anti‐M should be considered for newborns with hemolysis, a negative DAT, and prolonged anemia.