Timm, Michael

Abstract Objectives Fluorescence in situ hybridization (FISH) for plasma cell neoplasms (PCNs) requires plasma cell (PC) identification or purification strategies to optimize results. We compared the efficacy of cytoplasmic immunoglobulin FISH (cIg-FISH) and fluorescence-activated cell sorting FISH (FACS-FISH) in a clinical laboratory setting. Methods The FISH analysis results of 14,855 samples from individuals with a suspected PCN subjected to cytogenetic evaluation between 2019 and 2022 with cIg-FISH (n = 6917) or FACS-FISH (n = 7938) testing were analyzed. Results Fluorescence-activated cell sorting–FISH increased the detection rate of abnormalities in comparison with cIg-FISH, with abnormal results documented in 54% vs 50% of cases, respectively (P < .001). It improved the detection of IGH::CCND1 (P < .001), IGH::MAF (P < .001), IGH::MAFB (P < .001), other IGH rearrangements (P < .001), and gains/amplifications of 1q (P < .001), whereas the detection rates of IGH::FGFR3 fusions (P = .3), loss of 17p (P = .3), and other abnormalities, including hyperdiploidy (P = .5), were similar. Insufficient PC yield for FISH analysis was decreased between cIg-FISH and FACS-FISH (22% and 3% respectively, P < .001). Flow cytometry allowed establishment of ploidy status in 91% of cases. In addition, FACS-FISH decreased analysis times, workload efforts, and operating costs. Conclusions Fluorescence-activated cell sorting–FISH is an efficient PC purification strategy that affords significant improvement in diagnostic yield and decreases workflow requirements in comparison with cIg-FISH.

Abstract Objectives Patients with clonal cytopenia of undetermined significance (CCUS) are at increased risk of developing myeloid neoplasia (MN). We evaluated whether a simple flow cytometry immunophenotyping (FCIP) assay could differentiate the risk of development of MN in patients with CCUS. Methods Bone marrow aspirates were assessed by FCIP panel in a cohort of 80 patients identified as having CCUS based on next-generation sequencing or cytogenetics from March 2015 to May 2020, with available samples. Flow cytometric assay included CD13/HLA-DR expression pattern on CD34-positive myeloblasts; CD13/CD16 pattern on maturing granulocytic precursors; and aberrant expression of CD2, CD7, or CD56 on CD34-positive myeloblasts. Relevant demographic, comorbidity, and clinical and laboratory data, including the type and extent of genetic abnormalities, were extracted from the electronic health record. Results In total, 17 (21%) patients with CCUS developed MN over the follow-up period (median survival follow-up, 28 months [95% confidence interval, 19-31]). Flow cytometry immunophenotyping abnormalities, including the aberrant pattern of CD13/HLA-DR expression, as detected at the time of the diagnosis of CCUS, were significantly associated with risk of developing MN (hazard ratio, 2.97; P = .006). Additional FCIP parameters associated with the development of MN included abnormal expression of CD7 on myeloblasts and the presence vs absence of any FCIP abnormality. Conclusions A simple FCIP approach that includes assessment of CD13/HLA-DR pattern on CD34-positive myeloblasts can be useful in identifying patients with CCUS at higher risk of developing MN.

Abstract Background: Chronic myelomonocytic leukemia (CMML) is an aging disease that arises on the background of clonal hematopoiesis, commonly involving multiple TET2 mutations or TET2/SRSF2 co-mutations. In a prior study of 1084 CMML patients (Leukemia 2020; 34:1407-1421), we demonstrated a high prevalence of TET2 mutations (56%) in CMML, with approximately 30% of patients having >1 TET2 mutation. The cell-type and allele specific distribution of these mutations remains of great interest and are thus far poorly defined. To visualize the genotypic and immunophenotypic correlates, we performed single-cell DNA analysis and simultaneous protein profiling on 2 CMML patients with multiple TET2 mutations. Methods: After Mayo Clinic IRB approval, two CMML patients with dual TET2 mutations and available peripheral blood mononuclear cells were included in the study. Patient samples were labeled with TotalSeq TM -D Heme Oncology Cocktail (BioLegend, San Diego, CA) consisting of 45 unique oligo-conjugated cell surface markers. The samples were then washed and loaded into a Tapestri microfluidics cartridge, where single cells (3,500 cells/µL) were encapsulated, lysed and barcoded using the MissionBio Myeloid custom amplicon panel consisting of 312 amplicons in 45 genes relevant to myeloid disorders (MissionBio, San Francisco, CA). The individual barcoded DNA and protein for each cell was purified, recovered, and labeled with Illumina i5/i7 indices, quanitifed by Agilent Bioanalyzer and pooled for sequencing. The libraries were sequenced via Illumina NovaSeq by the Mayo Genomics Core, and data was recovered and analyzed using MissionBio Tapestri Insights v3.0.2. Results: Two dual TET2 mutated CMML patients were included in the study, both male, ages 79 and 83, and both with CMML-0. Case 1 had TET2 p.G1172Efs*53 (variant allele fraction/VAF-6%) and p.H1732Tfs*13 (VAF-13%) mutations, while case 2 had TET2 p.Y620Ffs* (VAF-85%), p.G898* (VAF-82%), and SRSF2 p.95H (VAF-45%) mutations, respectively. In both patients, TET2 mutations were found to be preferentially distributed in monocytes (CD11b+, CD14+, CD64+, CD13+, CD33+, HLA-DR+), with very few classically defined granulocyte (CD16+,CD13+.CD33+, HLA-DR neg), B (CD19+,CD22+)/T (CD2+,CD4+,CD5+,CD7+) lymphocytes, NK cells (CD16+/CD56+/HLA-DR neg) and plasma cells (CD38+/CD138+), demonstrating mutant TET2. Among monocytes, TET2 mutations were enriched in classical/M01 (CD11b+, CD14+,CD16-) and intermediate/M02 monocytes and were infrequent in the MO3 fraction, commensurate with monocyte repartitioning seen in CMML. Interestingly, a large number of single cells with TET2 mutations also demonstrated an aberrant surface phenotype, with universal expression of CD14+ and CD11b+, along with a mixture of other markers (CD163+, CD11c, CD4+, CD2+, and/or CD56+). With regards to allelic distribution, while several permutations and combination were seen involving heterozygosity and homozygosity for the TET2 mutations identified on bulk sequencing, data was corroborative for both TET2 mutations being located on trans alleles. Conclusions: In this study, we demonstrate that TET2 mutations in CMML are highly enriched in classical circulating monocytes and infrequently occur in classical granulocytes, B/T and NK cells; with multiple TET2 mutations most commonly co-existing on trans alleles. Analyses on additional samples along with copy number variation assessments are ongoing. Figure 1: Lollipop plot of TET2 exons with dual TET2 mutations labeled above and oxygenase domain labeled in green; below are number of cells identified with each genotype for Case 1 (A) and Case 2 (B). Mutations are labeled proximal (yellow) and distal (red) and alleles are in grey. The directionality of the arrows to different genotypes is based on decreasing cell number and genotype. Uniform manifold approximation and projection (UMAP) plot of Case 1 (C) and Case 2 (D) with cells clustered by immunophenotype and labeled by genotype (non TET-2 mutated purple, heterozygous TET2 in green, homozygous TET2 in yellow). Relative protein expression of T-cell phenotype (CD14-, CD3+, CD4+/-,CD8+/-, CD5+, CD7+, CD2+), or classical monocytes (CD14+/CD16-) for Case 1 (E & F) and Case 2 (G & H) overlayed on UMAP. Royal blue represents non TET2-mutated cells, and mutated cells are labeled in pastel blue, green and light orange by mutation type. Figure 1 Figure 1. Disclosures Durruthy-Durruthy: Mission Bio Inc.: Current Employment. Arribas-Layton: Mission Bio, Inc.: Current Employment. Patnaik: StemLine: Research Funding; Kura Oncology: Research Funding.

AbstractBackgroundDespite the unprecedented success of ibrutinib in lymphoma therapy, the development of ibrutinib resistance due to acquired BTK or PLCγ2 mutations has become a new clinical problem. However, not all resistance is mediated by these mutations and these mechanisms are poorly understood due to a lack of study tools that truly recapitulate this clinical scenario.MethodsWe established a novel patient‐derived ibrutinib‐resistant mantle cell lymphoma (MCL) line named MCIR1. Using immunological, molecular, and cytogenetic approaches, we comprehensively characterized MCIR1 and further demonstrated its utility in the study of resistance mechanisms and treatments to overcome this resistance.ResultsWe show that MCIR1 is a bona fide ibrutinib‐resistant MCL cell line with normal BTK‐/PLCγ2 but ibrutinib‐resistant ERK1/2 and AKT1 signaling. RNA‐Seq analysis revealed a robust non‐canonical NF‐kB signaling that drives the ibrutinib resistance. We also demonstrate the potential utility of a MCIR1‐based cell and mouse model for the discovery of new treatments to overcome BTK inhibitor resistance.ConclusionsWe have established the first patient‐derived ibrutinib‐resistant MCL cell line MCIR1 that lacks BTK or PLCγ2 mutations but exhibits a hyperactive non‐canonical NF‐kB pathway. We further demonstrate its utility in the discovery and validation of new drugs to overcome this resistance.

Background: Clonal cytopenias of Undetermined Significance (CCUS) have a higher risk of progression to myeloid neoplasm (MN) compared to idiopathic cytopenias of undetermined significance (ICUS) (Malcovati et al., Blood, 2017). The implementation of flow cytometric (FC) immune-phenotyping and next generation sequencing (NGS) in clinical practice has improved the diagnosis of these entities but the clinical significance and interaction of these abnormal results are still unknown. In this study we investigated predictive factors that play role in survival and progression to MN in patients with ICUS/CCUS. Methods: Patients (Pts) who had undergone evaluation for unexplained cytopenia and had undergone FC and bone marrow morphological assessment between 3/2015-3/2020 at Mayo Clinic Rochester were included. Pts were excluded if a malignant hematological disorder was diagnosed prior to the time of FC. FC included evaluation of the following parameters: Abnormal expression of HLA-DR/CD13, CD2, CD7, CD45, and CD56 on blasts; and abnormal CD13/CD16 expression and side scatter on granulocytic cells. NGS panel up to 2018 included 34 genes, ASXL1, BCOR, BRAF, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, MYD88, NOTCH1, NPM1, NRAS, PHF6, PTPN11, RUNX1, SETBP1, SF3B1, TERT, TET2, TP53, U2AF1, WT1, and ZRSR2; in 2018, BRAF was omitted and 12 genes were added, ANRD26, DDX41, ELANE, ETNK1, KDM6A, MYD88, NOTCH1, RAD21, SH2B3, SRP72, SMC3, and STAG2. Results: Characteristics: Of 490 consecutive pts, 238 (median age 69 years (range 19-92), 66% males) met our inclusion criteria as ICUS. 86 (36%) pts had CCUS (abnormal cytogenetics and/or tested positive for pathogenic mutations on NGS). After a median follow-up of 28 months (95% CI: 20 to 31 months), 21 (25%) patients developed MN and 23 (27%) died during follow-up. Comparing ICUS vs CCUS: upon comparing CCUS pts to ICUS, several factors were found to be significantly different: CCUS pts were older (p= .02); majority male (p= .04), had more abnormal HLA-DR/CD13 (p <.0001), more side-scattered light by FC (p <.0001), more pancytopenia on follow up (p= .02) and more morphologic atypia (<.0001). Overall survival (OS) outcomes in CCUS: Several covariates were significant in univariate models, and model selection was used to generate a risk score based on abnormal CD7, abnormal CD13/CD16, abnormal HLA-DR/CD13, splenomegaly and history of prior chemotherapy or radiotherapy (range: 0 - 4; HR=2.58, [95 CI: 1.69 - 3.94], p<.0001). Risk scores were grouped as low risk (score=0), intermediate risk (score=1), and high risk (score=2+), with estimated 2-yr-OS of 95%, 84%, 40%, respectively (Figure 1). An extended risk score model including NGS factors added ASXL1 and IDH1 mutation status to the previous model (range: 0 - 6; HR=2.72, [95 CI 1.82 - 4.06], p<0.0001). MN-free survival (MNFS) in CCUS: 37 pts either progressed to MN and/or died with a median follow up time for MNFS of 22.4 months. Similar for OS, model selection approaches yielded a composite risk score: splenomegaly, BCOR mutation status, mutation in RAS signaling pathway, abnormal expression of flow markers CD13/CD16, HLA-DR/CD13 or CD7 (HR=3.23, [95 CI: 1.90 - 5.49], p=<0.0001). Risk scores were grouped as low risk (score=0), intermediate risk (score=1), and high risk (score ≥ 2), with estimated 1-yr MNFS of 84%, 74%, and 31%, respectively (Figure 2). Conclusion: CCUS has unique features compared to ICUS. Several factors, including clinical characteristics, immune-phenotyping by FC, and somatic mutations have important impact on risk of progression to MN and on overall survival. Our findings serve as proof-of-concept that such integrated models could help identify CCUS patients at higher risks for progression to MN or death. They can guide treatment accordingly and should be evaluated further. Disclosures Shah: Dren Bio: Consultancy.

Key Points Systemic immune microenvironment signatures in CMML indicate an altered T- and natural killer cell balance. CMML bone marrow dendritic cell aggregates associate with disease progression and systemic regulatory T-cell phenotypic switch.

Abstract Constitutive activation of B-cell receptor (BCR) signaling is a major driving mechanism for the proliferation and survival of various B-cell lineage non-Hodgkin lymphomas (NHL). Blocking BCR signaling using the first-in-class Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, has proved effective and is currently FDA approved for the treatment of several B-cell lymphomas. Unfortunately, significant subsets of patients possess either primary or acquired resistance to ibrutinib, often resulting in worse prognosis and poorer responses to subsequent therapies. Therefore, overcoming ibrutinib resistance is an urgent clinical need requiring new tools to both delineate the molecular mechanisms of resistance and develop viable therapeutics. To that end, we developed a mantle cell lymphoma (MCL) cell line, MCIR1, from a patient with clinically acquired ibrutinib resistance. MCIR1 is a bona fide MCL cell line featuring CD19+/CD5+/CD10-/CD23-/t(11;14)+/TP53+/-; and lacking the previously described mutations in BTK (C481S) and phospholipase C-gamma-2 (R665W, L845F). Importantly, MCIR1 cells possess hallmarks of ibrutinib resistance, including the insensitivity of ERK1/2 phosphorylation to ibrutinib in vitro, and the irresponsiveness of MCIR1 xenograft tumors to ibrutinib treatment in mice as compared with ibrutinib-sensitive Jeko-1 tumors. To delineate the molecular mechanism(s) of ibrutinib resistance, we identified by RNA-Seq analysis that MCIR1 cells possess robust activity of the non-canonical NFkB pathway and elevated expression of NFkB target genes, suggesting that this pathway likely drives their resistance to ibrutinib. Finally, we demonstrated the utility of MCIR1 as a tool for testing new drugs to combat ibrutinib resistance in vitro by showing its sensitivity to the anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein inhibitor, venetoclax. To our knowledge, MCIR1 is the first patient-derived ibrutinib-resistant cell line to be established. Hence, MCIR1 is especially useful because uniquely possesses the relevant clinical and biological features of patients with ibrutinib-resistant MCL. We propose MCIR1 as a tool to further the understanding of molecular mechanisms conferring ibrutinib resistance by providing a clinically relevant system with the capacity for molecular and genetic manipulations. Further, these features make MCIR1 an ideal model to screen drugs for combating ibrutinib resistance through both in vivo and in vitro approaches. Citation Format: Kevin E. Nowakowski, Jithma P. Abeykoon, Mary J. Stenson, Michael M. Timm, Curtis A. Hanson, Daniel L. Van Dyke, Anne J. Novak, Xiaosheng Wu, Thomas E. Witzig. MCIR1: A patient-derived ibrutinib-resistant mantle cell lymphoma line for the study of ibrutinib resistance and drug discovery [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 650.

Abstract Objectives Distinguishing between T-cell and natural killer (NK)–cell neoplasms could be difficult given their overlapping immunophenotype. In this study, we investigated whether a flow cytometry assay with cytoplasmic staining for CD3 could be used for this purpose. Methods Flow cytometry immunophenotyping was performed on 19 surface CD3 (sCD3)–negative mature T-cell neoplasms, 10 sCD3-positive mature T-cell neoplasms, 13 mature NK-cell neoplasms, and 19 normal controls. In addition to routine antibody panels (CD2, sCD3, CD4, CD5, CD7, CD8, CD16, CD45, CD56, CD57, CD94, CD158a, CD158b, CD158e, NKG2A TCRγ/δ), cytoplasmic staining for a monoclonal CD3 antibody (clone SK7/Leu-4) was assessed in all cases. A molecular study for T-cell receptor (TCR) gene rearrangement and an immunohistochemical study for TCRβ were performed. Results Our data showed all T-cell neoplasms were uniformly positive for cytoplasmic CD3 (cCD3) regardless of sCD3 expression, whereas 85% of NK-cell neoplasms completely lacked cCD3 expression. The 2 cases with classic NK-cell immunophenotype but partial cCD3 expression showed no molecular genetic features of T-cell lineage by TCR gene rearrangement studies. Conclusions Uniform cCD3 positivity and homogeneous cCD3 negativity highly suggest T-cell and NK lineage, respectively. When partial cCD3 expression is encountered, additional confirmatory studies should be pursued for the most accurate lineage assignment.

Abstract [Background]: MCL is an aggressive non-Hodgkin lymphoma (NHL) with a median survival of 3-4 years. New agents with mechanisms of action different than standard chemotherapy are needed to improve the outcome of MCL. Recent studies have documented single agent activity of bortezomib and rituximab in MCL. Analogs of rapamycin (temsirolimus) have also shown single agent activity in MCL (J Clin Oncol, June 27, 2005 epub). In order to provide rationale for a clinical trial combining rapamycin analogs with bortezomib or rituximab, we performed in vitro studies of the effect of combinations of these drugs on human MCL cell lines. [Methods]: Two human lymphoma cell lines - Granta 519 and M0258 - were incubated with various concentrations of rapamycin, bortezomib, and rituximab to determine the LD50 of each drug alone. For the experiments with rituximab a secondary goat anti-human (GAH) IgG Fc antibody was used to crosslink cell bound rituximab and induce cell killing. Percent viability was assessed after 48 hours using annexin/propidium iodide staining and flow cytometry. Tests for drug synergy used combinations of rapamycin and bortezomib and the Granta 519 cell line. Fixed ratios of the two drugs were tested and the results were evaluated for synergism (combination index <1 in median effect analysis) in inducing cell death. The drug concentrations used spanned the LD50 of each drug determined from the single-agent studies. In the studies combining rituximab with bortezomib or rapamycin, a single fixed dose of rituximab and GAH Fc was tested with various concentrations of the other drugs to detect additive anti-tumor effects. Studies were performed at least in triplicate. [Results]: All three drugs exhibited single-agent activity in vitro against the two cell lines. The LD50 for rapamycin ranged from 17.5 – 25 micromolar for the Granta 519 cell line and 7.5–10 micromolar for M0258. The LD50 for bortezomib ranged from 2–5 ng/ml in both cell lines. Incubation of the cells with various concentrations of rituximab and GAH Fc had mild cytotoxic activity (reduced viability to 50–70% compared to GAH Fc control). This activity was not concentration dependent indicating saturation of CD20 by rituximab; therefore, a fixed concentration of rituximab was used for the combination experiments. When rituximab was added to bortezemib the % viability decreased by up to 37% (compared to bortezomib alone), and the bortezomib LD50 typically decreased to <1–2 ng/ml (LD50 was 2–5 ng/ml with bortezomib alone). The addition of rapamycin to rituximab increased the cytotoxicity by 23% compared to either drug alone and decreased the rapamycin LD50. When rapamycin and bortezomib were combined as treatment of Granta 519 cells, rapamycin was synergistic with bortezomib with a combination index <1. Sequencing the rapamycin either before or after the bortezomib treatment did not effect synergy in either direction. [Conclusion]: In vitro, rapamycin synergizes with bortezomib to enhance its cytotoxicity in MCL lines. Rituximab enhances the cytotoxicity of both of rapamycin and bortezomib. These in vitro studies provide the rationale to test combinations of these active agents in patients with MCL.