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Background Clonal cytopenia of undetermined significance (CCUS) is a newly described entity with a high probability to progress into myeloid disorders upon follow-up (Malcovati et al, Blood 2017). Little data is known on how to effectively treat patients with CCUS. We hereby describe a single institution experience in managing symptomatic CCUS patients. Methods Patients who had CCUS and underwent active treatment were identified after receiving IRB approval. CCUS diagnosis was rendered if patients had non-diagnostic bone marrow biopsy evaluation combined with evidence of pathogenic myeloid somatic mutation using our institution next generation sequencing (NGS) panel (OncoHeme, Mayo Clinic). "Symptomatic patients" is determined by patients who need therapy beyond blood transfusion. Treatment type is based on physicians' choice. Clinical and lab results were collected. Response and progression were graded based on the MDS IWG 2006 criteria. The date of treatment initiation was used for overall survival (OS) and progression (either to myeloid neoplasm or worsening cytopenias) free survival (PFS) calculation. Stata 14.1(StatCorp) was used for data analysis. Results 1) Cohort characteristics: Between 2015 and 2020, 24 patients met inclusion criteria with median age of 72 years (range: 24-87). Twenty (83%) were men. 15 (63%) were white, 1 (4%) African American, 1 (4%) Native American and 7 unknown (33%). Eighty percent had ECOG PS≤1. Four (17%) patients had prior other hematology disorders and 3 (13%) had solid tumor. Two (8%) patients had previous radiation therapy, 2 (8%) received chemotherapy and 1 (4%) received allogeneic stem cell transplantation. At the time of treatment, the median of hemoglobin was 8.9 (range 6.8-13.7) g/dL, white blood cell 2.9 (range 1.4-12.3) *109/L, and platelet 76 (range 8-407) *109/L. Red blood cell (RBC) and platelet transfusion dependency rate were 54% and 29%, respectively. We identified 23 different mutations. Most common mutations occurred in SRSF2 (N=5, 21%), TET2 (N=5, 21%), U2AF1 (N=5, 21%), and TP53 (N=4, 17%). Ten (42%) patients had 1 mutation, and 14 (58%) had ≥2 mutations. Treatment included hypomethylating agents (HMA) (N=10, 42%), growth factors (N=11, 46%), steroid (N=4, 17%), testosterone (N=2, 8%), cyclosporine (N=2, 8%), rituximab (N=1, 4%), immunoglobulin (N=1, 4%), vitamin B12 and iron (N=1, 4%). Seven (29%) patients received ≥2 treatments. Median follow up duration was 17.5 months (range 8.9-26.1). Median time from the diagnosis to treatment was 2.1 months (range 0-26.8). 2) Molecular correlation with response The overall response rate (RR) was 50% (hematological or symptomatic improvement). Most responders (67%) were treated with HMA (p=0.013). HMA treatment effect was not significantly associated with DNA methylation genes (DNMT3A, IDH1, IDH2 and TET2, all p>0.05). The RR was 100% for the three patients with IDH1 mutation (treated with HMA, steroid and erythropoietin stimulating agent (ESA), respectively). The RR was 100% for the 3 patients with ZRSR2 mutation (2 HMA, 1 testosterone). The RR was 0% for 3 patients with SF3B1 mutation (1 testosterone and ESA, 1 vitamin B12 and iron, and 1 HMA). The number of mutations had no impact on RR (p=0.41). Table demonstrates the RR for each treatment. 3) Survival and progression outcome At the last day of follow up, RBC and platelet transfusion dependency rate were 38% and 25%, respectively. The median PFS was 16.9 months (95% CI: 7.5-65.3). Six patients (25%) progressed to myeloid malignancy with 4 myelodysplastic syndrome (MDS) and 2 acute myeloid leukemia (AML). Five patients progressed with worsening cytopenias. Among the 4 MDS patients, they had SF3B1, TET2, and ASXL1 mutations at the diagnosis of CCUS. Within the 2 AML pts, 1 had RUNX1 mutation, 1 had BCOR, DNMT3A, PHF6, RUNX1, SF3B1, STAG2, and TET2 mutations at the diagnosis of CCUS. The median OS was not reached with an estimated 2 year OS 77%. Three (13%) patients progressed with cytopenia and died. Their NGS at CCUS diagnosis showed ASXL1, TP53 and U2AF1 mutations, respectively. Conclusion Patients with CCUS responded to available treatments used for myeloid diseases. HMA were effective in this cohort. Mutation profile may have an impact on treatment response. Studies of larger cohorts are needed to validate our findings. Additionally, development of response and progression criteria would be helpful for such patients for reporting treatment outcomes. Disclosures Shah: Dren Bio: Consultancy.

Abstract BackgroundOverexpression of c-Myc is required for the progression of pre-malignant plasma cells in monoclonal gammopathy of undetermined significance (MGUS) to malignant plasma cells in multiple myeloma (MM). c-Myc also increases glutamine anaplerosis into the tricarboxylic acid (TCA) cycle within cancer cells. Whether increased glutamine anaplerosis is associated with the progression of pre-malignant to malignant plasma cells is unknown.MethodsHuman volunteers (N = 7) and patients with MGUS (N = 11) and MM (N = 12) were prospectively recruited to undergo an intravenous infusion of 13C-labelled glutamine followed by a bone marrow aspiration to obtain bone marrow cells and plasma.ResultsDespite notable heterogeneity, stable isotope resolved metabolomics (SIRM) revealed that the mean 13C-labelled glutamine anaplerosis into the TCA cycle was higher in malignant compared to pre-malignant bone marrow plasma cells relative to the remainder of their paired bone marrow mononuclear cells. RNA sequencing demonstrated a higher relative mRNA expression of c-Myc and glutamine transporters such as ASCT2 and SN2 in malignant compared to pre-malignant bone marrow plasma cells. Finally, higher quantitative levels of TCA cycle intermediates in the bone marrow plasma differentiated MM from MGUS patients.ConclusionMeasurement of the in vivo activity of glutamine anaplerosis into the TCA cycle provides novel insight into the metabolic changes associated with the transformation of pre-malignant plasma cells in MGUS to malignant plasma cells in MM.

AbstractAcute undifferentiated leukemia (AUL) is a very rare hematologic neoplasm that expresses no markers specific for either myeloid or lymphoid lineages. While commonly observed in several acute leukemias, KMT2A rearrangements in AUL have been rarely reported in the literature. We report the third case to our knowledge of AUL harboring a KMT2A rearrangement. Furthermore, the KMT2A/GIMAP8 gene fusion identified in this case represents a novel KMT2A rearrangement.

Abstract Objectives The diagnosis of T-cell large granular lymphocytic leukemia (T-LGLL) is challenging because of overlapping immunophenotypic features with reactive T cells and limitations of T-cell clonality assays. We studied whether adding an antibody against T-cell receptor β constant region 1 (TRBC1) to a comprehensive flow cytometry panel could facilitate the diagnosis of T-LGLL. Methods We added TRBC1 antibody to the standard T-cell and natural killer (NK) cell panel to assess T-cell clonality in 56 T-LGLLs and 34 reactive lymphocytoses. In addition, 20 chronic lymphoproliferative disorder of NK cells (CLPD-NKs) and 10 reactive NK-cell lymphocytoses were analyzed. Results Clonal T cells were detected in all available T-LGLLs by monotypic TRBC1 expression and clonal/equivocal T-cell receptor gene rearrangement (TCGR) studies, compared with only 27% of T-LGLLs by killer-cell immunoglobulin-like receptor (KIR) restriction. Overall, 85% of T-LGLLs had a blood tumor burden greater than 500 cells/µL. Thirty-four reactive cases showed polytypic TRBC1 expression, except for 5 that revealed small T-cell clones of uncertain significance. All CLPD-NKs showed expected clonal KIR expression and negative TRBC1 expression. Conclusions Addition of TRBC1 antibody to the routine flow cytometry assay could replace the TCGR molecular study and KIR flow cytometric analysis to assess clonality, simplifying the diagnosis of T-LGLL.

e20510 Background: pPCL is a rare and aggressive form of multiple myeloma (MM) with dismal survival outcomes compared to the remainder of MM patients. Several studies have validated the optimal cutoff for defining pPCL to be >5% circulating plasma cells (cPCs) on a peripheral blood smear due to equally poor outcomes in this less restrictive cohort. We evaluated the clinical outcomes and cytogenetic features of patients diagnosed with pPCL at our institution that were treated with novel agent induction therapies. Methods: We evaluated patients with pPCL diagnosed between 2000 – 2018 (using the 5% cut off) at Mayo Clinic, Minnesota. Data was extracted from a prospectively maintained database and from the review of electronic medical records. Patients were categorized as having high risk (HR) cytogenetics if any of the following abnormalities were present: del 17p, t(4;14), t(14;16) or t(14;20). Chi-square tests and Fisher exact tests were used to compare differences between sub-groups. Survival analysis was performed by the Kaplan-Meier method and differences assessed using the log rank test. Results: This cohort consisted of 67 patients with pPCL with a median age of 62 years (range: 34-91) of which 33 (46%) were male. The median follow up was 46 months (95% CI: 41 – 90). The median bone marrow plasma cell involvement was 84% (Range: 10 – 100) and the median cPCs percentage on the peripheral blood was 23% (range: 5 - 93). Data on primary cytogenetic abnormalities were available in 60 (85%) patients and the distribution was as follows: t(11;14) – 27 (45%), t(4;14) – 5 (8%), t(14;16) – 8 (13%), t(14;20)- 3(5%) and del 17p- 15(25%). All patients received novel agent induction therapy with 36 (54%) having received autologous stem cell transplant and 4 undergoing an allogeneic stem cell transplant. The median time to next therapy (TTNT) and overall survival (OS) for all patients was 13 months (95% CI: 9 – 17) and 24 months (95% CI: 19 – 40) respectively; 16 months and 51 months for standard risk (SR) vs. 10 months and 19 months for HR (P = 0.005 for OS), when stratified by cytogenetic risk. There were only 17 (31%) patients who were alive for ≥48 months since diagnosis (i.e. twice the median OS of this cohort); The absence of high risk cytogenetics (P = 0.049) and non-elevated LDH level (P = 0.047) at diagnosis predicted for achieving this survival goal. Conclusions: The outcomes of patients diagnosed with pPCL remain poor despite the use of novel agent induction therapy. However, some patients appear to do better than expected and this phenomenon may be influenced by the presence of HR cytogenetics.

AbstractMetaphase cytogenetic abnormalities, plasma cell proliferation index (PCPro), and gain 1q by fluorescence in situ hybridization (FISH) are associated with inferior survival in newly diagnosed multiple myeloma (MM) treated with novel agents; however, their role in risk stratification is unclear in the era of the revised International Staging System (R-ISS). The objective of this study was to determine if these predictors improve risk stratification in newly diagnosed MM when accounting for R-ISS and age. We studied a retrospective cohort of 483 patients with newly diagnosed MM treated with proteasome inhibitors and/or immunomodulators. On multivariable analysis, R-ISS, age, metaphase cytogenetic abnormalities (both in aggregate and for specific abnormalities), PCPro, and FISH gain 1q were associated with inferior progression-free (PFS) and overall survival (OS). We devised a risk scoring system based on hazard ratios from multivariable analyses and assigned patients to low-, intermediate-, and high-risk groups based on their cumulative scores. The addition of metaphase cytogenetic abnormalities, PCPro, and FISH gain 1q to a risk scoring system accounting for R-ISS and age did not improve risk discrimination of Kaplan-Meier estimates for PFS or OS. Moreover, they did not improve prognostic performance when evaluated by Uno’s censoring-adjusted C-statistic. Lastly, we performed a paired analysis of metaphase cytogenetic and interphase FISH abnormalities, which revealed the former to be insensitive for the detection of prognostic chromosomal abnormalities. Ultimately, metaphase cytogenetics lack sensitivity for important chromosomal aberrations and, along with PCPro and FISH gain 1q, do not improve risk stratification in MM when accounting for R-ISS and age.

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 Flow cytometric detection of T‐cell clonality is challenging. The current available methodology for T‐cell receptor (TCR) Vβ repertoire evaluation is a complex assay and has limited sensitivity especially for detecting low levels of disease. Therefore, there is an unmet need for a reliable, simple, and rapid assay to identify T‐cell clonality. The rearrangement of the TCRB gene involves the random and mutually exclusive expression of one of two constant β chain genes (TRBC1 and TRBC2), analogous to the kappa and lambda gene utilization by B cells. Methods Here, we used a single TRBC1 antibody, in conjunction with other T‐cell associated markers, to detect T‐cell clonality in tissue biopsies and body fluids. A total of 143 tissue/body fluid specimens from 46 patients with a definitive diagnosis of a T‐cell neoplasm and 97 patients with no T‐cell malignancy were analyzed with a cocktail of monoclonal antibodies including CD2/CD3/CD4/CD5/CD7/CD8/CD45/TCRγδ/TRBC1. Results We examined TRBC1 expression on neoplastic T‐cell populations identified based on their immunophenotypic aberrancies, and monotypic TRBC1 expression was identified in all 46 known T‐cell lymphoma cases. We applied a similar gating strategy to the 97 cases without T‐cell neoplasms, and arbitrarily dissected T‐cell populations into immunophenotypically distinct subsets; in this group, we found that all cases revealed an expected polytypic TRBC1 expression in all subsets. Conclusions Single TRBC1 antibody detection of T‐cell clonality by flow cytometry is a simple, rapid, and robust assay that could be routinely utilized in flow cytometric laboratories.

Abstract INTRODUCTION: Adult mature T- and NK-cell neoplasms are a heterogeneous group of aggressive lymphomas comprising approximately 10% to 20% of all non-Hodgkin lymphomas (NHL) with an estimated 5-year overall survival of 40%. Staging and treatment strategies are guided by malignant involvement on PET/CT scan and bone marrow (BM) biopsy. The Lugano Criteria, established in 2014, suggested that focal FDG uptake within the bone marrow was highly sensitive for both Hodgkin (HL) and diffuse large B-cell lymphoma (DLBCL) potentially obviating the need for a bone marrow biopsy if no lesions were seen. Limited data exist regarding the sensitivity of PET for BM involvement in T-cell lymphoma. This study compares PET/CT scans and repeat BM biopsy in patients who have undergone treatment for peripheral T-cell lymphomas (PTCL) by evaluating BM avidity on PET scans. METHODS: This is a single institution study using the Mayo Clinic Lymphoma Database between January 1, 2001 and January 1, 2015. We retrospectively identified all patients with a diagnosis of PTCL, biopsy proven BM involvement at diagnosis, and a concomitant PET for staging. Patients were then reviewed to assess completion of induction therapy and availability of both PET/CT and bone marrow results post-therapy. Evaluation for concordance and discordant BM involvement were then determined using BM biopsy as the gold standard. RESULTS: Sixteen patients had both PET/CT and BM biopsy after completing induction therapy. Median age at diagnosis was 63 years (range 34-72) and 69% were male. PTCL subtype was peripheral T-cell lymphoma, not otherwise specified in seven patients; ALK negative anaplastic large cell lymphoma in one patient; and angioimmunoblastic in 8 patients. Pre-treatment PET/CT scans demonstrated eight patients (50%) with false negative scans. Post-treatment biopsy results demonstrated that ten (62.5%) had biopsy proven residual bone marrow involvement after induction. Eight patients (50%) were found to have BM biopsy proven disease with a negative PET scan. Two patients (12.5%) had both positive BM biopsy and PET scans; 5 patients (31.3%) had negative BM biopsies and PET scans. One patient (6.25%) had a negative BM biopsy, but had a PET scan that revealed positive disease. This patient was considered to have had a false positive PET scan and indeed has remained in remission since April 2009 without any further relapse or treatment. Sensitivity of PET for BM involvement was very poor at 20% (2/10) with a specificity of 50% (2/4) (Table 1 and 2). CONCLUSIONS: This study in PTCL indicates that PET scans at the completion of therapy have a 50% false negative rate. These patients should not be assumed to have negative bone marrow involvement based solely on PET/CT scans. A bone marrow biopsy at the end of therapy is necessary in PTCL patients to confirm complete response. Table 1. Bone Marrow Involvement Pre-Therapy PET Scan Imaging PET Negative PET Positive Total Patients Bone Marrow Negative NA NA NA Bone Marrow Positive 8 8 16 Total Patients 8 (50%) 8 (50%) 16 Abbreviations: NA, not applicable. The study defined that all patients had to have a positive bone marrow at baseline to be eligible for the study. Table 2. Bone Marrow Involvement PET Negative PET Positive Total Patients Bone Marrow Negative 4 2 6 (37.5%) Bone Marrow Positive 8 2 10 (62.5%) Total Patients 12 (75%) 4 (25%) 16 Table 1 and 2. Involvement of bone marrow with peripheral T-cell lymphoma based on evaluation by PET scans versus bone marrow biopsy. Disclosures Maurer: Kite Pharma: Research Funding.