When does a PNH clone have clinical significance?

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired blood disease caused by somatic mutations in the phosphatidylinositol glycan class A (PIGA) gene required to produce glycophosphatidyl inositol (GPI) anchors. Although PNH cells are readily identified by flow cytometry due to their deficiency of GPI-anchored proteins, the assessment of the clinical significance of a PNH clone is more nuanced. The interpretation of results requires an understanding of PNH pathogenesis and its relationship to immune-mediated bone marrow failure. Only about one-third of patients with PNH clones have classical PNH disease with overt hemolysis, its associated symptoms, and the highly prothrombotic state characteristic of PNH. Patients with classical PNH benefit the most from complement inhibitors. In contrast, two-thirds of PNH clones occur in patients whose clinical presentation is that of bone marrow failure with few, if any, PNH-related symptoms. The clinical presentations are closely associated with PNH clone size. Although exceptions occur, bone marrow failure patients usually have smaller, subclinical PNH clones. This review addresses the common scenarios that arise in evaluating the clinical significance of PNH clones and provides practical guidelines for approaching a patient with a positive PNH result.

Total Oral Anticoagulation for Classic PNH As Primary Prophylaxis Strategy in a Complement Inhibitor Restricted Scenario: A Retrospective Analysis

Introduction. Paroxysmal nocturnal hemoglobinuria (PNH) is associated with thrombosis and bone marrow failure. Thrombosis is the leading cause of death and morbidity, with 29-44% of patients presenting at least one thrombotic episode during their disease's natural course. Treatment with complement inhibitors is a strategy associated with reducing up to 80% of thrombosis risk, and improving survival of PNH patients. However, the high cost of these therapies limited their access in Low and Middle-Income Countries (LMIC). Mexico, like other LMIC, experience low access to complement inhibitors. Hence, we established a primary thromboprophylaxis strategy in classic PNH. The primary objective of the present study is to analyze thrombosis-free survival and all-cause mortality in patients with classic PNH subjected to this prophylaxis strategy compared to historical controls. Methods. Single-center, retrospective, case-control study. We included patients treated from 1991 to 2021, diagnosed with classic PNH (clone size ≥50% determined by flow cytometry). We defined the treatment group as patients who received total oral anticoagulation as primary prophylaxis. We excluded patients who had presented thrombosis prior to classic PNH diagnosis, sub-clinic PNH, bone marrow failure, platelets <50x10 3/µ, and patients who failed to maintain a therapeutic (2 to 3) INR value three months after starting total oral anticoagulation with vitamin K antagonists (VKA). We collected data regarding clone type (II, III, or mixed), clone size, LDH values (<1.5 vs ≥1.5 x ULN), platelet count, thrombosis occurrence, thrombosis site, bleeding cases and their severity according to the international society on thrombosis and haemostasis (ISTH) classification, and causes of death. Results. We found 44 patients with classic PNH diagnosed from 1990 to 2021. Median age at diagnosis was 39 years (28 -51), 50% were female, median LDH value 1669 U/L (926-2,241), and median clone size 95% (80-99). Clone type III was most prevalent, with 61.36% cases, followed by mixed type II-III with 38.64%; we found no type II clone patients. Median follow-up was 21.03 years. Four patients developed a site of thrombosis; three cases (75%) presented simultaneous venous and arterial thrombosis, and one (25%) venous only. The most common site for thrombosis was the portal vein (n=3), followed by supra-hepatic veins (n=1). A total 19 patients (43.2%) received primary prophylaxis with total oral anticoagulation, 17 (89.5%) used VKA and 2 (10.5%) direct oral anticoagulants. We found no differences regarding age, LDH value, clone size and clone type between the treatment and control groups. There were no cases of thrombosis in the treatment group, while 5 patients (20%) in the control group developed thrombosis at some point (p=0.038, Table 1). Thrombosis-free survival was not reached in the treatment group, vs 12.78 years (IC95%,6.19-19.37) in the control group (p=0.026, Figure 1). Bleeding episodes occurred in 15.8% (n=3) of the treatment group patients, all of them minor according to the ISTH classification. The global survival was 85% at 15 years in the anticoagulation group vs 74% at 15 years in the control group, (p=0.071, figure 2). Mortality was 15.8% (n=3) in the treatment group vs 28.0% (n=7) in the control group, p=0.338. The only cause of death in the treatment group was progression to aplastic anemia. Causes of death in the control group were thrombosis (28.6%, n=2), aplastic anemia (28.6%, n=2), acute leukemia (14.2%, n=1), sepsis (14.2%, n=1) and digestive tract bleeding (14.2%, n=1). Discussion and conclusions. Thrombosis is the most frequent cause of morbidity and mortality in classic PNH patients. In 2003, Hall et al. found that primary prophylaxis with full-dose oral anticoagulation drastically reduced the thrombotic risk in these patients (0 vs. 36.5%, p=0.01). Meanwhile, complement inhibitor has become the mainstay therapy, thromboprophylaxis with oral anticoagulation has virtually stopped in countries where this therapy is accessible. The present study replicated the Hall et al. results, and demonstrated that primary thromboprophylaxis is safe and effective in reducing the risk of thrombosis. In LMIC, where access to complement inhibitors is limited, the use of thromboprophylaxis could abate thrombosis and should be taken in consideration in all patients. Hence, the clonal progression would become the leading cause of death. Figure 1 Figure 1. Disclosures Apodaca Chavez: Abbvie: Speakers Bureau; Asofarma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Rangel-Patiño: Abbvie: Speakers Bureau; Bristol: Consultancy. Demichelis: Jazz: Consultancy; Novartis: Consultancy, Research Funding, Speakers Bureau; ASH: Research Funding; Astellas: Consultancy; Gilead: Consultancy; Abbvie: Consultancy, Speakers Bureau; Bristol/Celgene: Consultancy, Speakers Bureau; AMGEN: Consultancy, Speakers Bureau.

Detection of Clonal Plasma Cells in POEMS Syndrome Using Multiparameter Flow Cytometry

Introduction POEMS syndrome is a rare systemic disorder characterized by various symptoms, including polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy (M-protein), and skin changes, which is based on the presence of monoclonal plasma cells (PC). We previously reported the analysis of the clonal immunoglobulin λ light-chain variable region (IGLV) gene rearrangements in POEMS syndrome using next-generation sequencing (NGS), and the clone identification rate was 36.7% (Kawajiri-Manako C, Am J Hematol. 2018;93(9):1161). EuroFlow-based next-generation flow (EuroFlow-NGF) has also been standardized for detecting minimal residual disease in multiple myeloma (MM). However, multiparameter flow cytometry (MFC) utility in detecting clonal PC of POEMS syndrome remains unknown. Therefore, to clarify the feasibility of detecting the clonality with MFC, we analyzed PC of bone marrow samples in patients with POEMS syndrome by MFC and validated gating strategies of PC in POEMS syndrome. Methods Patients with newly diagnosed or relapsed POEMS syndrome (n=25) at Chiba University Hospital from April 2019 to July 2021 were included in this study. Primary bone marrow aspirations were performed after obtaining informed consent following the Declaration of Helsinki. The collected bone marrow samples were analyzed by the SRL-Flow protocols (single-tube 8-color: CD138/CD27/CD38/CD56/CD45/CD19/CyIgκ/CyIgλ), which is highly correlated with EuroFlow-NGF (Takamatsu H, Int J Hematol. 2019;109(4): 377). Furthermore, 21 cases for which data files were available from SRL Inc. were reanalyzed by gating PC with lower expression of CD45 regarding the previous study (Dao LN, Blood. 2011;117(24):6438). This study was approved by the ethics committee of Chiba University Graduate School of Medicine. Results Among 25 patients, the median age was 59 (25-77) years, and 13 (52.0%) were men. The types of M-proteins identified by immunofixation electrophoresis (IFE) were IgA-λ in 12 (48.0%), IgG-λ in 9 (36.0%), IgA-κ in 1 (4.0%), IgG-κ in 1 (4.0%), and negative in 2 patients (8.0%), respectively. The median serum VEGF level was 3,240 (747-9,060) pg/ml. SRL-Flow identified the clonal PC in 9 of 25 (36.0%, λ in 7 and κ in 2): 7 of 23 IFE-positive cases (30.4%) and 2 of 2 IFE-negative cases (100%). The median clone size was 0.03 (0.01-3.78)%, smaller than MM. Interestingly, the clone size was not correlated with the level of serum VEGF (r = -0.17). Aberrant expression of clonal PC in POEMS syndrome was similar to MM: CD19-56+ in 5 (55.6%), CD19-56- in 2 (22.2%), CD19dim56- in 1 (11.1%), and CD19-56dim in 1 (11.1%) patient, respectively. No clone was detected in PC with CD19+56-. The clone identification rate by single-tube 8-color SRL-Flow protocols was lower than expected. Therefore, we reanalyzed the data by gating PC with lower expression of CD45 and CD38. In the reanalysis, we gated broadly for CD38 dim-to-bright and CD138 dim-to-bright cells and narrowly for CD38 dim and CD45 negative-to-dim cells. As a result, we identified the clonal PC in 15 of 21 cases (71.4%, λ in 13 and κ in 2): 13 of 19 IFE-positive cases (68.4%) and 2 of 2 IFE-negative cases (100%)). Of the 15 cases in which monoclonal PC were identified by reanalysis, 8 were negative by SRL-Flow. Of these, 6 were identified by selective gating of CD45- and CD38 dim, and 2 were identified by selective gating of CD45- and CD38+. The median clone size was 0.008 (0.001-3.27)%, smaller than that of MM, and the clone size was not correlated with the level of serum VEGF (r = -0.15). The aberrant expression of the clonal PC in POEMS remained similar to MM for CD19 and CD56 expression: CD19-56+ in 3 (20.0%), CD19-56- in 7 (46.7%), CD19-56dim in 4 (26.7%), and CD19dim 56- in 1 (6.7%) patient, respectively. No clone was detected in PC with CD19+56-. Conclusions EuroFlow-based SRL-Flow protocols detected the clonal PC in about one-third of POEMS patients. Aberrant immunophenotype of clonal PC in POEMS syndrome was similar to MM for CD19 and CD56 expression; however, CD45 and CD38 expression of POEMS PC tended to be downregulated. Selective gating of PC with CD38dim and CD45 negative-to-dim detected clonal PC in 71.4% of cases. This novel gating strategy is more accessible and might improve the identification rate of clonal PC in POEMS syndrome. Disclosures Tsukamoto: Daiichi Sankyo: Honoraria. Yokote: Kowa Company, Ltd.: Honoraria, Other: Scholarship; MSD K.K.: Honoraria, Other: Scholarship, Courses endowed by company; Astellas Pharma Inc.: Honoraria, Other: Scholarship; Mitsubishi Tanabe Pharma Corporation: Honoraria, Other: Scholarship; Amgen K.K.: Honoraria; Taisho Pharmaceutical Co., Ltd.: Honoraria, Other: Scholarship, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Honoraria, Other: Scholarship; Janssen Pharmaceutical K.K.: Honoraria; Kao Corporation: Other: Schlarship; Novo Nordisk Pharma Ltd.: Honoraria, Other: Scholarship; Teijin Pharma Limited: Other: Scholarship; Pfizer Japan Inc.: Honoraria, Other: Scholarship; Kyowa Kirin Co., Ltd.: Honoraria; Eli Lilly Japan K.K.: Honoraria, Other: Scholarship; Takeda Pharmaceutical Company Limited: Honoraria, Other: Scholarship; Sanofi K.K.: Honoraria; Ono Pharmaceutical Co., Ltd: Honoraria, Other: Scholarship; AstraZeneca K.K.: Honoraria; Daiichi Sankyo Company, Limited: Honoraria, Other: Scholarship; Novartis Pharma K.K.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria, Other: Scholarship; Shionogi Co., Ltd.: Other: Scholarship; Bayer Yakuhin, Ltd.: Other: Scholarship. Nakaseko: Novartis Pharma KK.: Honoraria. Takamatsu: Adaptive Biotechnologies, Eisai: Honoraria; SRL: Consultancy; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Sakaida: Bristol Myers Squibb: Research Funding; Chugai: Research Funding; Ono: Research Funding; Kyowa Kirin: Research Funding.

FISH and WGS in Newly Diagnosed and Relapsed/Refractory Multiple Myeloma - WGS Will Affect Future Treatment Decisions

Background: FISH is the gold standard for genetic characterization and risk stratification of multiple myeloma (MM) at diagnosis. Retrospective studies have shown the potential of whole genome sequencing (WGS) to gain new insights into MM genetics. With respect to clinical application, we prospectively performed FISH and WGS in parallel in patients (pts) with newly diagnosed MM (NDMM) and relapsed/refractory MM (RRMM) to evaluate advantages and limitations. Patients and methods: From bone marrow (BM) samples of 280 MM pts CD138+ cells were enriched by MACS. In 100 pts (30 NDMM, 70 RRMM) FISH (del(1p32), del(13q), del(17p), t(4;14), t(11;14), t(14;16), t(14;20), t(6;14), 1q gain, MYC rearrangement (MYCr) and trisomies 3, 5, 9) and WGS were performed. WGS: 150bp paired-end sequences were generated on NovaSeq 6000 (Illumina); median coverage 104x. Single nucleotide variants (SNV) were called with Strelka software, copy number variations (CNV) with GATK4 and structural variants (SV) with Manta. Technical artefacts and germline calls were reduced by a tumor/unmatched normal workflow with genomic DNA from a mix of anonymous donors. Variants with global population frequency >0.5% (gnomAD) were excluded. Results: In 100/280 (36%) MM pts WGS could be performed in parallel to FISH in this cohort with a low median BM plasma cell infiltration (PCI) of 6% (range 1-95%). Likelihood of DNA yields sufficient for WGS increased with higher PCI (23% (1-95%) vs 3% (1-50%) in 100 pts with WGS vs 180 pts without sufficient DNA). Comparison of FISH and WGS results for 100 pts showed 100% concordance for recurrent SV (table 1): t(11;14) in 29%, t(4;14) in 17% and t(14;16) in 5%. No pts harbored a t(14;20) but in one case FISH indicated another alteration of MAFB and WGS revealed MAFB rearranged to FAM46C resulting in an elevated MAFB expression. Due to heterogeneous breakpoints FISH is less reliable for the detection of MYCr than WGS (24/42 (57%) detected by FISH vs 40/42 (95%) by WGS). Partners of MYC included IGH (n=10), FAM46C (n=4), IGK (n=3), IGL (n=3), FOXO3 (n=2) and IRF4 (n=1). Rare IGH rearrangements with partner genes such as IRF1, IRF4, ZBTB38 and ZFP36L1 were observed in 11 pts all identified by WGS, but only 8 (73%) were detected by FISH. Total frequencies of CNV were del(13q) in 50%, del(17p) in 29%, del(1p32) in 22%, 1q gains in 61% and hyperdiploidy in 36%. WGS detected 95% of CNV (details table 1). CNV that were missed by WGS included one del(17p) and were all identified in small subclones with a median clone size of 10% (6-25%) in FISH. Tetraploid MM, potentially harboring adverse prognostic implication, was identified in 11 pts by FISH but not in WGS. However, in 97 pts WGS depicted additional SV and CNV compared to FISH. This includes aberrations of prognostic significance such as a TP53 deletion that was located adjacent to a 451kb gain on 17p. The FISH probe encompassing TP53 confirmed the 17p gain but masked the TP53 deletion. Also 1p deletions, which have been described to be a negative prognostic factor, were found more frequently by WGS with 24 pts harboring a del(1p) other than del(1p32) (locus evaluated by FISH) which was detected in 22 pts by both methods. Furthermore, as WGS cannot only detect SV and CNV but also SNV, WGS allows screening for potentially clinically exploitable drug targets, e.g. BRAF V600E mutation (5 pts in our cohort) or resistance associated mutations such as in cereblon for immunomodulatory compounds (CRBN mutation in 3 pts) as well as biallelic events in TP53 (del(17p)+TP53 mutation), detected in 15 of our pts suggesting worst prognosis for affected pts. WGS in RRMM can shed light on the genetic background of relapses. Of note, homozygous del(16p) inducing loss of the treatment target were first described using our dataset in two patients with acquired resistance toward anti-BCMA CAR T cells or bispecific antibody therapy. Conclusions: FISH is the preferred approach in pts with BM PCI <10% and for the detection of clones <15% or variant ploidy levels. WGS is superior to FISH regarding the identification of biallelic events and rearrangements with heterogeneous breakpoints and rare partners. But even more, WGS can address various questions in follow up in terms of increase of complexity, target specific therapy, therapy failure and relapse. Thus, WGS allows a holistic insight into the genetics of MM and provides crucial information for clinical decision making. Figure 1 Figure 1. Disclosures Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Einsele: Janssen, Celgene/BMS, Amgen, GSK, Sanofi: Consultancy, Honoraria, Research Funding. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.

Clonal Heterogeneity and Evolutionary Phylogeny of Critical Cytogenetic Aberrations in Multiple Myeloma

Single-cell analysis is of significant importance in delineate the exact phylogeny of subclonal population and in discovering subtle diversification. So far studies of intratumor heterogeneity and clonal evolution in multiple myeloma (MM) were largely focused at the bulk tumor population level. Here, we performed quantitative multi-gene fluorescence in situ hybridization (QM-FISH) in 129 longitudinal samples of 57 MM patients. All the patients had newly-diagnosed and relapsed paired samples. An expanded cohort of 188 MM patients underwent conventional FISH (cFISH) to validate the cytogenetic evolution in bulk tumor level. 43 of 57 patients (75.4%) harbored three or four cytogenetic clones at diagnosis. We delineated the phylogeny of subclonal tumor population and derived the evolutionary architecture in each patient.13q deletion and the first 1q gain tended to be earlier cytogenetic alternation, whereas 16q and 17p deletion were acquired later. Patients with clonal stabilization had a significantly improved OS than those with other evolutionary patterns (median OS, 71.2 vs. 39.7 vs. 35.2 vs. 25.5 months, for stable, differential, branching and linear patterns, respectively, p=0.001). Besides, a high degree of consistency and complementarity across QM-FISH and cFISH was observed in evaluation of cytogenetic evolution pattern in MM. In total, at least two time-point cytogenetic evaluations by cFISH were underwent in 188 MM patients. The proportion of patients with high-risk cytogenetic features was 33% at diagnosis and 49% at relapse. The prognostic value of the presence of high-risk aberrations at diagnosis were attenuated over time (HR=1.79, p=0.002 for survival from diagnosis; HR=1.55, p=0.026 for survival from relapse, ). Survival from relapse were greater influenced by the presence of high-risk aberrations at relapse (HR=2.07, figure 5E) rather than present at diagnosis (HR=1.55). The present study investigated the prognostic value of evolution in copy number or clone size of 1q21 gain/amplification during follow-up. The incidence of patients carrying at least three copies of 1q21 was higher after relapse than at diagnosis (69% vs. 55%, p=0.004).Patients were categorized as six groups according to the change patterns in copy number and clone size of 1q21 gain between the two time-point samplings. Patients without 1q21 gain/amplification at both time points (group B) and patients who had obvious decrease in clone size or loss of 1q21 gain at relapse (group A) experienced similar superior outcome (Failure free survival after relapse (2 nd FFS), 18.1 vs. 27.8 months, p=0.469), whereas patients carrying 1q21 gain/amplification at both time points with or without increase (group C,D) in clone size relatively worse survival (2 nd FFS 12.4 and 10.5 months, respectively, p<0.05 compare to group A and B). The remaining patients who had an increase in copy number of 1q21 and those who developed de novo 1q21 gain at relapse were observed poorest outcome (group E and F,2 nd FFS 6.7 and 8.9 months). The interval time between two time-point samplings were similar among groups, whereas the different evolution pattern of 1q21 gain could clearly stratify both overall survival and post-relapse survival (p<0.001). This study shows that QM-FISH is a valuable tool to elucidate the clonal architecture at single cell level. Clonal evolution pattern is of prognostic significance, highlighting the need for repeated cytogenetic evaluation in relapsed MM. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Myeloma Patients with Deletion of 17p: Impact of Tandem Transplant and Clone Size

Introduction The presence of deletion 17p or monosomy 17 (del(17p)/-17) detected by fluorescence in situ hybridization (FISH) is well-established as a high-risk (HR) factor in multiple myeloma (MM). The del(17p)/-17 clone size and the clinical impact in patients that have ≥60% involved nuclei is controversial. Optimal treatment is also controversial, with tandem autologous stem cell transplantation (ASCT) explored but not uniformly adopted in the real world (ENM02, StaMMina). At our center, we have routinely offer tandem transplantation for del(17p)/-17 and now review our experience over a 10-year period, including the significance of del(17p)/-17 genetic variations on outcomes. Methods We performed a retrospective chart review of all patients identified with del(17p)/-17 patients who were offered tandem transplant and underwent at least one ASCT at Princess Margaret Cancer Centre from 2009-2019. Patient and disease characteristics, responses, and survival outcomes were collected from the Myeloma and Transplant databases and electronic patient records under REB approval. Results Patient, disease, and treatment characteristics. We identified 100 patients with del(17p)/-17 who underwent ASCT at our center. These patients were separated into three groups: those with a deletion of the TP53 locus (57%), those that had monosomy 14 (17%), and those with a relative loss the TP53 locus (eg. aneusomy or polyploidy,13%). The median % of nuclei with del(17p) was 39% (range 5-93%), with 27 (25%) over 60%. 28% of patients carried at least one other cytogenetic abnormality besides del(17p), most commonly t(4;14). All patients were intended for tandem transplant, but only 69 (69%) completed both transplants. Reasons for not proceeding to a second transplant were: patient declined (29%), toxicity (26%), clinical trial (19%), progression prior to the second transplant (13%), with one early death (3%). Median age at transplant was 61 years (range 40-72); most were male (61%) with advanced R-ISS II and III (97%). The most common induction regimen used was cyclophosphamide, bortezomib and dexamethasone (CYBORD)(87%), with 10% requiring a 2 nd induction regimen for inadequate response/progression. Standard conditioning was melphalan 200mg/m 2 for both first and second transplants. Median time from diagnosis to first ASCT was 5.9 months (range 3-24) and time from first to second transplant was 3.3 months (range 1-7). Most patients (90; 84%) received maintenance therapy post-transplant: 47% lenalidomide, 29% multiple agents (commonly lenalidomide and a proteasome inhibitor) Response and survival outcomes After induction, the rate of VGPR (very good partial response) or greater was 53%, increasing to 73% after 1 st ASCT, 85% after 2 nd ASCT. At a median follow-up of 32 months (range 3-130), the median PFS for all patients was 39.2 months 95% CI (24.1-46) and median OS was not yet reached (NYR) (95% CI 57.9-NYR). When analyzed by whether single or tandem transplant was performed, median PFS was 21.8 months (95% CI 10.3-43.3) for single ASCT, and 42.1 months (95% CI 32.8-NYR) for tandem ASCT (P=0.0096). The median OS was 57.6 months for single ASCT (95% CI 22.2-86.5), but NYR for tandem ASCT (95% CI 105.6-NYR) (p=0.0022). On subgroup analysis, patients with ≥60% del(17p) achieved a median PFS 45 months (95% CI 14.4-NYR) and median OS 68.5 months (95% CI 20.9-68.5), median was only PFS 24.9 months (95% CI0.4-NYR) and median OS 29.3 months (95% CI 0.4-68.5) for those who received single ASCT. (table1) TP53 signal pattern analysis (table 2) showed the median OS for patients with monosomy 17 was NYR, while it was 105.7 months for patients with deletion, and 86.5 months for those with relative loss. Median PFS was 43.3 months for the monosomy group, 32.8 months for deletion group, and NYR for relative loss. Conclusions Our experience shows that MM patients with del(17p)/-17 appear to have deeper responses and improved outcomes with more aggressive tandem transplantation. However, approximately 1/3 of our patients intended for tandem transplant did not undergo a second transplant, identifying a focus of future investigation and optimization. Our analysis also suggests that clone size of del(17p) >60% does impact OS negatively, but improved with tandem transplantation. The type of deletion also appears to affect outcomes with deletion having worse PFS but not worse OS. Figure 1 Figure 1. Disclosures Prica: Astra-Zeneca: Honoraria; Kite Gilead: Honoraria. Reece: Takeda: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Millennium: Research Funding; Sanofi: Honoraria; BMS: Honoraria, Research Funding; Karyopharm: Consultancy, Research Funding; GSK: Honoraria. Trudel: Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding; Genentech: Research Funding; Roche: Consultancy; Sanofi: Honoraria; Pfizer: Honoraria, Research Funding. Chen: Beigene: Membership on an entity's Board of Directors or advisory committees; Astrazeneca: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy.

Longitudinal Assessment and Prognostic Importance of Minimal Residual Disease (MRD) By Multiparametric Flow Cytometry in Patients with Systemic Light Chain (AL) Amyloidosis

Background: Minimal residual disease (MRD) assessment can provide an index of robust disease control, particularly if MRD negativity is sustained over time. Whereas MRD status has well-established prognostic implications in multiple myeloma, its role in light chain (AL) amyloidosis-a related but biologically distinct plasma cell disorder-is presently under investigation. Aims: The aims of our study were to evaluate (1) major organ deterioration-progression-free survival (MOD-PFS) based on MRD status and (2) MRD evolution patterns during disease surveillance for patients with AL amyloidosis in hematologic complete response (hemCR) after therapy. Methods: We established MRD status using multiparametric 10-color flow cytometry of bone marrow aspirates. At least 2 x 10 6 events were measured using a Beckman Coulter Navios Flow Cytometer and analyzed by Kaluza Software (sensitivity level of ≤10 -5). Sequential MRD testing was performed (≥12 months apart) for patients returning for follow-up. We evaluated hematologic and organ responses or progression based on consensus guidelines (Palladini, 2012). MOD-PFS was defined as time from hemCR achievement to major organ deterioration (i.e., the kidney or heart), hematologic progression or death from any cause (whichever occurred first). Presence of MRD was not used to guide treatment decisions. Results: A total of 96 patients with hemCR after therapy were tested, of whom 46 (48%) were MRD negative and 50 (52%) MRD positive on first assessment. The median estimated clone size for those with MRD positivity was 3.4 x 10 -4 (range 1.4 x 10 -5 to 5.7 x 10 -3). Baseline characteristics were similar between groups, except for greater use of daratumumab and ≥2 lines of therapy to achieve hemCR in the MRD-negative group. MRD positivity correlated with a higher level of involved free light chains (iFLC) in the serum at the time of MRD assessment (23.4 mg/L vs 17.3 mg/L, P = .011). In regard to organ responses, patients with MRD negativity had a significantly higher rate of renal response (91% vs 67%, P = .017) but a similar rate of cardiac response (74% vs 68%, P = .668). After a median follow-up of 50 months from hemCR achievement, the MRD-negative group demonstrated a superior MOD-PFS (HR 0.38, 95% CI 0.16-0.88, P = .034). Progression or organ deterioration events in the MRD-negative group occurred early after hemCR achievement, while in the MRD-positive group these events continued to occur over time. A total of 8 patients had died, including 4 in each group. Sequential MRD testing (median 12 months apart, range 12-28 months) was performed for 43 patients, of whom 16/19 (84%) had sustained MRD negativity; 21/24 (88%) had persistent MRD positivity; and 6/43 (14%) had MRD conversion. No patient received therapy between MRD assessments. Among those with sustained MRD negativity, there were 2 major organ deterioration events. The 3 patients who experienced loss of MRD negativity (which occurred at 12, 107 and 114 months after last treatment) had no progression or organ deterioration events. Of those with persistent MRD positivity, 13 (62%) had durable hemCR and organ responses; moreover, 4/14 (29%) and 1/11 (9%) of these patients attained even deeper renal and cardiac responses, respectively, in the interval between sequential MRD testing. Clone progression (defined as >1-log growth in MRD clone size) was observed in 3/21 (14%) patients with persistent MRD positivity, none of whom demonstrated hematologic relapse. Conclusions: We evaluated the dynamics of MRD status in relation to clinical outcomes and found that patients with MRD negativity had longer survival free from hematologic progression, major organ deterioration or death. Longer prospective follow-up is needed to determine whether this translates into an overall survival benefit. MRD conversion occurred in a small proportion (14%) of patients, but was not linked to adverse outcomes. Understanding the role of cross-sectional MRD testing for clinical disease tracking in AL amyloidosis requires further analysis with larger sample size. Figure 1 Figure 1. Disclosures Sanchorawala: Prothena: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Research Funding; Pfizer: Honoraria; Karyopharm: Research Funding; Sorrento: Research Funding; Celgene: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Caelum: Membership on an entity's Board of Directors or advisory committees, Research Funding; Regeneron: Membership on an entity's Board of Directors or advisory committees; Proclara: Membership on an entity's Board of Directors or advisory committees; Oncopeptide: Research Funding.

Loss of nr4a1 abrogates Fitness of asxl1-mutant Hematopoietic Clones

Clonal fitness of mutant hematopoietic stem and progenitor cells (HSPCs) underlies clonal hematopoiesis (CH), a state of clonal expansion associated with increased risk of blood malignancies and cardiovascular disease. Mechanisms by which acquired mutations lead to clonal fitness are not known. We used a zebrafish model to study the effect of acquired asxl1 mutations on HSPC clonality with TWISTR (Tissue editing With Inducible Stem cell Tagging via Recombination) that combined mosaic CRISPR-Cas9 mutagenesis with color labeling of HSPC clones. TWISTR asxl1 mutants showed clonal dominant states with the expansion of single-colored clones contributing to over 30% of myelopoiesis. These zebrafish had normal hematopoietic output and no major lineage skewing. Single-cell RNA sequencing analysis of TWISTR mutant marrow cells harboring asxl1 mutations showed >10-fold upregulation of inflammatory cytokines in mutant mature myeloid cells and >30-fold upregulation of anti-inflammatory modulators in immature progenitors. Increased inflammation has been widely documented in persons with CH with acquired mutations in DNMT3A, TET2, ASXL1 and other genes. Moreover, chronic inflammation due to infection was shown to promote relative clonal fitness in Dnmt3a mutant mice. Based on our results, we proposed the hypothesis that upregulation of the anti-inflammatory genes, including nr4a1, served as a mechanism of resistance to chronic inflammation created by the mutant HSPC progeny, resulting in a self-perpetuating cycle of clonal fitness in that environment. To test this, we used TWISTR to generate mosaic mutants of asxl1 and nr4a1 by injecting zebrafish with guide RNAs targeting exon 12 of asxl1 and exon 3 of nr4a1 together. Our model would predict that abrogation of nr4a1 expression in asxl1-mutant clones would weaken their fitness relative to clones that maintained nr4a1 expression. We sorted over 300 clones of various sizes from this cohort of zebrafish and sequenced the two targeted genes. We found that clones with frameshift mutations in asxl1 with either no nr4a1 mutations or heterozygous nr4a1 mutations had a clone size of 20%±14% or 19.7%±15% in myeloid cells, respectively. Asxl1-mutant clones with biallelic frameshift mutations in nr4a1 were significantly smaller with a clone size of 13.8±11.5% (p < 0.015). Clones with intact asxl1 did not differ in their clone size independent of nr4a1 genetic status (11.9% wildtype nr4a1 vs 11.5% homozygous nr4a1 mutant). Chemical inhibition of nr4a1 over 3 months resulted in reduced change of edited clones in asxl1-mutant zebrafish compared to vehicle-treated zebrafish, with median change in allelic fraction of 3%±4.8% vs 5.3%±7.5%, respectively. This suggested that upregulation of nr4a1 in asxl1-mutant clones maintains their fitness in inflammatory conditions, potentially by limiting HSPC exhaustion. We successfully used TWISTR to study asxl1 induced CH in zebrafish and identified nr4a1 upregulation as a critical pathway engaged for establishing clonal fitness. Disclosures Zon: Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder; CAMP4 Therapeutics: Current holder of individual stocks in a privately-held company, Other: Founder; Amagma Therapeutics: Current holder of individual stocks in a privately-held company, Other: Founder; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder; Branch Biosciences: Current holder of individual stocks in a privately-held company, Other: Founder; Celularity: Consultancy; Cellarity: Consultancy.

Eight-Color Flow Cytometry Phenotypic Markers and Disease Progression in Monoclonal Gammopathy of Unknown Significance

Introduction: Flow cytometric immunophenotyping is considered an indispensable tool for the diagnosis, classification, and monitoring of plasma cell disorders. Herein, we seek to study the clinical significance of expression of phenotype markers in monoclonal gammopathy of unknown significance (MGUS). Methods: We identified a cohort of patients with a diagnosis of MGUS from the institutional myeloma database. Bone marrow (BM) aspirate assessment was performed using 8-color immunophenotypic next-generation flow cytometric (NGF) analysis with a minimum sensitivity of 10 -5 cells at the time of diagnosis or first visit to our institution. BM aspirate samples were immunophenotyped on a FACSCanto II flow cytometer using antibodies (BD) to delineate normal and abnormal plasma cells [CD138 (V-500), CD38 (FITC), CD19 (PE-Cy7), CD45 (V-450), CD27 (PercpCy5.5), CD81 (APC-H-7), CD56 (APC) and CD20 (PE)]. The sensitivity or the Limit of Detection (LOD) for this assay was validated to 20 cells in 2 ×10 6 events (0.001%), and the reproducibility or Lower Limit of Quantitation (LLOQ) is 50 cells in 2 ×10 6 events. Clinical and laboratory variables were also collected. Based on previously published data, expression (CD19, CD45, CD81), and lack of expression (CD56, CD27, CD20) of the above-mentioned surface antigens were analyzed. Additional variables such as IgA isotype, size of M-protein (≥15 g/L), and abnormal free light chain ratio(abnFLR) (defined as <0.1 or >10) were included in regression fitting models. Results: A total of 157 patients with MGUS were included in this analysis. The median age at diagnosis was 60 years (range 24- 84), 84 (53 %) patients were female and 25 (16%) were African American. Overall, IgG Kappa (75/148, 50%) was the most common isotype. Fluorescent-in-situ hybridization (FISH) data were available in 35 patients with t (4:14) and t (14;16) seen in 3 patients each. At a median follow-up of 18.15 years (quantiles 11.35, 33.62), 28 patients experienced disease progression (25 to MM, 2 to Waldenstrom macroglobulinemia, and 1 Smoldering myeloma). The median progression-free survival of this cohort was 17.3 years. Among these, occurrences of the bone lesion (8/28; 28.6%) were the most common pattern of disease progression to MM. This analysis showed lower odds of progression with the expression of CD27 (OR-0.39, 95% CI 0.15-0.99) (figure 1A). Disease progression was more common in patients with an abnormal plasma cell clone size ≥ of 3.1% at diagnosis (60% vs. 12.5%, p=0.0005). An abnormal plasma cell clone of ≥3.1% at diagnosis, was associated with increased odds of progression (OR-10.79, 95% CI 4.02-28.98) and a shorter PFS (12.5 years versus NR, p=0.01) (figure 1B). Serum M-spike ≥1.5 g/dL (OR-3.54;95% 1.30-9.62) and abnFLR (OR-2.30, 95% CI 1.00-5.32) were also associated with a higher odds of progression. However, in this population, the presence of IgA isotype did not increase the odds of MGUS progression. In a stepwise regression model, serum M-spike≥1.5 g/dL, abnFLR, and the lack of expression of CD27 were associated with the risk of disease progression. Conclusion: In addition to previously published risk factors, our cohort shows that the expression of CD27 antigen by eight-color flow cytometry confers a lower risk of disease progression of MGUS. This is consistent with our previous report that CD27 is progressively down-regulated in the transition from normal plasma cells (NPC) to MGUS to MM (Zhan et al, Blood 2006). Furthermore, we show that size of the myeloma clone (≥ 3.1% ) is a possible surrogate marker for disease progression in MGUS. Figure 1: 1A shows forest plot of odds ratios for flow cytometry markers, IgA isotype, size of M protein, abnFLR and plasma cell clone size. 1B shows the Kaplan Meier estimates of PFS for patients stratifies by plasma cell clone size. Figure 1 Figure 1. Disclosures Mohan: Medical College of Wisconsin: Current Employment. Atrash: GSK: Research Funding; AMGEN: Research Funding; Jansen: Research Funding, Speakers Bureau.