Del 5q Research Articles

Differentiation Scoring (DS) Derived from Cellworks Computational Omics Biology Model (CBM) Predicts Response to Hypomethylating Agents (HMA) and Patient Survival in Myelodysplastic Syndrome (MDS)

Background: Differentiation arrest resulting from aberrant methylation represents a key pathogenic and phenotypic feature of MDS. Hypomethylating agents (HMA) can reverse epigenetic dysregulation, but as single agents fail to control the disease in approximately 50% of patients, many of whom progress to AML and carry a dismal prognosis. We hypothesized that Differentiation Scoring (DS) derived from Cellworks Computational Omics Biology Model (CBM) of each patient's genomic aberrations could serve as a biomarker to predict response to HMA. Methods: We studied 169 MDS patients treated with azacytidine (n = 86) or decitabine (n = 83) whose genomic aberrations and clinical outcomes were available from public databases. Biosimulation of the disease state was computationally derived from pathways that were up- or down-regulated by individual genomic aberrations. DS was defined as the ratio of the quantified impact of key differentiation biomarkers in myeloid malignancies (CEBPA, GATA1, SPI1 and ITGAM) divided by the impact of HOXA9 transcription, a known repressor of differentiation, and normalized on a log2 scale. We also used CBM to determine the change in DS attributable to HMA to define an efficacy score (ES) as a parameter of drug response. A Pearson correlation coefficient was obtained using the two variables. DS in clinical responders (R) and non-responders (NR) was evaluated with the Student t-test. We defined DS > 1.5 as DS-high and < 1.5 as DS-low. Survival data were available for 101 patients and correlated with DS-high (n=49) and DS-low (n=52) groups using a KM-curve. Results: Median age of this cohort was 69 years (range, 20-87 years), including 56 females, 111 males, and 2 of unknown sex. Regarding HMA response, 62 patients were R while 107 were NR. Prior to therapy, 86 patients had DS-high and 83 had DS-low scores. Post-treatment DS were correlated with HMA ES (R = 0.58, p < 2.2e-16), and showed a favorable HMA response for DS-high patients. Biosimulation showed the incremental benefit on DS of HMA was greater for people with DS-high than DS-low scores (44% vs 16%, p = 7.51 e-17). Median DS was higher in R than NR (p=0.00232). Patients with DS-high scores had superior survival compared to those with DS-low scores (log rank p = 0.0016). (Figure 1a) Different genomic aberrations contributed to the DS scores. DS-high patients had more TP53 mutations, 17p del, 8q amp, and 5q del. On the other hand, DS-low patients more often had mutations in ASXL1, SRSF2, RUNX1, DNMT3A, EZH2, NF1, NRAS, CBL, or 7q deletion. (Figure 1b) Loss of TP53 due to mutation or deletion upregulates DNMT1 and silences HOXA9 transcription, leading to DS-high scores. KAT6A (8q) amp also upregulates EZH2 and CpG methylation, while 5q del causes GATA1 transcription, increasing CpG methylation via SALL4 to produce high ß-catenin signaling that led to DS-high scores. By contrast, 7q loss, ASXL1, SRSF2, and EZH2 mutations downregulated EZH2, diminished CpG methylation and unsilenced HOXA9 transcription. As a result, HOXA9 arrested differentiation results in DS-low scores. Additionally, NF1 or NRAS mutations upregulate ERK signaling which inhibits CEBPA, and also caused DS-low scores. Conclusions: Computational biosimulation based on patients’ individual genomic aberrations reveals a spectrum of DS among patients with MDS. DS strongly predicted HMA response. In general, NR had lower DS and survival than DS-high patients. While patients with DS-low scores might have fared even worse without HMA therapy, we propose that DS-low patients should be considered for novel combination therapy at the time of diagnosis given their unsatisfactory outcomes with HMA therapy as a single agent. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

NOTCH1 Signaling Is Dysregulated in CLL By the Deubiquitinase USP28 That Is Recurrently Affected By Del(11q) and Can be Therapeutically Targeted

Active NOTCH1 signaling is present in approximately half of chronic lymphocytic leukemia (CLL) patients and was shown to be a marker for disease outcome. The NOTCH1 signaling pathway can be aberrantly activated through mutations in the NOTCH1 gene itself or co-factors such as FBXW7, resulting in protein stabilization of cleaved NOTCH1 (NICD). However, 50% of CLL cases with NOTCH1 activation do not harbor alterations in these genes, suggesting additional mechanisms for NOTCH1 hyperactivation. Since NICD can be stabilized due to defects in the ubiquitination/degradation machinery, we aimed to elucidate the role of the on chromosome 11q23 located deubiquitinase USP28 in the regulation of NOTCH1 activity and as a novel therapeutic target for NOTCH1-dependent CLL patients. First, we evaluated USP28 expression levels in a previously published cohort of 285 untreated CLL patients characterized via SNP array analysis and gene expression profiling (Edelmann et al, Blood 2012; Bloehdorn et al, Nat Commun 2021). The presence of monoallelic USP28 deletion was identified in 90% of CLL patients harboring a heterozygous deletion of chromosome 11q (del(11q); n=96), resulting in a significant decrease of USP28 RNA expression in comparison to non-del(11q) cases (n=199, P<0.0001). In non-del(11q) cases, USP28 expression levels were variable and assessment of protein levels of USP28 and its target proteins by western blotting in primary CLL cells (n=8) revealed a correlation between high protein levels of USP28 and remarkably high levels of NICD and other USP28 targets such as c-MYC or c-JUN. In addition, interaction of USP28 with NICD was shown by immunoprecipitation assays. In parallel, we implemented the CRISPR/Cas9 system to generate HG3 CLL-derived cell lines harboring USP28 biallelic deletion (HG3-USP28KO, n=5), monoallelic 11q deletion (HG3-del(11q), 17 Mb size including both ATM and USP28 genes, n=3), biallelic ATM truncation (HG3-ATMKO, n=3) or the combination of both events (HG3-del(11q) ATMKO, n=3). Overexpression of USP28 in HG3WT and HG3-USP28KO cells increased NOTCH1 activity measured by luciferase reporter assays (P<0.05, P<0.001, respectively). In contrast, in HG3-del(11q) cells decreased USP28 RNA and protein expression resulted in reduced NOTCH1 activity compared to HG3WT cells (P<0.05). Notably, HG3-del(11q) ATMKO cell lines showed even lower levels of NOTCH1 activity (P<0.01). Given that ATM phosphorylates USP28 (Zhang et al, Cell 2006), we hypothesized that in CLL cells ATM regulates USP28 activity and thereby also downstream NOTCH1 signaling. Indeed, NOTCH1 activity was reduced in HG3-ATMKO cells (P<0.01). However, USP28 overexpression in HG3-ATMKO cells did not lead to increased NOTCH1 signaling activity as observed in HG3WT cells. Moreover, pharmacological ATM inhibition (5 µM of KU-55933) also resulted in reduced NOTCH1 activity in HG3WT cells (P<0.0001) and reduced NICD levels in primary CLL cells (n=12; P<0.05). We finally explored the impact of the small molecule USP28 inhibitor AZ1 (alone or in combination with ibrutinib or venetoclax) on NICD protein levels and viability of primary CLL cells (n=15). AZ1 treatment (10 µM) resulted in decreased NICD levels, independent of mutations within NOTCH1 or FBXW7 (P<0.01). ATP quantification and 7-AAD staining revealed that increasing doses of AZ1 significantly reduced viability of NOTCH1/FBXW7-MUT primary cells (n=5) as well as of NOTCH1/FBXW7-WT cells (n=6) expressing high protein levels of USP28 and NICD (P<0.01; P<0.05, respectively). In contrast, AZ1 treatment did not affect viability of CLL patient cells expressing low NICD levels (n=4; P=0.56) Furthermore, AZ1 combined with venetoclax potentiated the reduction of cell viability especially in NOTCH1/FBXW7-MUT or patient cells expressing high USP28 and NICD levels (P<0.001; P<0.05). We propose that high levels of the deubiquitinase USP28 are responsible for hyperactivation of NOTCH1 signaling in CLL. This hypothesis is further confirmed in del(11q) CLL cells, where USP28 heterozygous deletion resulted in low NOTCH1 activity. In addition, our data reveal that USP28 activity is regulated by ATM, uncovering a novel mechanism of NOTCH1 modulation via the ATM-USP28-NOTCH1 axis (Figure 1). Finally, we demonstrate that USP28 inhibition by AZ1 suppresses USP28-mediated NOTCH1 activation and could represent a novel therapeutic strategy in CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Monosomy 7 and Del(7q) Cause Selective Sensitivity to Inhibitors of Nicotinamide Phosphoribosyltransferase in Acute Myeloid Leukemia

Monosomy 7 and deletion of chromosome arm 7q (-7/-7q) occur in up to 10% of patients with acute myeloid leukemia (AML). In AML, -7/-7q are associated with resistance to chemotherapy and adverse prognosis. As such, treatment outcomes for this subgroup of patients remain particularly dismal, and targeted therapies are urgently needed. To identify targeted therapies for AML with -7/-7q we analyzed ex vivo drug sensitivities in 158 AML samples. The presence of -7/-7q (n=18) was determined using karyotyping and exome-sequencing of bone marrow (BM) mononuclear cells and a matched skin biopsy control. Mononuclear cells from fresh bone marrow aspirates of AML patients were incubated with a library of up to 525 oncology drugs for 72h, with each drug pre-plated in 5 concentrations. Cell viability was measured using the CellTiter Glo (CTG) assay, and the response of each sample was summarized as a drug sensitivity score (DSS), which is a modified integration of the dose-response inhibition curve, directly proportional to the sensitivity of a sample. We found that AML samples with -7/-7q were highly sensitive to the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor daporinad (p-value ~ 4.2e-06) (Figure A). NAMPT is the rate-limiting enzyme in the NAD+ salvage pathway. The NAMPT gene is located at 7q22.3. Monosomy 7 and del(7q) lead to the deletion of one copy (i.e., haploinsufficiency) of NAMPT. Notably, a rare AML with del(7q) in which the deletion did not include the NAMPT gene (and which was thus diploid for NAMPT) was not sensitized to NAMPT inhibition. Taken together, this data demonstrates a significant association between the NAMPT haploinsufficiency in AML with -7/-7q and increased sensitivity to NAMPT inhibition. To further support our findings, we analyzed the sensitivity of AML cells without or with -7/-7q to four NAMPT inhibitors (daporinad, GMX1778, KPT-9274, and LSN3154567) using multiparametric flow cytometry. Viably frozen BM mononuclear cells from AML patients with (n=11) and without (n=51) -7/-7q were thawed and incubated with drugs in 8 concentrations for 72 hours. After incubation, cell viability was analyzed using the iQue Screener Plus flow cytometer with a panel consisting of CD45, CD3, CD34, CD117, CD14, and CD38 antibodies, to identify myeloid and lymphoid cell populations, as well as Annexin V and 7-AAD to discriminate live from dead cells. We found that CD34+ myeloblasts from AML with -7/-7q were selectively killed by all four NAMPT inhibitors, validating our initial results (p-value ~ 1.4e-02). An in-depth analysis of cellular subpopulations revealed that while CD34+CD38+ cells (representing more differentiated myeloblasts) were significantly more sensitive to NAMPT inhibition in -7/-7q samples compared to wild-type samples (p-value ~ 4.5e-04), the CD34+CD38- compartment (anticipated to contain the immature leukemic stem and progenitor cells) was not sensitized in this group (Figure B). Interestingly, the analysis of ex vivo drug sensitivities also revealed that while the majority of AML with -7/-7q are resistant to the FDA-approved BCL2 inhibitor venetoclax, they are highly sensitive to daporinad (Figure A). This finding proposes NAMPT inhibitors as a suitable alternative to venetoclax for -7/-7q patients. Additionally, since venetoclax has previously shown significant efficacy against the highly BCL2-dependent undifferentiated (CD34+CD38-) myeloblasts, its combination with NAMPT inhibitors represents a promising strategy for achieving total leukemic cell death in these patients. In conclusion, we have found that AML with NAMPT haploinsufficiency caused by -7/-7q shows selective sensitivity to NAMPT inhibition. This suggests that NAMPT inhibitors have the potential to be a potent targeted therapy for AML with -7/-7q, a disease with a particularly adverse prognosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Chromosome 7 Myeloid Tumor Suppressor Genes Cooperate in Chemotherapy Resistance

Loss of all or part of chromosome 7 [-7/del(7q)] is common in myeloid neoplasms and is associated with chemoresistance. -7/del(7q) occurs in de novo and therapy-related myeloid neoplasms (t-MN), high-risk secondary malignancies arising after prior exposure to chemotherapy or radiation. We previously reported that deficiency of CUX1, a 7q-encoded tumor suppressor gene, promotes hematopoietic stem and progenitor cell (HSPC) drug resistance, enabling HSPCs to persist during genotoxic insult, expand, and transform to t-MN. To comprehensively identify additional chromosome 7 genes putatively involved in drug resistance, we mined 13 chemotherapy resistance CRISPR-Cas9 genome-wide knockout screens performed in human cell lines. We included screens with daunorubicin, commonly used in the treatment of de novo acute myeloid leukemia, as well as screens with cisplatin and etoposide, chemotherapeutics that predispose to t-MN. Using loose thresholds for inclusion, we identified 113 genes that are potentially selected for by chemotherapy when edited. We prioritized candidates for validation based on those genes that were recurrent hits in more than one screen and genes with high expression levels in human HSPCs. Of the 9 top candidates tested in subsequent in vitro assays, we confirmed four genes to promote resistance when CRISPR-edited. This work yielded two 7q genes known to promote drug resistance when deficient, EZH2 and KMT2C/MLL3, validating our approach. We also identified novel drug resistance phenotypes for AHR and LUC7L2 deficiency. We demonstrate that combined deficiency in EZH2 and CUX1 leads to increased drug resistance in vitro and in vivo, to an extent greater than loss of either single gene alone. Transcriptome profiling additionally supports a genetic interaction between EZH2 and CUX1. In summary, chromosome 7 harbors multiple genes involved in chemotherapy resistance, supporting the concept of 7q as a contiguous gene syndrome. Furthermore, we reveal a previously unknown genetic interaction between the 7q genes CUX1 and EZH2. A refined understanding of the molecular pathways driving del(7q) pathogenesis and drug resistance will enable development of therapies designed to counter these treatment barriers.

Therapy-Related Myelodysplastic Syndromes with Isolated Del(5q): Comparative Analysis of Phenotype and Long-Term Survival

Background: Myelodysplastic syndromes (MDS) with chromosome 5q deletion [MDS-del(5q)] is a subcategory of MDS recognized by both the International Consensus (ICC) and World Health Organization (WHO) classification systems. Diagnostic criteria according to ICC-2022 include i) presence of del(5q) alone or with only one other additional cytogenetic abnormality, other than -7/del(7q), ii) absence of multi-hit TP53 mutation, iii) presence of ≥1 cytopenias, iv) presence of bone marrow myeloid dysplasia in ≥1 lineages, v) absence of ≥5% bone marrow blasts, and vi) absence of ≥2% circulating blasts (Arber et al. Blood 2022). We used WHO-2016 diagnostic criteria to identify Mayo Clinic patients with MDS-del(5q) and confirmed a study population that also met ICC-2022 criteria. The main objective of the current study was to describe the phenotype and long-term survival of patients with therapy-related MDS-del(5q) and compare the findings with a cohort of patients with primary MDS-del(5q), sourced from the same database. Methods: The current study was conducted under an institutional review board approved minimum risk protocol that allowed retrospective collection and analysis of data from patients with MDS-del(5q) who were seen at the Mayo Clinic. Diagnosis of MDS-del(5q) was retrofitted to comply with ICC-2022 criteria, with the exception that patients with additional cytogenetic abnormalities were excluded from the current report (Arber et al. Blood 2022). Most patients were treated with lenalidomide, often given at the recommended dose of 10 mg/day x 21-28 days, with dose adjustements at physician discretion. Erythroid response criteria included transfusion independence for at least 8 weeks for transfusion dependent patients and an increase in hemoglobin by 1.5 g/dl lasting for at least 8 weeks, in transfusion independent patients. Transfusion dependency was assigned to those patients who had received at least 3 units of packed red cells in the 8 weeks prior to diagnosis. Conventional statistical methods were applied using JMP Pro 16.0.0 software (SAS Institute, Cary, NC, USA). Results A total of 93 patients (median age 73 years; range 24-89; females 63%) were included in the current study, including primary (n=79) and therapy-related (n=14) MDS-del(5q). Cancer/treatment history for the latter group of patients included prostate cancer/radiotherapy (n=5), thyroid cancer/radiotherapy (n=2), breast cancer/radiotherapy (n=2), bladder cancer/chemotherapy (n=2), colon cancer/chemotherapy (n=1), lung cancer/chemotherapy (n=1), and non-Hodgkin's lymphoma/chemotherapy/radiotherapy (n=1). Median (range) time from radiotherapy/chemotherapy and recognition of MDS-del(5q) was 16 years (1.9-30). Comparison of presenting features for therapy-related vs primary MDS-del(5q) revealed the former to be associated with older age (median 77 vs 72 years; p=0.002), male gender (72% vs 30%; p=0.003), and lesser likelihood of transfusion-dependent anemia, at time of initial diagnosis (28% vs 68%; p=0.03). The two groups were otherwise similar in their leukocyte and platelet counts and mutational landscape, including prevalence of TP53 mutations (25% vs 13%, respectively). All 79 patients with primary vs 10 of the 14 with therapy-related disease received lenalidomide therapy with erythroid response rates of 62% vs 80% (p=0.2) and complete/partial cytogenetic response rates of 13%/10% vs 0/0, respectively (p=0.05); the latter information was available in a subset of 64 patients. After a median follow up of 4.8 years, 38 (41%) deaths and 16 (17%) leukemic transformations were documented; the corresponding figures for therapy related/primary disease were 50%/39% and 14%/18%, respectively (p=0.4 and 0.7). Figure 1 depicts similar (p=0.6) survival data for therapy-related vs primary MDS-del(5q), with corresponding median survival (3-year survival) rates of 6.3 years (60%) vs 7.7 years (63%). Conclusions: The current study describes the circumstances surrounding the development of therapy-related MDS-del(5q) and highlights differences in age and gender distribution, compared to primary MDS-del(5q). The two groups were otherwise similar in their response to lenalidomide therapy, overall survival, and leukemic transformation rates while their similarity in mutation distribution requires validation with higher number of informative cases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Predictors of Adverse Clinical Outcome in Diffuse Large B-Cell Lymphoma Richter Transformation of CLL/SLL: A Consortium of Richter Transformation Investigators (CORTI) Multicenter Study across 8 US, UK, and Australian Academic Centers

Background Richter transformation (RT) of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is a known but uncommon complication with dismal prognosis despite improvements in CLL outcomes with novel agents. Previously published studies are characterized by cohort heterogeneity with respect to CLL treatments (predating the rituximab era and novel agents) and RT histology [diffuse large B-cell lymphoma (DLBCL) and classic Hodgkin lymphoma (cHL)], limiting their generalizability in the modern era. To this end, we examined characteristics and outcomes in 222 DLBCL-RT cases, enriching for patients treated with novel agents. Methods We collected 247 RT cases diagnosed between 1995-2022 across eight academic institutions, excluding 23 with cHL-RT and 2 with "pseudo-transformation" after BTKi withdrawal (final n = 222; 41% of cases post-2010). We analyzed > 65 CLL/RT biologic, pathologic, and clinical parameters. Primary outcomes included complete response (CR) to first line of therapy [in CLL (iwCLL 2008/2018) and RT (Lugano 2014)] and 5-year overall survival (5yr-OS) after RT, estimated using Cox proportional hazards [yielding hazard ratios (HRs) and 95% confidence intervals (CIs)] and flexible parametric survival models (for prediction of standardized survival). Results The antecedent CLL included: 11% cases with atypical CLL, 84% IGHVUM, and 70% ZAP70+ with 46% and 43% of patients harboring del(13q) and del(17p), respectively. CLL/RT was synchronous (CLL diagnosis within 2 months of RT diagnosis) in 13% of cases. Only del(17p) correlated with C/HC (≥ 3 / ≥ 5 chromosome abnormalities) karyotype (p < 0.0001), and ZAP70/CD38 had no association with IGHV status. RT was primarily nodal (47.3%), non-GCB (90.3%), and EBV-negative (95%) with p53 IHC-positive large cells in 60% of cases and MYC translocation in 10%. Preceding CLL was treated with a median of 2 lines of therapy (range: 1-7) with 53% heavily-treated (≥ 2 lines). The most common first lines (LOT1) were chemotherapy (fludarabine-based or bendamustine/CHOP equivalents; 73.6%) and BTKi/novel agents (17%); 37% received BTKi as any LOT, while 44% received any novel agent (BTKi, BCL2i, PI3Ki, or SYKi). CLL CRLOT1 was achieved in 41.2% with overall response of 77%. Median time to RT from CLL diagnosis (sequential group) was 47.6 months pre-2010 (IQR: 87.8) vs. 21.8 months post-2010 (IQR: 34.6, p < 0.0001). During a median follow up of 10.2 months (IQR: 27.1 months) post-RT, 59% of patients died. Median OS was 17.4 months (range: 11.3-24.2) with 28% 5yr-OS. In the pre-rituximab era (i.e. prior to 2010), median OS was significantly shorter compared to the present era (8 vs. 19.9 months). Indicators of inferior 5yr-OS on univariable analysis included: sequential transformation (HR: 2.34 [95% CI: 1.25-4.35]), not achieving CRLOT1 for CLL (HR: 1.62 [95% CI: 1.01-2.58]), heavily-treated CLL (HR: 1.98 [95% CI: 1.36-2.88]), and C/HC karyotype (HR: 1.59 [95% CI: 1.02-2.46]). While trisomy 12 predicted superior outcome (HR: 0.54 [95% CI: 0.32-0.94]), del(13q) and del(17p) had no impact. Adverse baseline covariates at RT diagnosis were low (< 3.5 g/dL) serum albumin (HR: 3.13 [95% CI: 2.09-4.69]), low (< 150x103/uL) platelet count (HR: 2.38 [95% CI: 1.64-3.47]), LDH > 1.5x ULN (HR: 1.63 [95% CI: 1.12-1.38]), and ECOG-PS ≥ 2 (HR: 2.4 [95% CI: 1.51-3.81]). Notably, p53 IHC positivity in RT predicted increased all-cause mortality (HR: 2.13 [95% CI: 1.27-3.5]), retaining significance in both minimally- and heavily-treated CLL subsets. RT was treated using CHOP-R or EPOCH-R in 62%. Not achieving CRLOT1 for RT predicted worse 5yr-OS (HR: 3.90 [95% CI: 2.09-7.29]). A multivariable, main effects Cox model was constructed using five variables (age at transformation, CLL treatment number, LDH > 1.5x ULN, albumin, and p53 IHC). In this age-adjusted model, all variables retained adverse prognostic significance (Figure 1). Marginal OS was predicted by p53 IHC, standardizing over remaining covariates, and with a trend towards inferior 5yr-OS in p53-positive vs. -negative RT (13% vs. 38%; Figure 2). Conclusion This multicenter, international study is the largest cohort of RT in the era of novel therapies with solely DLBCL histology. We confirmed the utility of several traditional predictors. In addition, we identified p53 IHC as a promising predictor of adverse outcome (in univariable and multivariable analyses) based on these preliminary analyses. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Deletion of eif6 Partially Rescues Survival, but Not Neutropenia, of Zebrafish Model of Shwachman-Diamond Syndrome: The Roles of Sbds and Eif6 in Perturbing Lipid Metabolism

Introduction: Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic insufficiency, skeletal defects, neutropenia, and an increased risk of myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML). Biallelic mutations in SBDS account for ~90% of patients. SBDS interacts physically with EFL1 to release EIF6 from the pre-60S ribosomal subunit and promote the assembly of the mature 80S ribosome in the cytoplasm. A subset of SDS patients acquire del(20q), resulting in loss of EIF6. Additionally, somatic heterozygous EIF6 mutations have been found in SDS patients. SDS patients harboring del(20q) and EIF6 mutations are linked to a lower risk of developing MDS/AML when compared to other SDS patients. Together these factors point to a potential mechanism whereby a reduction in EIF6 levels may lead to a suppression in ribosome biogenesis defects. Methods and Results: We have created sbds knockout (KO) zebrafish strains that phenocopy the human syndrome with stunted growth, neutropenia and atrophy in the pancreas, liver, and intestine. Unlike the Sbds-null mice which die in early embryogenesis, sbds-null fish survive to early juvenile stages. The sbds KO fish display an accumulation of Eif6 in liver and intestine. Interestingly, we also found an accumulation of EIF6 in SDS patient-derived lymphoblasts. EIF6 has been described as a key regulator of metabolism, specifically stimulating glycolytic and fatty acid synthesis. Lipid metabolomics of sbds KO fish show a decrease in free fatty acids and phosphatidylcholine. Expression of several genes critical in lipid metabolism (srebp1, fasn and pparg) is upregulated in sbds KO fish. To study the role of Eif6 in the pathophysiology of SDS, we created an eif6 KO zebrafish with an insertion of one base pair resulting in a premature stop codon. By immunoblotting we demonstrated that mutants did not display Eif6 by 6 days post fertilization (dpf), and heterozygous fish displayed less protein than wildtype siblings. This new strain showed early mortality (~10 dpf), neutropenia, tp53 pathway activation, with a pronounced upregulation of cdkn1a as early as 5 dpf. RNA-Seq data of eif6 KO fish at 5 dpf showed a total of 2225 differentially expressed genes (DEGs): 668 downregulated and 1557 upregulated. These DEGs are involved in two main gene expression pathways: ribosome biogenesis and oxidative phosphorylation. We also observed a dysregulation of lipid metabolism markers in the eif6 KO fish. Furthermore, we bred the sbds KO with the eif6 KO, we observed a partial rescue on survival only in sbds-/- with one copy of eif6(eif6+/-) but failed to rescue their neutropenia. Conclusions: Our organismal models of sbds or eif6 deletion provide new insights into the pathophysiology of human SDS: 1) SBDS affects lipid metabolism possibly due to an accumulation of EIF6, 2) Loss of eif6 affects development/survival at an earlier stage than loss of sbds, and 3) Loss of either sbds or eif6 markedly upregulates cdkn1a, which is downstream of tp53. Interestingly, Eif6 partially rescues survival of sbds-null organisms, but only in the haploinsufficient state. EIF6 may offer a promising target for a novel therapeutic strategy in SDS.

Final Results of the Phase 1/2 Study of Acalabrutinib Monotherapy in Treatment-Naive Chronic Lymphocytic Leukemia with >6 Years of Follow-up

Background: Acalabrutinib is a next-generation, covalent Bruton tyrosine kinase inhibitor approved for the treatment of patients with treatment-naive (TN) or relapsed/refractory chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). A previous analysis of the phase 1/2, multicenter ACE-CL-001 study (NCT02029443) demonstrated durable responses and good long-term tolerability of acalabrutinib as first-line therapy in patients with CLL at a median follow-up of 53 months (Byrd et al. Blood. 2021;137:3327-38). Here, we report the final analysis in the TN cohort of that study, the longest running study of acalabrutinib, with over 6 years of follow-up. Methods: Patients included in the study were aged ≥18 years with TN CLL/SLL who met the 2008 iwCLL criteria for treatment, were inappropriate for or declined standard chemotherapy, and had ECOG performance status 0-2. All TN patients were treated in the phase 2 portion of the study and received acalabrutinib 100 mg twice daily (BID) or 200 mg once daily (QD), later switching to 100 mg BID, until progressive disease (PD) or unacceptable toxicity. The primary endpoint was safety. Secondary endpoints included investigator-assessed overall response rate (ORR), time to response (TTR), duration of response (DOR), and progression-free survival (PFS), with a post hoc analysis of event-free survival (EFS). Results: Ninety-nine patients (n=62 100 mg BID; n=37 200 mg QD) were treated. The median age was 64 years; 47% of patients had Rai stage 3-4 disease, 10% had del(17p), 14% had TP53 mutation, 17% had del(11q), 62% had unmutated IGHV, and 18% had complex karyotype. At the final data cut-off date of July 15, 2021, the median study follow-up was 73.7 months (range 0.92-82.4), and 70 (71%) patients remain on acalabrutinib treatment. The most common reasons for treatment discontinuation were adverse events (AEs) (10%) and PD (6%). The most common AEs (Table 1) were consistent with those reported in the prior update (53-month median follow-up). The only grade ≥3 AEs with a ≥50% increase in incidence since the prior update were pneumonia (4% [prior update] vs 8%) and syncope (4% [prior update] vs 6%). AEs that led to treatment discontinuation were amyotrophic lateral sclerosis, angiosarcoma, cardiac failure, cerebral hemorrhage, gastric hemorrhage, glioblastoma multiforme, non-cardiac chest pain, prostate cancer, sepsis, small cell lung cancer, and urinary tract infection (in 1 patient each). All-grade and grade ≥3 events of clinical interest included infection (87% and 19%, respectively), bleeding events (74%, 7%), major bleeding events (8%, 7%), and hypertension (29%, 13%). Atrial fibrillation/flutter occurred in 6% of patients (3% grade ≥3). Second primary malignancies excluding non-melanoma skin (all grades) occurred in 14% of patients Two deaths were reported (cardiac failure, n=1; multiple organ dysfunction syndrome, n=1); neither was considered treatment related. ORR was 97% (9% complete response; 88% partial response). Median TTR was 3.7 months (range 1.7-22.1). Median DOR was not reached; the estimated 66-month DOR rate was 89% (95% CI 80-94). ORR of 100% was seen across high-risk groups: del(17p) (9/9), del(11q) (15/15), unmutated IGHV (57/57), and complex karyotype (12/12). Median PFS was not reached; the estimated 72-month PFS rate was 87% (95% CI 77-93; Figure 1). The estimated 72-month EFS was 78% (95% CI 68-85). Reported EFS events overall included AEs (n=10/99, 10%), disease progression (n=9/99, 9%), and new anticancer therapy (n=3/99, 3%). Conclusions: The final data from ACE-CL-001 further support the long-term efficacy and safety of acalabrutinib monotherapy, with high response rates and rapid, durable responses seen in patients with TN CLL regardless of high-risk genomic features. With over 6 years of follow-up, this study firmly establishes the tolerability and safety profile of acalabrutinib seen consistently in subsequent studies, with a low incidence of atrial fibrillation/flutter seen and no new long-term safety issues identified. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Venetoclax Consolidation Achieves Durable Off-Treatment Remissions in Patients with High Risk CLL Who Have Been on Ibrutinib More Than a Year

Background Patients (pts) receiving ibrutinib (ibr) for CLL rarely achieve complete remission (CR) with undetectable minimal residual disease (U-MRD). Therefore, indefinite ibr maintenance therapy (Rx) is standard of care. Long-term Rx with ibr results in a cumulative risk of Rx discontinuation due to progression or toxicity. The risk of progression is highest in pts with complex karyotype and/or del(17p); some series suggest increased risk in pts with del(11q) or persistently elevated β2-microglobulin. Venetoclax (ven) is synergistic with ibr. We hypothesized that adding ven to ibr in pts at high-risk for CLL progression, who had been on ibr for more than 12 months (mo) would achieve U-MRD and allow Rx discontinuation. Methods We designed a phase II, investigator-initiated, response-adapted clinical trial with the addition of ven to ibr in pts (pts) with one or more high-risk features for disease progression who had received at least 1y of ibr regardless of line of Rx. Pts had detectable disease at time of study entry without meeting IWCLL criteria for progression. High-risk was defined as presence of at least one of: del(17p) or TP53 mutation; complex karyotype; del(11q); or elevated β2-microglobulin. Ibr was continued at 140-420mg/d and standard initiation and escalation of ven was performed to the target dose of 400mg/d. Rx with combined ibr and ven continued for a maximum duration of 24, 28-day cycles (C). Pts had bone marrow (BM) evaluation for MRD by flow cytometry (sensitivity 10-4) and CT scan for re-staging every 6mo. Pts in CR with U-MRD on two consecutive evaluations, 6mo apart, stopped ven, but could continue ibr at physician discretion. Pts who were MRD+ post-C24 continued ibr maintenance. Results: We report the results of the first 45 pts enrolled. All patients have completed ven treatment. Median number of prior therapies was 1 (range 0-4). 22/45 patients were receiving ibr as initial Rx. 63% of pts had del(17p) and/or mutated TP53. On intent to treat analysis, 17/45 pts (38%) post-C6 and 26/45 (57%) post-C12 achieved U-MRD. Three patients stopped ven prior to C12 (ineligible, n=1, metastatic solid tumor, n=2). Six of 16 (38%) patients with MRD+ at C12, converted to U-MRD by the end of the study. Best cumulative rate of U-MRD in BM was 33/45 (73%), Figure 1A. There was no difference in U-MRD4 rates or PFS according in those with TP53 abnormalities vs those without. Three pts were in CR prior to ven. Of the remaining pts, 24/42 (57%) pts improved response to CR/CRi during ven treatment. Median follow-up is 37.5 mo for PFS and 40.3 mo for OS. Forty-one patients completed treatment per protocol. Of the 32 patients with U-MRD at the completion of Rx, 10 stopped ven prior to C24 per protocol (5 after C12 and 5 after C18) after achieving U-MRD CR on two consecutive response assessments. At completion of ven, 10/32 pts with U-MRD4 continued ibr at physician discretion (6/10 with del(17p)). Five patients have progressed (Figure 1B): 1 during combination therapy; 3 during ibr maintenance (2 of whom had U-MRD4 at completion of ven); 1 with Richter's Syndrome during observation after achieving U-MRD4 CR. No pt died during ven Rx. Two patients died after receiving treatment for other cancers (metastatic melanoma, AML) and one after coming off protocol to receive acalabrutinib. Blood MRD analysis was obtained q6 mo (Figure 1C) in pts with U-MRD at the EOT; 10/32 have had confirmed MRD re-emergence (8/22 who stopped ibrutinib and 2/10 who continued); 3 subsequently progressed. Treatment was well-tolerated. The most common AE was diarrhea in 47 pts, grade 1-2 in all but 1 pt. The most common grade 3-4 AE was neutropenia, seen in 20% pts (Grade 3 in 8 and grade 4 in 1 patient). No febrile neutropenia was seen. Three patients developed grade 3 infections (pneumonia, pyelonephritis, skin abscess). No patient stopped ven due to toxicity. Seventeen patients experienced at least one SAE, most commonly non-melanoma skin cancers (n=9). Conclusions: Consolidation ven added to ibr in pts with high-risk CLL was well-tolerated and achieved cumulative BM U-MRD4 rate of 73% ibr discontinuation in 49% of pts. Only 1/45 pts progressed during combination Rx. At a median of 12 mo post-ven follow up, most pts who attained BM U-MRD have ongoing U-MRD in blood. A second cohort restricted to 45 high-risk pts with TP53 abnormalities or complex karyotype is accruing and there are plans to also include patients treated with acalabrutinib. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Long-Term Analysis of Anti-CD20mab + HDMP for the Treatment of Patients with Chronic Lymphocytic Leukemia

Background: The addition of an anti-CD20 monoclonal antibody (mAB) to high dose methylprednisolone (HDMP) has been demonstrated to be an effective, non-myelosuppressive treatment (Tx) regimen for patients (Pts) with chronic lymphocytic leukemia (CLL). (Castro et al., 2009) We performed a single institution retrospective analysis of Pts treated with an anti-CD20 mAB and HDMP evaluating long-term efficacy, safety, and immune reconstitution. Methods: 102 Pts treated between the years of 2001 and 2015 were evaluated. Tx consisted of HDMP 1gm/m2 IV on days 1, 2, and 3 of each cycle. Rituximab (R) was administered as either 375 mg/m2 weekly; 750 mg/m2 daily on days 1, 2 and 3; or 750 mg/m2 weekly times during cycle one and daily times three for cycle two and three. Obinutuzumab (G) and ofatumumab (O) were administered per FDA standard dosing. Pts received 3-6 cycles of Tx. Some Pts who achieved a partial response (PR) to initial therapy underwent alemtuzumab (Alem) consolidation, 10mg, administered three times a week for a total of eight weeks. All Pts received anti-microbial prophylaxis consisting of fluconazole, bactrim and acyclovir. Response assessment was performed using iwCLL criteria. Results: The Pts had a median age of 66 (range; 40-85). Rai stage at time of Tx initiation: I 6%; II 43%; III 17%; Stage IV 34%. 63% of Pts were previously treated. The median number of previous Txs was 2 (range; 1-5). 55% of Pts had been treated with immunotherapy alone, 67% with chemoimmunotherapy, and 13% of Pts with a BCL2 inhibitor. Pts harbored the following cytogenetic abnormalities: del(13q) 46%; trisomy 12 30%; del(11q) 22%; normal 13%; del(11q) 22%; del(17p) 19%; and complex in 26%. 58% of Pts harbored an unmutated immunoglobulin heavy chain (IGHV). 84% of Pts were treated with R, 11% with G, and 5% with O. 96 Pts (94%) had a response to Tx (OR). 18 Pts (18%) achieved a complete response (CR); 78 Pts (76%) achieved a PR; and six Pts (6%) had stable disease (SD). Seventeen Pts underwent Alem consolidation. Twelve Pts (71%) achieved a CR; four Pts (24%) achieved a PR; and one patient (6%) had SD after Alem consolidation. With a median follow-up of 114 months, the median progression free survival (PFS) for the entire cohort was 27.1 months. The median time to next treatment (TTNT) for the entire cohort was 30.0 months. For Pts who received Alem consolidation, the median PFS was 90.1 months, and the median TTNT was 97.8 months. (Figure 1A) Employing cox proportional hazards model, factors associated with improved PFS were IGHV mutated (HR 0.38; 95% CI 0.18 to 0.79); CR (HR 0.17; 95% CI 0.05 to 0.47); and Alem consolidation (HR 0.21; 95% CI 0.09 to 0.46). Factors associated with improved TTNT was presence of del13q (HR 0.41; 95% CI 0.18 to 0.93); IGHV mutated (HR 0.36; 95% CI 0.13 to 0.92); CR (HR 0.12; 95% CI 0.03 to 0.37); and Alem consolidation (HR 0.24; 95% CI 0.10 to 0.53). 9% Pts developed infection requiring hospitalization, and 5% Pts developed an opportunistic infection. Immediately after Tx, serum IgA, IgG and IgM levels decreased (mean IgA pre-Tx 69.4mg/dL vs post-Tx 43.0mg/dL; p <0.0001; mean IgG pre-Tx 738.0mg/dL vs post-Tx 494.3mg/dL; p <0.001; mean IgM pre-Tx 94.8mg/dL vs post-Tx 44.5mg/dL; p = 0.07). At approximately 36- and 48-months serum IgA and IgG levels returned to normal levels, respectively. (Figure 1B) 57% of Pts received supplemental intravenous immunoglobulin, thus we investigated factors associated with recovery of serum IgA levels at 36 months. We found that only disease status at 36 months impacted serum IgA levels. (Mean IgA without progression at 36 months 74.7mg/dL vs 44.9 mg/dL with progression at 36 months; p = 0.035). Conclusions: In our analysis of Pts with CLL treated with an anti-CD20 mAB + HDMP we found a high OR of 94% and median PFS of 27.1 months. This compares favorably to traditional low intensity chemoimmunotherapy approaches for CLL. Pts who received Alem consolidation had durable remissions, with a median PFS of 90.1 months. The overall incidence of infectious complications was manageable, with normalization of serum IgA and IgG three and four years after completion of therapy. Patients without progression at 36 months were found to have higher serum IgA levels. While the Tx landscape for CLL has dramatically changed, the addition of HDMP to an anti-CD20 mAB can be a Tx option for patients desiring short course of Tx, requiring bridging to more definite therapy, or debulking strategy prior to venetoclax based Tx. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Early Treatment with Ofatumumab in Patients with High-Risk CLL

Introduction Early treatment initiation for patients with high-risk CLL has not been shown to be associated with clinical benefit in studies utilizing rituximab and rituximab in combination with alemtuzumab. Ofatumumab is a fully human monoclonal antibody targeting CD20. It is well-tolerated and has improved target binding and cytotoxicity compared to rituximab. We therefore sought to evaluate the efficacy of ofatumumab as early treatment in patients with high-risk CLL. Methods This was a single-arm, open label, phase II trial for treatment-naïve, watch-and-wait patients with high-risk for progression to need treatment. Based on currently available models, high-risk CLL was defined as presence of one or more of the following: Rai stage 2 disease or Rai stage 0-1 with disease related fatigue, β2M ≥ 3mg/L, ALC ≥ 25K/µL, unmutated IGHV, ZAP70-positive, CD38-positive, or FISH with del(11q) or del(17p). Patients who met 2008 iwCLL criteria for treatment were excluded. The primary objective was overall response rate (ORR) with secondary objectives of progression-free survival (PFS), time-to-next treatment (TTNT), and overall survival (OS). Treatment consisted of a total of 8 weekly doses of ofatumumab. The first weekly dose was 300mg to reduce infusion reactions, subsequently ofatumumab 1000mg was then administered weekly for 7 weeks. Results Between 04/2011 and 06/2015, 44 pts were enrolled in the study and initiated treatment. The median age at study entry was 60 years with a median time from diagnosis to study entry of 15.3 mo. The median ALC, HGB and PLT counts were 20.1K/µL, 14mg/dL, and 166K/µL, respectively. The median LDH was 481U/L (range 345 - 855). When characterized based on FISH alterations, 19(43%) pts had del(13q), 7(16%) pts had trisomy 12, 8(18%) pts had del(11q), 3(7%) pts had del(17p), and 7(16%) pts had no abnormalities. When characterized based on other high-risk features: 24(55%) pts had unmutated IGHV, 21(48%) pts were ZAP70(+), 15(34%) pts were CD38(+), 6(14%) had elevated β2M, and 21(48%) had an ALC over 25K/µL. The ORR to ofatumumab (complete + partial remission) was 63% with 3(7%) pts attaining undetectable MRD status at 10-4 sensitivity. At a median follow up of 8 years, the median PFS, TTNT, and overall survival (OS) were 16.3 mo, 42.8 mo, and not reached. Thirty (68%) pts went on to receive further treatment (19 pts on BTK based regimens, 2 pts received venetoclax monotherapy, 5 pts received combined venetoclax and ibrutinib, 3 pts received chemoimmunotherapy, and 1 pt received obinutuzumab monotherapy). This regimen of early treatment with ofatumumab was well tolerated. The most common adverse reaction was infusion reactions occurring in 16 pts (36%) with all reactions being amendable to antihistamine and/or steroid treatment. In all of these pts the infusion was restarted. One pt had to discontinue ofatumumab treatment after the second infusion due to autoimmune hepatitis. Conclusion In a population of patients with CLL at high-risk for proceeding to treatment, early treatment with ofatumumab resulted in a PFS and TTNT of 16.3 mo and 42.8 mo, respectively. The PFS was similar to that of similarly designed trials with rituximab monotherapy and rituximab in combination with alemtuzumab, respectively. Further internal matched analysis is being conducted to compare time to second treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Phase 1/2 Study of Acalabrutinib Monotherapy in Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia: Final Results with >4 Years of Follow-up

Background: Treatment with Bruton tyrosine kinase (BTK) inhibitors has remarkably improved clinical outcomes for patients with chronic lymphocytic leukemia (CLL). Acalabrutinib is a highly selective, covalent BTK inhibitor approved for the treatment of CLL. Here we report the final analysis of the phase 1/2 study of acalabrutinib (multicenter, multicohort ACE-CL-001 study; NCT02029443) with median follow-up of 53 months in patients with relapsed/refractory (R/R) CLL/small lymphocytic lymphoma (SLL). Prior analyses were reported at median follow-up durations of 14 months (Byrd et al. N Engl J Med. 2016;374:323-32) and 41 months (Byrd et al. Blood. 2020;135:1204-13). Methods: Adult patients with CLL or SLL who had received ≥1 prior treatment and had an Eastern Cooperative Oncology Group performance status (ECOG PS) ≤2 were included in the R/R cohort of the study. In the dose-escalation portion of the study, patients were enrolled into dose cohorts at acalabrutinib dosages of 100, 175, 250, and 400 mg once daily (QD) and 100 mg or 200 mg twice daily (BID). In the phase 2 portion of the study, patients received oral acalabrutinib 100 mg BID or 200 mg QD, later switching to 100 mg BID, until progressive disease or unacceptable toxicity occurred. The primary study endpoint was safety. Secondary endpoints included overall response rate (ORR), duration of response (DOR), and progression-free-survival (PFS), with post hoc analysis of event-free survival (EFS). Response rates were based on the iwCLL 2008 criteria with modification for lymphocytosis. Results: In total, 134 patients with R/R CLL/SLL received ≥1 dose of acalabrutinib (dosing information was previously reported; Byrd et al. Blood. 2020;135:1204-13). Median age was 66 years, and 97% of patients had ECOG PS ≤1, 73% unmutated IGHV, 27% del(17p), 20% del(11q), and 35% complex karyotype. The median number of prior therapies was 2 (range 1-13). At the final data cutoff date of July 15, 2021, at a median follow-up of 52.6 (range 0.56-88.8) months, 31% of patients remained on acalabrutinib. The most common reasons for discontinuing treatment were progressive disease (37%) and adverse events (AEs; 15%). The most commonly reported AEs were consistent with those reported in the previous update (Table 1); no new AEs emerged with longer term follow-up. AEs leading to discontinuation that occurred in >1 patient were pneumonia (5 patients), anemia, neutropenia, diarrhea, and thrombocytopenia (2 patients each). Events of clinical interest (all grades; grade ≥3, respectively) included atrial fibrillation/flutter (9%; 3%), hypertension (23%; 11%), infection (88%; 33%), bleeding events (71%; 6%), major bleeding events (8%; 6%), and ventricular tachyarrhythmias (2%, 1%). No cases of sudden death were reported. The ORR (partial response or better) was 90% (95% confidence interval [CI] 84-95); 4% of patients had complete response and 87% partial response (PR). The median DOR was 60.1 months (range 2.7-81.0). The 66-month estimated DOR rate was 45% (95% CI 36-55). Responses were similar regardless of high-risk features, including unmutated IGHV (n=73/81; 90%), del(17p) (n=27/31; 87%), del(11q) (n=20/23; 87%), and complex karyotype (n=17/20; 85%). The median PFS was 66.1 months (range 0.4-87.8; Figure 1), and the median EFS was 53.8 months (range 44.1-66.1). The 72-month estimated PFS was 45% (95% CI 36-54) and the 72-month estimated EFS was 37% (95% CI 29-46). Reported EFS events overall included AEs (n=18/134, 13%), death (n=5/134, 4%), disease progression (n=54/134, 40%), and new anticancer therapy (n=3/134, 2%). Conclusions: The results of this final analysis confirm the earlier reports of acalabrutinib efficacy and were later confirmed by similar outcomes of response durability and long-term tolerability in the pivotal ASCEND phase 3 trial in patients with R/R CLL/SLL (Jurczak et al. J Clin Oncol. 2022;40[suppl 16]:7538). Reported AEs indicate a tolerable and consistent safety profile, with low rates of atrial fibrillation and major bleeding events with median follow-up of 53 months (up to 7.4 years). No new safety signals were identified. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Fludarabine, Cyclophosphamide and Rituximab (FCR) As First Line Treatment in Patients with Chronic Lymphocytic Leukemia (CLL): A Long-Term Analysis of the German CLL Study Group (GCLLSG) Registry

Introduction: Large randomized trials investigating targeted agents have resulted in a markedly improved progression-free survival (PFS) when compared to standard chemoimmunotherapy. However, for small subgroups with a favorable risk profile, overall survival was not consistently superior for targeted agents. Long-term follow up on FCR suggested that favorable risk patients (pts) benefit significantly from this therapy and can expect an exceptionally long PFS. In addition, targeted first line (1L) therapies for favorable risk pts are reimbursed only in a minority of countries. Here, we report long-term efficacy data of FCR treatment obtained from pts documented within the GCLLSG registry. Methods: Documentation within the GCLLSG registry started in 2013 and included both pts from prospective trials (29.7%), most importantly of the CLL8 trial (22.6%) and CLL10 trial (62.1%) as well as retrospective data. Pts with confirmed diagnosis of CLL who received FCR as 1L treatment were included. Patient characteristics were reported in median, interquartile ranges or frequencies and percentages. Response was reported as response or non-response. Event-free survival (EFS), which was defined as the time from start of (first) FCR treatment to disease progression, initiation of subsequent treatment for CLL or death. EFS was chosen as an endpoint for this report due to concerns that PFS might give unrealistically good results due to lack of documentation. However, PFS and TTNT data will be presented at the meeting. Overall survival was calculated from initiation of 1L treatment for all pts who received 1L treatment after 2013. Hazard ratios (HR) and 95% confidence intervals (CI) were calculated by Cox proportional hazards regression. Variables that were significantly associated with EFS in univariate analyses (significance level was set at 5%) were considered as candidates for the multivariate modelling to identify independent prognostic risk factors. Results: As of September 2021, a total of 5922 pts were enrolled in the registry of which 417 received FCR as 1L treatment between 2003 and 2021. 223 pts (53.5%) received treatment before 2013 and 194 (46.5%) after 2013. Median observation time from 1L treatment was 95.8 (interquartile range 58.7-126.8) months. Patient characteristics showed a median age of 58 (range 24-83) years. Unmutated IGHV (U-CLL) was present in 61.4% of the pts. Regarding genomic aberrations based on the hierarchical model, del(17p) was found in 1.9%, del(11q) in 15.2%, trisomy12 in 15.5%, mono-allelic del(13q) in 34.3% and none of these changes in 33.0% of the pts. 88.8% of the pts responded to first line therapy, 11.2% were documented as non-responder. Median EFS was 71.1 month, for 188 pts no event was documented at the time point of data cut-off. Age group (≤ 65 years, >65 years), del(11q), IGHV mutational status, TP53 status, and serum thymidine kinase (TK) were associated with EFS in univariate analyses. Overall, 116 pts with 67 events were included in the final multivariable model for EFS. U-CLL (HR 1.949 (95%CI: 1.170-3.247; p=0.010) and TK >10 U/l (HR 2.107 (95%CI: 1.153-3.849; p=0.015) were independent adverse prognostic risk factors for EFS. When low TK and M-CLL were combined, the median EFS was not reached, with a 5-year survival rate of 95.8%. In contrast, median EFS was 84.1 months for pts with U-CLL and high TK. In U-CLL pts, median EFS was 87.0 months for pts with TK ≤ 10 U/l, and 58.9 months for pts with TK > 10 U/l. Median overall survival was not reached with a 5-year survival rate of 92.7%. Conclusion: Within this cohort of pts with a long follow up in the GCLLSG registry, FCR treatment was associated with a long EFS especially in pts with a low risk profile exhibiting a mutated IGHV status und low TK. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Rapid Generation of Murine CLL/Richter Syndrome Models By Multiplexed CRISPR/Cas9 Editing of Common CLL Driver Genes

Background: Recent large-scale next generation sequencing studies have revealed a highly heterogeneous genetic landscape in CLL. However, the majority of the genetic lesions are relatively infrequent and their functional role and clinical relevance are still unknown. Moreover, in vivo models to study how these lesions cooperate between themselves and affect the response to novel therapeutic agents are still lacking. To further understand the relative contribution of individual genetic lesions to CLL development, progression and response to treatment, we performed multiplex CRISPR/Cas9-editing of normal and leukemic murine B cells by targeting a large set of CLL driver genes. Materials and Methods: Multiplexed CRISPR/Cas9 editing was done on normal B1 and B2 B cells isolated from the peritoneal cavity of young (<4 months) del(13q14)MDR-/- mice or on leukemic B1 B cells isolated from the spleen of old (>12 months) Eμ-TCL1 mice using a recently established procedure (Chakraborty et al, Blood 2021). The effects of the introduced genetic lesions on leukemia cell growth and treatment resistance were investigated by analyzing the mutant allele frequency (MAF) of the injected cells and cells recovered from the spleens of untreated or venetoclax-treated mice. Results: To investigate whether multiplex CRISPR/Cas9 editing can be used to transform normal murine B cells derived from del(13q14)MDR-/- mice, we simultaneously targeted 12 putative CLL driver genes representative of cellular pathways that are commonly affected in CLL, including DNA damage response (TP53, ATM and POT1), chromatin modifiers (CHD2, SETD2), cell cycle (RB1), inflammation (SAMHD1) and the NF-kB (BIRC3, NFKBIE, TRAIL-R), NOTCH (FBXW7) and MYC pathways (MGA) (n=4 experiments). The transfected B1 and B2 B cells were transplanted in the peritoneal cavity of B cell-deficient NSG mice and analyzed at different timepoints within a range of 1 to 5 months. Both cellular subsets were detected in the peritoneal cavity of NSG mice at the initial timepoint, but B1 B cells preferentially expanded at the later timepoints, giving rise to CD19+/CD5+ leukemias 3-5 months after transplantation. An increase in MAF was observed for all targeted genes, suggesting that all of the introduced genetic lesions provide a growth advantage to the leukemic cells. To investigate the impact of selected genetic lesions on CLL progression and Richter transformation (RT), we simultaneously introduced activating mutations in NOTCH1 (targeting of the PEST domain) or inactivating mutations in MGA, ATM, TP53, CDKN2A, CDKN2B, BIRC3 and NFKBIE in CLL cells derived from Eμ-TCL1 transgenic mice (n=3 independent experiments). A rise in MAF was observed for all 8 genes following in vivo propagation of the edited cells, suggesting that all of these mutations are positively selected during leukemia progression. However, selection of NOTCH1, TP53, ATM, BIRC3 and NFKBIE mutations occurred more rapidly, reaching the maximum MAF already after the 1st passage in vivo, whereas the MAF of genetic lesions specifically associated with Richter transformation, i.e. CDKN2A, CDKN2B and MGA, rouse more slowly and increased during serial passages (Figure 1). Interestingly, a substantial increase in MAF was observed for CDKN2A and CDKN2B when cells from the 1st passage were placed in culture (from 44% to 93%, p=0.017, and from 64% to 94%, p=0.043, after 6 weeks in culture for CDKN2A and CDKN2B, respectively), consistent with our recent finding that biallelic disruption of these genes allows for spontaneous proliferation of Eμ-TCL1 leukemia cells and may represent the transforming factor in a subset of cases with Richter Transformation. In contrast, the MAF of the other five driver genes showed no or only minor changes during culture. The MAF of CDKN2A and CDKN2B also increased to a greater extent in mice treated with venetoclax compared to untreated mice, indicating that cells with such genetic lesions are more resistant to venetoclax treatment. Conclusions: This study shows that multiplex CRISPR/Cas9 editing can be used to rapidly generate murine leukemias with the genetic make-up of human CLL or Richter Syndrome. These models can represent an important preclinical tool to study how individual genetic lesions cooperate during CLL development and progression and how they affect the response to treatment with novel therapeutic agents. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Chip-Derived Vs De Novo Treatment-Related Myeloid Neoplasms after Autologous Stem Cell Transplant for Lymphoid Disorders

Autologous stem cell transplant (ASCT) involves high intensity conditioning regimens with significant cytotoxicity. Therapy-related myeloid neoplasms (tMN) represent a serious complication characterized by high-risk features and poor treatment outcomes in patients potentially cured from their primary neoplastic disorders. While the 2016 WHO classification broadly divides tMN into MDS or AML, yet their heterogeneity warrants a more distinctive characterization based on specific prior exposures and subsequently-developed unique features, as emphasized by the updated 2022 WHO revision. We previously analyzed the genomics of tMN including features suggestive of their derivation from either CHIP or de novo driver mutations1. Specifically for tMN pathobiology, ASCT may be very instructive due to the high dose therapy and repopulation of the hematopoiesis out of a limited number of transplanted hematopoietic stem cells. Post-ASCT tMN may be unique and possibly related to antecedent CHIP, CHIP induced by the procedure, or driver lesions. We took advantage of a large volume of ASCT performed at our institution along with excellent clinical annotations and long follow up to study the dynamics of tMN in this setting. We accrued a total of 1507 patients undergone ASCT, of whom 35 (2.3%) developed tMN at a median follow up time of 5.4 years following the procedure. The clinical and molecular phenotype of these index cases were then compared to a cohort of tMN patients (n=263) without prior history of ASCT. Of note, both groups had similar MDS/AML proportions. Compared to the rest of the ASCT cohort, patients developing tMN were older at the time of the transplant procedure (66 vs 46% ≥60 years, P=.02) and had a higher male preponderance (91 vs 59%, P<.0001). Post-ASCT tMN was more common following non-Hodgkin's lymphoma (NHL) than multiple myeloma (MM) (2.5 vs 1.1%, P=.0002) but similar between NHL and Hodgkin's disease (2.5 vs 1.5%, P=0.13). We then studied whether graft size and mobilization regimens could increase the risk of tMN. The incidence of this complications was higher with a mobilization involving GCSF (G), Etoposide (E) and Plerixafor (P) (GEP) rather than G, G+E or G+P (P=.0001), as well as when a CD34+ dose ≤3.5 x106 (4.1 vs 1.9%, P=.03) was infused. No difference was noted with respect to time to engraftment following ASCT. Multivariate analysis confirmed the independent influence of age ≥60 (HR 2.4, 95% CI 1.1-5.6), male sex (HR 7.1, 2.5-29.9), diagnosis (prior NHL vs MM; HR 3.3, 1.5-8.2) and mobilization scheme (GEP vs G+P; HR 4.7, 1.3-13.7). The caveat was the lack of significant effect of the graft size on tMN risk. When compared to non-transplanted cases, tMN following ASCT still showed a male preponderance (91 vs 51%, P<.0001) and exhibited a higher prevalence of high-risk (IPSS-R>3.5) MDS (79 vs 57%, P=.05). Furthermore, tMN arising post-ASCT appeared to follow a more aggressive course with a significantly shorter latency duration following the procedure (median 2.6 vs 6.5 years, P<.0001) and shorter overall survival (median 16 vs 58 months, P<.0001; Fig1). Cytogenetic profiling revealed higher frequencies of complex karyotype (49 vs 31%, P=.05), del(5q)/5- (26 vs 20%, P=.5), del(7q)/7- (46 vs 27.5%, P=.03), and del(17p)/17- (27 vs 6%, P=.0025) in the ASCT-exposed as opposed to non-transplanted cases. Subsequent analysis of isolated cytogenetic aberrations showed that isolated del(7q)/7- was significantly enriched in post-ASCT tMN (20 vs 6%, P=.0129). Mutational analysis revealed similar patterns with the exception of TP53, which was significantly more frequent in the ASCT-exposed group (41 vs 13%, P=.0028), mostly displaying a multi-hit configuration (32 vs 11%, P=.0178). We have previously shown that ancestral hits in DNMT3A, TET2, ASXL1 and JAK2 (CHIP-mutations) indicate derivation of disease from CHIP vsU2AF1, RUNX1 and STAG2 highlighted de novo cases, thereby prompting us to investigate whether CHIP may have been the originator of tMN post ASCT2. However, the frequencies of CHIP- and de novo -mutations were similar across the post-ASCT tMN and the non-ASCT tMN groups. Interestingly, within the post-ASCT tMN, patients who had CHIP-derived tMN had lower survival compared to the rest of the group (median 9 vs 25 months, P=.0079). Sequencing experiments on serial specimens are ongoing to determine whether CHIP- derived mutations were present prior to ASCT constituting a risk for tMN. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clone Size By Conventional Cytogenetics in Myelodysplastic Syndromes Correlates with Clinical Features and Outcomes

Introduction Chromosomal abnormalities are detected in 30-40% of myelodysplastic syndromes (MDS) patients (pts) using conventional karyotyping of 20 metaphases. Conventional cytogenetics remain crucial for risk stratification and certain cytogenetic abnormalities are MDS defining. The Revised international prognostic scoring system (IPSS-R) and its new molecular version (IPSS-M) account for conventional cytogenetic abnormalities regardless of the number of metaphases those abnormalities are detected among (dichotomous assignment). We aimed to assess correlation of "clone size by conventional cytogenetics” defined as percentage of metaphases where chromosome abnormalities are detected with certain clinical features and outcomes in MDS. Methods We retrieved cases from Moffitt MDS database with cytogenetics of interest (i.e. del5q, del20q, del7q/-7, chromosome 3 (ch3), trisomy 8 (+8) and complex karyotype). Inclusion for this study required presence of at least 10 metaphase analysis of chromosome abnormalities by conventional cytogenetics at time of diagnosis or prior to any therapy. We assessed clinical features of interest and outcomes based on % of cells/metaphases harboring the chromosome abnormality using G-banding "conventional cytogenetics clone size". Results This study included 1096 pts, 151 with isolated del5q, 134 pts with del20q, 113 pts with del7q/-7, 38 pts with isolated ch3, 163 pts with +8, and 497 pts had complex karyotype. Detailed disease baseline characteristics, molecular/cytogenetic data, risk stratification and outcomes of all those cohorts were available and reviewed. Among 151 isolated del 5q, pts with >75% metaphases harboring del5q had more severe anemia (hgb < 8.0 g/dl), 29% compared to 10%,12%, and 18% for pts with <25%, 25-50, and 51-75% metaphases, respectively (p .03). Among pts who were treated with lenalidomide (n=98), there was a trend for higher response rate among pts with >75% del5q metaphases (61% vs 46%; p=.09). There was no difference in presence of TP53-m based on % of metaphases harboring del5q. We identified 134 pts with del20q, pts with >75% del20q metaphases had more thrombocytopenia (< 100 u/l), 65% compared to 42%, 26%, and 42% for pts with <25%, 25-50, and 51-75% metaphases, respectively (p .005). For 163 pts with +8, the median OS was 59 months (mo) for pts with <25% +8 detected metaphases compared to 36 mo for those >25%. (p=.05) For 113 pts with del7q/-7, the median OS was worse for -7 (n= 81) compared to del7q (n=32); (18.5 vs 29 mo, p =.024). The median OS was 98 mo for del7q/-7 with <25% metaphases compared to 18 mo for those > 25% metaphases, p=.009. There was no difference in response to hypomethylating agents based on 25% cutoff. We had 38 pts with isolated ch3 abnormality, the median OS was 122 mo for 2 pts with <25% metaphases compared to 24 mo for those >25%, p=.09. Finally, among 497 pts with complex karyotype, the median OS was 20 mo for those with <25% metaphases compared to 12 mo for >25% (p=.01). There was no difference in presence of TP53-m based on % of metaphases. Conclusions The number of cells harboring chromosome abnormalities in MDS using regular karyotyping "clone size by conventional cytogenetics” strongly correlated with clinical features and outcomes. A 25% cut off should be examined in context of IPSS-R and IPSS-M scoring for cytogenetic risk rather than dichotomous assignment to risk group. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Initial Results from SELECT-AML-1, a Phase 2 Study of Tamibarotene in Combination with Venetoclax and Azacitidine in RARA-Positive Newly Diagnosed AML Patients Ineligible for Standard Induction Chemotherapy

RARA-positive AML is a novel genomically-defined subset with an actionable target for treatment with tamibarotene, an oral and selective RARα agonist (McKeown, Cancer Discovery 2017). Approximately 30% of non-APL AML patients are RARA-positive based on overexpression of the RARA gene in peripheral blood blasts as determined by an RT-PCR based assay (de Botton, ESH 2019). In patients with newly diagnosed (ND) AML ineligible for standard induction chemotherapy, tamibarotene/azacitidine led to a CR/CRi rate of 61% in RARA-positive patients with a rapid onset of response. The combination was well-tolerated with no increase in myelosuppression compared to azacitidine (aza) alone (de Botton, ASH 2020). Responses were observed irrespective of mutations or cytogenetic risk. Approximately one-third of ND AML patients ineligible for standard induction therapy do not respond to venetoclax (ven) and nearly all eventually relapse (DiNardo, NEJM 2020). Translational data suggest that RARA-positive patients are enriched for monocytic features based on a positive Monocytic Expression Score (MES) that may be associated with ven resistance (Fiore, ASH 2020), suggesting that the blood-based RARA biomarker test selects for patients who may respond to tamibarotene and who may also be less likely to respond to ven/aza alone. SELECT-AML-1 (NCT04905407) is a Phase 2, open-label, multi-center study in the US and France comparing the activity of tamibarotene/ven/aza to ven/aza in treatment-naive RARA-positive ND AML patients ineligible for standard induction therapy. The 3-part study includes a safety lead-in of the tamibarotene/ven/aza combination, a randomized efficacy study versus ven/aza, and a salvage arm for ven/aza only patients with primary endpoints of safety, CR/CRi rate, and overall response rate, respectively. Following the safety lead-in, patients will be randomized 1:1 to receive tamibarotene/ven/aza or ven/aza. In the salvage arm, patients randomized to ven/aza only who experience progressive disease, treatment failure, or relapse, will have tamibarotene added to their ven/aza regimen. Patients currently enrolled in the safety lead-in are treated with starting doses of aza at 75 mg/m2 IV/SC daily on Days 1-7, ven on Days 1-28 per the ven USPI/SMPC, followed by tamibarotene at 6 mg twice per day PO on Days 8-28 of each 28-day cycle. Enrollment is ongoing. As of 22 June 2022, six patients have enrolled to date, including four who are response-evaluable. Baseline characteristics of the response-evaluable patients include 75% male; median age 57 (55-74); median bone marrow blasts 55% (39-100%); cytogenetically normal (n=2), inversion 16 (n=1), del 5q and -7 (n=1); and molecular genetic testing normal (n=3), mutations of IDH2, BCOR, SRSF2, CSF3R, RUNX1 (n=1). The MES was high in 3 of 4 (75%) response evaluable patients consistent with a prior report in RARA-positive AML (Fiore, ASH 2020). All 4 response-evaluable patients achieved a best response of CR/CRi (2 CR, 2 CRi). The onset of response was rapid with all patients achieving an initial response after Cycle 1 (2 CR, 1 CRi, 1 MLFS). Median duration of treatment was 68 days (21-110). Two patients discontinued, both in remission, one due to death from neutropenic sepsis and the other by physician decision. The adverse event (AE) profile of the triplet was consistent with reports of ven/aza in AML. AEs experienced by 2 or more patients (all grades/causality) included febrile neutropenia, pneumonia, anxiety, cough, diarrhea, fatigue, decreased appetite, myalgia, decreased neutrophil count, and decreased platelet count. The only serious AE (all causality) occurring in more than one patient was febrile neutropenia. A high response rate was observed in early results of tamibarotene/ven/aza in RARA-positive ND AML patients ineligible for standard induction chemotherapy with 100% (4 of 4) response evaluable patients achieving CR/CRi, 3 of whom may have features of ven-resistance based on high MES. The triplet demonstrated an initial safety profile similar to ven/aza. Data from the safety lead-in will inform triplet dosing for the randomized portion of the study.

Myeloid Sarcoma in Myelodysplastic Syndrome : Review

Introduction: Myeloid sarcoma (MS), Granulocytic sarcoma (GS), or Chloroma is defined in the World Health Organization (WHO) classification as a tumor mass consisting of myoblasts with or without maturation and involving any anatomic site other than the bone marrow. Myeloid sarcoma usually founds either concurrently or following a previously recognized (acute myeloid leukemia) AML. Common sites of occurrence are skin, sinuses, bone, and others. It rarely involves the central nervous system and Spinal cord involvement usually manifests as epidural mass causing cord compression. Methodology: A literature search was performed for English language articles using PubMed, Scopus, and Google Scholar, for any date up to June 2022. The following keywords were used "MDS and/or Myeloid sarcoma (granulocytic sarcoma) or Chloroma." Search results were initially reviewed by title and abstract, and articles were selected for more in-depth analysis if deemed relevant. Pertinent articles were examined in-depth for this report. Results: We reviewed 73 articles from the year 1981 to 2021. Males were almost twice the females, with 46 males and 24 females with aThe median age was 67 years. Median complete blood count (CBC) showed hemoglobin (Hgb) of 9.2 g/dL, white blood cells (WBC) of 7.45 x109/L, and platelets of 82 x109/L. The different subtypes of MDS reported were; 11 (15%) patients with CMML, 3 (4%) with del 5q-, and 1 patient had MDS with hypoblastic bone marrow. The most common site of involvement was the skin in 33 (45%) of the patients, followed by lymph nodes in 10 (13%) patients, spinal/para-spinal in 8 (10%) patients, face or ear-nose-throat (ENT) in 7 (9%) patients, Gastrointestinal area I in 6 (8%) patients. Other involved areas include prostate in three patients, bladder in three patients, breast in three patients, brain in two patients, and testicular, bronchial, mediastinum, and vaginal involvement in one patient each (some patients have more than one site involved). Twenty-six (35%) patients were treated with chemotherapy alone or in combination with surgery or radiotherapy, 14 (19%) patients underwent local surgical resection, while 12 (16%) patients received radiation therapy alone or in combination. In addition, two patients underwent an allogeneic bone marrow transplant. On the other hand, 17 (23%) patients did not receive any treatment. Eighteen (24%) patients progressed to acute myeloid leukemia (AML). For overall survival, 35 (47%) patients died. Conclusion: Myeloid sarcoma (MS) is rare with MDS. However, it should be thought of and kept in mind as a differential diagnosis with an initial investigation by imaging and biopsy. Multiple sites can be involved with or without the involvement of the bone marrow with a considerable number of patients progressing to to AML and die. There are no guidelines for treatment up to now. The use of new MDS risk stratification scores, along with cytogenetics and mutations profile, might help to predict who is at risk in the future. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Co-Existing ASXL1 and SF3B1 Mutations Carried Worse Outcome Than ASXL1 or SF3B1 Mutations Alone

Introduction: Somatic mutations in the ASXL1 gene (additional sex combs like 1) are frequently observed in myeloid malignancies, including Clonal cytopenia of unknown significance (CCUS), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), MDS/MPN, and acute myeloid leukemia (AML), and have been found to be independent predicative factor for poor overall survival. Mutations in SF3B1 (Splicing factor 3B subunit 1) were also frequently detected in the above hematopoietic disorders. It represents distinct molecular and clinical patient groups and shows particular association with ringed sideroblasts. Most studies have associated SF3B1 mutations with good clinical prognosis. Both ASXL1 and SF3B1 mutations have been reported to frequently coexist with the mutations in other genes. Co-mutation of ASXL1 with SF3B1 is less frequent and has even been reported to be mutually exclusive by some authors. There are very few and contradictory reports about the clinical significance of co-existing ASXL1 and SF3B1 mutations, which is the goal of this study. Previous studies were also confounded by patients with mutations in some other genes. Method: We identified 175 patients with only ASXL1 mutations, only SF3B1 mutations, or only ASXL1/SF3B1 co-mutations from our database of 8285 patients with NextGen sequencing (NGS) results, to study and compare their clinical features and outcomes. Baseline characteristics and other somatic mutation distribution were compared by chi square (categorical variables) and t-test (continuous variables). Overall survival was calculated using the Kaplan-Meier method log-rank test. Results: Approximately 8285 patients with diagnosed or suspected myeloid neoplasm were tested by NGS. There were1515 patients with ASXL1 mutations with or without other genes mutated, and 583 patients with SF3B1 mutations with or without other genes mutated. There were also 97 patients with concurrent ASXL1 and SF3B1 mutations, most with additional genes mutated. Out of these patients, we identified 69 patients with only ASXL1 mutations (ASXL1 group), 89 patients with only SF3B1 mutations (SF3B1 group), and 17 patients with only ASXL1 plus SF3B1 mutations (ASXL1/SF3B1 group). The median age of ASXL1 group (69) was much younger than SF3B1 (76) and ASXL1/SF3B1 group (p values 0.01208 and 0.0001, respectively). The female to male ratios were similar among these groups. ASXL1 mutations were more frequent in AML and CCUS (22.47% and 33.71%) than ASXL1/SF3B1 (11.76% and 0%) and SF3B1 (1.45% and 7.25%) (p values < 0.003) mutations. On the contrary, SF3B1 and ASXL1/SF3B1 mutations were more frequent in MDS patients (75.36% and 64.71%) than ASXL1 mutations. In MDS/MPN, ASXL1/SF3B1 mutations (23.53%) were more frequent than SF3B1 (15.94%) and ASXL1 mutations (5.62%, p value 0.03513). The three groups of patients showed similar cytogenetic changes, with similar percentages of patients with normal karyotype, del(5q), del(7/7q), del(17p/TP53), del(20q), or trisomy 8. Finally, we compared the overall survival of the three groups of patients with normal karyotype by Kaplan-Meier survival analysis. As expected, ASXL1 group showed worse overall survival than SF3B1 group with a hazard ratio of 5.834 (p value 0.0175). However, it is interesting to found that ASXL1/SF3B1 group showed poorer overall survival than both SF3B1 and ASXL1 groups with a hazard ratio of 9.840 (p value 0.0052) (Figure 1). Conclusion: Co-existing ASXL1 and SF3B1 mutations carried worse outcome than ASXL1 or SF3B1 mutation alone, likely due to abnormalities in both epigenetic regulatory pathway and RNA splicing pathway. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Validation of the 5th Edition of the World Health Organization Classification of Myelodysplastic Neoplasms on 852 Consecutive De Novo Patients from a Single Institute

Background: A summary of the World Health Organization (WHO) 5th edition (2022) classification of myelodysplastic neoplasms (MDS) has been published recently. As compared to the 2016 (revised 4th) WHO criteria, the newest proposal places greater emphasis on genomic and pathology features. In this study, we assess the validity of the WHO 2022 classification for MDS patients. Methods: Data from 852 consecutive subjects ≥ 18 years with newly-diagnosed MDS according to the WHO 2016 criteria in our center from August 30, 2016 to September 22, 2021 were interrogated. Subjects were re-diagnosed and re-classified according to WHO 2022 guidelines. The last follow-up was on June 4, 2022, with a median follow-up of 24 months. Results: Applying the WHO 2022 guidelines, 30 subjects with an NPM1 mutation were re-classified as acute myeloid leukemia (AML) who were previously classified as MDS with excess blast type2 (MDS-EB2; n = 13), MDS with multi-lineage dysplasia (MDS-MLD; n = 9), MDS with excess blast type1 (MDS-EB1; n = 6) and MDS unclassifiable (MDS-U; n = 2) according to WHO 2016 criteria. Nine subjects previously classified as (MDS-U) were re-classified to clonal cytopenia of undetermined significance (CCUS). Re-classification of remaining 813 subjects between the WHO 2016 to WHO 2022 classification are displayed in Figure1. Classification of the 11 subjects with MDS with low blasts and isolated del(5q) (MDS-5q) remained unchanged. In addition to prior MDS with ring sideroblasts (MDS-RS) patients (n=45), 25 subjects were reclassified as MDS with low blasts and SF3B1 mutation (MDS-SF3B1) because the new criteria have no limitation on numbers of ring sideroblasts. We re-classified 53 subjects as MDS-biTP53, most commonly those with excess blasts (34/53; 64%). Amongst subjects without the genetic abnormalities defined above, 80 previously classified as MDS with singe/multi-lineage dysplasia (MDS-SLD/MLD) or MDS-U were re-classified as hypoplastic MDS (MDS-h) and the remaining 293 as MDS with low blasts (MDS-LB). Subjects previously classified as MDS-EB1 or EB2 were re-classified as MDS with increased blasts type1 (MDS-IB1; n = 161) or MDS with increased blasts type2 (MDS-IB2; n = 103) after those with MDS with fibrosis (MDS-f; n = 42) were excluded. Survival according to WHO 2022 classification for MDS are shown in Figure2. Median survival of MDS subjects was 45 months (95% Confidence Interval [CI], [34, 56] months). There were significantly briefer median survivals of subjects with MDS-biTP53 (10 months [8, 12] months) and MDS-f (15 months [8, 23 months]) compared with other subtypes. Median survivals of MDS-h and MDS-LB cohorts were unreached but those with MDS-h had longer survival (P = 0.07). Median survivals for MDS-IB1 and MDS-IB patients were 24 months ([18, 30] months) and 26 months ([17, 35] months), respectively. Survival rate between MDS-IB1 and MDS-IB2 patients were comparable (P=0.707). We next explored the prognostic value of myelofibrosis in subjects without increased blasts. 40 subjects in our cohort had grade-2/-3 myelofibrosis. 37 were re-classified from the MDS-LB cohort and 3 from the MDS-h cohort. Median survival of subjects with low blasts and myelofibrosis (MDS-LBf) was 23 months (8, 38 months), significantly briefer compared with those with MDS-LB or MDS-h (unreached; P < 0.001) comparable to subjects with MDS-IB1 (24 months; P = 0.80) or MDS-IB2 (26 months; P = 0.79) and longer compared with subjects with MDS-f (15 months; P = 0.04). We also tested the prognostic value of dysplastic lineages in subjects with MDS-LB according to WHO 2022 classification. Based on bone marrow histology 28 subjects were considered MDS-LB with single lineage dysplasia (MDS-LB-SLD) and 228, MDS-LB with multiple lineage dysplasia (MDS-LB-MLD). Median survivals of both cohorts were unreached however MDS-LB-SLD had longer survival (P = 0.01). Based on these findings, we suggest the revised WHO 2022 classification divide subjects with low blasts in to MDS-LBf, MDS-h, MDS-LB-SLD and MDS-LB-MLD. The first supersedes other categories and MDS-h overrides the diagnosis of MDS-LB-SLD or MDS-LB-MLD. Conclusion: We validate the WHO 2022 classification of MDS as thoughtful and propose some refinements to provide more accurate prognostic information. Because our study is from 1 center, external validation is needed. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal