17p Deletion Research Articles

CDKN2A-p16 Deletion and Activated KRASG12D Drive Barrett’s-Like Gland Hyperplasia-Metaplasia and Synergize in the Development of Dysplasia Precancer Lesions

Background & AimsBarrett’s esophagus (BE) is the precursor of esophageal dysplasia and adenocarcinoma (EAC). CDKN2A-p16 deletions were reported in 34-74% of BE patients who progressed to dysplasia and EAC, suggesting that p16 loss may drive neoplastic progression. KRAS activation frequently occurs in EAC and pre-cancer lesions. LGR5+ stem cells in the squamocolumnar-junction (SCJ) of mouse stomach, contribute as BE progenitors. We aimed to determine the functional effects of p16 loss and KRAS activation in Barrett’s-like metaplasia and dysplasia development. MethodsWe established mouse models with conditional knockout of CDKN2A-p16 (p16KO) and/or activated KRASG12D expression in interleukin-1b transgenic mice targeting SCJ LGR5+ cells and characterized histologic alterations (mucous-gland hyperplasia/metaplasia, inflammation, and dysplasia) in mouse SCJ. Gene expression was determined by microarray, RNAseq, and immunohistochemistry of SCJ tissues and cultured 3D organoids. Resultsp16KO mice exhibited increased mucous-gland hyperplasia/metaplasia vs. controls (p=.0051). Combined p16KO+KRASG12D resulted in more frequent dysplasia and higher dysplasia scores with 82% of p16KO+KRASG12D mice developing high-grade dysplasia (p=0.0036). SCJ transcriptome analysis showed several activated pathways in p16KO vs. control mice (apoptosis, TNF-alpha/NFkB, proteasome degradation, p53 signaling, MAPK, KRAS, and G1-to-S transition). Conclusionsp16 deletion in LGR5+ cell precursors triggers increased SCJ mucous-gland hyperplasia/metaplasia. KRASG12D synergizes with p16 deletion resulting in higher grades of SCJ glandular dysplasia, mimicking Barrett’s high-grade dysplasia. These genetically modified mouse models establish a functional role of p16 and activated KRAS in the progression of Barrett’s like lesions to dysplasia in mice, representing an in vivo model of esophageal pre-cancer. Derived 3D organoid models further provide in vitro modeling opportunities of esophageal pre-cancer stages.

P53R245W Mutation Fuels Cancer Initiation and Metastases in NASH-driven Liver Tumorigenesis.

Using somatic NASH-driven mouse models with p53 deletion or mutant p53R245W only in hepatocytes, we discovered that p53R245W increased carcinoma initiation, fueled hepatocholangial carcinoma incidence, and tripled metastases.

Network targeting combination therapy of synthetic lethal vulnerabilities in 9p21-deficient glioblastoma: A case report.

Patients with relapsed or progressive glioblastoma only rarely respond to salvage therapies. Nevertheless, comprehensive genomic profiling can provide insight that can identify promising approaches. Signaling pathway analyses have revealed synthetic lethal partnerships, which create the possibility of targeting vulnerabilities arising from the loss of tumor suppressor genes. For synthetic lethal vulnerabilities that are not present in normal tissues, lethal cytotoxicity against cancer cells can be achieved without the necessity of causing normal tissue toxicity. This case report describes a patient with progressive glioblastoma with homozygous deletion of chromosome 9p21. Vulnerabilities created by CDKN2A and MTAP loss were exploited with pemetrexed, bevacizumab, and candesartan to achieve a clinically meaningful remission by targeting multiple synthetic lethal nodes. Synthetic lethality can reveal the basis for exceptional responsiveness, thus extending the utility of molecular profiling and fulfilling the promise of precision medicine.

10084-TB-2 IN VITRO CHARACTERIZATION OF COMBINATION THERAPY IN GENETICALLY-ENGINEERED MURINE DIFFUSE MIDLINE GLIOMA MODELS

Abstract BACKGROUND Diffuse midline glioma (DMG) is an incurable pediatric brain cancer without a FDA-approved drug that prolongs survival. Recent molecular analyses have suggested the potential efficacy of CDK4/6 inhibitors and MEK inhibitors. We recently established a genetically engineered murine DMG model that could capitulate the characteristics of human DMG. Here, we evaluated the features of DMG cells and the efficacy of the CDK4/6 inhibitor ribociclib and the MEK inhibitor trametinib in vitro. METHODS We used the RCAS/Tv-a avian retroviral system to generate DMG in Nestin-expressing progenitors in the neonatal mouse brainstem. We dissociated the DMG tissue to single cells and conducted the spheroid culture. We incubated the DMG cells with a hindbrain organoid to evaluate invasiveness. We treated the DMG cells with CDK4/6 inhibitor ribociclib and MEK inhibitor trametinib to evaluate the synergistic effect. RESULTS Induction of PDGF-A overexpression, H3.3K27M mutation, and p53 deletion resulted in gliomas in murine brainstem. Median survival of the murine DMG model was 62.5 days. From the DMG tissue, we successfully established the DMG cell line with spheroid culture. By incubating with the organoid, DMG cells could invade into the organoid. There was a mild synergistic effect with ribociclib and trametinib both in the cytotoxicity assay and the TUNEL apoptosis assay. CONCLUSIONS We could establish the DMG cell line that maintained the proliferative capability and invasiveness.

Predicting Stage Progression in Binet Stage a Chronic Lymphocytic Leukemia.

The variable clinical course of chronic lymphocytic leukemia (CLL) and the lack of consensus on follow-up and treatment strategies have necessitated a prognostic model for identifying high-risk patients at the time of diagnosis. We involved a retrospective analysis of demographic and clinical characteristics of 212 patients diagnosed with Binet stage A CLL and thus eligible for risk stratification by both the International Prognostic Score for Early-stage CLL (IPS-E) and the alternative IPS-E (AIPS-E). We evaluated the applicability of these prognostic indices in our young, Middle Eastern cohort (median age 59 at diagnosis). During the study period with a median follow-up of 3.5 years, 67 patients (32 %) experienced progression to first treatment and cumulative incidence of treatment was 13 % at 1 year and 28 % at 3 years after diagnosis. Sixty-nine (51 % of the 136 with a known value) patients harbored an unmutated immunoglobulin heavy chain gene (IGHV) and 21 (10 %) an 11q or 17p deletion with 11 % lacking FISH results. For each early-stage CLL prognostic index, more patients were identified as high-risk for disease progression (51 % of 124 patients evaluable for IPS-E; 42 % of 109 patients evaluable for AIPS-E) than intermediate-risk and low-risk. Multivariable models involving the IPS-E and AIPS-E components revealed that unmutated IGHV and elevated absolute lymphocyte count were significant predictors of earlier treatment requirement. Both prognostic scores were discriminative of time to first treatment (log-rank p < 0.001; c-statistics of 0.74 for IPS-E and 0.69 for AIPS-E). Although clarity on clinical behavior with regard to initiation of treatment remains elusive, IPS-E and AIPS-E are valuable tools for identifying high-risk patients.

Bcl-2 inhibition in the treatment of hematologic malignancies

Apoptosis is a tightly regulated process of cell death occurring through extrinsic and intrinsic pathways. The Bcl-2 family of proteins is implicated in the intrinsic pathway and encompasses both pro-apoptotic and anti-apoptotic proteins. Anti-apoptotic Bcl-2 proteins are frequently overexpressed in hematologic malignancies and so Bcl-2 inhibitors have been developed to combat these malignancies. The first and so-far only FDA-approved Bcl-2 inhibitor has been venetoclax, initially for treatment of chronic lymphocytic leukemia (CLL) with 17-p deletion as a second-line agent, followed by later expansion to all CLL and selected acute myeloid leukemia (AML) indications. Venetoclax and inhibitors of other Bcl-2 family members have demonstrated significant potential. However, their use requires careful consideration of disease indication, along with biomarkers associated with disease and optimal drug combinations. Side-effect profiles and specific patterns of resistance must be considered as well. In this review, we examine in detail the characteristics of the Bcl-2 family of proteins and their role in apoptosis. We discuss the drug development process that led to the first-in-class approval of venetoclax, along with relevant use considerations. Finally, we examine future directions in this domain of pharmaceutical development.

Impact of Cytogenetic Abnormalities, Induction and Maintenance Regimens on Outcomes After High-Dose Chemotherapy and Autologous Stem Cell Transplantation in Patients With Newly Diagnosed Multiple Myeloma: A Decade-Long Real-World Experience.

High-dose chemotherapy and autologous stem cell transplant (HDT-ASCT) has become a standard of care for transplant eligible newly diagnosed multiple myeloma (NDMM) patients. While cytogenetic abnormalities have been shown to affect outcomes after HDT-ASCT in clinical trials, these trials often exclude or underrepresent elderly patients with comorbidities and those belonging to ethnic minorities. We describe our institutional experience highlighting the impact of high-risk cytogenetic abnormalities (HRCAs) on outcomes after HDT-ASCT for NDMM patients. A total of 449 patients with NDMM who underwent HDT-ASCT between February 2012 and August 2022 were included in this retrospective analysis. HRCAs included the presence of one or more of: deletion 17p, t(14;16), t(4;14), and amplification 1q. Survival analyses, including progression-free survival (PFS) and overall survival (OS), were performed using Kaplan-Meier estimator. With a median follow-up of 29 (1 - 128) months for the entire patient population, the best overall response rate for the patients with HRCAs was lower compared to those with standard risk cytogenetics (90% vs. 96%; P = 0.01). Patients with HRCAs had an inferior PFS compared to patients with standard-risk cytogenetics (29 vs. 58 months; P < 0.001) without a difference in OS (70 months vs. not reached; P = 0.13). In a multivariable analysis adjusting for factors including age, race, and comorbidities, HRCAs, non-lenalidomide-based maintenance, non-proteasome inhibitor-based maintenance, and age greater than 65 were associated with inferior PFS. Amongst these factors, only non-lenalidomide-based maintenance was associated with inferior OS.

Clinical and genomic differences in supratentorial versus infratentorial NF2 mutant meningiomas.

Mutations in NF2 are the most common somatic driver mutation in sporadic meningiomas. NF2 mutant meningiomas preferentially arise along the cerebral convexities-however, they can also be found in the posterior fossa. The authors investigated whether NF2 mutant meningiomas differ in clinical and genomic features based on their location relative to the tentorium. Clinical and whole exome sequencing (WES) data for patients who underwent resection of sporadic NF2 mutant meningiomas were reviewed and analyzed. A total of 191 NF2 mutant meningiomas were included (165 supratentorial, 26 infratentorial). Supratentorial NF2 mutant meningiomas were significantly associated with edema (64.0% vs 28.0%, p < 0.001); higher grade-i.e., WHO grade II or III (41.8% vs 3.9%, p < 0.001); elevated Ki-67 (55.0% vs 13.6%, p < 0.001); and larger volume (mean 45.5 cm3 vs 14.9 cm3, p < 0.001). Furthermore, supratentorial tumors were more likely to harbor the higher-risk feature of chromosome 1p deletion (p = 0.038) and had a larger fraction of the genome altered with loss of heterozygosity (p < 0.001). Infratentorial meningiomas were more likely to undergo subtotal resection than supratentorial tumors (37.5% vs 15.8%, p = 0.021); however, there was no significant difference in overall (p = 0.2) or progression-free (p = 0.4) survival. Supratentorial NF2 mutant meningiomas are associated with more aggressive clinical and genomic features as compared with their infratentorial counterparts. Although infratentorial tumors have higher rates of subtotal resection, there is no associated difference in survival or recurrence. These findings help to better inform surgical decision-making in the management of NF2 mutant meningiomas based on location, and may guide postoperative management of these tumors.

Comparison of World Health Organization and International Consensus Classification Guidelines for Myeloid Neoplasms Harboring TP53-Mutations Using an Independent International Cohort

BACKGROUND: Recently proposed World Health Organization (WHO, 5 th edition) and International Consensus Classification (ICC) recognized myeloid neoplasms (MN) harboring TP53-mutated ( TP53mut) as a separate entity. However, there are critical differences between the two classifications regarding allelic-status, variance allele frequency (VAF) cut-off and blast categories, that may lead to under- or overestimation of the risk. METHODS: We analyzed MN harboring TP53mut (VAF ≥2%, n=536) managed at Mayo Clinic (USA) and South Australia Health Network (Australia). ICC (Arber et al, Blood 2022) and WHO (Bluebook, Beta v2) definitions were used to stratify MN harboring TP53mut. RESULTS: Of 536 cases, 379 (71%) had myelodysplastic syndromes (MDS) (&amp;lt;20% blasts) and 157 (29%) had acute myeloid leukemia (AML). Using WHO, 160 (42%) of 379 were classified as MDS-bi TP53 as they harbored 2 TP53mut (n=115; 30%) or 1 TP53mut plus confirmed loss of heterozygosity (LOH) or copy neutral LOH (cnLOH) of TP53 locus (n=45; 12%). Sixty-two (16%) TP53mut MDS with VAF &amp;gt;49% were classified as presumptive MDS-bi TP53. Of the 157 cases with 1 TP53mut VAF ≤49%, 17 confirmed to have an intact wild-type TP53 copy and were classified as monoallelic. While 140 (89%) cases did not have copy number variation (CNV) analysis. We next evaluated the number of TP53mut cases classified differently by WHO and ICC. As WHO does not recognize TP53mut AML as a separate entity, 157 (29%) TP53mut AML were not classifiable. 46% (174/379) of the TP53mut MDS including 48 (30%), 12 (19%) and 79 (65%) of bi TP53, presumptive bi TP53 and monoallelic TP53mut MDS according to WHO were classified differently by ICC ( Figure 1A). Discordance between the two classifications were predominantly driven by: (i) The difference in VAF cut-off. Sixty patients (11%) with TP53mut VAF &amp;lt;10% were excluded from the ICC classification of TP53mut MN as it mandates TP53mut VAF cut-off ≥10%. 11 (18%) and 41 (68%) of these had bi TP53, monoallelic TP53mut MDS by WHO criteria while 8 (13%) had AML. Importantly, within the VAF &amp;lt;10% cohort, median overall survival (OS) of 2 TP53mut or 1 TP53mut with 17p13.1 (hereafter 17p) deletion or complex karyotype (CK, n=30) was significantly poor compared to 1 TP53mut VAF &amp;lt;10% (11.6 vs 32.4 months; P =0.002, Figure 1B), suggesting that ICC may underestimate the poor prognosis of multi-hit or multi-hit equivalent TP53mut MN with VAF &amp;lt;10%. (ii) The difference in bone marrow blast cut-off. The underlying assumption by WHO is that prognosis of bi TP53mut MDS is poor irrespective of blast percentage category (0-9% vs 10-19%). In contrast, ICC considers that TP53mut MDS/AML (blasts 10-19%) is associated with poor prognosis irrespective of TP53 allelic status. Median OS of TP53mut MDS with 10-19% blasts was significantly poor compared to blast 0-9% (6.4 vs 11.6 months, P &amp;lt;0.0001). Second, median OS of bi TP53mut was significantly poor than monoallelic TP53mut in MDS- blast 0-9% (10.1 vs 15.9 months; P =0.0001) but not in blasts 10-19% (6.8 vs 6.2 months; P =0.19). Hence, exclusion of monoallelic TP53mut MDS with blast 10-19% as TP53mut MN from the WHO classification may underestimate the poor prognosis. (iii) The difference in the definition of biallelic and multi-hit TP53 loss. 35 cases with 1 TP53mut VAF ≤49%, had 17p deletion but did not have CNV analysis to confirm the loss of TP53 locus and could not be classified further using WHO. ICC classified 18 (50%) of these cases as multi-hit. Median OS of 1 TP53mut VAF ≤49%, and 17p deletion on karyotype was significantly poor than 1 TP53mut (VAF ≤49%without 17p deletion on karyotype (6.4 vs 14.6 months, P =0.001). (iv) The difference in the definition of multi-hit equivalent. 32% (33/104) WHO defined monoallelic cases without available CNV data, were classified as multi-hit-equivalent by ICC as they harbored CK. However, WHO does not consider CK as presumptive evidence of bi TP53. Median OS of 1 TP53mut VAF ≤49% with CK was significantly poor compared to 1 TP53mut without CK (8.2 vs 26.9 months, P =0.0008). CONCLUSION: Our study provides evidence that poor prognosis of ‘multi-hit’ TP53 with VAF &amp;lt;10% is underestimated by ICC. On the other hand, poor prognosis of monoallelic TP53 MDS with 10-19% blasts as well as 1 TP53mut VAF ≤49% in the presence of CK is underestimated by WHO. These data suggest that both classifications can be further refined.

TP53 Mutational Status in Myelodysplastic Neoplasm and Acute Myeloid Leukemia: Impact of Reclassification By Who 2022 and ICC Criteria - a Korean Multi-Center Study

Introduction: The WHO 2022 classification (WHO 2022) introduced the category of myelodysplastic neoplasm (MDS) with biallelic TP53 inactivation (MDS-bi TP53), while the international consensus classification (ICC) included the categories of MDS, MDS/acute myeloid leukemia (AML), and AML with mutated TP53. Particularly in MDS, multi-hit TP53 mutations are clinically important, but there were differences in the criteria defining multi-hit TP53 mutations between the WHO 2022 and ICC classifications. In this study, we investigated the TP53 mutational status in Korean adult patients with MDS and AML and evaluated the impact of TP53 mutation-related criteria of WHO 2022 and ICC on the reclassification of MDS. Methods: This study included patients aged 18 years or older who were diagnosed with MDS and AML from January 2017 to December 2022 at six institutions. The results of bone marrow examination, chromosome analysis, TP53 fluorescence in situ hybridization, and targeted next-generation sequencing including TP53 were analyzed in 1264 patients with MDS and 2112 patients with AML. Multi-hit TP53 mutation was defined based on WHO 2022 and ICC criteria as having any of the following: one TP53 mutation with variant allele frequency (VAF) ≥ 50%, one TP53 mutation with VAF ≥ 10% with 17p deletion, one TP53 mutation with VAF ≥ 10% with complex karyotype (only for ICC), two TP53 mutations with VAF ≥ 10% (only for ICC), or two TP53 mutations (only for WHO 2022). Results: TP53 mutations were observed in 9.3% (118/1264) and 9.1% (192/2112) of patients with MDS and AML, respectively. Among these, 90% of patients had a single TP53 mutation and multiple TP53 mutations were observed in 10% of patients. There was no difference according to MDS or AML ( P=0.478), and 91% of patients with a single TP53 mutation had a VAF of ≥ 10%. Median VAFs of TP53 mutations were 42.7% and 44.4% in MDS and AML, respectively ( P=0.899), and complex karyotype was observed in 76.1% (236/310) of all patients with TP53 mutations without differences between MDS and AML ( P=0.534). In contrast, 17p deletion was identified in 40.3% of AML patients, significantly higher than in MDS (27.4%, P=0.02). Within MDS, 17p deletion was observed in 46.7% in MDS with 10-19% blasts (MDS-H), significantly higher than in MDS with 0-9% blasts (MDS-L, 20.7%, P=0.008), but similar to AML ( P=0.514). When each TP53 mutation-related criterion based on WHO 2022 and ICC criteria were applied to MDS, 55.7% (49/88) and 75% (66/88) of MDS-L met the criteria for multi-hit TP53 mutation and were classified as MDS-bi TP53 (WHO 2022) and MDS with mutated TP53 (ICC), respectively. Notably, more patients were classified into this category based on ICC criteria ( P&amp;lt;0.001), all due to the complex karyotype-related criterion included only in the ICC. Among MDS-H, 63.3% (19/30) of patients were classified as MDS-bi TP53 (WHO 2022) based on multi-hit TP53 mutation criteria, whereas 86.7% (26/30) of patients were classified as MDS/AML with mutated TP53 (ICC) based on the single TP53 mutation criterion with VAF ≥ 10%. Overall, 57.6% (68/118) and 78% (92/118) of patients with MDS and TP53 mutations were reclassified into the TP53 mutation-related MDS or MDS/AML subgroup according to WHO 2022 and ICC, respectively. Conclusions: TP53 mutation status was not different between MDS and AML, but 17p deletion was observed at approximately 40% in MDS-H and AML, much higher than in MDS-L. Multi-hit TP53 mutation based on WHO 2022 and ICC was present at a relatively high frequency at 60-80% in MDS and AML with TP53 mutations. In particular, when ICC criteria were applied to MDS, 20% more patients were reclassified into TP53 mutation-related MDS or MDS/AML subgroup than when WHO 2022 criteria were applied.

Phase I Study of Response-Adapted Treatment with Venetoclax, Obinutuzumab, and Magrolimab (VENOM) in Relapsed and Refractory Indolent B-Cell Malignancies

Background and Significance Targeted agents and immunotherapy have changed the treatment landscape for follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and chronic lymphocytic leukemia (CLL), but indefinite therapy leads to cumulative toxicity and excessive cost. CD47 is a checkpoint for the innate immune system providing an inhibitory signal to phagocytic macrophages and a rational therapeutic target in B-cell lymphoma. Magrolimab is a fully humanized IgG4 anti-human CD47 antibody, with high activity in FL when combined with rituximab (Advani NEJM 2018). Our objective is to test the safety of adding escalating doses of venetoclax to magrolimab and obinutuzumab in a response-adapted study design with planned dose expansion cohorts (www.clinicaltrials.govidentifier NCT04599634). Study Design and Methods Patients with grade 1-3A FL, MCL, MZL, or CLL relapsed after and/or refractory to ≥2 prior lines of therapy are eligible. Pts with FL are eligible after 1 prior therapy if FLIPI ≥2 or disease progression within 24 months of the end of the last therapy. Pts with MCL are eligible after 1 prior therapy if blastoid histology, 17p deletion, TP53 mutation, Ki67 ≥30%, or received Bruton's tyrosine kinase inhibitor (BTKi) as first-line therapy. Pts with CLL are eligible after 1 prior therapy if 17p deletion, TP53 mutation, or received both a BTKi and a BCL2 inhibitor. Eligibility includes age ≥18 and adequate organ function unless dysfunction secondary to lymphoma. A positive direct antiglobulin test without hemolytic anemia is allowed. Patients with a history of hemolytic anemia or autoimmune thrombocytopenia within 3 months of enrollment are excluded. No washout period is required for prior anti-lymphoma treatment. Active HIV, CMV, and hepatitis B or C are excluded. The safety of venetoclax, obinutuzumab, and magrolimab will first be determined in 2 dose-finding cohorts of up to 24 pts (Cohort 1: 6-12 pts with FL and Cohort 2: 6-12 pts with MZL, MCL, or CLL). During dose-finding, all pts start triplet therapy with venetoclax, obinutuzumab, and magrolimab (Figure 1). Magrolimab IV is administered at a 1mg/kg priming dose on D1 of the first cycle, followed by 30mg/kg x 3 weekly loading doses, then 30 mg/kg on D1 of each 4-wk cycle. Obinutuzumab IV is administered at 100mg on D1, 900mg on D2, and 1000mg on D8 and D15 of the first cycle, followed by 1000mg on D1 of each 4-wk cycle. Pts with FL receive venetoclax 800mg (DL1) daily throughout therapy duration, whereas pts with MZL, MCL, and CLL start venetoclax at 20mg with weekly escalation to 400mg (DL1) over 5 weeks. Dose-limiting toxicity is assessed during the first 4 wks for FL, and the first 5 wks for MZL, MCL, and CLL. If ≥2 of 6 pts in either arm experience DLT, 6 additional pts will be enrolled to the respective arm at DL(-1) of venetoclax (600mg for FL and 200mg for MZL, MCL, and CLL). After dose-finding is completed, expansion cohorts of each histology will first receive magrolimab and obinutuzumab for two 4-wk cycles in a window for translational research. On-treatment tumor biopsies are optional during the window. After the window, venetoclax will be added at the dose determined during dose-finding. FDG-PET and CT scans are performed at baseline, after window, and after cycles 3 and 6 of triplet therapy. During both dose-finding and dose-escalation, pts with complete response (CR) after 6 cycles of triplet therapy will stop therapy. If these pts relapse, they can be retreated with an additional 6 cycles of triplet therapy. Pts with partial response (PR) after 6 cycles of triplet therapy receive an additional 6 cycles of triplet therapy. All pts stop treatment after 12 cycles. The primary objective is safety, with secondary endpoints of overall response rate, duration of response, and PFS. Exploratory objectives include the identification of a molecular signature that predicts response to magrolimab and obinutuzumab, and correlation of response with circulating tumor DNA levels.

1q21+ Is Associated with Poor Prognosis in Newly Diagnosed Multiple Myeloma Patients with Extramedullary Disease: A Retrospective Study

Abstract: Background: 1q21+ is a common cytogenetic abnormality in multiple myeloma (MM) and is considered as an independent factor for poor prognosis, but its impact on extramedullary disease (EMD) remains unknown. Method: In our retrospective analysis, patients who met the diagnostic criteria of IMWG and first diagnosed with EMD from January 2011 to May 2022 were enrolled in the study. EMD patients were divided into 2 subgroups: 1q21+ and 1q21- according to the FISH analysis. 1q21+ abnormality was regarded as gain of 1q21 (gain [1q21], 3 copies) and amplification of 1q21 (amp [1q21], ≥4 copies). Continuous variables were analyzed by independent-sample t test or Mann-Whitney U test and categorical variables were compared using chi-square test or Fisher's exact test. Kaplan-Meier method was applied for PFS and OS curve made and differences were evaluated by Log-rank test. Cox proportional hazard models were constructed for prognostic factors analyze. Result: Patients with 1q21+ presented with advanced International Staging System stages (stage III percentage: 68.2% vs. 30.0%, P=0.006), lower level of hemoglobin (91g/dL vs. 113.5g/dL, P=0.004), higher tumor burden (BMPC infiltration≥50% : 45.0% vs. 11.9%, P＜0.001), higher level of serum β2-microglobulin (7.24 g/L vs. 3.85 g/L, P=0.003) and lactic dehydrogenase (LDH) (206.5 U/L vs. 177 U/L, P=0.019). Higher incidence of soft tissue related EMD (54.5% vs. 18.6%, P＜0.001), renal dysfunction (50.5% vs. 17.7%, P=0.002) and hypercalcemia (27.3% vs. 7.1%, P=0.011) was also observed in 1q21+ subgroup. 1q21+ was found to be strongly associated with other high risk cytogenetic abnormalities including IgH/FGFR3 translocation (22.7% vs. 4.3%, P=0.007) and IgH/MAF translocation (22.7% vs. 1.4%, P＜0.001). Patients with 1q21+ had a significantly shorter overall survival (OS) and progression-free survival (PFS) (OS: 24 months vs. 47 months, P=0.002; PFS: 14months vs. 38months, P＜0.001), and the poor survival outcome couldn't be reversed by autologous hematopoietic stem cell transplantation (auto-HSCT). Further investigation estimated the impact of cooccurrence of 1q21+ and del17p on survival outcome. For median OS, 1q21- patients without del17p was 47 months, 1q21+ patients without del17p were 28 months, 1q21- patients with del17p was 22 months, and patients presented with both 1q21+ and del17p was 18 months (p=0.02). The median PFS for 1q21- without del17p, 1q21+ without del17p, 1q21+ with del17p was 38 months, 20 months and 6.5 months respectively, while not reached for 1q21- patients (P＜0.001). Multivariate analysis suggested 1q21+ along with EMD-S, elevated LDH level and P53 deletion were independent risk factors for poor prognosis in EMD patients. For 1q21+ EMD patients, hypercalcemia, elevated LDH level and P53 deletion were defined as independent adverse risk prognostic factors. Conclusion: Taken together, our findings emphasized that 1q21+ increased the possibility of disease progression and predicted poor survival in EMD patients. 1q21+ EMD tends to be associated with other high risk disease factors. ASCT may not overcome the adverse effect of 1q21+ in EMD patients. We suggested 1q21+ should be considered as a high-risk factor and added into routine assessment of risk stratification in EMD patients.

MS-553 Therapy Inhibits BCR Signaling and Increases Survival Dependence on BCL-2 in BTK-Inhibitor Resistant CLL

B-cell receptor (BCR) signaling pathway inhibitors and B-cell lymphoma-2 (BCL-2) antagonist venetoclax (Ven) have transformed the treatment of chronic lymphocytic leukemia (CLL), including in high-risk patients with 17p deletion and/or TP53 mutations and complex karyotype. Despite the durable remissions observed with inhibitors of Bruton's Tyrosine Kinase (BTKi), a significant proportion of patients develop resistance with acquired mutations in BTK and its downstream target phospholipase Cγ2 ( PLCG2). Patients who progress on BTKi including ibrutinib (ibr) often receive Ven but eventually relapse, underscoring an urgent need for new treatment strategies. Protein kinase C-β (PKC-ß) is a protein downstream of BCR signaling (BTK-PLCG2) and is essential for CLL cell survival and proliferation in vivo. Here we investigate MS-553, an inhibitor of PKC-ß, with a focus of combination strategies in CLL patient samples including relapsed/refractory CLL. We show that CLL cells in contact with stroma upregulate PKC-ßII, its immediate downstream effectors, pGSK and pERK as well as ß-catenin in CLL patients including those with mutant TP53 and 17p (n=8). Correspondingly, MS-553 (5µM, 48h) exposure reduced levels of p-PKC-ßII and its downstream effectors (p-ERK, p-GSK) in a dose dependent manner regardless of TP53 mutation in both CLL cells and those supported by NK.Tert (stromal cell line derived from bone marrow, BM, and mimics BM microenvironment). Pre-treatment (48h, 5µM MS-553) of stromal NK.Tert cells with MS-553 prevented the upregulation of PKC-ßII within adjacent CLL cells implicating the stroma as a major source of PKC-ß activation in CLL cells. We also tested whether MS-553 could inhibit PKC-ß signaling in CLL cells exposed to phorbol myristate acetate (PMA-400nM, 90 min), which simulates BCR activation. MS-553 (5µM, 48h) exposure caused downregulation of BTK effectors (pPKC-ßII, pERK, pGSK), BCL-2 family member proteins (MCL-1 and BCLxl), and ß-catenin (n=10). In parallel experiments, we used BH3 profiling to show that CLL cells' (n=8) exposure to MS-553 in vitro developed increased dependence on BCL-2 and BCLxl for survival (Bim, Bad, XXA1_y4ek, 5-fold± 2-fold increase) in both wild type (WT) and mutant (Mut) CLL cells, suggesting a mechanistic rationale for synergy with Ven. To confirm this, CLL cells were exposed to MS-553, Ven or a combination of MS-553 and Ven. ~3-fold and 9-fold synergistic increase in the mitochondrial dysfunction (measured by cytochrome C release, 12h, 5µM MS-553, 1nM Ven) was observed in stromal supported WT and Mut CLL respectively in MS-553 versus Ven+MS-553 treated group. Similarly, ~3-fold increase was observed in WT and Mut CLL alone group and more than 10-fold increase was found in stromal supported WT and Mut group in cell death determined as percent apoptotic index (measured by annexin V-PI staining, 18h, 5µM MS-553, 1nM Ven) in MS-553 versus Ven+MS-553 group respectively. Finally, we validated our preclinical findings in CLL samples obtained from four patients on MS-553 therapy (NCT03492125, Range = cycles 0-19, MS-553 28 days/cycle). All patients showed an increase in survival dependence towards BCL-2 and BCLxl by Cycle 3-6 of MS-553 therapy. This was accompanied by downregulation of ß-catenin, pPKC-ßII, pGSK, pERK along with BCLxl and MCL-1 after Cycle 10 or later of MS-553 therapy (data summary table1). Overall, our data provides clear evidence that MS-553 inhibits BCR signaling in vitro and in patients on MS-553 therapy. It also provides a rationale for combinations with Ven as a treatment partner based on its ability to increase survival dependence on BCL-2.

Spatially Resolved Transcriptomics Unveils Unique T-Cell Dysfunctional States and Prognostic Gene Expression Signatures in TP53-Mutated Acute Myeloid Leukemia

Introduction Somatic TP53 mutations and 17p deletions with genomic loss of TP53 occur in 8% to 10% of AML and correlate with primary induction failure, high risk of relapse, and dismal prognosis. We have previously shown that TP53 abnormalities are associated with a pro-inflammatory and immunosuppressive tumor microenvironment (TME), including high CD274 and FoxP3 expression, with dysfunctional natural killer (NK)/CD8 + T-cell states and with response to flotetuzumab, a CD123 × CD3 bispecific T-cell engager. In the current study, we explore the cellular neighborhoods of TP53-mutated ( TP53-m) AML using spatially resolved transcriptomics and we employ machine learning to identify prognostic RNA signatures. Methods We analyzed the expression of &amp;gt;1,800 immune and TME genes in bone marrow (BM) formalin-fixed paraffin-embedded (FFPE) sections (5-µm) from a discovery cohort of 11 patients with chemotherapy-naïve AML (N=5 TP53-m and N=6 TP53-wt cases) using the GeoMx™ Digital Spatial Profiling (DSP) platform (Cancer Transcriptome Atlas with next generation sequencing read-out; NanoString Technologies, Seattle, WA). Imaging was performed at 20× magnification and a total of 151 circular regions of interest (ROIs) with a 300-600 µm diameter were selected (Fig. 1A). We then generated a ROI-by-gene count matrix for deconvolution and downstream pathway analysis. For each ROI, enrichment scores reflecting immune senescence/dysfunction and leukemia stemness were calculated with gene set variation analysis (GSVA), using a manually curated compendium of published gene signatures as input. We also employed public bulk and single-cell RNA-sequencing (sc-RNA-seq) AML cohorts for in silico modeling and validation. Results We initially captured transcripts from ROIs of TP53-m and TP53-wt AML with varying degrees of BM infiltration with T cells (CD3 hotspots) and AML blasts, and we found that TP53-m is enriched in T cell-specific, type I/II interferon (IFN) and pro-inflammatory genes ( IL1B), as well as leukemia stem cell (LSC)-related gene programs (LSC17). After dichotomizing ROIs based on a median split of CD3D RNA expression, we observed that T-cell low ROIs express higher levels of monocyte-macrophage and myeloid derived suppressor cell (MDSC)-related genes, including S100A9 and S100A8. These findings point to previously unappreciated links between BM infiltration with immune-modulatory myeloid cell types and leukemia stemness / T-cell abundance. We then tested for differential cell density in neighborhoods (regions of transcriptionally similar cells) across TP53-m and TP53-wt AML using MiloR in a sc-RNA-seq AML dataset annotated by cell type (N=42 cases, of which N=3 with TP53-m; Lasry A, et al. Nat. Cancer 2023). This orthogonal approach highlighted a significantly higher frequency (FDR &amp;lt;0.05) of multipotent and granulocyte-macrophage progenitor neighborhoods in TP53-m AML, alongside suppression of adaptive immune system pathways (Fig. 1B). We next inferred active receptor-ligand pairs mediating the crosstalk between T cells and leukemic blasts in the TME of TP53-m AML, and we uncovered decreased MHC class I/II-mediated signaling, curbed CD8 +/CD4 + T cell-to-AML interactions and enhanced CD6/ CDCP1( CD318) signaling (Fig. 1C). We also found that CD8 + and CD4 + T cells from TP53-m AML are enriched in effector memory (T EM) / terminally differentiated effector memory (T EMRA) and regulatory T-cell gene programs, respectively, compared with their cellular counterparts from TP53-wt patients (Fig. 1D). In particular, T EM cells from TP53-m AML expressed genes known to regulate T-cell dysfunctionality, foremost PRDM1, and transcription factors ( ZEB2) expected to drive the differentiation of killer cell lectin-like receptor (KLR) + exhausted T-cell states with residual cytolytic potential. Finally, we applied the least absolute shrinkage and selection operator (LASSO) statistical method for feature selection to our discovery AML dataset, and we constructed a TP53-m-related ‘spatial metagene’ that predicted poor overall survival in TCGA-LAML (N=170) and Beat-AML2 cases (N=444; Fig. 1E). Conclusions Spatial gene programs of leukemia stemness, T-cell dysfunction and immune suppression are enriched in TP53-m AML. It remains to be determined whether TP53-m AML with a pre-existing but ineffective T-cell response may be amenable to T cell-targeting immunotherapies.

Immune Dynamics Associated with Patient Outcomes Identified By Multimodal Single-Cell Analysis in Multiple Myeloma Patients Receiving Talquetamab Monotherapy

Background: Talquetamab (Tal) is a CD3xGPRC5D bispecific antibody (BiAb) that redirects endogenous T cells to mediate killing of GPRC5D-expressing multiple myeloma (MM) cells. In the recent phase I/II MonumenTAL-1 trial (NCT03399799), Tal showed ≥ 70% response rate when given at the recommended phase 2 doses (RP2D) in heavily pretreated MM patients (Chari A et al., NEJM 2022). However, there is still a need to identify predictive biomarkers and better understand the immune dynamics associated with treatment response and durability with this novel agent. Methods:We identified 30 patients who received Tal monotherapy and had bone marrow (BM) and peripheral blood (PB) samples collected at baseline and PB collected at cycle 3 day 1 (C3D1). Cellular indexing of transcriptomes and epitopes (CITE)-seq was performed to understand transcriptional changes and cell surface proteomics. Total cells were isolated by Ficoll separation from PB and BM samples and were then analyzed separately and only samples of good quality were included in the analysis. Samples were filtered for good quality based on total cell count: at least 1000 cells with more than 500 unique molecular identifiers (UMIs) or genes and less than 4-5 standard deviations above median for UMI, genes and mitochondrial transcripts. A total of 60,851 BM cells (15 pts at baseline) and 299,996 PB cells (23 pts at baseline and 17 pts on C3D1) were analyzed. Downstream analysis was performed using the Seurat R package. All patients included in this study had signed consent for an IRB-approved institutional sub-study (GCO # 18-00456). Results: The 30 patients had a median age of 66.2 years and 57% were female. They received a median of 6 prior lines of therapy, with 83% being triple-class refractory and 50% penta-drug refractory. Only 2 patients were previously treated with anti-BCMA CAR-T cell therapy and none were treated with other BiAbs. 43% of patients had high-risk cytogenetics (including t(4;14), t(14;16) and 17p deletion) and 13% had extramedullary disease at time of initiating therapy. Ten patients (30%) received the recommended phase 2 dose (RP2D) of Tal (0.4 mg/kg weekly or 0.8 mg/kg every other week). Patients were followed for a median of 32.5 months and had a median progression-free survival (PFS) of 6.7 months. 22/30 patients (73%) achieved a partial response (PR) or better, and 14 patients had a short PFS of &amp;lt;150 days. There was no association between the prevalence of different immune cell subsets and depth of response, but rather with PFS. In the baseline BM, a longer PFS was associated with a higher percentage of CD8+ cytotoxic T cells (central memory p=0.00031, effector memory p=0.0022, EMRA p=0.014) and a trend towards higher CD4+ cytotoxic T cells (p=0.072). Of note, we did not find the same associations in the baseline PB samples, which may indicate that the PB may not always reflect the tumor microenvironment in the BM. When we next looked at immune changes in the PB of patients with longer PFS, we found that they exhibited higher percentage of NK cells (p=0.0018 for CD56 low, p=0.04 for CD56 high) and Gamma Delta T cells (p=0.04) at C3D1. Gamma Delta T cells are novel players that warrant further investigation, as they only constitute 1-5% of the T cell repertoire yet are involved in early immune response and do not require major histocompatibility complex (MHC) antigen presentation to function. Profiling of activation and exhaustion markers of these immune subsets is underway. Conclusion: Our findings highlight that the baseline BM immune repertoire and the PB immune changes during Tal treatment may predict long term outcomes. Further in vitro mechanistic studies of T cell function and fitness and validation of these results in independent cohorts will be critical to develop these into predictive biomarkers of long-term response to Tal.

Extended Follow-up and Resistance Mutations in CLL Patients Treated with Acalabrutinib

Background: Covalent Bruton tyrosine kinase inhibitor (cBTKi) is a standard treatment for chronic lymphocytic leukemia (CLL). Understanding of acquired resistance to cBTKi therapy has largely come from data on patients (pts) treated with the first-in-class cBTKi ibrutinib. Mutations in BTK C481 and to a lesser extent PLCG2 are frequently detected in ibrutinib-treated pts at the time of progressive disease (PD). In contrast, much less is known about genetic mechanisms of drug resistance in pts treated with next generation cBTKi acalabrutinib and zanubrutinib. A recent study identified 3 pts with BTK C481 mutations and no pts with PLCG2 mutations among 8 acalabrutinib-treated pts with progressive disease (PD) (Black, et al. Blood 2022). A second study found a kinase-dead BTK L528W mutation in 7/13 zanubrutinib-treated pts (Blombery, et al. Blood Adv 2022). Although cBTKi share the same mechanism of action by binding to BTK C481, the latter report discloses differences in clonal selection and underscores the need for further investigation in pts treated with next generation cBTKi. In a phase 2 study of acalabrutinib in pts with treatment naïve (TN) CLL or small lymphocytic lymphoma (SLL) with 17p deletion, or TP53 or NOTCH1 mutation, or relapsed/refractory (R/R) CLL/SLL regardless of cytogenetics or somatic mutations (Sun, et al. Blood 2020), we sought to understand clonal evolution during treatment. Methods: Peripheral blood mononuclear cells (PBMCs) were collected from 48 pts at baseline, after 6 months, then annually on treatment until PD. Current analysis includes 14 PD pts [excluding 3 Richter transformation (RT) pts] comprising 10 R/R and 4 TN CLL pts. Additionally, paired baseline and PD bone marrow (BM) from 6 pts and lymph node (LN) samples from 2 pts were included. Targeted next-generation sequencing of ~700 recurrently mutated genes in hematological malignancies (Collins, et al. Leuk Lymphoma. 2021) was performed using DNA isolated from CD19 + enriched cryopreserved PBMCs and BM aspirates, as well as fresh-frozen core LN biopsies. Filtered variants in putative CLL driver genes (Knisbacher, et al. Nat Genet. 2022) with variant allele frequencies (VAF) ≥1% for BTK and PLCG2, ≥5% for variants with known oncological significance, and ≥7.5% for all other variants were included in downstream analysis. Results: After a median follow-up of 6.5 years (IQR 2.9-6.3), 23/48 (48%) pts developed PD (20 CLL, 3 RT). PD occurred in 17/32 (53%) R/R pts and 6/16 (38%) TN pts. Median progression-free survival was reached at 6.0 years. Best response was complete remission in 2/20 (10%) pts and partial remission in 18/20 (90%) pts with CLL PD. IGHV was unmutated in all but one PD patient. At baseline, 34 CLL driver genes were mutated in 41/45 pts (91%; excluding 3 RT pts) with most mutations in NOTCH1, TP53, ATM, SF3B1, BIRC3, and NFKBIE. During acalabrutinib treatment but before PD, 14/45 pts (31%; 8 R/R and 6 TN) acquired mutations in 11 driver genes, most commonly in BRAF (n=4), CREBBP (n=4), and KMT2D (n=2). At the time of PD, 11/14 pts (79%) had newly acquired mutations in 4 genes, predominantly BTK (n=6) and PLCG2 (n=4). BTK C481 mutations (VAF 2-42%) were detected in 6/14 (43%; 4 R/R and 2 TN) pts and T474I mutation (VAF 4-46%) in 3/14 (21%; 1 R/R and 2 TN) pts. Paired BM samples in 2 pts and LN samples in 2 pts showed the same BTK mutations as PBMCs with similar VAFs. PLCG2 mutations (VAF 1-8%) were found in 4/14 (29%; 2 R/R and 2 TN) pts. BTK C481 co-occurred with BTK T474I in 3 pts, but on different sequencing reads suggesting independent subclones, and with a PLCG2 mutation in 1 pt. There have been no pts with BTK L528 mutations. In conclusion, mutations in BTK and PLCG2 are the predominant resistance mechanism to acalabrutinib. Among BTK mutations, C481 was the most common. All T474I mutations co-occurred with C481.

Real-World Utilization of Daratumumab in Front-Line Treatment of Newly Diagnosed Multiple Myeloma: A Retrospective Observational Study

Introduction: Daratumumab (DARA), a CD38 monoclonal antibody, has demonstrated remarkable efficacy among newly diagnosed (ND) and relapsed/refractory multiple myeloma (MM) patients. The introduction of a quadruplet therapy with an addition of DARA per the GRIFFIN trial has improved the treatment paradigm. However, the real-world data on treatment patterns and utilization of DARA in front-line treatment are limited. This study aimed to evaluate the real-world utilization of DARA in front-line treatment and examine factors that may impact the utilization of DARA-based regimen in NDMM. Methods: We conducted a retrospective observational study of all patients, age ≥ 18, with clinical MM who were newly diagnosed and treated within the Yale New Haven Health (YNHH) system between August 1, 2020 and August 31, 2022. We manually reviewed clinical presentation, age at diagnosis, gender, race, ethnicity, Revised International Staging System (R-ISS) stage, Eastern Cooperative Oncology Group (ECOG) performance status (PS), insurance type, practice setting, immunoglobulin (Ig) isotype, hemoglobin (Hb), creatinine (Cr), and calcium (Ca) at diagnosis, presence of bone lesion, bone marrow plasma cell (BMPC) %, cytogenetic abnormalities, transplant eligibility, and treatment response based on the International Myeloma Working Group (IMWG) criteria at 3 months after front-line therapy. Descriptive statistics were performed with Pearson's χ²-test using GraphPad PRISM version 9.0.0. Results: We identified 241 patients with NDMM during the study period within the YNHH system. Overall median age at diagnosis was 69 years (interquartile range: 61-78). 60.2% (n=145) were male, 68% (n=164) identified themselves as white and 90% (n=217) as non-Hispanic. 51.9% (n=125) had R-ISS stage II and 65.6% (n=158) had ECOG PS of 0-1. 63.1% (n=152) were treated at a community practice setting and 53.9% (n=130) had commercial insurance. 55.6% (n=134) had IgG isotype and 41.1% (n=99) had unfavorable risk cytogenetics which was defined as a 17p deletion, t(4;14), t(14;16), t(14;20), gain or amplification of 1q, 1p deletion, and chromosome 1 translocations. 28.2% (n=68) received front-line quadruplet therapy: DARA+lenalidomide (LEN)+bortezomib (BORTE)+dexamethasone (DEX) (n=54), DARA+cyclophosphamide+BORTE+DEX (n=10), or DARA+LEN+carfilzomib (CARFIL)+DEX (n=4). 58.5% (n=141) received triplet therapy, 8.3% (n=20) received doublet therapy, 0.83% (n=2) received monotherapy, and 4.1% (n=10) opted for hospice care. 41.5% (n=100) received DARA in front-line treatment: quadruplet as above (n=68), triplet of DARA+LEN+DEX, DARA+BORTE+DEX, or DARA+CARFIL+DEX (n=31), and doublet of DARA+DEX (n=1). Factors including gender (male vs. female, p=0.018), practice setting (academic vs community, p=0.014), R-ISS stage (I vs II vs III vs unknown, p=0.003), Ig isotype (IgG vs IgA vs light chain vs other, p=0.006), cytogenetic abnormalities (standard risk vs unfavorable risk, p=0.02), and transplant eligibility (transplant eligible vs transplant ineligible, p=0.001) were associated with the adoption of DARA in front-line treatment. While 51.7% (n=46) of the patients treated at an academic practice setting received DARA in front-line treatment, only 35.5% (n=54) of the patients treated at a community practice setting received such treatment. 69% (n=69) of those who received DARA-based regimen in front-line treatment achieved at least a very good partial response (VGPR) at 3 months compared to 54.6% (n=77) of those who received non-DARA-based regimen. There were no significant differences in age, race, ethnicity, ECOG PS, insurance type, laboratory data (Hb, Cr, and Ca) at diagnosis, presence of bone lesion, and BMPC % between the group treated with DARA-based regimen and the group treated with non-DARA-based regimen. Conclusion: This analysis of real-world data from patients with NDMM highlighted underutilization of DARA in front-line treatment, especially at a community practice setting. Gender, practice setting, R-ISS stage, Ig isotype, cytogenetic abnormalities, and transplant eligibility were significantly associated with the adoption of DARA in front-line treatment. Additionally, utilization of DARA in front-line treatment was associated with a better treatment response at 3 months, suggesting that interventions to promote the utilization of DARA will help improve outcomes of NDMM patients.

Isatuximab-Based Regimens As Salvage Therapy for Patients with Multiple Myeloma: Sardinian, Multicenter, Retrospective Clinical Practice Experience with 27 Cases Outside of Controlled Clinical Trials

Introduction: Isatuximab (Isa), a humanized monoclonal antibody directed against the surface antigen CD38, has been available in Italy since October 2021 for the treatment of relapsed or refractory Multiple Myeloma (RRMM) in combination with Pomalidomide (P) and Dexamethasone (d) (IsaPd) after two lines of therapy with lenalidomide and a proteosome inhibitor, and from April 2022 in combination with carfilzomib (K) after one line of treatment. Patients eligible for these therapies must not be refractory to Daratumumab. Here, we report preliminary efficacy and safety data for Isatuximab in a Sardinian study of RRMM patients treated with IsaPd and IsaKda as salvage therapy outside of controlled clinical trials. The primary objective of this study was to evaluate the toxicity profile and quality of response to Isatuximab-based regimens administered as salvage therapy in a real-world setting. Methods: The 2 cohorts included 27 patients with RRMM from 4 Sardinian centers who received at least one cycle of IsaPd (16 patients) or IsaKd (11 patients) as salvage therapy between January 2022 and March 2023. Patients received Isa 10 mg/kg IV weekly for the first 4 weeks (wks), then every 2 weeks in combination with Pomalidomide and Dexamethasone (4mg PO days 1-21; 40mg [20mg at &amp;gt; 75 years] PO or IV weekly) every 28 days for the IsaPd regimen. The IsaKd regimen consisted of administration of 10 mg/kg Isatuximab intravenously once weekly in the first cycle, then once every 2 weeks in subsequent cycles. Both arms received Carfilzomib at a dose of 20 mg/m2 on days 1 and then 56 mg/m2 on days 8, 9, 15, and 16 of the first cycle, then 56 mg/m2 on days 1, 2, 8, 9, 15, and 16 in subsequent cycles, while 20 mg of Dexamethasone was administered on days 1, 2, 8, 9, 15, 16, 22, and 23 of each cycle. Results: The median age of the 16 IsaPd-treated patients was 67.5 years (range 48-89 years) and 61 years (range 49-84 years) for the IsaKd population; 44% and 64% were male, respectively (Table 1). The median number of prior therapies was 2 (range 2-4) for the IsaPd and 1 (range 1-4) for the IsaKd cohort; 11 (68.75%) and 9 (82%) received prior autologous stem cell transplantation (ASCT), respectively. Four patients in both populations started treatment because of biochemical relapse, and no refractory patients were recorded in the IsaKd cohort. Three patients (2 in the IsaPd population and 1 in the IsaKd cohort) had creatinine clearance ≤30 mL/min. Among the IsaKd-cohort, 5 (46%) had R-ISS 2 stage and three had a 17p deletion. At the last data collection, 13 cases in the IsaPD population were evaluable for response. The median number of courses administered to date was 4 (range, 1-14). The overall response rate (ORR) was 69%, with 2 stringent complete remissions (sCR) (15%) and 1 very good partial remissions (VGPRs) (7.7%). The median time to first response was 1 month, while the median time to best response was 2 months. In the IsaKd population, 10 patients were evaluable with an overall response rate (ORR) of 100%, with 1 complete remission (sCR) (10%), 1 complete remission (10%), and 5 very good partial remissions (VGPRs) (50%). A median time to first response of 1 month was also recorded in this population, whereas the median time to best response was 3 months. Renal insufficiency and age &amp;gt; 70 years had no significant impact on the likelihood of achieving a response in either population.In the IsaPd cohort, after a median follow-up of 6 months (range 1-14 months), 9 patients discontinued treatment, 7 due to disease progression, 1 due to toxicity, and 1 due to patient choice) Six patients died (5 patients from progressive disease and 1 from secondary acute myeloid leukemia). Common grade 3 or 4 adverse events were fatigue (57%), thrombocytopenia (35%), neutropenia (57%), lymphocytopenia (15.9%), anemia (29%), and infection (21%). Infusion reactions occurred in only 2 patients (12.5%) and were grade 1 or 2 without treatment discontinuation. In the IsaKd population, the median follow-up time was 4 months (range 1-8 months). 1 patient discontinued treatment and died due to disease progression. Grade 3 or 4 adverse events were fatigue (18%), thrombocytopenia (18%), neutropenia (18%), anemia (36%), and infection (63%) No infusion reactions occurred. Conclusions: Despite the limited number of cases, our preliminary real-world data confirm that Isa-based treatment is an effective and safe therapy for RRMM patients, broadly consistent with results from controlled clinical trials.

Seven-Day Vein-to-Vein Point-of-Care Manufactured CD19 CAR T Cells (GLPG5201) in Relapsed/Refractory CLL/SLL Including Richter's Transformation: Results from the Phase 1 Euplagia-1 Trial

Background Despite treatment advances, chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) remain incurable. Patients (pts) with concurrent Richter's transformation (RT) have poor prognoses with limited treatment options. Chimeric antigen receptor (CAR) T-cell products targeting CD19 have shown activity in pts with CLL/SLL or RT, yet data are limited and no therapies are approved. Due to limited treatment options and short survival of pts with RT, rapid treatment delivery is essential. A novel decentralized and automated point-of-care (PoC) manufacturing model was developed to administer fresh, autologous CAR-T treatments within 7 days of apheresis. An update on the Phase (Ph) 1 Euplagia-1 trial of PoC manufactured GLPG5201 is presented. Methods Euplagia-1 (CTIS: 2022-501686-47-00) is a Ph1/2 study of PoC-manufactured GLPG5201 in pts with R/R CLL or SLL, including pts with RT. Pts with CD19+ R/R CLL/SLL with ≥2 prior therapy lines are eligible. Pts with RT are eligible regardless of prior therapy. GLPG5201, an anti-CD19/41BB CAR-T therapy, is administered as a fresh product following fludarabine/cyclophosphamide lymphodepleting chemotherapy. Upon screening, pts receive daily ibrutinib until leukapheresis. The primary objectives are safety and determining a recommended Ph2 dose (Ph1), and objective response rate (ORR; Ph2). Secondary objectives include additional efficacy measures, pharmacokinetics and feasibility of PoC manufacturing. Results As of April 26, 2023, 12 pts were enrolled in Ph1 at dose level (DL) 1 (35×10 6 CAR+ T cells, n=6) or DL2 (100×10 6 CAR+ T cells, n=6) at a single institution. All pts were diagnosed with R/R CLL, 7/12 with concurrent RT. Median age was 66 years (range 58-71); 8/12 pts were male. Median prior lines of therapy were 4 (range 2-10); 10/12 pts received a BTK inhibitor, 9/12 pts venetoclax, and 1 pt an allogeneic stem-cell transplant. Five of 7 pts with RT relapsed after, or were refractory to, R-CHOP. Six of 12 pts had a TP53 mutation, 1 pt had a 17p deletion; 11 pts had an unmutated IGHV status and 2 had a complex karyotype reported. GLPG5201 was manufactured for all pts and administered as a fresh infusion. Median vein-to-vein time was 7 days (range 7-14). Due to lower manufactured CAR+ T cell yield, 3 pts received DL1 instead of the intended DL2. A preserved early memory phenotype for CD4+ and CD8+ CAR T cells was observed in the final product, compared with apheresis starting material ( Figure). Most treatment emergent adverse events (TEAEs) were Grade 1-2; most Grade ≥3 events were hematological ( Table). Six (50%) pts had Grade 1-2 cytokine release syndrome (CRS), with no CRS Grade ≥3. No neurotoxicity was reported. A dose limiting toxicity occurred in 1 pt (Grade 4 neutropenia, DL2), which was manageable and within expectations in this pt population. No unexpected GLPG5201-related toxicities were observed; no deaths occurred while pts were on study. Eleven pts responded to treatment (best ORR 92%). Complete response (CR) was observed for 9 pts (CR rate [CRR] 75%). ORR and CRR were 83% and 67% at DL1, and 100% and 83% at DL2, respectively. All but 1 pt with RT responded (ORR 86%); CR was achieved by 5/7 pts with RT (CRR 71%). One pt with RT was refractory with CD19-negative disease. At the time of analysis, 9/11 (82%) pts had ongoing responses; duration was up to 9 months post-infusion (median follow-up 3.2 months; range 1-9). Two pts progressed after an initial response, 1 pt with CD19-negative disease. Robust CAR T-cell expansion was observed in all pts by qPCR, with a median maximum expansion of 4.4×10 5 copies/µg DNA (C max). Median T max was 14 days (range 9-20 days). Median AUC 0-28d was 5.3×10 6 copies/µg DNA × days. GLPG5201 could be detected in peripheral blood up to 9 months post-infusion. Conclusion Data from 12 pts enrolled in the Ph1 Euplagia-1 study demonstrate that PoC CAR-T manufacturing with a short vein-to-vein time is feasible. GLPG5201 is administered as a fresh product; median vein-to-vein time is 7 days. There are no unexpected safety findings, and no CRS Grade ≥3 or any ICANS are reported. Efficacy data are encouraging, with a 92% best ORR and 75% CRR; 6/7 pts with RT responded. With this novel PoC manufacturing model, an early phenotype of less differentiated CAR T cells is preserved in the final product. GLPG5201 demonstrates rapid in vivo expansion and durable persistence post-infusion . Euplagia-1 is ongoing, and updated data will be presented.

Beyond the Primary: Unveiling the Prognostic Value of Secondary Cytogenetic Abnormalities in Multiple Myeloma

Introduction Fluorescent In-Situ Hybridization (FISH) has been an indispensable tool to characterize the cytogenetic landscape of multiple myeloma (MM), mainly for identifying primary abnormalities such as heavy chain gene (IgH) translocations and odd-number chromosome trisomies. These aberrations play a crucial role in risk stratifying the disease given their correlation with disease aggressiveness, treatment response and overall prognosis. High-risk (HR) cytogenetics are identified through the presence of t(14;16), t(14;20) or loss of the p53 gene locus. Less is known about the prognostic implication of secondary abnormalities, in particular gain of 1q21. We aimed to evaluate the association between primary and secondary cytogenetic abnormalities and their impact on overall survival (OS) in MM patients. Methods We performed a retrospective analysis on a FISH records database from three Mayo Clinic campuses. Cohort included patients &amp;gt;18 years of age, diagnosed with multiple myeloma between 2005 and 2022, with FISH performed within two years of diagnosis. Demographics including age, date of diagnoses and OS data were retrieved from electronic medical records. Statistical analyses were done using BlueSky Statistics software. Study was conducted in accordance with the principles of the Declaration of Helsinki; informed consent was obtained from all participants. Results We analyzed a total of 2221 patients, among which 59.8% were male, with a median age of diagnosis 65 years. Hyperdiploidy was the most prevalent primary cytogenetic abnormality observed in 44.3% of patients, followed by t(11;14) as the most common translocation (23%), and t(4;14) (8.2%). Gain(1q21) was assessed in 925 patients, among which 64%, 30.3% and 5.7% had 2, 3 and ≥4 copies respectively. Furthermore, 11.4% (n=253) exhibited either mutated TP53 or del17p, and 37.8% (n=839) presented with 13q loss or monosomy 13. The median follow-up for the overall population was 102 months (8.5 years), with a median OS of 75.5 months. Patients with high-risk translocations had significantly shorter median OS of 40.01 months compared to 80.65 months for those with standard risk translocations (p &amp;lt;0.001). The presence of ≥4 1q copies was associated with significantly reduced overall survival (2 copies: not reached, 3 copies: 69.4 months, ≥4 copies: 40.1774; p &amp;lt;0.001). This was consistent even when considering the presence of high-risk translocations (Figure 1). To confirm the independent significance of these findings, we performed multivariate Cox regression models, incorporating age at diagnosis, year of diagnosis, gender, presence of hyperdiploid disease, and presence of high-risk translocations. Given that overall survival outcomes were analyzed irrespective of treatment received, we incorporated year of diagnosis as a variable to control for potential impact of evolving treatment regimens over the years. The presence of ≥4 1q copies was associated with reduced OS, with a hazard ratio (HR) of 2.12 (CI: 1.38-3.26; p &amp;lt;0.001). The presence of 3 copies also showed prognostic significance (HR: 1.43; p: 0.012). Similarly, both the presence of TP53mut/mon17 (HR: 1.96, CI: 1.66-2.31; p &amp;lt;0.001) and 13qdel/mon13 (HR: 1.21, CI: 1.08-1.38) were associated with decreased OS. On the other hand, 1p deletion did not have any significant prognostic implications (HR: 1.84, CI: 0.38-1.59; p 0.113). When incorporating all cytogenetic events into a multivariate model, variables associated with reduced OS included age at diagnosis, the presence of both of 3 and ≥4 1q copies, TP53mut/mon17, t(4;14), t(14;16) and t(14;20). However, 13qDel/mon13 lost significance when incorporated with other abnormalities (Table 1). Conclusion Our investigation sheds light on the less explored realm of secondary cytogenetic abnormalities and confirm a strong association between increasing number of 1q copies and reduced overall survival, independent of other cytogenetic abnormalities and treatment regimens. These results underscore the utility of FISH-based comprehensive cytogenetic profiling at baseline, which enables reliable identification of high-risk patients. Future prospective trials should further elucidate the pathogenic mechanism for these findings. Our analysis used OS as primary endpoint and included over 2000 MM patients, reinforcing the clinical relevance of our findings.