Unmutated IGHV Status Research Articles

Unmutated IGHV at diagnosis in patients with early stage CLL independently predicts for shorter follow-up time to first treatment (TTFT)

The mutational status of the IGHV gene is routinely assessed in patients with chronic lymphocytic leukaemia (CLL), since it is both prognostic of clinical outcome and predictive of response to treatment. This study evaluates the IGHV mutational status, assessed in newly diagnosed CLL patients, as a stand-alone predictor of time to first treatment (TTFT). We analysed the data of 236 CLL patients, diagnosed at our centre between January 2004 and September 2020, with a minimum follow-up period of 3.0 years, Binet A-B and Rai 0-II stages. IGHV was unmutated in 38.1 % and mutated in 61.9 % of cases. The univariate analysis showed a statistically significant difference (p < 0.001) in TTFT based on unmutated (85.2 % at 14 years, 95 % CI = 63.3–94.5 %) or mutated (41.3 % at 14 years, 95 % CI = 29.5–51.8 %) and the need for treatment at 1, 3 and 5 years was of 20.0 % vs 4.1 % (p < 0.001), 42.7 % vs 11.4 % (p < 0.001) and 55.8 % vs 20.0 % (p < 0.001) in unmutated and mutated IGHV patients, respectively. Multivariate analysis confirmed that unmutated IGHV status negatively affects TTFT (p < 0.001), in addition to high-risk genomic aberration (p = 0.025), Rai stage I (p = 0.007) and II (p-value < 0.001). The difference in TTFT based on unmutated or mutated IGHV status remains statistically significant also when considering the subgroups by the genomic aberrations and Rai stages. Our findings suggest that, with the single analysis of the IGHV mutational status at CLL diagnosis, along with clinical and laboratory data, and without karyotype and TP53 data, clinicians will have prognostic and predictive indications for the first clinical treatment and appropriate follow-up of patients.

IGHV Mutational Status in a Cohort of Bulgarian CLL Patients: High Unmutated CLL Prevalence in North-East Bulgaria.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. One of the best established CLL prognostic markers is the somatic hypermutational status of the IGHV gene which is a part of the immunoglobulin heavy chain variable region. Technology for IGHV genotyping has been optimized and has been applied in routine diagnostics for the first time in Bulgaria. A total of 105 patients with CLL from different Bulgarian regions were tested. IGHV mutational status was determined by Sanger sequencing on total genomic DNA (gDNA) or RNA extracted from mononuclear cells. All sequencing profiles were analyzed with the IMGT/V-QUEST tool. Within the course of the analysis a high percentage of IGHV unmutated status was established in the Varna district on the Black Sea (Northeast Bulgaria). In addition, the IGHV genotyping performed on gDNA revealed a rare case with multiple rearrangements. The present data from IGHV genotyping will help in choosing the proper treatment for the benefit of Bulgarian CLL patients.

Abstract P13: Genetic and epigenetic characterization of B-cell chronic lymphocytic leukemia with IG::BCL3-translocation provides evidence for a distinct biological entity

Abstract IG translocations occur in various B-cell neoplasms and result in the deregulation of (onco)genes due to their juxtaposition with regulatory elements in the immunoglobulin loci, predominantly the immunoglobulin heavy chain (IGH) locus (14q32). Some of these IGH translocations are pathognomonic for distinct disease entities, like those involving MYC, BCL2 or CCND1 in Burkitt, Follicular or Mantle Cell Lymphomas, respectively. In Chronic Lymphocytic Leukemia (CLL), the BCL3 gene (19q13), a transcriptional coactivator of the nuclear factor-kappaB (NF-κB) family, is a known partner gene in these translocations. Two subsets of IG::BCL3 translocation-positive B-cell neoplasms have been described that exhibit differences in chromosomal aberrations, IGHV mutation status, and histopathology [Martín-Subero et al. 2007]. In CLL, IG::BCL3 translocation is linked to a younger age at diagnosis, a more aggressive clinical course, and an atypical tumor cell phenotype [Michaux et al. 1997; Au et al. 2002]. A total of 89 B-cell neoplasms, primarily diagnosed as CLL, displaying IG::BCL3 translocation detected by fluorescence in situ hybridization (FISH), were examined. Analysis of DNA methylation and copy number aberrations (CNA) was conducted for 86 IGH::BCL3-translocated B-cell neoplasms using 850K EPIC BeadChip array data. Evaluation of chromosomal aberrations typical for CLL was moreover performed by FISH and the IGHV mutation status was determined. IGH::BCL3 breakpoint junctions were sequenced in 23 samples using targeted capture and whole-genome sequencing approaches. RNA expression of BCL3 was assessed using the HTG mRNA pan B-cell panel. In UMAP analysis of DNA methylation, CLLs with IG::BCL3 translocation exhibited segregation from other CLLs, irrespective of IGH translocations. Supervised analysis revealed global hypomethylation of B-cell neoplasms with IG::BCL3 translocation compared to other CLL. A total of 66/69 CLLs with IG::BCL3 translocation showed an unmutated IGHV status (96%). Copy number determination using BeadChip data (n=86) and FISH analysis (n=85) showed a significantly higher incidence of trisomy 12 in IG::BCL3-translocated cases compared to the general CLL population (p=0.011). Breakpoint sequencing indicated IG::BCL3 junctions to be recurrently associated with aberrant class-switch recombination at the IGH locus, involving IGHA (n=11/23), IGHG (n=5/23) and IGHM segments (n=5/23). Breakpoints at the BCL3 locus displayed two groups. 80 cases (all CLL) were located upstream of BCL3 and two other B-cell neoplasms (NHL, DLBCL) were located downstream of BCL3. BCL3 expression analysis in 15 IG::BCL3-translocated CLLs confirmed its transcriptional upregulation compared to the general CLL population. Our genetic and epigenetic analyses support the view that CLL with IG::BCL3 translocation may constitute a genetically and epigenetically distinct subtype of B-cell neoplasms, diverging from the typical CLL. Citation Format: Cosima Drewes, Cristina López, Nnamdi Okeke, Sina Hillebrecht, Petra Schütz, Anja Fischer, Anja Mottok, Susanne Bens, Christof Schneider, Stephan Stilgenbauer, Eugen Tausch, Reiner Siebert. Genetic and epigenetic characterization of B-cell chronic lymphocytic leukemia with IG::BCL3-translocation provides evidence for a distinct biological entity [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P13.

A Single Center Real World Study of Outcome and Resistance of Bruton Tyrosine Kinase Inhibitors (BTKi) in Chinese Patients with Chronic Lymphocytic Leukeima/Small Lymphocytic Lymphoma

Introduction Acquired BTK/PLCG2 mutations remain to be the driver of Bruton Tyrosine Kinase inhibitor (BTKi) resistance. But the underlying mechanisms need to be further explored. In this study, we retrospectively analyzed clinical and biological characteristics of BTKi-treated CLL patients and report the outcome and resistance in our center (the First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital). Method 262 patients treated with single BTKis continuously were collected and follow up the outcome in our center. Progression-free survival (PFS) was defined as from BTKi initiation to disease progression on BTKi. Next-generation sequencing (NGS) and High sensitivity droplet digital PCR (ddPCR) acquired at treatment initiation /or at the time of disease progression were used to describe mutational landscape. Kaplan-Meier method and the log-rank test were used for analysis. Results A total of 262 patients were enrolled from January 2014 to March 2023, and the median age was 63 (range 28-88) years. Ibrutinib, Zanubrutinib, Orelabrutinib were administered in 55.3%,18.3% and 17.9% of patients respectively, in addition to 6 patients with Acalabrutinib and one with Loxo-305, among them 47.7% (125/262) received BTKi as first line treatment. With a median follow up of 27.3 months, 208 patients were in durable remission while 54 patients relapsed, the median exposure of BTKi was 26.8 (range, 1-73.8) sand 27.7 (range, 3-69.8) months respectively. The median PFS was 5.8 years (5.0-NA, 95% CI) for the whole cohort. Clinical and biological characteristics of both cohorts at initiation of BTKi treatment were described and compared in Table 1. Higher proportion of bulky disease (50.0% vs 23.5%), IGHV unmutated status (84.3% vs 50.5%), del(17p) (44.7% vs 12.9%), TP53 mutation (37.8% vs 15.2%), TP53 aberrations (62.5% vs 24.3%) and complex karyotype (40.5% vs 21.1%) were shown in the BTKi relapsed cohort as compared to responsive cohort. Inferior PFS was observed in patients with TP53 aberration vs without TP53 aberration ( 45.6 mos vs not reached, p&amp;lt;0.0001), unmutated IGHV vs mutated IGHV (56.2 mos vs not reached, p=0.0005), with del(17p) vs without del(17p) (36.1 mos vs not reached, p&amp;lt;0.0001), with complex karyotype and without complex karyotype (69.8 mos vs not reached, p=0.0401), bulky disease vs no bulky disease (45.6 mos vs 69.8 mos, p=0.0006) and elevated LDH vs normal LDH (66.5 mos vs not reached, p=0.0083). Moreover, patients with del(11q) or BTKi given as second or greater line showed a tendency of inferior PFS ( p= 0.1963, p= 0.0735, respectively). Regarding the relapsed cohort, 43 patients displayed CLL progression while 11 patients displayed Richter transformation (RT, including 1 Hodgkin lymphoma [HL]) , the median time from initiation to progression were 33.7 months and 9.5 months, respectively（p&amp;lt;0.0001). BTK/PLCG2 mutations were the most common mutation at the timepoint of disease progression. By integrating NGS and ddPCR results, 54.9% (28/51) relapsed patients acquired BTK/PLCG2 mutation. BTKC481S mutation was the dominant hotspot mutation in all kinds of BTKi relapsed cases. Other mutations at the C481 site included C481R, C481Y, C481F. However, BTKT474 mutation was only detected in Orelabrutinib-resistant cases (Figure 1). 6 patients presented with multi-hit mutations. Treatment after disease progression on BTKis is still challenging. In our relapsed cohort, the median PFS was 5.7 months and poorer in RT as compared with CLL progression (3.0 months vs 6.7 months, p= 0.0874). Venetoclax-based regimen might be an effective salvage therapy which could showed a benefit trend in PFS as compared to other post BTKi therapy (10.1 months vs 3.1 months, p= 0.1818). Conclusion In our real world study, patients with IGHV unmutated status, TP53 aberrations, bulky disease and elevated LDH showed inferior PFS. Acquired BTK/PLCG2 mutations remained to be key drivers of BTKis resistance and BTKC481S mutation was the dominant mutation, while BTKT474 mutation was only detected in Orelabrutinib-resistant patients. The prognosis was rather poor for relapsed patients especially for RTs, treatment strategy after disease progression remains to be optimized.

Minimal Residual Disease-Guided Combination of Ibrutinib and Venetoclax Compared to FCR in Untreated Patients with CLL of Intermediate Risk : Interim Results of MRD Kinetics in the Eradic Trial from the Filo Group

With the emergence of targeted therapies, defining the best strategy for first-line treatment in chronic lymphocytic leukemia (CLL) patients has become challenging. The aim of the ERADIC phase 2 study was to compare the efficacy of a standard FCR regimen to that of an MRD-guided combination of ibrutinib and venetoclax (IV), in fit patients with CLL of intermediate risk defined by either unmutated IGHV status, 11q deletion or complex karyotype in the absence of TP53 alteration. MRD was assessed in bone marrow (BM) or peripheral blood (PB) by flow cytometry. After a lead-in phase of ibrutinib as a single agent from month (M)1 to M3, the total duration of treatment with the IV combination was based on BM M9 MRD. If it was &amp;lt;0.01% (uMRD) at that time, treatment was continued for 6 additional months (M15) then stopped. If M9 BM-MRD was ≥0.01%, IV treatment was continued for 18 additional months (M27). BM-MRD was reassessed at that time-point in both arms. Additionally, PB-MRD evaluation was performed every 6 months.The primary endpoint will be the percentage of patients with BM uMRD at M27. Here, intermediate safety data and MRD kinetics (M9 to M21) are presented. Between September 2019 and February 2021, 120 patients were randomized 1:1 between the two treatment arms. The median age was 59 [34-72] and 61 [34-74] year-old in the FCR and IV arms, respectively. Patient characteristics were well balanced between the 2 arms in terms of gender (male 72% FCR, 74% IV), PS ECOG 0-1 (59% FCR, 68% IV) and Binet stage (A, B and C 15%, 64%, 21% for FCR ; 8.5%, 59% and 32% for IV). An11q deletion was found in 20% and 24% of the cases in the FCR and IV arms, respectively and all patients but one had unmutated IGHV. At the time of data cut-off for this analysis, the median follow-up was 29.7 months [range: 25.3- 32.8]. Sixty-three serious adverse events (SAE) have been reported so far, 32 in the FCR arm and 31 in the IV arm. In the FCR arm, the most frequent SAE were infections (N=12 including 4 COVID-19), febrile neutropenia (N=5) biological tumour lysis syndrome (N=3), and secondary malignancies (N=2, including 1 myelodysplastic syndrome and 1 acute myeloid leukemia). In the IV arm, the most frequently reported SAE were infections (N=8 including 4 COVID-19), cardiovascular events (N=7), biological tumour lysis syndrome (N=5), acute renal failure (N=2) and secondary malignancies (N=2, including 1 colorectal cancer and 1 skin basal cell carcinoma). Four grade 5 adverse events were reported, respectively 2 in the FCR arm (1 septic shock and 1 AML) and 3 in the IV arm (2 sudden deaths and one death COVID-19-related). In the FCR arm (intention to treat [ITT] n=57), 59.6% of the patients had BM-uMRD at M9. The kinetics of PB-uMRD was 68% at M9, 65% at M15 and 52% at M21. In the IV arm (ITT n=54), the rate of BM-uMRD at M9 was much lower at 33%. PB-MRD kinetics showed levels of uMRD of 52%,70% and 67% at M9, M15 and M21 respectively. In terms of response, CR/CRi rates were 56% for the FCR arm and 66% for the IV arm. Three patients progressed in the FCR arm (1 at M9 and 2 at M15) and 2 in the IV arm, at respectively M13 and M39. Among the 13 patients who achieved BM-uMRD at M9 in the IV arm, 10 have indeed stopped according to the protocol and only 1 has progressed at M39. In conclusion, monitoring MRD kinetics in this trial showed a stable level of PB-uMRD in the FCR arm yet a clear increase in the IV arm between M9 and M15. Toxicity remains an important parameter in both treatment arms that will have to be taken into account when determining whether treatment should be continued because of detectable BM-MRD at M9 (IV arm). Upcoming data of the primary enpoint analysis at M27 will be of great interest to try to determine the best strategy.

Extended Follow up of a Phase 2 Study of Early Intervention with Lenalidomide in Patients with High-Risk Chronic Lymphocytic Leukemia

CONCLUSIONS Lenalidomide (LEN) is an effective therapy for chronic lymphocytic leukemia (CLL) and has immunomodulatory effects that were hypothesized to improve immune function in patients (pts) with untreated early stage CLL. A NCI/CTEP sponsored phase 2 study (NCT01351896) randomized asymptomatic pts with high-risk CLL to receive LEN either concurrent with or delayed from (3 months after 1st dose) two doses of 13-valent protein-conjugated pneumococcal vaccine (PCV13). The primary endpoint was the proportion of pts who achieve an antibody response to the 2nd PCV13 vaccine and was not significantly different between groups (Thangavadivel et al., Clinical Cancer Research 2020). Despite this, many pts experienced effective CLL disease control with LEN. Here we report long-term results from this trial to examine the impact of LEN early treatment on CLL disease course, complicating infections (INF), and second neoplasms (SN). Pts ≥18 and &amp;lt;80 years-old who had CLL with a high-risk feature, defined as FISH positive for del(17)(p13.1) and/or del(11)(q22.3), complex karyotype (≥3 abnormalities), or unmutated IGHV status, no prior treatment, and no iwCLL 2008 criteria for treatment were eligible. LEN was started at 2.5mg daily and increased to 5mg as tolerated. Data on INF and SN was determined from study adverse events and graded (gr) using CTCAE v4.0 and 5.0. Overall survival (OS) and progression-free survival (PFS) were calculated from the start of LEN and discontinuation of LEN, and analyzed using the Kaplan-Meier method. Time to next therapy (TTNT) was calculated from start or discontinuation of LEN to the initiation of next therapy treating death without new therapy as a competing risk. Data cut for analysis was 6/12/2023. A total of 49 pts were enrolled between 11/2011 and 7/2014. The median age was 59 (range 40-70) years, 69% were men, and 96% were Caucasian. In terms of CLL features, 94% (46/49) were IGHV unmutated, 47% (23/49) had complex karyotype, 14% (7/49) had del(17)(p13.1), and 31% (15/49) had del(11q)(q22.2). The median follow-up from LEN start was 4.6 (range 0.4-11.2) years and median time on LEN treatment was 3.7 (range 0.02-10.1) years with 2 pts remaining on LEN at 9.7 and 10.1 years. The median PFS after starting LEN was 5.7 (95% CI: 3.2-6.4) years with an estimated 8-year PFS of 25.9% (95% CI: 13.9%-39.6%) (Figure). Eleven pts had progressive disease (PD) at the time of LEN discontinuation and their median time from first dose of LEN to PD was 3.7 (range 0.3-6.0) years. For those who discontinued without PD, the median PFS from discontinuation was 1.5 (95% CI: 0.6-2.6) years. For those who initiated a subsequent therapy, the median TTNT from starting LEN was 4.4 (range 0.3-9.1) years and from discontinuing LEN was 1.1 (range 0.1-7.8) years. There were 7 deaths, and the median OS was not reached with an estimated 8-year OS of 73.9% (95% CI: 51.6%-87.2%). There were 217 episodes of INF that occurred in 88% (43/49) of pts. Patients experienced multiple INF: 24 (49%) pts had 1-3 INFs and 19 (39%) had ≥3 INFs with 5 of those having &amp;gt;10 INFs. The most frequent INFs were upper respiratory (n=71), sinus (n=37), skin and soft tissue (n=27), and lower respiratory (n=22) (Table). INFs were gr 1-2 in 95% of cases. Gr ≥3 INF were experienced by 18% (9/49) of patients with 10 events. There were 9 gr 3 INFs (lower respiratory tract n=4, kidney n=1, urinary tract n=1, appendix n=1, joint n=1, meninges n=1) and 1 gr 5 COVID-19 INF which occurred in a pt who discontinued LEN &amp;gt;2 years prior. The rate of gr ≥3 INF was 3.8 (95% CI: 1.8-7.0) per 100-person-years. SN were experienced by 8 pts who had 19 SNs. The most common SN were non-melanoma skin cancers (n=15). Other SN were lung adenocarcinoma (n=2 in 1 pt), melanoma in situ (n=1), and squamous cell carcinoma in a parotid lymph node from a skin SCC (n=1). The rate SN was 7.2 (95% CI 4.3-11.3) per 100-person-years. There were no cases of acute leukemia. In this population of pts with CLL with high-risk disease features predictive of need for earlier treatment, LEN resulted in a prolonged time to subsequent therapy, even in cases of LEN discontinuation. The incidence of gr ≥3 INFs and SN were as expected for this population supporting that LEN does not increase risk for these complications.

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.

Outcomes and Treatment Sequences of Therapies with BCL2- and BTK Inhibitors in Chronic Lymphocytic Leukemia (CLL): An Analysis of Patient Data within the German CLL Study Group (GCLLSG) Registry

Introduction: Targeted agents, in particular BCL2-inhibitors (BCL2i) and BTK-inhibitors (BTKi), have profoundly improved the outcome of patients (pts) with chronic lymphocytic leukemia (CLL) and are considered standard treatment. However, the transition from treatments evaluated within clinical trials into routine clinical practice can be complex. We here present real-world data from the German CLL Study Group (GCLLSG) registry and report outcomes and treatment sequences of pts treated with either BCL2i (venetoclax) or BTKi (acalabrutinib, ibrutinib, spebrutinib, tirabrutinib, or zanubrutinib) as first administered targeted agent. Methods: Pts from multiple German sites with confirmed diagnosis of CLL and with at least one documented first-line CLL treatment between July 1 st 2014 and January 30 th 2023 were included in this analysis. Pts participating in an active clinical trial were excluded. Treatments were categorized according to the backbone of therapy and pts were allocated to either the venetoclax (Ven) or the BTKi cohort depending on their first treatment with one of these substances. Time to next treatment (TTNT), event-free survival (EFS) and overall survival (OS) were analyzed using Kaplan-Meier methods. Results: A total of 274 pts were treated with a Ven-based regimen while 915 pts were treated with a BTKi as first therapy. Median observation time from the first documented treatment (irrespective if given as first-line or in a later therapy line) was 26 months (range 0 - 296) in the Ven cohort and 78 months (range 0-266) in the BTKi cohort. For the Ven cohort, median age at the time of first Ven treatment was 71 years (range 32 - 87) with 172 pts (62.8%) being male. Median CIRS score was 2 (range 0-13) and 12 pts (7.5%) had an ECOG performance status &amp;gt; 1. Among 57 pts with known molecular genetics, 32 pts (56.1%) had an unmutated IGHV status and 9 out of 51 evaluable pts (17.6%) had a deletion and/or mutation TP53. Applying the CLL-IPI, 11 out of 58 evaluable pts (19.0%) were in the very high-risk group. In the BTKi cohort, median age at time of first BTKi treatment was 72 (range 32 - 92), with 628 pts (68.6%) being male. Median CIRS score was 3 (range 0-17) and 45 pts (9.5%) had an ECOG performance status &amp;gt; 1. Among 162 pts with available data, 108 pts (66.7%) had an unmutated IGHV status and 59 out of 130 evaluable pts (45.4%) had a deletion and/or mutation TP53. Using the CLL-IPI, 46 out of 145 evaluable pts (31.7%) were in the very high-risk group. A total of 152 of 274 pts (55.5%) in the Ven cohort received Ven as first-line treatment (median number of prior treatments: 0, range 0-9) and 352 of 915 pts (38.5%) in the BTKi cohort received a BTKi as first-line treatment (median number of prior treatments: 1, range 0-12). Among all 222 administered treatments prior to first Ven and 1090 treatments administered prior to first BTKi, chemoimmunotherapy (70.3% and 68.3%, respectively) and chemotherapy (10.4% and 17.8%, respectively) were the most frequent. For subsequent therapy following the first documented Ven or BTKi treatment, most pts were treated with targeted agent regimens. In the Ven cohort, 37 subsequent treatments were documented in 26 pts, of which 10 (27.0%) were a Ven-containing regimen and 13 (35.1%) a BTKi-containing regimen. Within the BTKi cohort, 393 subsequent treatments were administered in 264 pts, of which 146 (37.2%) included Ven and 106 (27.0%) a BTKi Treatment sequences were visualized with a sankey-diagram ( Figure 1). Median EFS from the start of the first documented Ven treatment was 31.8 months, with an estimated 2-year EFS rate of 66.6%. Median TTNT was 63.7 months and median OS was 96.5 months with an estimated 2-year OS rate of 89.5%. When Ven was given as first-line treatment, the estimated 2-year OS rate was 93.6% versus 86.5% when given in a later therapy line for the first time. For the BTKi cohort, median EFS from start of first documented BTKi treatment was 23.2 months, with an estimated 2-year EFS rate of 48.6%. Median TTNT was 68.4 months and median OS was 85.9 months with an estimated 2-year OS rate of 84.9%. When BTKi were given as first-line treatment, the estimated 2-year OS rate was 92.3% versus 81.3% when given in a later therapy line for the first time. Conclusion: EFS, TTNT and OS were comparable following Ven versus BTKi-based therapy in pts documented in the GCLLSG registry between 2014 and 2023.

Outcomes of CLL Patients Exposed to Venetoclax+/-R after Ibrutinib in France: The Resist Retrospective Study from the Filo-CLL Group

Background: In chronic lymphocytic leukemia (CLL), despite the fact that Bruton tyrosine kinase inhibitors (BTKi) are increasingly used 1st or 2nd line therapy, and the Bcl-2 inhibitor (Bcl-2i) venetoclax +-rituximab (VEN-R) relapses, based on the good results of the MURANO trial (although it did not include BTKi-exposed patients [pts]), multicentric retrospective studies describing the real world outcomes of BTKi exposed pts under Bcl-2i treatment are still lacking. Most of the data published so far, extracted from USA databases, lack granularity to explain which clinical or biological factors define prognosis in the modern therapeutic era of CLL. Here, results of the experience gathered by 10 FiLO-CLL-affiliated centers in France are presented, monitored by physicians in charge of the pts. The RESIST registry assembled outcomes from 150 pts, among whom 117 are presented here as a first efficacy analysis (the complete set of pts will be presented at the congress). Results: Analysis of these 117 pts shows a predominance of men (68%) and a median age of 59 years (29-85). A median of two previous lines of treatment (range 1-5] was given before initiating VEN, with a majority of immunochemotherapy (FCR) in first line and progressive increase of ibrutinib use. As expected, the rate of complete response (CR) decreased over the lines, yet remained over 30%. Intolerance to ibrutinib was the main cause for discontinuation in 27.4%, and 66.7% were refractory or relapsed patients (45% presenting with BTK mutations before VEN, the other patients stopped due to other causes). At the time of VEN treatment (75% VEN-R), 35.3% of the pts had bulky disease, 63% elevated lactate dehydrogenase and 24.8% a bad performans status (3-4). High risk cytogenetic was found in nearly all patients: 63.8% del(17p) and/or TP53 mutation and 94% unmutated IGHV status. The sequence of VEN therapy was terminated as planned for 56.3% of the pts, while it was stopped for progression/relapse in 27.6% and for intolerance in 16.1%. The most common grade ≥ 3 adverse events observed with VEN, reported in 92 pts, were hematological (30.4%), gastro-intestinal (8.7%), infectious (3.3%) and cutaneous (4.4%). Tumor lysis syndrome during the ramp-up phase was infrequent (grade 1 = 8.4%, grade 2 = 1.9% and grade 3 = 0.9%). At the time of analysis, the best response was CR for 69% of the pts (CR+Cri+PR: 95%). MRD assessment was done in 52 pts, 50% of which obtained MRD undetectable responses. With a median follow-up of 37.7 months (mo; 95% confidence interval [CI] 32.7-40.1), 27.6% of the pts had progressed. The mPFS is 35.9 mo (95%CI 29.8-53), mOS 59.4 mo (95%CI 44-Not Reached) and median time to next treatment (TNTT) 45.9 mo (95%CI 31.7-58.8), while in the 7-year update of the VenR arm of the MURANO trial the latter outcomes were much higher (54.7 mo, not reached, and 63 mo respectively). On univariate and multivariate analyses, only TP53 mutations tended to be significantly associated with worse PFS (HR = 1.83 [0.99; 3.38], p=0.05) and OS (HR = 2.11 [0.91; 4.86], p=0.07). A total of 44 patients relapsed after VEN, with a median time to next treatment-2 of 18.0 mo (95%CI 5.5-21.1). The updated results of this subset of patients will be presented at the congress. Conclusion: Although slightly improved as compared to other real-life series in Italy or in the US, these data indicate that VEN or VEN+R after progression, relapse or intolerance to BTKi is moderately efficient with only about 35.9 months median PFS (in spite of undetectable MRD in 26/52 available patients). TP53 abnormalities were confirmed to be an important risk factor, yet not BTK mutations, or. This work is important to help designing clinical trials aiming at improving the outcomes with next generation BTKi/Bcl-2i, or even CAR-T cells.

Investigating the Influence of Germline ATM Variants on Susceptibility to Chronic Lymphocytic Leukemia and Other Malignancies

Introduction: Chronic lymphocytic leukemia (CLL) patients (pts) face an increased susceptibility to secondary cancers. Furthermore, the hereditary predisposition to CLL is influenced by factors that have yet to be fully elucidated by GWAS-identified common variants. Among the hereditary cancer risk mutations observed, germline Ataxia-telangiectasia mutated ( ATM) aberrations are among the most prevalent. Rare germline ATM variants are detected in 24% of CLL pts, surpassing the frequency observed in other hematologic neoplasms and the general population. In CLL, loss of ATM via 11q23 deletion is linked to an unfavorable prognosis and a shortened progression-free survival. The extent to which germline ATM variants predispose CLL pts to other malignancies remains unclear; to examine this potential risk we conducted a retrospective study to assess the influence of germline ATM variants on the predisposition to secondary neoplasms in CLL pts and their relatives. Methods: Five hundred and eighty-five pts seen at Dana-Farber Cancer Institute and who had NGS performed to evaluate germline ATM status, either through direct germline sequencing of saliva or by inference according to the hierarchical algorithm we have previously published (Lampson, 2022), were mailed a questionnaire that investigated: demographics; personal and family history of any cancer; non-medical radiation and Agent Orange exposure; and Ataxia-Telangiectasia syndrome (AT). Of these, 333 replied (57%). Information collected from our CLL database included FISH, IGHV status and treatment status. European ancestry was categorized based on the United Nations geoscheme (UNSD, 2019). Pt characteristics are in Table 1. Pts were stratified into 4 groups, based on ATM mutational status and del(11q): Group-1, ATM germline variants alone (N=63); Group-2, ATM germline with somatic ATM mutation and/or del(11q)(N=22); Group-3, ATM somatic aberration alone (including del(11q))(N=41); and Group-4, no ATM aberration (N=207). Results: Of the 333 pts in our cohort, baseline demographic and clinical characteristics among the 4 groups were balanced, except for unmutated IGHV status. Nineteen (86%) and 36 (86%) pts of groups 2 and 3, carrying somatic ATM aberrations, had unmutated IGHV, compared to 21 (33%) and 76 (37%) of groups 1 and 4, without somatic ATM aberrations(p&amp;lt;0.01). Regarding treatment, 60% of our cohort had received at least one line of therapy (median: 1 (1-7)). Among the 164 pts (49%) with history of an additional non-CLL neoplasm, 221 cancers were reported. Excluding pts with only non-melanoma skin cancers, the incidence is reduced to 31% (104 pts). The median number of second cancers per pt is 1 (1-4). Of the 134 pts with available age of onset data for the secondary malignancy, 80 (60%) were diagnosed with the secondary cancer after CLL. Comparing pts with germline ATM variants (N=85) to the ones without germline ATM variants (N=248), no significant difference in the incidence of secondary cancer was found (p=0.73). The median age of secondary cancer onset was also similar between the groups (median: 63 and 64 years in germline ATM present vs absent, p=0.28). Of the whole cohort, 283 pts (85%) reported at least one relative with a cancer history (median 2 relatives affected (1-17)). The incidence of B-cell lymphoproliferative disorders was significantly higher (p=0.02) in pts with germline ATM variants (32%) compared to those without germline ATM variants (21%), including familial CLL (25% vs18%) (p=0.04). Familial CLL was also strongly associated with Ashkenazi Jewish ethnicity (39% incidence in Ashkenazi pts vs 16% in non-Ashkenazi, p=0.004). No difference in the incidence of other hematologic or solid malignancies was found between the relatives in the two groups. No pts reported a familial or personal history of AT. Compared to pts without any ATM aberration, time to first treatment (TTFT) was shorter in pts harboring somatic ATM events, while no difference was observed between pts with germline ATM variants (median TTFT: 82, 59, 52, 90 months for group 1, 2, 3 and 4 respectively, p=0.01) (Figure 1). Conclusions: Our results suggest a higher incidence of B-cell lymphoproliferative disorders, including familial CLL, in the relatives of CLL pts carrying germline ATM variants. The presence of these germline variants did not impact TTFT, compared to pts harboring somatic ATM mutations.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: Patient stratification based on genomic biomarkers is critical to optimize treatment of chronic lymphocytic leukemia (CLL). Using digitized, genomically annotated peripheral blood smears (PBS) of patients with CLL, we evaluated whether deep learning can predict presence of TP53 aberrations (del[17p] and/or TP53 mutation) and unmutated IGHV status solely based on cytomorphology. Methods: The workflow consisted of sample collection and digitization, manual annotation, segmentation, morphological feature extraction, and aberration classification. Whole slide images were generated from PBS of 414 patients with previously untreated CLL (CLL14, GIVe, BAAG). Manual annotation was conducted using Qupath software (v0.3.2) with ImageJ. A tailored Stardist model was used for semantic segmentation. Morphological features were collected from each segmented white blood cell (WBC) and fed into the classification model. Two neural network (NN) models were generated for each aberration (TP53 and IGHV) based on a cohort split into 70/30% training and testing datasets. The models were evaluated using AUC (Area Under the Curve of ROC [Receiver Operating Curve]). Results: In preparation of model training, 4,962 manual cells from 18 randomly selected patients were manually annotated. For the TP53 classifier, 141,873 WBC from 52 patients were segmented using the Stardist model. For the IGHV classifier, 131,410 WBC from 57 patients were segmented. The model was trained on 256 × 256 pixels patches obtained from original images with 200 epochs and 100 batch_size whilst making use of data augmentation. An intersection over union (IoU) value of 89.6% confirmed good model performance, i.e. accurate WBC segmentation. Lymphocyte cells were then segmented on each frame image. In total, 28 single-cell morphological features were captured and 8 best representatives fed into the NN model. The collected metadata vector features were randomly assigned to non-overlapping training and testing sets. Separate datasets were used for the TP53 and IGHV classifier. On a single-cell level, the models achieved an AUC of 86% for TP53 aberrations and IGHV status, respectively. For independent validation, the TP53 and IGHV classifiers were tested on a set of unseen images of 33 and 22 patients, respectively. AUC values of 78% and 72%, respectively, were achieved, confirming good model performance. To classify individual patients, a threshold of ≥50% positive cells/image was used to either classify a patient as TP53 aberrated or IGHV unmutated. Using this approach, 70% of patients with TP53 aberration and 68% with IGHV unmutated status were accurately classified. Keywords: Bioinformatics, Chronic Lymphocytic Leukemia (CLL), Computational and Systems Biology, Diagnostic and Prognostic Biomarkers No conflicts of interests pertinent to the abstract.

Analysis of racial disparities accounting for treatment received in chronic lymphocytic leukemia: A real-world cohort analysis.

7533 Background: Recent population-based studies have shown that Black patients (pts) with CLL have worse overall survival (OS) than White pts with CLL. These studies could not account for the use of modern therapies, CLL prognostic variables, or comorbidity and thus the root cause of this disparity remains unclear. Here, we use the Flatiron Health Database (FHD) to study this disparity, while accounting for disease-specific variables and treatment (tx) received. Methods: Retrospective analysis of pts with CLL in the FHD treated between 2002 to 2022 was performed. Pt characteristics such as age at diagnosis (dx), year (yr) of dx, months (mos) from dx to first tx, race, sex, IGHV status, ECOG performance status (PS), and use of small molecule inhibitors at any time (including BTK inhibitors, venetoclax, PI3K inhibitors, and lenalidomide) were collected and analyzed. OS was measured from tx start. Multivariable cox regression (MVA) was used to determine adjusted odds of survival. Results: 13646 pts with CLL were identified. Mean age at dx was 65.3 yrs, and mean time from dx to tx was 32.3 mos. 81.2%, 8.4%, and 10.4% of pts were White, Black, or other race, respectively, and 63.2% of pts were male. 61.4% of pts were IGHV unmutated, and 89.1% of pts had a ECOG PS 0/1. 58.7% of pts were treated with a small molecule inhibitor at any point during their tx. After a median follow-up of 5.3 yrs, the median OS was 10.5 yrs (95% CI: 10.2-10.8). Black pts were younger at dx (63.1 vs 65.4), had shorter time to first tx (23.6 mo vs 34 mo), were more likely to be IGHV unmutated than White pts (83% vs 60.1%), and had more small molecule inhibitor use (69% vs 58.5%). These differences were all statistically significant (p &lt;0.01). 90.8% and 89.1% of Black and White pts had an ECOG PS of 0/1, respectively. Median OS for Black pts was 10 yrs (95% CI: 9.1-11.1) vs 10.5 yrs (95% CI: 10.2-10.8) for White pts. On MVA, Black pts had higher risk of death than White pts of the same age despite increased use of small molecule inhibitor (Table). However, the interaction between age and race was also significant, indicating a decreasing disparity between Black and White race as pts aged. Time to first tx, IGHV unmutated status, and ECOG PS were also independently associated with worse OS. Conclusions: Black race especially at younger age was independently associated with worse OS despite the use of small molecule inhibitors, IGHV status, and ECOG PS. [Table: see text]

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Background: One-year fixed-duration venetoclax-obinutuzumab (Ven-Obi) is a standard-of-care for patients (pts) with previously untreated chronic lymphocytic leukemia (CLL). Due to its ongoing follow-up, the CLL14 study provides unique insights into long-term outcomes of pts after Ven-Obi therapy. Methods: Pts with previously untreated CLL and coexisting conditions were randomized 1:1 to Ven-Obi or chlorambucil-obinutuzumab (Clb-Obi). Primary endpoint was investigator-assessed progression-free survival (PFS). Secondary endpoints included safety, rates of minimal residual disease (MRD), time to next treatment (TTNT) and overall survival (OS). Results: Of 432 enrolled pts, 216 were randomly assigned to Ven-Obi, 216 to Clb-Obi. At a median follow-up of 76.4 months (interquartile range 52.5–80.5), PFS remained superior for Ven-Obi compared to Clb-Obi (median 76.2 vs. 36.4 months; HR 0.40 [95% CI 0.31–0.52], p < 0.0001). Progressive disease (PD) occurred in 67 cases in the Ven-Obi arm with 39 second-line treatments, and in 141 cases in the Clb-Obi arm (with 103 second-line treatments). TTNT was significantly longer after Ven-Obi (6-year TTNT 65.2% vs. 37.1%; HR 0.44, 95% CI 0.33–0.58, p < 0.0001). In both arms, the most frequent second-line treatments were BTK inhibitors (61.5% in the Ven-Obi arm, 55.4% in the Clb-Obi arm). The PFS and TTNT difference between the two arms was maintained across all risk groups, including pts with TP53 mutation/deletion (median PFS 51.9 vs. 20.8 months; median TTNT 57.3 vs. 29.0 months) and unmutated IGHV status (median PFS 64.8 vs. 26.9 months; median TTNT 85.4 vs. 40.6 months). Multivariate analysis identified TP53 deletion/mutation, unmutated IGHV and lymph node size ≥5 cm as independent negative prognostic factors for PFS in pts treated with Ven-Obi. Five years after treatment completion, 17 (7.9% of the intention-to-treat population) pts in the Ven-Obi arm still had uMRD (<10−4 by NGS in peripheral blood), 22 (10.2%) had low (L)-MRD (≥10-4 and <10-2) and 23 (10.6%) high (H)-MRD (≥10-2), compared to 4 (1.9%) uMRD, 9 (4.2%) L-MRD and 18 (8.3%) H-MRD in the Clb-Obi arm. Overall, 48 deaths were reported in the Ven-Obi arm (9 PD related) and 70 in the Clb-Obi arm (26 PD related); 6-year-OS rate was 78.7% in the Ven-Obi and 69.2% in the Clb-Obi arm (HR 0.69 [0.48–1.01], p = 0.052). Second primary malignancies were reported in 30 pts in the Ven-Obi and 18 in the Clb-Obi arm; cumulative incidences 6 years after randomization were 14.2% and 8.5%, respectively (p = 0.071). No new safety signals were observed. Encore Abstract - previously submitted to EHA 2023 The research was funded by: Roche, AbbVie Keywords: chronic lymphocytic leukemia (CLL), combination therapies Conflicts of interests pertinent to the abstract O. Al-Sawaf Consultant or advisory role: AbbVie, Ascentage, AstraZeneca, BeiGene, Eli Lilly, Gilead, Janssen, Roche Honoraria: AbbVie, Adaptive, AstraZeneca, BeiGene, Eli Lilly, Gilead, Janssen, Roche Research funding: AbbVie, BeiGene, Janssen, Roche Educational grants: AbbVie, AstraZeneca, Janssen, Gilead, Roche

Characterization of the genetic and epigenetic landscape of B‐cell neoplasms with IG::BCL3‐translocation

Introduction: The BCL3 gene (19q13) encodes a transcriptional coactivator of the nuclear factor-kappaB (NF-κB) family. It is recurrently deregulated in B-cell neoplasms by a juxtaposition to regulatory elements of one of the immunoglobulin loci, mostly the immunoglobulin heavy chain (IGH) locus (14q32). Two distinct subsets of IG::BCL3 translocation-positive B-cell neoplasms, differing in the number of chromosomal aberrations, IGHV mutation status and histopathology, have been described [Martín-Subero et al., 2007]. In B-cell chronic lymphocytic leukaemia (CLL), the IG::BCL3 translocation has been associated with younger age at diagnosis, a more aggressive clinical course and an atypical tumour cell phenotype [Michaux et al., 1997; Au et al., 2002]. Methods: A total of 89 B-cell neoplasms with IG::BCL3 translocation detected by fluorescence in situ hybridisation (FISH), including predominately cases diagnosed as CLL, were studied. Breakpoints at the BCL3 and IGH loci were sequenced in a subset of samples using targeted capture and breakpoint-spanning PCR approaches. Chromosomal aberrations detected by FISH as well as the IGHV mutation status were analysed. In addition, DNA methylation and copy number aberration (CNA) analyses were performed for 48 samples with CLL using the 850K EPIC BeadChip array. Results: Targeted capture-based sequencing data showed that in the majority of cases BCL3 translocations are associated with aberrant class-switch recombination at the IGH locus, often involving IGHA segments (n = 4/10). From the 69 IG::BCL3-translocated cases with available IGHV mutation status, a total of 51 (74%) were classified as unmutated and 18 (26%) as mutated. This indicates an overrepresentation of cases with unmutated IGHV status among the IG::BCL3-translocated cases compared to the general CLL population. Based on EPIC BeadChip data (total n = 48) 25 (52%) IG::BCL3-translocated cases carried a trisomy 12, 11 cases (23%) a deletion 13q, 7 cases (15%) a deletion 17p and 3 cases (6%) a deletion 11q. FISH analysis of a partially overlapping set of neoplasms (n = 54) was in line with the EPIC BeadChip data and revealed 17 IG::BCL3-translocated cases (32%) with trisomy 12, 7 cases (13%) with deletion 17p and deletion 13q each, and 4 cases (7%) with deletion 11q. Thus, IG::BCL3-translocated cases showed a striking skewing towards the presence of trisomy 12 compared to the general CLL population (p = 0.011). In the DNA methylation analyses, the IG::BCL3-translocated CLLs segregated separately from other CLLs with and without IGH translocations. Conclusions: Our large multi-OMICs study of IG::BCL3-translocated B-cell neoplasms corroborates previous reports on the pathogenetic heterogeneity of these lymphomas and moreover provides evidence that IG::BCL3-translocated CLL might form a genetically and epigenetically distinct subtype of B-cell neoplasms distinct from common CLL. Keywords: Chronic Lymphocytic Leukemia (CLL), Genomics, Epigenomics, and Other -Omics No conflicts of interests pertinent to the abstract.

CTP synthase 2 predicts inferior survival and mediates DNA damage response via interacting with BRCA1 in chronic lymphocytic leukemia

BackgroundCytidine triphosphate synthase 2 (CTPS2) is an essential metabolic enzyme that catalyzes the biosynthesis of CTP. CTP synthases contribute to lymphocytes proliferation and tumorigenesis, but the role of CTPS2 in chronic lymphocytic leukemia (CLL) remains undefined.MethodsIn silico analysis was performed to quantified the expression and clinical analysis of CTPS2 and BRCA1. The expression was then validated on the internal sets. Loss-and gain-of-function assays were conducted to investigate the physiological phenotypes in CLL. RNA-seq was employed to probe the molecular mechanism of CTPS2.ResultsHerein, significant elevated expression of CTPS2 was observed in CLL patients compared to normal CD19 + B cells, which was verified in three independent cohorts. Furthermore, overexpression of CTPS2 was closely associated with undesired prognostic indicators, including unmutated IGHV status and chromosome 11q23 deletion. Additionally, elevated CTPS2 expression predicted adverse overall survival and treatment-free survival with independent prognostic significance. Downregulation of CTPS2 in CLL cells exhibited attenuated cell proliferation, arrested G2/M cell cycle and increased apoptosis. The addition of CTP or glutamine could reverse the above effects. Since RNA-seq showed the enrichment in DNA damage and response signaling, we subsequently found that silence of CTPS2 remarkably elevated DNA damage and decreased DNA repair. It was demonstrated that CTPS2 mediated DNA damage response via interacting with Breast Cancer 1 (BRCA1) protein in CLL through CoIP assays and rescued experiments.ConclusionsCollectively, our study generated the novel findings that CTPS2 promoted CLL progression via DNA damage response and repair pathway. Targeting nucleotide metabolism potentially became an attractive strategy for treatment against CLL.

Obinutuzumab in combination with chlorambucil in first line treatment of elderly patients with chronic lymphocytic leukemia

Introduction. Optimal therapy for elderly patients with chronic lymphocytic leukemia (CLL) is the subject of intensive research.Aim – to study the safety of obinutuzumab, as well as the selection of the optimal scheme of its use in patients with CLL, complicated by diabetes mellitus, renal insuffi ciency, cardiac comorbidity.Materials and methods. The study included primary patients with CLL having indications requiring therapy. The inclusion criteria were: Cumulative Illness Rating Scale, CIRS) (CIRS) &gt; 6 and or glomerular fi ltration rate (GFR) &lt; 70 mL/min, the lower limit of GFR was not restricted. The study focused on patients with diabetes mellitus, renal failure and signifi cant cardiac pathology. Patients with Richter’s syndrome, CNS involvement, HBs-antigen, and 17p deletion were not included. In the fi rst cycle obinutuzumab was administered at a dose of 100 or 25 mg on the fi rst day and 900 or 975 mg on the second day, then at a dose of 1000 mg on days 8 and 15. For all subsequent cycles, obinutuzumab was given at a dose of 1000 mg on day 1. The dosage of chlorambucil was 10 mg/m2 from days 1 to 7. Treatment cycles in totals of 6 were repeated every 28 days.Results. The study included 90 patients. Median age was 73.5 years, range – 60–89 years, there were 49 men (54 %) and 41 women (46 %). Twenty-four patients (27 %) had stage C, IGHV unmutated status was detected in 76 % of patients. The median creatinine clearance was 48.6 mL/min (25–110). The median CIRS score was 3 (range – 1–14). Thirty-one patients (34 %) had signifi cant cardiovascular comorbidity (previous myocardial infarction, coronary artery stenting or bypass, HF ≥ II NYHA, peripheral artery disease) as well as hemodynamically signifi cant valvular disease. Fifteen patients (17 %) had diabetes mellitus and 71 patients (79 %) had creatinine clearance &lt; 70 ml/min. Infusion reactions to obinutuzumab grade ≥ II were reported in 29 patients (32 %). Hospitalization on the day of administration or the next day after the fi rst administration was required in 5 cases (5.5 %). Twenty-seven (30 %) patients could not complete 6 cycles. The largest number of patients (14 people, 15.5 %) stopped treatment after 1 course. The causes were the development of persistent cytopenia (n = 4), grade IV reaction to obinutuzumab (n = 3), patient’s refusal (n = 2), infectious complications (n = 2), severe tumor lysis syndrome (n = 1), acute pancreatitis (n = 1) and toxicodermia (n = 1). The leading cause of premature discontinuation on subsequent cycles was persistent neutropenia. Progression during treatment occurred in 3 patients only. Overall survival was signifi cantly predicted by CIRS (maximum discriminatory value of 3, p = 0.013) as well as GFR &lt; 50 mL/min (p = 0.03). No other associations were identifi ed. At least 1 episode of grade III–IV neutropenia occurred in 41 % of patients. Grade IV neutropenia was associated with creatinine clearance &lt; 60 mL/min (p = 0.05), baseline neutrophil level &lt; 2 × 109/L (p = 0.0001), baseline monocyte level &lt; 0.3 × 109/L (p = 0.007) and age &gt; 70 years (p = 0.01). The effectiveness of treatment was evaluated in patients who completed at least 3 cycles of therapy. Complete remission was achieved in 26 patients (35 %), partial remission – in 41 (54 %), stabilization – in 4 (5 %), progression was noted in 3 (4 %). Sixteen patients (18 %) were not available to respond to the assessment. Minimal residual disease &lt; 0.01 % in the bone marrow after completion of treatment was found in 17 patients (19 %), within 0.01–0.9 % – in 25 (28 %) patients. The median follow-up from the date of therapy initiation was 39.7 months (range – 0.6–72 months). The median relapse-free survival was not reached, and 2- and 3-year survival rates were 81 and 62 %, respectively. Poor relapse-free survival signifi cantly correlated with unmutated IGHV genes (HR = 2.4, 95 % CI: 1.12–5.0, p = 0.02) and partial response as opposed to complete response (HR = 3.35; 95 % CI: 1.45–7.7, p = 0.03).Conclusion. The results of our study have practical implications as obinutuzumab is actively integrated into modern treatment regimens. Infusion reactions pose a high risk of complications in elderly patients. The dose of obinutuzumab on day 1 of administration in elderly patients, should not exceed 25 mg. The G-Clb regimen may not be optimal in patients over 75 years of age due to the unpredictable risk of complications. In patients at high risk of neutropenia, it may be appropriate to consider primary prophylaxis. ClbG is an effective regimen that resulted in high rate of MRD-negative responses and prolonged relapse-free survival.

Richter transformation in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia can evolve to an aggressive lymphoma-in most of the cases diffuse large B cells lymphoma, rarely Hodgkin lymphoma-and this complication is defined Richter syndrome (RS). Immunogenotypic features that characterize RS include unmutated IgHV status with high prevalence of IgHV4-39/D6-13/J5 sequence; deletion of chromosome 17p or 11q; activation of oncogenes as NOTCH1 and c-MYC; inactivation of onco-suppressors as TP53 and CDKN2A; high expression of CD38 in lymph-nodes. The prognosis of this condition is very poor: patients experience a rapid clinical deterioration with frequent therapeutic failure since the current options include suboptimal strategies as standard chemo-immunotherapy followed by hematopoietic stem cells transplantation or enrollment in clinical trials which investigate the efficacy of target drugs. Understanding the biology of such a heterogeneous condition is crucial to personalize the treatment and improve patient's survival.

Treatment Patterns and Outcomes of Patients with Chronic Lymphocytic Leukemia with TP53 Mutations

Background: Aberration of the TP53 gene in chronic lymphocytic leukemia (CLL) is a well-established marker of poor prognosis and a predictor of chemorefractory disease. Identification of a TP53 gene mutation is clinically useful in determining which patients may benefit from small-molecule inhibitors such as Bruton tyrosine kinase (BTKi) and BCL2 inhibitors (BCL2i). However, the treatment patterns in routine practice and outcomes of patients harboring TP53 mutations in CLL is not well reported. We present a descriptive analysis of CLL patients treated at Mayo Clinic with TP53 mutations including their demographics, treatment patterns, and outcomes. Methods: Patients with CLL who had a TP53 mutation detected by Sanger sequencing between June 2014 and April 2021 at Mayo Clinic Rochester were identified from a hematopathology database. Patient demographics, clinical and pathologic characteristics, treatment, and follow-up information were abstracted from the Mayo Clinic CLL database and by chart review. Date of TP53 mutation test was baseline. Discrete variables were compared using Chi-square or Fisher's exact tests; continuous variables were compared using Kruskal-Wallis test. Overall survival (OS) was calculated from date of TP53 mutation test until date of death from any cause or last known date alive. Rates of Richter transformation (RT) by prior treatment exposure were estimated by cumulative incidence accounting for competing risk of death. Results: A total of 145 patients were identified; 70% were male, and the median age at diagnosis was 59 years (range: 36-92). At diagnosis, 15% of patients had Rai stage III-IV, 77% had unmutated IGHV, 38% had del17p, 92% had high or very high CLL-IPI, and 62% had ≥3 cytogenetic abnormalities (Table 1). A TP53 gene mutation was identified prior to any CLL treatment in 63 patients (43%, Group 1) and after at least one prior line of treatment in 82 patients (57%, Group 2). The median time from CLL diagnosis to the first detected TP53 mutation was 3 months in Group 1 and 96 months in Group 2. There was significant heterogeneity in types of TP53 mutations with the majority of variants (n=102) being unique to an individual. At the time of TP53 mutation detection, Group 2 patients more frequently had advanced Rai stage and unmutated IGHV status (Table 1). Del17p by FISH was present prior to or at time of TP53 mutation identification (+/- 3 months) in 54% of patients in Group 1 and 52% in Group 2. Group 1 had a median follow-up of 26 months (from TP53 mutation detection date); 46/63 patients had initiated a first-line (1L) treatment (39 BTKi-based, 1 BCL2i-based, 2 BTKi+BCL2i-based, 4 chemoimmunotherapy [CIT]). In Group 2, the median number of prior lines of therapy was 2 (range 1-16). Prior treatment exposure included 38 (46%) CIT only, 41 (50%) BTKi-based ± CIT, and 3 (4%) BTKi-based + BCL2i-based ± CIT. Median follow-up was 21 months; 56/82 patients had initiated a new line of therapy (subsequent 1L). For 38 patients with prior CIT only exposure, 28 had subsequent 1L including 21 BTKi-based, 2 BCL2i-based, and 5 CIT. For 44 patients with prior BTKi exposure, 28 had subsequent 1L including 5 BTKi-based, 5 BCL2i-based, 9 BTKi+BCL2i-based, and 9 CIT. The median OS was not reached in Group 1 and was 40.6 months (95% CI 30.1-NE) in Group 2 (HR=0.21, 95% CI=0.10-0.45) (Figure 1). In Group 2, the median OS for patients with prior CIT only exposure was 55.5 (95% CI 30.1-NE) months and for patients with prior BTKi exposure was 33.3 (95% CI 14.7-NE) months (HR=0.59, 95% CI=0.31-1.12) (Figure 1). Three patients in Group 1 and 13 patients in Group 2 (3 prior CIT only, 10 prior BTKi) developed RT during the follow-up period; the cumulative incidence rates of RT at 2- and 5-year were 1.7% and 4.6%, respectively, in Group 1, and 10.8% and 21.0%, respectively, in Group 2. Conclusions: Regardless of prior treatment exposure, enrichment for high-risk features was present at time of TP53 mutation detection. Majority of patients were treated with novel agents after known TP53 mutations. OS after TP53 mutation was inferior in patients who had been previously treated, particularly those who had BTKi exposure already prior to TP53 mutation detection, compared to previously untreated patients. Future studies are needed to understand the characteristics of clonal evolution before and after treatment and the clinical impact of specific TP53 mutations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Chronic Lymphocytic Leukemia Treatment: Single Center Experience

Background: Chronic lymphocytic leukemia (CLL) is relatively uncommon in the Middle East, with a younger age at diagnosis and different clinical characteristics compared to the West. There is little research on CLL in this region. This study aims to evaluate widely used prognostic markers and examine treatment outcomes of CLL patients in the Kuwait Cancer Control Center (KCCC), the only cancer center in Kuwait. Methods: This is a retrospective analysis of unselected 141 CLL patients receiving treatment in the KCCC. Diagnostic and treatment guidelines in our center were in accordance with the International Workshop on CLL criteria. Results: The median age of those patients at diagnosis was 58 years (IQR, 51-65), with 60% of patients under the age of 60. The male to female ratio in this group was 3.3:1. Median absolute lymphocyte count was 51x109/L (IQR, 51-65), and the median hemoglobin and platelet count were 11.8 g/dL (IQR, 10.1-13.8) and 170x109/L (IQR, 129-222). FISH analysis was performed in 118 patients, of which 9% (11) demonstrated del17p. Del13q and trisomy 21 were detected in 31% (36) and 26% (31) patients, respectively. IGHV mutational status was determined in 80 patients, 25% (20) were mutated, and 75% (60) were unmutated. When it comes to treatment, 47% (65) of patients were on FCR regimen, while 11% (15) and 12% (16) were receiving BR and CR, respectively. Approximately 15% (21) of patients in our study were on a BTKi. In 105 patients, the median progression-free survival (PFS) was 44.2 months [95% CI: 36-61.5], with a PFS rate of 48% and 32% at 25 and 50 months, respectively. In 121 patients with no history of progression or treatment in our study cohort, 41.5% and 21% progressed or received treatment at 25 and 50 months, respectively, with a median time to first progression or treatment around 37.6 months [95% CI: 29.1-49.5]. Examining 103 patients with prior history of progression or treatment, 34% and 13% progressed again or required a second course of treatment at 25 and 50 months, respectively, with a median time to second progression or treatment around 65.5 months [95% CI: 40.5-NA]. Those receiving chemoimmunotherapy exhibited a higher median PFS of 93 months [95% CI: 83-NA] compared to those receiving a BTKi with a median PFS of 42 months [95% CI: NA-NA]. However, the difference was not statistically significant (p=0.34). IGHV mutation status was significantly associated with OS (p=0.044) but not PFS (p=0.28). Those with unmutated IGHV had an inferior outcome compared to their mutated counterparts with OS rates at 50 months, around 15% and 25%, respectively, PFS rates were also lower in the unmutated compared to a mutated group, 21% vs. 31% at 50 months, respectively, and PFS medians around 49 [95% CI: 36.4-NA] and 94.5 [95% CI: 94.5-NA], respectively. Multivariate cox regression analysis on OS was not significant (p=0.160). None of the individual treatment regimens were significant predictors of OS compared to the reference (BR), except FCR. Treatment with FCR was associated with a hazard ratio of 0.11 (p = 0.011). Unmutated IGHV status, presence of del17p, and age greater than 60 were all associated with greater hazard ratios, but none were significant predictors of OS. Conclusion: Our CLL treatment group is younger. Unmutated IGHV was associated with a poor prognosis, consistent with the literature. However, treatment with BTKis was not associated with superior outcomes compared to CIT; however, this study is not powered to detect a difference between the two arms. Those receiving FCR had the best survival outcome compared to those receiving other therapies. Keywords: Chronic Lymphocytic Leukemia, Treatment Outcomes, Kuwait, Middle East Funding Agency: Kuwait Foundation for the Advancement of Science, Grant number: PR1713MM03 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Final Analysis of the Prospective Multicenter CLL2-Give Trial of Obinutuzumab (GA101, G), Ibrutinib (I), and Venetoclax (Ve) in Untreated Patients with CLL with 17p Deletion/TP53 Mutation

Background There is still need for improvement in outcome of high-risk chronic lymphocytic leukemia (CLL) with del(17p) and/or TP53 mutation. The CLL2-GIVe trial tested time limited, response-adapted, fixed duration combination treatment of obinutuzumab, ibrutinib and venetoclax ("GIVe" regimen) in high-risk CLL patients (pts) with a combination of three different agents with distinct mechanisms of action. Here we present the final analysis at the end of the study. Methods CLL2-GIVe is an open-label, multicenter phase 2 trial including pts with previously untreated CLL with del(17p) and/or TP53 mutation. Induction therapy contained 6 cycles (C) of obinutuzumab (GA-101), ibrutinib, and venetoclax, venetoclax and ibrutinib were continued up to C12 as consolidation. Ibrutinib monotherapy was given for C13 to C36 in pts not achieving a complete response (CR) and undetectable minimal residual disease (uMRD) at cycles 9 and 12. The primary endpoint was CR rate at C15 (final restaging) with a null-hypothesis of 25%. Secondary endpoints included MRD, survival, and safety. Results Between September 2016 and August 2018, 41 pts were enrolled. The last pt reached end of study in January 2022. Median age was 62 (range 35-85) years, del(17p) was present in 26 pts, TP53mut in 39 pts and 32 pts had an unmutated IGHV status. With a CR rate of 58.5% (24 pts) at final restaging (C15), the primary endpoint was met (95% CI: 42.1-73.7, p<0.001), rate of partial remission (PR) was 41.5% (17 pts). Peripheral blood uMRD at C15 was achieved in 32 pts (78%). After a median observation time of 38.4 (3.7 - 44.9) months, 9 progression-free survival (PFS) events and 3 overall survival (OS) events had occurred. The 36-month OS is 92.6 % (Figure 1a) and median OS is not reached. Three pts died (1 pt at C3 due to retrospectively pre-existing ovarian cancer, 1 pt at C9 due to cardiac failure unrelated to study treatment, 1 pt due to respiratory tract infection after Richter transformation (RT) under subsequent treatment). The PFS rate at 36 months is 79.9% and median PFS is not reached (Figure 1b). Progressive disease (PD) was observed in 7 pts between C27 and C42. PD was nodal (n=3) or lymphocytosis (n=3), and 1 case of RT occurred. Subsequent treatments were administered in 4 pts (1 pt with 3 therapies: venetoclax, idelalisib/rituximab, ibrutinib; obinutuzumab (n=1); acalabrutinib/venetoclax/obinutuzumab (n=2)). Six pts received complete ibrutinib maintenance treatment after C15 due to PR at final restaging, 3 of these had also detectable MRD. None of these pts progressed. 6 pts with PR and uMRD discontinued treatment at C15 not according to protocol: 2 pts stopped due to adverse event (AE); 4 pts due to other reasons. 5 pts with PR who discontinued treatment at C15 not according to protocol had no uMRD: 2 pts stopped due to AE, 1 pt due to death, 1 pt due to other reasons, 1 pt refused ibrutinib maintenance. At C36, 18 (43.9%) of pts had uMRD in peripheral blood, while 12 pts (29.3%) had detectable MRD at C36: 8 pts were MRD intermediate (≥ 10-4 and < 10-2) (19.5%), 4 pts positive (≥10-2) (9.8%). In 11 pts (26.8%) analysis was not performed due death (n=2), beginning of a new CLL therapy (n=2), 1 withdrawal of informed consent, missed visits during Covid19 pandemic (n=3), 3 pts were lost to follow-up. The most frequent grade 3-5 AEs up to the end of study were blood and lymphatic system disorders (58.5%), with neutropenia (48.8%) and thrombocytopenia (17.1%), and infections (19.5%). Most frequent AEs of any grade were gastrointestinal disorders in 85.4% of pts, most of them were low grade. Most common cardiac disorder of any grade was atrial fibrillation in 14.6% of pts. Conclusion The CLL2-GIVe regimen is a potent and promising fixed-duration, first-line treatment for pts with high-risk CLL with a manageable safety profile worthy of further exploration in phase 3 studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal