Cohort Of Chronic Lymphocytic Leukemia Patients Research Articles

A Practical Guideline for MicroRNA Sequencing Data Analysis in Chronic Lymphocytic Leukemia.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. They have been associated with several diseases and cancers, including chronic lymphocytic leukemia (CLL). CLL is the most common form of adult leukemia, and its pathogenesis is driven by the deletion of miRNAs, such as the miR-15a/16-1 cluster. In addition to initiating the development of CLL, the function of miRNAs in regulating the progression of this tumor remains to be investigated. Here, we present a computational pipeline, from the processing of miRNA sequencing files to functional analysis, including differential gene expression and gene set enrichment analysis.We exemplified the utility of the pipeline by applying it to genome-wide small RNA sequencing data from a cohort of CLL patients. The analysis revealed dysregulated expression profiles of miRNAs in CLL. The target genes of these miRNAs are not only associated with the response of CLL patients to current therapies but also involved in several cancer hallmarks, including the avoidance of cell death, the deregulation of cellular energetics, the activation of invasion and metastasis, and genome instability. The identified miRNA-gene interactions offer valuable insights for developing targeted therapies for CLL. In addition, we underscored the importance of a practical and robust computational pipeline to ensure the reliability and reproducibility of miRNA sequencing data analysis.

Conditional survival to assess prognosis in patients with chronic lymphocytic leukemia

Biomarkers in chronic lymphocytic leukemia (CLL) allow assessment of prognosis. However, the validity of current prognostic biomarkers based on a single assessment point remains unclear for patients who have survived one or more years. Conditional survival (CS) studies that address how prognosis may change over time, especially in prognostic subgroups, are still rare. We performed CS analyses to estimate 5-year survival in 1-year increments, stratified by baseline disease characteristics and known risk factors in two community-based cohorts of CLL patients (Freiburg University Hospital (n = 316) and Augsburg University Hospital (n = 564)) diagnosed between 1984 and 2021. We demonstrate that 5-year CS probability is stable (app. 75%) for the entire CLL patient cohort over 10 years. While age, sex, and stage have no significant impact on CS, patients with high-risk disease features such as non-mutated IGHV, deletion 17p, and high-risk CLL-IPI have a significantly worse prognosis at diagnosis, and 5-year CS steadily decreases with each additional year survived. Our results confirm that CLL patients have a stable survival probability with excess mortality and that the prognosis of high-risk CLL patients declines over time. We infer that CS-based prognostic information is relevant for disease management and counseling of CLL patients.

PRMT1 Promotes Chronic Lymphocytic Leukemia Progression Via Modulating Methylation of MAST1

Introduction As a member of the protein arginine N-methyltransferase family, protein arginine methyltransferase 1 (PRMT1) plays a crucial regulatory role in the etiology of malignant neoplasms. PRMT1-mediated protein methylation promoted the maintenance of acute myeloid leukemia, which revealed the promising potency of PRMT1 inhibitors. Recent study suggested that dysregulation of PRMT5 promotes Richter's transformation in chronic lymphocytic leukemia (CLL). However, the effects of PRMT1 in the tumorigenesis and progression of CLL still remained ill-defined. Hence, the aim of this study was to investigate the clinical significance and mechanisms of PRMT1 underlying the development of CLL. Methods Peripheral blood samples were collected from 79 newly diagnosed CLL patients (47 males and 32 females; age range 39-85 years, median 63 years) in Shandong Provincial Hospital CLL (SPHCLL) cohort with informed consent. CRISPR-Cas9 technology was used to stably knockout PRMT1 in CLL cells. A label free quantitative proteomics analysis was implemented to reveal the protein methylation mediated by PRMT1. Assessment of cell viability, apoptosis and cell cycle were analyzed by cell counting kit-8, annexin V-PE/7AAD and PI/ RNase staining, respectively. This study was approved by the Medical Ethics Committee of Shandong Provincial Hospital. Results This study examined the expression of PRMT1 in SPHCLL and GEO databases, and discovered the upregulation of PRMT1 mRNA in CLL cells. Aberrantly elevated expression of PRMT1 was observed in a cohort of newly diagnosed CLL patients than healthy donors in mRNA level (donors vs. CLL patients, 0.07±0.06 vs. 0.20 ±0.21, p=0.013). Furthermore, increased expression of PRMT1 was correlated with inferior prognosis in two long-term follow-up cohorts of CLL patients (HR=2.80, p=0.002, and HR=1.551, p=0.009). To elucidate the functional significance of PRMT1 in CLL, we established stable PRMT1 knockdown cells using lentiviral shRNAs and PRMT1 knockout cells using CRISPR/Cas9 technology. The suppression of PRMT1 remarkably inhibited cell proliferation, induced cell apoptosis and blocked cell cycle at G1/S phase in CLL cells. Furthermore, it was observed that C7280948, a selective inhibitor of PRMT1, resulted in the defective proliferation of CLL primary cells in a dose-dependent manner. We subsequently performed pre-clinical investigations of C7280948 in the orthotopic CLL xenograft murine model. Notably, the administration of C7280948 significantly diminished the leukemia burden and induced the lessened degree of splenomegaly in comparison to the control group (Fig. 1A). Moreover, a reduction in the population of CLL cells was detected in bone marrow and spleen of mice treated with C7280948. To decipher the role of PRMT1 in the pathogenesis of CLL, the label free quantitative proteomics analysis was conducted on PRMT1-deficent and control MEC1 cells. Knockdown of PRMT1 diminished asymmetric dimethylarginine of some proteins in CLL cells. In accordance with the results of proteomics analysis, microtubule associated serine/threonine kinase 1 (MAST1) was selected as a candidate target for further investigation. The methylation site of MAST1 protein was identified as R802 (Fig. 1B). Increased expression of MAST1 was correlated with inferior prognosis of CLL patients ( p<0.001), indicating the essential role of MAST1 in the progression of CLL. These results supported the hypothesis that PRMT1 interacted with MAST1 and enhanced the activity of MAST1 via methylating this oncoprotein in CLL cells. Conclusion The present study provides robust evidence for the oncogenic function of PRMT1 in the pathogenesis of CLL, highlighting the potential therapeutic efficacy of the selective PRMT1 inhibitor C7280948. Collectively, the targeted inhibition of PRMT1 exhibits promising prospects for the management of CLL patients.

Expression signature of human endogenous retroviruses in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is one of the most diagnosed forms of leukemia worldwide and it is usually classified into two forms: indolent and aggressive. These two forms are characterized by distinct molecular features that drive different responses to treatment and clinical outcomes. In this context, a better understanding of the molecular landscape of the CLL forms may potentially lead to the development of new drugs or the identification of novel biomarkers. Human endogenous retroviruses (HERVs) are a class of transposable elements that have been associated with the development of different human cancers, including different forms of leukemias. However, no studies about HERVs in CLL have ever been reported so far. Here, we present the first locus-specific profiling of HERV expression in both the aggressive and indolent forms of CLL. Our analyses revealed several dysregulations in HERV expression occurring in CLL and some of them were specific for either the aggressive or indolent form of CLL. Such results were also validated by analyzing an external cohort of CLL patients and by RT-qPCR. Moreover, in silico analyses have shown relevant signaling pathways associated with them suggesting a potential involvement of the dysregulated HERVs in these pathways and consequently in CLL development.

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

Chronic lymphocytic leukemia (CLL) patients with hepatitis B virus (HBV) infection have a poor prognosis, underlying mechanism remains unclear. NOTCH mutations are frequent in CLL and associated with disease progression and drug resistance. It is also reported to be associated with hepatitis infection in lymphoid malignancies. In order to investigate the relation between NOTCH pathway and HBV-associated CLL, we studied 98 previously untreated HBV positive CLL patients and 244 HBV-negative CLL. A total of 342 treatment naïve patients diagnosed with CLL at the First Affiliated Hospital of Nanjing Medical University from 1 January 2010 to 31 October 2021 were enrolled in our study. The mutation hotspots of NOTCH pathway genes were analyzed by Next Generation Sequencing (NGS) of DNA that used PCR for the identification of genomic mutation. Fisher exact and χ2 tests were used to assess the correlation between HBV infection and clinical, demographic factors in CLL patients. Survival curves were generated by Graphpad 9.5. NOTCH mutations were more frequent in HBV positive CLL subgroup (17.3% vs. 7.4%, p = 0.033). By survival analysis, HBV infection was associated with disease progression and poor survival (p = 0.0099 for overall survival [OS] and p = 0.0446 for time-to-treatment [TTT]). Any lesions of the NOTCH pathway (NOTCH1, NOTCH2 and SPEN) aggravated prognosis. In multivariate analysis, NOTCH mutation retained an independent significance for HBV-infected patients (p = 0.016 for OS and p = 0.023 for TTT). However, HBV positive with NOTCH unmutated had no statistical difference in prognosis compared with HBV negative patients (p = 0.1706 for OS and p = 0.2387 for TTT), which indicated that NOTCH pathway mutation contributed to inferior prognosis in HBV-infected CLL. In conclusion, a cohort of CLL patients with HBV positive displayed a worse clinical outcome and the status of NOTCH signaling pathway might play a crucial role. Keywords: chronic lymphocytic leukemia (CLL), diagnostic and prognostic biomarkers No conflicts of interests pertinent to the abstract.

Cost-Utility of Venetoclax Plus Obinutuzumab for Treatment-Naïve Patients with Chronic Lymphocytic Leukemia without Del17p/TP53-Mutation Compared to Chlorambucil Plus Obinutuzumab

INTRODUCTION: Recently presented 6-year follow-up data from the CLL14 trial for chronic lymphocytic leukemia (CLL) showed increased overall survival (OS) and progression-free survival (PFS) using venetoclax plus obinutuzumab (VenO) as 1st line treatment when compared to chlorambucil plus obinutuzumab (ClbO) (Al-Sawaf, 2022). However, the higher treatment cost of VenO compared to off-patent chemotherapy requires evaluation of the potential trade-offs of added costs and improved health outcomes to inform decision-making. In Denmark, targeted agents are only reimbursed for treatment-naïve CLL patients with del17p/TP53-mutation. The objective of this study was to investigate the cost-effectiveness of VenO compared to ClbO in treatment-naïve CLL without del17p/TP53-mutation, including the course of disease until death. METHODS: A cost-utility analysis was conducted to simulate a hypothetical cohort of CLL patients with characteristics corresponding to the CLL14 cohort without del17p/TP53-mutation receiving either VenO or ClbO as 1st line treatment followed by either venetoclax plus rituximab (VenR) or ibrutinib (Ibr) as 2nd line treatment or no treatment after progression. Second line treatment was assumed to begin one month after progression. Model outcomes were the expected cost, quality-adjusted life-years (QALYs) and life-years (LY) per patient. Outcomes were used to calculate the incremental cost-effectiveness ratio (ICER) i.e. the added cost per extra QALY gained. A Markov model was constructed with a lifetime time horizon (Figure 1) and was set to replicate recently presented data for OS and PFS for 72 months from the CLL14 study. Parametric survival functions were estimated for the remaining life years, assuming a log-normal and exponential distribution for OS and PFS, respectively in the base-case. A Danish health sector perspective was applied with all costs estimated in EUR. Costs and quality of life decrements related to administration of both 1st and 2nd line treatment, as well as adverse event treatment, were included. Best available published data from the MURANO trial (VenR) (Kater et al., 2020) and Danish real-world evidence (Ibr) (Aarup et al., 2020) for 2nd line treatment was utilized to reflect current clinical practice in Denmark. Deterministic and probabilistic sensitivity analyses (PSA) were performed. Due to the absence of an official threshold of marginal cost per QALY in Denmark, results were compared to the British National Institute for Health and Care Excellence (NICE) threshold of GBP 20,000-30,000 (EUR 23,600-35,600). RESULTS: First-line treatment with VenO provided 5.28 QALY (7.54 LY) for the average patient compared to 3.80 QALY (6.12 LY) in the ClbO group (Table 1). Total costs per patient were EUR 202,821 for VenO and EUR 195,781 for ClbO, resulting in a base-case ICER of EUR 4,808 per QALY. The PSA showed that VenO was recommendable in 97% of the simulations at the lower NICE threshold. Results were robust to the assumptions on choice of 2nd line treatment, while exclusion of all costs and effects of 2nd line treatment showed cost-effectiveness results favoring ClbO. Cost of 2nd line treatment constituted up to 70% of the total patient cost, and especially the use of Ibr in 2nd line was associated with a higher total cost per patient. At a discount of 15% on the list price of venetoclax, 1st line VenO provided savings compared to ClbO. CONCLUSIONS: VenO was more effective in terms of QALY but at a higher cost than ClbO. VenO was cost-effective with inclusion of costs and effects of 2nd line treatment when using the NICE threshold indicating that VenO may also be cost-effective in Danish, and potentially other European, healthcare settings. While additional sensitivity analyses are awaited, these findings, along with previously published OS and PFS data, suggest that moving VenO into 1st line treatment may be beneficial and cost-effective also for patients without del17p/TP53-mutation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Chronic Lymphocytic Leukemia in African Ancestry Population Is Characterized By Increased Telomere Erosion

Background: Chronic lymphocytic leukemia (CLL), a neoplastic disease of relatively mature B-cells, is characterized by a variable clinical course. Although initial studies suggesting more aggressive disease phenotype and inferior clinical outcomes among CLL patients of African ancestry (AA) were published a decade ago, biological features contributing towards this augmented disease severity remain largely unknown. Telomere erosion in a cohort of CLL patients of European Ancestry (EA) has been previously associated with high-risk molecular markers including unmutated immunoglobulin heavy-chain variable region (u-IGHV) status, and genetic alterations such as del(17p) and del(11q) by FISH. Here we characterized telomere erosion in AA CLL patients compared to CLL patients of EA. Methods: We extracted DNA from CD5+/CD19+ cells peripheral blood mononuclear cells and utilized it to measure the average telomere length through quantitative-PCR (qPCR). Briefly, primers for telomere and the single copy gene (SCG) HBB were used for amplification of 20 ng of DNA in a total volume of 20 µL in triplicates using the KAPA SYBR® FAST optimized for LightCycler®480 and analyzed on a LightCycler®480. Absolute telomere lengths were then obtained by generating standard curves using synthetic oligonucleotide PCR standards for telomere and the SCG gene. The qPCR results were validated by terminal restriction fragment length (TRF) analysis and Southern hybridization (N=28; r=0.8531; p<0.0001) using the TeloTTAGGG Telomere Length Assay. TRF was used as the gold standard of telomere length quantification; however, the high quantity of genomic DNA required for the assay (2µg) limited its broad use. Five CLL samples with known telomere lengths through TRF analysis were included in every qPCR plate in order to perform a linear regression to account for variability between qPCR plates as described previously. Results: Telomere erosion was evaluated in a cohort of 133 AA (mean age at sample collection 62.7 years ±13.6) and 147 age-matched EA CLL patients (mean age at sample collection 61.6 years ±10.2). The AA samples were collected from 95 (71%) male patients and 71% presented u-IGHV, while EA samples were collected from 110 (77%) male patients and 66% u-IGHV. As expected, we observed shorter telomere length in u-IGHV CLL (median 3.7 kb; range 0.8-44.9) compared to mutated immunoglobulin heavy-chain variable region (m-IGHV) CLL (median 6.8 kb; range 1.8-44.7; p<0.0001) in the full cohort. AA CLL presented shorter telomere lengths (median 4.2 kb; range 0.6-13.1) when compared to EA CLL (median 4.7 kb; range 1.1-44.9; p=0.005; Figure 1A). We highlight that the differences in telomere length observed were restricted to the CD5+/CD19+ cells, with no difference observed in germline samples collected from AA and EA patients. When stratifying by IGHV mutation status, shorter telomere length was observed in u-IGHV when compared to m-IGHV among both AA CLL (median 3.5 kb and 6.3, respectively; p<0.0001) and EA CLL (median 3.9 kb and 7.5, respectively; p<0.0001; Figure 1B). The significant shorter telomere lengths in AA CLL remained significant (p=0.006) after accounting for IGHV status, age, sex, number of mutated CLL driver genes, and Rai stage in multivariate analyses. Conclusions: Our results demonstrated increased telomere erosion in AA CLL compared to EA CLL, even after accounting for other known prognostic factors. Telomere length has been described as an independent prognostic factor in multiple cancers. In CLL, telomere shortening can lead to a selective pressure for loss of function of DNA damage checkpoint genes, becoming permissive to further cell proliferation and telomere shortening. Future studies are needed to see if these findings contribute towards the severe clinical outcomes in AA CLL compared to EA CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract A04: Targeted protein degradation as a strategy to overcome non-covalent BTK inhibitor resistance in lymphoma

Abstract Bruton’s tyrosine kinase (BTK) is a target for multiple generations of covalent (irreversible) and non-covalent (reversible) inhibitors due to its critical role in the proliferation and survival of B-cell malignancies. Similar to drug resistance mechanisms in other cancers, resistance to covalent BTK inhibitors (BTKi) in chronic lymphocytic leukemia (CLL), such as ibrutinib, arise through on-target BTK mutations at the BTK C481 residue (the binding site of ibrutinib), which allow escape from BTK inhibition. Non-covalent BTKi represent a new avenue to overcome resistance to the clinically approved covalent BTKi; however, we have recently discovered mechanisms of resistance to non-covalent BTK inhibition in patients with CLL (Wang, Xi, Thompson, Montoya et al NEJM 2022). We used bulk and single cell genomic analyses that identified a series of acquired BTK mutations in a cohort of CLL patients that relapsed on the phase I/II clinical trial of pirtobrutinib. We discovered mutations (BTK V416L, A428D, M437R, T474I, L528W) that occur at critical residues within the catalytic kinase domain of BTK and conferred resistance to both non-covalent and covalent BTKis. Based on structural models of BTK, we observed that these mutations physically impede drug binding, and disrupt the normal kinase activity of BTK but can, upon B-cell receptor stimulation, sustain AKT, ERK, and NF𝜅B signaling as well intracellular Ca2+ release in the presence of pirtobrutinib. In addition, CITE-seq analyses of 53,722 cells from this cohort identified pre-existing leukemic and immune cell states associated with development of resistance in patients. These findings identify novel mechanisms to engender clinical resistance to non-covalent BTKis, including contribution of the immune microenvironment to response to BTK inhibition. Together our data suggest that mutations at the BTK kinase domain may alter conformation of BTK’s non-kinase protein interaction domains thus allowing BTK to be used as a scaffold for other signaling molecules to phosphorylate phospholipase C gamma 2 (PLC𝛾2), the direct downstream target of BTK. We have performed mass spectrometry phosphoproteomics to evaluate potential signaling molecules that are active and could bypass BTK in catalytically inactive BTK mutant cells. Given the above, we decided to target BTK’s non-kinase function by investigating targeted protein degraders of BTK using BTK-degrading protein targeting chimeras (PROTACS). The immediate advantages of using protein degraders are (1) Binding may occur at any site of the target protein, and (2) PROTACS can act catalytically to bind to and degrade multiple target proteins. Our studies have shown that several of the BTK mutants that engendered clinical resistance to covalent and non-covalent BTKi are sensitive to these degraders, elucidating an exciting alternative therapeutic approach for patients who become resistant to BTKi therapies. We are continuing to test the efficacy of BTK PROTAC degraders in overcoming acquired resistance mechanisms in CLL as well as other B-cell lymphomas. Citation Format: Skye Montoya, Eric Wang, Jessie Bourcier, Sana Chaudhry, Tulasigeri Totiger, Alejandro Pardo, Gabriel Pardo, Maurizio Affer, Jacob Jahn, Anthony Mato, Omar Abdel-Wahab, Justin Taylor. Targeted protein degradation as a strategy to overcome non-covalent BTK inhibitor resistance in lymphoma [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A04.

The spectrum of subclonal TP53 mutations in chronic lymphocytic leukemia: A next generation sequencing retrospective study.

Chronic lymphocytic leukemia (CLL) is a hematological disorder with complex clinical and biological behavior. TP53 mutational status and cytogenetic assessment of the deletion of the corresponding locus (17p13.1) are considered the most relevant biomarkers associated with pharmaco-predictive response, chemo-refractoriness, and worse prognosis in CLL patients. The implementation of Next Generation Sequencing (NGS) methodologies in the clinical laboratory allows for comprehensively analyzing the TP53 gene and detecting mutations with allele frequencies ≤10%, that is, "subclonal mutations". We retrospectively studied TP53 gene mutational status by NGS in 220 samples from 171 CLL patients. TP53 mutations were found in 60/220 (27.3%) samples and 47/171 (27.5%) patients. Interestingly, subclonal mutations could be detected in 31/60 samples (51.7%) corresponding to 25 patients (25/47, 53.2%). We identified 44 distinct subclonal TP53 mutations clustered in the central DNA-binding domain of p53 protein (exons 5-8, codons 133-286). Missense mutations were predominant (>80%), whereas indels, nonsense, and splice site variants were less represented. All subclonal TP53 variants but one [p.(Pro191fs)] were already described in NCI and/or Seshat databases as "damaging" and/or "probably damaging" mutations (38/44, 86% and 6/44, 14%, respectively). Longitudinal samples were available for 37 patients. Almost half of them displayed at least one TP53 mutant subclone, which could be alone (4/16, 25%) or concomitant with other TP53 mutant clonal ones (12/16, 75%); different patterns of mutational dynamics overtimes were documented. In conclusion, utilization of NGS in our "real-life" cohort of CLL patients demonstrated an elevated frequency of subclonal TP53 mutations. This finding indicates the need for precisely identifying these mutations during disease since the clones carrying them may become predominant and be responsible for therapy failures.

P596: CLINICAL IMPACT OF TP53 DISRUPTION IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS TREATED WITH A BCR INHIBITOR. A CAMPUS CLL EXPERIENCE

Background: In chronic lymphocytic leukemia (CLL), the presence of TP53 aberration (mutations and/or deletion) predicts an increased risk of relapse and death after chemo-immunotherapy (CIT). Although TP53 deletion and mutations mostly co-occur and are considered equal prognosticators, the clinical impact of isolated or concomitant mutations and deletions remains unclear in the context of targeted agents. Aims: To investigate the clinical relevance of isolated or concomitant TP53 aberrations in a large cohort of CLL patients treated with BCR inhibitors. Methods: In the framework of an institutional Italian multicenter working group on CLL (Campus CLL), a retrospective analysis of 229 CLL patients (51 treatment naïve, and 178 relapsed/refractory) treated with ibrutinib was carried out. All patients referred to a single institution for del17p analyses by FISH (167-kb 17p13 orange probe, MetaSystems) and TP53 mutations by NGS, both analyses carried out on CD19-purified (>85% pure) CLL samples within 6 months prior the start of ibrutinib treatment. The median follow-up from ibrutinib treatment was 36.3 months (95% CI 29.5-41.5 months). Overall survival (OS) and progression free survival (PFS) were computed from the start of ibrutinib treatment. Results: In the CLL cohort, 76 patients with del17p showed a trend for inferior OS respect to wt cases (P=0.0662, Fig.1A), while a significant correlation was found with PFS (P=0.0162). With regard to TP53 mutations, 296 TP53 mutations were found in 126 patients (range of mutations/patients 1-11). As with CIT, TP53-mutated patients, irrespective of VAF (Fig.1B), experienced a significantly worse OS and PFS than wt cases also by univariate analyses (P=0.0160, and P=0.0378, respectively). The combination of del17p with TP53 mutation data identified 95 cases with no TP53 aberrations, 8 cases del17p only, 58 cases TP53-mutated only, and 68 cases bearing both del17p deletion and TP53 mutation (Fig.1CD). Only patients with concomitant TP53 mutations and del17p experienced significantly shorter OS and PFS compared to TP53wt cases (P=0.0122, and P=0.0076, respectively; Fig.1CD). Conversely, patients presenting a single aberration showed no significant differences compared to wt patients (Fig.1CD). The simultaneous presence of TP53 mutations and del17p remained an independent predictor factor both for OS and PFS by multivariate analysis, together with the previous therapy lines (0-1 vs. >1), and anemia. The evolution of TP53-mutated clones was assessed by longitudinal NGS analysis of sequential PB samples collected from 38 patients (16 relapsed, and 22 on treatment) corresponding to 127 TP53 mutations. Among relapsed cases, 7 showed a prominent expansion of the TP53-mutated clone, 8 remained stable, and the remaining case displayed an evident decrease (Fig.1E). In non-relapsed patients, 3 cases presented an increase of TP53 mutations, 13 remained stable, and 6 showed a reduction (Fig.1F), with no significant difference compared to relapsed cases (P=0.0623, c2 test). BTK and PLCG2 mutations were found in 9/16 (56%) relapsed cases and in 3/22 (14%) patients still on ibrutinib (P=0.0492, c2 test). Of note, only 3/7 relapsed cases that presented a positive selection for TP53 mutations showed the presence of BTK mutations at the time of relapse. Image:Summary/Conclusion: This retrospective study indicates that only the concomitant presence of TP53 mutations and deletion is an independent negative prognostic factor for OS and PFS in patients with CLL on ibrutinib treatment; the simultaneous investigation of del17p and TP53 mutations may lead to a more precise risk assessment.

P624: CHROMOTHRIPSIS IN PATIENTS WITH CLL AND COMPLEX KARYOTYPE: PATTERNS OF ABERRATIONS AND PROGNOSTIC VALUE

Background: Chromothripsis is a unique catastrophic event found in 1-5% of chronic lymphocytic leukemia (CLL) patients, associated with poor prognostic factors and short survival (Edelmann et al, 2012; Puente et al, 2015; Leeksma et al, 2021). It is characterized by the presence of multiple genomic rearrangements in one chromosome, resulting in oscillating switches between 2-3 copy number states by chromosomal microarrays (CMA) or next-generation sequencing (Stephens et al, 2011). Optical genome mapping (OGM) is a new method based on the imaging of long DNA molecules (>250kb) labelled at specific motifs, generating a unique pattern. This allows mapping of the genomic location of each molecule and detection of structural and numerical chromosomal abnormalities with high resolution and sensitivity. Aims: 1. To analyze the genome complexity associated with chromothripsis and the patterns of rearrangements detected by OGM; 2. To determine the prognostic impact of chromothripsis in a cohort of CLL patients enriched in complex karyotypes (CK). Methods: A total of 33 patients with chromothripsis detected by CMA were included: 11 with chromothripsis-like patterns (≥7 switches), 22 with classical chromothripsis (≥10 switches). Nine of the cases were studied with OGM (Bionano Genomics). Clinico-biological features and time to first treatment (TTFT) were analyzed. Results were compared with a cohort of 129 patients showing CK without chromothripsis (control group) (Ramos-Campoy et al, 2021). Results: All patients with chromothripsis showed a high genomic complexity (30/33 with CK by chromosome banding analysis and 3/33 classified as complex by CMA [range: 10-24 CNV, being at least three of them >5Mb (range: 3-6)]. Forty-six chromothriptic events were identified. These affected 1-4 chr/patient, but a single chromosome was involved in 25/33 patients (76%). Chromothripsis was distributed throughout all chromosomes, most frequently affecting 3 (n=5), 6 (n=5) and 13 (n=5). The events were mainly multiple losses (25/46) or alternated gains and losses (19/46). Size and coordinates of CNVs detected by OGM and CMA were highly concordant. OGM detected multiple rearrangements cryptic by CMA (median: 9/chromothriptic event). Two patterns of chromothripsis-related rearrangements were identified: one clustered in the chromothriptic region (3/9) and the other involving both chromothriptic and non-chromothriptic chromosomes (range: 1-6 chr) (6/9). One patient showed 36 rearrangements throughout the genome, suggesting the coexistence of chromothripsis and chromoplexy, characterized by the presence of multiple chained translocations (Figure). Patients with chromothripsis showed a higher frequency of TP53del/mut (70% vs 39%; p=0.001) and a shorter TTFT (15m vs 2m; p=0.013) than the control group. No differences were observed for IGHV and del(11q) status. When stratifying patients based on TP53 status, the presence of chromothripsis was not associated with shorter TTFT. In the multivariate analysis including TP53, genomic complexity by CMA and chromothripsis, only TP53 was statistically significant (HR=1.6; p=0.029). Image:Summary/Conclusion: 1. Chromothriptic events involve multiple loci in CLL patients; 2. OGM allows not only the detection of CNV but also the identification of two rearrangement patterns associated with chromothripsis (clustered or involving non-chromothriptic chromosomes) whose clinical impact should be further explored. 3. In CLL patients with CK, the adverse prognosis of chromothripsis is associated with a higher frequency of TP53del/mut.

P615: BCL2 RESISTANCE MUTATIONS IN A REAL-WORLD COHORT OF PATIENTS WITH VENETOCLAX-TREATED CHRONIC LYMPHOCYTIC LEUKAEMIA

Background: The Bcl-2 inhibitor venetoclax has transformed the therapeutic landscape of therapy refractory and treatment naïve chronic lymphocytic leukaemia (CLL). Despite the remarkable response rates in both patient groups, a subset of CLL patients receiving venetoclax treatment experience disease progression, frequently associated with pathogenic variants of the BCL2 gene. Apart from the most common hotspot G101V and D103Y mutations, pathogenic variants affecting several different loci of the coding region of BCL2 have been described, however data and international recommendations on the sensitive detection and monitoring of these mutations are still absent to date. Aims: Our aim was to develop a droplet digital PCR (ddPCR) based assay for the detection of BCL2 G101V and D103Y mutations and to reveal further resistance mutations emerging at relapse by performing targeted ultra-deep next-generation sequencing (NGS). Methods: Peripheral blood samples from 71 patients treated with venetoclax-rituximab or first line venetoclax-obinutuzumab combination were collected from 11 Hungarian oncohematological centres. Genomic DNA was extracted from peripheral blood mononuclear cells and leukemic cell purity was assessed by flow cytometry. Fractional abundance of the most common hotspot BCL2 mutations G101V and D103Y was assessed using the QX200 digital droplet PCR system. Following ddPCR analyses, targeted ultradeep NGS was performed on samples obtained at relapse using a custom-designed panel covering the whole coding regions of BCL2, BTK, PLCG2 and TP53 genes. Fractional abundance of BCL2 mutations identified by NGS was assessed retrospectively in serial peripheral blood samples by ddPCR using custom-designed, allele-specific assays. Results: Lower quantitative limit of detection of the BCL2 ddPCR assays was tested and could ubiquitously be established as 0.01%. With a median follow up of 14 months, BCL2 G101V and D103Y mutations were detected in 8.5% (6/71) and 9.9% (7/71) of the patients, respectively, with 90% (9/10) of them experiencing relapse during the follow-up period. Three patients harbored both resistance mutations as detected by ddPCR. BCL2 G101V and/or D103Y were observed in 43.5% (10/23) of the cases showing signs of disease progression. In four cases, emergence of the mutations predated the first clinical signs of relapse with a median 4 months. In samples obtained at relapse, targeted ultradeep NGS uncovered two additional BCL2variants (V156D and A113G), which were successfully backtracked and detected by ddPCR. Both V156D and A113G were detected in patients previously found to harbor G101V or D103Y, with no further BCL2 resistance mutations identified in G101V and D103Y wild type cases. Summary/Conclusion: In a real-world cohort of CLL patients receiving venetoclax in combination therapy, comprehensive and sensitive screening of BCL2 mutations can identify molecular mechanisms of venetoclax resistance in nearly half of the patients experiencing relapse. In patients harboring multiple BCL2 mutations, convergent evolution of the CLL subclones may contribute to the driver mechanisms of resistance, justifying the comprehensive approach for the detection of these variants. In secondary venetoclax resistant cases displaying wild type BCL2, further molecular screening methods are required to reveal alternative genetic or non-genetic reasons for disease progression.

Structure and prognostic signifi cance of 13q14 deletion in chronic lymphocytic leukemia

Introduction. 13q14 deletion is the most common chromosomal abnormality in chronic lymphocytic leukemia (CLL), and as the sole abnormality determines the most favorable prognosis of the disease. Using molecular genetic methods two subtypes of 13q14 deletion were identifi ed based on the size of the lost chromosomal material: small (type I) with the involvement of the D13S319 segment containing MIR15A/MIR16-1 and DLEU1 genes and large (type II) containing centromeric region of 13q14 involving RB1 gene. Data on the impact of type I and II deletions on the course of CLL are controversial.Aim — to evaluate the prognostic signifi cance of different variants of 13q14 deletion in CLL.Patients and methods. The study enrolled two cohorts of CLL patients. Cohort 1: 256 patients who were studied by FISH with DNA probes for detection of 13q14/D13S319, 11q23/ATM, 17p13/TP53 deletions, and trisomy 12 before immunochemotherapy. 101 patients with identifi ed 13q14/D13S319 deletion were analyzed with a DNA probe for RB1 locus for determination of deletion size (type I or type II). Cohort 2: 28 patients at different stages of the disease with deletion 13q14 detected by FISH were studied by using combination of standard and molecular cytogenetic methods (mFISH, mBAND, arrayCGH) to clarify the structure of 13q abnormalities.Results. In Cohort 1 chromosomal aberrations were detected in 75 % of patients: 13q deletion — 52 % (isolated — 36 % of all cases and 48 % of cases with deletion), 11q deletion — 19 %, +12 — 13 %, 17p deletion — 6 %. 13q14 deletion type I was detected in 56 %, type II — in 44 % of patients. Type II deletion correlated with the presence of 11q deletion (p = 0.05). Isolated deletions of type I and II were found in 61 and 39 %, respectively. Biallelic deletion was identifi ed in 12.7 % of patients with 13q deletion. Statistically signifi cant differences in OS were obtained in type I and II groups of patients with isolated 13q14 deletions: median OS was not reached and made 67.5 months, respectively, p = 0.05. In Cohort 2 structural abnormalities of chromosome 13 by conventional cytogenetic analysis (CCA) were identifi ed in 50 % of cases: 13q deletion — 11 cases; translocations involving 13q14 — 6 cases. In 5 cases with biallelic deletion identifi ed by FISH, 13q14 deletion by CCA was detected in two patients, and only in one allele.Conclusion. In general, 13q14 deletion is a cytogenetic factor of favorable prognosis for CLL but its structure is heterogeneous. Loss of tumor suppressor RB1 (type II deletion) negatively affects OS in patients treated with immunochemotherapy

Notch2 Increases the Resistance to Venetoclax-Induced Apoptosis in Chronic Lymphocytic Leukemia B Cells by Inducing Mcl-1.

Chronic lymphocytic leukemia (CLL) has experienced a clinical revolution—thanks to the discovery of crucial pathogenic mechanisms. CLL is still an incurable disease due to intrinsic or acquired resistance of the leukemic clone. Venetoclax is a Bcl-2 inhibitor with a marked activity in CLL, but emerging patterns of resistance are being described. We hypothesize that intrinsic features of CLL cells may contribute to drive mechanisms of resistance to venetoclax. We analyzed the expression of Interferon Regulatory Factor 4 (IRF4), Notch2, and Mcl-1 in a cohort of CLL patients. We evaluated CLL cell viability after genetic and pharmaceutical modulation of Notch2 expression in patients harboring trisomy 12. We tested venetoclax in trisomy 12 CLL cells either silenced or not for Notch2 expression or in combination with an inhibitor of Mcl-1, AMG-176. Trisomy 12 CLL cells were characterized by low expression of IRF4 associated with high levels of Notch2 and Mcl-1. Notch2 and Mcl-1 expression determined protection of CLL cells from spontaneous and drug-induced apoptosis. Considering the involvement of Mcl-1 in venetoclax resistance, our data demonstrated a contribution of high levels of Notch2 and Mcl-1 in a reduced response to venetoclax in CLL cells carrying trisomy 12. Furthermore, reduction of Mcl-1 expression by silencing Notch2 or by treatment with AMG-176 was able to restore the response of CLL cells to venetoclax. The expression of Notch2 identifies a subset of CLL patients, mainly harboring trisomy 12, characterized by high levels of Mcl-1. This biological mechanism may compromise an effective response to venetoclax.

Incidence of Atrial Fibrillation and Bleeding in CLL Patients Treated with Ibrutinib: Evidence from the Veterans Health Administration

Background: Chronic Lymphocytic Leukemia (CLL) is the second most common type of leukemia in adults, with an estimated 62,130 new cases expected to be diagnosed in 2017 (Siegel, Miller, and Jemal 2017). The introduction of novel oral agents in CLL, starting with ibrutinib (IB) in 2013, has revolutionized the therapeutic landscape, with efficacy and safety that compare favorably overall with prior mainstays of treatment such as chemoimmunotherapy. However, novel agents are associated with specific toxicities that may limit their utility. Several clinical trials have suggested that there is an association between IB and the risk of bleeding-related adverse events and atrial fibrillation (Afib) in CLL patients (Leong et al. 2016; McMullen et al. 2014; Lipsky et al. 2015). The risk of bleeding and Afib in CLL patients treated with IB in a real-world clinical setting is unknown.Aims: This study examines the real-world incidence of bleeding and Afib in CLL patients receiving IBR in the Veterans Health Administration (VHA). We also report the utilization of anticoagulant and antiplatelet medications following treatment with IB. For comparison, the incidence of bleeding and Afib and use of anticoagulant and antiplatelet medications during treatment in a cohort of CLL patients treated with bendamustine and rituximab (BR) is also reported.Methods: Patients who were diagnosed and treated for CLL at the VHA from 2010-2014 were identified through the VA Clinical Cancer Registry (VACCR). Patients were excluded if they had a prior malignancy. Patients were followed until the end of study observation period, death, or lack of utilization of hematology/oncology services for 18 months, or incidence of another cancer. For the IB cohort, patients were indexed at the time of the first dispensation of IB. Comorbidities and medications that could influence incidence of bleeding or Afib were extracted in the six months prior to the index date while the incidence of bleeding and Afib was extracted in the six months following the index date. Charlson Comorbidity Index (CCI),(Deyo 1992; D'Hoore, Bouckaert, and Tilquin 1996) liver disease (cirrhosis, alcoholic liver disease, HCV infection)(Kramer et al., n.d.), and diabetes (controlled and uncontrolled) were examined (Guzman et al. 2014). Treatments dispensed, evidence of VHA system use, bleeding events, and Afib were determined from the administrative records, lab records, pharmacy dispensation records, and clinical notes in the electronic medical record. Descriptive statistics were used to report estimates.Results: From 2010-2014, 2,796 patients were diagnosed and received care for their CLL within the VHA. We identified 172 patients receiving IB and 291 patients receiving BR (table 1).Of the BR patients, 282 (97%) were male. Similarly, 167 (97%) of IB patients were male. The median age at diagnosis was 67 years for BR patients and 69 years for IB patients. The CCI, liver disease and end-stage liver disease comorbidities were higher in patients receiving BR than IB, however, there were significantly more patients with diabetes (both controlled and uncontrolled) who received IB (24%) compared to those who received BR (8%). The use of anticoagulants following induction therapy did not differ between BR and IB patients (9% v 8%), nor did the use of antiplatelets (6% v 2%). Incidence of anticoagulants and antiplatelet usage prior to BR or IB induction therapy was minimal (fewer than 10 patients) and is therefore not reported. Of the 291 patients that received BR, 12 (4%) developed a bleeding event compared to 20 (12%) of the 172 patients that received IB. Additionally, 13 (8%) of IB patients developed Afib compared to only 3% of BR patients.Conclusions: Real-world evidence from a nationwide cohort of CLL patients suggest that while IB is associated with increased bleeding-related adverse events and Afib it is comparable to those reported in previous clinical trials (Leong et al. 2016; Lipsky et al. 2015). Additionally, these findings suggest that patients in real-world clinical care settings with higher levels of comorbidities may be at an increased risk for bleeding events and Afib. [Display omitted] DisclosuresHalwani:Kyowa Hikko Kirin: Research Funding; Bristol Myers Squib: Research Funding; Seattle Genetics: Research Funding; Roche/Genentech Inc.: Research Funding; Genetech Inc.: Research Funding; Takeda: Research Funding; Pharmacyclics: Research Funding; Amgen: Research Funding; AbbVie: Research Funding; Immune Design: Research Funding; Miragen: Research Funding. Sauer:AbbVie: Research Funding; Genentech Inc.: Research Funding; Pharmacyclics: Research Funding; Amgen: Research Funding.

The Effect of Immune Checkpoint Inhibitors in Patients with Concomitant Advanced Melanoma and Chronic Lymphocytic Leukaemia

Immune checkpoint inhibitors (ICI's) have revolutionized the management of patients with advanced melanoma (AM). However, the data on the effectiveness of ICI's has been largely restricted to patients on clinical trials, thus excluding patients with second malignancies. Chronic Lymphocytic Leukemia (CLL) is the most prevalent adult leukemia and is associated immune dysregulation and a 3.8 fold increase in melanoma incidence. We report a multi-center, retrospective case-control study examining differences between patients who received checkpoint therapy in the context of the dual diagnoses of CLL and advanced melanoma (n = 20) compared to those with advanced melanoma alone (n = 154).Prognostic features for AM (including age, brain metastasis, visceral involvement, LDH and neutrophil count) were comparable between the two cohorts, however absolute lymphocyte count (ALC) was higher in CLL patients (p <0.0001) as anticipated. 3/20 of the CLL cohort experienced haematological autoimmune adverse events (AIHA, ITP, HLH), which necessitated cessation of ICI therapy; these events were not reported in the non-CLL cohort.There was not a significant difference between response rates to ICI's, 1-year survival or overall survival between CLL and non-CLL cohorts (Fig 1A). All recorded mortality (8/8) in the CLL cohort was attributable to AM progression or complications of ICI therapy rather than progressive CLL. The most significant predictor of survival in CLL cohort was prior exposure to fludarabine which showed a significantly shorter OS (median 2.3 vs 29.23 months, HR 7.406, p < 0.0001) than those who had not (Fig 1B).This cohort also offers a unique opportunity to observe the effects of ICI therapy on the progression of CLL. To our knowledge, this is the largest reported cohort of CLL patients treated with checkpoint inhibitors to date. The diagnosis of CLL pre-dated the diagnosis of AM in 18/20 cases. 5 cases had required prior treatment for CLL, while the remaining 15 cases were under observation alone. All 20 cases were Stage 0-1 according to the Rai classification at the time of melanoma diagnosis and no cases demonstrated a transformation to an aggressive subtype. 0/20 cases experienced a haematological response of their CLL while on ICI therapy and 6/20 cases progressed clinically either on the basis of tumour burden or absolute lymphocyte count within 12 months of ICI therapy (Fig 1C).This retrospective data demonstrates that ICI's have equivalent therapeutic efficacy in AM in patient with concurrent CLL This was unexpected as it was anticipated that the immune dysregulation associated with CLL would impair the ICI-mediated anti melanoma immunological response. The exception to this trend, was the poor efficacy of ICI's in patients with prior fludarabine exposure, presumably owing to the profound T cell depletion associated with this therapy. Additionally, ICI therapy alone appears ineffective in the management of CLL in keeping with recent phase II trial data. [Display omitted] DisclosuresGill:Janssen: Consultancy, Honoraria, Other: travel expenses.

Hypogammaglobulinemia in Chronic Lymphocytic Leukemia: A Predictor of Outcome?

BACKGROUNDHypogammaglobulinemia (HYPO) is a typical feature of chronic lymphocytic leukemia (CLL), with an incidence of 20-60% at diagnosis and a relationship with infections occurrence. Predictive and prognostic factors are well known (IGHV, del11q, del17p, TP53); CLL-IPI score including age, clinical stage, beta2microglobulin, IGHV and deletion 17p and/or TP53 mutation has been recently validated. It identifies 4 risk groups with significantly different time to first treatment (TTFT) and overall survival (OS). Prognostic significance of HYPO at diagnosis has not been extensively evaluated in terms of OS and TTFT. Only IgG serum levels have been reported to be associated with TTFT but no data are available on other immunoglobulin classes (Ig).AIM To evaluate the impact of HYPO and single Ig classes on TTFT in a retrospective cohort of CLL patients and to assess the relationship between HYPO and CLL-IPI.METHODSWe retrospectively evaluated 698 consecutive CLL patients diagnosed at our Institution from 1983 till 2016. Data from laboratory, biological analysis and clinical stage were collected. We also evaluated immunoglobulin (Ig) status (i.e. IgG, IgM and IgA) at diagnosis and calculated CLL-IPI. HYPO was defined basing on our laboratory cut-offs (IgA 70 mg/dl, IgG 700 mg/dl, IgM 40 mg/dl). However, as no recognized prognostic/predictive Ig cut off has been reported to date, we aimed to identify a prognostic threshold for each Ig class.RESULTSFrom 698 patients assessed, 410 cases were evaluable for Ig values at diagnosis. IgA levels were lower than 70 mg/dl in 17,4%, IgG lower than 700 mg/dl in 22,2%, and IgM lower 40 mg/dl in 33.7%. Forty-six percent of patients presented deficit of at least one Ig class, while 7.8% of patients had all Ig low. Each Ig deficit was related with a shorter TTFT with the following hazard ratios (HRs): 2.09 (1.45-3.03) for IgA (P<0.0001), 1.58 (1.10-2.27) for IgG (P=0.008) and 1.52 (1.09-2.13) for IgM (P=0.01) (Figure 1, A-B-C). However, only IgA deficit maintains statistical significance in multivariate analysis [HR 1.59 (1.08-2.35)].A prognostic threshold for each Ig class was identified maximizing the differences in TTFT and the following values were obtained: 80 mg/dl for IgA, 410 mg/dl for IgG and 18 mg/dl for IgM (Figure 1, D-E-F).Considering CLL-IPI, 186 patients presented IPI 0-1, 99 had IPI 2-3, 32 patients IPI 4-6, and 12 patients had IPI 7-10. Even in our series, CLL-IPI separated four risk groups with different TTFT and OS, suggesting that our cohort may be suitable to evaluate new prognostic factors. As regards the relationship between HYPO and CLL-IPI, we observed a correlation with IgA levels, using our laboratory cut-off. Moreover, we found a relationship among CLL-IPI and both IgA and IgM values, when using the newly validated Ig cut-off. Finally, CLL-IPI was a stronger prognostic factor for TTFT than HYPO in our analysis. However, the addition of IgA deficit to CLL-IPI appears to further improve CLL prognostication.CONCLUSIONOur analysis supports the impact of HYPO on CLL prognosis. Although CLL-IPI has a stronger prognostic value for TTFT compared to HYPO, the inclusion of IgA deficit appears to further improve accuracy on CLL prognostication. [Display omitted] DisclosuresBarcellini:Alexion: Honoraria; Agios: Honoraria, Research Funding; Novartis: Honoraria.

Use of Artificial Intelligence Electrocardiography to Predict Atrial Fibrillation (AF) in Patients with Chronic Lymphocytic Leukemia (CLL)

Use of Artificial Intelligence Electrocardiography to Predict Atrial Fibrillation (AF) in Patients with Chronic Lymphocytic Leukemia (CLL)

Abstract 1516: Deep profiling of T-cell repertoire and tumor heterogeneity in chronic lymphocytic leukemia patients following allogeneic T-cell therapy

Abstract Chemotherapy/ targeted therapy are both known to trigger evolution of treatment resistant clones that can lead to relapse. Allogeneic stem cell transplant (alloSCT) for refractory Chronic Lymphocytic Leukemia (CLL) patients is associated with better outcomes. We hypothesized that allogeneic T-cell immunotherapies, including alloSCT and donor lymphocyte infusion (DLI) would impact tumor evolution through the application of selective immunologic pressure with reciprocal changes in the T-cell compartment. Here, we tested a cohort of 24 heavily pre-treated CLL patients treated. Treatments consisted of alloSCT alone, or with follow-up DLI, which are two established mediators of effective Graft versus Leukemia (GVL). Our cohort included 11 patients who relapsed (denoted as non-responder, NR) after alloSCT and 13 patients who had complete response (CR) after alloSCT, with 11/13 patients showing durable CR with a median post-transplant overall survival (OS) of 9.8 years. We mapped the evolutionary trajectories of tumor cells by whole exome sequencing (WES) of sort purified CLL in post-transplant relapsed patients. To investigate changes in immune repertoire and gene expression post-transplant, CD3 positive T-cells from peripheral blood and bone marrows of CLL patients at complete donor chimerism were analyzed both at bulk and at the single cell level. We found evidence of subclonal leukemic evolution in the majority of our CLL patient cohort after nonmyeloablative HLA-matched alloSCT. Different patterns of CLL evolution were observed, and these changes included putative CLL drivers in every case. In all of the 11 patients with longitudinal post-alloSCT samples available, we observed branched CLL evolution in 4 patients, linear evolution in 4 patients, and no evolution in 3 patients. These data suggest that differential sensitivity of leukemic subclones to allogeneic T cell killing may underlie the branched and linear evolution that we observed, and therefore can shape leukemic subclonal architecture after transplant. Of note, we found that clonal CLL was more responsive to alloSCT in comparison to CLL with subclonal disease architecture.To identify T-cells with GVL potential, we first cataloged potential neoantigens by screening mutated regions in CLL with in silico HLA binding prediction models. Neoantigen specific T-cells were then sorted from longitudinal peripheral blood samples using tetramers, followed by identification of GVL specific TCR in both bulk and single cell setting. We were able to identify T-cells that coevolved with specific tumorigenic lesions in a subset of CLL patients. Taken together, our results suggest that donor-derived antigen-specific T-cells mediate clonal selection of CLL with concurrent changes in allogeneic T-cells, and that these changes can be monitored in longitudinal patient samples. Citation Format: Celine Kerros, John P. Miller, Xizeng Mao, Haven R. Garber, Hannah C. Beird, Jianhua Zhang, Jason Roszik, Paul Leonard, Li Zhao, Sahil Seth, Pei Lin, Huandong Sun, William G. Wierda, Issa F. Khouri, Karen Clise-Dwyer, Andrew Futreal, Shoudan Liang, Koppikar Priya, Jeffrey Molldrem. Deep profiling of T-cell repertoire and tumor heterogeneity in chronic lymphocytic leukemia patients following allogeneic T-cell therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1516.

Cause of death in patients with newly diagnosed chronic lymphocytic leukemia (CLL) stratified by the CLL-International Prognostic Index (CLL-IPI).

8026 Background: CLL progression and CLL-related complications (infections and second malignancies) were the leading cause of death (COD) in a prospective cohort of CLL patients (Strati, BJH 2017). The CLL-IPI integrates major clinical and molecular prognostic factors and stratifies patients into 4 risk groups with distinct prognosis. It is unknown if COD differs according to CLL-IPI risk group in patients with newly diagnosed CLL. Methods: Patients diagnosed with CLL between 1/2000-12/2019 and seen within 1 year of diagnosis were identified from the Mayo Clinic CLL database. Cumulative incidences of cause-specific death were analyzed using Gray’s test, with deaths from different causes treated as competing events and deaths from unknown causes excluded. Results: 1276 patients were included in this study. The median age at diagnosis was 63 years (range 24-92), and 880 (69%) were male. Based on CLL-IPI score, 449 (35%) had low risk disease, 443 (35%) had intermediate risk disease, and 384 (30%) had high/very high risk disease. Median follow-up time for the study was 6 years; 286 deaths occurred. The COD was CLL progression in 99 (35%), infection in 16 (6%), second malignancy in 47 (16%), CLL-unrelated in 59 (21%), and unknown in 65 (23%) patients. The rates of death due to CLL progression were higher (17.3% at 5 years; 30.3% at 10 years) than the rates due to CLL-related complications (5.7% at 5 years; 12.9% at 10 years) or due to CLL-unrelated causes (8.6% at 5 years; 16.9% at 10 years) in the CLL-IPI high/very high risk group, but not the CLL-IPI low or intermediate risk group (Table). A higher CLL-IPI risk group was associated with a higher rate of death due to CLL progression ( P &lt; 0.001), as well as a higher rate of death due to CLL-related complications ( P = 0.013), and CLL-unrelated causes ( P &lt; 0.001). Conclusions: Causes of death in newly diagnosed CLL patients differ according to their CLL-IPI risk group. In patients with high/very high risk CLL, improving CLL disease control with novel agents seems justified. In patients with low/intermediate risk CLL, there should be increased efforts to reverse immune dysfunction to reduce infections and second malignancies. [Table: see text]