ALBERT

Description: Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B cell receptor immunoglobulins (BcR IG). Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR IG stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR IG stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. In order to address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29,856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed 'satellites', were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.

Description: Background Richter's transformation (RT) refers to the development of an aggressive lymphoma in patients with chronic lymphocytic leukemia (CLL) or small lymphocytic leukemia (SLL). Roughly, 2-10 % of patients with CLL develop RT most often as diffuse large B-cell lymphoma (DLBCL) or Hodgkin lymphoma (HL). Aim This study aimed to assess the incidence rate and risk factors for RT for patients with CLL in a nationwide cohort. Furthermore, we want to assess prognostic risk factors for patients with RT. Methods All patients diagnosed with CLL in Denmark between 2008 and 2016 were included in this study. Clinical data was retrieved from the Danish National CLL Registry (DCLLR), whereas all histologically verified DLBCL, HL and/or transformation diagnoses for patients with CLL were retrieved from the Danish National Pathology Registry. Patients were followed from date of CLL diagnosis until date of RT, death or end of follow-up, whichever came first. The time to RT was estimated as cumulative incidence considering death as a competing risk. Stepwise Cox analysis with backward elimination was applied to identify independent risk factors for RT in patients with CLL. Results A total of 3771 CLL patients were identified, and followed for 14165 person-years. With a median follow-up of 4.3 (IQR (2.4;6.6)) years, 120 (3%) CLL patients had a transformation diagnosis, of which 4 patients were excluded due to misdiagnosis. DLBCL accounted for 78/116 (67%) cases, HL for 15/116 (13%) cases and one patient presented with both DLBCL and HL. In the remaining 22/116 (19%) cases the subtype of the transformation was either unspecified or unclassified RT. The median time to RT was 3.4 (IQR (1.8;5.7)) years from CLL diagnosis and the median overall survival (OS) after development of RT was 4.9 (IQR (0.7;8.4)) years. The cumulative incidence of RT, calculated by Aalen-Johansen estimator, at 5 and 8 years post-CLL diagnosis were 3.3% and 7.9% respectively (Figure 1). The annual crude incidence rate of RT was approximately 0.7% per year for all CLL patients. In all, 918 (24%) patients received CLL-related treatment, of whom 59 (6.4%) patients developed RT, resulting in a cumulative incidence of RT of 7% after 5 years and 11% after 8 years. At the time of CLL diagnosis, patients treated for CLL prior to RT diagnosis had a worse median OS (1.49 years) compared to RT patients who were untreated for CLL (6.16 years). In the univariate analysis, RT was significantly associated with male gender, advanced Binet stage (B or C), unmutated IGHV status (CLL-U), elevated beta-2-microglobulin (〉3.5 mg/L) and elevated lactate dehydrogenase (〉205 U/L). Of cytogenic aberration, deletion 13q (del(13q)) had a protective effect on the risk of RT, whereas deletion 11q (del(11q)) and deletion 17p (del(17p)) increased the risk. In the multivariable model, advanced Binet stage (HR 2.86 (1.82;4.51), p

Description: Introduction/background: Clonal TP53 aberrations (del(17p) and/or TP53 mutations) in patients with chronic lymphocytic leukemia (CLL) correlates with a poor prognosis. Similar outcome has been demonstrated for patients with subclonal TP53 aberrations (Rossi, Blood, 2014). In order to guide novel targeted therapies approved in frontline treatment of TP53 aberrated (TP53+) CLL, development and clinical validation of robust assays for subclonal TP53 aberrations is warranted. Methods: DNA extracted from peripheral blood of CLL patients were diluted 1:5 in DNA derived from a cell line containing a known, but rare TP53 point mutation. TP53 exons 2-10 were PCR amplified from undiluted and diluted DNA in parallel using Phusion proofreading DNA polymerase and subsequently sequenced by targeted Next Generation Sequencing (tNGS) on Illumina MiSeq and HiSeq 2500. Sensitivity and specificity of the assay was tested by serial dilution of patient samples with known TP53 insertions, deletions or substitutions. Consecutive biobanked samples from CLL patients at a single institution were used for validation of the clinical impact of subclonal TP53 aberrations. Nucleotide variants were called by CLC Biomedical Genomics Workbench 3.0. An algorithm for detection of true mutations was developed based on comparison of the diluted and undiluted samples analyzed in parallel. Overall survival (OS) was analyzed using Kaplan-Meier. Results: The sensitivity and specificity of the assay was validated by detection of all 8 known TP53 aberrations in serial dilutions with the threshold of the assay established at 0.2% allelic burden. Known mutations were still detectable at 0.02% at the highest dilution. A test sample of patients with known del(17p) demonstrated TP53 mutations in 6 out of 7 patients (5 clonal and 1 subclonal TP53+) in accordance with previously reported frequencies of TP53 mutations among patients with del(17p). In total, 92 samples from 46 consecutive patients were analyzed. With a median coverage of 93,272 reads (98% above 20,688 reads, range: 5,153-720,025), 27 TP53 aberrations were found in 10 (22%) of the patients. Twenty-six (96%) mutations were subclonal with a median allelic burden of 0.9% (range: 0.2-97.5%). Seven patients had a single aberration. In two previously treated patients, 8 and 10 subclonal aberrations were detected. One patient with a known del(17p) also had subclonal TP53 mutations. Three patients had solely subclonal mutations below 1% allelic burden. Considered the hot spot region of TP53 in CLL, exons 4-8 harbored 93% of the detected mutations. The median (IQR) survival for TP53+ patients and patients with wild type TP53 (wt-TP53) was 37.5 (range: 2-106) and 104 (range: 9-113) months, respectively. In Kaplan-Meier analyses, TP53+ patients had a significantly poorer OS versus patients with wt-TP53 (p=0.00002, Figure 1). The two TP53+ long-term survivors both had an allelic burden below 1%, and the only other TP53+ patient with less than 1% allelic burden survived for 53 months, indicating that the prognostic impact of very low allelic burden TP53 aberrations needs further investigation. Conclusion: We have developed a robust assay for TP53 aberrations with a sensitivity of 0.2% allelic burden based on an algorithm including a dilution step. In an initial test cohort of 46 patients, 10 patients demonstrated TP53 mutations as low as 0.2% allelic burden that significantly affected OS. Validation of the clinical impact of subclonal TP53 aberrations is ongoing based on our consecutive biobank with 600+ patients. Establishment of a new cut-off for clinical treatment decisions based on subclonal TP53 aberrations is warranted. Figure 1 Overall survival in 46 consecutive CLL patients based on TP53 mutation status shows significant difference (p=0.00002) between patients withwild type TP53 (wt-TP53) and TP53 mutations (TP53+) as low as 0.2% allelic burden. Figure 1. Overall survival in 46 consecutive CLL patients based on TP53 mutation status shows significant difference (p=0.00002) between patients withwild type TP53 (wt-TP53) and TP53 mutations (TP53+) as low as 0.2% allelic burden. Disclosures Niemann: Gilead: Consultancy; Abbvie: Consultancy; Roche: Consultancy; Janssen: Consultancy.

Description: Introduction In chronic lymphocytic leukemia (CLL), TP53 aberration (TP53ab: del(17p) and/or TP53 mutation) remains the only biomarker directly influencing clinical practice (Blood. 2018;131(25):2745-2760). Further, TP53abs are early events that can lead to acquisition of additional somatic mutations, which suggest genomic instability caused by loss of p53, especially upon therapy. Single-agent ibrutinib is an effective therapy for both treatment-naïve (TN) and relapsed or refractory (R/R) CLL patients including those with del(17p). However, long-term progression-free survival (PFS) and overall survival are inferior for ibrutinib treated patients with TP53abs and R/R CLL due to early (10%) and 102 low burden TP53muts (VAF ≤10%) including 67 minor TP53muts (VAF T, which was predicted to encode functional p53 (http://p53.iarc.fr). Based on the number of TP53abs, 6 patients had only 1 TP53ab and 23 had 〉1 TP53ab. All patients with 〉1 TP53ab had biallelic TP53 disruption including 1 patient harboring a total of 38 TP53muts without concomitant del(17p). With a median follow-up time of 5.0 years (IQR: 4.9-6.0), PFS was 100% for patients with only 1 TP53ab. Among the 23 patients with 〉1 TP53ab, 9 patients had PD and 1 patient had died without progression (Figure 1A). There was no difference in PFS between patients with 2 TP53ab and patients with 〉2 TP53ab (Figure 1B). The cumulative incidence of PD for patients with 〉1 TP53ab was 39%, while no patients with only 1 TP53ab had progressed. Even though 5/6 patients with only 1 TP53ab were also TN, having 〉1 TP53ab impacted PFS negatively among both patients with TN (4 with PD and 1 death) and R/R CLL (5 with PD). Conclusion Patients with monoallelic TP53 aberration demonstrate excellent outcome on single-agent ibrutinib regardless of prior treatment status. Biallelic TP53 aberration identifies patients who have inferior outcome with ibrutinib monotherapy and may benefit from combination therapy. Among patients with biallelic TP53 disruption, having more than two TP53 aberrations does not portend a worse prognosis. The prognostic role of biallelic TP53 aberration calls for validation in independent, prospective studies and in clinical trials using targeted combination regimens. Disclosures Brieghel: Rigshospitalet, Denmark: Research Funding; Arvid Nilson's Fund: Research Funding. Wiestner:Pharmacyclics LLC, an AbbVie Company: Research Funding. Niemann:Janssen: Consultancy, Research Funding; Novo Nordisk Foundation: Research Funding; Gilead: Consultancy; CSL Behring: Consultancy; Danish Cancer Society: Research Funding; Roche: Consultancy; AstraZeneca: Consultancy; Abbvie: Consultancy, Research Funding; Novartis: Consultancy.