Abstract: Abstract 1371 Background: IGHV1-69 gene identifies the most frequent IGHV gene in chronic lymphocytic leukemia (CLL) and identifies the paradigm of unmutated CLL (U-CLL), being used in roughly 1/3 U-CLL. It is often rearranged to form subsets of stereotyped HCDR3 patterns, likely selected and transformed from the natural naïve B-cell repertoire (Blood. 2010; 115:71-7). Being unmutated, IGHV1-69 CLL are hypothetically expected to have competent tumor B-cell receptors (BCR) and to progress more rapidly. However, it has not been investigated if progression occurs similarly in all the subsets. Aims: we aimed to investigate the prognostic significance of mutational status and of stereotypic B-cell receptors in IGHV1-69+ CLL. Methods: Nucleotide sequences of the tumor IGHV1-69/D/J rearrangement, clinical and molecular prognostic parameters at diagnosis and clinical status at follow-up of 294 IGHV1-69+ CLL patients were obtained from 22 hematological Institutions in Italy. CLL B-cell derived IGHV1-69 rearrangements were scanned for HCDR3 stereotypic patterns and assigned to subsets according to the criteria by Murray et al (Blood. 2008; 111: 1524–1533). Enpoint of outcome was time to progression requiring first treatment according to NCI criteria (TTFT) in Rai stage 0 CLL. Results: Of 294 IGHV1-69+ CLL, 264 (89,8%) were unmutated, 168 (57,1%) were assigned to subsets, of which subsets 7 (n=23, 7,8%), 6 (17, 5,8%), 3 (13, 4,4%), 5 (10, 3,4%) and 9 (10, 3,4%) were the most frequent. CD38, ZAP70, normal or sole del13, +12, del11 and del17p scored positive in 109/264 (58,7%), 139/245 (56,7%), 128/248 (50,5%), 51/248 (20,6%), 43/248 (17,3%) and 34/248 (13,7%). CLLs were reclassified as 18 (6,1%) clinical MBL, 101 (34,4%) Rai 0, 155 (52,7%) Rai I-II and 20 (6,8%) Rai III-IV CLL. Subset 6 was also UM in 16/17 (94,1%) cases. Prevalence of CD38 (p 〈 .001), ZAP70 (p=.016), normal or sole del13 (p 〈 .001), +12 (p=.026), del11 (p=.011), and clinical high risk CLL (p=.025) were lower in IGHV1-69 M-CLL than in IGHV1-69 U-CLL. TTFT was significantly shorter in stage 0 IGHV1-69 U-CLL than in IGHV1-69 M-CLL (49 vs 144 months, p 〈 .001, while it was not different between CLL assigned or not to subsets (65 vs 55 months, p=.346). However, specific analysis of individual subsets revealed differential outcomes (p=.005). Among all, it emerged that subset 6 had a TTFT equivalent to IGHV1-69 M-CLL (p=.29) and significantly longer than stage 0 IGHV1-69 U-CLL (median not reached vs 48 months, p=.017). Conclusions: our analysis documents and confirms that unmutated status of IGHV, and not stereotypy, is a relevant prognosticator of outcome (TTFT) in CLL. In the IGHV1-69 CLL it exclude a role of IGHV gene use for CLL progression. However, the good prognosis of Rai 0 U-CLL assigned to subset 6 suggests a differential clinical benign course of this particular subset, irrelevant of unmutated status. One possibility is that the IGHV1-69/D3-16/J3 rearrangements of subset 6 produce a tumor-specific BCR with stereotypic HCDR3 patterns that are anergized by antigen while circulating in the peripheral blood in early stage (Rai 0) CLL. Disclosures: No relevant conflicts of interest to declare.

Abstract: Novel therapies targeting BTK (ibrutinib), PI3Kδ (idelalisib) and BCL2 (venetoclax) are active in poor-risk chronic lymphocytic leukemia (CLL) and are widely administered to patients with relapsed/refractory (R/R)-CLL. Given the activity of ibrutinib in high-risk CLL patients, including those with del17p/TP53 mutation or germline IGHV genes, we assumed that this drug could diminish the prognostic utility of the CLL-IPI, because the outcome of patients with high- and very high-risk CLL-IPI scores may improve. Recently, Soumerai et al (Lancet Hematology, 2019) proposed a new risk score for overall survival (OS) based on four accessible markers, called BALL (β2-microglobulin, anemia, LDH, last therapy), in the setting of R/R-CLL patients receiving chemo-immunotherapy or targeted therapies in clinical trials. This model segregates CLL patients into three groups with significantly different OS. This multicenter, observational retrospective study aimed at validating the proposed BALL score for R/R-CLL real-world patients treated with ibrutinib. The primary objectives were to determine whether: i) the BALL score is of prognostic value for ibrutinib R/R-CLL patients, ii) the BALL score is predictive of progression-free survival (PFS); and iii) the CLL-IPI retains its prognostic power also in R/R patients treated with ibrutinib. This study, from an institutional Italian multicenter working group on CLL ('Campus CLL'), included CLL patients collected from 18 Italian centers and 1 Israeli center, who received ibrutinib 420 mg/day outside of clinical trials as salvage therapy with available data for the calculation of the BALL and CLL-IPI scores at the time of the start of treatment. OS was estimated for all subgroups according to both scores. Additionally, risk-specific PFS was assessed. Kaplan-Meier curve, log-rank test and Cox regression analyses were performed. The prognostic accuracy of the predictive model was assessed by the Harrell's C-index. Overall, 541 CLL patients were included in this analysis. The majority of patients were Binet stages B and C (87.2%). The median age was 70.4 years (range 37 - 88) and 353 cases (65.2%) were male. The median number of previous therapies was 2 (range 1-9). The baseline patients' features are listed in Table 1. After a median follow-up of 1.8 years (range 1 month to 5.8 years), 101 patients had died and 206 experienced an event (death or progression). According to the BALL score, 372 patients (68.8%) were classified as low-risk, 132 (24.4%) as intermediate-risk and 37 (6.8%) as high-risk. Stratification of patients according to the BALL score predicted significant differences in terms of OS. Thus, low-risk patients had a 2-year OS probability of 89.2% (HR=1), intermediate-risk of 79.9% (HR=2.8, 95%CI 1.8-4.4, P 〈 0.0001) and high-risk of 48.2% (HR=6.6, 95%CI 3.9-11, P 〈 0.0001) (Figure 1). The C-statistic was 0.66 (P 〈 0.001) for OS prediction. The CLL-IPI score indicated that 32 patients (5.9%) were classified as low-risk, 123 (22.7%) as intermediate-risk, 252 (46.6%) as high-risk and 134 (24.8%) as very high-risk. Stratification of patients according to the CLL-IPI score did not allow to predict significant differences in OS. Indeed, low-risk patients had a 2-year OS probability of 92.7% (HR=1), intermediate-risk patients of 88.4% (HR=2.3, 95%CI 0.5-9.9, P=0.26), high-risk of 84% (HR=3.3, 95%CI 0.8-13.7, P=0.1) and very high-risk of 76.4% (HR=4.3, 95%CI 1.038-17, P=0.046). Both the BALL and CLL-IPI scores failed to stratify patients significantly in terms of PFS. Our multicenter retrospective study confirms the prognostic power of the BALL score in this real-world series of R/R-CLL patients treated with ibrutinib, as previously reported in the setting of clinical trials (Soumerai et al, Lancet Hematology 2019). Furthermore, the CLL-IPI did not retain a discriminative power in the current retrospective study of ibrutinib-treated patients, possibly due to i) the well-known activity of ibrutinib in high-risk CLL patients, i.e. those with TP53 disruption and/or germline IGHV genes, which represent the most heavily weighted adverse risk factors contributing to the CLL-IPI, and to ii) the fact that the CLL-IPI was designed for patients receiving front-line chemo-immunotherapy. Conversely, the BALL score may identify higher risk R/R-CLL patients potentially requiring alternative and more effective therapeutic strategies. Disclosures Mauro: Jannsen: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Shire: Consultancy, Research Funding; Roche: Consultancy, Research Funding. Coscia:Gilead: Membership on an entity's Board of Directors or advisory committees; Karyopharm Therapeutics: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees. Herishanu:Roche: Honoraria; Janssen: Honoraria; AbbVie: Honoraria. Varettoni:ABBVIE: Other: travel expenses; Roche: Consultancy; Gilead: Other: travel expenses; Janssen: Consultancy. Rigolin:AbbVie: Speakers Bureau; Gilead: Speakers Bureau; Gilead: Research Funding. Rossi:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Other: Scientific advisory board; Janseen: Honoraria, Other: Scientific advisory board; Roche: Honoraria, Other: Scientific advisory board; Astra Zeneca: Honoraria, Other: Scientific advisory board. Gaidano:AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sunesys: Consultancy, Honoraria; Astra-Zeneca: Consultancy, Honoraria. Cuneo:Amgen: Honoraria; Abbvie: Honoraria, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Roche: Honoraria, Speakers Bureau. Foà:Roche: Consultancy, Speakers Bureau; Celltrion: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Speakers Bureau; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Membership on an entity's Board of Directors or advisory committees.

Abstract: To compare the capacity of ibrutinib (IB) and idelalisib‐rituximab (IDELA‐R) of prolonging overall survival (OS) as in CLL patients, previously treated with chemotherapy only. Methods A real‐life cohort of 675 cases has been identified and investigated in the database of the groups participating in the study. Results At an unadjusted univariate analysis, a significant death risk reduction was observed favoring IB (IDELA‐R vs IB HR = 0.5, 95% CI = 0.36‐0.71) although with some limitations due to the non‐randomized and retrospective nature of the study and to the lower number of patients in the IDELA‐R group (112 cases) related to the current prescribing practice. To overcome the potential problem of confounding by indication, we adjusted the association between the type of therapy and mortality for all variables significantly associated with OS at Cox univariate analysis. Furthermore, those variables, differently distributed between the two study groups, were introduced into the multivariate Cox model to improve the effectiveness of the analysis. By introducing all these variables into the multiple Cox regression model, we confirmed the protective effect of IB vs IDELA‐R (HR = 0.67, 95% CI = 0.45‐0.98, P  = .04) independent of potential confounders. Conclusions Although our analysis presents some constraints, that is, the unavailability of additional potential confounders, and the retrospective nature of the study, this observation may be of help for the daily clinical practice, particularly in the absence of randomized trials comparing the two schedules.

Abstract: Here we evaluated the epigenomic and transcriptomic profile of XPO1 mutant chronic lymphocytic leukaemia (CLL) and their clinical phenotype. By ATAC‐seq, chromatin regions that were more accessible in XPO1 mutated CLL were enriched of binding sites for transcription factors regulated by pathways emanating from the B‐cell receptor (BCR), including NF‐κB signalling, p38‐JNK and RAS‐RAF‐MEK‐ERK. XPO1 mutant CLL, consistent with the chromatin accessibility changes, were enriched with transcriptomic features associated with BCR and cytokine signalling. By combining epigenomic and transcriptomic data, MIR155HG , the host gene of miR‐155, and MYB , the transcription factor that positively regulates MIR155HG, were upregulated by RNA‐seq and their promoters were more accessible by ATAC‐seq. To evaluate the clinical impact of XPO1 mutations, we investigated a total of 957 early‐stage CLL subdivided into 3 independent cohorts ( N  = 276, N  = 286 and N  = 395). Next‐generation sequencing analysis identified XPO1 mutations as a novel predictor of shorter time to first treatment (TTFT) in all cohorts. Notably, XPO1 mutations maintained their prognostic value independent of the immunoglobulin heavy chain variable status and early‐stage prognostic models. These data suggest that XPO1 mutations, conceivably through increased miR‐155 levels, may enhance BCR signalling leading to higher proliferation and shorter TTFT in early‐stage CLL.

Abstract: Background. Approximately 70% of newly diagnosed chronic lymphocytic leukemia (CLL) patients present in early Binet or Rai stage, may never require treatment, and may have a life expectancy similar to that of the general population. Two independent and recent studies have identified the clinical and immunogenetic variables associated with shorter time to first treatment (TTFT) in Binet A and Rai 0 CLL (Condoluci et al., Blood 2020; Cohen et al., Haematologica 2020). However, the clinical impact of gene mutations in predicting TTFT is not completely understood. Purpose. Using a training/validation approach, we aimed at identifying new molecular biomarkers that may predict early treatment requirement and may help clinicians to better plan the watch and wait strategy in asymptomatic early stage CLL patients. Methods. The training cohort included 295 CLL in Binet A stage who did not require treatment for at least 3 months after diagnosis. The two validation multicenter cohorts included 402 treatment-naïve Binet A CLL patients (Binet A validation cohort) and 395 untreated Rai 0 CLL patients (Rai 0 validation cohort), respectively. In the training cohort, tumor genomic DNA was isolated from peripheral blood mononuclear cells at the time of diagnosis and was analyzed in the coding exons plus splice sites of the most frequently mutated genes in CLL with a next-generation-sequencing (NGS) approach using a variant allele frequency (VAF) threshold of 5%. In the validation series, the XPO1 gene (exons 15 and 16) was analyzed by NGS or by Sanger sequencing. The primary endpoint was TTFT defined as the time interval between the date of CLL diagnosis and the date of first CLL treatment. Results. In the training cohort, NGS mutational analysis showed that XPO1 was mutated in 7 (2.4%) patients. In univariate analysis, trisomy 12 (HR 2.42; 95% CI 1.43-4.15; p=0.001), unmutated IGHV genes (HR 4.51; 95% CI 2.83-7.05; p & lt;0.0001) and mutations of XPO1 (HR 8.88; 95% CI 3.77-20.95; p & lt;0.0001) (Fig. 1A), NOTCH1 (HR 3.02; 95% CI 1.66-5.51; p & lt;0.001) and SF3B1 (HR 2.65; 95% CI 1.15-6.10; p=0.022) were associated with a shorter TTFT. By multivariate analysis, XPO1 mutations (HR 4.24; 95% CI 1.72-10.44; p=0.002) and unmutated IGHV genes (HR 3.43; 95% CI 2.08-5.67; p & lt;0.0001) maintained an independent association with a shorter TTFT. XPO1 mutational analysis was subsequently investigated in 2 independent multicenter cohorts of early stage CLL patients. In the Binet A validation cohort (N=402 patients), XPO1 was mutated in 15 (3.7%) patients and was associated with a shorter TTFT (HR 2.59; 95% CI 1.36-4.96; p=0.004) (Fig. 1B). Similarly, also in the Rai 0 validation cohort, (N=395 patients), XPO1was mutated in 8 (2.0%) patients and was associated with a shorter TTFT (HR 6.02; 95% CI 15.03-4.96; p & lt;0.001) (Fig. 1C). Moreover, in the Rai 0 validation cohort, XPO1 mutations maintained an independent association with a shorter TTFT when corrected in multivariate analysis by the IGHV mutational status (HR 3.31; 95% CI 1.30-8.44; p=0.012). By combining the training and the validation cohorts (N=1092 patients), a total of 30 somatically acquired XPO1 mutations were identified (2.7% of patients). More precisely, 27 (90.0%) mutations affected XPO1 codon E571 and 3 (10.0%) codon D624. This finding suggests that a target sequencing or an allele-specific polymerase chain reaction based method may be used to identify XPO1 mutations in a simple and time-effective manner. From a clinical perspective, patients carrying either XPO1 E571 or D624 mutations showed superimposable outcome in terms of TTFT (p=0.345) (Fig. 1D). Conclusions. Mutations of the XPO1 gene, encoding for exportin 1 which mediates the nuclear export of proteins and RNAs, are an independent predictor of shorter TTFT validated in independent series of early stage treatment-naïve CLL patients. XPO1 mutations are conceivably gain-of-function and may enhance cell proliferation by exporting out of the nucleus with a greater extent proteins that physiologically downregulate cell proliferation. Based on these results, XPO1 mutational analysis might be incorporated in other prognostic scores and help clinicians to refine the management of the watch and wait strategy for early stage CLL. In addition, XPO1 inhibitors are particularly active in XPO1E571 mutated cells (Taylor et al., Cancer Discov 2019) providing initial pre-clinical rational for their usage in XPO1 mutated CLL patients. Figure Disclosures Scarfo: AstraZeneca: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Del Giudice:AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Roche: Other: grant for meeting partecipation; Janssen: Other: grant for meeting participation; Tolero: Membership on an entity's Board of Directors or advisory committees. Sportoletti:Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Marasca:Shire: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees. Ghia:Acerta/AstraZeneca: Consultancy, Honoraria; ArQule: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; BeiGene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; Novartis: Research Funding; Celgene/Juno: Consultancy, Honoraria; Lilly: Consultancy, Honoraria; MEI: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Adaptive, Dynamo: Consultancy, Honoraria. Foà:Roche: Membership on an entity's Board of Directors or advisory committees; Incyte: Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Gaidano:Sunesys: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astrazeneca: Membership on an entity's Board of Directors or advisory committees. Rossi:AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding.