Abstract: Background. Chronic lymphocytic leukemia (CLL) is the most common leukemia of adults in western countries. CLL is a highly heterogeneous disease; some patients may never require treatment, whereas other relapse early after frontline therapy. In approximately 70% of newly diagnosed cases, CLL presents at an early clinical stage and is managed with a watch & wait strategy. Until now, few clinical and molecular predictors inform on the risk of treatment requirement and the impact of CLL gene mutations is not completely understood. Purpose. We aimed at identifying new molecular markers that may predict early treatment requirement and may help clinicians to better plan the watch & wait strategy in asymptomatic early stage CLL patients. Methods. This study includes 295 Binet A CLL patients referring at our institution who did not require treatment for at least 3 months after diagnosis. Tumor genomic DNA (gDNA) was isolated from peripheral blood mononuclear cells at the time of diagnosis. gDNA was analyzed in the coding exons plus splice sites of the most frequently mutated genes in CLL with a next-generation-sequencing (NGS) approach. NGS analysis was performed on the Illumina MiSeq instrument (coverage 〉 2000x in 〉 80% of the target region). The somatic function of VarScan2 was used for variant calling and a stringent bioinformatic pipeline was developed to protect against the false call of polymorphisms and sequencing errors. A threshold of 5% of variant allele frequency was set for variant calling. The primary endpoint was time to first treatment (TTFT) defined as the timeinterval between the date of CLL diagnosis and the date of first CLL treatment. Statistical analysis was performed using SPSS version 24.0. Results. The median age of the study cohort was 70.8 years old, 136 (46.1%) patients were female, 71 (24.1%) harbored unmutated IGHV genes, 44 (14.9%) had trisomy 12, 12 (4.1%) had 17p deletion, 15 (5.1%) had 11q deletion and 150 (50.8%) had 13q deletion. NGS mutational analysis showed that NOTCH1 was the most frequently mutated gene occurring in 25 (8.5%) patients, followed by ATM in 18 (6.1%), TP53 in 17 (5.8%), MYD88 in 12 (4.1%), SF3B1 in 10 (3.4%), XPO1 in 7 (2.4%), EGR2 in 6 (2.0%), NFKBIE in 4 (1.4%), POT1 in 2 (0.7%), and BIRC3 in 1 (0.3%) patients. After a median follow-up of 9.5 years, 80 (27.1%) patients required treatment. In univariate analysis, molecular characteristics associated with a shorter TTFT were 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 〈 0.0001) and mutations of XPO1 (HR: 8.88; 95% CI 3.77-20.95; p 〈 0.0001), NOTCH1 (HR: 3.02; 95% CI 1.66-5.51; p 〈 0.001) and SF3B1 (HR: 2.65; 95% CI 1.15-6.10; p=0.022). Interestingly, neither 17p deletion nor TP53 mutations associated with a shorter TTFT, in line with the notion that TP53 disruption interacts with treatment but not with a watch & wait strategy. By multivariate analysis, XPO1 mutations (HR: 4.24; 95% CI 1.72-10.44; p=0.002) maintained an independent association with a shorter TTFT (Table 1). At 10 years, XPO1 mutated patients had a probability of remaining free from treatment of 0% compared to 69.3% for wild type cases (p 〈 0.0001) (Figure 1). Conclusions. Mutations of the XPO1 gene, encoding for exportin 1 which mediates the nuclear export of proteins and RNA, are an independent predictor of shorter TTFT and, if validated, might help clinicians in a better management of the watch & wait strategy for Binet A CLL patients. Moreover, since most of mutations, approximately 90%, affect the glutamic acid in position 571, polymerase chain reaction based methods may be used to identify XPO1 mutations in a simple and time effective manner. Disclosures Rossi: 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; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Gaidano:AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astra-Zeneca: Consultancy, Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sunesys: Consultancy, Honoraria.

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.

Abstract: Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in western countries, with an incidence of approximately 5.1/100,000 new cases per year. Some patients may never require treatment, whereas others relapse early after front line therapeutic approaches. Recent whole genome and whole exome sequencing studies have allowed a better understanding of CLL pathogenesis and the identification of genetic lesions with potential clinical relevance. Consistently, precision medicine plays a pivotal role in the treatment algorithm of CLL, since the integration of molecular biomarkers with the clinical features of the disease may guide treatment choices. Most CLL patients present at the time of diagnosis with an early stage disease and are managed with a watch and wait strategy. For CLL patients requiring therapy, the CLL treatment armamentarium includes both chemoimmunotherapy strategies and biological drugs. The efficacy of these treatment strategies relies upon specific molecular features of the disease. TP53 disruption (including both TP53 mutation and 17p deletion) is the strongest predictor of chemo-refractoriness, and the assessment of TP53 status is the first and most important decisional node in the first line treatment algorithm. The presence of TP53 disruption mandates treatment with biological drugs that inhibit the B cell receptor or, alternatively, the B-cell lymphoma 2 (BCL2) pathway and can, at least in part, circumvent the chemorefractoriness of TP53-disrupted patients. Beside TP53 disruption, the mutational status of immunoglobulin heavy variable (IGHV) genes also helps clinicians to improve treatment tailoring. In fact, patients carrying mutated IGHV genes in the absence of TP53 disruption experience a long-lasting and durable response to chemoimmunotherapy after fludarabine, cyclophosphamide, and rituximab (FCR) treatment with a survival superimposable to that of a matched general population. In contrast, patients with unmutated IGHV genes respond poorly to chemoimmunotherapy and deserve treatment with B cell receptor inhibitors. Minimal residual disease is also emerging as a relevant biomarker with potential clinical implications. Overall, precision medicine is now a mainstay in the management and treatment stratification of CLL. The identification of novel predictive biomarkers will allow further improvements in the treatment tailoring of this leukemia.

Abstract: We aimed at molecularly dissecting the anatomical heterogeneity of small lymphocytic lymphoma (SLL), by analysing a cohort of 12 patients for whom paired DNA from a lymph node biopsy and circulating cells, as well as plasma‐circulating tumour DNA (ctDNA) was available. Notably, the analyses of the lymph node biopsy and of circulating cells complement each other since a fraction of mutations (20·4% and 36·4%, respectively) are unique to each compartment. Plasma ctDNA identified two additional unique mutations. Consistently, the different synchronous sources of tumour DNA complement each other in informing on driver gene mutations in SLL harbouring potential prognostic and/or predictive value.