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<0.0001) and mutations of XPO1 (HR 8.88; 95% CI 3.77-20.95; p<0.0001) (Fig. 1A), 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) 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<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<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.

Fixed-duration treatment with venetoclax (Ven), a highly selective Bcl-2 inhibitor combined with an anti-CD20 monoclonal antibody, showed high efficacy inducing high rates of deep responses with undetectable minimal residual disease (uMRD) in patients with previously treated and untreated chronic lymphocytic leukemia (CLL). The efficacy and safety of the Ven and rituximab (VenR) combination have been investigated in a multicenter, prospective study of the GIMEMA group that included young patients with previously untreated CLL (LLC 1518, VERITAS, NCT03455517). The primary endpoint of this study was the CR rate assessed according to the iwCLL criteria. Inclusion criteria were: treatment requirement per iwCLL criteria, age ≤65 years, cumulative Illness rating scale score ≤6, creatinine clearance ≥30 mL/min, and an unfavorable biologic profile with IGHV unmutated and or TP53 disruption. Treatment consisted of the Ven dose ramp-up (from 20 to 400 mg daily, during 5-weeks) followed by Ven 400 mg daily, combined with R for six 28-day courses (375 mg/m2, course 1; 500 mg/m2, courses 2-6). Patients continued with Ven single agent, 400 mg daily, until month 13. Tumor lysis syndrome (TLS) prophylaxis measures included hydration, allopurinol, or rasburicase. All patients received PneumocystisJirovecii prophylaxis. G-CSF was given in patients with recurrent and severe granulocytopenia. Adverse events (AEs) were graded according to the CTCAE criteria v.5, TLS events were classified according to Howard's criteria. Response was assessed at months 7 and 15 and included clinical examination, PB evaluation, BM aspirate, BM biopsy, and CT scan. MRD was checked centrally in the PB and BM by a 6/4-color flow-cytometry assay with a sensitivity of at least 10-4 according to the internationally standardized European Research Initiative on CLL. Quantitative MRD results assessed by flow-cytometry were categorized as uMRD (uMRD4; <10-4), intermediate MRD, or high MRD (≥10-2). MRD was further evaluated by allele-specific oligonucleotide PCR with a sensitivity up to 10-5 in the PB and BM of patients who showed uMRD4 by flow-cytometry. During the follow-up, MRD was monitored every 6 months. Between October 2018 and May 2020, 77 patients with CLL were included in this study. Two patients were off study before the start of treatment (withdrawal of consent, 1; Covid-19 infection, 1) and were not included in the analysis. The median age was 53.5 years (range 38-65). Binet stage B/C was present in 84% of patients, increased beta-2 microglobulin in 41%. Seventy-one (96%) of patients were IGHV unmutated, while 3 (4%) were IGHV mutated and showed TP53 mutation (Table 1). At the data cutoff of June 30, 2020, 65 (87%) patients completed the ramp-up phase. The planned 400 mg dose of Ven was reached within 5 weeks in 78.5% of patients. Response was assessed in 34 patients at the end of the VenR combination therapy. A response was achieved by 32 (94%) patients. Responses included 20 (59%) CRs, 1 CRi (3%) and 11 (32%) PRs due to residual enlarged nodes (median maximum size, 1.9 cm). Treatment failure due to toxicity was recorded in 2 (6%) patients. Overall, a response with uMRD4 by flow-cytometry in the PB was observed in 26 (76.5%) cases, and in the PB and BM, in 17 (50.0%). The rates of patients with CR and uMRD4 by flow-cytometry in the PB, and both in the PB and BM, were 44%, and 35%, respectively (Table 2). No detectable disease by PCR, both in the PB and BM, was observed in 4 (12%) patients. With a median follow-up of 4.5 months from the start of therapy, no patient has progressed or died. Fifty-three percent of patients were hospitalized during the first seven days of the Ven ramp-up phase. A transient laboratory TLS was observed in 3 patients. Treatment was discontinued after the first dose of Ven in 1 patient with evidence of laboratory TLS associated with severe neurologic toxicity due to the concomitant administration of fentanyl. Selected grade ≥3 AEs included neutropenia in 10 patients (ramp-up phase, 5) and neutropenic fever in 4. Grade ≥3 infection was recorded in 3 patients and was the reason for treatment discontinuation in 1 who developed COVID-19 pneumonia. In conclusion, the preliminary results of this study demonstrate the high efficacy of the front-line VenR combination, which resulted in a high proportion of CRs and responses with uMRD4 in young patients with CLL and an unfavorable biologic profile. Disclosures Mauro: Astrazeneca: Membership on an entity's Board of Directors or advisory committees; Takeda: 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; Jannsen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Octopharma: Consultancy. Reda:Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees. Trentin:Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shire: Honoraria; Takeda: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees. Coscia:Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm Therapeutics: Research Funding; Abbvie: Honoraria, 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, Research Funding, Speakers Bureau. 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. Laurenti:Roche: 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; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Gaidano:Astrazeneca: 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; Sunesys: Membership on an entity's Board of Directors or advisory committees. Marasca: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; Shire: Honoraria; Roche: Membership on an entity's Board of Directors or advisory committees. Murru:Gilead: 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; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Rigolin:Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Scarfo: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: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees. Marchetti:Pfizer: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees. Levato:Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: 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. Galieni:Celgene: Honoraria; Takeda: Honoraria; AbbVie: Honoraria; Janssen: Honoraria. Liberati:Verastem: Research Funding; Onconova: Research Funding; Janssen: Honoraria, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Research Funding; Pfizer: Research Funding; Karyopharm: Research Funding; Morphosys: Research Funding; Novartis: Research Funding; GSK: Research Funding; Incyte: Honoraria; Oncopeptides: Research Funding; Takeda: Research Funding. Molica: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; Gilead: 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. Visentin:Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: 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. Vitale:Janssen: Honoraria. Del Giudice:Janssen: Other: grant for meeting participation; Tolero: Membership on an entity's Board of Directors or advisory committees; Roche: Other: grant for meeting partecipation; AstraZeneca: Membership on an entity's Board of Directors or advisory committees. Cuneo: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; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astra Zeneca: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Foà:Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: 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; Novartis: Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees.

Background: Single agent Ibrutinib (IBR), an inhibitor of Bruton's tyrosine kinase, is approved for the front-line treatment of patients with chronic lymphocytic leukemia (CLL). Aims: As in CLL patients, the addition of anti-CD20 monoclonal antibody provides superior efficacy to chemotherapy, in a prospective multicenter, study of the GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) group the potential for improved efficacy with the addition of rituximab (R) to IBR was investigated in previously untreated and unfit patients with CLL. Methods: Eligible patients were CLL patients requiring front-line treatment who showed a Cumulative Illness Rating Scale (CIRS) score >6 and/or creatinine clearance <70 mL/min. During the first phase of this study patients received IBR, 420 mg once daily continuously, and R, 375 mg/sqm, on day 1 of months 1–6. Then, IBR was given as a single agent until progression, or severe toxicity, or complete response (CR) with no evidence of minimal residual disease (MRD) or for a maximum duration of 6 years. The primary endpoint of this study was progression-free survival (PFS). Results: Between March 2015 and April 2017 143 unfit patients with CLL were included in this study. The median follow-up was 22.4 months (range, 1–31.3). The median age of patients was 73 years, (range, 37–88), median CIRS score 6 with an ECOG 1–2 performance status in 37% of patients. Binet stage B/C was present in 81% of patients, increased beta-2 microglobulin in 71% and bulky nodes (size ≥ 5 cm) in 14%. The presence of del(17p) or del(11q) was recorded in 9% and 14% of cases respectively, TP53 mutations in 15% and 51% of patients were IGHV unmutated. Response to IBR+R combination was assessed in 141 patients. On an intention-to-treat basis, 114 (81%) patients achieved a response that was a CR in 20%, an L-PR in 13% and a PR in 52%. MRD was undetectable by flow cytometry (<10–4) in the peripheral blood and bone marrow of 3 patients and by PCR also in 2. Seven (5%) patients showed no response while a treatment failure was considered in 14% who experienced early discontinuation of treatment due to other reasons than no response. After the first phase of the study with IBR+R combination, 106 patients continued treatment with IBR as a single agent. PFS and OS rates at 24 months were 91% (95% CI, 85.45–97.16) and 93% (95% CI, 88.54–98.52) respectively. No significant differences in PFS and OS were observed according to the response to IBR+R combination (PFS and OS at 12 months from response, CR vs L-PR/PR: 100% vs 98.5%; p = 0.44); and the IGHV mutational status (mutated vs unmutated IGHV, PFS at 24 months: 92% vs 89%; p = 0.7; OS at 24 months: 93% vs 92%; p = 0.7), Patients with del(17p) and/or TP53 mutations showed a lower, though not significantly lower, survival (TP53 disruption, absent vs present, PFS at 24 months: 94% vs 77%; p = 0.08; OS at 24 months: 95% vs 87%; p = 0.2). Serious adverse events (AEs) were recorded in 18 (12%) patients. Most frequent severe AEs were infections (7 patients); cardiac disorders (7 patients, atrial fibrillation in 5); pyrexia/ neutropenic fever (5 patients), bleeding events (3, subdural hematoma in 2). Fatal AEs were recorded in 4 cases (leukoencephalopathy, 2; sepsis,1; myocardial infarction, 1). Summary/Conclusion: The addition of R to IBR resulted in a higher rate of CR than those seen with IBR given as a single agent. Front-line treatment with IBR+R given to old and unfit patients with CLL was relatively well tolerated and an effective treatment option with durable responses.

Background:The somatic hypermutation (SHM) of immunoglobulin heavy chain variable (IGHV) region genes classifies chronic lymphocytic leukemia (CLL) patients into two subgroups: unmutated CLL (UM‐CLL, ≥98%) associated with an adverse prognosis and mutated CLL (M‐CLL, <98%) showing a good outcome. In cases with a % of IGHV identity close to the 98% cut‐off, defined as “borderline” (BL‐CLL), the prognosis remains controversial.Aims:To investigate the clinico‐biologic features and prognosis of BL‐IGHV CLL in view of their undefined outcome.Methods:We retrospectively analyzed 811 untreated CLL patients, including 327 at diagnosis, sequenced at our Institution. BL‐CLL were defined as cases with a 97–97.9% IGHV identity, according to the 2017 European Research Initiative on CLL (ERIC) recommendations. IGHV were amplified using family‐specific VH primers (framework region 1 [FR1] or leader primers) and sequenced using the 3500 Series Genetic Analyzer. Following the ERIC recommendations, we updated the IGHV analysis with two different approaches in order to accurately determine the SHM rate: 1) re‐sequencing of 82 old FR1 cases with leader primers; 2) realignment of all cases to the updated IMGT/V‐QUEST tool. From the re‐analysis with leader primers of 82 old FR1 sequenced cases, none shifted the UM/M IGHV category, while 6 (7.3%) were reclassified as follows: 4 M‐CLL as BL‐CLL and 2 BL‐CLL as UM‐CLL. No case shifted across the three categories after the realignment to the updated IMGT/V‐QUEST tool. Major stereotyped subset analysis was determined according to the ARResT/AssignSubsets tool (bat.infspire.org/arrest/ericll.org/pages/services/tool). Time‐to‐first treatment (TFT) was evaluated as a clinical endpoint. Two further independent cohorts of 308 and 465 newly diagnosed CLL patients were used to validate the data.Results:In the 811 untreated patients, 418 (51.5%) UM‐CLL, 39 (5%) BL‐CLL and 354 (43.5%) M‐CLL were identified, with a higher frequency of UM‐CLL due to the enrichment of cases enrolled in first line clinical trials. Sixteen cases (2%) belonged to stereotyped subset #2. BL‐CLL showed a similar proportion of TP53 deletion and mutation compared to UM‐CLL, but a significant lower frequency of other unfavorable prognostic factors (stage, CD38/ZAP70 expression) (Table 1). Moreover, BL‐CLL were enriched in subset #2 (5/39, 13%), in agreement with the higher incidence of subset #2 among these cases. Applying the conventional 98% IGHV cut‐off, TFT was significantly shorter in UM‐CLL than M‐CLL (p < 0.0001), as well as in subset #2 CLL vs M‐CLL (p = 0.017), as expected. When analyzing the 97–97.9% IGHV subgroup, BL‐CLL showed a significantly longer TFT than UM‐CLL (p < 0.0001), similar to that of M‐CLL (Fig. 1a). The TFT of BL‐CLL cases remained comparable to that of M‐CLL, also when the analysis was restricted to the 327 CLL patients at diagnosis (80% Binet stage A) (125 UM‐CLL, 19 BL‐CLL and 183 M‐CLL) (Fig. 1b), as well as excluding subset #2 cases. These findings were validated in two independent cohorts of CLL at diagnosis (105 UM‐CLL, 16 BL‐CLL, 187 M‐CLL; TFT BL vs UM‐CLL: p < 0.0001 and 143 UM‐CLL, 13 BL‐CLL, 309 M‐CLL; p = 0.024), that included 719 Binet stage A CLL.Summary/Conclusion:Our data suggest that the prognosis of BL‐CLL is better than that of UM‐CLL and similar to that of M‐CLL, thus supporting the 98% cut‐off for clinical purposes. The issue of BL‐CLL remains open to further investigations.image

Background Chronic lymphocytic leukemia (CLL) has a heterogeneous clinical course. Beside patients requiring immediate treatment, others show an initial indolent phase followed by progression and others do not progress for decades. The latter two subgroups usually display mutated IGHV genes and a favorable FISH profile. Patients and methods Patients with absence of disease progression for over 10 years (10-34) from diagnosis were defined as ultra-stable CLL (US-CLL). Forty US-CLL underwent extensive characterization including whole exome sequencing (WES), ultra-deep sequencing and copy number aberration (CNA) analysis to define their unexplored genetic landscape. Microarray analysis, comparing US-CLL with non-US-CLL with similar immunogenetic features (mutated IGHV/favorable FISH), was also carried out to recognize US-CLL at diagnosis. Results WES was carried out in 20 US-CLL and 84 non-silent somatic mutations in 78 genes were found. When re-tested in a validation cohort of 20 further US-CLL, no recurrent lesion was identified. No clonal mutations of NOTCH1, BIRC3, SF3B1 and TP53 were found, including ATM and other potential progression driving mutations. CNA analysis identified 31 lesions, none with known poor prognostic impact. No novel recurrent lesion was identified: most cases showed no lesions (38%) or an isolated del(13q) (31%). The expression of 6 genes, selected from a gene expression profile analysis by microarray and quantified by droplet digital PCR on a cohort of 79 CLL (58 US-CLL and 21 non-US-CLL), allowed to build a decision-tree capable of recognizing at diagnosis US-CLL patients. Conclusions The genetic landscape of US-CLL is characterized by the absence of known unfavorable driver mutations/CNA and of novel recurrent genetic lesions. Among CLL patients with favorable immunogenetics, a decision-tree based on the expression of 6 genes may identify at diagnosis patients who are likely to maintain an indolent disease for decades.

Spontaneous Clinical Regression In Chronic Lymphocytic Leukemia : Clinical And Biologic Features Of 9 Cases From The ERIC Registry

Refined karyotype-based prognostic stratification of chronic lymphocytic leukemia with a low- and very-low-risk genetic profile

SummaryWhole exome sequencing and copy number aberration (CNA) analysis were performed on cells taken from peripheral blood (PB) and lymph nodes (LN) of patients with chronic lymphocytic leukaemia (CLL). Of 64 non‐silent somatic mutations, 54 (84·4%) were clonal in both compartments, 3 (4·7%) were PB‐specific and 7 (10·9%) were LN‐specific. Most of the LN‐ or PB‐specific mutations were subclonal in the other corresponding compartment (variant frequency 0·5–5·3%). Of 41 CNAs, 27 (65·8%) were shared by both compartments and 7 (17·1%) were LN‐ or PB‐specific. Overall, 6 of 9 cases (66·7%) showed genomic differences between the compartments. At subsequent relapse, Case 10, with 6 LN‐specific lesions, and Case 100, with 6 LN‐specific and 8 PB‐specific lesions, showed, in the PB, the clonal expansion of LN‐derived lesions with an adverse impact: SF3B1 mutation, BIRC3 deletion, del8(p23·3‐p11·1), del9(p24·3‐p13·1) and gain 2(p25·3‐p14). CLL shows an intra‐patient clonal heterogeneity according to the disease compartment, with both LN and PB‐specific mutations/CNAs. The LN microenvironment might contribute to the clonal selection of unfavourable lesions, as LN‐derived mutations/CNAs can appear in the PB at relapse.