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Purpose:In chronic lymphocytic leukemia (CLL), TP53 mutations are associated with reduced survival and resistance to standard chemoimmunotherapy (CIT). Nevertheless, the clinical impact of subclonal TP53 mutations below 10% to 15% variant allele frequency (VAF) remains unclear.Experimental Design:Using a training/validation approach, we retrospectively analyzed the clinical and biological features of TP53 mutations above (high-VAF) or below (low-VAF) the previously reported 10.0% VAF threshold, as determined by deep next-generation sequencing. Clinical impact of low-VAF TP53 mutations was also confirmed in a cohort (n = 251) of CLL treated with fludarabine-cyclophosphamide-rituximab (FCR) or FCR-like regimens from two UK trials.Results:In the training cohort, 97 of 684 patients bore 152 TP53 mutations, while in the validation cohort, 71 of 536 patients had 109 TP53 mutations. In both cohorts, patients with the TP53 mutation experienced significantly shorter overall survival (OS) than TP53 wild-type patients, regardless of the TP53 mutation VAF. By combining TP53 mutation and 17p13.1 deletion (del17p) data in the total cohort (n = 1,220), 113 cases were TP53 mutated only (73/113 with low-VAF mutations), 55 del17p/TP53 mutated (3/55 with low-VAF mutations), 20 del17p only, and 1,032 (84.6%) TP53 wild-type. A model including low-VAF cases outperformed the canonical model, which considered only high-VAF cases (c-indices 0.643 vs. 0.603, P < 0.0001), and improved the prognostic risk stratification of CLL International Prognostic Index. Clinical results were confirmed in CIT-treated cases (n = 552) from the retrospective cohort, and the UK trials cohort.Conclusions:TP53 mutations affected OS regardless of VAF. This finding can be used to update the definition of TP53 mutated CLL for clinical purposes.

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CD49d, the α4 chain of the VLA-4 integrin, is a negative prognosticator in chronic lymphocytic leukemia (CLL) with a key role in CLL cell-microenvironment interactions mainly occurring via its ligands VCAM-1 and fibronectin. In the present study, we focused on EMILIN-1 (Elastin-MIcrofibriL-INterfacer-1), an alternative VLA-4 ligand whose role has been so far reported only in non-hematological settings, by investigating: i) the distribution of EMILIN-1 in CLL-involved tissues; ii) the capability of EMILIN-1 to operate, via its globular C1q (gC1q) domain, as additional adhesion ligand in CLL; iii) the functional meaning of EMILIN-1 gC1q/VLA-4 interactions in CLL. EMILIN-1 is widely present in the CLL-involved areas of bone marrow biopsies (BMBs) without difference between CD49d negative and positive cases, displaying at least three different expression patterns: "fibrillar", "dot-like" and "mixed". The lack in CLL-BMB of neutrophil elastase, whose proteolytic activity degrades EMILIN-1 and impairs EMILIN-1 function, suggests full functional EMILIN-1 in CLL independently of its expression pattern. Functionally, EMILIN-1 gC1q domain promotes adhesion of CLL cells through specific interaction with VLA-4, and releases pro-survival signals for CLL cells, as demonstrated by enhanced ERK and AKT phosphorylation and impairment of in-vitro-induced apoptosis. EMILIN-1/VLA-4 interaction can efficiently contribute to the maintenance of the neoplastic clone in CLL. This article is protected by copyright. All rights reserved.

Purpose: In chronic lymphocytic leukemia (CLL), TP53 mutations are associated with reduced survival and resistance to standard chemoimmunotherapy (CIT). Nevertheless, the clinical impact of subclonal TP53 mutations below 10% to 15% variant allele frequency (VAF) remains unclear. Experimental Design: Using a training/validation approach, we retrospectively analyzed the clinical and biological features of TP53 mutations above (high-VAF) or below (low-VAF) the previously reported 10.0% VAF threshold, as determined by deep next-generation sequencing. Clinical impact of low-VAF TP53 mutations was also confirmed in a cohort (n = 251) of CLL treated with fludarabine-cyclophosphamide-rituximab (FCR) or FCR-like regimens from two UK trials. Results: In the training cohort, 97 of 684 patients bore 152 TP53 mutations, while in the validation cohort, 71 of 536 patients had 109 TP53 mutations. In both cohorts, patients with the TP53 mutation experienced significantly shorter overall survival (OS) than TP53 wild-type patients, regardless of the TP53 mutation VAF. By combining TP53 mutation and 17p13.1 deletion (del17p) data in the total cohort (n = 1,220), 113 cases were TP53 mutated only (73/113 with low-VAF mutations), 55 del17p/TP53 mutated (3/55 with low-VAF mutations), 20 del17p only, and 1,032 (84.6%) TP53 wild-type. A model including low-VAF cases outperformed the canonical model, which considered only high-VAF cases (c-indices 0.643 vs. 0.603, P < 0.0001), and improved the prognostic risk stratification of CLL International Prognostic Index. Clinical results were confirmed in CIT-treated cases (n = 552) from the retrospective cohort, and the UK trials cohort. Conclusions: TP53 mutations affected OS regardless of VAF. This finding can be used to update the definition of TP53 mutated CLL for clinical purposes.

KRAS mutations account for the most frequent mutations in human cancers, and are generally correlated with disease aggressiveness, poor prognosis, and poor response to therapies. KRAS is required for adult hematopoiesis and plays a key role in B cell development and mature B cell proliferation and survival, proved to be critical for B cell receptor-induced ERK pathway activation. In mature B cell neoplasms, commonly seen in adults, KRAS and RAS-MAPK pathway aberrations occur in a relevant fraction of patients, reaching high recurrence in some specific subtypes like multiple myeloma and hairy cell leukemia. As inhibitors targeting the RAS-MAPK pathway are being developed and improved, it is of outmost importance to precisely identify all subgroups of patients that could potentially benefit from their use. Herein, we review the role of KRAS and RAS-MAPK signaling in malignant hematopoiesis, focusing on mature B cell lymphoproliferative disorders. We discuss KRAS and RAS-MAPK pathway aberrations describing type, incidence, mutual exclusion with other genetic abnormalities, and association with prognosis. We review the current therapeutic strategies applied in mature B cell neoplasms to counteract RAS-MAPK signaling in pre-clinical and clinical studies, including most promising combination therapies. We finally present an overview of genetically engineered mouse models bearing KRAS and RAS-MAPK pathway aberrations in the hematopoietic compartment, which are valuable tools in the understanding of cancer biology and etiology.

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The Notch signaling pathway plays a fundamental role for the terminal differentiation of multiple cell types, including B and T lymphocytes. The Notch receptors are transmembrane proteins that, upon ligand engagement, undergo multiple processing steps that ultimately release their intracytoplasmic portion. The activated protein ultimately operates as a nuclear transcriptional co-factor, whose stability is finely regulated. The Notch pathway has gained growing attention in chronic lymphocytic leukemia (CLL) because of the high rate of somatic mutations of the NOTCH1 gene. In CLL, NOTCH1 mutations represent a validated prognostic marker and a potential predictive marker for anti-CD20-based therapies, as pathological alterations of the Notch pathway can provide significant growth and survival advantage to neoplastic clone. However, beside NOTCH1 mutation, other events have been demonstrated to perturb the Notch pathway, namely somatic mutations of upstream, or even apparently unrelated, proteins such as FBXW7, MED12, SPEN, SF3B1, as well as physiological signals from other pathways such as the B-cell receptor. Here we review these mechanisms of activation of the NOTCH1 pathway in the context of CLL; the resulting picture highlights how multiple different mechanisms, that might occur under specific genomic, phenotypic and microenvironmental contexts, ultimately result in the same search for proliferative and survival advantages (through activation of MYC), as well as immune escape and therapy evasion (from anti-CD20 biological therapies). Understanding the preferential strategies through which CLL cells hijack NOTCH1 signaling may present important clues for designing targeted treatment strategies for the management of CLL.

The introduction of agents inhibiting the BCR-associated kinases such as ibrutinib has dramatically changed treatments algorithms of chronic lymphocytic leukaemia (CLL) as well as the role of different adverse prognosticators. We evaluated the efficacy of ibrutinib as single agent, in a real-life context, on 180 patients with CLL mostly pre-treated, recruited from three independent cohorts from Italy. Patients received 420 mg oral ibrutinib once daily until progression or occurrence of unacceptable side effects. Seventy-three patients discontinued ibrutinib for progression or for adverse events. NOTCH1 mutations (M) were correlated with a reduced redistribution lymphocytosis, calculated at 3 months on ibrutinib (p=0.022). Moreover, NOTCH1 mutated patients showed inferior nodal response at 6 months on ibrutinib compared to NOTCH1 wild type patients (p

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In chronic lymphocytic leukemia (CLL), the mechanisms controlling cell growth and proliferation in the presence of NOTCH1 mutations remain largely unexplored. By performing a gene expression profile of NOTCH1-mutated (NOTCH1-mut) versus NOTCH1 wild-type CLL, we identified a gene signature of NOTCH1-mut CLL characterized by the upregulation of genes related to ribosome biogenesis, such as nucleophosmin 1 (NPM1) and ribosomal proteins (RNPs). Activation of NOTCH1 signaling by ethylenediaminetetraacetic acid or by coculture with JAGGED1-expressing stromal cells increased NPM1 expression, and inhibition of NOTCH1 signaling by either NOTCH1-specific small interfering RNA (siRNA) or γ-secretase inhibitor reduced NPM1 expression. Bioinformatic analyses and in vitro activation/inhibition of NOTCH1 signaling suggested a role of MYC as a mediator of NOTCH1 effects over NPM1 and RNP expression in NOTCH1-mut CLL. Chromatin immunoprecipitation experiments performed on NOTCH1 intracellular domain (NICD)-transfected CLL-like cells showed the direct binding of NOTCH1 to the MYC promoter, and transfection with MYC-specific siRNA reduced NPM1 expression. In turn, NPM1 determined a proliferation advantage of CLL-like cells, as demonstrated by NPM1-specific siRNA transfection. In conclusion, NOTCH1 mutations in CLL are associated with the overexpression of MYC and MYC-related genes involved in protein biosynthesis including NPM1, which are allegedly responsible for cell growth and/or proliferation advantages of NOTCH1-mut CLL.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a variable clinical course. Novel biomarkers discovered over the past 20 years have revolutionized the way clinicians approach prognostication and treatment especially in the chemotherapy-free era. Herein, we review the best established prognostic and predictive biomarkers in the setting of chemoimmunotherapy (CIT) and novel targeted therapy. We propose that TP53 disruption (defined as either TP53 mutation or chromosome 17p deletion), unmutated immunoglobulin heavy chain variable region gene status (UM IGHV), NOTCH1 mutation, and CD49d expression are the strongest prognosticators of disease progression and overall survival in the field of novel biomarkers including recurrent gene mutations. We also highlight the predictive role of TP53 disruption, UM IGHV, and NOTCH1 mutation in the setting of CIT and TP53 disruption and CD49d expression in the setting of novel targeted therapy employing B-cell receptor (BCR) and B-cell lymphoma-2 (BCL2) inhibition. Finally, we discuss future directions in the field of biomarker development to identify those with relapsed/refractory disease at risk for progression despite treatment with novel therapies.

In chronic lymphocytic leukemia (CLL), NOTCH1 mutations have been associated with clinical resistance to the anti-CD20 rituximab, although the mechanisms behind this peculiar behavior remain to be clarified. In a wide CLL series (n=692), we demonstrated that CLL cells from NOTCH1-mutated cases (87/692) were characterized by lower CD20 expression and lower relative lysis induced by anti-CD20 exposure in vitro. Consistently, CD20 expression by CLL cells was upregulated in vitro by gamma-secretase inhibitors or NOTCH1-specific small interfering RNA and the stable transfection of a mutated (c.7541- 7542delCT) NOTCH1 intracellular domain (NICD-mut) into CLL-like cells resulted in a strong downregulation of both CD20 protein and transcript. By using these NICD-mut transfectants, we investigated protein interactions of RBPJ, a transcription factor acting either as activator or repressor of NOTCH1 pathway when respectively bound to NICD or histone deacetylases (HDACs). Compared with controls, NICD-mut transfectants had RBPJ preferentially complexed to NICD and showed higher levels of HDACs interacting with the promoter of the CD20 gene. Finally, treatment with the HDAC inhibitor valproic acid upregulated CD20 in both NICD-mut transfectants and primary CLL cells. In conclusion, NOTCH1 mutations are associated with low CD20 levels in CLL and are responsible for a dysregulation of HDAC- mediated epigenetic repression of CD20 expression.

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In chronic lymphocytic leukemia (CLL), stabilizing mutations of NOTCH1, affecting up to 10–15% of cases, have been associated to poor prognosis, disease progression and refractoriness to chemotherapy. NOTCH1 mutations are significantly overrepresented in trisomy 12 CLL, a disease subset frequently expressing CD49d, the α4 chain of the very-late-activation-4 integrin, a well-known key regulator of microenviromental interactions, and negative prognosticator in CLL. In the present study, by analysing a wide cohort of 1180 CLL, we observed a very strong association between the presence of NOTCH1 mutations and the expression of CD49d (Po0.0001), occurring also outside the trisomy 12 CLL subset. Using both the MEC-1 CLL-like cells stably transfected with the NOTCH1 intracellular domain and primary CLL cells bearing a mutated or wild-type NOTCH1 gene configuration, we provide evidence that triggering of the NOTCH1 pathway resulted in a positive CD49d expression regulation, which was driven by a NOTCH1-dependent activation of nuclear factot-κB (NF-κB). Consistently, pharmacological inhibition of the NOTCH1 and/or of the NF-κB pathways resulted in impaired NF-κB nuclear translocation with consequent down-modulation of CD49d expression. Altogether, our data link for the first time NOTCH1 mutations to CD49d expression regulation through the involvement of the NF-κB pathway in CLL.

Background. TP53 mutations (TP53mut) along with 17p13 deletion (del17p) are strong predictors of poor survival and refractoriness to chemo-immunotherapies (CIT) in chronic lymphocytic leukemia (CLL), and their analyses should be always performed before treatment. Studies based upon ultra-deep-NGS have shown that TP53mut can be present at very low level in CLL cell populations, although their detrimental clinical impact in this setting is still matter of debate (Rossi, Blood 2014), and ERIC recomendations discourage to report TP53 mutations if subclonal Malcikova, Leukemia 2018). Aim. To investigated the presence and clinical relevance of clonal/subclonal TP53 aberrations in a large CLL cohort. Methods. The study includes 1,058 out of 1,613 CLL patients (509 treated with standard CIT) diagnosed between 1991 and 2018, and consecutively referred to a single institution for del17p analyses by FISH (167-kb 17p13 orange probe, MetaSystems), and TP53mut by ultra-deep NGS (MiSeq Illumina; median coverage >2,000X with an amplicon-based strategy covering exons 2-11 using 40ng DNA/test) in CD19-purified (>85% pure) CLL samples, collected before treatment (as per ERIC recommendations). For TP53mut analyses, FASTQ files were aligned to the Hg19 reference with Burrows-Wheeler Aligner-MEM algorithm, and allele variants called by FreeBayes (Garrison & Marth, arXiv 2102) with non-stringent parameters. To calculate random/systematic errors we generated a specific database with all the variant allele frequencies (VAF) observed in a subset of TP53 wild type (wt) subjects (n=362). TP53mut were accepted if: i) validated by Fisher exact test after Bonferroni correction (p Results. A total of 248 TP53mut (Fig.A) were found in 154 patients (13.5%, Fig.B) with a median mutations/patient of 1.65 (range 1-11). According to the 12.5% VAF cutoff for TP53mut (Nadeu, Blood 2016), 87 cases were clonal (at least one clone > 12.5%) and 67 subclonal (all clones 0.4% for the clinical impact of TP53mut (Fig.D), and the c-index of combined clonal/subclonal TP53mut (0.645) was significantly higher than the c-index of clonal TP53mut alone (0.602; P10% of nuclei had significantly shorter OS than cases with del17p in 0.5% as the best cutoff. In keeping with this cutoff, TP53 mutated patients experienced a significantly shorter OS than wt patients. Again cases with clonal and subclonal mutation experienced the same OS (Fig.I). Importantly, the cutoff found in the training cohort was able to reproduce the very same results also in the validation cohort both in term of mutation per se and in terms of clonal and subclonal TP53 mutations (Fig.J). Conclusion. i) By applying ERIC recommendations and a rigorous pipeline of analysis, TP53mut impacted on OS also with VAF 10% of nuclei. These cutoffs may be employed for the clinical management of CLL patients. Figure Disclosures Di Raimondo: Takeda: Consultancy; Amgen: Consultancy, Honoraria, 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.

The advent of agents inhibiting the BCR-associated kinases (ibrutinib and idelalisib) and the antiapoptotic protein Bcl-2 (venetoclax) has dramatically changed treatments algorithms of CLL as well as the role of different adverse prognosticators. The marked benefit of these new drugs has been investigated in the context of NOTCH1 mutations (M). In fact, NOTCH1 M were significantly correlated with CD49d overexpression identifying CLL with reduced lymphocytosis and inferior nodal response (Tissino, 2018). Moreover, NOTCH1 M were characterized by overexpression and increased activity of the NF-kB pathway genes, promoting tumor cell proliferation and survival (Benedetti, 2017). The primary aims of our clinical research were: i) to correlate NOTCH1 M with CD49d expression and bax/bcl-2 ratio in ibrutinib-treated patients; ii) to verify the impact of NOTCH1M on the peripheral lymphocytes redistribution and on the nodal response calculated as percentual reduction from baseline (SPD); iii) to evaluate the impact of NOTCH1 M on overall response rate (ORR), progression free survival (PFS) and overall survival (OS); iiii) finally, to assess NOTCH1 M as an independent prognostic factor. Therefore, we evaluated the efficacy of ibrutinib as single agent, in a real-life contest, on 180 patients recruited from three independent cohorts from Italy, median age 69 years (36-90), median number of previous regimens 2 [range 0-4; 26 patients (14.4%) previously untreated]. Noteworthy, 24/64 (37.5%) TP53 mutated patients were treated in first line with ibrutinib (p30%. Bax/bcl-2 ratio, evaluated in 113 patients, was calculated by flow cytometry, dividing mean florescence intensity (MFI) of bax by MFI of bcl-2 on CLL cells. The threshold of positivity was set at the median value >1.5 (range 0.41-5.10). Sixty-five patients were NOTCH1 M (36.11%). NOTCH1 M were strongly correlated both with CD49d >30% (51/65; p=0.0001) and bax/bcl-2 ratio Disclosures No relevant conflicts of interest to declare.

Trisomy 12 is a recurrent cytogenetic abnormality that occurs in 15–20% of Chronic Lymphocytic Leukemia (CLL).[1][1],[2][2] Within the hierarchical model proposed by Dohner et al .,[3][3] trisomy 12 CLL (tris12 CLL) carry an intermediate prognostic risk, with median overall survival (OS) and time

In chronic lymphocytic leukemia (CLL), the mechanisms controlling cell growth and proliferation in the presence of NOTCH1 mutations remain largely unexplored. By performing a gene expression profile of NOTCH1-mutated (NOTCH1-mut) versus NOTCH1 wild-type CLL, we identified a gene signature of NOTCH1-mut CLL characterized by the upregulation of genes related to ribosome biogenesis, such as nucleophosmin 1 (NPM1) and ribosomal proteins (RNPs). Activation of NOTCH1 signaling by ethylenediaminetetraacetic acid or by coculture with JAGGED1-expressing stromal cells increased NPM1 expression, and inhibition of NOTCH1 signaling by either NOTCH1-specific small interfering RNA (siRNA) or γ-secretase inhibitor reduced NPM1 expression. Bioinformatic analyses and in vitro activation/inhibition of NOTCH1 signaling suggested a role of MYC as a mediator of NOTCH1 effects over NPM1 and RNP expression in NOTCH1-mut CLL. Chromatin immunoprecipitation experiments performed on NOTCH1 intracellular domain (NICD)-transfected CLL-like cells showed the direct binding of NOTCH1 to the MYC promoter, and transfection with MYC-specific siRNA reduced NPM1 expression. In turn, NPM1 determined a proliferation advantage of CLL-like cells, as demonstrated by NPM1-specific siRNA transfection. In conclusion, NOTCH1 mutations in CLL are associated with the overexpression of MYC and MYC-related genes involved in protein biosynthesis including NPM1, which are allegedly responsible for cell growth and/or proliferation advantages of NOTCH1-mut CLL.

Introduction. The clinical course of patients with chronic lymphocytic leukemia (CLL) is highly heterogeneous. The deletions/mutations of 17p/TP53 are predictors of chemorefractoriness, and for this reason, in the management algorithm of CLL patients, when present, indicate treatment with with chemo-free regimens also in the context of first-line therapy. Recent studies based on ultra-deep-next generation sequencing (NGS) have shown that TP53 mutations can be present at very low clonal abundance in tumor cell populations, although whether these mutations may have a detrimental clinical impact on disease course is still to be established. Aim. To investigate the presence of clonal and subclonal mutations of TP53 in a large cohort of CLL cases using an ultra-deep NGS strategy, and determined their clinical relevance for patients outcome. Methods. The study includes 590 CLL patients characterized for the deletion at chromosome 17p13 (FISH analysis) and TP53 mutations in samples before treatment. In all cases, analyses were carried out on DNA extracted from nearly pure (>90%) tumor cells. TP53 mutational status was investigated by NGS with an amplicon based strategy. Sequencing reads analysis was made by the Burrows-Wheeler Aligner-MEM algorithm and by SAMtools. Variant calling was performed using the entire pipeline established on the MiSeq Reporter software. Results were expressed as percentage of mutated DNA. The minimal allelic fraction for mutation calling was set at 1%. Synonymous variants and polymorphisms described in the Single Nucleotide Polymorphism Database (dbSNP138) were removed. Outcome variable was overall survival (OS). Clinical correlations were made using Kaplan-Meier plots and log-rank test. Results. FISH and mutational analyses were performed in samples within 2 years from diagnosis in 92% of the cases (Figure 1A). A total of 125 TP53 mutations (Figure 1B) were found in 96 patients (11.7%). Subclonal mutations have similar molecular characteristics as their respective high frequency allele mutations supporting a comparable pathogenic effect (Figure 1B). According to a 15% cutoff of variant allele frequency (VAF), 78 cases were considered clonal and 18 subclonal (Figure 1C) for TP53 mutations (1% < VAF < 15%). In this context, cases with subclonal and clonal TP53 mutations experienced significant shorter OS than TP53 wild-type (wt) cases, without differences between clonal and subclonal cases (Figure 1E). Accordingly, ROC analysis on the same cohort identified a cutoff of >0% for the clinical impact of TP53 mutations (Figure 1E inset). Deletion of chromosome 17p was found in 180 out of 574 patients (31.3%), and using a 10% cutoff, 61 patients presented a percentage of deleted nuclei above the cutoff (Figure 1D). Using only 17p deletion data and considering the above mentioned cutoff, patients with 17p13 deletion ≥10% experienced shorter OS than wt cases, while patients with 17p13 deletion 9% of deleted nuclei as optimal cutoff for OS discrimination (Figure 1F inset). Given the frequent co-occurrence of TP53 mutations with 17p deletions, we also evaluated the impact of isolated TP53 mutations and 17p deletions. By using the ROC cutoffs for the definition of mutated/deleted cases, 466 cases (81.1%) presented no TP53 disruption (TP53 mutations and deletion), 47 cases (8.2%) were TP53 mutated only, 15 cases (2.6%) were 17p deleted only and 46 cases (8.1%) presented a concomitant TP53 mutation and 17p deletion. Kaplan-Meier curves demonstrated comparable significant shorter OS intervals for TP53 mutated and/or deleted CLL cases respect to wt cases, while no differences were observed between these three groups (Figure 1G). Conclusion. By using a highly sensitive NGS approach, we have detected small subclones of TP53 in a relative high proportion of patients. TP53 mutations conferred a significant shorter OS irrespectively of VAF percent, while deletion of chromosome 17p impacted on OS only when detectable in more than 10% of nuclei. These cutoffs, once validated by prospective studies, may be employed in daily practice for the clinical management of CLL patients. Disclosures Zaja: Novartis: Honoraria, Research Funding; Abbvie: Honoraria; Celgene: Honoraria, Research Funding; Amgen: Honoraria; Janssen: Honoraria; Takeda: Honoraria; Sandoz: Honoraria.

Background Extensive sequencing data highlighted the recurrence of mutations resulting in constitutive MAP kinase signaling in chronic lymphocytic leukemia (CLL), and proposed these mutations as late drivers of CLL progression (X.S. Puente, Nature, 2015; D.A. Landau, Nature, 2015). Although some preliminary studies suggested KRAS, NRAS and BRAF mutations as possibly associated with trisomy 12 (TRI12) and/or unmutated (UM) IGHV genes (C.D. Herling, Blood, 2016; K. Takahashi, Blood, 2018), dedicated analysis of these aspects is still missing. Aim To correlate the occurrence of KRAS, NRAS and BRAF mutations with specific clinico-biological features in CLL. Methods The study included 534 primary CLL purposely enriched with 300 TRI12 cases: 190 cases with TRI12 as sole chromosomal aberration (TRI12only) and 110 cases harboring TRI12 plus other chromosomal aberrations (TRI12plus). The cohort was also enriched in cases with NOTCH1 aberrations (n=214) to evaluate their possible role in RAS-RAF mutations incidence. Amplicon-based targeted next-generation sequencing assay was performed using Miseq Illumina on DNA from purified CLL samples. Analyzed amplicons mapped in the hotspot regions of KRAS, NRAS (exons 2, 3 and 4) and BRAF (exons 11 and 15) genes. Variant allele frequency (VAF) was obtained by Miseq report. CLL cases were characterized for IGHV mutational status, main cytogenetic abnormalities, NOTCH1 and TP53 aberrations. Time to first treatment (TTFT) data were correlated with molecular findings. Results We found 90 missense point mutations (Fig.1) in 64 CLL cases, with prevalence of KRAS (44 mutations in 38 patients), followed by BRAF (31 mutations in 24 patients) and NRAS (15 mutations in 13 patients). Nearly all mutations have been previously associated with gain-of-function phenotype and increased RAS/ERK downstream signaling. The co-occurrence of 2 mutated genes (mainly KRAS and BRAF) were observed in 11 patients. Mutations were mainly subclonal (median VAF 6.15%, range 1.3%-61.6%) with only one third of mutations (27/90) above 15% VAF. A high association between the presence of KRAS/NRAS/BRAF mutations and UM IGHV and the presence of TRI12 was observed. Overall, 87.3% of KRAS/NRAS/BRAF mutated cases had a UM IGHV (p Furthermore, a higher prevalence of KRAS/NRAS/BRAF mutations was found in NOTCH1 wild type (wt) (22.1%) when compared to NOTCH1 mutated (11.2%) cases, in the context of the UM IGHV group (p=0.008), pointing to a mutual exclusivity of these mutations in the pathogenesis of disease. No other significant associations with clinical variables as RAI stage at diagnosis, presence of TP53 mutations or gender were observed. We finally evaluated if KRAS/NRAS/BRAF mutations had an impact on TTFT in the context of UM IGHV/TRI12only/NOTCH1wt, that showed the highest incidence of mutations (26/70, 37%). Both KRAS mutations alone (p=0.005) and KRAS/NRAS mutations (p=0.04) were associated with shorter TTFT (Fig.3). Conclusions Taken together our data show that KRAS, NRAS and BRAF mutations are almost exclusively found in UM IGHV/TRI12/NOTCH1wt CLL and, in the context of this CLL group the presence of KRAS and NRAS mutations is associated with unfavorable prognosis. The high incidence of KRAS, NRAS and BRAF activating mutations in TRI12 CLL together with the observation of higher level of ERK phosphorylation (S. Decker, Blood, 2012) and higher sensitivity to MEK/ERK inhibitors (S. Dietrich, J Clin Invest, 2018) described in the TRI12, reinforce the evidence of an essential role for MEK/ERK signaling in TRI12 CLL, pointing to this pathway as the most appropriate therapeutic target. Disclosures Zaja: Novartis: Honoraria, Research Funding; Janssen: Honoraria; Abbvie: Honoraria; Celgene: Honoraria, Research Funding; Amgen: Honoraria; Takeda: Honoraria; Sandoz: Honoraria.

Background Altough the inhibitor of Bruton's tyrosyne kinase (BTK) ibrutinib has transformed the management of patients with CLL obtaining decreased tumor burden and improved progression free survival (PFS), it does not induce substantial apoptosis in vitro (Ponader, 2012). Dysregulation of the mitchondrial pathway of apoptosis is one of the hallmarks of CLL cell pathophysiology and thus targeting the anti-apoptotic protein bcl-2 with venetoclax is a promising new therapeutic strategy (Souers, 2013; Roberts, 2016). Moreover ibrutinib increases bcl-2 dependence (Deng, 2017) enhancing sensitivity to venetoclax in CLL. Therefore the basal level of apoptosis measured through bax/bcl-2 ratio before therapy may be crucial to test sensitivity to ibrutinib. Aims The primary aims of our research were: i) to verify the correlations of bax/bcl-2 ratio with other well-known biological and clinical prognosticators in patients treated with ibrutinib; ii) to evaluate the impact of bax/bcl-2 ratio on overall response rate (ORR), PFS and overall survival (OS); iii) to test bax/bcl-2 ratio as an independent prognostic factor. Patients and Methods Therefore, we evaluated the efficacy of ibrutinib, in a real-life contest, on 100 patients, median age 60 years (38-85), median number of previous regimens 2 (0-4; 14% previously untreated). Patients received 420 mg oral ibrutinib once daily until progression or occurrence of unacceptable side effects. Median follow up on ibrutinib was 16 months. ORR was 92% [complete response (CR): 27%, partial response (PR): 32%, PR with lymphocytosis (PR-L): 33%]. The estimate 1-year PFS and OS were 74% and 77%, respectively. Twenty-nine patients (29%) discontinued ibrutinib therapy due to progression (n=11) and serious adverse events (n=18). Five out of 6 patients with Richter's syndrome (RS) had baseline del(17p)/TP53 mutations (M). Interestingly, PFS was significantly better for patients in first/second lines (n=38) vs later lines of therapy (n=55; p=0.022). Bax/bcl-2 ratio was calculated by flow cytometry, dividing mean florescence intensity (MFI) of bax by MFI of bcl-2 on peripheral CLL cells before treatment with ibrutinib. The threshold of positivity was set at the median value higher than 1.5 (range 0.38-5.10). Results Sixty-four patients had bax/bcl-2 ratio 20% (40/64; p=0.005), but not with CD49d or CD38 >30%. There was only a slight significant correlation between bax/bcl-2 ratio Conclusions In our hands, bax/bcl-2 ratio was a powerful prognosticator for patients treated with ibrutinib, particularly useful to identify subsets of CLL patients with different levels of spontaneus apoptosis and consequent different clinical outcome under ibrutinib treatment. At the light of these our recent observations, bcl-2 antagonists, such as ABT-199, should be reasonably combined with BTK inhibitors, as it is already underway in clinical protocols, in order to overcome apoptosis resistance in patients treated with ibrutinib, particularly within the CLL subset characterized by lower bax/bcl-2 ratio e poor response to therapy with ibrutinib. Figure. Figure. Disclosures Del Principe: Gilead: Membership on an entity's Board of Directors or advisory committees.

Background Chronic lymphocytic leukemia (CLL) is characterized by a CD20 expression lower than other lymphoproliferative disorders, hampering treatment efficacy of anti-CD20 antibodies. Although most mechanisms behind this phenomenon are still obscure, we demonstrated that mutations of NOTCH1 associate with a lower CD20 expression, ascribed to a NOTCH1 mutation-driven repression of CD20 transcription by hystone deacetylases (Pozzo et al. Leukemia 2016). This may explain the association between NOTCH1 mutations and clinical resistance to anti-CD20 immunotherapy in CLL patients treated with FCR (Stilgenbauer et al. Blood 2014). SF3B1 mutations affect ~10% CLL cases at diagnosis; a recent characterization of their functional meaning highlighted transcriptome-wide alterations of splicing patterns affecting several pathways, including the NOTCH1 pathway (Wang et al. Cancer Cell 2016). In this context, one of the most aberrantly-spliced genes is DVL2, a key component of the Wnt pathway and negative regulator of the NOTCH1 pathway. Aim To investigate CD20 expression in SF3B1-mutated CLL and the correlation with a putative secondary activation of the NOTCH1 pathway through splicing alterations of the NOTCH1 negative regulator DVL2. Methods Mutational status of NOTCH1 and SF3B1 was evaluated by next generation sequencing. Mutations with VAF-variant allele frequency below 2% were discarded. CD20 expression was evaluated by flow cytometry on the CD19+5+ population. Percentage of low-CD20 expressing (CD20dim) population was defined as %P1-(100-%P1), P1 being a linear gate spanning from zero to CD20 mode. Transcript levels of MS4A1 (encoding for CD20 protein) and spliced DVL2 were evaluated by QRT-PCR in cases with at least 75% of CD19+5+ cells. Statistical analyses were performed using Mann-Whitney rank-test. Results In a cohort of 537 unselected CLL cases, 93 cases (17.3%) carried NOTCH1 mutations: 62 delCT, 20 other truncating, 11 3′UTR (VAF range 3-84%, mean 32%; NOTCH1-mut); 46 cases carried SF3B1 mutations (8.5%, VAF range 5-53%, mean 32%; SF3B1-mut), the hotspot surrounding K700 being the most frequent (50%) followed by the hotspot surrounding G742 (30%); 7 cases (1,3%) carried both NOTCH1 and SF3B1 mutations. As trisomy 12 CLL is characterized by increased expression of CD20 and mutation of SF3B1 co-occurred in only 5/121 cases, the trisomy 12 subset was excluded from further analyses. Confirming previous findings, NOTCH1-mut cases were characterized by a lower CD20 mean fluorescence intensity (MFI) compared to WT cases (p=0.0154). In agreement with the hypothesis of a more active NOTCH1 pathway, CD20 expression was found diminished also in those cases carrying SF3B1 mutations, compared to WT cases (p=0.0059). Since CD20 expression is highly variable between cases, spanning a 2/3-log range of fluorescence intensity, we evaluated the percentage of the CD20dim population, this quantity being independent from the actual MFI. This analysis confirmed that CLL cases with NOTCH1 or SF3B1 mutations show a greater proportion of CD20dim cells (NOTCH1-mut p Presence of the alternatively spliced isoform of DVL2 (altDVL2) was identified only in those CLL cases with SF3B1 mutations (p35%) to isolate CD20high and CD20low populations. MS4A1 expression (higher in the CD20high subset) inversely correlated with altDVL2 expression (Figure 1c). As DVL2 can act as negative regulator of NOTCH1, we followed the hypothesis that loss of WT DVL2 due to alternative splicing may result in a more active NOTCH1 pathway. To simulate loss of DVL2 in the SF3B1-wild type CLL-like MEC1 cell line, cells were trasfected with siRNA for DVL2 itself and screened for CD20 expression. After 24 hours from transfection, CD20 expression was found downregulated at both protein (p=0.0062; Figure 1d) and transcript (p=0.0129) level. Conclusions In addition to NOTCH1-mut cases, also CLL cases bearing SF3B1 mutations are characterized by a lower CD20 expression, allegedly through a more active NOTCH1 pathway, potentially resulting in clinical resistance to anti-CD20 monoclonal antibodies. Disclosures Zaja: Amgen: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria; Sandoz: Honoraria; Abbvie: Honoraria.

Background Ibrutinib, inhibitor of Bruton's tyrosyne kinase (BTK), demonstrated exceptional activity both in untreated and relapsed/refractory CLL patients, obtaining overall response rate (ORR), progression-free survival (PFS) and overall survival (OS) far superior to chemoimmunotherapy (Byrd, 2014). Interestingly, NOTCH1 mutations (M) were significantly correlated with CD49d overexpression which identified cases with reduced lymphocytosis and inferior nodal response (Tissino, 2018). Moreover, NOTCH1 M were characterized by remarkable expression of the NF-kB pathway genes (Benedetti, 2017) and thus may confer poor response to ibrutinib. Aims On this background, the primary aims of our research were: i) to correlate NOTCH1 M with biological and clinical prognosticators in ibrutinib-treated patients; ii) to verify the impact of NOTCH1M both on the peripheral lymphocytes redistribution and on the nodal response calculated as percentual reduction from baseline (SPD); iii) to evaluate the impact of NOTCH1 M on ORR, PFS and OS; iiii) finally, to assess NOTCH1 M as an independent prognostic factor. Patients and Methods Therefore, we evaluated the efficacy of ibrutinib as single agent, in a real-life contest, on 166 patients recruited from three independent cohorts from Italy, median age 61 years (27-85), median number of previous regimens 2 (0-4; 15% previously untreated). Noteworthy, 23/60 (38%) TP53M patients were treated in first line with ibrutinib (p Results Sixty-one patients were NOTCH1 M (36.75%). NOTCH1 M were significantly correlated with trisomy 12 (p=0.00045) and with CD49d >30% (p=0.001). Absolute lymphocyte counts (ALCs) were collected before treatment and at days ranging from 30 to 90 on ibrutinib. NOTCH1 M were significantly correlated with lower median ALCs measured both before (28.9 x 106/ml vs 57.0 x 106/ml; p=0.0006) and during (28.0 x 106/ml vs 54.6 x 106/ml; p=0.0005) ibrutinib therapy. Also the median SPD, calculated at 3-6 months on ibrutinib, was significantly lower in NOTCH1 M patients (51.8% vs 75.7%; p or Conclusions NOTCH1 M patients were characterized by a reduced redistribution lymphocytosis and by a lower and/or slower nodal response under ibrutinib treatment. The clinical consequences were a significant higher number both of PR without lymphocytosis and relapses. Therefore, evaluation of NOTCH1 M in patients initiating ibrutinib may identify those cases that would benefit from combination therapy approaches, which are already underway in experimental protocols, using new molecules working through different pathways, such as venetoclax or obinutuzumab. Figure. Figure. Disclosures Del Principe: Gilead: Membership on an entity's Board of Directors or advisory committees.

CD49d, the alpha-chain of the integrin heterodimer α4β1, was identified among the strongest predictors of overall survival (OS) in chronic lymphocytic leukemia (CLL), along with IGHV mutational status and deletion of the 17p chromosome involving TP53. In addition to TP53, the clinical relevance of NOTCH1, SF3B1 and BIRC3 gene mutations has been recently emphasized. By analyzing a cohort of 778 unselected CLL patients, we assessed the clinical relevance of CD49d as an OS predictor in subgroups defined by mutation/deletion of the TP53, NOTCH1, SF3B1 and BIRC3 genes. In this context, CD49d emerged as an independent predictor of OS in multivariate Cox analysis (Hazard ratio =1.88, P

Background. CD49d, the alpha-chain of the integrin heterodimer VLA-4, has been identified among the strongest predictors of overall survival (OS) in chronic lymphocytic leukemia (CLL), along with IGHV mutational status and deletion of the 17p (17p-) chromosome involving TP53 (Bulian et al, JCO, 2014). In addition to TP53, the clinical relevance of NOTCH1, SF3B1 and BIRC3 gene mutations has been recently emphasized. Aim. To test the relevance of CD49d in molecular subgroups of CLL defined by NOTCH1, SF3B1 and BIRC3 gene mutations. Methods. The study was based on a cohort of 778 unselected CLL (422 untreated and 356 treated cases) all characterized for CD49d expression (CD49dhigh, ≥30% positive cells by flow cytometry in 229 cases), IGHV mutational status (unmutated, UM, in 262 cases), karyotype abnormalities according to the hierarchical stratification (13q- in 308 cases, +12 in 103 cases, 11q- in 64 cases), tested at diagnosis, along with mutations/deletions (hereinafter, disruption) of TP53 (disrupted in 84 cases, including 58 cases with 17p-), BIRC3 (disrupted in 59 cases), and mutationsof NOTCH1 (mutated in 81 cases), SF3B1 (mutated in 54 cases)tested at diagnosis in 65% of cases, in all cases before therapy. Chromosome abnormalities by FISH were defined by using a 5% cut-off; IGHV, TP53, BIRC3, NOTCH1 and SF3B1 mutations were investigated by Sanger sequencing. Median follow-up of patients was 80 months with 173 deaths. Results. i) association with CD49d: a CD49dhigh phenotype associated with age ≥65 years (p=0.0001), Rai stage I or higher (p>0.0001), UM IGHV status (p>0.0001), absence of 13q- (p=0.0001), presence of +12 (p Conclusion. CD49d was confirmed as independent negative OS prognosticator in CLL also when the novel recurrent alterations of NOTCH1, BIRC3, and SF3B1 genes were considered. In this setting, TP53 disruption and NOTCH1 mutations remained independent OS prognosticators, allegedly for their physiopathological roles in CLL cell immuno-chemoresistance. Figure 1. Figure 1. Disclosures Gaidano: Morphosys, Roche, Novartis, GlaxoSmith Kline, Amgen, Janssen, Karyopharm: Honoraria, Other: Advisory boards; Celgene: Research Funding.

CD49d expression identifies a chronic-lymphocytic leukemia subset with high levels of mobilized circulating CD34 + hemopoietic progenitors cells

Background. NOTCH1 mutations are found in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, and with higher frequencies in chemorefractory CLL, CLL in advanced disease phases and in Richter Syndrome transformations (Rossi et al. Blood, 2013). In CLL, NOTCH1 mutations have been recently associated with clinical resistance to anti-CD20 immunotherapy (Stilgenbauer et al. Blood, 2014, Dal Bo et al. AHO, 2014). In lymphoproliferative disorders, susceptibility to rituximab is determined by CD20 levels (Golay et al. Blood, 2001), which are in turn epigenetically modulated via HDAC (Shimizu et al., Leukemia, 2010). Aim. To investigate whether NOTCH1 mutations could affect CD20 expression in CLL. Methods. NOTCH1 mutations and NOTCH1 mutational burden were evaluated by ARMS PCR, QRT-PCR, conventional and next generation sequencing. NOTCH1 protein levels were evaluated by western blot. CD20 expression was evaluated by flow cytometry. Transcript levels of MS4A1, encoding for CD20 protein, were evaluated by QRT-PCR. CLL cells and CLL-like cell line MEC-1 cells were treated with the HDAC inhibitor valproic acid (VPA) at a concentration of 3mM. Susceptibility to rituximab was evaluated by complement dependent cytotoxicity (CDC) assay. MEC-1 cells were transfected with a vector containing a NOTCH1 intracellular domain (NICD) carrying either the 7544-7545delCT or a stop codon at the beginning of NICD sequence (c.5304G>A), as control. Results. i) In a cohort of 452 CLL, 54 cases carried NOTCH1 mutations. NOTCH1-mutated cases (NOTCH1-mut) with high mutational burden showed 3.0- fold higher transmembrane NOTCH1 and 2.1- fold higher NICD protein expression compared to NOTCH1 wild-type cases (NOTCH1-wt). NOTCH1-mut showed a lower Mean Fluorescent Intensity (MFI) of CD20 expression than NOTCH1-wt both in trisomy 12 (tris12, 18 NOTCH1-mut vs. 44 NOTCH1-wt, p Conclusions. CLL cases carrying NOTCH1 mutations are characterized by low CD20 expression levels that may confer resistance to anti-CD20 immunotherapy. The low CD20 expression, at least in part due to HDAC-dependent repression mechanism(s), can be reverted by HDAC inhibitor therapy. Disclosures No relevant conflicts of interest to declare.