Unmutated Status Research Articles

Additional value of quantitative expression of ZAP-70 and CD38 for prognostic factors in chronic lymphocytic leukemia.

e19006 Background: Chronic lymphocytic leukemia (CLL) has heterogeneous clinical course. Identification of prognostic markers is important for proper management of disease. Cytogenetics, IgVH mutation status, TP53 functional status, CD38, ZAP-70 and CD49d expression are accepted prognostic factors in CLL. ζ-chain-associated protein kinase 70 (ZAP-70) is associated with disease progression. However, measurement of ZAP-70 and CD38 expression relies on qualitative flow cytometry. Authors tested hypothesis whether quantitative analysis of markers can improve information about prognosis of CLL. Methods: 217 CLL patients were included; diagnosis was made from peripheral blood by morphology and immunophenotyping by flow cytometry with common antibodies. Expressions of intracellular ZAP-70 and surface CD38 were measured using quantitative fluorescence cytometry. Standardized fluorescent microparticles were used to quantify molecules of equivalent soluble fluorochrome (MESF units). Analysis of rearrangement of the IgVH gene was performed according to the European recommendation. Deletions at 11q22-q23 ( ATM), 17p13 ( TP53), 13q34 ( RB1) loci and trisomy of chromosome 12 were detected by I-FISH. Results: Patients’ ages ranged from 33 to 86 years and included all Rai stages. Expression of ZAP-70 ranged from 546 to 5,955 MESF, CD38 from 1,886 to 31,619 MESF. 48% patients embodied mutated and 52% unmutated IgVH status. In cytogenetics, del(13q) was detected in 59%, trisomy 12 in 13%, del(11q) in 21%, and del(p53) in 8% of patients. IgVH (in %) was plotted against ZAP-70 (in MESF) and patients were divided into high (HR) and low risk (LR) groups by cluster analysis based on Kaplan-Meier overall survival curves. Patients in HR group had significantly poorer prognoses (p = 0.0001) than those in LR group. When patients from HR group were clustered according to CD38 expression, very high risk (VHR) group with high CD38 and significantly worse prognosis (p = 0.035) was identified. Conclusions: We have demonstrated that quantitative ZAP-70 and CD38 expressions cannot replace IgVH analysis, but, contrary to that, addition of quantitative data can further specify prognoses of patients with CLL.

Prognostic impact of NOTCH1, MYD88 and SF3B1 mutations in Polish population of chronic lymphocytic leukemia patients

INTRODUCTION Currently available prognostic factors determining the course of chronic lymphocytic leukemia (CLL) are not fully efficient, especially for newly diagnosed patients. Investigation of molecular changes may help clarify the reasons for the heterogeneity of the disease. Apart from already confirmed TP53 mutations, the novel candidates: NOTCH1, SF3B1, and MYD88 might represent clinically relevant biomarkers. OBJECTIVES The aim of this study was to evaluate the mutational status of NOTCH1, MYD88, and SF3B1 and to compare the results with confirmed prognostic factors: ZAP‑70, CD38, and immunoglobulin heavy‑chain variable region (IGHV) mutation in CLL. The study assessed also prognostic significance in terms of the time to first treatment (TTFT) and subset analysis. PATIENTS AND METHODS The study was conducted on samples of 370 newly diagnosed patients with CLL. The analysis was performed using high‑resolution melting, Sanger sequencing, and polymerase chain reaction methods. RESULTS Patients harboring the NOTCH1 mutation were significantly more often found among patients with an unmutated IGHV gene status and high expression of CD38 and ZAP‑70. The MYD88 mutation was equally distributed in patients with mutated and unmutated IGHV status (5 vs 7 patients). For MYD88 and SF3B1, there were no significant differences in the levels of CD38 and ZAP‑70 expression. The tendency for lower median TTFT was revealed in patients with mutated SF3B1 (P = 0.08). The analysis showed the presence of 14 different types of the subsets of IGHV in 50 of 345 patients (14.5%). The most frequent were subsets #1 and #2. CONCLUSIONS The NOTCH1 and SF3B1 mutations accompany biological markers of unfavorable prognosis in patients with CLL. The mutations may contribute to the identification of patients with high‑risk CLL.

Cell-Intrinsic Determinants of Ibrutinib-Induced Apoptosis in Chronic Lymphocytic Leukemia.

Purpose: Ibrutinib, a Bruton tyrosine kinase (BTK) inhibitor, is approved for the treatment of relapsed chronic lymphocytic leukemia (CLL) and CLL with del17p. Mechanistically, ibrutinib interferes with B-cell receptor (BCR) signaling as well as multiple CLL cell-to-microenvironment interactions. Given the importance of ibrutinib in the management of CLL, a deeper understanding of factors governing sensitivity and resistance is warranted.Experimental Design: We studied 48 longitudinally sampled paired CLL samples, 42 of which were procured before and after standard CLL chemotherapies, and characterized them for well-studied CLL molecular traits as well as by whole-exome sequencing and SNP 6.0 array profiling. We exposed these samples to 0.25 to 5 μmol/L of ibrutinib ex vivo and measured apoptosis fractions as well as BCR signaling by immunoblotting. We disrupted TP53 in HG3, PGA1, and PG-EBV cell lines and measured BCR signaling and ibrutinib responses.Results: CLL samples demonstrated a surprisingly wide range of ex vivo sensitivities to ibrutinib, with IC50 values ranging from 0.4 to 9.7 μmol/L. Unmutated IGVH status, elevated ZAP70 expression, and trisomy 12 were associated with heightened sensitivity to ibrutinib treatment. Five CLL samples were substantially more resistant to ibrutinib following relapse from chemotherapy; of these, three had acquired a del17p/TP53-mutated status. A validation sample of 15 CLL carrying TP53 mutations, of which 13 carried both del17p and a TP53 mutation, confirmed substantially less sensitivity to ibrutinib-induced apoptosis.Conclusions: This study identifies that CLL harboring del17p/TP53-mutated cells are substantially less sensitive to ibrutinib-induced apoptosis than del17p/TP53 wild-type cells. Clin Cancer Res; 23(4); 1049-59. ©2016 AACR.

V(D)J Recombination Excision Circles of B- and T-cells as Prognostic Marker in B-Cell Chronic Lymphocytic Leukemia

Background & Aims. T-cell receptor excision circles (TREC) and к-deleting recombination excision circles (KREC) are extrachromosomal DNA segments generated during V(D)J recombination process that characterize the diversity of the antigen repertoire of T- and B-cells. The aim of our study is to identify the prognostic value of the excision circles in the chronic lymphocytic leukemia (CLL) setting. Methods. The excision circles' levels were assessed by means of real time PCR in 109 patients with high-risk CLL and 16 matched healthy individuals. Results. KREC levels were significantly (p < 0.001) lower in CLL patients vs. the reference group. TREC levels were lower in groups with unmutated status of immunoglobulin heavy chain variable region genes (p < 0.05) and 11q deletions (p < 0.1). Moreover, the KREC levels were higher in NOTCH1 mutation carriers than in noncarriers (p < 0.05). The comparison of treatment outcomes demonstrated a correlation between a high TREC level and achievement of complete remission. The prognostic value of the bio-marker was confirmed by ROC-analysis: AUC<sub>TREC</sub> = 0.713 (p = 0.001) Conclusion. Association between excision circles' le' and clinical/laboratory CLL prognostic factors, as well complete remission achievement, makes possible the plementation of the test for early prediction of the treatm outcome.

Characterization and Prognostic Impact of Multiple Productive IGHV Rearrangements in CLL

Background:In chronic lymphocytic leukemia (CLL) one of the strongest prognostic factors is IGHV mutational status. Infrequently, patients present not only with a single IGHV rearrangement but with multiple productive rearrangements. In about 2% of all CLL patients analyzed on cDNA level multiple rearrangements display the same mutational status and are categorized accordingly following ERIC recommendations. In another 1% rearrangements with discordant IGHV mutational status are detected and preclude a definite risk assignment. Only limited data exist on these rare subgroups.Aim:To characterize treatment-naive CLL patients with multiple productive IGHV rearrangements and determine the impact on prognosis.Patients and Methods:Out of 8,016 treatment-naive CLL patients between 2005 and 2015 and with data on IGHV mutational status we identified 204 (3%) with multiple productive rearrangements. IGHV mutational status was analyzed on cDNA and in all cases according to ERIC recommendations. IGHV mutated status (M) was defined by sequence identity <98% and unmutated status (U) by ≥98%. Chromosome banding analysis was available in 102 cases and interphase FISH with probes for 17p13, 13q14, 11q22 and centromeric region of chromosome 12 in 191. Male:female ratio was 3:1 and median age 68 years (range: 38-89). Additionally, data on SF3B1 and TP53 mutations was present in all cases. Follow-up data on time to first treatment (TTT) and overall survival (OS) was available in 105 cases with a median follow-up of 4 years. For statistical comparison we used a cohort of 1,262 untreated CLL patients with single IGHV rearrangement (median age: 67 years; range: 30-91, median follow-up: 6 years).Results:Out of 204 patients with multiple, productive rearrangements 199 (98%) presented with two and 5 patients (2%) with three IGHV rearrangements. Concordant IGHV mutated status (MM) was present in 120 cases (59%), whereas concordant unmutated status (UU) was seen in 34 patients (17%). In 50 cases (25%) a mixed IGHV status (UM) was detected.We analyzed frequencies of complex karyotype by CBA, biclonality according to immunophenotype (concurrent kappa restricted and lambda restricted subpopulations) and/or CBA, TP53 disruption (TP53mut and/or del(17p)), SF3B1mut, del(11q), trisomy 12, and del(13q). Overall, a higher frequency of biclonality was detected in patients with multiple vs. single IGHV rearrangements (16% vs. 1%, p<0.001). However, association to neither MM, UU nor UM existed. MM presented with molecular and cytogenetic characteristics similar to M. Correspondingly, UU showed similar frequencies of mutations and aberrations to U, except for higher frequency of trisomy 12 in UU vs. U (42% vs. 19%, p=0.003). Interestingly, UM presented with characteristics similar to U and UU. UM was associated with TP53 disruption vs. M (16% vs. 5%, p=0.003) and vs. MM (5%, p=0.035) as well as with SF3B1mut vs. M (16% vs. 5%, p=0.008). Furthermore, UM cases showed high frequency of del(11q) vs. M (29% vs. 3%, p<0.001) and vs. MM (1%, p<0.001) and less frequently del(13q) sole vs. M (41% vs. 60%, p=0.011) and MM (41% vs. 69%, p=0.001).No significantly differences in TTT were observed between MM and M (median: 13 vs. 14 years) and between UU and U (6 vs. 4 years), respectively. However, the difference between MM vs. UU (p=0.022) and M vs. U (p<0.001) was significant. The UM subgroup presented with a TTT (median: 4 years) similar to U and UU, whereas it was significantly shorter vs. M (p=0.003) and MM (p=0.006), respectively. A similar picture emerged for survival. 5-year OS of MM was not different vs. M (94% vs. 90%) but vs. U (78%, p=0.001). The statistical analysis of OS in UU was hampered by low case numbers. UM presented again with similar 5-year OS vs. U (81% vs. 78%, n.s.) and significantly worse OS vs. M (90%, p=0.049) and vs. MM (94%, p=0.014).Conclusions: (1) Patients with multiple productive IGHV rearrangements and concordant IGHV status show similar prognosis and characteristics to patients with single rearrangement with the respective IGHV status. (2) Cases with mixed IGHV status show similar prognosis to patients with IGHV unmutated status and accordingly are characterized by high frequencies of adverse prognostic factors like TP53 disruption, SF3B1mut, and del(11q), whereas del(13q) sole is less frequent. DisclosuresJeromin:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Dicker:Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Lenalidomide Maintenance after Front Line Therapy Substantially Prolongs Progression Free Survival in High Risk CLL: Interim Results of a Phase 3 Study (CLL M1 study of the German CLL Study Group)

Lenalidomide Maintenance after Front Line Therapy Substantially Prolongs Progression Free Survival in High Risk CLL: Interim Results of a Phase 3 Study (CLL M1 study of the German CLL Study Group)

Detailed Clinical, Immunological and Molecular Analysis of NOTCH1, SF3B1 and MYD88 mutations in Chronic Lymphocytic Leukemia Patients Reveals Accumulation of Negative Prognostic Features in NOTCH1 and SF3B1 mutated Individuals

The clinical course of chronic lymphocytic leukemia (CLL) is highly heterogeneous, from stable to a rapidly progressive. The variety of prognostic factors has been already described, nevertheless they are not fully efficient in predicting the course of CLL, especially when the disease is diagnosed at an early stage. Recently, beside well established cytogenetic prognostic factors, novel molecular mutations of predictive value have been identified. Many of them have been thoroughly characterized, including TP53 mutation, which is commonly considered as a strong, negative prognostic factor. The use of next-generation sequencing technology has also revealed previously unknown genomic alterations, such as neurogenic locus notch homolog protein1 (NOTCH1), splicing factor 3B subunit 1 (SF3B1) or myeloid differentiation primary response 88 (MYD88). These new mutations could partly explain the CLL heterogeneity and help in identifying clinically relevant groups of patients.The aim of the study was to characterize CLL patients with NOTCH1, MYD88 and SF3B1 mutations with regard to molecular and immunological prognostic markers in CLL. Peripheral blood mononuclear cells (PBMCs) were obtained from 369 CLL patients at the moment of diagnosis and the median age reached 65 years. Sixty percent of the patients were male. Distribution of disease stages according to the Rai classification was the following: 0 stage=93, I stage=52, II stage=69, III stage=14, IV stage=26. Clinically, the prognostic significance in terms of time to first treatment (TTFT) was assessed for 202 CLL patients.DNA samples were extracted from PBMCs obtained after Ficoll density gradient centrifugation. NOTCH1 c.7544_7545delCT (n=316) in PEST domain (exon 34) and MYD88 L265P (n=323) mutations were investigated by ARMS PCR. Screening for SF3B1 (n=364) mutations K700, E622/R625 and H662/K666 (exons 14 and 15) were performed using HRM analysis and the results were confirmed by Sanger sequencing. The IGHV gene mutations were investigated by Sanger sequencing.NOTCH1 mutations were found in 19/316 (6.0%) patients. Patients harbouring NOTCH1 mutations prevalently have unfavourable prognostic factors including unmutated IGHV gene status, expression of CD38 (>30%) and expression of ZAP-70 (>20%). The complete analysis of correlations between NOTCH1, MYD88 and SF3B1 mutations and prognostic markers in CLL are presented in Table 1. Analysis of IGHV subsets in patients with NOTCH1 mutation revealed frequent presence of subset #1 in n=2/19 (10.5%), which is associated with particularly poor prognosis in CLL. Patients belonging to subsets #5, #6, #201 and #202 were also present, each in single NOTCH1 mutated CLL case (5.2%). MYD88 mutation occurred in 12/323 (3.7%), of whom one patient was characterized as subset #2 and another as subset #4. MYD88 mutations were nearly equally distributed in patients with mutated/unmutated IGVH status (5 vs. 7). SF3B1 mutations occurred in 17/364 (4.7%) patients, furthermore two of them carrying negative prognostic features of subsets #2 and one subset #1. Patients belonging to subsets #3 and #6 were also present.Certain negative prognostic factors accompany poor clinical outcome. The assessment of median TTFT revealed the significant differences between patients from various prognostic groups (Fig. 1). Patients with unmutated IGHV gene status were characterized by significantly shorter TTFT than patients harbouring the mutation (p<0.0001). The similar correlation occurs in patients with ZAP-70 positive (p=0.04) and CD38 positive (p=0.0003). There were no significant differences in patients with mutated and unmutated NOTCH1 and MYD88, while in patients harbouring SF3B1 mutation the tendency to lower median TFTT was revealed (p=0.08). Interestingly, the significant difference in median TTFT was observed in groups of men and women, showing the better outcome in female patients (median 10 vs 28 months, p=0.01).NOTCH1 and SF3B1 mutations accompany certain biological markers of unfavourable prognosis. Undeniably, the mutations may contribute to the identification of poor-risk CLL patients and in combination with conventional lesions of CLL may be the key to accurate estimation of the disease prognosis.Acknowledgments: This work was supported by Polish Ministry of Science and Higher Education Scientific Grant "The best from the best" ("Najlepsi z Najlepszych") No. 506-0000-39-0000. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

High Peripheral Blood Circulating BDNF Levels Are Associated with Good Prognosis in CLL Patients; A CXCR-4 Dependent Effect?

Introduction: BDNF is a neuronal growth factor that previously showed to exert survival effect in B cells. The role of BDNF in CLL is unknown.Objectives: To study the circulating levels of BDNF in CLL patients and their association with disease prognosis. We also looked for CXCR-4 as a possible mechanisms underlying BDNF effect in CLL.Design and methods: The total BDNF levels and the levels of the BDNF precursor; proBDNF were quantified in the serum of 36 CLL patients and 5 healthy controls by commercial ELISA kits (DY248 and DY3175 DuoSet, respectively, from R&D Systems, Minneapolis, MN, USA). The patients’ BDNF levels were correlated to the disease characteristics and clinical course. mRNA and protein expression of the high affinity receptor for BDNF; TrkB were evaluated by real time PCR and flow cytometry respectively. The effect of BDNF on CXCR4 surface expression and migration of CLL cells towards SDF-1 were studied in-vitro.Results: The total serum levels of BDNF in CLL patients was not statistically different compared to healthy controls (mean±SD; 19.9±15.1ng/ml vs. 10.5±9.5ng/ml respectively, p=0.19). Serum proBDNF levels in both CLL patients and healthy controls were under the detection limit of the kit. Within the CLL group we found higher total BDNF levels in patients with mutated immunoglobulin variable heavy-chain (IGHV) status than in patients with unmutated IGHV (25.2±14.6ng/ml vs. 14.1±13.0 in mutated and unmutated status, respectively, p=0.028). We also found higher serum levels of total BDNF in CLL patients in Binet stage A compared to those with a more advance clinical stage (mean±SD ; 24.5±14.6ng/ml vs. 17.0±16.9 and 10.2±9.6 in A,B and C, respectively, p=0.013). Accordantly, higher BDNF levels were detected in patients who had a clinically stable disease compared to patients who had disease progression and required treatment (23.1±14.7ng/ml vs. 13.4±14.5, respectively, p=0.013). The mRNA levels of the BDNF receptor; TrkB, were twofold lower in CLL cells compared to normal B cells. However, the overall TrkB mRNA transcripts were very low in CLL cells and normal B cells compared to the normalized housekeeping genes of Beta-2-microglobulin and CD19. Likewise, surface expression of TrkB in CLL was undetectable using several commercial monoclonal Abs by flow cytometry. In vitro culture of CLL cells in serum free media for 24h resulted in an increased CXCR4 surface expression and greater apoptosis. Culture of CLL cells in the presence of recombinant BDNF (50ng/ml) resulted in downregulation CXCR-4 surface levels and protection from spontaneous apoptosis, irrespectively of the IGHV mutational status. The effect of BDNF on CXCR4 downregulation was also confirmed at 1h culture as it shows to induce similar effect to SDF-1 (50ng/ml) and additive effect when combined with SDF-1. Finally, possible competition between BDNF and SDF-1 was indicated as pretreatment of CLL cells with BDNF inhibit their migration toward SDF-1.Discussion: Our findings show association between high BDNF levels and favorable disease prognosis in CLL. Undetected TrkB expression in CLL cells is compatible with previous reports showing sequestration of TrkB in normal and malignant B cells and suggests TrkB independent effect of BDNF in CLL. The effect of BDNF on the survival and migration of CLL cells via CXCR-4 needs to be further investigated. DisclosuresNo relevant conflicts of interest to declare.

Predictive Value of Mutation Analysis in the Diagnostic Approach to Patients with Unexplained Cytopenia

▪The diagnostic approach to unexplained cytopenia is hampered by the poor specificity of dysplastic changes that may complicate the distinction between myeloid neoplasms (MN) and non-malignant cytopenias. In the last years, several somatic mutations were identified in MN; however, the diagnostic value of mutation analysis needs to be defined. In this study, we performed a mutation screening in a prospective cohort of patients with unexplained cytopenia undergoing a comprehensive diagnostic work-up, with the aim to estimate the predictive value of somatic mutations.This study included two cohorts: a learning cohort that consisted of 683 consecutive patients investigated for unexplained cytopenia at the Policlinico San Matteo & University of Pavia, Italy, between 2003 and 2015; and a validation cohort, including 190 patients referred as second opinion for suspected MDS. A set of 42 genes was analyzed on DNA from peripheral blood granulocytes using Illumina HiSeq (Illumina Inc., CA, USA). The diagnosis of patients in the learning cohort was MN in 409 cases (233 MDS, 86 MDS/MPN, 35 MPN; 55 AML), other cytopenia in 120 cases, whereas in 154 patients a provisional diagnosis of Idiopathic Cytopenia of Undetermined Significance (ICUS) was adopted. After a median follow-up of 22 months (range 3-136), 38 patients in this category developed a MN (ICUS-MN).The most frequently mutated genes were TET2 (171/683, 25%), ASXL1 (15%), SRSF2 (14%), SF3B1 (11%), DNMT3A (10%), RUNX1 (9%). Significantly higher number of mutations per subject and variant allele frequency (VAF) were observed in MN (n=2, range 0-9; VAF=0.39, 0.03-0.57) compared with ICUS (n=0, 0-7; VAF=0.31, 0.03-0.51) or other cytopenia (n=0, 0-2; VAF=0.06, 0.03-0.44) (P<0.001). Fifty-seven of 409 (14%) MN patients did not carry any mutation in the set of analyzed genes. Among these, 6 had MDS del(5q), while 18 received a diagnosis of MDS based on mild dysplasia with normal karyotype, and showed a 5-year probability of progression of 0%, suggesting that a diagnosis of MDS might have been not appropriate despite WHO criteria. When accounting for these cases, an unmutated status had a negative predictive value for MN of 0.83. Conversely, having two or more mutations had a positive predictive value (PPV) for MN of 0.94. We then performed ROC analyses to explore the cut-off value of VAF with the highest predictive value for MN, and found that a VAF equal or higher than 0.10 had a PPV for having or developing MN of 0.92.Next, we calculated the predictive value for diagnosis of MN of the most frequently mutated genes. In multivariable logistic regression, having two or more mutations (OR=4.07, P<0.001) or carrying SF3B1 mutation (OR=3.56, P=0.016) were independent predictors for MDS or other MN. SF3B1 mutation showed a specificity for myelodysplasia of 0.99, suggesting that this lesion may provide presumptive evidence of MDS even in the absence of definitive morphological features. RNA splicing genes (SF3B1, SRSF2, U2AF1) and RUNX1 had the highest predictive value for MN irrespective of co-occurring mutations, PPVs ranging from 0.88 to 0.93. PPVs increased to 0.97-0.99 when accounting for ICUS-MN as true positive cases. Conversely, PPVs of mutations in genes of DNA methylation (TET2, DNMT3A) and chromatin (ASXL1) as isolated lesions ranged from 0.39 to 0.69, while having one or more co-mutated genes significantly increased PPVs to 0.88-0.96.Among patients with a diagnosis of ICUS, 57 of 154 (37%) carried one or more mutations (Clonal Cytopenia of Undetermined Significance, CCUS). Patients with CCUS showed a significantly higher probability of developing MDS compared with those without evidence of clonality (HR=7.48, 10-year cumulative probabilities of progression: 96% vs 15% respectively, P<.001).Finally, the predictive values of mutation analysis were tested in the independent validation cohort of patients referred to our institution for suspected MDS, and were fully confirmed.In conclusion, selected mutated genes or co-mutation patterns may identify patients with high likelihood of having MDS or other MN. The definition of a category of CCUS allows to recognize with high sensitivity patients who do not fulfill diagnostic criteria but are at high risk of developing MDS. Taken together, these data suggest that mutation analysis on peripheral blood cells may significantly improve current diagnostic approach to patients with unexplained cytopenia. DisclosuresOgawa:Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Kan research institute: Consultancy, Research Funding.

Lack of Prognostic Significance of the Conventional and Novel Prognostic Markers in Trisomy 12 Chronic Lymphocytic Leukemia (CLL)

Background. Trisomy 12 (tris12) is a recurrent cytogenetic abnormality in chronic lymphocytic leukemia (CLL), occurring in approximately 15-20% of cases, often as the unique cytogenetic alteration, that is usually considered a clonal driver lesion occurring early in CLL evolution. In the Dohner hierarchical categorization, tris12 CLL are identified as having an intermediate prognostic risk, although recent reports suggest a more complex and heterogeneous clinical behavior. Compared to CLL lacking this cytogenetic abnormality, tris12 CLL show more atypical morphology and immunophenotype, more frequent expression of the negative prognostic markers CD49d and CD38, and presence of NOTCH1 mutations and an unmutated (UM) IGHV gene status. The increased fraction of tris12 CLL carrying adverse prognostic features is in contrast to the intermediate clinical behavior associated with most tris12 CLL cases.Aim. To perform a comprehensive evaluation of the clinical impact of the major genetic, immunogenetic and immunophenotypic prognostic markers in tris12 CLL.Methods. The study was based on a multicenter series of tris12 CLL defined according to Dohner (n=283, including 73 cases also bearing del13q), and a comparison group (control) of 553 cases with either del13q (n=308) or without any cytogenetic abnormality (no del17p, del11q, tris12, del13q, n=245). Median follow-up of patients in the tris12 and control groups were 4 years (range 0-22) and 7 years (range 0-28), with 54% and 57% treated patients, and 18% and 15% deaths, respectively. Patient characterization included modified Rai stage, CD49d (CD49dhigh, ≥30% positive cells by flow cytometry), CD38 (CD38high, ≥30% positive cells by flow cytometry) and ZAP-70 (ZAP-70high, ≥20% positive cells by flow cytometry) expression, and IGHV mutational status (mutated, M, or UM according to the 2% cutoff). TP53, BIRC3, NOTCH1 and SF3B1 mutations were screened either at diagnosis or before therapy by NGS with at least 1000X coverage and 1% of sensitivity. Groups were compared by chi-square test; overall survival (OS) was computed from diagnosis to death or censored at last observation, and analyzed by Cox regression analysis.Results. Comparing the tris12 and the control groups, median age was 64 years (range 30-92) vs 66 years (range 33-92), male gender 55% vs 56% (p=0.86), the modified Rai stage was early in 52% vs 54%, intermediate in 41% vs 42% and advanced in 7% vs 4% (p=0.20). As previously reported, tris12 CLL were characterized by a higher prevalence of cases expressing CD49d (85% vs 31%) and CD38 (62% vs 17%; all p<0.0001), and of UM IGHV cases (55% vs 25%, p<0.0001). Analysis of recurrent mutations highlighted a higher prevalence of NOTCH1 mutations (26% vs 8%, p<0.0001) and of BIRC3 mutations (21% vs 1%, p<0.0001) in tris12 vs control group CLL. Conversely, no differences were found in the fraction of cases with TP53 mutations (3% vs 4%, p=0.38) or SF3B1 mutations (7% vs 7%, p=0.89), and in cases expressing ZAP-70 (62% vs 52%, p=0.09). The impact of these features on OS was tested by univariate analysis: in tris12 CLL, only the UM IGHV gene status predicted shorter OS (HR=2.37, p=0.0063), while none of the other characteristics reaching statistical significance as OS predictors (CD49d HR=1.36, p=0.36; CD38 HR=0.42, p=0.052; ZAP-70 HR=3.12, p=0.07; TP53 HR=2.33, p=0.25; NOTCH1 HR=1.40, p=0.22; SF3B1 HR=2.05, p=0.17; BIRC3 HR=1.22, p=0.61). On the other hand, in the control cohort, a significantly higher HR was found for CD49d (HR 3.11, p<0.0001) and CD38 (HR 3.45, p<0.0001) expression, TP53 (HR 2.88, p=0.0026), NOTCH1 (HR 3.57, p<0.0001), and SF3B1 (HR 2.57, p=0.0038) mutations, as well as for the UM IGHV gene status (HR=2.81, p<0.0001), but not for ZAP-70 expression and BIRC3 mutations (HR=1.74 and HR=1.91, p=0.15 and p=0.37, respectively).Conclusions. Mutational status of IGHV genes was the sole prognostic factor able to stratify OS in tris12 CLL. Despite the high frequency of NOTCH1 and BIRC3 mutations, as well as of CD49d and CD38 overexpression, these markers failed to convey a prognostic risk in tris12 CLL. The lack of a significant clinical impact for TP53 and SF3B1 mutations might be partly explained by the low number of mutated cases combined with a relative short follow up in our tris12 cohort. These findings are in keeping with the hypothesis of a different patho-biological mechanism occurring in tris12 CLL, which however remains to be fully elucidated. DisclosuresD'Arena:Janssen-Cilag: Honoraria. Rossi:Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria. Gaidano:Janssen: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Shanafelt:Genentech: Research Funding; Janssen: Research Funding; Celgene: Research Funding; GlaxoSmithkKine: Research Funding; Pharmacyclics: Research Funding; Cephalon: Research Funding; Hospira: Research Funding.

The B-Cell Receptor Signaling Inhibitor Molecules CD305 and CD307b Are Markers of Favorable Prognosis in Chronic Lymphocytic Leukemia with Both Mutated and Unmutated IGHV Gene Status

Background. The regulation of the B cell receptor (BCR)-mediated response to antigenic stimulation in chronic lymphocytic leukemia (CLL) is balanced by competing activating and inhibitory signals derived from specific co-receptors. In particular, the BCR inhibitory molecules Leukocyte associated Ig like receptor 1 (LAIR-1)/CD305 and human Fc receptor-like 2 (FCRL2)/CD307b have been independently reported as molecules associated with longer time to first treatment in CLL, although their synergic functional role and combined prognostic relevance remains to be explored. Aim. To assess the functional role and prognostic value of CD305 and CD307b as OS predictors in CLL. Methods. The study included 467 CLL cases all characterized at diagnosis for Rai stage (stages 0-I: 355 cases), CD49d expression (CD49d- CLL, <30% of positive cells by flow cytometry: 266), IGHV mutational status (mutated, M: 270), karyotype abnormalities according to the hierarchical stratification (normal/13q-/+12: 365). Median follow-up of patients was 70 months with 82 deaths. Immunophenotypic analysis was performed using a combination of anti-CD5 FITC, -CD19PE-Cy7, -CD305PerCPCy5.5, CD307bAPC mAbs. For functional assays, mouse anti-human IgM, and agonistic mouse anti-human CD305 and CD307b were used. Fab goat anti-mouse was used as cross linker. Phosphorylation state of ERK (pERK) and ATK (pATK) were evaluated by either flow cytometry or western blotting. Calcium flux was analysed by flow cytometry. Results.A significantly higher (p<0.0001) CD305 and CD307b expression in M versus UM CLL was documented, with mean % of expression of 58% vs. 33% (CD305), and 88% vs. 74% (CD307b). The best cut-off levels for OS, calculated using a ROC analysis, were 10% for CD305 and 85% for CD307b. Using these cut-offs, 331 (70.9%) and 296 (63.4%) were classified CD305+ and CD307bbright, respectively. The clinical impact of both markers as OS predictors was confirmed in both univariate (hazard ratio/confidence interval (HR/CI)= 0.39/0.25-0.60; p<0.0001 for CD305; HR/CI= 0.26/0.16-0.41; p<0.0001 for CD307b), and multivariate (HR/CI=0.41/0.26-0.63; p=0.0001 for CD305;HR/CI=0.27/0.17-0.42; p<0.0001 for CD307b) analyses. Therefore, we dichotomized CLL cases according to the expression of 2 markers (n=220) versus 0/1 markers (n=247). The prognostic impact of this combined markers expression was tested in univariate analysis (HR/CI=0.23/0.13-0.40; p<0.0001 ) and in a multivariate model including: IGHV mutational status, CD49d expression, Rai stage (stage 0-I versus stages II-IV), karyotype abnormalities (normal/del13/+12 versus del11/del17). The combined markers expression retained its prognostic impact (HR/CI=0.37/0.21-0.68; p=0.0013), along with the UM IGHV (HR/CI=2.54/1.51-4.26; p=0.0004), CD49d expression (HR/CI=1.83/1.13-2.97; p=0.015) del11/del17 (HR/CI=1.86/1.14-3.05; p=0.014). Consistently, expression of 2 markers identified subsets with longer OS in the context of both M (p=0.009) and UM CLL (p=0.004; see Figure). To functionally explain the peculiar clinical behavior of CLL expressing these molecules, we evaluated CLL cell response to BCR triggering in CD305+/CD307bbright CLL, with either a M (n=9) or a UM (n=7) IGHV gene status. A significant increase of anti-IgM response was found in 24-hour cultures compared to 2-hour cultures, irrespective of the IGHV mutational status: p-ERK, 41.2% vs 20% (p=0.006); pAKT, 18% vs 7% (p=0.037); calcium flux, mean AUC 2.0x106 vs 1.6x106 (p=0.01). This increased BCR response was paralleled by a significant down-regulation of both CD305 and CD307b after 24-hour culture compared to 2-hour culture (mean MFI 40 vs 92.5 for CD305, p=0.0009; 95.5 vs 414 for CD307b, p=0.001) with no difference between M and UM cases. We next tested the effects on BCR signaling of anti-CD305 and anti-CD307b ligation (n=4). While BCR engagement induced pERK (mean fold increase compared to the control=12), concomitant engagement of BCR and CD305 or CD307b reduced the pERK level to 68% or 40%, respectively. Moreover, simultaneous engagement of both CD305 and CD307b further inhibited pERK level to 28%. Conclusions.A CD305+/ CD307bbright phenotype predicts longer OS in CLL, in both M and UM IGHV CLL. The synergic effect of the B-cell receptor signaling inhibitors CD305 and CD307b may functionally explain this peculiar clinical behavior. [Display omitted] DisclosuresChiarenza:Gilead: Consultancy; Janssen: Consultancy; Roche: Consultancy.

Prognostic Impact of the New CLL-IPI Index in a Single Center CLL Spanish Cohort

The hallmark for Chroniclymphocytic leukemia (CLL) is a highly variable clinical course. An international prognostic index (CLL-IPI) is a promising tool to improve the precision of prognostic counseling and to identify patients who deserve closed monitoring. The CLL-IPI is a risk-weighted model comprising the risk factors age, stage, del(17p)/TP53 mutation, IGHV mutation status, and β2-microglobulin (β2-M)(LancetOncol 2016).The aims of the study were 1) to determine the IGHV status as well as the IGHV repertoire 2) to assess/validate the applicability of the CLL-IPI in general practice.We included all patients diagnosed of CLL according to the National Cancer Institute Working Group guidelines in our institution between 1986 and 2014 who had at least a 24 months follow-up. Amplification and sequence analysis of IGH rearrangements were performed on either DNA or cDNAusing the BIOMED-2 protocol. Sequence data were analyzed using the IMGT database and tools. Clinical and biological data were extracted from medical records and included age, stage, CD38 and ZAP-70 expression, serum LDH, β2-M, cytogenetics and lines of treatment. Overall survival (OS) was calculated from diagnosis to last follow-up or death, time to first treatment (TFT) from diagnosis to first treatment administration or last follow-up.209 CLL patients were originally included but complete data to calculate the CLL-IPI was only available in 176 pts. Median age of the series was 65 years (range, 33 to 92), and a slight male predominance 102 (58.5%). The main clinical characteristics are detailed in Table 1. Median follow-up of patients was 71.5 months (range, 24-315).We identified 105/209 patients (50%) with unmutated IGHV. Somatic mutations among IGHV gene subgroups display a hierarchy of mutations (IGHV3>IGHV1>IGHV4). Among the functional IGHV genes, the most frequently encountered were IGHV1-69 (31; 14.6%). It was the most recurrently used in the unmutated group.The most represented IGHV gene within the mutated subset was IGHV4-34, which was used in 15 cases (7.1%). We have observed 39 IGHV genes. The most frequents are showed in Figure1.As previously described, patients with unmutated status showed a higher expression of CD38 and ZAP-70, unfavorable cytogenetics and a higher proportion of treated patients. The CLL-IPI index identified four groups of patients: low risk (0-1 points) n=74 (42%), intermediate (2-3) 67 (38.1%), high (H) (4-6) 29 (16.5%) and very high (VH) 6 (3.4 %). The 5-year OS and 5-year TFT of the CLL-IPI risk groups differed significantly (p< 0.0001, log-rank test) between the low (OS 92.2%, TFT 74.1%), intermediate (OS 83.2%, TFT 34.6%) and high-very high groups (OS 61.5%, TFT 22.8%). We only identified 6 patients (3.4%) with a VH, with no difference in terms of OS and TFT between the VH and high (H) risk groups, probably due to the small number of patients. When we considered the H and VH altogether, the CLL-IPI identified three groups with significantly different TFT and OS (Figure 2)In summary, in our cohort the frequencies of the IGHV genes used in BCR rearrangements were similar to those described in the Mediterranean area and confirm a geographical-dependent leukaemic repertoire.We confirm that the CLL-IPI is a useful tool for real-life practice as it identifies three risk groups with significantly different time to first treatment and overall survival curves. In our experience, the CLL-IPI applied to the whole CLL population at diagnosis discriminates a smaller proportion of patients in the high (16.4%) and very high groups (3.4 %) compared to the original training cohort based on treated patients included in clinical trials. Our results are closely similar to the MAYO cohort that included patients consecutively diagnosed and observed. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Low Bax/Bcl-2 Ratio and NOTCH1 Mutations Represent Powerful and Synergistic Adverse Prognostic Factors within Trisomy 12 Chronic Lymphocytic Leukemia (CLL)

Trisomy 12 is the third most common cytogenetic abnormality in CLL with several distinguishing features including abormal morphology, high prevalence of NOTCH1 mutations (NOTCH1 M) and increased expression of the alpha-integrins CD11a and CD49d (Balatti, 2012; Zucchetto, 2013). Trisomy 12 marks a disease subset characterized by high rates of cell proliferation, disease progression and Richter syndrome transformation (Rossi, 2013). Noteworthy, NOTCH1 M implicate constitutive activation of NOTCH1 signaling which triggers apoptosis resistance and increased survival of CLL cells (Rosati, 2009). Moreover, bax/bcl-2 ratio (bax/bcl-2), marker of apoptosis, predicts chemoresistance and progressive disease in CLL (Del Principe, 2016). The today availability in clinical use of venetoclax (ABT-199), a potent oral anti-bcl-2 peptidomimetic (Seymour, 2014), prompted us to analyze the real impact of the apoptosis (bax/bcl-2) on trisomy 12 CLL prognosis. The primary aims of our research were: 1) to demonstrate a significant association between NOTCH1 M and bax/bcl-2; 2) to correlate NOTCH1 M and bax/bcl-2 with biological and clinical prognosticators; 3) to determine progression-free survival (PFS) and overall survival (OS) upon NOTCH1 M and bax/bcl-2; 4) to evaluate NOTCH1 M and bax/bcl-2 as independent prognostic factors. Therefore we investigated 653 CLL patients, median age 66 years, 363 males and 290 females. Using the 5% cutoff for the FISH cytogenetic abnormalities, 140 (21.4%) cases were classified as trisomy 12 CLL. Bax/bcl-2 was calculated by flow cytometry, dividing mean florescence intensity (MFI) of bax by MFI of bcl-2 on CLL cells. The threshold was set at the median value >1.5 (range 0.32-5.30). The presence of NOTCH1 M was investigated with ARMS PCR for c.75447545delCT and by Sanger sequencing of NOTCH1 exon 34. CD49d was >20% in almost all trisomy 12 pts (116/140; 83%). Forty-five patients were NOTCH1 M (32.1%) and 45 were bax/bcl-2 positive (32.1%). There was a very strong correlation between bax/bcl-2 <1.5 and NOTCH1 M (42/45; p<0.0001), corroborating the close dependence of the lacking apoptosis on NOTCH1 M. Both lower bax/bcl-2 and NOTCH1 M were significantly associated with lymphocyte doubling time <12 months (p=0.0077 and p=0.0006), with beta-2 microglobulin >2.2 mg/dl (p=0.0001 and p=0.0007) and LDH >300 U/L (p=0.004 and p=0.00001), thus confirming their significant association both with a high proliferative rate and a high tumor burden. Strong correlations were found between NOTCH1 M or bax/bcl-2 <1.5 and IGHV unmutated status (p<0.0001 and p=0.0005) or ZAP-70 >30% (p<0.0001 and p=0.00002). With regard to clinical outcome, significant shorter PFS and OS were observed in patients with NOTCH1 M vs NOTCH1 wild type [WT] (0% vs 27% at 10 years, p=0.001 and 26% vs 78% at 14 years, p=0.005) or lower bax/bcl-2 (9% vs 38% at 10 years, p=0.0007 and 45% vs 89% at 14 years, p=0.002). Interestingly, NOTCH1 M and bax/bcl-2 showed synergistic prognostic properties, since NOTCH1 M and bax/bcl-2 <1.5 identified a trisomy 12 CLL subset at worst prognosis with regard to PFS (0% vs 40% at 10 years, p=0.00004, Figure) and OS (20% vs 100% at 14 years, p=0.0001, Figure). Therefore, bax/bcl-2 and NOTCH1 M are powerful prognosticators, showing synergistic clinical effects. In multivariate analysis of OS, bax/bcl-2 (p=0.004) together with age (p=0.0006), CD49d (p=0.01) and IGHV status (p=0.03) was confirmed as an independent prognostic factor. In conclusion, the modern strategies to trigger apoptosis and block proliferation, such as venetoclax, may be very effective in this CLL subset, also taking into account that trisomy 12 CD49d+ CLL has abbreviated lymphocytosis with retention of CLL cells within tissues (Thompson, 2014) and consequent poor reduction of lymphadenopathy during treatment with ibrutinib. [Display omitted] DisclosuresLo Coco:Teva: Consultancy, Honoraria, Speakers Bureau; Lundbeck: Honoraria, Speakers Bureau; Novartis: Consultancy; Baxalta: Consultancy; Pfizer: Consultancy.

Extensive next-generation sequencing analysis in chronic lymphocytic leukemia at diagnosis: clinical and biological correlations.

BackgroundIn chronic lymphocytic leukemia (CLL), next-generation sequencing (NGS) analysis represents a sensitive, reproducible, and resource-efficient technique for routine screening of gene mutations.MethodsWe performed an extensive biologic characterization of newly diagnosed CLL, including NGS analysis of 20 genes frequently mutated in CLL and karyotype analysis to assess whether NGS and karyotype results could be of clinical relevance in the refinement of prognosis and assessment of risk of progression. The genomic DNA from peripheral blood samples of 200 consecutive CLL patients was analyzed using Ion Torrent Personal Genome Machine, a NGS platform that uses semiconductor sequencing technology. Karyotype analysis was performed using efficient mitogens.ResultsMutations were detected in 42.0 % of cases with 42.8 % of mutated patients presenting 2 or more mutations. The presence of mutations by NGS was associated with unmutated IGHV gene (p = 0.009), CD38 positivity (p = 0.010), risk stratification by fluorescence in situ hybridization (FISH) (p < 0.001), and the complex karyotype (p = 0.003). A high risk as assessed by FISH analysis was associated with mutations affecting TP53 (p = 0.012), BIRC3 (p = 0.003), and FBXW7 (p = 0.003) while the complex karyotype was significantly associated with TP53, ATM, and MYD88 mutations (p = 0.003, 0.018, and 0.001, respectively). By multivariate analysis, the multi-hit profile (≥2 mutations by NGS) was independently associated with a shorter time to first treatment (p = 0.004) along with TP53 disruption (p = 0.040), IGHV unmutated status (p < 0.001), and advanced stage (p < 0.001). Advanced stage (p = 0.010), TP53 disruption (p < 0.001), IGHV unmutated status (p = 0.020), and the complex karyotype (p = 0.007) were independently associated with a shorter overall survival.ConclusionsAt diagnosis, an extensive biologic characterization including NGS and karyotype analyses using novel mitogens may offer new perspectives for a better refinement of risk stratification that could be of help in the clinical management of CLL patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0320-z) contains supplementary material, which is available to authorized users.

SLP76 integrates into the B-cell receptor signaling cascade in chronic lymphocytic leukemia cells and is associated with an aggressive disease course.

SLP76 is ectopically expressed in chronic lymphocytic leukemia cells where it plays a role in B-cell receptor signaling.

Impact of IgVH mutational status in patients with chronic lymphocytic leukemia with isolated good and intermidiate risk genetic aberrations.

7534Background: Genetic abnormalities in chronic lymphocytic leukemia (CLL) have prognostic impact. The presence of trisomy 12 (T12) and normal karyotype (NK), and 13q deletion (del13q) are associated with an intermediate and good risk profile in CLL, respectively (Dohner, 2000). The unmutated (unmut) status of the immunoglobulin variable heavy chain gene (IgVH) correlates with survival in sole del13q CLL (Gladstone, 2011). The prognostic relevance of IgVH mutational status in intermediate risk genetic variants (T12 and NK) has not been described. We investigated the impact of the IgVH mutational status in CLL patients (pts) with isolated Del13q, T12 or NK in a large cohort. Methods: From January 2000 to January 2013 we identified 1267 CLL patients using the Moffitt Cancer Center and Total Cancer Care databases. Descriptive data was reported in patients with both unmut ( 2% from the germline sequence). Overall survival (OS) was estimated using the ...

Combination of bendamustine and rituximab as front-line therapy for patients with chronic lymphocytic leukaemia: multicenter, retrospective clinical practice experience with 279 cases outside of controlled clinical trials

Recently, encouraging results in terms of safety and efficacy have been obtained using bendamustine–rituximab (BR) in untreated chronic lymphocytic leukaemia (CLL) patients enrolled in a phase II study. Here, we report a retrospective international multicenter study of CLL patients treated with BR as front-line therapy.The cohort included 279 patients with progressive CLL from 33 centers (29 Italian, 3 Israeli and 1 German) who received at least 1 cycle of BR as first-line treatment during the 2008–2014 period. The primary objective of this study was to evaluate the efficacy and safety of BR administered as front-line therapy, outside of controlled clinical trials.Median age was 70 years (range, 43–86 years); 62.4% were males and 35.8% had Binet stage C. Forty-two patients (15.2%) were unfit (cumulative illness rating scale [CIRS] score ≥7), and 140 (50.2%) had creatinine clearance ≤70 ml/min. Fluorescent in situ hybridisation analysis, available for 192 cases, showed that 21 (10.9%) had del11q and 18 (9.4%) del17p. The overall response rate (ORR) was 86.4%, with a complete remission rate of 28%. Patients with del17p had an ORR of 66.7%. After median follow-up of 24 months, the 2-year progression-free survival (PFS) was 69.9%; CIRS ≥7, immunoglobulin heavy-chain variable-region (IGHV) unmutated status, del17p and BR dose intensity <80% were independently associated with shorter PFS. Grade III or IV neutropenia, thrombocytopenia, and anaemia were observed in 25.9%, 15.4%, and 15.1% of patients, respectively. Twenty-four patients (8.6%) had severe infections.BR is also an effective and safe regimen for untreated CLL patients, outside of controlled clinical trials.

Evans syndrome secondary to chronic lymphocytic leukaemia: presentation, treatment, and outcome.

Evans syndrome (ES) is defined by the combination (either simultaneous or sequential) of immune thrombocytopenia (ITP) and autoimmune haemolytic anaemia (AIHA). When related to secondary conditions, ES may arise in patients with chronic lymphocytic leukaemia (CLL), which is frequently associated to autoimmune cytopenias (AIC). We analysed 25 patients with ES secondary to CLL, which were identified from a large series of consecutive patients with CLL, diagnosed and followed up in two institutions. They represented 2.9% of the whole series. Thirteen patients presented with concurrent ITP and AIHA (simultaneous ES), while others developed the two AIC sequentially. Occurrence of ES was associated with unfavourable biological prognostic factors like ZAP-70 expression, unmutated immunoglobulin heavy chain variable region gene status, 17-p13 deletion and TP53 gene mutations. Of note, the majority of patients with ES (66%) had stereotyped B cell receptor configuration. Most patients had short-lasting remissions and required second-line treatments to control the autoimmune manifestations of ES. Patients with ES were associated with inferior survival compared to patients not developing AIC, especially when ES developed early in the course of CLL, although the reduced survival was not confirmed by multivariate analysis. In conclusion, ES secondary to CLL is a difficult-to-treat complication, characterised by adverse biological features and clinical outcome.

Prognostic Impact and Risk Factors of Reducing Prescribed Doses of Fludarabine, Cyclophosphamide and Rituximab (FCR) during Frontline Treatment of Chronic Lymphocytic Leukemia (CLL)

Prognostic Impact and Risk Factors of Reducing Prescribed Doses of Fludarabine, Cyclophosphamide and Rituximab (FCR) during Frontline Treatment of Chronic Lymphocytic Leukemia (CLL)

Elevated Basal Autophagy in SF3B1 Mutated Myelodysplastic Syndromes: Relationship with Survival Outcomes and Therapeutic Implications

About 60-80% of patients with myelodysplastic syndromes (MDS) manifest with anemia. Red blood cell (RBC) transfusions are the most commonly used therapy to alleviate anemia in patients that are ineligible for other curative approaches. Transfusion-dependent patients frequently develop iron overload, which correlates with infections, mortality, leukemia, and organopathy. At the subcellular level, long-term iron exposure produces iron-catalyzed hydroxyl radicals that induce oxidative damage to mitochondria and disrupt bioenergetic homeostasis. The use of iron-chelating drugs to counter transfusion-related iron overload remains controversial due to the significant side effects that these agents cause. New therapies that effectively address iron overload in transfusion-dependent MDS patients are clearly needed. Mitochondrial dysfunction frequently occurs during MDS cell maturation and leads to abnormal iron distribution. However, the mechanistic basis of this biological phenomenon has not been rigorously studied. We previously linked the presence of Splicing factor 3b subunit 1 (SF3B1) mutations, which are frequent in patients with refractory anemia with ring sideroblasts (RARS), with abnormalities in mitochondrial iron. Transmission electron microscopy and flow cytometry showed that mitochondria of SF3B1 mutant RARS patients have higher iron content than those of wild type (WT) RARS patients and expressed an increased mRNA level of the iron transporter, Mitoferrin 1. Despite these prevalent mitochondrial abnormalities and transfusional dependence, SF3B1 mutant lower-risk MDS patients experienced significantly longer median survival compared to SF3B1 WT lower-risk MDS patients (34 mos vs. 13 mos; P = .002; N=16 vs. 101). Autophagy is an evolutionarily conserved lysosomal mechanism of protein degradation that plays a critical role in the elimination of damaged mitochondria and other organelles. We hypothesized that autophagic clearance of defective mitochondria may contribute to the superior survival of SF3B1 mutant patients suffering from transfusion-mediated iron overload. We conducted RNA sequencing analyses on a group of fresh bone marrow (BM) cells of SF3B1 mutant and WT RARS patients and healthy donors (n = 11) to investigate the basal autophagy status in this distinct patient population. In addition to confirming increased levels of mitochondrial transporters such as Mitoferrin 1 and 2 (FC = 2.0), we detected a striking increase in multiple genes involved in the proximal and distal regulation of autophagy in cells of SF3B1 mutant RARS compared to WT RARS patients. Genes controlling the early stages of autophagy including the protein kinases ULK1 (FC = 2.0) and ULK3 (FC = 3.9; P=.05), ATG complexes ATG2A/B (FC = 1.9), ATG9A (FC = 5.5; P=.05), ATG18 (FC = 4.8; P=.02) and the cysteine proteases ATG4A/C (FC = 2.0) were all elevated in SF3B1 mutant RARS vs. SF3B1 WT RARS patients. Key components of the late stages of the autophagic degradation cascade, including multiple members of the cathepsin family of lysosomal proteases [CTSL1: FC = 20.9; CTSD: FC = 5.8 (P=.06); CTSB: FC = 2.1; CTSE: FC = 5.9 (P=.01); CTSD: FC = 1.8], were also significantly increased. qRT-PCR confirmed higher expression levels in the BM cells of RARS patients carrying sole SF3B1 mutations compared to cells of SF3B1 WT RARS patients. The link between SF3B1, mitochondrial iron, and elevated autophagy was specific as evidenced by the unmutated status and lack of significant mRNA changes in any other splicing factor genes including PRPF8. Our data demonstrate that autophagy may play an important, previously unreported role in SF3B1 mutant RARS. Based on our findings, we hypothesize that SF3B1 mutant RARS cells stimulate autophagy to eliminate damaged mitochondria and alleviate iron overload and that further stimulation of autophagy will diminish the pathogenic effects of chronic transfusions. We are currently investigating the therapeutic benefit and pharmacodynamics of autophagy-modulating drugs (temsirolimus, metformin, arsenic trioxide) in in vitro (primary cells) and in vivo (SF3B1 haploinsufficient mice) models of MDS to facilitate the design of an investigator-initiated clinical trial that will test autophagy modulation as a new precision strategy for the treatment of transfusion-dependent patients with low-risk MDS carrying SF3B1 mutations. DisclosuresSekeres:TetraLogic: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Carew:Boehringer Ingelheim: Research Funding.