Prognostic Impact of NOTCH1 Mutations in Chronic Lymphocytic Leukemia (CLL): A Study On 538 Patients.

Abstract Abstract 2873 Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Recurrent activating mutations of NOTCH1 have been reported, including the relevance of NOTCH1 mutations as independent negative prognostic marker (Rossi et al., Blood 2012). Aims: 1. Determine the frequency and prognostic impact of NOTCH1 mutations (NOTCH1 mut) in a large unselected cohort of adult CLL patients. 2. Evaluate the range of mutational burden by amplicon deep-sequencing. Patients and Methods: We investigated 538 patients (189 female, 349 male; median age: 66.1 years, range: 37.8 – 90.5 years) with untreated CLL by FISH and for NOTCH1 mut (Transcript ID ENST00000277541) using next-generation amplicon deep-sequencing (454 Life Sciences, Branford, CT). Additionally, TP53 (n=45 mut/523 screened, 8.6%) and IGHV mutation status were analyzed. IGHV status was unmutated in 39.2% (209/533) and mutated in 60.8% (324/533) cases. Since NOTCH1 mut in CLL are known to be located predominantly within the C-terminal PEST domain (Rossi et al., Blood 2012) we sequenced exons 33–34 (covering codons 2029 to 2556), represented by 7 distinct PCR reactions with a median amplicon length of 345 bp. In median, 608 bidirectional reads (range 140–2,117) were generated per amplicon, thereby allowing a sensitive detection of variants, i.e. at a cut-off value of 5% ∼30 independent reads were sequenced. Results: All patients were investigated by FISH: del(17p) (30/538, 5.6%), del(11q) (57/538, 10.6%), +12 (103/538, 19.1%), del(6q) (8/538, 1.5%), normal karyotype according to FISH (NK) (111/538, 20.6%) and del(13q) as sole abnormality (229/538, 42.6%). In total, 81 NOTCH1 mut were observed in 71/538 (13.2%) patients. The vast majority of mutations (98.8%) were found to be heterozygous, only 1/81 mutation (1.2%) was homozygous. We identified 23 point mutations (6 missense and 17 nonsense; 28.4%) and 58 frame-shift alterations (57 deletions and 1 indel; 71.6%). The most frequently occurring mutation was as previously described p.Pro2514ArgfsX4 (c.7541_7542delCT), which was identified in 51/81 (62.7%) variants. The median mutational burden as assessed by deep-sequencing read counts was 28% of sequence reads carrying the mutation (range: 2% - 69%). Of note, in 54/81 (66.7%) variations the detected mutation load was ≤20% and therefore would be below the detection level of Sanger sequencing. In detail, a mutational burden ≤20% was observed in 32/81 (39.5%) variations and ≤10% in 22/81 (27.2%) mutations. 10/71 (14.1%) NOTCH1 mut patients carried 2 mutations. In 9/10 patients a different mutational load between the 2 NOTCH1 mut was detected, indicating the presence of 2 independent clones or clonal evolution with acquisition of a second mutation in the initially NOTCH1 single mutated clone. Mutations mainly clustered in the C-terminal part, i.e. codons 2,385 to 2,555 of exon 34 where 72/81 (88.8%) alterations were located. Confirming published data, statistical analyses revealed NOTCH1 mut being associated with unmutated (unmut) IGHV status (unmut vs mut: 59/209, 28.2% vs 11/324, 3.4%; P<0.001), TP53 mut (mut vs unmut: 10/45, 22.2% vs 58/478, 12.1%, P=0.064) and +12 as sole cytogenetic aberration (+12 sole vs remainder: 23/64, 35.9% vs 48/472, 10.2%; P<0.001). We did not detect any difference in NOTCH1 mut frequency between cases harboring +12 sole and +12 with other aberrations (+12 sole vs +12: 23/64, 35.9% vs 13/45, 28.9%; P=0.54). In contrast, NOTCH1 mut were rare events in patients with del(13q) (del(13q) vs remainder: 24/296, 8.1% vs 47/240, 19.6%; P<0.001). No associations with other cytogenetic subgroups were detected. Univariable cox regression analyses revealed an adverse prognostic impact for NOTCH1 mut (P=0.056) and IGHV unmut (P<0.001). With respect to patients of the favorable prognostic risk group (IGHV mut, TP53 unmut, n=146), NOTCH1 mut patients (n=9) showed a significantly shorter time to treatment (TTT) than NOTCH1 wild-type cases (n=137) (median TTT n.r. vs. 9.4 years, P=0.042). Conclusion: 1. We present the first deep-sequencing study of NOTCH1 mutations in a large unselected CLL cohort and report an overall frequency of 13.2%. 2. The mutational burden of 66.7% of NOTCH1 mutations in CLL patients was ≤20%. 3. NOTCH1 mutations are an adverse prognostic parameter associated with shorter TTT and represent yet another novel important biomarker in CLL. Disclosures: Weissmann: MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment.

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Translocations Involving the IGH@locus Occur In 3.7% of Chronic Lymphocytic Leukemia and Are Associated with Unmutated IGHV Status and a Shorter Time to Treatment: A Study on 2,135 Cases

Blood ◽

10.1182/blood.v116.21.3596.3596 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3596-3596

Author(s):

Claudia Haferlach ◽

Frank Dicker ◽

Susanne Schnittger ◽

Wolfgang Kern ◽

Torsten Haferlach

Keyword(s):

Multivariate Analysis ◽

Regression Analysis ◽

Chronic Lymphocytic Leukemia ◽

Cox Regression ◽

Lymphocytic Leukemia ◽

Employment Equity ◽

Mutation Status ◽

Cox Regression Analysis ◽

Igh Locus ◽

Equity Ownership

Abstract Abstract 3596 Introduction: Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a variable clinical course and a large spectrum of treatment options. Based on FISH data, a prognostic classification system has been established with 13q deletions as sole abnormality associated with a favorable prognosis and 17p and 11q deletions correlating with an unfavorable outcome. Recently, the combined evaluation of FISH data, IGHV mutation status and chromosome banding analysis (CBA) revealed that the impact of distinct genetic parameters differs with respect to overall survival (OS) and time to treatment (TTT). Thus far only few data is available on less frequent genetic abnormalities such as 14q deletions and translocations involving the IGH@ locus (tIGH). Therefore, we analyzed CLL with tIGH in detail with respect to frequency, partner genes and impact on prognosis. Methods/Patients: 78 CLL cases with tIGH were identified from 2,135 CLL sent to our laboratory for diagnostic work-up. All cases had been evaluated by immunphentotyping, FISH and CBA. Result: The most frequent tIGH was t(14;19)(q32;q13) (BCL3, n=21) followed by t(14;18)(q32;q21) (BCL2, n=19), t(8;14)(q24;q32) (CMYC, n=7) and t(11;14)(q13;q32) (CCND1, n=6). In the remaining 25 cases 5 recurrent translocations (t(2;14)(p13;q32), n=3; t(4;14)(p16;q32), FGFR3, n=2; t(11;14)(p15;q32), n=2; t(14;17)(q32;q25), n=2; and t(7;14)(q21;q32), n=2) were observed while the remaining 14 translocations were identified in single cases only. In 9/78 cases (11.5%) the tIGH was the sole abnormality. Recurrent additional chromosome abnormalities were +12 (n=7), del(13q) (n=9), del(11q) (n=3). A 17p deletion was observed in 1 case. In two cases tIGH was present only in a subclone and was a secondary abnormality occurring in addition to an del(11q) and a +12, respectively. CLL with tIGH were compared to 401 CLL without tIGH comprising all other genetic subgroups (subdivided according to Döhner et al.: del(17p) n=26, del(11q) n=42, +12 n=42, “normal” n=88, del(13q) sole n=177 and del(14q) n=26). An unmutated IGHV status was more frequent in CLL with tIGH as compared to all others (26/46 (54.3%) vs 128/353 (36.3%); p=0.023). For 53 cases with tIGH and all cases of the non-tIGH cohort clinical follow-up data was available. Median OS was 143.8 months (mo) in CLL with tIGH and 72.9 mo in patients with del(17p) while it was not reached in all other subgroups. In Cox regression analysis only del(17p) and mutated IGHV status were significantly associated with OS (p<0.0001, relative risk (RR)=7.0; p=0.014, RR=0.38). Median TTT was as follows: total cohort: 60.9 mo; tIGH: 27.8 mo; del(17p): 58.9 mo; del(11q): 19.7 mo; +12: n.r.; “normal” 63.9 mo; del(13q) sole: 83.0 mo and del(14q): 21.0 mo. In univariate Cox regression analysis the following parameters were significantly associated with shorter TTT: tIGH (p=0.004, RR=1.82), del(11q) (p<0.0001, RR=2.55), and del(14q) (p=0.007, RR=2.1), while del(13q) sole and mutated IGHV status were associated with longer TTT (p<0.0001, RR=0.40; p<0.0001, RR=0.23). In multivariate analysis including tIGH, del(11q), del(14q) and del(13q) sole all parameters retained their impact on TTT. However, if IGHV mutation status was included in the model only the mutated IGHV mutation status retained an impact on TTT (p<0.0001, RR=0.26). Next, patients with tIGH were subdivided according to their partner genes. Median OS was not reached in all subgroups, while median TTT was as follows: t(11;14): 101.2 mo, t(14;18): 47.9 mo, t(14;19): 11.0 mo, t(8;14): 18.5 mo and other partner genes: 27.8 mo. In univariate Cox regression analysis only t(14;19) was significantly associated with shorter TTT (p<0.001, RR=3.1). Including t(14;19) into multivariate analysis revealed a significant impact of both mutated IGHV mutation status and t(14;19) on TTT (p<0.0001, RR=0.286; p=0.004, RR=3.60). Conclusion: Translocations involving the IGH@ locus occur at low frequency in CLL. They are associated with unmutated IGHV status and a shorter TTT. TTT is especially short in cases with t(14;19). The prognostic impact of t(14;19) is independent of IGHV mutation status. In contrast CLL with t(11;14) and t(14;18) are neither associated with shorter OS nor shorter TTT. This data supports the application of CBA in CLL in order to identify all clinically relevant chromosomal aberrations, including those not detected by routine FISH analysis. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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High-Throughput Sequencing For The Identification Of NOTCH1 mutations In Early Stage Chronic Lymphocytic Leukemia: Biological and Clinical Implications

Blood ◽

10.1182/blood.v122.21.1622.1622 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1622-1622

Author(s):

Marta Lionetti ◽

Sonia Fabris ◽

Giovanna Cutrona ◽

Luca Agnelli ◽

Carmela Ciardullo ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Allele Frequency ◽

Deep Sequencing ◽

Mutant Allele ◽

Sanger Sequencing ◽

Early Stage ◽

Lymphocytic Leukemia ◽

Mutant Allele Frequency ◽

Mutational Activation ◽

Notch1 Mutations

Abstract NOTCH1 mutations have recently emerged as new genetic lesions significantly correlated with survival in chronic lymphocytic leukemia (CLL). NOTCH1 c.7541_7542delCT is by far the most frequently observed NOTCH1 mutation in the disease. To estimate the prevalence and clonal evolution of NOTCH1 c.7541_7542delCT mutation, and prospectively investigate its clinical significance in early stage CLL and clinical monoclonal B cell lymphocytosis (cMBL), we analyzed by next generation sequencing (NGS) 384 cases at diagnosis enrolled in the GISL O-CLL1 multicenter trial. The patient cohort included 100 cMBL and 284 Binet stage A CLL cases, 48 of whom were also longitudinally investigated at progression or during follow-up (32 and 16, respectively) in absence of treatment. Deep sequencing of the NOTCH1 mutation hotspot was performed by Roche 454 pyrosequencing on the Genome Sequencer Junior instrument. NOTCH1 mutation was validated by an extremely sensitive PCR-based approach and Sanger sequencing. The association between NOTCH1c.7541_7542delCT and clinical, molecular and biological variables, as well as its impact on progression free survival (PFS), were tested. Deep sequencing analysis of NOTCH1 mutation hotspot in our cohort (median depth of coverage 1510x, ranging from 605 to 2842) revealed a mutant allele frequency ranging from 0.02% to 75% of total reads in 145 cases. The occurrence of the mutation was subsequently assessed by an extremely sensitive ARMS (ampliﬁcation refractory mutation system)-PCR, which allowed to confirm the presence of delCT in the 49 cases with frequency of mutated sequencing reads greater than 0.7%, specifically in 11% of cMBL (11/100) and 13.4% of CLL patients (38/284). Furthermore, mutated samples were subjected to DNA Sanger sequencing: in line with the expected sensitivity of the method, the mutation was identified only in samples with higher mutation loads according to NGS (mutant allele frequency ≥ 7%, n=25). Our data revealed that often NOTCH1 mutational activation affected a neoplastic sub-clone, especially in cMBL patients. NOTCH1 mutated patients utilized unmutated IGHV genes more frequently, and had higher expression of CD38 and ZAP-70 (P=3.2e-11, P=2.6e-08, P=3.4e-05, respectively). Trisomy 12 was more frequent in this patient group (P=5.4e-04), whereas 13q14 deletion was less represented than in the NOTCH1 wild-type patients (P=2.8e-03). NOTCH1 mutation was associated with the occurrence of stereotyped HCDR3 (P=5.6e-03); in addition, compared with other major BCR subsets, CLL subset #10 was significantly enriched in NOTCH1 mutations (P=0.032). The prevalence of the analyzed dinucleotide deletion was not significantly different between cMBL and CLL patients, even if only Rai 0 cases (28/197 cases, 14.2% mutation frequency) were considered. The percentages of variant sequencing reads in NOTCH1-mutated cases were slightly higher in CLL (median 19.6%) than in cMBL (median 4.2%), a finding confirmed by a regression analysis that highlighted the association of the CLL presentation with higher percentages of NOTCH1 delCT reads (P=0.033). NOTCH1-mutated cases, both at sub-clonal and clonal levels, displayed a significant reduction in median PFS (P=0.0018), although NOTCH1 mutation prognostic value, in multivariate analysis, was not independent if 11q and/or 17p deletion, IGHV mutational status, and cMBL or CLL status were considered. Finally, sequential analyses in a representative fraction of cases of our dataset indicated that (i) NOTCH1 mutation did not occur during the course of the disease and that (ii) the mutational load in positive cases was stable over time. These findings highlight the importance of using high sensitive methods for an accurate detection of NOTCH1 mutation in cMBL/early stage CLL. This is required for a better prognostic stratification and also to obtain useful information for potential therapeutic approaches, since sub-clonal mutations in untreated CLL can possibly anticipate the dominant genetic composition of the relapsing tumor. Disclosures: No relevant conflicts of interest to declare.

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EGR2 Mutations in Chronic Lymphocytic Leukemia: A New Bad Player

Blood ◽

10.1182/blood.v126.23.4126.4126 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4126-4126

Author(s):

Daniel Noerenberg* ◽

Emma Young* ◽

Viktor Ljungström ◽

Larry Mansouri ◽

Karla Plevova ◽

...

Keyword(s):

Overall Survival ◽

General Practice ◽

Chronic Lymphocytic Leukemia ◽

Deep Sequencing ◽

Research Funding ◽

Sanger Sequencing ◽

Lymphocytic Leukemia ◽

Prognostic Impact ◽

Bcr Signaling ◽

The Uk

Abstract *Contributed equally as first authors. **Contributed equally as senior authors. Recurrent mutations within EGR2, a versatile transcription factor involved in differentiation of hematopoietic cells, were recently reported in 8% of advanced-stage chronic lymphocytic leukemia (CLL) patients, where they appear to be associated with a worse outcome. EGR2 is activated through ERK phosphorylation upon B-cell receptor (BcR) stimulation, and we have previously shown that EGR2 -mutated CLL patients display altered expression of EGR2 down-stream target genes compared to wildtype (wt) patients, thereby pointing to a pathogenic role for EGR2 mutations in dysregulating BcR signaling. To gain further insight into the incidence and prognostic impact of EGR2 mutations in CLL, we screened samples from a well-characterized series of 1430 patients, either by Sanger sequencing (n=1019) or targeted deep-sequencing (n=370), both covering the recently reported EGR2 hotspot in exon 2. In addition, whole-exome data was available for an additional 43 patients. Different cohorts were included in our analysis ranging from 'general practice' CLL (33% IGHV-unmutated (U-CLL), 6% TP53 -aberrant (TP53abn), n=693), to adverse-prognostic CLL (89% U-CLL, 26% TP53abn, n=325), patients belonging to clinically aggressive stereotyped subsets #1-3 & #5-8 (n=342), patients relapsing after FCR therapy (n=41) and Richter transformed cases (n=31), thus reflecting the heterogeneous nature of CLL. Nineteen EGR2 mutations were detected by Sanger sequencing, while 22 additional mutations were identified with deep-sequencing using a 5% variant allele frequency (VAF) cutoff (median 39%, range 5.6-63.9%, median coverage 43,000X). With the exception of one in-frame deletion, all mutations were missense alterations located within the three zinc-finger domains. Significant enrichment of EGR2 mutations was observed in adverse-prognostic (18/325, 5.5%) and FCR-relapsing (4/41, 9.8%) CLL compared to the 'general practice' cohort (18/693, 2.6%, Figure 1A). A surprisingly low frequency was observed among clinically aggressive stereotyped subsets (5/342, 1.5%), although the cause for this observation is currently unknown. Finally, 2/31 (6.5%) cases with Richter transformation carried an EGR2 mutation. Of the 4 FCR-relapsing, EGR2 -mutated cases with available overtime samples, all demonstrated a significant expansion of the EGR2 -mutated clone at relapse (VAF-increase between 15-41%). In addition, subclonal levels of EGR2 hotspot mutations (VAF 0.5-5%) were detected in an additional 13/370 (3.5%) cases by deep-sequencing. The majority of EGR2 -mutated CLL patients (32/39, 82%) concerned U-CLL and the following aberrations co-occurred: 11q-deletions (n=10), TP53abn (n=6), NOTCH1 (n=3)or SF3B1 (n=3) mutations. EGR2 -mutated patients displayed a significantly worse overall survival compared to wt patients (median survival 59 vs. 141 months, p=0.003, using a conservative 10% VAF cutoff), and a poor outcome similar to cases with TP53abn (Figure 1B). In multivariate analysis (n=583), EGR2 status remained an independent factor (p=0.038), along with stage (p=0.048) and IGHV status (p<0.0001), while TP53abn and del(11q) showed borderline significant values (p=0.069 and p=0.059, respectively). To investigate the impact of EGR2 mutations in a homogeneously treated patient cohort, EGR2 mutation analysis of the UK CLL4 trial is underway. To date, 8/247 patients have been identified as EGR2 -mutated by deep-sequencing and they show a decrease of their median overall survival (42 vs. 77 months) compared to wt patients; however, this did not reach statistical significance, probably due to the low number of EGR2 -mutated cases. Final results of the UK CLL4 trial will be presented at the ASH meeting. In summary, EGR2 -mutant cases appear to constitute a novel poor-prognostic subgroup of CLL, with mutations occurring either as disease-initiating aberrations, i.e. in cases where mutations were found in the entire clone, or as subclonal driver events linked to progressive disease. The latter is reflected by the enrichment of EGR2 mutations in aggressive CLL and the association of EGR2 mutations with an overall dismal prognosis. Considering the potential role of mutated EGR2 in altering BcR signaling, it will be particularly relevant to study the efficacy of BcR inhibitors in this patient group. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Langerak: Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam); InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics.; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL. Schuh:Acerta Pharma BV: Research Funding. Strefford:Roche: Research Funding.

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Prognostic impact and landscape of NOTCH1 mutations in chronic lymphocytic leukemia (CLL): a study on 852 patients

Leukemia ◽

10.1038/leu.2013.218 ◽

2013 ◽

Vol 27 (12) ◽

pp. 2393-2396 ◽

Cited By ~ 44

Author(s):

S Weissmann ◽

A Roller ◽

S Jeromin ◽

M Hernández ◽

M Abáigar ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Lymphocytic Leukemia ◽

Prognostic Impact ◽

Notch1 Mutations

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Small Subclones Harboring NOTCH1, SF3B1 or BIRC3 Mutations Are Clinically Irrelevant in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v124.21.295.295 ◽

2014 ◽

Vol 124 (21) ◽

pp. 295-295

Author(s):

Davide Rossi ◽

Hossein Khiabanian ◽

Silvia Rasi ◽

Carmela Ciardullo ◽

Lodovico Terzi di Bergamo ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Sanger Sequencing ◽

Lymphocytic Leukemia ◽

Sensitivity Threshold ◽

Consecutive Series ◽

Prognostic Impact ◽

Newly Diagnosed ◽

Tp53 Mutations ◽

Clonal Abundance ◽

Notch1 Mutations

Abstract Introduction. Ultra-deep next generation sequencing (NGS) allows sensitive detection of mutations and estimation of their clonal abundance in tumor cell populations. TP53 mutations identified by ultra-deep NGS significantly impact on survival of chronic lymphocytic leukemia (CLL) patients, independent of their representation in the tumor clone and even if restricted to a small fraction of leukemic cells. Purpose. Here we aim at assessing the frequency, prognostic impact and evolution during disease course of NOTCH1, SF3B1 and BIRC3 mutations identified by ultra-deep NGS in newly diagnosed CLL. Methods. The study was based on a consecutive series of 304 newly diagnosed and previously untreated CLL (median age: 71 years; Binet A/B/C: 79/12/9%; unmutated IGHV genes: 35%; del13q/+12/del11q/del17p: 51/21/8/9%; median follow-up: 8.1 years). By ultra-deep NGS, TP53 mutations (exons 4-8) occurred in 15% of CLL with a median clonal abundance of 18% (range 0.2-95%). NOTCH1 (hg19 g.chr9:139390596-139390829), SF3B1 (exons 14, 15) and BIRC3 (exons 6, 9) mutations were screened on peripheral blood samples by amplicon-based ultra-deep NGS (454 Life Sciences) (average depth: 2437). A bioinformatic algorithm was applied to call non-silent variants out of background NGS noise. Variant calling by the algorithm was validated by Sanger sequencing or, if the variant was below the sensitivity threshold of Sanger sequencing, by both duplicate ultra-deep NGS and allele specific PCR (AS-PCR). Variant allele frequency (VAF) was corrected for tumor representation. Results. Ultra-deep NGS identified 46 NOTCH1 mutations (median VAF 24%; range: 1.4-64%) in 14% (43/304) CLL, 43 SF3B1 mutations (median VAF 16%; range: 0.5-48%) in 11% (35/304) CLL and 37 BIRC3 mutations (median VAF 5.6%; range: 0.2-47%) in 8% (26/304) CLL. In cases harboring more than one mutation (NOTCH1=3; SF3B1=2; BIRC3=5), the variants mapped on distinct sequencing reads from the same amplicon suggesting that they belonged to different CLL subclones. Out of the variants identified by ultra-deep NGS, a significant fraction of NOTCH1 (n=14; median VAF: 3.9%; range: 1.4-9%), SF3B1 (n=25; median VAF: 4.3%; range: 0.5-17%) and BIRC3 (n=30; median VAF: 1.4%; range: 0.2-6%) mutations were missed by Sanger sequencing because their VAF was below the sensitivity threshold of the assay (~10%). The molecular spectrum of these small subclonal mutations was highly consistent with that of mutations that were visible by Sanger sequencing. The overall survival of cases harboring solely small subclonal mutations of NOTCH1, SF3B1 and BIRC3 was similar to that of wild type cases and longer than that of cases with clonal lesions (Fig. 1). Outcome-driven statistical approaches (maxstat and recursive partitioning) were applied to identify the optimal cut-off of the VAF in order to test whether the clonal representation of mutations correlated with CLL survival. Application of these approaches to TP53 mutations failed in identifying a clear cut-off, suggesting that TP53 variants are relevant to the outcome of CLL independent of their VAF. Conversely, in the case of NOTCH1, SF3B1 and BIRC3 mutations, these approaches consistently identified 25%, 35% and 1%, respectively, as the best VAF cut-off values for outcome prediction (Fig. 1), thus suggesting that small subclones harboring NOTCH1, SF3B1 or BIRC3 mutations are clinically irrelevant. By multivariate analysis, NOTCH1 mutations >25% of VAF (HR: 1.8; p=.038), SF3B1 mutations >35% of VAF (HR: 2.9; p=.005) and BIRC3 mutations >1% of VAF (HR: 1.8; p=.044) were significantly associated with an increase in the hazard of death, after adjusting for TP53 lesions (HR: 2.9; p<.001), del11q (HR: 2.2; p=.007), +12 (HR: 1.3, p=.174) and del13q (HR: 0.8; p=.512). Among cases harboring small mutated subclones, longitudinal deep NGS of sequential samples documented the outgrowth of the variants to a clonal level in 1/5 cases for NOTCH1, in 5/12 for SF3B1 and in 3/10 for BIRC3. While selection of NOTCH1 and BIRC3 variants did not associate neither with disease feature nor with treatment, the outgrowth of subclonal SF3B1 mutations was restricted to cases showing unmutated IGHV genes (p=.015). Conclusions. Small mutated subclones of the NOTCH1, SF3B1 and BIRC3 genes appear to be clinically irrelevant. Mutations of NOTCH1 and SF3B1 require a substantial clonal representation to be harmful. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Mutations of NOTCH1 Are An Independent Predictor of Survival in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v118.21.283.283 ◽

2011 ◽

Vol 118 (21) ◽

pp. 283-283

Author(s):

Davide Rossi ◽

Silvia Rasi ◽

Giulia Fabbri ◽

Valeria Spina ◽

Marco Fangazio ◽

...

Keyword(s):

Multivariate Analysis ◽

High Risk ◽

Chronic Lymphocytic Leukemia ◽

Univariate Analysis ◽

Lymphocytic Leukemia ◽

Prognostic Impact ◽

Prognostic Role ◽

Trisomy 12 ◽

Notch1 Mutations

Abstract Abstract 283 The clinical course of chronic lymphocytic leukemia (CLL) ranges from very indolent, with a nearly normal life expectancy, to rapidly progressive leading to death and occasionally undergoing transformation to Richter syndrome (RS). TP53 disruption identifies a fraction of high risk CLL destined to experience a very short survival. High risk CLL, however, cannot be fully recapitulated by TP53 disruption and other lesions of cancer genes may be implicated in this aggressive phenotype. Analysis of the CLL coding genome has recently disclosed that the NOTCH1 proto-oncogene is recurrently mutated at CLL presentation. Here we assessed the prognostic role of NOTCH1 mutations in CLL. Two series of previously untreated CLL were utilized as training (n=309, median follow-up 6 years) and validation (n=230, median follow-up 7 years) cohorts. NOTCH1 mutations were analyzed by DNA Sanger sequencing in blind with respect to clinical data. In the training series, NOTCH1 mutations occurred in 34/309 (11.0%) patients, being mostly represented (26/34, 76.5%) by a recurrent two bp frameshift deletion (c.7544_7545delCT). The remaining NOTCH1 mutations (8/34, 23.5%) were frameshift deletions other than c.7544_7545delCT (n=7) and frameshift insertions (n=1). All mutations were predicted to disrupt the NOTCH1 PEST domain. CLL with NOTCH1 mutations preferentially carried unmutated IGHV genes (76.5%, p<.001). Other characteristics at presentation associated with NOTCH1 mutations were advanced Rai stage (26.5%, p=.006) and trisomy 12 (44.1%, p<.001). By univariate analysis, NOTCH1 mutations associated with an increase in the hazard of death (HR: 3.77; 95% CI: 2.14–6.66) and a significant overall survival OS shortening (p<.001) (Fig. 1A). Multivariate analysis selected NOTCH1 mutations as an independent risk factor of OS (HR: 4.22; 95% CI: 2.15–8.28; p<.001), after adjusting for age (p<.001), Rai stage (p=.005), IGHV mutation status (p=.465), 11q22-q23 deletion (p=.128), trisomy 12 (p=.183) and TP53 disruption (p<.001). The poor prognosis conferred by NOTCH1 mutations was attributable, at least in part, to a shorter time to progression requiring treatment (p<.001), and a higher cumulative probability of RS development (p=.026). Although NOTCH1 mutated patients were devoid of TP53 disruption in 31/34 (91.2%) cases, the OS predicted by NOTCH1 mutations was similar to that of TP53 mutated/deleted CLL (Fig. 1C). Analysis of the validation series confirmed: i) the prevalence of NOTCH1 mutations at CLL presentation (26/230, 11.3%); ii) the spectrum of NOTCH1 mutations at CLL presentation (c.7544_7545delCT: 21/26, 80.7%; other mutations: 5/26, 19.3%) iii) the adverse prognostic impact of NOTCH1 mutations in CLL both by univariate analysis (Fig. 1B) and by multivariate analysis (HR: 2.08; 95% CI: 1.10–3.93; p=.023); iv) the preferential mutually exclusive distribution of NOTCH1 mutations and TP53 disruption (25/26, 96.2%); v) that OS of NOTCH1 mutated CLL is similarly poor as that of TP53 disrupted CLL (Fig. 1D). The current study on 539 CLL documents that NOTCH1 mutations: i) represent one of the most frequent cancer gene mutations known to be involved at CLL presentation; ii) identify a subgroup of patients showing poor OS similar to that of TP53 disrupted cases; iii) exert a prognostic role independent of widely accepted clinical and genetic risk factors; iv) predict OS in series from different institutions, as documented by the training-validation approach chosen for the design of this study. Disclosures: No relevant conflicts of interest to declare.

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SF3B1 Mutations Have Adverse Impact On Time to Treatment Especially in Patients with 13q Deletions: A Study On 1,124 Chronic Lymphocytic Leukemia (CLL) Patients

Blood ◽

10.1182/blood.v120.21.709.709 ◽

2012 ◽

Vol 120 (21) ◽

pp. 709-709 ◽

Cited By ~ 1

Author(s):

Sabine Jeromin ◽

Claudia Haferlach ◽

Katharina Bayer ◽

Frank Dicker ◽

Sandra Weissmann ◽

...

Keyword(s):

Adverse Effect ◽

Cox Regression ◽

Multivariable Analysis ◽

Normal Karyotype ◽

Lymphocytic Leukemia ◽

Prognostic Impact ◽

Complex Karyotype ◽

Time To Treatment ◽

Cox Regression Analysis ◽

Equity Ownership

Abstract Abstract 709 Introduction: Mutations in the spliceosome gene SF3B1 (splicing factor 3b, subunit 1; SF3B1mut) have recently been described in CLL and occur in 5–15% of CLL patients. They are suggested to be associated with a more aggressive course of disease, reflected by a shorter time to treatment (TTT) and overall survival. However, validation in a larger cohort is lacking. Aims: 1. Determine the frequency of SF3B1mut in a large CLL cohort. 2. Evaluate the association of SF3B1mut with established prognostic markers and its prognostic impact in relation to these parameters. Patients and Methods: 1,124 newly diagnosed CLL patients were included. The cohort comprised 64.4% (724/1124) males and 35.6% (400/1124) females with a median age of 66.8 years (range: 29.6 – 90.5 years). In all cases, the coding region of SF3B1 (exon 11 – 16) was analyzed by Sanger sequencing. Low mutation/wildtype ratios (<10%) were confirmed by an amplicon next-generation deep-sequencing assay (454 Life Sciences, Branford, CT). Additionally, TP53 (n= 83 mut/1,098 screened, 7.4%) and IGHV mutation status were analyzed. IGHV status was unmutated in 37.6% (423/1,124) and mutated in 62.4% (701/1,124).The IGHV3-21 gene was present in 63 patients, 41 of whom had a mutated status and were assigned together with the cases with unmutated IGHV (unfavorable IGHV). For all cases data on immunophenotype, FISH and chromosome banding analyses (CBA) were available. FISH categories were defined according to Döhner et al. (NEJM, 2000): del(17p) (48/1,124, 4.3%), del(11q) (123/1,124, 10.9%), +12 (139/1,124, 12.4%), normal karyotype (NK) according to FISH (273/1,124, 24.3%) and del(13q) as sole abnormality (324/1,124, 28.8%). According to CBA normal karyotype was present in 19.2% (216/1,124) and complex karyotype (at least 3 chromosomal abnormalities) in 16.2% (182/1,124). Clinical follow-up data were available in 56 SF3B1mut and 505 SF3B1 wt patients. Results: The frequency of SF3B1mut was 9.3% (105/1,124) with a median mutation/wildtype ratio of 35% (range: 5 – 60%). In 105 patients 110 SF3B1mut were detected. The most frequent mutation was Lys700Glu (47/110, 42.7%) followed by Gly742Asp (12/110, 10.9%) and Lys666Asn/Glu/Ser/Thr (11/110, 10.0%). SF3B1mut showed a strong association with prognostic unfavorable IGHV status (unfavorable vs favorable: 76/464, 16.4% vs 29/660, 4.4%, p<0.0001). They were more frequent in cases with IGHV3-21 (19.0% vs 8.7%, p=0.012) and IGHV1-69 (19.7% vs 7.9%, p<0.0001), but were mutually exclusive of IGHV1-2 (0% vs 9.7%, p=0.027). However, SF3B1mut were evenly distributed between patients with TP53mut and TP53 wt. Furthermore, SF3B1mut were associated with NK according to FISH (NK vs aberrant by FISH: 40/273, 14.7% vs 65/851, 7.6%, p=0.001) or CBA (NK vs aberrant: 29/216, 13.4% vs 76/908, 8.4%, p=0.027) and were less frequent in +12 (2/139, 1.4% vs 103/985, 10.5%, p<0.0001) and homozygous del(13q) cases (12/224, 5.4% vs 93/900, 10.3%, p=0.021). SF3B1mut were rarely detected in cases with IGH translocations identified by FISH and/or CBA (2/55, 3.6% vs. 103/1069, 9.6%, p=0.159). In contrast, SF3B1mut were very frequent in patients with del(11q) (25/123, 20.3% vs. 80/1,001, 8.0%, p<0.0001). Patients with SF3B1mut had a significantly shorter TTT than SF3B1wt patients (median TTT: 4.8 vs. 7.5 years, p<0.0001). Particularly in the subgroup with del(13q) sole the adverse effect of SF3B1mut was very prominent (median TTT: 1.1 vs 7.6 years, p<0.0001). In univariable cox regression analysis parameters associated with a shorter TTT were SF3B1mut (relative risk (RR): 2.05, p=0.001), complex karyotype (RR: 1.47, p=0.026), aberrant karyotype (RR: 1.66, p=0.036), and del(11q) (RR: 2.54, p<0.0001). Parameters associated with a longer TTT had favorable IGHV status (RR: 0.31, p<0.0001) and del(13q) sole (RR: 0.60, p=0.005). Multivariable analysis revealed an independent impact for SF3B1mut (RR: 1.54, p=0.048) besides aberrant karyotype (RR: 1.91, p=0.012), favorable IGHV status (RR: 0.35, p<0.0001) and del(13q) sole (RR: 0.63, p=0.017). Conclusions: 1. SF3B1mut are associated with unfavorable IGHV status (unmutated, IGHV3-21) and del(11q), which are known to have adverse effect on outcome in CLL. 2. Besides, SF3B1mut is an independent prognostic parameter associated with shorter TTT, with an especially striking impact in the prognostically favorable subgroup of patients with del(13q) as sole abnormality. Disclosures: Jeromin: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Bayer:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership.

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Surface IgM Levels Independently Influence Clinical Behavior and Associate with Altered Phenotype and Genetics in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v124.21.830.830 ◽

2014 ◽

Vol 124 (21) ◽

pp. 830-830

Author(s):

Samantha J Drennan ◽

Annalisa D'Avola ◽

Ian Tracy ◽

Isla Henderson ◽

Marta Larrayoz ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Kinase Inhibitors ◽

Cox Regression ◽

Conflicts Of Interest ◽

Dominant Role ◽

Critical Role ◽

Lymphocytic Leukemia ◽

Clinical Behavior ◽

Notch1 Mutations

Abstract The tumor B-cell receptor (BCR) is the key to survival and proliferation in chronic lymphocytic leukemia (CLL) and is now a therapeutic target of very effective BCR-associated kinase inhibitors. CLL cells are typically characterized by a variable state of anergy, defined by low surface IgM (sIgM) levels and signaling ability with consequent relatively low proliferation rate. The subset with unmutated IGHV (U-CLL) is generally less anergic with a poorer prognosis than that with mutated IGHV (M-CLL), and appears more sensitive to BCR-inhibitors (Byrd et al., NEJM, 2013). However it remains unclear if the variable anergy reflects the nature of the cell of origin and/or the functional state of the BCR, thereby determining clinical behavior. In this study we investigated if variations of BCR sIgM levels and signaling correlate with clinical behavior of CLL and assessed the phenotypic and genetic associations with the variations. The study included samples at diagnosis or prior to treatment from 222 patients with sIgM/D CLL diagnosed according to the iWCLL2008 criteria (99 months median follow up). sIgM levels on the CD19+/CD5+ CLL cells and signaling [% intracellular Ca2+ mobilization (iCa2+)] were determined using soluble F(ab’)2 polyclonal antibodies (Mockridge at al, Blood, 2007). sIgM levels were analyzed for their association with i) survival, ii) phenotype (CD38, ZAP70 and BCR regulators CD5, CD19, CD20, CD22) and iii) genetic lesions (Del13q, Trisomy12, Del11q/ATM, Del17p/TP53, or NOTCH1 and SF3B1 mutants). Time from diagnosis to progression requiring first treatment (TTFT) was used as primary endpoint, while overall survival (OS) was used as a secondary endpoint to avoid the influences of chemotherapy. Best cut-offs for progression requiring treatment were determined with ROC and Youden’s T-tests (treatment as a state variable). Levels of sIgM (5th-95th percentile 12-378, median 52) varied markedly between patients. Levels were higher at more advanced stage of disease and sIgM expression (MFI>56) and signaling (iCa2+>6%) associated with significantly more rapid TTFT. Although sIgD levels and signaling also associated with shorter TTFT, multivariate Cox regression adjusted for IGHV status, sIgM levels, sIgM signaling, sIgD levels and sIgD signaling revealed that only U-IGHV (p<0.001, 95% CI 1.64-3.97) and high sIgM levels (p=0.004, 95% CI 1.24-3.16) were independent predictors of shorter TTFT. Also, high sIgM levels and not high sIgD associated with shorter OS, despite chemotherapy. These data confirmed the specific influence of anergy which drives down sIgM and not sIgD, and is associated with less aggressive tumor behavior in CLL. By focusing on sIgM, a multivariate analysis adjusted for IGHV status, sIgM levels, CD38 and ZAP70 confirmed U-IGHV (p=0.002, 95% CI 1.35-3.56) and high sIgM (p=0.003, 95% CI 1.25-3.04) as the only independent predictors of shorter TTFT. Consistently, high sIgM levels associated with a more aggressive CLL even when U-CLL or M-CLL were investigated separately. Phenotypic analyses showed that high sIgM CLL had higher CD20, CD38, ZAP70, and CD22, than low sIgM CLL. Genetic analysis revealed that CLL subsets harboring Trisomy12 or Del17p/TP53 had significantly higher sIgM levels and signaling capacity than Del13q, even when U-CLL or M-CLL were analyzed separately. Trisomy12 U-CLL cases were enriched for NOTCH1 mutations. Consistently, U-CLL with NOTCH1 mutations (with or without Trisomy12) expressed higher sIgM than Del13q U-CLL. This study has several biological and clinical implications. First, it supports the critical role of sIgM in CLL-associated anergy. Our previous studies had shown dynamic variations of sIgM on circulating CLL cells following (auto)antigen engagement in tissue (Mockridge et al., Blood, 2007; Coelho et al. Blood, 2013). The subpopulation with higher sIgM is potentially the most dangerous and the most sensitive to kinase-inhibitors. Second, it documents the dominant role of sIgM levels on cell signaling and on tumor progression in CLL, with relevance even within U-CLL and M-CLL. Third, this study suggests influences of altered genetics on sIgM levels. However BCR-inhibitors overcome the influence of genetic lesions like Del17p/TP53. This points to a dominant role of reversed sIgM anergy in the behavior of CLL in vivo and claims the need of studies to verify sensitivity to kinase-inhibitors in CLL patients with different sIgM levels. Disclosures No relevant conflicts of interest to declare.

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NOTCH1 Mutations Are Associated with Low CD20 Expression in Chronic Lymphocytic Leukemia: Evidences for a NOTCH1-Mediated Epigenetic Regulatory Mechanism

Blood ◽

10.1182/blood.v124.21.296.296 ◽

2014 ◽

Vol 124 (21) ◽

pp. 296-296

Author(s):

Federico Pozzo ◽

Tamara Bittolo ◽

Pietro Bulian ◽

Erika Tissino ◽

Francesca Maria Rossi ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Hdac Inhibitor ◽

Lymphocytic Leukemia ◽

Transcript Levels ◽

Expression Levels ◽

Qrt Pcr ◽

Mutational Burden ◽

Cd20 Expression ◽

Anti Cd20 ◽

Notch1 Mutations

Abstract 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<0.001) and in non-tris12 (36 NOTCH1-mut vs. 354 NOTCH1-wt, p=0.005) cases. MS4A1 transcript levels were lower in NOTCH1-mut than in NOTCH1-wt (tris12 CLL, p=0.024; non-tris12 CLL, p=0.002). By performing cell sorting to isolate CD20low and CD20high subpopulations in 4 cases with subclonal NOTCH1 mutations (range 26%-45%), the CD20low subpopulation always showed an enrichment in NOTCH1 mutational burden when compared to the CD20high counterpart, along with lower MS4A1 expression levels. NICD mutated MEC-1 cells, expressing higher NICD transcript (2.2 over the control) and NICD (1.3 over the control) protein, were characterized by lower CD20 (0.1 over the control) and MS4A1 transcript (0.77 over the control) expression. ii) In keeping with CD20 levels, primary NOTCH1-mut CLL cells showed lower relative lysis induced by rituximab than NOTCH1-wt CLL cells (mean relative lysis: NOTCH1-mut, 5% vs. NOTCH1-wt, 30%, p=0.008). This phenomenon was confirmed by using the MEC-1 cell model (mean relative lysis: NICD mutated, 3% vs. control, 30%, p=0.006). iii) NICD mutated MEC-1 cells expressed higher levels of both HDAC1 (2.0 over the control) and HDAC2 (1.4 over the control) transcripts. Moreover, in co-immunoprecipitation experiments, NICD mutated MEC-1 cells were characterized by lower levels of HDAC1/HDAC2 bound to the transcriptional factor CBF-1, due to a competition for the binding to CBF1 with the high NICD levels present in these cells, and suggesting a nuclear interplay between NOTCH1 and HDAC where CBF-1 is the connecting element. The dependency of low CD20 expression by HDAC activity was demonstrated by the capability of VPA to increase CD20 expression in primary NOTCH1-mut CLL cells (1.3 over the control) and NICD mutated MEC-1 transfectants (1.4 over the control). 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.

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Risk Stratification In Chronic Lymphocytic Leukemia Patients With IGHV3-21 Gene Usage According To Presence Of Stereotypy and Mutations In SF3B1

Blood ◽

10.1182/blood.v122.21.4114.4114 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4114-4114

Author(s):

Sabine Jeromin ◽

Frank Dicker ◽

Katharina Bayer ◽

Sandra Weissmann ◽

Christiane Eder ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Risk Stratification ◽

Lymphocytic Leukemia ◽

Prognostic Impact ◽

Employment Equity ◽

Mutation Status ◽

Mutational Status ◽

Trisomy 12 ◽

Equity Ownership

Abstract Introduction Chronic Lymphocytic Leukemia (CLL) patients with monoclonal IGHV3-21 gene rearrangements have been described to have adverse prognosis independent of mutational status. Heterogeneous data exists whether only patients with a stereotyped motif in the junctional region (designated as subset #2, Stamatopoulos K. et al., Blood 2007) suffer from worse prognosis. Furthermore, it was recently suggested that co-occurrence of subset #2 and mutations (mut) in SF3B1 are indicative of a shorter time to treatment (TTT). Aims 1. Determine the prognostic impact of IGHV3-21 and subset #2 rearrangements. 2. Evaluate the association with SF3B1mut and its prognostic impact. Patients and Methods IGHV3-21 positive (n=213) and independently 1,094 unselected CLL patients without prior treatment were analyzed. The whole cohort comprised 63.9% (835/1,307) males and 36.1% (472/1,307) females with a median age of 66.8 years (range: 27.5 – 90.5 years). In all cases IGHV mutation status was analyzed. IGHV unmutated (unmut) status was present in 38.6% (504/1,307) and mutated status in 61.4% (803/1,307). Stereotypy of IGHV3-21 was classified according to published criteria (Agathangelidis A. et al., Blood 2012). SF3B1 was analyzed in all and TP53 in 1,262 cases for mutations. For all patients data on immunophenotype was available. Cases were further analyzed by FISH using probes for del(17p) (n=1,305), del(11q) (n=1,303), trisomy 12 (n=1,303) and del(13q) (n=1,305). Clinical follow-up data was available in 1,040 patients with a median follow-up of 4.4 years (IGHV3-21: n=160, 4.2 years). Results Of 213 IGHV3-21 positive patients, 111 (52.1%) cases were classified as subset #2 B-cell receptor. The frequency of IGHVmut was significantly higher in subset #2 vs. non-subset #2 (78/111, 70.3% vs. 49/102, 48.0%, p=0.001). IGHV3-21 was highly associated with SF3B1mut (52/213, 24.4% vs. 92/1,094, 8.4%, p<0.001), which were particularly frequent in subset #2 cases (38/111, 34.2% vs. 14/102, 13.7%, p=0.001). Furthermore, IGHV3-21 was associated with del(11q) (35/210, 16.7% vs. 122/1,093, 11.2%, p=0.028) and was rare in patients with trisomy 12 (8/210, 3.8% vs. 168/1,093, 15.4%, p<0.001). Accordingly, del(11q) was particularly frequent in subset #2 patients (25/110, 22.7% vs. 10/100, 10.0%, p=0.016), whereas trisomy 12 (1/110, 0.9% vs. 7/100, 7.0%, p=0.029) and del(17p) (1/111, 0.9% vs. 8/101, 7.9%, p=0.015) were nearly absent. Kaplan-Meier analysis revealed no significant difference in TTT between IGHV3-21mut vs. unmutated cases. However, IGHV3-21mut cases had slightly longer TTT compared to IGHVunmut (5.3 years vs. 3.4 years, p=0.039). Taking stereotypy into account, subset #2 patients showed nearly identical TTT compared to IGHVunmut patients (3.5 vs. 3.4 years). Further stratification according to IGHV mutational status presented mutated non-subset #2 patients with a similar TTT compared to IGHVmut cases (9.2 vs. 10.2 years), whereas all other subgroups assorted together with IGHVunmut (Fig. 1A). Additionally, there was a trend to a shorter TTT in subset #2 in combination with SF3B1mut vs. SF3B1wt (1.2 vs. 4.4 years, p=0.056) (Fig. 1B). In univariate Cox regression analysis, following parameters were analyzed and showed significant impact on TTT: IGHVmut (p<0.001, HR 0.33), IGHV3-21 (p=0.002, HR 1.51), subset #2 (p=0.005, HR 2.04), SF3B1mut (p<0.001, HR 2.06). A multivariate analysis including IGHV3-21, IGHVmut and SF3B1mut revealed independent impact on TTT only for the latter two parameters: IGHVmut (p<0.001, HR 0.35) and SF3B1mut (p=0.001, HR 1.59). In contrast, analyzing subset #2, IGHVmut and SF3B1mut in a multivariate model, only subset #2 (p=0.011, HR 1.93) and SF3B1mut (p=0.023, HR 1.82) retained their prognostic effect, whereas IGHV mutational status had no independent impact. Conclusions 1. Our data suggests to prognostically stratify IGHV3-21 patients according to the presence of stereotypy, since only subset #2 patients showed shorter TTT, whereas mutated non-subset #2 cases had a TTT similar to IGHVmut cases. 2. Mutation status of SF3B1 further refines the risk stratification of subset #2 patients, as co-occurrence of subset #2 with SF3B1mut leads to shorter TTT compared to subset #2/SF3B1wt cases. Disclosures: Jeromin: MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Bayer:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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