NOTCH1, SF3B1, and TP53 mutations in fludarabine-refractory CLL patients treated with alemtuzumab: results from the CLL2H trial of the GCLLSG

Genomic features are important pathogenic and prognostic factors in chronic lymphocytic leukemia (CLL).^1,2  Recently, mutations in NOTCH1 and SF3B1 have been identified in CLL by next-generation sequencing.^3-6 NOTCH1^mut occurred in 5% to 15% of patients and resulted in a truncated, yet constitutively active protein.⁷ SF3B1^mut have been identified in 10% to 20% of patients and have also notably been reported in myelodysplastic syndrome, pointing to the pathogenic role of pre-mRNA splicing in hematologic malignancies.^5,6,8  Correlations have been described in CLL for NOTCH1^mut with trisomy 12 and for SF3B1^mut with deletion 11q. Both mutations have been associated with unmutated immunoglobulin heavy chain variable gene (IGHV) status, rapid progression, refractory disease, and short survival in initial studies on heterogeneous cohorts outside clinical trials.^3-5,8-10  Refractory CLL can be explained only in about half of all patients by 17p deletion and/or TP53 mutation, but initial data have pointed to NOTCH1^mut and SF3B1^mut as alternative causes of resistance.^3-5,8-11  Here, we evaluated NOTCH1^mut and SF3B1^mut compared with TP53^mut in the CLL2H trial, a multicenter phase 2 trial of subcutaneous alemtuzumab in fludarabine-refractory CLL.^12,13 

Clinical results of the CLL2H trial of the German CLL Study Group (GCLLSG) have been reported.¹³  Genomic aberrations, IGHV status, TP53^mut, β2-microglobulin, and thymidine kinase were analyzed in the central reference laboratories of the GCLLSG.^14-17 NOTCH1 was studied by Sanger sequencing of 2 polymerase chain reaction fragments from the PEST domain (exon 34, chr9:139 390,619-139 391,290), whereas SF3B1 (exons 13-16) was analyzed by DHPLC (WAVE 3500HT; Transgenomic Inc.), with a limit of detection of 5% to 10% mutant allele, followed by sequencing (Big Dye Terminator Kit, ABI 3100 sequencer; Applied Biosystems, Germany). Details are available on request. Tumor load was at least 80%. Somatic origin of mutations was confirmed in CD19 negative fractions wherever available. Statistical analyses were performed by Fisher’s exact test, Kaplan-Meier estimates, and Cox proportional hazards regression with significance as P < .05 (two-sided). The study was approved by the ethics committee of Ulm University, and was conducted in accordance with the Declaration of Helsinki.

The incidences of NOTCH1^mut, SF3B1^mut, and TP53^mut were 13.4% (13 of 97), 17.5% (17 of 97), and 39.0% (39 of 100), respectively. The mutation frequencies of NOTCH1 and SF3B1, therefore, were not markedly increased in the CLL2H cohort of F-refractory CLL, as compared with recent data from large trials of patients in need of first treatment.^18,19  This is in contrast to the much higher incidence of TP53^mut in CLL2H as compared with the first-line trials, pointing to a greater role of p53 as a cause of refractoriness to F-based therapy.

Of note, in none of our 97 patients concurrent NOTCH1^mut and SF3B1^mut were found (Figure 1). This is consistent with previous studies on nonrefractory patients^18-20  and points to different driver mutations in CLL. In contrast, TP53^mut was found together with NOTCH1^mut in 3 of 13 (23.1%; P = .349) cases and with SF3B1^mut in 7 of 17 (41%; P = 1.000) cases, whereas in some previous studies mutual exclusivity was reported.^8,10 

The most common NOTCH1^mut was the published deletion of 2 base pairs in exon 34 (c.7541_7542delCT; n = 11).³  Furthermore, a single base pair deletion was identified (c.7444delC; n = 2). The most common SF3B1^mut was p.K700E (c.A2098G; n = 7). In addition, 10 different point mutations were found in 1 case each (supplemental Table 1, available on the Blood Web site). All SF3B1^mut were heterozygous missense mutations in exons 14, 15 and 16, resulting in 10 different amino acid changes. No patient harbored more than 1 mutation and the mutation profile observed was in-line with previous studies on nonrefractory cohorts (supplemental Table 1).^{3,5,6,10,18,20} 

Sequential samples, obtained prior to first therapy and at the time of inclusion into CLL2H (refractory disease), were available for 13 patients (supplemental Table 2). NOTCH1^mut were detected in 3 of 13 patients prior to therapy and 1 of these patients harbored 2 mutations (c.7541_7542delCT and c.7444delC). Interestingly, this patient had lost the delCT mutation at the time of refractory disease, whereas the other 3 NOTCH1^mut remained present. Of note, 1 patient had a SF3B1^mut prior to first therapy that was not detected anymore at inclusion into CLL2H. TP53^mut were present in 3 of 13 patients before treatment and remained unchanged.

IGHV was unmutated in 71 of 90 (79%) cases overall, and in 100% of the NOTCH1^mut (P = .062) cases, but only in 60.0% of the SF3B1^mut patients (P = .078) (supplemental Table 3). We observed the expected increased incidences of 17p deletion in TP53^mut (63%; P < .0001), and 11q deletion in SF3B1^mut (47%; P = .029), but only a weak association of trisomy 12 with NOTCH1^mut, which was likely due to small numbers (23%; P = .201).^6,9,18,21  Of note, no SF3B1^mut case harbored trisomy 12 (see also Oscier et al¹⁸ ), and there was a high rate of sole TP53^mut (without 17p deletion) in this group (Figure 1).

Comparing baseline characteristics between patients with or without mutations, no significant differences were found for age, sex, stage, “B”-symptoms, prior lines of treatment, blood cell counts, lactate dehydrogenase, β2-microglobulin, thymidine kinase levels, or lymphadenopathy (supplemental Table 3).

The overall response rate (ORR) among the 97 cases was 34% (3% complete remissions [CRs] and 31% partial remissions [PRs]). ORR was 50% (0% CR, 50% PR) and 41.2% (0% CR, 41.2% PR) for NOTCH1^mut and SF3B1^mut cases, respectively. After a median follow-up time of 33.9 months, the entire cohort had a median progression-free survival (PFS) of 7.7 months and a median overall survival (OS) of 18.3 months. NOTCH1^mut cases showed a significantly longer PFS compared with wild-type cases (median 15.5 months vs 6.7 months; P = .025), but there was no significant difference in OS (median not reached vs 18.3 months; P = .181) (Figure 2). The PFS benefit may not translate into OS prolongation due to lack of efficacy of salvage chemotherapy after alemtuzumab failure, in particular in the NOTCH1^mut subgroup. Moreover, the prognostic impact of NOTCH1^mut seems to be dependent on the treatment context: other trials showed no impact on outcome after allo-stem cell transplantation²²  and a differential impact on outcome after fludarabine/cyclophosphamide or fludarabine/cyclophosphamide/rituximab treatment.¹⁹  For SF3B1^mut, there was no significant difference in PFS (median 4.8 vs 7.7; P = .974) and OS (median 29.0 vs 17.1; P = .243). There was no significant impact of TP53^mut on ORR (P = .19), PFS (P = .80), or OS (P = .48). Confirmation of these findings is pending from other trials and the discrepancy between different series may be due to patient characteristics and treatments.

To identify markers of independent prognostic impact, we performed multivariable analyses, including clinical and biological parameters (age, Eastern Cooperative Oncology Group [ECOG] performance status, white blood cell count, thymidine kinase, β2-microglobulin, IGHV status, 11q deletion, 17p deletion, NOTCH1^mut, SF3B1^mut, TP53^mut). Regarding PFS, ECOG performance status > 1 (hazard ratio [HR] 2.080; P = .057), increased thymidine kinase (>26.5 U/L [median], HR 1.479; P = .007), and elevated β2-microglobulin (>4.41 mg/L [median], HR 1.188; P = .0003) were identified as independent adverse factors, whereas NOTCH1^mut (HR 0.375; P = .049) was a favorable marker. Regarding OS, none of the gene mutations showed significant prognostic impact, whereas age (at increments of 10 years) (HR 1.035; P = .024), ECOG performance status > 1 (HR 2.575; P = .017), and increased thymidine kinase (HR 1.568, P = .002) were identified as independent unfavorable factors.

In summary, our analysis performed in a homogenous and uniformly treated cohort of fludarabine-refractory CLL patients derived from a multicenter prospective trial showed NOTCH1^mut in 13.4%, SF3B1^mut in 17.5%, and TP53^mut in 39.0% of the cases, with NOTCH1^mut and SF3B1^mut being mutually exclusive. As compared with CLL cohorts in need of first treatment, the incidence of NOTCH1^mut and SF3B1^mut appeared not to be markedly increased in contrast with TP53^mut. NOTCH1^mut was found as a favorable prognostic marker for PFS, but none of the mutations impacted OS in this trial.

This work was supported by grants from the Else Kröner Fresenius Stiftung (Else Kröner Forschungskolleg Ulm), the CLL Global Research Foundation (Alliance), the Virtual Helmholtz Institute (VH-VI-404, TP2), and the DFG (SFB 1074 project B2).

Contribution: A.S., M.C., K.D., D.M., R.B., S.M., M.H., H.D., and S.S. designed research; T.Z., H.D., and S.S. provided patients and samples; A.S., P.P., M.R., T.Z., A.B., D.W., M.C., K.D., J.E., S.K., D.M., R.B., S.M., H.D., M.H., and S.S. performed research and analyzed data; M.H., H.D., and S.S. gave administrative support; and S.S. wrote the paper.

Conflict-of-interest disclosure: S.S. received research support and honoraria from Genzyme and Sanofi. The remaining authors declare no competing financial interests.

Correspondence: Stephan Stilgenbauer, Department of Internal Medicine III, Ulm University, Albert Einstein Allee 23, Ulm, 89081 Germany; e-mail: stephan.stilgenbauer@uniklinik-ulm.de.