NOTCH1, SF3B1 and TP53 Mutations in Fludarabine-Refractory CLL Patients Treated with Alemtuzumab: Results From the CLL2H Trial of the Gcllsg

Abstract ⁷¹⁰

Genetic alterations play a major role in stratifying prognostic risk groups in chronic lymphocytic leukemia (CLL). Recently, next generation sequencing strategies revealed novel recurrent mutations in genes such as NOTCH1 and SF3B1 in CLL. Correlations have been reported for SF3B1 mutations (SF3B1^mut) with deletion 11q and for NOTCH1 mutations (NOTCH1^mut) with trisomy 12. Both mutations have been associated with unmutated IGHV, refractory disease and inferior outcome in heterogeneous cohorts outside clinical trials.

We assessed the incidence and prognostic impact of NOTCH1, SF3B1 and TP53 mutations in the CLL2H trial of the GCLLSG, a multicenter phase II trial of subcutaneous alemtuzumab (3 × 30mg/week, max. 12 weeks, n=103, NCT00274976) for fludarabine-refractory CLL patients. Genetic studies were performed in the central reference laboratory of the GCLLSG on 97/103 (94%) trial patients based on availability of DNA. NOTCH1 was analyzed by Sanger sequencing of a PCR fragment from the PEST domain (exon 34, chr9:139,390,619–139,391,290), while SF3B1 (exons 13–16) and TP53 (exons 2–11) were analyzed by DHPLC (WAVE® 3500HT, Transgenomic Inc.) followed by sequencing of aberrant fragments.

NOTCH1^mut were found in 13/97 (13.4%), SF3B1^mut in 17/97 (17.5%), and TP53^mut in 37/99 (37.4%) cases. Two different types of NOTCH1^mut (c.7541_7542delCT, n=11; c.7444delC, n=2) and 13 different point mutations of SF3B1 were identified. All SF3B1^mut were heterozygous missense mutations resulting into 11 different amino acid changes in exons 14, 15 and 16. Of note, in none of the 97 cases concurrent NOTCH1^mut and SF3B1^mut was found. IGHV was unmutated in 71/90 (79%) cases overall, in 100% of NOTCH1^mut (p=.062) and in 60.0% of SF3B1^mut (p=.078). Moreover, SF3B1^mut was associated with 11q deletion (p=.042), but we observed no significant correlation of NOTCH1^mut with trisomy 12 (p=.321).

TP53^mut was found together with NOTCH1^mut in 3/12 (25%, p=.349) and with SF3B1^mut in 7/17 (41%, p=1.000) cases.

The overall response rates (ORR) among the 97 cases was 34% (3% CR and 31% PR). After a median follow-up time of 33.9 months, the median progression-free survival (PFS) was 7.7 months, and the median overall survival (OS) was 18.3 months. The ORR was not significantly different when comparing the groups with or without mutations (50.0% vs. 34.2% for NOTCH1, p=.341 and 41.2% vs. 35.1 % for SF3B1, p=.781, respectively). PFS in NOTCH1^mut cases was significantly superior as compared to wild type cases (median 15.47 months vs. 6.74 months, p=.025, see Figure) but there was no significant difference in OS (median not reached vs. 18.3 months, p=.181). For SF3B1^mut, we found no significant difference between mutated and wild type cases in PFS (median 4.76 vs. 7.72, p=.974) and OS (median 29.0 vs. 17.1, p=.243). Multivariate analysis of PFS and OS was performed including age, ECOG performance status, WBC, thymidine kinase, β2-microglobulin, IGHV status, 11q deletion, 17p deletion, NOTCH1^mut, SF3B1^mut and TP53^mut as potential factors. Regarding PFS, ECOG performance status > 1 (HR 2.080, p=.057), increased thymidine kinase (cont.) (HR 1.479, p=.007) and elevated β2-microglobulin (cont.) (HR 1.188, p=.0003) emerged as independent unfavorable prognostic 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 multicenter trial of fludarabine-refractory CLL patients showed NOTCH1^mut in 13.4%, SF3B1^mut in 17.5% and TP53^mut in 37.4% cases, with NOTCH1^mut and SF3B1^mut being mutually exclusive. As compared to non-refractory CLL cohorts, the incidence of NOTCH1^mut and SF3B1^mut do not appear to be increased which is in contrast to TP53^mut. NOTCH1^mut was found as a favorable prognostic marker for PFS, but none of the mutations impacted OS in this trial.

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