Contributions
Abstract: S1574
Type: Oral Presentation
Presentation during EHA23: On Sunday, June 17, 2018 from 09:00 - 09:15
Location: Room A6
Background
Beyond TP53, no genetic alteration has so far clearly been linked with the pathogenesis of primary high‑risk or refractory chronic lymphocytic leukemia (CLL). TP53 dysfunction and defects in other DNA‑damage response systems such as ATM lead to chromosomal instability with secondary events not necessarily associated with adverse prognosis. This constitutes the challenge, to differentiate between passenger alterations without survival advantage and driver alterations.
Aims
We aimed to identify pivotal genomic alterations contributing to high‑risk CLL biology beyond TP53 aberrations.
Methods
We performed high‑resolution SNP‑array profiling and targeted sequencing (NOTCH1 and SF3B1 among other candidates with potential functional relevance) on 75 relapsed/refractory CLL cases including 18 cases without TP53 dysfunction. Samples were derived from the CLL2O trial of the German / French CLL Study Groups (GCLLSG/FCLLSG). Our cohort was extended by 71 treatment‑naïve TP53‑deficient primary high-risk cases enrolled on the CLL2O trial or the CLL8 or CLL11 trial of the GCLLSG. All samples were CD19 tumor cell enriched. The GISTIC 2.0 algorithm was used to identify significantly enriched copy number alterations (CNAs). Expression of NOTCH1 target genes was quantified by TaqMan‑probe-based quantitative PCR.
Results
Increased genomic complexity was a hallmark of relapsed/refractory and treatment‑naïve high‑risk CLL, and was associated with TP53 and ATM dysfunction. In relapsed/refractory cases previously exposed to the selective pressure of chemo(immuno)therapy, only gain(8)(q24.21) and del(9)(p21.3) were found particularly enriched beyond CNAs routinely assessed in CLL (17% and 11%, respectively). Both of these copy number alterations affected key regulators of cell cycle progression, namely c‑MYC and CDKN2A/B. Gains in 8q24.21 were either focal gains in a c‑MYC enhancer region or larger gains directly affecting the c‑MYC locus, but only the latter type was highly enriched in relapsed/refractory CLL. Loss of CDKN2A/B, which never occurred in standard-risk CLL cases at treatment initiation, was found frequently to co‑occur with gain of c‑MYC. In this combination it was likely associated with Richter transformation, whereas monoallelic loss of CDKN2A in the absence of c-MYC gain was probably not. Mutations in SF3B1 (23%) and NOTCH1 (23%) were found enriched in high‑risk CLL with a comparable distribution between relapsed/refractory and treatment-naïve cases. In addition to the high frequency of activating NOTCH1 mutations, we found recurrent genetic alterations in SPEN (4% mutated), RBPJ (8% deleted) and SNW1 (8% deleted), all affecting a protein complex that represses transcription of NOTCH1 target genes. We investigated the functional impact of these alterations on HES1, DTX1 and c‑MYC gene transcription. De‑repression of these NOTCH1 target genes was particularly observed with SPEN mutations, but our results also suggested functional impact of RBPJ loss.
Conclusion
In summary, we show that highly complex genomic aberrations are a hallmark of high‑risk CLL with TP53 and ATM alteration. We provide a registry of significantly enriched CNAs, define novel recurrent CNAs and identify genomic alterations likely contributing to disease refractoriness. Enrichment of c-MYC gain and CDKN2A/B loss in relapsed/refractory CLL points to the significance of de‑regulated cell cycle in high-risk biology and alterations in a protein complex repressing NOTCH1 target genes likely add to the pathogenic role of aberrant NOTCH1 signaling in CLL.
Session topic: 5. Chronic lymphocytic leukemia and related disorders – Biology & Translational Research
Keyword(s): Cell cycle progression, Chronic Lymphocytic Leukemia, Notch, Refractory
Abstract: S1574
Type: Oral Presentation
Presentation during EHA23: On Sunday, June 17, 2018 from 09:00 - 09:15
Location: Room A6
Background
Beyond TP53, no genetic alteration has so far clearly been linked with the pathogenesis of primary high‑risk or refractory chronic lymphocytic leukemia (CLL). TP53 dysfunction and defects in other DNA‑damage response systems such as ATM lead to chromosomal instability with secondary events not necessarily associated with adverse prognosis. This constitutes the challenge, to differentiate between passenger alterations without survival advantage and driver alterations.
Aims
We aimed to identify pivotal genomic alterations contributing to high‑risk CLL biology beyond TP53 aberrations.
Methods
We performed high‑resolution SNP‑array profiling and targeted sequencing (NOTCH1 and SF3B1 among other candidates with potential functional relevance) on 75 relapsed/refractory CLL cases including 18 cases without TP53 dysfunction. Samples were derived from the CLL2O trial of the German / French CLL Study Groups (GCLLSG/FCLLSG). Our cohort was extended by 71 treatment‑naïve TP53‑deficient primary high-risk cases enrolled on the CLL2O trial or the CLL8 or CLL11 trial of the GCLLSG. All samples were CD19 tumor cell enriched. The GISTIC 2.0 algorithm was used to identify significantly enriched copy number alterations (CNAs). Expression of NOTCH1 target genes was quantified by TaqMan‑probe-based quantitative PCR.
Results
Increased genomic complexity was a hallmark of relapsed/refractory and treatment‑naïve high‑risk CLL, and was associated with TP53 and ATM dysfunction. In relapsed/refractory cases previously exposed to the selective pressure of chemo(immuno)therapy, only gain(8)(q24.21) and del(9)(p21.3) were found particularly enriched beyond CNAs routinely assessed in CLL (17% and 11%, respectively). Both of these copy number alterations affected key regulators of cell cycle progression, namely c‑MYC and CDKN2A/B. Gains in 8q24.21 were either focal gains in a c‑MYC enhancer region or larger gains directly affecting the c‑MYC locus, but only the latter type was highly enriched in relapsed/refractory CLL. Loss of CDKN2A/B, which never occurred in standard-risk CLL cases at treatment initiation, was found frequently to co‑occur with gain of c‑MYC. In this combination it was likely associated with Richter transformation, whereas monoallelic loss of CDKN2A in the absence of c-MYC gain was probably not. Mutations in SF3B1 (23%) and NOTCH1 (23%) were found enriched in high‑risk CLL with a comparable distribution between relapsed/refractory and treatment-naïve cases. In addition to the high frequency of activating NOTCH1 mutations, we found recurrent genetic alterations in SPEN (4% mutated), RBPJ (8% deleted) and SNW1 (8% deleted), all affecting a protein complex that represses transcription of NOTCH1 target genes. We investigated the functional impact of these alterations on HES1, DTX1 and c‑MYC gene transcription. De‑repression of these NOTCH1 target genes was particularly observed with SPEN mutations, but our results also suggested functional impact of RBPJ loss.
Conclusion
In summary, we show that highly complex genomic aberrations are a hallmark of high‑risk CLL with TP53 and ATM alteration. We provide a registry of significantly enriched CNAs, define novel recurrent CNAs and identify genomic alterations likely contributing to disease refractoriness. Enrichment of c-MYC gain and CDKN2A/B loss in relapsed/refractory CLL points to the significance of de‑regulated cell cycle in high-risk biology and alterations in a protein complex repressing NOTCH1 target genes likely add to the pathogenic role of aberrant NOTCH1 signaling in CLL.
Session topic: 5. Chronic lymphocytic leukemia and related disorders – Biology & Translational Research
Keyword(s): Cell cycle progression, Chronic Lymphocytic Leukemia, Notch, Refractory