Abstract Topic: 15. Myeloproliferative neoplasms - Biology & Translational Research
Background: Phenotype driver mutations (mut.) in JAK2, CALR, and MPL and JAK-STAT pathway dysregulation characterize MPN. Germline SNV have been associated with susceptibility to various cancers. Little is known about germline SNV in the JAK-STAT genome as causal mediators of BCR::ABL negative MPN.
Aims: To present germline SNV in the JAK2, CALR, MPL, and 7 STAT-family genes in AYA and Non-AYA from the MARY project of the East German Study Group for Hematology and Oncology (OSHO)
Methods: After signed informed consent, phenotype data and blood samples were collected [n=122: AYA (<39 y)=45, Non-AYA (>40 y)=77]. Besides whole transcriptome sequencing (RNA-seq) from blood derived RNA, WES from blood and FACS sorted CD3+ cells derived DNA was performed on an Illumina NovaSeq 6000 at Novogene. At the Core Facilty Imaging of the MLU Halle-Wittenberg (www.medizin.uni-halle.de/cfi), raw reads were quality checked (FastQC, version 0.11.9), low quality reads clipped off (Cutadapt, version 2.8), processed reads aligned (HiSat2 [https://doi.org/10.1038/nmeth.3317],version 2.1.0) to the human genome (UCSC hg38), indexed (samtools, version 1.10), and used for variant calling (bcftools, version 1.10.2) mpileup pipeline. Annotation was performed (Ensembl VEP [doi:10.1186/s13059-016-0974-4], version 108.1) using Ensembl GRCm38.108 [https://doi.org/10.1093/nar/gkab1049], and merging/summarizing of variant and annotation data was performed via an in-house R script. MARY was approved by the ethical committee and funded by Incyte.
Results: Median age in AYA and Non-AYA was 32.5 and 52 years respectively. Diagnoses were ET in 54 (44%), PV in 33 (27%), and PMF in 28 (23%) pts. JAK2 exon 14, CALR, and MPL mut. were detected in 77%, 11%, and 1.7%. 11 (9%) pts were triple negative. Thyroid dysfunction (23%), thrombosis (37%) and a family history of thrombosis (43%) were similar across age. ET (p<0.001), thrombosis after diagnosis (p=0.05), CALR mut., and a triple negative genotype (p=0.004) were higher in AYA.
All pts harbored JAK2 protein coding SNV (median n=44). In 93% and 71% of pts, a median of 27 and 8 SNV in CALR and MPL were detected respectively. MPL SNV correlated with triple negative genotype (p=0.03). Irrespective of phenotype driver mut., JAK2 SNV correlated negatively with CALR and MPL SNV in AYA (p=0.02) and positively in non-AYA (p=0.03). Of 5 germline CALR and MPL SNV detected in >50% of pts, 3 were purely germline while somatic rs2974752 and rs1049481,COSV57131545 were found in further pts without the germline counterpart (Table 1). Median STAT1 (n=370), STAT2 (n=70), STAT4 (n=75), STAT5A (n=28), STAT5B (n=127), and STAT6 (n=127) SNV were comparable across age. STAT3 SNV were less in AYA (mean n=451) vs Non-AYA (mean n=573) (p=0.02). In AYA, CALR SNV correlated with STAT1 (p=0.04) and STAT5B SNV (p=0.02) while in Non-AYA, JAK2 SNV correlated with STAT3 and STAT4 SNV (p=0.02) and a negative association between STAT1 and STAT5B SNV was found (p=0.04). Irrespective of the number of STAT SNV, the percentages of protein coding to nonsense-mediated-decay, and intron retention biotypes within a gene were entirely constant across all pts. A negative association between protein coding STAT3 SNV and protein coding STAT4 SNV in non-AYA was found (p<0.001).
Summary/Conclusion: The recurrent germline mut. in JAK-STAT pathway genes establish a linkage to map certain DNA regions that may be responsible for the MPN trait in AYA and Non-AYA. The integration of RNA-seq data will document the functional role of these genes and their potential cooperative activity with the classical phenotype driver mut.
Keywords: Myeloproliferative disorder