Exon-Level Genomic Abnormalities Are Frequent in Cytogenetically Normal Patients with Philadelphia-Chromosome Negative Myeloproliferative Neoplasms (MPNs)

Polycythemia vera (PV) and essential thrombocythemia (ET) exhibit an abnormal karyotype in 10%-28% of patients (pts) while the frequency increases in up to 90% of pts with advanced forms of myelofibrosis (PMF) and MPN accelerated/blast phase. Similarly, Klampfl et al. (Blood,118(1) 167-176) demonstrated that an increase in the number of genomic abnormalities detected by chromosomal microarrays (CMA) is associated with disease progression. Although genomic aberrations in cytogenetically normal pts with MPN have been previously reported, this study focused on identifying cryptic genomic alterations with exon-level precision to genomically characterize pts with MPN that were karyotypically normal at the time of CMA analysis.

We prospectively evaluated a cohort of 151 cytogenetically normal pts (23% PV, 35% ET, 21% PMF, and 21% post PV/ET MF) using a high-resolution chromosomal microarray (CMA) platform that detects both regions of copy-neutral loss of heterozygosity (cnLOH) and copy number alterations (CNAs) with exon-level resolution (Agilent's cancer-specific 2x400k aCGH+SNP). In order to uncover regions harboring genes that may have biological and/or clinical implications, we identified the minimal overlapping regions (MORs) containing CMA abnormalities occurring in at least 4 pts, using the "GRanges" package (Bioconductor).

Genomic aberrations were documented in 69% (n=104) of pts. Of the 238 CMA abnormalities identified, 52 were gains, 38 were losses, and 148 were regions of cnLOH. Next-generation sequencing (NGS) was performed in 97% of these pts and 65% had JAK2, 15% had CALR, and 2% had MPL mutations. The remaining pts (n=25) were triple-negative, and 12 pts harbored other non-driver gene mutations. Only CMA lesions were identified in 6 pts (4%) and both NGS and CMA could not genomically detect abnormalities in 7 pts (5%). The most frequent MOR mapping to a single gene harbored JAK2 cnLOH which occurred in 25% of pts, consistent with previous reports. A statistical difference in the JAK2V617F variant allele frequency (VAF) in pts with 9p cnLOH (VAF=80%) and without 9p cnLOH (VAF=18%) (p<0.001) was observed. In pts with PV, a high JAK2 allele burden is associated with an aggressive phenotype (Vannucchi et al. Therapeutic Advances in Hematology 2010 2, 21-32). Alterations of 1q32.1 involving MDM4, a negative regulator of TP53, was the second most frequent MOR occurring in 14% of pts. We have recently demonstrated that gain of 1q results in overexpression of MDM4 and is associated with advanced forms of MPN (Marcellino et al, Blood Advances, 2018 2(24), 3581-3589). Four other recurrent MORs harboring a single gene, TAF15, RASA3, RHOC, and NCOR2 occurred in 8%, 5%, 4%, and 3% of pts, respectively. Each had intragenic gains. We previously reported alterations in these genes in the spleens of pts with MF (Zimran et al., Haematologica, 2018 (10)103). Our current results indicate that these same alterations are present in the bone marrow cells of cytogenetically normal MPN pts. Since these abnormalities are present in pts with advanced forms of MPN, alterations of these genes may represent markers for disease progression. Of the MORs harboring multiple genes, 3 regions involved 1p, 17q, and 18q in 4%, 3%, and 6% of pts, respectively. All pts had cnLOH of these regions and each harbored relevant genes that may be implicated in MPN; CDKN2C (1p), PPM1D (17q), and ASXL3(18q).

Our results demonstrated that CMA abnormalities were present in 51% of pts with ET, 66% with PV, 87% with PMF, and 70%-100% with post ET/PV MF. CMA lesions were the sole alteration in 4% while 5% of chromosomally normal pts with an initial diagnosis of ET had neither somatic mutations nor CMA abnormalities. We provided evidence that recurrent genomic alterations can be identified in regions associated with disease progression, as well as novel regions harboring genes with potential pathobiological implications. We conclude that CMA is a vital tool in identifying cryptic genomic aberrations in cytogenetically normal pts with chronic phase MPN.