In 2008, Pardanani et al., based on an analysis of SNPs in four candidate genes (EPOR, MPL, GCSFR, JAK2), confirmed a significant association between the specific SNPs in the JAK2 gene (46/1 haplotype or GGCC haplotype) and the onset of sporadic MPNs (47). The term JAK2 haplotypeGGCC_46/1 refers to the combination of particular alleles of four SNPs inherited together and generating a GGCC allele’s combination (rs3780367 T/G in intron 10, rs10974944 C/G intron 12, rs12343867 T/C intron 14, and rs1159782 T/C intron 15). All four SNPs spanning the region of about 250kb from JAK2 intron 10 to the INSL4 gene, are in complete linkage disequilibrium (48). Now, the presence of 46/1 haplotype is considered a factor predisposing to the JAK2V617F acquisition and MPN development (49–51). The allele frequency of the JAK2 haplotypeGGCC_46/1 in the healthy population is about 24%. Its presence is significantly increased (40 to 80%) in JAK2V617F positive MPN pts (50–54). Recently, other germline variations of HBS1L-MYB, MECOM, and THRB-RARB have been also considered as factors determining the genetic predisposition to MPN (55, 56).
In our study, the distribution of different genotypes of the JAK2 haplotypeGGCC_46/1 in ET pts was similar to the previously published data (43). However, we showed that the JAK2 haplotypeGGCC_46/1 was significantly more frequent in JAK2V617F-positive than in CALR-positive ET pts. The latter observation is in agreement with the data previously reported by others (57).
The results of the study of an association between the presence of the JAK2 haplotypeGGCC_46/1 and the predisposition to CALR positive or MPL positive MPN are still inconclusive. Also, the association between the JAK2 haplotypeGGCC_46/1 and triple-negative MPN occurrence is not fully determined (58).
Our results confirmed a significantly higher JAK2V617F VAF in homozygous carriers of JAK2 haplotypeGGCC_46/1 (C/C genotype) and a significant increase of the C/C genotype in ET pts with JAK2V617F VAF > 50%.
An analysis of the total expression of the JAK2 gene in our ET pts group led to interesting results. The total JAK2 mRNA level did not significantly differ between pts defined by JAK2 haplotypeGGCC_46/1, but it was significantly increased in JAK2V617F positive pts. The association was evident, despite the confirmed correlation between the JAK2V617F VAF and JAK2V617F mRNA level. In our opinion, the increased total JAK2 expression in ET pts may result from different factors, including the allelic expression imbalance of JAK2 V617F mutation, MPN associated chronic inflammation, the presence of other non-coding SNP affecting the JAK2 expression, or mutations of epigenetic genes regulators [DNMT3A, TET2, EZH2, ASXL1, and IDH1/2 (via effects on TET2-mediated methylation)].
In 2013, Kim et al. demonstrated the JAK2V617F allelic imbalance expression by comparing the VAF in the total RNA (cDNA) and genomic DNA. They showed an increase of mutant allele at the RNA level, especially in ET (3 fold increase) and polycythaemia vera (PV) (2 fold increase) pts. It should be mentioned that the latter phenomenon was not observed in PMF pts (1.1 fold increase) (59). Another possibility of an increased total JAK2 mRNA expression in ET pts is abnormal JAK-STAT signaling. The potential link between a chronic inflammation and the development of myeloproliferative neoplasm has been postulated by Hasselbalch in 2012 (60). Later, it was shown that MPN driver mutations, such as JAK2V617F and MPL, were responsible for continuous, increased signaling via the JAK2-STAT pathway and the promotion of cytokine production by malignant and non-malignant cells (61–64). Probably, the abnormal JAK-STAT signaling resulted also in an abnormal total JAK2 expression. The above-mentioned hypothesis may be supported by the data concerning another JAK2 co-expressed gene – programmed death-ligand 1 (PD-L1). In 2019, Guru et al. showed that JAK2 V617F mutation was accompanied by an increased PD-L1 expression. An increased expression of PD-L1 may be caused by excessive activation of STAT3/5 which are the regulators of PD-L1. It was also shown that in the case of JAK2V617F, the PD-L1 expression was mainly mediated by STAT3 (65). The overexpression of PD-L1 may also be caused by the acquisition of 9q UPD, which was confirmed in 6–18% of cases with ET (32, 66).
Recently published data confirmed that the mRNA level of STAT3 was significantly higher in JAK2V617F positive pts with PV and ET. Moreover, the up-regulation of STAT3 and STAT5 was associated with JAK2V617F VAF (67). It should be noted that the PD-L1 mRNA level is not affected by the PD-L1 or JAK2 gene copy number variations, which has been shown in our study.
It cannot be excluded that another gene, SNP, also affects the JAK2 expression in pts with JAK2V617F positive ET. Recently, Cardinale et al. postulated that the rs1887428 SNP located in the promoter region of the JAK2 gene might influence the JAK2 expression in another JAK2V617F associated disease – inflammatory bowel disease. The study confirmed a very modest impact of the above-mentioned SNP on the JAK2 expression and downstream amplification effect through the expression of the pathway member STAT5B and epigenetic modification of the JAK2 locus (68).
In 2020, Jacquelin et al. confirmed the mutational cooperation between the JAK2V617F expression and the loss of DNA methyltransferase 3A in hematopoietic cells due to monoallelic or biallelic mutations of the DNMT3A gene. The coexistence of the above-mentioned mutations resulted in an aberrant self-renewal, inflammatory signaling, driven by increased accessibility at enhancer elements, and finally the progression of PV to the fibrotic phase (69). Another possibility includes the presence of other JAK2 gene mutations affecting the mRNA splicing machinery. mRNA investigations showed no splicing defects around exon 14, and a constant level of mRNA accumulation per JAK2 gene copy, regardless of the presence or absence of the exon 14 JAK2V617F mutation (48).
Another important question concerning the fluctuation of the total JAK2 mRNA level during a natural disease outcome and evolution into the fibrotic phase (post-ET-MF). The results of our study showed a lower total JAK2 mRNA level (JAK2V617F + wild type) in post-ET-MF, in comparison to ET pts. The detailed analysis has shown that the decrease in JAK2 and PD-L1 mRNA expression is gradual and depends on the bone marrow fibrosis grade. A similar analysis concerning JAK2V617F VAF in ET and post-ET-MF pts did not reveal significant differences. This is incompatible with the previous data confirming high JAK2V617F VAF in post-ET-MF pts (70). This discrepancy results likely from other criteria used previously for the diagnosis of PV, ET, and post-ET-MF, which might result in over diagnosis of ET, even instead of PV (71, 72). Such a possibility was confirmed by the Swedish National MPN Registry data, documenting the increased frequency of newly diagnosed ET and reduced frequency of newly diagnosed PV from 2008 to 2015 (73). The interpretation of the obtained data is difficult due to the fact that the fibrotic transformation in ET is rarely observed and occurs in 9–15% of pts during a long-term follow-up (74, 75). On the other hand, it should be noted that the clinical MPN manifestation is not only related to the type of the driver mutation, but also depends on the profile of other coexisting mutations modifying the disease phenotype. The frequency and VAF of specific coexisting mutation(s) differ between pts, contributing to a specific disease phenotype in individual cases (1, 11, 76). Among others, the mutations involved in the DNA methylation (ASXL1, TET2, DNMT3A, IDH1, IDH2), histone modification (EZH2, ASXL1), and splicing (SF3B1, SRSF2, U2AF1, and ZRSR2), and mutations in the transcription factors genes (RUNX1, NFE2, PPM1D, and TP53) are most frequently found in ET pts (77, 78). An adverse prognostic relevance of some of them (SH2B3/LNK, SF3B1, U2AF1, TP53, IDH2, and EZH2) on overall, leukemia-free and myelofibrosis-free survival of ET pts was recently demonstrated (76, 79–81). Furthermore, the ASXL1 mutations (most frequently found in post-ET-MF pts) have been also identified as a genetic risk factor for the fibrotic transformation of ET (21). Similarly, the co-occurrence of variants/mutations of SRSF2 and U2AF1 increased the risk of myelofibrotic evolution in PV and ET pts, respectively (80). The results of our study confirmed the co-occurrence of ASXL1, SRSF2, U2AF1 mutations in the studied ET pts group. Their frequency was below 10% and was similar to that reported by others (79, 82). However, it should be mentioned that its frequency in our JAK2V617F positive cases was 2-times higher in post-ET-MF than in ET individuals.
According to the limited published results, the risk of fibrotic transformation in ET pts carrying MPL mutation is higher than in other driver mutation types (83). Another factor predisposing to fibrotic transformation which has been postulated by Rumi et al. is a copy-neutral loss of heterozygosity (CN-LOH) of chromosome 1p34.2 and high MPL allele burden (84). Recently, Ferrer-Marín et al. showed that the rs2431697 TT genotype affected the expression of miR-146a, a brake in NF-κB signaling, as a risk factor for the fibrotic transformation of PV and ET (85). It cannot be ruled out that epigenetic regulators affect the PD-L1 expression, as well. It was documented that PD-L1 expression might be downregulated by the abundance of miR-513, miR- 570, miR-34a, and miR-200 (86)(87)(88).
The interpretation of the lowered PD-L1 and JAK2 mRNA expression levels detected in our study in ET patients transforming to the fibrotic phase is difficult. The bone marrow failure in the advanced ET phase is likely associated with bone marrow fibrosis, reduction of bone marrow cellularity, bone marrow myeloid metaplasia, and diversity in molecular characteristics of the emerging subclones. Wang et al. showed that MF spleens contained greater numbers of malignant primitive hematopoietic cells than peripheral blood, and the significant increase in total CD34 + cells counts in JAK2V617F negative vs. positive samples (89). The latter may result in a distinct pattern of expression of JAK2 and PD-L1 genes (90)(91)(92). The association between PD-L1 expression and JAK2V617F mutation was recently documented by Hara et al. in a patient with a coexisting JAK2V617F‑positive ET and lung carcinoma in whom pembrolizumab (a drug directly blocking the interaction between PD-1 and its ligands, PD-L1, and PD-L2) treatment resulted in simultaneous normalization of the platelet count and a decrease of JAK2V617F VAF (93).
Despite the progress in the last years in the treatment of ET, none of the available therapies can change the outcome of the disease (94). Up to date, none of the therapies improve overall survival and prevent leukemic or fibrotic transformation (95). For these reasons, there is a need to identify a new potential molecular mechanism affecting the drug resistance to improve the ET outcome. One of them is the PD1/PD-L1 axis. In 2018, Holmström et al. documented that PD-L1 specific T cell response was stronger in pts with ET and PV and weaker and rarer in pts with pre-PMF and PMF MPN (96).
The results of our study can explain, at least in part, the lack of efficacy (clinical or bone marrow pathologic response) of the pembrolizumab treatment in patients with advanced primary, post-ET-MF and post-PV myelofibrosis (97) and shed more light on the relationship between the types of driver mutations, the PD-L1 expression and the ET progression to the fibrotic phase.