Combined Effects of Clonal Hematopoiesis and Carotid Stenosis on Cardiovascular Mortality.

BACKGROUND: The expansion of hematopoietic stem cells caused by acquired somatic mutations (clonal hematopoiesis [CH]) is a novel cardiovascular risk factor. The prognostic value of CH in patients with carotid atherosclerosis remains to be evaluated. OBJECTIVES: This study assessed the prognostic significance of CH in patients with atherosclerosis as detected by ultrasound of the carotid artery. METHODS: We applied deep sequencing of selected genomic regions within the genes DNMT3A, TET2, ASXL1, and JAK2 to screen for CH in 968 prospectively collected patients with asymptomatic carotid atherosclerosis evaluated by duplex sonography. RESULTS: We detected clonal markers at variant allele frequency ≥2% in 133 (13.7%) of 968 patients (median age 69.2 years), with increasing prevalence at advanced age. Multivariate analyses including age and established cardiovascular risk factors revealed overall presence of CH to be significantly associated with increased risk of cardiovascular death (HR: 1.50; 95% CI: 1.12-2.00; P = 0.007), reflected also at the single gene level. The effect of CH was more pronounced in older patients and independent of the patients' inflammatory status as measured by high-sensitivity C-reactive protein. Simultaneous assessment of CH and degree of carotid stenosis revealed combined effects on cardiovascular mortality, depicted by a superior risk for patients with >50% stenosis and concomitant CH (adjusted HR: 1.60; 95% CI: 1.08-2.38; P = 0.020). CONCLUSIONS: CH status in combination with the extent of carotid atherosclerosis jointly predict long-term mortality. Determination of CH can provide additional prognostic information in patients with asymptomatic carotid atherosclerosis.

Evaluation of the ATM L2307F germline variant in 121 Italian pedigrees with familial myeloproliferative neoplasms.

Not available.

Effects of Tulp4 deficiency on murine embryonic development and adult phenotype.

Genetically engineered mouse models have the potential to unravel fundamental biological processes and provide mechanistic insights into the pathogenesis of human diseases. We have previously observed that germline genetic variation at the TULP4 locus influences clinical characteristics in patients with myeloproliferative neoplasms. To elucidate the role of TULP4 in pathological and physiological processes in vivo, we generated a Tulp4 knockout mouse model. Systemic Tulp4 deficiency exerted a strong impact on embryonic development in both Tulp4 homozygous null (Tulp4-/-) and heterozygous (Tulp4+/-) knockout mice, the former exhibiting perinatal lethality. High-resolution episcopic microscopy (HREM) of day 14.5 embryos allowed for the identification of multiple developmental defects in Tulp4-/- mice, including severe heart defects. Moreover, in Tulp4+/- embryos HREM revealed abnormalities of several organ systems, which per se do not affect prenatal or postnatal survival. In adult Tulp4+/- mice, extensive examinations of hematopoietic and cardiovascular features, involving histopathological surveys of multiple tissues as well as blood counts and immunophenotyping, did not provide evidence for anomalies as observed in corresponding embryos. Finally, evaluating a potential obesity-related phenotype as reported for other TULP family members revealed a trend for increased body weight of Tulp4+/- mice. RESEARCH HIGHLIGHTS: To study the role of the TULP4 gene in vivo, we generated a Tulp4 knockout mouse model. Correlative analyses involving HREM revealed a strong impact of Tulp4 deficiency on murine embryonic development.

PD-L1 overexpression correlates with JAK2-V617F mutational burden and is associated with 9p uniparental disomy in myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPN) are chronic stem cell disorders characterized by enhanced proliferation of myeloid cells, immune deregulation, and drug resistance. JAK2 somatic mutations drive the disease in 50-60% and CALR mutations in 25-30% of cases. Published data suggest that JAK2-V617F-mutated MPN cells express the resistance-related checkpoint PD-L1. By applying RNA-sequencing on granulocytes of 113 MPN patients, we demonstrate that PD-L1 expression is highest among polycythemia vera patients and that PD-L1 expression correlates with JAK2-V617F mutational burden (R = 0.52; p < .0001). Single nucleotide polymorphism (SNP) arrays showed that chromosome 9p uniparental disomy (UPD) covers both PD-L1 and JAK2 in all MPN patients examined. MPN cells in JAK2-V617F-positive patients expressed higher levels of PD-L1 if 9p UPD was present compared to when it was absent (p < .0001). Moreover, haplotype-based association analyses provided evidence for germline genetic factors at PD-L1 locus contributing to MPN susceptibility independently of the previously described GGCC risk haplotype. We also found that PD-L1 is highly expressed on putative CD34+ CD38- disease-initiating neoplastic stem cells (NSC) in both JAK2 and CALR-mutated MPN. PD-L1 overexpression decreased upon exposure to JAK2 blockers and BRD4-targeting agents, suggesting a role for JAK2-STAT5-signaling and BRD4 in PD-L1 expression. Whether targeting of PD-L1 can overcome NSC resistance in MPN remains to be elucidated in forthcoming studies.

Germline genetic factors influence the outcome of interferon-α therapy in polycythemia vera.

Interferon-α (IFN-α)-based treatments can induce hematologic and molecular responses (HRs and MRs, respectively) in polycythemia vera (PV); however, patients do not respond equally. Germline genetic factors have been implicated in differential drug responses. We addressed the effect of common germline polymorphisms on HR and MR after treatment of PV in the PROUD-PV and CONTINUATION-PV studies in a total of 122 patients who received ropeginterferon alfa-2b. Genome-wide association studies using longitudinal data on HR and MR over a 36-month follow-up did not reveal any associations at the level of genome-wide statistical significance. Furthermore, we performed targeted association analyses at the interferon lambda 4 (IFNL4) locus, well known for its role in hepatitis C viral clearance and recently reported to influence HR during treatment of myeloproliferative neoplasms. We did not observe any association of IFNL4 polymorphisms with HR in our study cohort; however, we demonstrated a statistically significant effect of the functionally causative IFNL4 diplotype (haplotype pair, including the protein-coding variants rs368234815/rs117648444) on MR (P = 3.91 × 10-4; odds ratio, 10.80; 95% confidence interval, 2.39-69.97) as reflected in differential JAK2V617F mutational burden changes according to IFNL4 diplotype status. Stratification of patients with PV based on IFNL4 functionality may allow for optimizing patient management during IFN-α-based therapy.

Myelomonocytic Skewing In Vitro Discriminates Subgroups of Patients with Myelofibrosis with A Different Phenotype, A Different Mutational Profile and Different Prognosis.

Normal hematopoietic function is maintained by a well-controlled balance of myelomonocytic, megaerythroid and lymphoid progenitor cell populations which may be skewed during pathologic conditions. Using semisolid in vitro cultures supporting the growth of myelomonocytic (CFU-GM) and erythroid (BFU-E) colonies, we investigated skewed differentiation towards the myelomonocytic over erythroid commitment in 81 patients with myelofibrosis (MF). MF patients had significantly increased numbers of circulating CFU-GM and BFU-E. Myelomonocytic skewing as indicated by a CFU-GM/BFU-E ratio ≥ 1 was found in 26/81 (32%) MF patients as compared to 1/98 (1%) in normal individuals. Patients with myelomonocytic skewing as compared to patients without skewing had higher white blood cell and blast cell counts, more frequent leukoerythroblastic features, but lower hemoglobin levels and platelet counts. The presence of myelomonocytic skewing was associated with a higher frequency of additional mutations, particularly in genes of the epigenetic and/or splicing machinery, and a significantly shorter survival (46 vs. 138 mo, p < 0.001). The results of this study show that the in vitro detection of myelomonocytic skewing can discriminate subgroups of patients with MF with a different phenotype, a different mutational profile and a different prognosis. Our findings may be important for the understanding and management of MF.

Mutational landscape of the transcriptome offers putative targets for immunotherapy of myeloproliferative neoplasms.

Ph-negative myeloproliferative neoplasms (MPNs) are hematological cancers that can be subdivided into entities with distinct clinical features. Somatic mutations in JAK2, CALR, and MPL have been described as drivers of the disease, together with a variable landscape of nondriver mutations. Despite detailed knowledge of disease mechanisms, targeted therapies effective enough to eliminate MPN cells are still missing. In this study of 113 MPN patients, we aimed to comprehensively characterize the mutational landscape of the granulocyte transcriptome using RNA sequencing data and subsequently examine the applicability of immunotherapeutic strategies for MPN patients. Following implementation of customized workflows and data filtering, we identified a total of 13 (12/13 novel) gene fusions, 231 nonsynonymous single nucleotide variants, and 21 insertions and deletions in 106 of 113 patients. We found a high frequency of SF3B1-mutated primary myelofibrosis patients (14%) with distinct 3' splicing patterns, many of these with a protein-altering potential. Finally, from all mutations detected, we generated a virtual peptide library and used NetMHC to predict 149 unique neoantigens in 62% of MPN patients. Peptides from CALR and MPL mutations provide a rich source of neoantigens as a result of their unique ability to bind many common MHC class I molecules. Finally, we propose that mutations derived from splicing defects present in SF3B1-mutated patients may offer an unexplored neoantigen repertoire in MPNs. We validated 35 predicted peptides to be strong MHC class I binders through direct binding of predicted peptides to MHC proteins in vitro. Our results may serve as a resource for personalized vaccine or adoptive cell-based therapy development.

Whole-exome sequencing identifies novel MPL and JAK2 mutations in triple-negative myeloproliferative neoplasms.

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed "triple negative." We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis analyzed by WES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1 germline (R321W) MPL mutation were detected. All of the identified MPL mutations were gain-of-function when analyzed in functional assays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders.

Capture Hi-C identifies the chromatin interactome of colorectal cancer risk loci.

Multiple regulatory elements distant from their targets on the linear genome can influence the expression of a single gene through chromatin looping. Chromosome conformation capture implemented in Hi-C allows for genome-wide agnostic characterization of chromatin contacts. However, detection of functional enhancer-promoter interactions is precluded by its effective resolution that is determined by both restriction fragmentation and sensitivity of the experiment. Here we develop a capture Hi-C (cHi-C) approach to allow an agnostic characterization of these physical interactions on a genome-wide scale. Single-nucleotide polymorphisms associated with complex diseases often reside within regulatory elements and exert effects through long-range regulation of gene expression. Applying this cHi-C approach to 14 colorectal cancer risk loci allows us to identify key long-range chromatin interactions in cis and trans involving these loci.

Common germline variation at the TERT locus contributes to familial clustering of myeloproliferative neoplasms.

The C allele of the rs2736100 single nucleotide polymorphism located in the second intron of the TERT gene has recently been identified as a susceptibility factor for myeloproliferative neoplasms (MPN) in the Icelandic population. Here, we evaluate the role of TERT rs2736100_C in sporadic and familial MPN in the context of the previously identified JAK2 GGCC predisposition haplotype. We have confirmed the TERT rs2736100_C association in a large cohort of Italian sporadic MPN patients. The risk conferred by TERT rs2736100_C is present in all molecular and diagnostic MPN subtypes. TERT rs2736100_C and JAK2 GGCC are independently predisposing to MPN and have an additive effect on disease risk, together explaining a large fraction of the population attributable fraction (PAF = 73.06%). We found TERT rs2736100_C significantly enriched (P = 0.0090) in familial MPN compared to sporadic MPN, suggesting that low-penetrance variants may be responsible for a substantial part of familial clustering in MPN.

Genome integrity of myeloproliferative neoplasms in chronic phase and during disease progression.

Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) are clonal myeloid disorders with increased production of terminally differentiated cells. The disease course is generally chronic, but some patients show disease progression (secondary myelofibrosis or accelerated phase) and/or leukemic transformation. We investigated chromosomal aberrations in 408 MPN samples using high-resolution single-nucleotide polymorphism microarrays to identify disease-associated somatic lesions. Of 408 samples, 37.5% had a wild-type karyotype and 62.5% harbored at least 1 chromosomal aberration. We identified 25 recurrent aberrations that were found in 3 or more samples. An increased number of chromosomal lesions was significantly associated with patient age, as well as with disease progression and leukemic transformation, but no association was observed with MPN subtypes, Janus kinase 2 (JAK2) mutational status, or disease duration. Aberrations of chromosomes 1q and 9p were positively associated with disease progression to secondary myelofibrosis or accelerated phase. Changes of chromosomes 1q, 7q, 5q, 6p, 7p, 19q, 22q, and 3q were positively associated with post-MPN acute myeloid leukemia. We mapped commonly affected regions to single target genes on chromosomes 3p (forkhead box P1 [FOXP1]), 4q (tet oncogene family member 2 [TET2]), 7p (IKAROS family zinc finger 1 [IKZF1]), 7q (cut-like homeobox 1 [CUX1]), 12p (ets variant 6 [ETV6]), and 21q (runt-related transcription factor 1 [RUNX1]). Our data provide insight into the genetic complexity of MPNs and implicate new genes involved in disease progression.

Molecular pathogenesis of Philadelphia chromosome negative chronic myeloproliferative neoplasms.

Clonal hematopoiesis triggered by somatic mutations plays a central role in the pathogenesis of Philadelphia chromosome negative chronic myeloproliferative neoplasms (MPNs). After the discovery of JAK and MPL mutations, continual technological advances have led to the identification of increasing numbers of genetic defects in MPN patients, most of them chromosomal aberrations such as deletions and acquired uniparental disomies. Although efforts to map the genetic lesions to single genes resulted in the discovery of defects in the TET2 and CBL genes, most of the target genes comprised in the chromosomal lesions still remain to be identified. In this review, the different genetic defects found in MPN and their relationships to each other and to disease pathogenesis are critically evaluated. At present, most of the evidence points to a random acquisition of phenotypic and non phenotypic mutations contributing to clonal heterogeneity in MPN. The origin of genetic instability in context of hereditary factors and the common JAK2 haplotype predisposing for the disease are discussed. Furthermore, we address how the choice of therapeutic approaches could be influenced by the genetic complexity.

The role of the JAK2 GGCC haplotype and the TET2 gene in familial myeloproliferative neoplasms.

BACKGROUND: Myeloproliferative neoplasms constitute a group of diverse chronic myeloid malignancies that share pathogenic features such as acquired mutations in the JAK2, TET2, CBL and MPL genes. There are recent reports that a JAK2 gene haplotype (GGCC or 46/1) confers susceptibility to JAK2 mutation-positive myeloproliferative neoplasms. The aim of this study was to examine the role of the JAK2 GGCC haplotype and germline mutations of TET2, CBL and MPL in familial myeloproliferative neoplasms. DESIGN AND METHODS: We investigated patients with familial (n=88) or sporadic (n=684) myeloproliferative neoplasms, and a control population (n=203) from the same demographic area in Italy. Association analysis was performed using tagged single nucleotide polymorphisms (rs10974944 and rs12343867) of the JAK2 haplotype. Sequence analysis of TET2, CBL and MPL was conducted in the 88 patients with familial myeloproliferative neoplasms. RESULTS: Association analysis revealed no difference in haplotype frequency between familial and sporadic cases of myeloproliferative neoplasms (P=0.6529). No germline mutations in TET2, CBL or MPL that segregate with the disease phenotype were identified. As we observed variability in somatic mutations in the affected members of a pedigree with myeloproliferative neoplasms, we postulated that somatic mutagenesis is increased in familial myeloproliferative neoplasms. Accordingly, we compared the incidence of malignant disorders between sporadic and familial patients. Although the overall incidence of malignant disorders did not differ significantly between cases of familial and sporadic myeloproliferative neoplasms, malignancies were more frequent in patients with familial disease aged between 50 to 70 years (P=0.0198) than in patients in the same age range with sporadic myeloproliferative neoplasms. CONCLUSIONS: We conclude that the JAK2 GGCC haplotype and germline mutations of TET2, CBL or MPL do not explain familial clustering of myeloproliferative neoplasms. As we observed an increased frequency of malignant disorders in patients with familial myeloproliferative neoplasms, we hypothesize that the germline genetic lesions that underlie familial clustering of myeloproliferative neoplasms predispose to somatic mutagenesis that is not restricted to myeloid hematopoietic cells but cause an increase in overall carcinogenesis.

A common JAK2 haplotype confers susceptibility to myeloproliferative neoplasms.

Genome-wide association studies have identified a number of new disease susceptibility loci that represent haplotypes defined by numerous SNPs. SNPs within a disease-associated haplotype are thought to influence either the expression of genes or the sequence of the proteins they encode. In a series of investigations of the JAK2 gene in myeloproliferative neoplasms, we uncovered a new property of haplotypes that can explain their disease association. We observed a nonrandom distribution of the somatic JAK2(V617F) oncogenic mutation between two parental alleles of the JAK2 gene. We identified a haplotype that preferentially acquires JAK2(V617F) and confers susceptibility to myeloproliferative neoplasms. One interpretation of our results is that a certain combination of SNPs may render haplotypes differentially susceptible to somatic mutagenesis. Thus, disease susceptibility loci may harbor somatic mutations that have a role in disease pathogenesis.

Clonal analysis of deletions on chromosome 20q and JAK2-V617F in MPD suggests that del20q acts independently and is not one of the predisposing mutations for JAK2-V617F.

We developed a real-time copy number polymerase chain reaction assay for deletions on chromosome 20q (del20q), screened peripheral blood granulocytes from 664 patients with myeloproliferative disorders, and identified 19 patients with del20q (2.9%), of which 14 (74%) were also positive for JAK2-V617F. To examine the temporal relationship between the occurrence of del20q and JAK2-V617F, we performed colony assays in methylcellulose, picked individual burst-forming units-erythroid (BFU-E) and colony-forming units-granulocyte (CFU-G) colonies, and genotyped each colony individually for del20q and JAK2-V617F. In 2 of 9 patients, we found that some colonies with del20q carried only wild-type JAK2, whereas other del20q colonies were JAK2-V617F positive, indicating that del20q occurred before the acquisition of JAK2-V617F. However, in colonies from 3 of 9 patients, we observed the opposite order of events. The lack of a strict temporal order of occurrence makes it doubtful that del20q represents a predisposing event for JAK2-V617F. In 2 patients with JAK2-V617F and 1 patient with MPL-W515L, microsatellite analysis revealed that del20q affected chromosomes of different parental origin and/or 9pLOH occurred at least twice. The fact that rare somatic events, such as del20q or 9pLOH, occurred more than once in subclones from the same patients suggests that the myeloproliferative disorder clone carries a predisposition to acquiring such genetic alterations.