Mutations in cancer-relevant genes are ubiquitous in histologically normal endometrial tissue.

OBJECTIVE: Endometrial cancer (EndoCA) is the most common gynecologic cancer and incidence and mortality rate continue to increase. Despite well-characterized knowledge of EndoCA-defining mutations, no effective diagnostic or screening tests exist. To lay the foundation for testing development, our study focused on defining the prevalence of somatic mutations present in non-cancerous uterine tissue. METHODS: We obtained ≥8 uterine samplings, including separate endometrial and myometrial layers, from each of 22 women undergoing hysterectomy for non-cancer conditions. We ultra-deep sequenced (>2000× coverage) samples using a 125 cancer-relevant gene panel. RESULTS: All women harbored complex mutation patterns. In total, 308 somatic mutations were identified with mutant allele frequencies ranging up to 96.0%. These encompassed 56 unique mutations from 24 genes. The majority of samples possessed predicted functional cancer mutations but curiously no growth advantage over non-functional mutations was detected. Functional mutations were enriched with increasing patient age (p = 0.045) and BMI (p = 0.0007) and in endometrial versus myometrial layers (68% vs 39%, p = 0.0002). Finally, while the somatic mutation landscape shared similar mutation prevalence in key TCGA-defined EndoCA genes, notably PIK3CA, significant differences were identified, including NOTCH1 (77% vs 10%), PTEN (9% vs 61%), TP53 (0% vs 37%) and CTNNB1 (0% vs 26%). CONCLUSIONS: An important caveat for future liquid biopsy/DNA-based cancer diagnostics is the repertoire of shared and distinct mutation profiles between histologically unremarkable and EndoCA tissues. The lack of selection pressure between functional and non-functional mutations in histologically unremarkable uterine tissue may offer a glimpse into an unrecognized EndoCA protective mechanism.

The Role of Hypoxia-Inducible Factor 1 in Mild Cognitive Impairment.

Neuroinflammation and reactive oxygen species are thought to mediate the pathogenesis of Alzheimer's disease (AD), suggesting that mild cognitive impairment (MCI), a prodromal stage of AD, may be driven by similar insults. Several studies document that hypoxia-inducible factor 1 (HIF-1) is neuroprotective in the setting of neuronal insults, since this transcription factor drives the expression of critical genes that diminish neuronal cell death. HIF-1 facilitates glycolysis and glucose metabolism, thus helping to generate reductive equivalents of NADH/NADPH that counter oxidative stress. HIF-1 also improves cerebral blood flow which opposes the toxicity of hypoxia. Increased HIF-1 activity and/or expression of HIF-1 target genes, such as those involved in glycolysis or vascular flow, may be an early adaptation to the oxidative stressors that characterize MCI pathology. The molecular events that constitute this early adaptation are likely neuroprotective, and might mitigate cognitive decline or the onset of full-blown AD. On the other hand, prolonged or overwhelming stressors can convert HIF-1 into an activator of cell death through agents such as Bnip3, an event that is more likely to occur in late MCI or advanced Alzheimer's dementia.

Thrombotic Thrombocytopenic Purpura Associated With Pegylated Interferon Alfa-2a Use in a Patient With Polycythemia Vera.

Pegylated interferon alfa-2a (pegIFNa) is being increasingly used for treatment of myeloproliferative neoplasms; however, its side effects, including autoimmune complications, are not unusual. We report on a 47-year-old woman with polycythemia vera (PV) treated with pegIFNa and in complete hematologic remission who developed thrombotic thrombocytopenic purpura (TTP). To our knowledge, thrombotic microangiopathy has been reported as a side effect of interferon (IFN) use in patients with hepatitis and chronic myeloid leukemia, but not in those with PV. Our patient had a low ADAMTS13 level due to an inhibitor, which normalized after withholding pegIFNa and initiating treatment for TTP with therapeutic plasma exchange and corticosteroids. She experienced refractory TTP, necessitating treatment with rituximab and splenectomy. Postsplenectomy, she developed a high platelet count, warranting the need to restart treatment for PV. However, JAK2V617F allelic burden by real-time PCR was 0.7%, indicating that the increased platelet count was likely secondary to splenectomy. Therefore, we elected to monitor her counts and JAK2V617F allelic burden closely. With this case report, we hope to alert treating physicians that TTP should be considered as a complication of pegIFNa therapy in PV and that prompt discontinuation of the drug with necessary treatment should be instituted to prevent fatal complications.

The evolution of cellular deficiency in GATA2 mutation.

Constitutive heterozygous GATA2 mutation is associated with deafness, lymphedema, mononuclear cytopenias, infection, myelodysplasia (MDS), and acute myeloid leukemia. In this study, we describe a cross-sectional analysis of 24 patients and 6 relatives with 14 different frameshift or substitution mutations of GATA2. A pattern of dendritic cell, monocyte, B, and natural killer (NK) lymphoid deficiency (DCML deficiency) with elevated Fms-like tyrosine kinase 3 ligand (Flt3L) was observed in all 20 patients phenotyped, including patients with Emberger syndrome, monocytopenia with Mycobacterium avium complex (MonoMAC), and MDS. Four unaffected relatives had a normal phenotype indicating that cellular deficiency may evolve over time or is incompletely penetrant, while 2 developed subclinical cytopenias or elevated Flt3L. Patients with GATA2 mutation maintained higher hemoglobin, neutrophils, and platelets and were younger than controls with acquired MDS and wild-type GATA2. Frameshift mutations were associated with earlier age of clinical presentation than substitution mutations. Elevated Flt3L, loss of bone marrow progenitors, and clonal myelopoiesis were early signs of disease evolution. Clinical progression was associated with increasingly elevated Flt3L, depletion of transitional B cells, CD56(bright) NK cells, naïve T cells, and accumulation of terminally differentiated NK and CD8(+) memory T cells. These studies provide a framework for clinical and laboratory monitoring of patients with GATA2 mutation and may inform therapeutic decision-making.

Methylation of AR locus does not always reflect X chromosome inactivation state.

Clonality can be established by a lack of mosaicism in a female because of random inactivation of either the maternal or paternal X chromosome early in embryogenesis. The methylation status of CpG sites close to the trinucleotide repeats in exon 1 of the human androgen receptor (AR) X chromosome gene assay (HUMARA) has been used to determine clonality. This HUMARA at times indicated clonal hematopoiesis in healthy elderly women, thus precluding its applicability. We used a clonality assay based on quantitative expression of polymorphic X chromosome genes (qTCA) and found no evidence of clonal hematopoiesis in healthy nonanemic elderly persons. We found instances of discordance between HUMARA results and those obtained by pyrosequencing and qTCA methods, as well as by directly quantifying AR gene expression. To determine the basis of this discrepancy we examined the methylation pattern of the AR locus subject to HUMARA. Notably, we found the extent of DNA methylation to be highly variable at the AR gene in granulocytes of persons with discordant results and also in erythroid burst-forming unit colonies but not in those with clonal hematopoiesis. These data provide the molecular basis of incomplete correlation with the pattern of DNA methylation of this X chromosome AR gene locus.

The homozygous VHL(D126N) missense mutation is associated with dramatically elevated erythropoietin levels, consequent polycythemia, and early onset severe pulmonary hypertension.

von Hippel-Lindau (VHL) protein is the principal negative regulator of hypoxia sensing mediated by transcription factors. Mutations in exon 3 of the VHL gene lead to Chuvash (VHL(R200W)) and Croatian (VHL(H191D)) polycythemias. Here, we describe an infant of Bangladesh ethnicity with a novel homozygous VHL(D126N) mutation with congenital polycythemia and dramatically elevated erythropoietin (EPO) levels, who developed severe fatal pulmonary hypertension. In contrast to Chuvash polycythemia, erythroid progenitors (BFU-Es) did not reveal a marked EPO hypersensitivity. Further, NF-E2 and RUNX1 transcripts that correlate with BFU-Es EPO hypersensitivity in polycythemic mutations were not elevated.

Combined de-novo mutation and non-random X-chromosome inactivation causing Wiskott-Aldrich syndrome in a female with thrombocytopenia.

OBJECTIVE: Disorders linked to mutations in the X chromosomes typically affect males. The aim of the study is to decipher the mechanism of disease expression in a female patient with a heterozygous mutation on the X-chromosome. PATIENTS AND METHODS: Clinical data was extracted from the Canadian Inherited Marrow Failure Registry. Genomic ribonucleic acid (DNA) and complementary DNA (cDNA) underwent Sanger sequencing. Protein analysis was performed by flow cytometry. X-inactivation patterns were analyzed by evaluating the DNA methylation status and cDNA clonal expression of several genes on the X-chromosome. SNP array was used for molecular karyotyping of the X-chromosome. RESULTS: A female with thrombocytopenia, eczema and mild T-lymphocyte abnormalities with extensive negative diagnostic testing, was suspected to have Wiskott-Aldrich syndrome (WAS)/X-linked thrombocytopenia. Although the girl had a mutation (c.397G > A, p.E133K) in only one allele, she was found to have an extremely skewed X-inactivation pattern and no expression of the WAS protein. Family studies using DNA methylation analysis and cDNA clonal expression of several genes on the X-chromosome demonstrated that the patient developed de-novo non-random inactivation of the X-chromosome that does not carry the mutation. Genome-wide high-density molecular karyotyping excluded deletions and amplifications as a cause for the non-random inactivation of one X-chromosome. CONCLUSIONS: Our study emphasizes the need to test selected female patients with complete or incomplete disease expression for X-linked disorders even in the absence of a family history.

Down-regulation of the RUNX1-target gene NR4A3 contributes to hematopoiesis deregulation in familial platelet disorder/acute myelogenous leukemia.

RUNX1 encodes a DNA-binding α subunit of the core-binding factor, a heterodimeric transcription factor. RUNX1 is a master regulatory gene in hematopoiesis and its disruption is one of the most common aberrations in acute leukemia. Inactivating or dominant-negative mutations in the RUNX1 gene have been also identified in pedigrees of familial platelet disorders with a variable propensity to develop acute myeloid leukemia (FPD/AML). We performed analysis of hematopoiesis from 2 FPD/AML pedigrees with 2 distinct RUNX1 germline mutations, that is, the R139X in a pedigree without AML and the R174Q mutation in a pedigree with AML. Both mutations induced a marked increase in the clonogenic potential of immature CD34(+)CD38(-) progenitors, with some self-renewal capacities observed only for R174Q mutation. This increased proliferation correlated with reduction in the expression of NR4A3, a gene previously implicated in leukemia development. We demonstrated that NR4A3 was a direct target of RUNX1 and that restoration of NR4A3 expression partially reduced the clonogenic potential of patient progenitors. We propose that the down-regulation of NR4A3 in RUNX1-mutated hematopoietic progenitors leads to an increase in the pool of cells susceptible to be hit by secondary leukemic genetic events.

Novel exon 2 α spectrin mutation and intragenic crossover: three morphological phenotypes associated with four distinct α spectrin defects.

Hereditary pyropoikilocytosis is a severe hemolytic anemia caused by spectrin deficiency and defective spectrin dimer self-association, typically found in African populations. We describe two Utah families of northern European ancestry including 2 propositi with atypical non-microcytic hereditary pyropoikilocytosis, 7 hereditary elliptocytosis members and one asymptomatic carrier. The underlying molecular defect is a novel mutation in the alpha(α) spectrin gene, SPTA(R34P) that impairs spectrin tetramer formation. It is inherited in trans to the hypomorphic SPTA(αLELY) in the 2 propositi and 5 of 7 hereditary elliptocytosis individuals indicating that SPTA(αLELY) is not the sole determinant of the variable clinical expression. α Spectrin mRNA was mildly decreased in all hereditary elliptocytosis subjects, whereas both hereditary pyropoikilocytosis propositi had a severe decrease to ~10% of normal. Genotyping identified a unique SPTA intragenic crossover and uniparental disomy in one hereditary elliptocytosis individual. Two additional crossover events demonstrated the susceptibility of SPTA gene to rearrangement and revealed a novel segregation of the two SPTA(αLELY) mutations. We conclude that the profound phenotypic heterogeneity in these families can be attributed to the SPTA(R34P) mutation in combination with: 1) inheritance in trans of either SPTA(αLELY); or 2) the wild-type SPTA; 3) a decrease of α spectrin mRNA; and 4) SPTA intragenic crossover.

Sickle cell disease resulting from uniparental disomy in a child who inherited sickle cell trait.

Sickle cell disease (SCD) is a classic example of a disorder with recessive Mendelian inheritance, in which each parent contributes one mutant allele to an affected offspring. However, there are exceptions to that rule. We describe here the first reported case of conversion of inherited sickle cell trait to SCD by uniparental disomy (UPD) resulting in mosaicism for SS and AS erythrocytes. A 14-year-old boy presented with splenomegaly and hemolysis. Although his father has sickle cell trait, his mother has no abnormal hemoglobin (Hb). DNA sequencing, performed to rule out Hb S/ß-thalassemia, detected homozygous Hb SS. Further studies revealed mosaic UPD of the ß-globin locus, more SS erythroid progenitors than AS, but a reverse ratio of erythrocytes resulting from the survival advantage of AS erythrocytes. This report exemplifies non-Mendelian genetics wherein a patient who inherited sickle cell trait has mild SCD resulting from postzygotic mitotic recombination leading to UPD.

Extent of hematopoietic involvement by TET2 mutations in JAK2V6¹7F polycythemia vera.

TET2 mutations are found in polycythemia vera and it was initially reported that there is a greater TET2 mutational burden than JAK2(V617F) in polycythemia vera stem cells and that TET2 mutations precede JAK2(V617F). We quantified the proportion of TET2, JAK2(V617F) mutations and X-chromosome allelic usage in polycythemia vera cells, BFU-Es and in vitro expanded erythroid progenitors and found clonal reticulocytes, granulocytes, platelets and CD34(+) cells. We found that TET2 mutations may also follow rather than precede JAK2(V617F) as recently reported by others. Only a fraction of clonal early hematopoietic precursors and largely polyclonal T cells carry the TET2 mutation. We showed that in vitro the concomitant presence of JAK2(V617F) and TET2 mutations favors clonal polycythemia vera erythroid progenitors in contrast with non-TET2 mutated progenitors. We conclude that loss-of-function TET2 mutations are not the polycythemia vera initiating events and that the acquisition of TET2 somatic mutations may increase the aggressivity of the polycythemia vera clone.

Hematopoiesis is not clonal in healthy elderly women.

Clonality assays, based on X-chromosome inactivation, discriminate active from inactive alleles. Skewing of X-chromosome allelic usage, based on preferential methylation of one of the HUMARA alleles, was reported as evidence of clonal hematopoiesis in approximately 30% of elderly women. Using a quantitative, transcriptionally based clonality assay, we reported X-chromosome-transcribed allelic ratio in blood cells of healthy women consistent with random X-inactivation of 8 embryonic hematopoietic stem cells. Furthermore, we did not detect clonal hematopoiesis in more than 200 healthy nonelderly women. In view of the susceptibility of aging hematopoietic stem cells to epigenetic dysregulation, we reinvestigated the issue of clonality in elderly women. Forty healthy women (ages 65-92 years; mean, 81.3 years) were tested by a novel, quantitative polymerase chain reaction (qPCR) transcriptional clonality assay. We did not detect clonal hematopoiesis in any of the tested subjects. We also tested DNA from the same granulocyte samples using the methylation-based HUMARA assay, and confirmed previous reports of approximately 30% extensively skewed or monoallelic methylation, in agreement with likely age-related deregulated methylation of the HUMARA gene locus. We conclude that the transcriptionally based X-chromosome clonality assays are suitable for evaluation of clonal hematopoiesis in elderly women.

Clonal hematopoiesis in hematological disorders: Three different scenarios.

Clonality studies can establish the single-cell origin of tumors and thus differentiate clonal malignant and premalignant processes from reactive polyclonal processes. Detection of clonal cells may be based on direct tracking of cell lineage-specific sequences or disease-specific somatic mutations identifying the clonal population. Historically, clonal hematopoiesis was defined using the principle of X-chromosome inactivation based on observation that in circulating clonal cells, only one of the active chromosomes was expressed. In myeloproliferative neoplasms (MPNs) virtually all circulating erythrocytes, platelets, and granulocytes are products of single mutated stem cells that preferentially differentiate into the myeloid rather than lymphoid lineage. Thus, clonal differentiated myeloid cells co-exist in circulation with polyclonal long-lived T lymphocytes that originated before the MPN-initiating somatic clonal event. Chronic lymphocytic leukemia (CLL) starts in a differentiating B cell, but other lymphoid lineages and myeloid cells remain polyclonal. Normal T and B cells co-exist with the CLL clone, but are diluted by the massively expanded CLL population, which outnumbers the residual normal cells. Clonal hematopoiesis of undetermined potential (CHIP) has been identified by whole-genome sequencing of healthy individuals. These clones contain a specific somatic mutation previously considered to be disease defining but are detected in only a small proportion of circulating leukocytes, and there is no obvious suppression of normal hematopoietic stem cells. However, more studies are needed to properly define these clones, their persistence or disappearance, and their relative propensity for transforming into leukemias, myeloproliferative neoplasms, or other clonal hematological malignancies.

Erythropoiesis in polycythemia vera is hyper-proliferative and has accelerated maturation.

Polycythemia vera (PV) is an acquired myeloproliferative clonal disorder, characterized by augmented erythropoiesis. To better define PV pathogenesis, we performed an in vitro erythroid expansion from peripheral blood mononuclear cells of controls and PV patients and evaluated the cells for proliferation, apoptosis, erythroid differentiation, and morphology at the defined time points. PV erythroid progenitors exhibited increased proliferation at days 9-14 and accelerated maturation at days 7-14, with a larger S-phase population (40%) than controls (20%) at day 11; however, the proportion of apoptotic cells was comparable to controls. Previously, we have identified PV-specific dysregulation of several microRNAs (i.e. miR-150, 451, 222, 155, 378). We had analyzed expression profiles of selected target genes of these microRNAs based on in silico prediction and their known function pertinent to the observed PV-specific erythropoiesis differences. p27, cMYB and EPOR showed differential expression in PV erythroid progenitors at the specific stages of erythroid differentiation. In this study, we identified accelerated maturation and hyper-proliferation at early stages of PV erythropoiesis. We speculate that aberrant expression of p27, c-MYB, and EPOR may contribute to these abnormal features in PV erythropoiesis.

Concordance of assays designed for the quantification of JAK2V617F: a multicenter study.

BACKGROUND: Many different techniques have been designed for the quantification of JAK2V617F allelic burden, sometimes producing discrepant results. DESIGN AND METHODS: JAK2V617F quantification techniques were compared among 16 centers using 11 assays based on quantitative polymerase chain reaction (with mutation-specific primers or probes, or fluorescent resonance energy transfer/melting curve analysis), allele-specific polymerase chain reaction, conventional sequencing or pyrosequencing. RESULTS: A first series of blinded samples (granulocyte DNA, n=29) was analyzed. Seven assays (12 centers) reported values inside the mean +/- 2SD; the mean coefficient of variation was 31%. Sequencing techniques lacked sensitivity, and strong discrepancies were observed with four techniques, which could be attributed to inadequate standards or to different modes of expression of results. Indeed, quantification of JAK2V617F in relation to another control gene produced higher than expected values, suggesting the possibility of more than two JAK2 copies/cell. After calibration of assays with common 1% to 100% JAK2V617F standards (dilutions of UKE-1 cells in normal leukocytes), 14 centers tested ten new samples. JAK2V617F allelic burdens greater or equal than 1% were then reliably quantified by five techniques -- one allele specific-polymerase chain reaction and four TaqMan allele-specific quantitative polymerase chain reaction assays, including one previously giving results outside the mean +/- 2SD -- with a lower mean coefficient of variation (21%). Of these, only the two TaqMan allele-specific quantitative polymerase chain reaction assays with primer-based specificity could detect 0.2% JAK2V617F. CONCLUSIONS: Techniques expressing the allelic burden as JAK2V617F/total JAK2 and using a common set of standards produced similar quantification results but with variable sensitivity. Calibration to a reference standard improved reproducibility.

Nuclear-Cytoplasmic Transport Is a Therapeutic Target in Myelofibrosis.

PURPOSE: Myelofibrosis is a hematopoietic stem cell neoplasm characterized by bone marrow reticulin fibrosis, extramedullary hematopoiesis, and frequent transformation to acute myeloid leukemia. Constitutive activation of JAK/STAT signaling through mutations in JAK2, CALR, or MPL is central to myelofibrosis pathogenesis. JAK inhibitors such as ruxolitinib reduce symptoms and improve quality of life, but are not curative and do not prevent leukemic transformation, defining a need to identify better therapeutic targets in myelofibrosis. EXPERIMENTAL DESIGN: A short hairpin RNA library screening was performed on JAK2V617F-mutant HEL cells. Nuclear-cytoplasmic transport (NCT) genes including RAN and RANBP2 were among top candidates. JAK2V617F-mutant cell lines, human primary myelofibrosis CD34+ cells, and a retroviral JAK2V617F-driven myeloproliferative neoplasms mouse model were used to determine the effects of inhibiting NCT with selective inhibitors of nuclear export compounds KPT-330 (selinexor) or KPT-8602 (eltanexor). RESULTS: JAK2V617F-mutant HEL, SET-2, and HEL cells resistant to JAK inhibition are exquisitely sensitive to RAN knockdown or pharmacologic inhibition by KPT-330 or KPT-8602. Inhibition of NCT selectively decreased viable cells and colony formation by myelofibrosis compared with cord blood CD34+ cells and enhanced ruxolitinib-mediated growth inhibition and apoptosis, both in newly diagnosed and ruxolitinib-exposed myelofibrosis cells. Inhibition of NCT in myelofibrosis CD34+ cells led to nuclear accumulation of p53. KPT-330 in combination with ruxolitinib-normalized white blood cells, hematocrit, spleen size, and architecture, and selectively reduced JAK2V617F-mutant cells in vivo. CONCLUSIONS: Our data implicate NCT as a potential therapeutic target in myelofibrosis and provide a rationale for clinical evaluation in ruxolitinib-exposed patients with myelofibrosis.

Kinetic and spectroscopic analysis of the catalytic role of H79 in the methionine aminopeptidase from Escherichia coli.

To gain insight into the role of the strictly conserved histidine residue, H79, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli ( EcMetAP-I), the H79A mutated enzyme was prepared. Co(II)-loaded H79A exhibits an overall >7000-fold decrease in specific activity. The almost complete loss of activity is primarily due to a >6000-fold decrease in k cat. Interestingly, the K m value obtained for Co(II)-loaded H79A was approximately half the value observed for wild-type (WT) EcMetAP-I. Consequently, k cat/ K m values decreased only 3000-fold. On the other hand, the observed specific activity of Mn(II)-loaded H79A EcMetAP-I decreased by approximately 2.6-fold while k cat decreased by approximately 3.5-fold. The observed K m value for Mn(II)-loaded H79A EcMetAP-I was approximately 1.4-fold larger than that observed for WT EcMetAP-I, resulting in a k cat/ K m value that is lower by approximately 3.4-fold. Metal binding, UV-vis, and EPR data indicate that the active site is unperturbed by mutation of H79, as suggested by X-ray crystallographic data. Kinetic isotope data indicate that H79 does not transfer a proton to the newly forming amine since a single proton is transferred in the transition state for both the WT and H79A EcMetAP-I enzymes. Therefore, H79 functions to position the substrate by hydrogen bonding to either the amine group of the peptide linkage or a backbone carbonyl group. Together, these data provide new insight into the catalytic mechanism of EcMetAP-I.