NGS-defined measurable residual disease (MRD) after initial chemotherapy as a prognostic biomarker for acute myeloid leukemia.

Treated AML patients often have measurable residual disease (MRD) due to persisting low-level clones. This study assessed whether residual post-treatment somatic mutations, detected by NGS, were significantly prognostic for subsequent clinical outcomes. AML patients (n = 128) underwent both pre-and post-treatment testing with the same 42-gene MRD-validated NGS assay. After induction, 59 (46%) patients were mutation-negative (0.0024 VAF detection limit) and 69 (54%) had ≥1 persisting NGS-detectable mutation. Compared with NGS-negative patients, NGS-positive patients had shorter overall survival (17 months versus median not reached; P = 0.004; hazard ratio = 2.2 [95% CI: 1.3-3.7]) and a shorter time to relapse (14 months versus median not reached; P = 0.014; HR = 1.9 [95% CI: 1.1-3.1]). Among 95 patients with a complete morphologic remission (CR), 43 (45%) were MRD-positive by NGS and 52 (55%) were MRD-negative. These MRD-positive CR patients had a shorter overall survival (16.8 months versus median not reached; P = 0.013; HR = 2.1 [95% CI: 1.2-3.9]) than did the MRD-negative CR patients. Post-treatment persisting MRD positivity, defined by the same NGS-based test used at diagnosis, is thus a more sensitive biomarker for low-level leukemic clones compared to traditional non-molecular methods and is prognostic of subsequent relapse and death.

Jun blockade of erythropoiesis: role for repression of GATA-1 by HERP2.

Although Jun upregulation and activation have been established as critical to oncogenesis, the relevant downstream pathways remain incompletely characterized. In this study, we found that c-Jun blocks erythroid differentiation in primary human hematopoietic progenitors and, correspondingly, that Jun factors block transcriptional activation by GATA-1, the central regulator of erythroid differentiation. Mutagenesis of c-Jun suggested that its repression of GATA-1 occurs through a transcriptional mechanism involving activation of downstream genes. We identified the hairy-enhancer-of-split-related factor HERP2 as a novel gene upregulated by c-Jun. HERP2 showed physical interaction with GATA-1 and repressed GATA-1 transcriptional activation. Furthermore, transduction of HERP2 into primary human hematopoietic progenitors inhibited erythroid differentiation. These results thus define a novel regulatory pathway linking the transcription factors c-Jun, HERP2, and GATA-1. Furthermore, these results establish a connection between the Notch signaling pathway, of which the HERP factors are a critical component, and the GATA family, which participates in programming of cellular differentiation.

R634W KIT Mutation in an Adult With Systemic Mastocytosis.

Mastocytosis is a clonal neoplasm with the potential to affect various organs within the body. It can range in clinical severity from benign to extremely aggressive. Mastocytosis can be separated into cutaneous, systemic, and leukemic forms, as well as mast-cell sarcoma and extracutaneous mastocytoma. It is most often an acquired condition but can be inherited; the most commonly identified genetic aberrations leading to mastocytosis are activating mutations involving codon 816 of the KIT gene. Herein, we present the case of a 30-year-old Caucasian man with systemic mastocytosis discovered to have a p.Arg634Trp mutation involving KIT. To our knowledge, this mutation has previously only been identified in children with familial urticarial pigmentosa. Ours is the the first case report in the literature of an adult with systemic mastocytosis likely due to a p.Arg634Trp KIT mutation.

Control of myeloid dendritic cell differentiation and function by CD1d-restricted (NK) T cells.

While regulating a wide variety of immunologic responses, the precise immunologic functions of CD1d-restricted (NK) T cells are not well defined. Notably, In vitro activation of human NK T cell clones results in the secretion of multiple cytokines important for the recruitment and differentiation of myeloid dendritic cells (DC). Once differentiated, these DC strongly activate NK T cells. In humans, CD1d is expressed by myeloid DC and on tumor cells of this lineage. Another specialized myeloid antigen presenting cell, the epithelioid histiocyte seen in granulomatous inflammation, also expresses CD1d. Because myeloid DC are important regulators of Th1/Th2 T cell responses, cross talk between human NK T cells and myeloid DC would be expected to have significant impact on many immune responses. Consistent with this hypothesis, NK T cells are required for myeloid DC-controlled antitumor responses in mice, and regulate diabetes in nonobese diabetic (NOD) mouse by locally controlling the frequency and function of DC subsets. Thus, regulation of myeloid DC by NK T cells controls both the transition from innate to adaptive immunity and the Th-phenotype of subsequent T cell responses.

Submicroscopic deletion of 5q involving tumor suppressor genes (CTNNA1, HSPA9) and copy neutral loss of heterozygosity associated with TET2 and EZH2 mutations in a case of MDS with normal chromosome and FISH results.

Advances in genome-wide molecular cytogenetics allow identification of novel submicroscopic DNA copy number alterations (aCNAs) and copy-neutral loss of heterozygosity (cnLOH) resulting in homozygosity for known gene mutations in myeloid neoplasms. We describe the use of an oligo-SNP array for genomic profiling of aCNA and cnLOH, together with sequence analysis of recurrently mutated genes, in a patient with myelodysplastic syndrome (MDS) presenting with normal karyotype and FISH results. Oligo-SNP array analysis revealed a hemizygous deletion of 896 kb at chromosome 5q31.2, representing the smallest 5q deletion reported to date. The deletion involved multiple genes, including two tumor suppressor candidate genes (CTNNA1 and HSPA9) that are associated with MDS/AML. The SNP-array study also detected 3 segments of somatic cnLOH: one involved the entire long arm of chromosome 4; the second involved the distal half of the long arm of chromosome 7, and the third encompassed the entire chromosome 22 (UPD 22). Sequence analysis revealed mutations in TET2 (4q), EZH2 (7q), ASXL1 (20q11.21), and RUNX1 (21q22.3). Coincidently, TET2 and EZH2 were located at segments of cnLOH resulting in their homozygosity. Loss of heterozygosity affecting these two chromosomes and mutations in TET2 and EZH2 are indicative of a myelodysplastic syndrome with a poor prognosis. Deletion of the tumor suppressor genes CTNNA1 and HSPA9 is also likely to contribute to a poor prognosis. Furthermore, the original cnLOHs in multiple chromosomes and additional cnLOH 14q in the follow-up study suggest genetic evolution of the disease and poor prognosis. This study attests to the fact that some patients with a myelodysplastic syndrome who exhibit a normal karyotype may have underlying genetic abnormalities detectable by chromosomal microarray and/or targeted mutation analyses.

Unique in vitro and in vivo thrombopoietic activities of ingenol 3,20 dibenzoate, a Ca(++)-independent protein kinase C isoform agonist.

Thrombopoiesis following severe bone marrow injury frequently is delayed, thereby resulting in life-threatening thrombocytopenia for which there are limited treatment options. The reasons for these delays in recovery are not well understood. Protein kinase C (PKC) agonists promote megakaryocyte differentiation in leukemia cell lines and primary cells. However, little is known about the megakaryopoietic effects of PKC agonists on primary CD34+ cells grown in culture or in vivo. Here we present evidence that the novel PKC isoform-selective agonist 3,20 ingenol dibenzoate (IDB) potently stimulates early megakaryopoiesis of human CD34+ cells. In contrast, broad spectrum PKC agonists failed to do so. In vivo, a single intraperitoneal injection of IDB selectively increased platelets in mice without affecting hemoglobin or white counts. Finally, IDB strongly mitigated radiation-induced thrombocytopenia, even when administered 24 hours after irradiation. Our data demonstrate that novel PKC isoform agonists such as IDB may represent a unique therapeutic strategy for accelerating the recovery of platelet counts following severe marrow injury.

Aberrant overexpression of IL-15 initiates large granular lymphocyte leukemia through chromosomal instability and DNA hypermethylation.

How inflammation causes cancer is unclear. Interleukin-15 (IL-15) is a pro-inflammatory cytokine elevated in human large granular lymphocyte (LGL) leukemia. Mice overexpressing IL-15 develop LGL leukemia. Here, we show that prolonged in vitro exposure of wild-type (WT) LGL to IL-15 results in Myc-mediated upregulation of aurora kinases, centrosome aberrancies, and aneuploidy. Simultaneously, IL-15 represses miR-29b via induction of Myc/NF-κBp65/Hdac-1, resulting in Dnmt3b overexpression and DNA hypermethylation. All this is validated in human LGL leukemia. Adoptive transfer of WT LGL cultured with IL-15 led to malignant transformation in vivo. Drug targeting that reverses miR-29b repression cures otherwise fatal LGL leukemia. We show how excessive IL-15 initiates cancer and demonstrate effective drug targeting for potential therapy of human LGL leukemia.

RUNX1 and GATA-1 coexpression and cooperation in megakaryocytic differentiation.

Megakaryocytic and erythroid lineages derive from a common bipotential progenitor and share many transcription factors, most prominently factors of the GATA zinc-finger family. Little is known about transcription factors unique to the megakaryocytic lineage that might program divergence from the erythroid pathway. To identify such factors, we used the K562 system in which megakaryocyte lineage commitment is dependent on sustained extracellular regulatory kinase (ERK) activation and is inhibited by stromal cell contact. During megakaryocytic induction in this system, the myeloid transcription factor RUNX1 underwent up-regulation, dependent on ERK signaling and inhibitable by stromal cell contact. Immunostaining of healthy human bone marrow confirmed a strong expression of RUNX1 and its cofactor, core-binding factor beta (CBFbeta), in megakaryocytes and a minimal expression in erythroblasts. In primary human hematopoietic progenitor cultures, RUNX1 and CBFbeta up-regulation preceded megakaryocytic differentiation, and down-regulation of these factors preceded erythroid differentiation. Functional studies showed cooperation among RUNX1, CBFbeta, and GATA-1 in the activation of a megakaryocytic promoter. By contrast, the RUNX1-ETO leukemic fusion protein potently repressed GATA-1-mediated transactivation. These functional interactions correlated with physical interactions observed between GATA-1 and RUNX1 factors. Enforced RUNX1 expression in K562 cells enhanced the induction of the megakaryocytic integrin proteins alphaIIb and alpha2. These results suggest that RUNX1 may participate in the programming of megakaryocytic lineage commitment through functional and physical interactions with GATA transcription factors. By contrast, RUNX1-ETO inhibition of GATA function may constitute a potential mechanism for the blockade of erythroid and megakaryocytic differentiation seen in leukemias with t(8;21).

Stromal inhibition of megakaryocytic differentiation is associated with blockade of sustained Rap1 activation.

Coculture with stromal cells tends to maintain normal hematopoietic progenitors and their leukemic counterparts in an undifferentiated, proliferative state. An example of this effect is seen with megakaryocytic differentiation, wherein stromal contact renders many cell types refractory to potent induction stimuli. This inhibitory effect of stroma on megakaryocytic differentiation correlates with a blockade within hematopoietic cells of protein kinase C-epsilon (PKC-epsilon) up-regulation and of extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase activation, both of which have been implicated in promoting megakaryocytic differentiation. In this study K562DeltaRafER.5 cells, expressing an estradiol-responsive mutant of the protein kinase Raf-1, were used to determine the relevance and stage of ERK/MAPK pathway blockade by stromal contact. Activation of DeltaRafER by estradiol overrode stromal blockade of megakaryocytic differentiation, implicating the proximal stage of the ERK/MAPK pathway as a relevant control point. Because stromal contact blocked delayed but not early ERK activation, the small guanosine triphosphatase (GTPase) Rap1 was considered as a candidate inhibitory target. Activation assays confirmed that Rap1 underwent sustained activation as a result of megakaryocytic induction, as previously described. As with ERK activation, stromal contact selectively blocked delayed but not early Rap1 activation, having no effect on Ras activation. Enforced expression of either wild-type Rap1 or the GTPase (GAP) resistant mutant Rap1 V12 failed to override stromal inhibition, suggesting that the inhibitory mechanism does not involve GAP up-regulation but rather may target upstream guanine nucleotide exchange factor (GEF) complexes. Accordingly, coimmunoprecipitation demonstrated stromally induced alterations in a protein complex associated with c-Cbl, a scaffolding factor for Rap1-GEF complexes.

Defective human MutY phosphorylation exists in colorectal cancer cell lines with wild-type MutY alleles.

Oxidative DNA damage can generate a variety of cytotoxic DNA lesions such as 8-oxoguanine (8-oxoG), which is one of the most mutagenic bases formed from oxidation of genomic DNA because 8-oxoG can readily mispair with either cytosine or adenine. If unrepaired, further replication of A.8-oxoG mispairs results in C:G to A:T transversions, a form of genomic instability. We reported previously that repair of A.8-oxoG mispairs was defective and that 8-oxoG levels were elevated in several microsatellite stable human colorectal cancer cell lines lacking MutY mutations (human MutY homolog gene, hmyh, MYH MutY homolog protein). In this report, we provide biochemical evidence that the defective repair of A.8-oxoG may be due, at least in part, to defective phosphorylation of the MutY protein in these cell lines. In MutY-defective cell extracts, but not extracts with functional MutY, A.8-oxoG repair was increased by incubation with protein kinases A and C (PKA and PKC) and caesin kinase II. Treatment of these defective cells, but not cells with functional MutY, with phorbol-12-myristate-13-acetate also increased the cellular A.8-oxoG repair activity and decreased the elevated 8-oxoG levels. We show that MutY is serine-phosphorylated in vitro by the action of PKC and in the MutY-defective cells by phorbol-12-myristate-13-acetate but that MutY is already phosphorylated at baseline in proficient cell lines. Finally, using antibody-isolated MutY protein, we show that MutY can be directly phosphorylated by PKC that directly increases the level of MutY catalyzed A.8-oxoG repair.

CD1d and invariant NKT cells at the human maternal-fetal interface.

Invariant CD1d-restricted natural killer T (iNKT) cells comprise a small, but significant, immunoregulatory T cell subset. Here, the presence of these cells and their CD1d ligand at the human maternal-fetal interface was investigated. Immunohistochemical staining of human decidua revealed the expression of CD1d on both villous and extravillous trophoblasts, the fetal cells that invade the maternal decidua. Decidual iNKT cells comprised 0.48% of the decidual CD3+ T cell population, a frequency 10 times greater than that seen in peripheral blood. Interestingly, decidual CD4+ iNKT cells exhibited a striking Th1-like bias (IFN-gamma production), whereas peripheral blood CD4+ iNKT clones exhibited a Th2-like bias (IL-4 production). Moreover, compared to their peripheral blood counterparts, decidual iNKT clones were strongly polarized toward granulocyte/macrophage colony-stimulating factor production. The demonstration of CD1d expression on fetal trophoblasts together with the differential pattern of cytokine expression by decidual iNKT cells suggests that maternal iNKT cell interactions with CD1d expressed on invading fetal cells may play an immunoregulatory role at the maternal-fetal interface.