TP53 suppression promotes erythropoiesis in del(5q) MDS, suggesting a targeted therapeutic strategy in lenalidomide-resistant patients.

Stabilization of p53 in erythroid precursors in response to nucleosomal stress underlies the hypoplastic anemia in myelodysplastic syndromes (MDS) with chromosome 5q deletion [del(5q)]. We investigated whether cenersen, a clinically active 20-mer antisense oligonucleotide complementary to TP53 exon10, could suppress p53 expression and restore erythropoiesis in del(5q) MDS. Cenersen treatment of ribosomal protein S-14-deficient erythroblasts significantly reduced cellular p53 and p53-up-regulated modulator of apoptosis expression compared with controls, accompanied by a significant reduction in apoptosis and increased cell proliferation. In a two-stage erythroid differentiation assay, cenersen significantly suppressed nuclear p53 in bone marrow CD34+ cells isolated from patients with del(5q) MDS, whereas erythroid burst recovery increased proportionally to the magnitude of p53 suppression without evidence of del(5q) clonal suppression (r = -0.6; P = 0.005). To explore the effect of p53 suppression on erythropoiesis in vivo, dexamethasone, a glucocorticoid receptor-dependent p53 antagonist, was added to lenalidomide treatment in eight lower-risk, transfusion-dependent, del(5q) MDS patients with acquired drug resistance. Transfusion independence was restored in five patients accompanied by expansion of erythroid precursors and decreased cellular p53 expression. We conclude that targeted suppression of p53 could support effective erythropoiesis in lenalidomide-resistant del(5q) MDS.

Differential regulation of effector- and central-memory responses to Toxoplasma gondii Infection by IL-12 revealed by tracking of Tgd057-specific CD8+ T cells.

Production of the pro-inflammatory cytokine IL-12 by innate phagocytes drives the differentiation of IFN-gamma-producing effector T cells during Toxoplasma gondii infection. However, the role of IL-12 in the regulation of memory CD8+ T cell differentiation and function during murine toxoplasmosis is unclear. To track memory CTL development, we identified a novel H-2K(b)-restricted CTL population specific for the Toxoplasma antigen tgd057. Tgd057-specific CTLs were induced by both vaccination and natural peroral infection, and were representative of the polyclonal CTL population. Tgd057-specific primary effector cells required IL-12 for the differentiation of KLRG1+ effector subpopulations and IFN-gamma production in response to restimulation with parasite-infected cells, but not to restimulation with cognate peptide. The effect of IL-12 deficiency during the primary response was profoundly imprinted on memory CTLs, which continued to show defects in cell numbers, KLRG1+ effector memory subpopulation differentiation, and IFN-gamma recall responses. Importantly, isolated CD62L(hi) KLRG1- CD8+ T cells differentiated in the absence of IL-12 were enhanced in their ability to generate IFN-gamma-producing secondary tgd057-specific effector cells. Our data, for the first time, demonstrate the negative impact of IL-12 signaling on the quality of the central memory CTL compartment. Thus, despite the beneficial role of IL-12 in promoting effector differentiation, excessive exposure to IL-12 during CTL priming may limit the development of long-term protective immunity through the decreased fitness of central memory CTL responses.

FISH Signal Pattern for an APL Variant Translocation with a PRKAR1A-RARA Fusion.

OBJECTIVES: Fluorescence in situ hybridization (FISH) is a quick and reliable test to detect the reciprocal t(15;17)(q22;q21) translocation in acute promyeloid leukemia (APL). The typical signal pattern for positive t(15;17) is one red, one green, and two fusion when using a PML/RARA dual fusion translocation probe. However, for variant translocations leading to the fusion of a RARA gene with an alternate gene partner, a RARA break-apart probe should be used to verify the RARA rearrangement. The typical signal pattern for a positive RARA break-apart probe is one red, one green, and one fusion. In this study, we report a rare APL case with a PRKAR1A-RARA fusion gene with a signal pattern distinct from that of t(15;17) and its other variants.

A case report and case review: Chronic myeloid leukemia (CML) blast phase with myelomastocytic differentiation.

We presented a patient with CML who progressed to unclassical blast phase after Tyrosine Kinase Inhibitors (TKIs) therapy. The patient presented with 2 populations of blasts: one with no cytoplasmic granules and was CD117 weak+/tryptase-/CD34- (typical myeloblasts), and another with metochromatic granules in the cytoplasm and was CD117 strong+/tryptase+/CD25+/CD34 subset+ (myelomastocytic blasts). Almost all the cells were positive for BCR/ABL1 fusion and no KT V816F mutation was detected. The patient was misdiagnosed as having blast phase CML with coexisting mast cell leukemia at an outside institute. Three similar cases and previously described myelomastocytic leukemia are reviewed and discussed.

Macrocystic (Mammary Analogue) Secretory Carcinoma: An Unusual Variant and a Pitfall in the Differential Diagnosis of Cystic Lesions in the Head and Neck.

Mammary analogue secretory carcinoma (MASC) is a relatively recently described salivary gland adenocarcinoma characterized by ETV6-NTRK3 gene fusion and in most cases indolent clinical behavior. The majority of tumors show an admixture of microcystic, solid, and tubular growth patterns but only a few cases with dominant macrocystic growth have been reported. We report 15 cases of macrocystic MASC. There were 11 men and 4 women (17 to 88 y age range, average 47 y). The patients presented with a painless cystic mass, the majority in the region of the parotid gland (n=13), as well as in submandibular gland (n=1) and the neck (n=1). All tumors were circumscribed measuring 1.0 to 4.0 cm in greatest diameter (mean: 1.75 cm). Twelve tumors were unilocular, while 3 were multilocular. The cystic spaces were predominantly lined by a single epithelial cell layer with focal areas in which the epithelium was multilayered with papillary and hobnail features. In 3 of the cases there were more solid foci of intracystic tumor characterized by papillary and/or microcystic growth. The neoplastic cells were round to oval with hyperchromatic to vesicular nuclei with centrally located nucleoli and eosinophilic or vacuolated cytoplasm. Tumor cells showed strong positivity for S100 protein and mammaglobin, while DOG1 was uniformly negative. A minority of cases showed focal p63 reactivity predominantly limited to the periphery of the cystic lining. ETV6 gene rearrangement was identified in 9 cases. Macrocystic MASC can simulate benign and malignant salivary gland lesions and needs to be included in the differential diagnosis of cystic lesions in the head and neck. To the best of our knowledge, our report represents the first series of macrocystic MASCs wholly focusing on this unusual variant.

BCL2 Amplicon Loss and Transcriptional Remodeling Drives ABT-199 Resistance in B Cell Lymphoma Models.

Drug-tolerant "persister" tumor cells underlie emergence of drug-resistant clones and contribute to relapse and disease progression. Here we report that resistance to the BCL-2 targeting drug ABT-199 in models of mantle cell lymphoma and double-hit lymphoma evolves from outgrowth of persister clones displaying loss of 18q21 amplicons that harbor BCL2. Further, persister status is generated via adaptive super-enhancer remodeling that reprograms transcription and offers opportunities for overcoming ABT-199 resistance. Notably, pharmacoproteomic and pharmacogenomic screens revealed that persisters are vulnerable to inhibition of the transcriptional machinery and especially to inhibition of cyclin-dependent kinase 7 (CDK7), which is essential for the transcriptional reprogramming that drives and sustains ABT-199 resistance. Thus, transcription-targeting agents offer new approaches to disable drug resistance in B-cell lymphomas.

Early expression of p107 is associated with 3T3-L1 adipocyte differentiation.

In response to hormonal stimulation quiescent 3T3-L1 preadipocyte cells reenter the cell cycle and undergo a mitotic expansion phase prior to terminal differentiation. The cell cycle regulatory proteins p130 and p107 undergo dramatic changes in protein levels within 24 h of differentiation. The role of these proteins in regulating adipocyte mitotic clonal expansion and/or differentiation are unclear. It has recently been demonstrated that adipocyte proliferation can be uncoupled from adipocyte differentiation through the use of the pharmacological MEK inhibitor PD98059 or the tyrosine phosphatase inhibitor, sodium vanadate. We examined the expression of p130 and p107 in stimulated 3T3-L1 cells in the presence of either PD98059, U0126 or sodium vanadate. While inhibition of MEK blocked proliferation, the cells underwent differentiation normally. In contrast, vanadate blocked differentiation without affecting proliferation. Inhibition of MEK did not affect the increase in p107 expression in stimulated cells indicating that induction of p107 is independent of MAP kinase signaling. Vanadate treatment caused a significant delay in p107 expression in the first 24 h following stimulation. Under these conditions, p130 expression was relatively unchanged. Our results indicate that a rapid increase in p107 expression correlates with a commitment to undergo adipocyte differentiation. The data further suggest that the rapid induction of p107 is not required for cellular proliferation during the mitotic clonal expansion phase. Taken together, these findings provide correlative data that implicate p107 in the terminal differentiation, but not proliferation, of quiescent preadipocytes following hormonal stimulation.

Cytogenetics of neurofibromas: two case reports and literature review.

Only a few karyotypes of neurofibromas have been documented in the literature. In this report, we describe two new cases in which conventional cytogenetics demonstrated the presence of abnormal clones. Combining karyotypes of the nine previously reported cases, we found that the most frequent structural rearrangements involved chromosome 9p. Including the two cases reported here, 5/11 cases involved 9p, and four of these involved the 9p21 approximately p22 region.

Direct visualization of the endomitotic cell cycle in living megakaryocytes: differential patterns in low and high ploidy cells.

Endomitosis in megakaryocytes (MKs) involves repeated DNA replication in the absence of cytokinesis and is a crucial part of MK development. However, chromosomal dynamics have never been observed in living MKs. We developed a new transgenic mouse model in which the expression of human histone H2B fused in-frame to green fluorescent protein is targeted to MKs. Ex vivo time-lapse microscopy analysis indicated that chromosomal condensation occurs at early mitosis in all MKs. In high ploidy MKs (>or=8N), late anaphase was marked by a ring-type alignment of chromosomes with multiple territories formed between them. By contrast, in low ploidy MKs mitotic chromosomes segregated to form two groups separated by a clear space before re-joining to one cluster. This is the first study to document chromosomal segregation patterns during endomitosis ex vivo and to indicate their potential differential regulation in low and high ploidy cells.

Estrogen receptor inhibits interleukin-6 gene expression by disruption of nuclear factor kappaB transactivation.

The estrogen receptor (ER) suppresses interleukin-6 (IL-6) gene expression through interaction with nuclear factor kappaB (NF-kappaB) in a hormone-dependent manner. Classic ER binding to DNA is not required and the mechanism of repression is unclear. Previously reported studies suggest that the interference of NF-kappaB binding to DNA by ER may play an important role. An alternative model for repression would be the disruption of NF-kappaB transactivation. In the present study, gel shift assays were used to examine the binding of RelA and p50 dimers to the IL-6 promoter in the presence of ER. The effect of ER on NF-kappaB transactivation was studied independent of NF-kappaB binding to DNA using the mammalian one-hybrid system. ER had little effect on the binding of homodimers or heterodimers of RelA and p50 to the IL-6 promoter. In transfection experiments, both ERalpha and ERbeta inhibited NF-kappaB-mediated expression in a hormone dependent manner with repression also dependent upon dimerization of RelA with p50. Mutant ER that is unable to transactivate failed to repress NF-kappaB expression, but deletion of the N-terminal portion of the receptor had no effect. Taken together, these results suggest that the disruption of NF-kappaB-mediated transactivation plays a significant role in ER inhibition of IL-6 gene expression.