EBER-Negative, Double-Hit High-Grade B-Cell Lymphoma Responding to Methotrexate Discontinuation.

Background: First classified in 2016, high-grade B-cell lymphoma (HGBCL) is a lymphoid neoplasm that is typically seen as an aggressive lymphoproliferative disorder (LPD). In most patients with HGBCL, various oncogene rearrangements present with advanced clinical features, such as central nervous system involvement. Patients with underlying autoimmune and rheumatologic conditions, such as rheumatoid arthritis, are at higher risk for developing LPDs, including highly aggressive subtypes of non-Hodgkin lymphomas such as HGBCL. Case Presentation: We present a case of stage IV double-hit HGBCL with the presence of MYC and BCL6 gene rearrangements in an older veteran with rheumatoid arthritis treated with methotrexate. An excellent sustained response was observed for the patient's disease within 4 weeks of methotrexate discontinuation. To our knowledge, this is the first reported response to methotrexate discontinuation for a patient with HGBCL. Conclusions: Reducing immunosuppression should be considered in all patients with LPDs associated with autoimmune conditions or immunosuppressive medications, regardless of additional multiagent systemic therapy administration.

Stathmin 1 deficiency induces erythro-megakaryocytic defects leading to macrocytic anemia and thrombocythemia in Stathmin 1 knock out mice.

Stathmin 1 (STMN1) is a cytosolic phosphoprotein that was discovered as a result of its high level of expression in leukemic cells. It plays an important role in the regulation of mitosis by promoting depolymerization of the microtubules that make up the mitotic spindle and, aging has been shown to impair STMN1 levels and change microtubule stability. We have previously demonstrated that a high level of STMN1 expression during early megakaryopoiesis is necessary for proliferation of megakaryocyte progenitors and that down-regulation of STMN1 expression during late megakaryopoiesis is important for megakaryocyte maturation and platelet production. In this report, we examined the effects of STMN1 deficiency on erythroid and megakaryocytic lineages in the mouse. Our studies show that STMN1 deficiency results in mild thrombocytopenia in young animals which converts into profound thrombocythemia as the mice age. STMN1 deficiency also lead to macrocytic changes in both erythrocytes and megakaryocytes that persisted throughout the life of STMN1 knock-out mice. Furthermore, STMN1 knock-out mice displayed a lower number of erythroid and megakaryocytic progenitor cells and had delayed recovery of their blood counts after chemotherapy. These studies show an important role for STMN1 in normal erythro-megakaryopoietic development and suggests potential implications for disorders affecting these hematopoietic lineages.

Benchmarks for Academic Oncology Faculty.

The role of clinical researchers is vital to cancer progress. The teaching, research, and leadership roles that academic oncologists hold need to be accounted for and appropriately compensated. National metrics are currently inexistent, but are necessary to move the oncology research field forward. Clinical research and routine clinical care must be harmoniously integrated without competing. This article reviews the national landscape of clinical cancer research and proposes a call for action.

Paraproteinemic keratopathy in monoclonal gammopathy of undetermined significance treated with primary keratoprosthesis: Case report, histopathologic findings, and world literature review.

RATIONALE: We report a case of paraproteinemic keratopathy associated with monoclonal gammopathy of undetermined significance, treated with keratoprosthesis as a primary penetrating procedure. Histopathological findings and a world literature review are presented. PATIENT CONCERNS: A 74 year old female recently diagnosed with monoclonal gammopathy undetermined significance presented with progressive blurry vision bilaterally. DIAGNOSES: Examination revealed corneal opacities consistent with paraproteinemic keratopathy. INTERVENTIONS: Corneal transplantation with the Boston Type I keratoprosthesis was performed on the right and, a year later, on the left. OUTCOMES: Visual outcomes were good. Histopathological staining of host corneal buttons were consistent with monoclonality, and electron microscopy revealed fibrillar extracellular aggregates within intervening normal stroma. LESSONS: Corneal deposits may be the only manifestation of monoclonal gammopathy of undetermined significance in patients who are otherwise systemically asymptomatic. Ophthalmologists who encounter corneal opacities may order the appropriate diagnostic studies to determine the presence of occult systemic disease. Risk of graft failure after penetrating keratoplasty from recurring opacities is high, so keratoprosthesis as a primary penetrating procedure may afford superior long-term outcomes. Host corneal buttons retrieved from penetrating keratoplasty or corneal biopsy may be sent for histopathological examination to confirm the diagnosis.

Biomarkers for early detection of sickle nephropathy.

Renal complications affect nearly 30-50% of adults with sickle cell anemia (SCA), causing significant morbidity and mortality. Standard renal function tests like serum creatinine and glomerular filtration rate become abnormal in this disease only when renal damage has become extensive and largely irreversible. Moreover, not all patients develop sickle nephropathy (SN). Therefore, noninvasive biomarkers that predict early onset of SN are necessary. We performed a cross-sectional analysis for nephropathy in 116 patients with sickle cell disease, analyzing urinary kidney injury molecule-1 (KIM-1), liver-type fatty acid binding protein (L-FABP), N-acetyl-b-D-glucosaminidase (NAG), neutrophil gelatinase-associated lipocalin (NGAL) and transforming growth factor-ß1 (TGF-ß), together with conventional renal biomarkers (urine albumin and osmolality, and serum creatinine and cystatin C estimated GFR) during routine clinic visits when patients were at steady-state/baseline. We observed a distinct biomarker pattern: KIM-1 and NAG emerged as biomarkers with a strong association with albuminuria. Surprisingly, and in contrast to other acute/chronic renal disorders, NGAL, L-FABP, and TGF-ß levels did not show any relationship with albuminuria in patients with SCA. Our study identifies potential biomarkers for SN, and suggests longitudinal validation of these biomarkers for early detection of SN, so that therapeutic interventions can be applied before renal damage becomes irreversible.

Down-regulation of stathmin expression is required for megakaryocyte maturation and platelet production.

The final stages of of megakaryocyte (MK) maturation involve a series of steps, including polyploidization and proplatelet formation. Although these processes are highly dependent on dynamic changes in the microtubule (MT) cytoskeleton, the mechanisms responsible for regulation of MTs in MKs remain poorly defined. Stathmin is a highly conserved MT-regulatory protein that has been suggested to play a role in MK differentiation of human leukemic cell lines. However, previous studies defining this relationship have reached contradictory conclusions. In this study, we addressed this controversy and investigated the role of stathmin in primary human MKs. To explore the importance of stathmin down-regulation during megakaryocytopoiesis, we used a lentiviral-mediated gene delivery system to prevent physiologic down-regulation of stathmin in primary MKs. We demonstrated that sustained expression of constitutively active stathmin delayed cytoplasmic maturation (ie, glycoprotein GPIb and platelet factor 4 expression) and reduced the ability of MKs to achieve high levels of ploidy. Moreover, platelet production was impaired in MKs in which down-regulation of stathmin expression was prevented. These studies indicate that suppression of stathmin is biologically important for MK maturation and platelet production and support the importance of MT regulation during the final stages of thrombopoiesis.

Pharmacologic induction of fetal hemoglobin production.

Reactivation of fetal hemoglobin (HbF) expression is an important therapeutic option in adult patients with hemoglobin disorders. The understanding of the developmental regulation of γ-globin gene expression was followed by the identification of a number of chemical compounds that can reactivate HbF synthesis in vitro and in vivo in patients with hemoglobin disorders. These HbF inducers can be grouped in several classes based on their mechanisms of action. This article focuses on pharmacologic agents that were tested in humans and discusses current knowledge about the mechanisms by which they induce HbF.

Histone deacetylase inhibitors and hemoglobin F induction in beta-thalassemia.

Epigenomic modifiers, such as histone deacetylase inhibitors, are compounds that regulate gene expression by interfering with the enzymatic machinery that maintains the proper chromatin structure of the nucleus. These compounds are at the forefront of novel therapeutic agents for the treatment of several diseases including cancer and genetic disorders such as beta-thalassemia and sickle cell disease. Here we review the current understanding of the mechanism of action of epigenomic modifiers in the treatment of beta-thalassemia and sickle cell anemia. We also discuss how the lessons learned from the study of the effects of these compounds on the beta-globin locus, one of the best characterized regions of the human genome, might contribute to the understanding of the mechanism of action of these same compounds in cancer, where the specific regions of the genome that are responsible for the pathophysiology of the disease are often poorly defined.

Epigenetic analysis of the human alpha- and beta-globin gene clusters.

K562 erythroleukemia cells have been widely used as a model for the study of globin gene regulation. A number of agents have been shown to activate or suppress globin gene expression in these cells. However, the molecular effects of these agents on the epigenetic configuration of the alpha- and gamma-globin genes that encode HbF are not known. In this report, we investigated the relationship between globin expression and histone acetylation of the human alpha- and beta-globin clusters in the fetal erythroid environment of K562 cells. Our studies suggest that acetylation of histone H3 may be important in regulating developmental stage-specific expression of the different beta-like globin genes while acetylation of both histones H3 and H4 may be important for the regulation of tissue-specific expression of these genes. In contrast, acetylation of both histones H3 and H4 at the alpha-like globin promoters appears to be important for both developmental stage- and tissue-specific expression. Interestingly, butyrate-induced activation of alpha-globin gene expression in K562 cells is associated with significant increase in histone acetylation levels while TPA-induced inhibition is associated with decreased histone acetylation at its promoters. In contrast, changes in histone acetylation and DNA methylation do not appear to be important in the regulation of gamma-globin gene expression by the same agents. These data suggest that the butyrate-mediated induction of the fetal gamma-globin genes in K562 cells is not a direct result of its histone deacetylase inhibitor activity of butyrate on the chromatin of the gamma-globin promoters, while the induction of the alpha-globin genes could be a result of a direct effect of butyrate on chromatin at its promoters. This is another example of the important differences in the molecular mechanisms of regulation of the genes of the alpha- and beta-like globin clusters.

Synergistic antiangiogenic effects of stathmin inhibition and taxol exposure.

Stathmin is one of the key regulators of the microtubule cytoskeleton and the mitotic spindle in eukaryotic cells. It is expressed at high levels in a wide variety of human cancers and may provide an attractive target for cancer therapy. We had previously shown that stathmin inhibition results in the abrogation of the malignant phenotype. The microtubule-interfering drug, taxol, has both antitumorigenic and antiangiogenic properties. We had also shown that the antitumor activities of taxol and stathmin inhibition are synergistic. We hypothesized that taxol and stathmin inhibition may also have synergistic antiangiogenic activities. A replication-deficient bicistronic adenoviral vector that coexpresses green fluorescent protein and an anti-stathmin ribozyme was used to target stathmin mRNA. Exposure of endothelial cells to anti-stathmin adenovirus alone resulted in a dose-dependent inhibition of proliferation, migration, and differentiation into capillary-like structures. This inhibition was markedly enhanced by exposure of transduced endothelial cells to very low concentrations of taxol, which resulted in a virtually complete loss of proliferation, migration, and differentiation of endothelial cells. In contrast, exposure of nontransduced endothelial cells to taxol alone resulted in a modest inhibition of proliferation, migration, and differentiation. Our detailed analysis showed that the antiangiogenic effects of the combination of stathmin inhibition and taxol exposure are synergistic. Our studies also showed that the mechanism of this synergistic interaction is likely to be mediated through the stabilization of microtubules. Thus, this novel combination may provide an attractive therapeutic strategy that combines a synergistic antitumor activity with a synergistic antiangiogenic activity.

Role of epigenetic modifications in normal globin gene regulation and butyrate-mediated induction of fetal hemoglobin.

Butyrate is a prototype of histone deacetylase inhibitors that is believed to reactivate silent genes by inducing epigenetic modifications. Although butyrate was shown to induce fetal hemoglobin (HbF) production in patients with hemoglobin disorders, the mechanism of this induction has not been fully elucidated. Our studies of the epigenetic configuration of the beta-globin cluster suggest that DNA methylation and histone H3 acetylation are important for the regulation of developmental stage-specific expression of the beta-like globin genes, whereas acetylation of both histones H3 and H4 seem to be important for the regulation of tissue-specific expression. These studies suggest that DNA methylation may be important for the silencing of the beta-like globin genes in nonerythroid hematopoietic cells but may not be necessary for their silencing in nonhematopoietic cells. Furthermore, our studies demonstrate that butyrate exposure results in a true reversal of the normal developmental switch from gamma- to beta-globin expression. This is associated with increased histone acetylation and decreased DNA methylation of the gamma-globin genes, with opposite changes in the beta-globin gene. These studies provide strong support for the role of epigenetic modifications in the normal developmental and tissue-specific regulation of globin gene expression and in the butyrate-mediated pharmacologic induction of HbF production.

Therapeutic interactions between stathmin inhibition and chemotherapeutic agents in prostate cancer.

Limitations of prostate cancer therapy may be overcome by combinations of chemotherapeutic agents with gene therapy directed against specific proteins critical for disease progression. Stathmin is overexpressed in many types of human cancer, including prostate cancer. Stathmin is one of the key regulators of the microtubule network and the mitotic spindle and provides an attractive therapeutic target in cancer therapy. We recently showed that adenovirus-mediated gene transfer of anti-stathmin ribozyme could suppress the malignant phenotype of prostate cancer cells in vitro. In the current studies, we asked whether the therapeutic effects of stathmin inhibition could be further enhanced by exposure to different chemotherapeutic agents. Exposure of uninfected LNCaP human prostate cancer cells or cells infected with a control adenovirus to Taxol, etoposide, 5-fluorouracil (5-FU), or Adriamycin resulted in modest decrease in proliferation and clonogenicity. Interestingly, exposure of cells infected with an anti-stathmin adenovirus to Taxol or etoposide resulted in a complete loss of proliferation and clonogenicity, whereas exposure of the same cells to 5-FU or Adriamycin potentiated the growth-inhibitory effects of the anti-stathmin ribozyme, but the cells continued to proliferate. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis of uninfected cells or cells infected with a control adenovirus showed modest induction of apoptosis in the presence of different drugs. In contrast, cells infected with the anti-stathmin adenovirus showed a marked increase in apoptosis on exposure to Taxol or etoposide and a modest increase on exposure to 5-FU or Adriamycin. Overall, the effects of combinations of anti-stathmin ribozyme with Taxol or etoposide were synergistic, whereas the effects of combinations of anti-stathmin ribozyme with 5-FU or Adriamycin were additive. Moreover, triple combination of anti-stathmin ribozyme with low noninhibitory concentrations of Taxol and etoposide resulted in a profound synergistic inhibition of proliferation, clonogenicity, and marked induction of apoptosis. This synergy might be very relevant for the treatment of prostate cancer because Taxol and etoposide are two of the most effective agents in this disease. Thus, this combination may provide a novel form of prostate cancer therapy that would avoid toxicities associated with the use of multiple chemotherapeutic agents at full therapeutic doses.

Targeting stathmin in prostate cancer.

Stathmin is the founding member of a family of microtubule-destabilizing proteins that regulate the dynamics of microtubule polymerization and depolymerization. Stathmin is expressed at high levels in a variety of human cancers and provides an attractive molecule to target in cancer therapies that disrupt the mitotic apparatus. We developed replication-deficient bicistronic adenoviral vectors that coexpress green fluorescent protein and ribozymes that target stathmin mRNA. The therapeutic potential of these recombinant adenoviruses was tested in an experimental androgen-independent LNCaP prostate cancer model. Adenovirus-mediated transfer of anti-stathmin ribozymes resulted in efficient transduction and marked inhibition of stathmin expression in these cells. Cells that were transduced with the anti-stathmin adenoviruses showed a dramatic dose-dependent growth inhibition. This was associated with accumulation of LNCaP cells in the G2-M phases of the cell cycle. A similar dose-dependent inhibition of clonogenic potential was also observed in cells infected with anti-stathmin adenoviruses. Morphologic and biochemical analysis of infected cells showed a marked increase in apoptosis characterized by detachment of the cells, increased chromatin condensation, activation of caspase-3, and fragmentation of internucleosomal DNA. If these findings are confirmed in vivo, it may provide an effective approach for the treatment of prostate cancer.

Stathmin prevents the transition from a normal to an endomitotic cell cycle during megakaryocytic differentiation.

Physiological polyploidy is a characteristic of several cell types including the megakaryocytes (MK) that give rise to circulating blood platelets. MK achieve polyploidy by switching from a normal to an endomitotic cell cycle characterized by the absence of late mitotic stages. During an endomitotic cycle, the cells enter into mitosis and proceed normally through metaphase and early anaphase. However, late anaphase, telophase and cytokinesis are aborted. This abortive mitosis is associated with atypical multipolar mitotic spindles and limited chromosome segregation. Stathmin is a microtubule-depolymerizing protein that is important for the regulation of the mitotic spindle and interfering with its expression disrupts the normal mitotic spindle and leads to aberrant mitotic exit. As cells enter mitosis, the microtubule depolymerizing-activity of stathmin is switched-off, allowing microtubules to polymerize and assemble into a mitotic spindle. Reactivation of stathmin in the later stages of mitosis is necessary for the disassembly of the mitotic spindle and the exit from mitosis. Previous studies had shown that stathmin expression is downregulated as MK become polyploid and inhibition of its expression in K562 cells increases their propensity to become polyploid. In this report, we describe our studies of the mechanism by which stathmin plays its role in MK polyploidization. We show that stathmin overexpression prevents the transition from a mitotic cycle to an endomitotic cycle as determined by a decrease in the number of multipolar mitotic spindles. These observations support a model in which downregulation of stathmin expression in megakaryocytes and other polyploid cells may be a critically important factor in endomitosis and polyploidy.

p27(Kip1) and stathmin share the stage for the first time.

Cell migration is essential for development, morphogenesis, tissue repair and tumor metastasis. p27(Kip1) and stathmin are two cell-cycle-regulatory proteins that were recently shown to play important roles in the regulation of cell migration. In this article, we discuss a new study that places p27(Kip1) and stathmin in the same pathway by showing that stathmin, a microtubule-regulatory protein, mediates the effects of p27(Kip1) on cell motility. These findings provide new insights into migration and metastasis of tumor cells and the relationship of these processes to cell proliferation.

The role of stathmin in the regulation of the cell cycle.

Stathmin is the founding member of a family of proteins that play critically important roles in the regulation of the microtubule cytoskeleton. Stathmin regulates microtubule dynamics by promoting depolymerization of microtubules and/or preventing polymerization of tubulin heterodimers. Upon entry into mitosis, microtubules polymerize to form the mitotic spindle, a cellular structure that is essential for accurate chromosome segregation and cell division. The microtubule-depolymerizing activity of stathmin is switched off at the onset of mitosis by phosphorylation to allow microtubule polymerization and assembly of the mitotic spindle. Phosphorylated stathmin has to be reactivated by dephosphorylation before cells exit mitosis and enter a new interphase. Interfering with stathmin function by forced expression or inhibition of expression results in reduced cellular proliferation and accumulation of cells in the G2/M phases of the cell cycle. Forced expression of stathmin leads to abnormalities in or a total lack of mitotic spindle assembly and arrest of cells in the early stages of mitosis. On the other hand, inhibition of stathmin expression leads to accumulation of cells in the G2/M phases and is associated with severe mitotic spindle abnormalities and difficulty in the exit from mitosis. Thus, stathmin is critically important not only for the formation of a normal mitotic spindle upon entry into mitosis but also for the regulation of the function of the mitotic spindle in the later stages of mitosis and for the timely exit from mitosis. In this review, we summarize the early studies that led to the identification of the important mitotic function of stathmin and discuss the present understanding of its role in the regulation of microtubules dynamics during cell-cycle progression. We also describe briefly other less mature avenues of investigation which suggest that stathmin may participate in other important biological functions and speculate about the future directions that research in this rapidly developing field may take.

Hemoglobinopathies.

The outlook for patients with sickle cell disease has improved steadily during the last two decades. In spite of these improvements, curative therapies are currently available only to a small minority of patients. The main theme of this chapter is to describe new therapeutic options that are at different stages of development that might result in further improvements in the outlook for patients with these disorders. Dr. Joseph DeSimone and his colleagues had previously made the important observation that the hypomethylating agent 5-azacytidine can reverse the switch from adult to fetal hemoglobin in adult baboons. Although similar activity was demonstrated in patients with sickle cell disease and beta-thalassemia, concern about the toxicity of 5-azacytidine prevented its widespread use in these disorders. In Section I, Dr. DeSimone discusses the role of DNA methylation in globin gene regulation and describe recent clinical experience with decitabine (an analogue of 5-azacytidine) in patients with sickle cell disease. These encouraging studies demonstrate significant fetal hemoglobin inducing activity of decitabine in patients who fail to respond to hydroxyurea. In Section II, Dr. George Atweh continues the same theme by describing recent progress in the study of butyrate, another inducer of fetal hemoglobin, in patients with sickle cell disease and beta-thalassemia. The main focus of his section is on the use of a combination of butyrate and hydroxyurea to achieve higher levels of fetal hemoglobin that might be necessary for complete amelioration of the clinical manifestations of these disorders. Dr. Atweh also describes novel laboratory studies that shed new light on the mechanisms of fetal hemoglobin induction by butyrate. In Section III, Dr. Ronald Nagel discusses the different available transgenic sickle mice as experimental models for human sickle cell disease. These experimental models have already had a significant impact on our understanding of the pathophysiology of sickle cell disease. Dr. Nagel describes more recent studies in which transgenic sickle mice provide the first proof of principle that globin gene transfer into hematopoietic stem cells inhibits in vivo sickling and ameliorates the severity of the disease. Although stroke in adult patients with sickle cell disease is not as common as in children, adult hematologists, like their pediatric colleagues, need to make management decisions in adult patients with a stroke or a history of stroke. Dr. Robert Adams has led several large clinical studies that investigated the role of transfusions in the prevention of stroke in children with sickle cell disease. Much less is known, however, about the prevention of first or subsequent strokes in adult patients with sickle cell disease. In Section IV, Dr. Adams provides some general guidelines for the management of adult patients with stroke while carefully distinguishing between recommendations that are evidence-based and those that are anecdotal in nature.

Stathmin expression and megakaryocyte differentiation: a potential role in polyploidy.

OBJECTIVE: Megakaryopoiesis is characterized by two major processes, acquisition of lineage-specific markers and polyploidization. Polyploidy is a result of endomitosis, a process that is characterized by continued DNA replication in the presence of abortive mitosis. Stathmin is a major microtubule-regulatory protein that plays an important role in the regulation of the mitotic spindle. Our previous studies had shown that inhibition of stathmin expression in human leukemia cells results in the assembly of atypical mitotic spindles and abnormal exit from mitosis. We hypothesized that the absence of stathmin expression in megakaryocytes might be important for their abortive mitosis. MATERIALS AND METHODS: The experimental models that we used were human K562 and HEL cell lines that can be induced to undergo megakaryocytic differentiation and primary murine megakaryocytes generated by in vitro culture of bone marrow cells. The megakaryocytic phenotype was evaluated by flow cytometry and light microscopy. The DNA content (ploidy) was analyzed by flow cytometry. Stathmin expression was analyzed by Western and Northern blotting and by RT-PCR. RESULTS: Our studies showed an inverse correlation between the level of ploidy and the level of stathmin expression in megakaryocytic cell lines and in primary cells. More importantly, inhibition of stathmin expression in K562 cells enhanced the propensity of these cells to undergo endomitosis and to become polyploid upon induction of megakaryocytic differentiation. In contrast, inhibition of stathmin expression interfered with the ability of the cells to acquire megakaryocyte-specific markers of differentiation. CONCLUSION: Based on these observations, we propose a model of megakaryopoiesis in which stathmin expression is necessary for the proliferation and differentiation of early megakaryoblasts and its suppression in the later stages of megakaryocytic maturation is necessary for polyploidization.

Role of stathmin in the regulation of the mitotic spindle: potential applications in cancer therapy.

Stathmin is a member of a novel class of microtubule-destabilizing proteins that regulate the dynamics of microtubule polymerization and depolymerization. Stathmin promotes microtubule depolymerization during interphase and late mitosis. This microtubule depolymerizing activity of stathmin is regulated by changes in its level of phosphorylation that occur during cell cycle progression. These modifications allow it to play a critical role in the regulation of the dynamic equilibrium of microtubules during different phases of the cell cycle. Stathmin is expressed at high levels in a wide variety of human cancers. Inhibition of stathmin expression in malignant cells interferes with their orderly progression through the cell cycle and abrogates their transformed phenotype. Thus, stathmin provides an attractive molecular target for disrupting the mitotic apparatus and arresting the growth of malignant cells. In this review, we describe the current understanding of the role of stathmin in the regulation of the mitotic spindle and discuss its potential as a therapeutic target of cancer therapy.