Identifying monoclonal gammopathy of undetermined significance in electronic health data.

PURPOSE: Monoclonal gammopathy of undetermined significance (MGUS) is a prevalent yet largely asymptomatic precursor to multiple myeloma. Patients with MGUS must undergo regular surveillance and testing, with few known predictors of progression. We developed an algorithm to identify MGUS patients in electronic health data to facilitate large-scale, population-based studies of this premalignant condition. METHODS: We developed a four-step algorithm using electronic health record and health claims data from men and women aged 50 years or older receiving care from a large, multispecialty medical group between 2007 and 2015. The case definition required patients to have at least two MGUS ICD-9 diagnosis codes within 12 months, at least one serum and/or urine protein electrophoresis and one immunofixation test, and at least one in-office hematology/oncology visit. Medical charts for selected cases were abstracted then adjudicated independently by two physicians. We assessed algorithm validity by positive predictive value (PPV). RESULTS: We identified 833 people with at least two MGUS diagnosis codes; 429 (52%) met all four algorithm criteria. We randomly selected 252 charts for review, including 206 from patients meeting all four algorithm criteria. The PPV for the 206 algorithm-identified charts was 76% (95% CI, 70%-82%). Among the 49 cases deemed to be false positives (24%), 33 were judged to have multiple myeloma or another lymphoproliferative condition, such as lymphoma. CONCLUSIONS: We developed a simple algorithm that identified MGUS cases in electronic health data with reasonable accuracy. Inclusion of additional steps to eliminate cases with malignant disease may improve algorithm performance.

Cooperative Activity of GABP with PU.1 or C/EBPε Regulates Lamin B Receptor Gene Expression, Implicating Their Roles in Granulocyte Nuclear Maturation.

Nuclear segmentation is a hallmark feature of mammalian neutrophil differentiation, but the mechanisms that control this process are poorly understood. Gene expression in maturing neutrophils requires combinatorial actions of lineage-restricted and more widely expressed transcriptional regulators. Examples include interactions of the widely expressed ETS transcription factor, GA-binding protein (GABP), with the relatively lineage-restricted E-twenty-six (ETS) factor, PU.1, and with CCAAT enhancer binding proteins, C/EBPα and C/EBPε. Whether such cooperative interactions between these transcription factors also regulate the expression of genes encoding proteins that control nuclear segmentation is unclear. We investigated the roles of ETS and C/EBP family transcription factors in regulating the gene encoding the lamin B receptor (LBR), an inner nuclear membrane protein whose expression is required for neutrophil nuclear segmentation. Although C/EBPε was previously shown to bind the Lbr promoter, surprisingly, we found that neutrophils derived from Cebpe null mice exhibited normal Lbr gene and protein expression. Instead, GABP provided transcriptional activation through the Lbr promoter in the absence of C/EBPε, and activities supported by GABP were greatly enhanced by either C/EBPε or PU.1. Both GABP and PU.1 bound Ets sites in the Lbr promoter in vitro, and in vivo within both early myeloid progenitors and differentiating neutrophils. These findings demonstrate that GABP, PU.1, and C/EBPε cooperate to control transcription of the gene encoding LBR, a nuclear envelope protein that is required for the characteristic lobulated morphology of mature neutrophils.

Factors influencing survival among Kenyan children diagnosed with endemic Burkitt lymphoma between 2003 and 2011: A historical cohort study.

Discovering how to improve survival and establishing clinical reference points for children diagnosed with endemic Burkitt lymphoma (eBL) in resource-constrained settings has recaptured international attention. Using multivariate analyses, we evaluated 428 children with eBL in Kenya for age, gender, tumor stage, nutritional status, hemoglobin, lactate dehydrogenase (LDH), Epstein-Barr virus (EBV) and Plasmodium falciparum prior to induction of chemotherapy (cyclophosphamide, vincristine, methotrexate and doxorubicin) to identify predictive and prognostic biomarkers of survival. During this 10 year prospective study period, 22% died in-hospital and 78% completed six-courses of chemotherapy. Of those, 16% relapsed or died later; 31% achieved event-free-survival; and 31% were lost to follow-up; the overall one-year survival was 45%. After adjusting for covariates, low hemoglobin (<8 g/dL) and high LDH (>400 mU/ml) were associated with increased risk of death (adjusted Hazard Ratio (aHR) = 1.57 [0.97-2.41]) and aHR = 1.84, [0.91-3.69], respectively). Anemic children with malaria were 3.55 times more likely to die [1.10-11.44] compared to patients without anemia or malarial infection. EBV load did not differ by tumor stage nor was it associated with survival. System-level factors can also contribute to poor outcomes. Children were more likely to die when inadvertently overdosed by more than 115% of the correct dose of cyclophosphamide (a HR = 1.43 [0.84-2.43]) or doxorubicin (a HR = 1.25, [0.66-2.35]), compared with those receiving accurate doses of the respective agent in this setting. This study codifies risk factors associated with poor outcomes for eBL patients in Africa and provides a benchmark by which to assess improvements in survival for new chemotherapeutic approaches.

GABP transcription factor is required for development of chronic myelogenous leukemia via its control of PRKD2.

Hematopoietic stem cells (HSCs) are the source of all blood lineages, and HSCs must balance quiescence, self-renewal, and differentiation to meet lifelong needs for blood cell development. Transformation of HSCs by the breakpoint cluster region-ABL tyrosine kinase (BCR-ABL) oncogene causes chronic myelogenous leukemia (CML). The E-twenty six (ets) transcription factor GA binding protein (GABP) is a tetrameric transcription factor complex that contains GABPα and GABPß proteins. Deletion in bone marrow of Gabpa, the gene that encodes the DNA-binding component, caused cell cycle arrest in HSCs and profound loss of hematopoietic progenitor cells. Loss of Gabpα prevented development of CML, although mice continued to generate BCR-ABL-expressing Gabpα-null cells for months that were serially transplantable and contributed to all lineages in secondary recipients. A bioinformatic screen identified the serine-threonine kinase protein kinase D2 (PRKD2) as a potential effector of GABP in HSCs. Prkd2 expression was markedly reduced in Gabpα-null HSCs and progenitor cells. Reduced expression of PRKD2 or pharmacologic inhibition decreased cell cycling, and PRKD2 rescued growth of Gabpα-null BCR-ABL-expressing cells. Thus, GABP is required for HSC cell cycle entry and CML development through its control of PRKD2. This offers a potential therapeutic target in leukemia.

Epigenetic landscape of acute myelogenous leukemia--moving toward personalized medicine.

Acute myeloid leukemia (AML) is an aggressive hematologic cancer that is characterized by accumulation of immature myeloid cells in the blood and bone marrow. The malignant cells in AML have reduced capacity to mature fully, and often exhibit chromosomal abnormalities, defects in cell signaling, and abnormal cell cycle control. Genetic and epigenetic changes are implicated in the onset and progression of AML. While progress has been made in using genetic and epigenetic changes as prognostic features of AML, these findings have not yet been effectively translated into novel treatment strategies. Disappointingly, rates of recurrence in AML remain high and overall survival is poor. Research strategies should focus on developing a comprehensive landscape of genetic and epigenetic changes in individual patients with AML to expand the clinicians' therapeutic armamentarium and to individualize and optimize treatment.

The Ets transcription factor GABP is required for cell-cycle progression.

The transition from cellular quiescence (G0) into S phase is regulated by the mitogenic-activation of D-type cyclins and cyclin-dependent kinases (Cdks), the sequestration of the Cdk inhibitors (CDKIs), p21 and p27, and the hyperphosphorylation of Rb with release of E2F transcription factors. However, fibroblasts that lack all D-type cyclins can still undergo serum-induced proliferation and key E2F targets are expressed at stable levels despite cyclical Rb-E2F activity. Here, we show that serum induces expression of the Ets transcription factor, Gabpalpha, and that its ectopic expression induces quiescent cells to re-enter the cell cycle. Genetic disruption of Gabpalpha prevents entry into S phase, and selectively reduces expression of genes that are required for DNA synthesis and degradation of CDKIs, yet does not alter expression of D-type cyclins, Cdks, Rb or E2Fs. Thus, GABP is necessary and sufficient for re-entry into the cell cycle and it regulates a pathway that is distinct from that of D-type cyclins and CDKs.

GABP transcription factor is required for myeloid differentiation, in part, through its control of Gfi-1 expression.

GABP is an ets transcription factor that regulates genes that are required for myeloid differentiation. The tetrameric GABP complex includes GABPα, which binds DNA via its ets domain, and GABPß, which contains the transcription activation domain. To examine the role of GABP in myeloid differentiation, we generated mice in which Gabpa can be conditionally deleted in hematopoietic tissues. Gabpa knockout mice rapidly lost myeloid cells, and residual myeloid cells were dysplastic and immunophenotypically abnormal. Bone marrow transplantation demonstrated that Gabpα null cells could not contribute to the myeloid compartment because of cell intrinsic defects. Disruption of Gabpa was associated with a marked reduction in myeloid progenitor cells, and Gabpα null myeloid cells express reduced levels of the transcriptional repressor, Gfi-1. Gabp bound and activated the Gfi1 promoter, and transduction of Gabpa knockout bone marrow with Gfi1 partially rescued defects in myeloid colony formation and myeloid differentiation. We conclude that Gabp is required for myeloid differentiation due, in part, to its regulation of the tran-scriptional repressor Gfi-1.

PTEN is a tumor suppressor in CML stem cells and BCR-ABL-induced leukemias in mice.

The tumor suppressor gene phosphatase and tensin homolog (PTEN) is inactivated in many human cancers. However, it is unknown whether PTEN functions as a tumor suppressor in human Philadelphia chromosome-positive leukemia that includes chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL) and is induced by the BCR-ABL oncogene. By using our mouse model of BCR-ABL-induced leukemias, we show that Pten is down-regulated by BCR-ABL in leukemia stem cells in CML and that PTEN deletion causes acceleration of CML development. In addition, overexpression of PTEN delays the development of CML and B-ALL and prolongs survival of leukemia mice. PTEN suppresses leukemia stem cells and induces cell-cycle arrest of leukemia cells. Moreover, PTEN suppresses B-ALL development through regulating its downstream gene Akt1. These results demonstrate a critical role of PTEN in BCR-ABL-induced leukemias and suggest a potential strategy for the treatment of Philadelphia chromosome-positive leukemia.

Retrospective analysis of the efficacy of gemcitabine for previously treated AIDS-associated Kaposi's sarcoma in western Kenya.

OBJECTIVES: Evaluation of outcomes in the use of single-agent gemcitabine for the treatment of AIDS-associated Kaposi's sarcoma (KS) in a western Kenyan cancer treatment program. METHODS: Retrospective chart review of all patients with KS treated with single agent gemcitabine following failure of first-line Adriamycin, bleomycin, and vincristine (ABV). Baseline demographics were collected, and clinicians' assessments of response were utilized to fill out objective criteria for both response as well as symptom benefit assessment. RESULTS: Twenty-three patients with KS who had previously failed first-line therapy with ABV were evaluated. Following treatment, 22 of the 23 patients responded positively to treatment with stable disease or better. Of the 18 patients who had completed therapy, with a median follow-up of 5 months, 12 patients had no documented progression. CONCLUSIONS: Treatment options in the resource-constrained setting are limited, both by financial constraints as well as the need to avoid myelotoxicity, which is associated with high morbidity in this treatment setting. This work shows that gemcitabine has promising activity in KS, with both objective responses and clinical benefit observed in this care setting. Gemcitabine as a single agent merits further investigation for AIDS-associated KS.

GA-binding protein and p300 are essential components of a retinoic acid-induced enhanceosome in myeloid cells.

Expression of CD18, the beta chain of the leukocyte integrins, is transcriptionally regulated by retinoic acid (RA) in myeloid cells. Full RA responsiveness of the CD18 gene requires its proximal promoter, which lacks a retinoic acid response element (RARE). Rather, RA responsiveness of the CD18 proximal promoter requires ets sites that are bound by GA-binding protein (GABP). The transcriptional coactivator, p300, further increases CD18 RA responsiveness. We demonstrate that GABPalpha, the ets DNA-binding subunit of GABP, physically interacts with p300 in myeloid cells. This interaction involves the GABPalpha pointed domain (PNT) and identifies p300 as the first known interaction partner of GABPalpha PNT. Expression of the PNT domain, alone, disrupts the GABPalpha-p300 interaction and decreases the RA responsiveness of the CD18 proximal promoter. Chromatin immunoprecipitation and chromosome conformation capture demonstrate that, in the presence of RA, GABPalpha and p300 at the proximal promoter recruit retinoic acid receptor/retinoid X receptor from a distal RARE to form an enhanceosome. A dominant negative p300 construct disrupts enhanceosome formation and reduces the RA responsiveness of CD18. Thus, proteins on the CD18 proximal promoter recruit the distal RARE in the presence of RA. This is the first description of an RA-induced enhanceosome and demonstrates that GABP and p300 are essential components of CD18 RA responsiveness in myeloid cells.

Transcriptional regulation in myelopoiesis: Hematopoietic fate choice, myeloid differentiation, and leukemogenesis.

Myeloid cells (granulocytes and monocytes) are derived from multipotent hematopoietic stem cells. Gene transcription plays a critical role in hematopoietic differentiation. However, there is no single transcription factor that is expressed exclusively by myeloid cells and that, alone, acts as a "master" regulator of myeloid fate choice. Rather, myeloid gene expression is controlled by the combinatorial effects of several key transcription factors. Hematopoiesis has traditionally been viewed as linear and hierarchical, but there is increasing evidence of plasticity during blood cell development. Transcription factors strongly influence cellular lineage during hematopoiesis and expression of some transcription factors can alter the fate of developing hematopoietic progenitor cells. PU.1 and CCAAT/enhancer-binding protein alpha (C/EBPalpha) regulate expression of numerous myeloid genes, and gene disruption studies have shown that they play essential, nonredundant roles in myeloid cell development. They function in cooperation with other transcription factors, co-activators, and co-repressors to regulate genes in the context of chromatin. Because of their essential roles in regulating myeloid genes and in myeloid cell development, it has been hypothesized that abnormal expression of PU.1 and C/EBPalpha would contribute to aberrant myeloid differentiation, i.e. acute leukemia. Such a direct link has been elusive until recently. However, there is now persuasive evidence that mutations in both PU.1 and C/EBPalpha contribute directly to development of acute myelogenous leukemia. Thus, normal myeloid development and acute leukemia are now understood to represent opposite sides of the same hematopoietic coin.

Hypercalcemia due to Primary Hepatic Lymphoma.

A 65-year-old female with a history of mixed connective tissue disease and pulmonary fibrosis on azathioprine, hydroxychloroquine, and prednisone (osteoporosis on teriparatide) presented with a 1-month history of hypercalcemia. After discontinuation of teriparatide, the patient's hypercalcemia persisted. Further evaluation revealed primary hepatic lymphoma as the source of her hypercalcemia.

Sp1 control of gene expression in myeloid cells.

Gene transcription plays a critical role in the differentiation of myeloid cells. However, there is no single, master regulator of all myeloid genes. Rather, myeloid gene transcription is regulated by the combinatorial effects of a limited number of key transcription factors. Sp1 is a powerful activator of gene transcription in many cell types. Although it is wildly expressed, Sp1 binds and activates the promoters of a large number of important myeloid genes. This presents the paradox of how a widely expressed transcription factor can regulate lineage-specific gene transcription. This review discusses the structure, function, and expression patterns of Sp1 and its related Sp family members. Illustrative examples of the tissue-specific regulation of myeloid target genes are presented. The roles of post-translational modifications of Sp1, alterations in target gene chromatin structure, and important cooperating transcription factors are discussed. Thus, Sp1 serves as a model of how a widely expressed transcription factor regulates the expression of tissue-specific genes.

Sp1 and the ets-related transcription factor complex GABP alpha/beta functionally cooperate to activate the utrophin promoter.

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease caused by the absence of dystrophin. Utrophin is the autosomal homolog of dystrophin and capable of compensating for the absence of dystrophin, when overexpressed. In skeletal muscle, utrophin plays an important role in the formation of neuromuscular junctions. This selective enrichment occurs, in part by transcriptional regulation of the utrophin gene at the sub-synaptic nuclei in muscle. Utrophin's complex transcriptional regulation is not yet fully understood, however, GABP alpha / beta has recently been shown to bind the N box and activate the utrophin promoter in response to heregulin. In this study, we show that the transcription factor Sp1 binds and activates the utrophin promoter in Drosophila S2 cells as well as define a Sp1 response element. We demonstrate that heregulin treatment of cultured muscle cells activates the ERK pathway and phosphorylates serine residue(s) in the consensus ERK recognition site of Sp1. Finally, Sp1 is shown to functionally cooperate with GABP alpha / beta and cause a 58-fold increase of de novo utrophin promoter transcription. Taken together, these findings help define mechanisms used for transcriptional regulation of utrophin expression as well as identify new targets for achieving potentially therapeutic upregulation of utrophin in DMD.

GABP transcription factor (nuclear respiratory factor 2) is required for mitochondrial biogenesis.

Mitochondria are membrane-bound cytoplasmic organelles that serve as the major source of ATP production in eukaryotic cells. GABP (also known as nuclear respiratory factor 2) is a nuclear E26 transformation-specific transcription factor (ETS) that binds and activates mitochondrial genes that are required for electron transport and oxidative phosphorylation. We conditionally deleted Gabpa, the DNA-binding component of this transcription factor complex, from mouse embryonic fibroblasts (MEFs) to examine the role of Gabp in mitochondrial biogenesis, function, and gene expression. Gabpα loss modestly reduced mitochondrial mass, ATP production, oxygen consumption, and mitochondrial protein synthesis but did not alter mitochondrial morphology, membrane potential, apoptosis, or the expression of several genes that were previously reported to be GABP targets. However, the expression of Tfb1m, a methyltransferase that modifies ribosomal rRNA and is required for mitochondrial protein translation, was markedly reduced in Gabpα-null MEFs. We conclude that Gabp regulates Tfb1m expression and plays an essential, nonredundant role in mitochondrial biogenesis.

Why does my patient have leukocytosis?

Leukocytosis is one of the most common laboratory abnormalities in medicine, and one of the most frequent reasons for hematologic consultation. Effective evaluation of leukocytosis requires an attentive history, careful physical examination, meticulous review of the complete blood count and peripheral blood smear, judicious application of laboratory and radiologic testing, and thoughtful analysis. Definitive diagnosis may require bone marrow aspiration and biopsy, imaging studies, and specialized molecular tests. The differential diagnosis of leukocytosis includes physiologic responses to a broad range of infectious and inflammatory processes, as well as numerous primary hematologic disorders such as leukemias, lymphomas, and myeloproliferative neoplasms.

PU.1 is essential for CD11c expression in CD8(+)/CD8(-) lymphoid and monocyte-derived dendritic cells during GM-CSF or FLT3L-induced differentiation.

Dendritic cells (DCs) regulate innate and acquired immunity through their roles as antigen-presenting cells. Specific subsets of mature DCs, including monocyte-derived and lymphoid-derived DCs, can be distinguished based on distinct immunophenotypes and functional properties. The leukocyte integrin, CD11c, is considered a specific marker for DCs and it is expressed by all DC subsets. We created a strain of mice in which DCs and their progenitors could be lineage traced based on activity of the CD11c proximal promoter. Surprisingly, we observed levels of CD11c promoter activity that were similar in DCs and in other mature leukocytes, including monocytes, granulocytes, and lymphocytes. We sought to identify DNA elements and transcription factors that regulate DC-associated expression of CD11c. The ets transcription factor, PU.1, is a key regulator of DC development, and expression of PU.1 varies in different DC subsets. GM-CSF increased monocyte-derived DCs in mice and from mouse bone marrow cultured in vitro, but it did not increase CD8(+) lymphoid-derived DCs or B220(+) plasmacytoid DCs. FLT3L increased both monocyte-derived DCs and lymphoid-derived DCs from mouse bone marrow cultured in vitro. GM-CSF increased the 5.3 Kb CD11c proximal promoter activity in monocyte-derived DCs and CD8(+) lymphoid-derived DCs, but not in B220(+) plasmacytoid DCs. In contrast, FLT3L increased the CD11c proximal promoter activity in both monocyte-derived DCs and B220(+) plasmacytoid DCs. We used shRNA gene knockdown and chromatin immunoprecipitation to demonstrate that PU.1 is required for the effects of GM-CSF or FLT3L on monocyte-derived DCs. We conclude that both GM-CSF and FLT3L act through PU.1 to activate the 5.3 Kb CD11c proximal promoter in DCs and to induce differentiation of monocyte-derived DCs. We also confirm that the CD11c proximal promoter is not sufficient to direct lineage specificity of CD11c expression, and that additional DNA elements are required for lineage-specific CD11c expression.

Phase I trial examining addition of gemcitabine to CHOP in intermediate grade NHL.

PURPOSE: Gemcitabine induces a 20% response as single-agent therapy in patients with relapsed or refractory NHL. We report phase I findings of gemcitabine in combination with standard CHOP chemotherapy with G-CSF support for intermediate grade NHL. The protocol was modified during enrollment to include rituximab in CD 20+ lymphomas. METHODS: Patients received CHOP plus gemcitabine at 500 mg/m(2) (Cohort 1) or 750 mg/m(2) (Cohort 2) on days 1 and 4 of each 21-day cycle. Accrual was suspended once each cohort was filled. Dose escalation occurred after all patients in the cohort were determined to not have a dose-limiting toxicity. RESULTS: Between April 2002 and May 2004, 10 patients were enrolled and completed the study treatment (6 in Cohort 1, 4 in Cohort 2). In Cohort 1, grade 3 toxicities included neutropenia, anemia, neuropathy, and constipation. Grade 4 toxicities were febrile neutropenia and thrombocytopenia. In Cohort 2, grade 3 toxicities included neutropenia, thrombocytopenia, mucositis, anemia, and intestinal obstruction. Grade 4 toxicities included febrile neutropenia, neutropenia, and thrombocytopenia. One patient developed MDS 36 months after chemotherapy. Three of four patients in Cohort 2 developed dose-limiting toxicities (mucositis and thrombocytopenia) requiring dose reduction in gemcitabine after cycle 1. Overall, the survival rate at 2.5 years was 71%. CONCLUSIONS: This Phase I trial concludes that gemcitabine 500 mg/m(2) on days 1 and 4 of each 21-day cycle is the maximum tolerated dose when combined with standard CHOP chemotherapy with G-CSF support for intermediate grade NHL.

Retinoic acid signaling in myelopoiesis.

PURPOSE OF REVIEW: For decades, retinoic acid has been known to alter the proliferation and differentiation of myeloid cells. Currently, retinoic acid is a front-line agent in the treatment of certain forms of acute myelogenous leukemia. In this review, we focus on recent advances in our understanding of the mechanisms by which retinoids affect growth and proliferation of myeloid cells and contribute to the pathogenesis of leukemia. We have not attempted to summarize the related clinical literature. RECENT FINDINGS: The past 2 years have yielded important understanding of the mechanisms by which retinoids and their nuclear receptors interact with other signal transduction pathways and transcription factors to modify chromatin, alter gene expression, and participate in normal myeloid differentiation and leukemogenesis. Important advances regarding cell biology, molecular biology, biochemistry, and animal studies of retinoids and myeloid differentiation are reviewed. SUMMARY: Greater understanding of the role of retinoids and their receptors in myeloid cell growth and differentiation provides important insight into normal myelopoiesis. These findings have resulted in successful rational approaches to the treatment of acute leukemia and provide the promise of improved treatments in the near future.