Caregivers' Out-of-Pocket Expenses and Time Commitment Following Hematopoietic Stem Cell Transplantation at a Rural Cancer Center.

The emotional and physical toll on caregivers of cancer patients is well documented, but research evaluating the financial burdens and time commitments of caregivers is limited. We suspected that the rural location of our cancer center would intensify these burdens for caregivers. We conducted a prospective trial to assess the out-of-pocket expenses and time commitment of caregivers of hematopoietic stem cell transplantation recipients within the first 4 weeks after discharge from the hospital from a National Cancer Institute (NCI)-designated comprehensive cancer center. These results show that caregivers of autologous recipients paid out-of-pocket expenses of $196 over 4 weeks. If lost wages were included, the expenses increased to $736 during this period. Caregivers of allogeneic recipients had out-of-pocket expenses of $110 in 4 weeks, or a total of $610 when lost wages were included. In the month after discharge from the hospital, caregivers traveled a median distance of 450 miles or 560 miles, depending on whether the patient received an autologous transplant or an allogeneic transplant, respectively. These results demonstrate a compelling need to address caregiver support, given the significant financial out-of-pocket expenses and time commitment.

Down the repressors! Up the fetal hemoglobin!

One of the major goals of hemoglobinopathy research has been to devise improved pharmacologic strategies for the induction of fetal hemoglobin (HbF) in people with sickle cell disease and ß-thalassemia. In this issue of Blood, Dulmovits and colleagues report that pomalidomide, a drug approved by the US Food and Drug Administration (FDA) for treatment of multiple myeloma, induces HbF production by decreasing levels of several key transcriptional repressors of fetal globin gene expression. In addition, they show that pomalidomide induces HbF in differentiating erythroid cells from people with sickle cell disease and in myeloma patients.

Extracorporeal photopheresis for graft versus host disease: Identifying a clinical pathway and associated resource utilization.

Extracorporeal photopheresis (ECP) is an established therapy for the treatment of graft-versus-host-disease (GVHD) following an allogeneic stem cell transplant. We performed a prospective analysis of patients receiving ECP treatment for GVHD to identify a clinical pathway and resource utilization of this process. The cohort included consecutive allogeneic stem cell recipients with GVHD. ECP was performed using the CELLEX Photopheresis System or the UVAR XTS Photopheresis System (Therakos, Inc, Exton, PA). A clinical pathway was developed and a time and motion study was conducted to define the resource utilization and costs associated with ECP. Patients were treated with either CELLEX (n = 18 procedures) or UVAR (n = 4 procedures). Total time commitment for each procedure for the 2 machines differed. The time for ECP was 117 min (median, range: 91-164 min) using CELLEX and 161 min (median; range: 140-210) using the UVAR-XTS machine. Total costs of each ECP procedure were $3420.50. There is a considerable time commitment of the patient and the clinical staff when employing ECP to treat GVHD. ECP costs are significant considering this is a prolonged therapy continued for several months. With this finalized pathway and costs, we have a standardized clinical pathway for the treatment of GVHD. We are addressing minimizing resource utilization while emphasizing quality care for these patients.

Induction of fetal hemoglobin through enhanced translation efficiency of γ-globin mRNA.

Fetal hemoglobin (HbF) induction can ameliorate the clinical severity of sickle cell disease and ß-thalassemia. We previously reported that activation of the eukaryotic initiation factor 2α (eIF2α) stress pathway increased HbF through a posttranscriptional mechanism. In this study, we explored the underlying means by which salubrinal, an activator of eIF2α signaling, enhances HbF production in primary human erythroid cells. Initial experiments eliminated changes in globin messenger RNA (mRNA) stability or cellular location and reduction of adult hemoglobin as possible salubrinal mechanisms. We then determined that salubrinal selectively increased the number of actively translating ribosomes on γ-globin mRNA. This enhanced translation efficiency occurred in the recovery phase of the stress response as phosphorylation of eIF2α and global protein synthesis returned toward baseline. These findings highlight γ-globin mRNA translation as a novel mechanism for regulating HbF production and as a pharmacologic target for induction of HbF.

Eukaryotic initiation factor 2α phosphorylation mediates fetal hemoglobin induction through a post-transcriptional mechanism.

Strategies to increase fetal hemoglobin (HbF) levels can ameliorate symptoms and improve the lives of ß-hemoglobinopathy patients. Although most studies have focused on induction of γ-globin gene expression as an approach to induce HbF, we hypothesized that post-transcriptional regulation of HbF plays an underappreciated yet important role in controlling HbF levels. In the present study, we investigated whether increasing eukaryotic initiation factor 2α (eIF2α) phosphorylation, a key regulator of protein translation, could enhance HbF post-transcriptionally in human primary erythroid cells. Initial analysis using a known inhibitor of eIF2α dephosphorylation, salubrinal, revealed that elevated eIF2α phosphorylation enhanced HbF production without changing globin gene expression, proliferation, or cell differentiation. These results were further supported by the post-transcriptional induction of HbF by other pharmacologic activators of the eIF2α pathway and by genetic inactivation of the negative regulators, GADD34 and CReP. Additionally, we found that this novel mechanism of increasing HbF could be combined with clinically relevant transcriptional activators of γ-globin gene expression to additively enhance HbF. Taken together, these findings identify eIF2α phosphorylation as a post-transcriptional regulator of HbF induction that may be pharmacologically targeted, either alone or in combination, in ß-hemoglobinopathy patients.

Simvastatin and t-butylhydroquinone suppress KLF1 and BCL11A gene expression and additively increase fetal hemoglobin in primary human erythroid cells.

UNLABELLED: Although increased fetal hemoglobin (HbF) levels have proven benefit for people with ß-hemoglobinopathies, all current HbF-inducing agents have limitations. We previously reported that drugs that activate the NRF2 antioxidant response signaling pathway increase HbF in primary human erythroid cells. In an attempt to increase HbF levels achieved with NRF2 activators, in the present study, we investigated potential complementary activity between these agents and HMG-CoA reductase inhibitors (statins) based on their ability to induce KLF2 protein levels. Experiments in K562 cells showed that simvastatin increased KLF2 mRNA and protein and KLF2 binding to HS2 of the ß-globin locus control region and enhanced -globin mRNA production by the NRF2 activator Tert-butylhydroquinone (tBHQ). When tested in differentiating primary human erythroid cells, simvastatin induced HbF alone and additively with tBHQ, but it did not increase KLF2 mRNA or locus control region binding above levels seen with normal differentiation. Investigating alternative mechanisms of action, we found that both simvastatin and tBHQ suppress ß-globin mRNA and KLF1 and BCL11A mRNA and protein, similar to what is seen in people with an HPFH phenotype because of KLF1 haploinsufficiency. These findings identify statins as a potential class of HbF-inducing agents and suggest a novel mechanism of action based on pharmacologic suppression of KLF1 and BCL11A gene expression. KEY POINTS: Simvastatin and tBHQ suppress KLF1 and BCL11 gene expression and additively increase fetal hemoglobin in primary human erythroid cells. Because both drugs are FDA-approved, these findings could lead to clinical trials in the relatively near future.

Multiple physical stresses induce γ-globin gene expression and fetal hemoglobin production in erythroid cells.

Increased fetal hemoglobin (HbF) expression is beneficial for ß-hemoglobinopathy patients; however, current inducing agents do not possess the ideal combination of efficacy, safety and ease of use. Better understanding the mechanisms involved in γ-globin gene induction is critical for designing improved therapies, as no complete mechanism for any inducing agent has been identified. Given the cytotoxic nature of most known inducing drugs, we hypothesized that γ-globin is a cell stress response gene, and that induction occurs via activation of cell stress signaling pathways. We tested this hypothesis by investigating the ability of physical stresses including heat-shock (HS), UV- and X-irradiation and osmotic shock to increase γ-globin gene expression in erythroid cells. Experiments in K562 and KU812 cells showed that each of these stresses increased steady-state γ-globin mRNA levels, but only after 3-5days of treatments. HS and UV also increased γ-globin mRNA and HbF levels in differentiating primary human erythroid cells. Mechanistic studies showed that HS affects γ-globin mRNA at multiple levels, including nascent transcription and transcript stability, and that induction is dependent on neither the master regulator of the canonical HS response, HSF1, nor p38 MAPK. Inhibitor panel testing identified PI3K inhibitor LY294002 as a novel inducing agent and revealed potential roles for NFκB and VEGFR/PDGFR/Raf kinases in HS-mediated γ-globin gene induction. These findings suggest that cell stress signaling pathways play an important role in γ-globin gene induction and may provide novel targets for the pharmacologic induction of fetal hemoglobin.

Laboratory tests for paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematological disorder that is often suspected in a patient presenting with non-immune hemolytic anemia associated with pancytopenia or venous thrombosis. This disorder is a consequence of acquired somatic mutations in the phosphatidylinositol glycan class A (PIG-A) gene in the hematopoietic stem cells (HSC) of patients. The presence of these mutations leads to production of blood cells with decreased glycosyl phosphatidylinositol-anchored cell surface proteins, making red blood cells derived from the clone more sensitive to complement mediated hemolysis. The diagnosis of PNH may be difficult in some cases due a low proportion of PNH cells in the blood and occasionally due to difficulties in selecting the most appropriate diagnostic studies. The latest generation of tests allow for detection of very small populations of PNH cells, for following the natural course and response to therapy of the disease, and for helping to decide when to initiate therapy with monoclonal antibody targeting the terminal complement protein C5 (Eculizumab), anticoagulation, and in some cases allogeneic HSC transplant. In this article, we review the different diagnostic tests available to clinicians for PNH diagnosis.

Induction of human fetal hemoglobin via the NRF2 antioxidant response signaling pathway.

Although hematopoietic stem cell transplantation and gene therapy have the potential to cure ß-thalassemia and sickle cell disease, they are not currently available to most people with these diseases. In the near term, pharmacologic induction of fetal hemoglobin (HbF) may offer the best possibility for safe, effective, and widely available therapy. In an effort to define new pathways for targeted drug development for HbF induction, we evaluated the nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant response element signaling pathway. We found that 3 well-known activators of this pathway increased γ-globin mRNA at nontoxic doses in K562 cells. Tert-butylhydroquinone (tBHQ), the most active of these compounds, increased cellular levels and nuclear translocation of NRF2 and binding of NRF2 to the γ-globin promoter. siRNA knockdown of NRF2 inhibited γ-globin induction by tBHQ. When tested in human primary erythroid cells, tBHQ induced NRF2 binding to the γ-globin promoter, increased γ-globin mRNA and HbF, and suppressed ß-globin mRNA and HbA, resulting in a > 3-fold increase in the percentage of HbF. These results suggest that drugs that activate the NRF2/antioxidant response element signaling pathway have the potential to induce therapeutic levels of HbF in people with ß-hemoglobinopathies.

The core-binding factor beta subunit is required for bone formation and hematopoietic maturation.

Core-binding factor beta (Cbfbeta) is the common non-DNA-binding subunit of the Cbf family of heterodimeric transcription factors. Mice deficient in Cbfbeta have a severe block in fetal liver hematopoiesis at the stage of hematopoietic stem cell (HSC) emergence. Here we show that by providing Cbfbeta function in endothelial cells and hematopoietic progenitors we can rescue fetal liver hematopoiesis in Cbfbeta-deficient embryos. The rescued mice die at birth, however, with severe defects in skeletal development, though intramembranous ossification occurs to some extent. Fetal liver hematopoiesis is restored at embryonic day (E) 12.5, but by E17.5 significant impairments in lymphopoiesis and myelopoiesis are observed. Thus, we conclude that the Cbfbeta subunit is required for HSC emergence, bone formation and normal differentiation of lymphoid and myeloid lineage cells.

Novel therapeutic candidates, identified by molecular modeling, induce γ-globin gene expression in vivo.

The ß-hemoglobinopathies and thalassemias are serious genetic blood disorders affecting the ß-globin chain of hemoglobin A (α(2)ß(Α)(2)). Their clinical severity can be reduced by enhancing expression of fetal hemoglobin (γ-globin), producing HbF (α(2)γ(2,)). In studies reported here, γ-globin induction by 23 novel, structurally-unrelated compounds, which had been predicted through molecular modeling and in silico screening of a 13,000 chemical library, was evaluated in vitro in erythroid progenitors cultured from normal subjects and ß-thalassemia patients, and in vivo in transgenic mice or anemic baboons. Four predicted candidates were found to have high potency, with 4- to 8-fold induction of HbF. Two of these compounds have pharmacokinetic profiles favorable for clinical application. These studies thus effectively identified high potency γ-globin inducing candidate therapeutics and validated the utility of in silico molecular modeling.

CpG hypomethylation in a large domain encompassing the embryonic beta-like globin genes in primitive erythrocytes.

There is little evidence addressing the role of CpG methylation in transcriptional control of genes that do not contain CpG islands. This is reflected in the ongoing debate about whether CpG methylation merely suppresses retroelements or if it also plays a role in developmental and tissue-specific gene regulation. The genes of the beta-globin locus are an important model of mammalian developmental gene regulation and do not contain CpG islands. We have analyzed the methylation status of regions in the murine beta-like globin locus in uncultured primitive and definitive erythroblasts and other cultured primary and transformed cell types. A large ( approximately 20-kb) domain is hypomethylated only in primitive erythroid cells; it extends from the region just past the locus control region to before beta-major and encompasses the embryonic genes Ey, beta h1, and beta h0. Even retrotransposons in this region are hypomethylated in primitive erythroid cells. The existence of this large developmentally regulated domain of hypomethylation supports a mechanistic role for DNA methylation in developmental regulation of globin genes.

A cell stress signaling model of fetal hemoglobin induction: what doesn't kill red blood cells may make them stronger.

A major goal of hemoglobinopathy research is to develop treatments that correct the underlying molecular defects responsible for sickle cell disease and beta-thalassemia. One approach to achieving this goal is the pharmacologic induction of fetal hemoglobin (HbF). This strategy is capable of inhibiting the polymerization of sickle hemoglobin and correcting the globin chain imbalance of beta-thalassemia. Despite this promise, none of the currently available HbF-inducing agents exhibit the combination of efficacy, safety, and convenience of use that would make them applicable to most patients. The recent success of targeted drug therapies for malignant diseases suggests that this approach could be effective for developing optimal HbF-inducing agents. A first step in applying this approach is the identification of specific molecular targets. However, while >70 HbF-inducing agents have been described, neither molecular mechanisms nor target molecules have been definitively verified for any of these compounds. To help focus investigation in this area, we have reviewed known HbF-inducing agents and their proposed mechanisms of action. We find that in many cases, current models inadequately explain key experimental results. By integrating features of the erythropoietic stress model of HbF induction with data from recent intracellular signaling experiments, we have developed a new model that has the potential to explain several findings that are inconsistent with previous models and to unify most HbF-inducing agents under a common mechanism: cell stress signaling. If correct, this or related models could lead to new opportunities for development of targeted therapies for the beta-hemoglobinopathies.

Severe acquired platelet dysfunction because of primary myelofibrosis with full functional and morphological recovery after allogeneic hematopoietic cell transplantation.

: Primary myelofibrosis (PMF) is a clonal hematopoietic stem cell disorder characterized by fibrosis of the marrow cavity, marked megakaryocyte atypia and progressive cytopenias. Although thrombosis predominates, bleeding is the primary manifestation in up to 20% of patients and may be life-threatening. In this report, we document restoration of megakaryocyte and platelet structure and function in PMF after allogeneic hematopoietic cell transplantation (HCT). A 59-year-old man presented with recurrent episodes of postoperative bleeding preceding a diagnosis of primary myelofibrosis (PMF). Platelet aggregation and secretion studies showed abnormal responses to all agonists tested (epinephrine, ADP, arachidonic acid, U46619, collagen, ristocetin) despite the presence of thrombocytosis. After an allogeneic HCT, platelet morphology and function studies were all normal. The pathophysiology of platelet dysfunction in myeloid neoplasia is not well understood but, as highlighted in our report, restoration of platelet function by HCT supports a clonal process involving an early hematopoietic progenitor cell.

Ecological effects of fear: How spatiotemporal heterogeneity in predation risk influences mule deer access to forage in a sky-island system.

Forage availability and predation risk interact to affect habitat use of ungulates across many biomes. Within sky-island habitats of the Mojave Desert, increased availability of diverse forage and cover may provide ungulates with unique opportunities to extend nutrient uptake and/or to mitigate predation risk. We addressed whether habitat use and foraging patterns of female mule deer (Odocoileus hemionus) responded to normalized difference vegetation index (NDVI), NDVI rate of change (green-up), or the occurrence of cougars (Puma concolor). Female mule deer used available green-up primarily in spring, although growing vegetation was available during other seasons. Mule deer and cougar shared similar habitat all year, and our models indicated cougars had a consistent, negative effect on mule deer access to growing vegetation, particularly in summer when cougar occurrence became concentrated at higher elevations. A seemingly late parturition date coincided with diminishing NDVI during the lactation period. Sky-island populations, rarely studied, provide the opportunity to determine how mule deer respond to growing foliage along steep elevation and vegetation gradients when trapped with their predators and seasonally limited by aridity. Our findings indicate that fear of predation may restrict access to the forage resources found in sky islands.

Hypoxia-Induced VISTA Promotes the Suppressive Function of Myeloid-Derived Suppressor Cells in the Tumor Microenvironment.

Tumor hypoxia is a negative prognostic factor that is implicated in oncogenic signal activation, immune escape, and resistance to treatment. Identifying the mechanistic role of hypoxia in immune escape and resistance to immune-checkpoint inhibitors may aid the identification of therapeutic targets. We and others have shown that V-domain Ig suppressor of T-cell activation (VISTA), a negative checkpoint regulator in the B7 family, is highly expressed in the tumor microenvironment in tumor models and primary human cancers. In this study, we show that VISTA and HIF1α activity are correlated in a cohort of colorectal cancer patients. High VISTA expression was associated with worse overall survival. We used the CT26 colon cancer model to investigate the regulation of VISTA by hypoxia. Compared with less hypoxic tumor regions or draining lymph nodes, regions of profound hypoxia in the tumor microenvironment were associated with increased VISTA expression on tumor-infiltrating myeloid-derived suppressor cells (MDSC). Using chromatin immunoprecipitation and genetic silencing, we show that hypoxia-inducible factor (HIF)-1α binding to a conserved hypoxia response element in the VISTA promoter upregulated VISTA on myeloid cells. Further, antibody targeting or genetic ablation of VISTA under hypoxia relieved MDSC-mediated T-cell suppression, revealing VISTA as a mediator of MDSC function. Collectively, these data suggest that targeting VISTA may mitigate the deleterious effects of hypoxia on antitumor immunity.

Expression of GATA-1 in a non-hematopoietic cell line induces beta-globin locus control region chromatin structure remodeling and an erythroid pattern of gene expression.

GATA-1 is a hematopoietic transcription factor expressed in erythroid, megakaryocytic, mast cell and eosinophil lineages. It is required for normal erythroid differentiation, the expression of erythroid-specific genes and for the establishment of an active chromatin structure throughout the beta-globin gene locus. GATA-1 is also necessary for the formation and function of the locus control region DNase I hypersensitive site (HS) core elements. To determine whether GATA-1 was sufficient to direct formation of the locus control region (LCR) and an erythroid pattern of gene expression, we expressed GATA-1 in the non-hematopoietic HeLa cell line that does not express other hematopoietic transcription factors but does express GATA-2, GATA-3, and GATA-6. We found that production of the GATA-1 protein resulted in the formation of LCR DNase I HSs 1-4 in their normal locations, and that histones became hyperacetylated within these regulatory elements. Transcription of several erythroid-specific genes was activated in HeLa cells expressing GATA-1, including those coding for alpha-globin, beta-globin, the erythropoietin receptor, the erythroid krüpple-like factor and p45 NF-E2. Despite increased expression of these genes at the mRNA level, their protein products were not detected. These results imply that GATA-1 is sufficient to direct chromatin structure reorganization within the beta-globin LCR and an erythroid pattern of gene expression in the absence of other hematopoietic transcription factors.

Hospital readmission following transplantation: identifying risk factors and designing preventive measures.

BACKGROUND: About 1 in 7 of all hospitalized patients is readmitted within 30 days of discharge. The cost of readmissions is significant, with Medicare readmissions alone costing the health care system an estimated $28 billion a year. OBJECTIVE: To identify the rates of and causes for readmission within 100 days of patients receiving a hematopoietic stem cell transplant. METHODS: We performed a retrospective review of 235 consecutive transplant recipients (autologous, n = 144; allogeneic, n = 91) to determine rates and causes for readmission within 100 days of patients receiving a transplant. Medical records and hospital readmissions were reviewed for each patient. RESULTS: 36 allogeneic patients accounted for 56 readmissions. 23 autologous patients accounted for 26 readmissions. Autologous transplant recipients were most commonly readmitted for the development of a fever (n = 15 patients) or cardiopulmonary issues (n = 4). The most prevalent reasons for readmission in the allogeneic recipients included a fever (n = 21) or the development or exacerbation of graft-versus-host disease (n = 5). The readmission length of stay was 6 days (median range, 1-91 days) for allogeneic patients and 4 days (median range, 1-22 days) for autologous patients. There was no difference in survival between the readmitted and the non-readmitted cohorts (𝑃 = .55 for allogeneic patients; 𝑃 = .24 for autologous patients). Although allogeneic graft recipients demonstrated a higher readmission rate (39.6%) compared with autologous recipients (16%), none of the variables examined, including age, gender, performance status, diagnosis, remission status at the time of transplant, comorbidities, type of preparative chemotherapy regimen or donor type, identified patients at increased risk for readmission. LIMITATIONS: Variations in clinical care, physician practices, and patient characteristics need to be considered when examining readmission rates. Most of the allogeneic patient population included unrelated donor recipients (65%) who received nonmyeloablative conditioning regimens (81% of allogeneic recipients). These features may not be characteristic of other centers. CONCLUSIONS: In these high-risk patients, readmissions following a transplant are common. Enhanced predischarge education by nurses and pharmacists, along with ongoing outpatient education and rigorous outpatient follow-up through phone calls or social media may decrease readmission rates.

Defensive applications of gene transfer technology in the face of bioterrorism: DNA-based vaccines and immune targeting.

Gene transfer involves the introduction of an engineered gene into a person's cells with the expectation that the protein expressed from the gene will produce a therapeutic benefit. Strategies based on this principle have led to the approval of > 600 clinical trials and enrollment of approximately 3500 subjects worldwide in attempts to treat diseases ranging from cancer to AIDS to cystic fibrosis. While gene therapy has met with limited success and still has many hurdles to overcome before it sees wide application, it may be useful as a defensive strategy against bioterrorism agents including infectious microbes and toxins. Although many defensive strategies are possible, immunological strategies are currently the most developed and are being actively applied to the development of strategies against several of the most virulent potential bio-weapons. While most of these strategies are not yet ready for human application, DNA-based vaccines appear to be among the most promising in the fight against bioterrorism.

Vinblastine rapidly induces NOXA and acutely sensitizes primary chronic lymphocytic leukemia cells to ABT-737.

Proteins of the BCL2 family provide a survival mechanism in many human malignancies, including chronic lymphocytic leukemia (CLL). The BCL2 inhibitor ABT-263 (navitoclax) is active in clinical trials for lymphoid malignancies, yet resistance is expected on the basis of preclinical models. We recently showed that vinblastine can dramatically sensitize several leukemia cell lines to ABT-737 (the experimental congener of ABT-263). The goal of these experiments was to determine the impact of vinblastine on ABT-737 sensitivity in CLL cells isolated from peripheral blood and to define the underlying mechanism. Freshly isolated CLL cells from 35 patients, as well as normal lymphocytes and platelets, were incubated with various microtubule-disrupting agents plus ABT-737 to assess sensitivity to the single agents and the combination. ABT-737 and vinblastine displayed a range of sensitivity as single agents, and vinblastine markedly sensitized all CLL samples to ABT-737 within six hours. Vinblastine potently induced the proapoptotic protein PMAIP1 (NOXA) in both time- and dose-dependent manner and this was required for the observed apoptosis. Combretastatin A4, which dissociates microtubules by binding to a different site, had the same effect, confirming that interaction of these agents with microtubules is the initial target. Similarly, vincristine and vinorelbine induced NOXA and enhanced CLL sensitivity to ABT-737. Furthermore, vinblastine plus ABT-737 overcame stroma-mediated resistance to ABT-737 alone. Apoptosis was induced with clinically achievable concentrations with no additional toxicity to normal lymphocytes or platelets. These results suggest that vinca alkaloids may improve the clinical efficacy of ABT-263 in patients with CLL.