Treatment of relapsed/refractory severe aplastic anemia in children: Evidence-based recommendations.

Severe aplastic anemia (SAA) is a rare potentially fatal hematologic disorder. Although overall outcomes with treatment are excellent, there are variations in management approach, including differences in treatment between adult and pediatric patients. Certain aspects of treatment are under active investigation in clinical trials. Because of the rarity of the disease, some pediatric hematologists may have relatively limited experience with the complex management of SAA. The following recommendations reflect an up-to-date evidence-based approach to the treatment of children with relapsed or refractory SAA.

Treatment of newly diagnosed severe aplastic anemia in children: Evidence-based recommendations.

Severe aplastic anemia (SAA) is a rare potentially fatal hematologic disorder. Although overall outcomes with treatment are excellent, there are variations in management approach, including differences in treatment between adult and pediatric patients. Certain aspects of treatment are under active investigation in clinical trials. Because of the rarity of the disease, some pediatric hematologists may have relatively limited experience with the complex management of SAA. The following recommendations reflect an up-to-date evidence-based approach to the treatment of children with newly diagnosed SAA.

A murine model to evaluate immunotherapy effectiveness for human Fanconi anemia-mutated acute myeloid leukemia.

Fanconi anemia (FA)-mutated acute myeloid leukemia (AML) is a secondary AML with very poor prognosis and limited therapeutic options due to increased sensitivity to DNA-damaging agents. PD-1 immune checkpoint inhibitors upregulate T-cell killing of cancer cells and is a class of promising treatment for FA-AML. Here, we developed a novel FA-AML murine model that allows the study of human AML with a humanized immune system in order to investigate immunotherapeutic treatments in vivo. FA-AML1 cells and non-FA-mutated Kasumi-1 cells were injected into 8-10 week old NSG mice. Once leukemic engraftment was confirmed by HLA-DR expression in the peripheral blood, human peripheral blood mononuclear cells (hPBMCs) were injected into the mice. One week post-hPBMCs injection, Nivolumab (PD-1 inhibitor) or PBS vehicle control was administered to the mice bi-weekly. In our Nivolumab treated mice, FA-AML1, but not Kasumi-1-engrafted mice, had significantly prolonged overall survival. Both FA-AML1 and Kasumi-1 engrafted mice had decreased spleen weights. Higher leukemic infiltration into vital organs was observed in FA-AML1 engrafted mice compared to Kasumi-1 engrafted mice. In conclusion, our novel humanized murine model of FA-mutated AML is an attractive tool for supporting further studies and clinical trials using PD-1 inhibitors to treat FA-mutated AML.

Hematologic complications with age in Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure syndrome with leukemia predisposition. An understanding of the hematologic complications of SDS with age could guide clinical management, but data are limited for this rare disease. We conducted a cohort study of 153 subjects from 143 families with confirmed biallelic SBDS mutations enrolled on the North American Shwachman Diamond Registry or Bone Marrow Failure Registry. The SBDS c.258 + 2T>C variant was present in all but 1 patient. To evaluate the association between blood counts and age, 2146 blood counts were analyzed for 119 subjects. Absolute neutrophil counts were positively associated with age (P < .0001). Hemoglobin was also positively associated with age up to 18 years (P < .0001), but the association was negative thereafter (P = .0079). Platelet counts and marrow cellularity were negatively associated with age (P < .0001). Marrow cellularity did not correlate with blood counts. Severe marrow failure necessitating transplant developed in 8 subjects at a median age of 1.7 years (range, 0.4-39.5), with 7 of 8 requiring transplant prior to age 8 years. Twenty-six subjects (17%) developed a myeloid malignancy (16 myelodysplasia and 10 acute myeloid leukemia) at a median age of 12.3 years (range, 0.5-45.0) and 28.4 years (range, 14.4-47.3), respectively. A lymphoid malignancy developed in 1 patient at the age of 16.9 years. Hematologic complications were the major cause of mortality (17/20 deaths; 85%). These data inform surveillance of hematologic complications in SDS.

Diagnostic work-up for severe aplastic anemia in children: Consensus of the North American Pediatric Aplastic Anemia Consortium.

The North American Pediatric Aplastic Anemia Consortium (NAPAAC) is a group of pediatric hematologist-oncologists, hematopathologists, and bone marrow transplant physicians from 46 institutions in North America with interest and expertise in aplastic anemia, inherited bone marrow failure syndromes, and myelodysplastic syndromes. The NAPAAC Bone Marrow Failure Diagnosis and Care Guidelines Working Group was established with the charge of harmonizing the approach to the diagnostic workup of aplastic anemia in an effort to standardize best practices in the field. This document outlines the rationale for initial evaluations in pediatric patients presenting with signs and symptoms concerning for severe aplastic anemia.

Distinct genetic pathways define pre-malignant versus compensatory clonal hematopoiesis in Shwachman-Diamond syndrome.

To understand the mechanisms that mediate germline genetic leukemia predisposition, we studied the inherited ribosomopathy Shwachman-Diamond syndrome (SDS), a bone marrow failure disorder with high risk of myeloid malignancies at an early age. To define the mechanistic basis of clonal hematopoiesis in SDS, we investigate somatic mutations acquired by patients with SDS followed longitudinally. Here we report that multiple independent somatic hematopoietic clones arise early in life, most commonly harboring heterozygous mutations in EIF6 or TP53. We show that germline SBDS deficiency establishes a fitness constraint that drives selection of somatic clones via two distinct mechanisms with different clinical consequences. EIF6 inactivation mediates a compensatory pathway with limited leukemic potential by ameliorating the underlying SDS ribosome defect and enhancing clone fitness. TP53 mutations define a maladaptive pathway with enhanced leukemic potential by inactivating tumor suppressor checkpoints without correcting the ribosome defect. Subsequent development of leukemia was associated with acquisition of biallelic TP53 alterations. These results mechanistically link leukemia predisposition to germline genetic constraints on cellular fitness, and provide a rational framework for clinical surveillance strategies.

Diagnosis and treatment of pediatric myelodysplastic syndromes: A survey of the North American Pediatric Aplastic Anemia Consortium.

BACKGROUND: Myelodysplastic syndromes (MDS) represent a group of clonal hematopoietic stem cell disorders that commonly progress to acute myeloid leukemia (AML). The diagnostics, prognostics, and treatment of adult MDS are established but do not directly translate to children and adolescents. Pediatric MDS is a rare disease, characterized by unique cytogenetics and histology compared with adult MDS, and often arises secondary to germline predisposition or cytotoxic exposures. Our objective was to highlight aspects of diagnosis/management that would benefit from further systematic review toward the development of clinical practice guidelines for pediatric MDS. PROCEDURE: The North American Pediatric Aplastic Anemia Consortium (NAPAAC) is composed of collaborative institutions with a strong interest in pediatric bone marrow failure syndromes and hematologic malignancies. The NAPAAC MDS working group developed a national survey distributed to 35 NAPAAC institutions to assess data on (1) clinical presentation of pediatric MDS, (2) diagnostic evaluation, (3) criteria for diagnosis, (4) supportive care and treatment decisions, and (5) role of hematopoietic stem cell transplantation (HSCT). RESULTS: Twenty-eight of 35 institutions returned the survey. Most centers agreed on a common diagnostic workup, though there was considerable variation regarding the criteria for diagnosis. Although there was consensus on supportive care, treatment strategies, including the role of cytoreduction and HSCT, varied across centers surveyed. CONCLUSIONS: There is lack of national consensus on diagnosis and treatment of pediatric MDS. This survey identified key aspects of MDS management that will warrant systematic review toward the goal of developing national clinical practice guidelines for pediatric MDS.

A Child With Pancytopenia and Optic Disc Swelling.

A previously healthy 16-year-old adolescent boy presented with pallor, blurry vision, fatigue, and dyspnea on exertion. Physical examination demonstrated hypertension and bilateral optic nerve swelling. Laboratory testing revealed pancytopenia. Pediatric hematology, ophthalmology and neurology were consulted and a life-threatening diagnosis was made.

Hedgehog/GLI1 activation leads to leukemic transformation of myelodysplastic syndrome in vivo and GLI1 inhibition results in antitumor activity.

Myelodysplastic syndromes (MDSs) are stem cell disorders with risk of transformation to acute myeloid leukemia (AML). Gene expression profiling reveals transcriptional expression of GLI1, of Hedgehog (Hh) signaling, in poor-risk MDS/AML. Using a murine model of MDS we demonstrated that constitutive Hh/Gli1 activation accelerated leukemic transformation and decreased overall survival. Hh/Gli1 activation resulted in clonal expansion of phenotypically defined granulocyte macrophage progenitors (GMPs) and acquisition of self-renewal potential in a non-self-renewing progenitor compartment. Transcriptome analysis of GMPs revealed enrichment in gene signatures of self-renewal pathways, operating via direct Gli1 activation. Using human cell lines we demonstrated that in addition to canonical Hh signaling, GLI1 is activated in a Smoothened-independent manner. GLI1 knockdown or inhibition with GANT61 resulted in decreased proliferation and clonogenic potential. Our data suggest that GLI1 activation is frequent in MDS during disease progression and inhibition of GLI1 is an attractive therapeutic target for a subset of patients.

High prognostic value of pre-allogeneic stem cell transplantation minimal residual disease detection by WT1 gene expression in AML transplanted in cytologic complete remission.

We analyzed the outcome of allogeneic stem cell transplantation (allo-SCT) in acute myeloid leukemia (AML) patients according to molecular Minimal Residual Disease (MRD) status prior to allo-SCT. MRD was assessed by the quantitative expression of the pan-leukemic marker Wilms' tumor (WT1) gene, according to the validated LeukemiaNet method. Between 2005 and 2016, 122 consecutive AML patients, WT1 positive at diagnosis, received allo-SCT in cytologic complete remission (cCR). The median age at SCT was 53 years (range 18-70). Quantitative analysis of WT1 gene expression (bone marrow samples) was available in all cases both at diagnosis (100% of samples overexpressed WT1 with a mean of 8607±8187 copies/104 Abelson) and immediately before allo-SCT. Eighty one cases (66%) were MRD-WT1 negative (WT1 <250 copies) and 41/122 (44%) cases were MRD-WT1 positive (WT1 >250 copies) prior to allo-SCT. We evaluated post-SCT overall survival (OS), disease free survival (DFS) and relapse rate (RR), according to MRD-WT1 status pre-SCT. Both post-allo-SCT OS and DFS were significantly improved in patients who were MRD-WT1 negative at the time of SCT compared with those who were MRD-WT1 positive, with a median OS and DFS not reached in the MRD-WT1 negative group and 9 and 8 months, respectively, in the WT1 positive group (OS log-rank p<0.0001; hazard ratio [HR] 3.9, 95% confidence interval [95% CI] 2.0-7.38; DFS log-rank p<0.0001; HR 3.73, 95% CI 2.0-6.72). The RR after SCT was 15% (12/81) in pre-SCT MRD-WT1 negative cases and 44% (18/41) in MRD-WT1 positive cases (p=0.00073). Univariate analysis showed that MRD-WT1 negativity pre-SCT and grade <2 acute GVHD were significant prognostic factors for improved OS and DFS. However multivariate analysis showed MRD-WT1 negativity pre-SCT was the only independent prognostic factor for improved OS and DFS. These data show that pre allo-SCT molecular MRD evaluation using WT1 expression is a powerful predictor of post allo-SCT outcomes in AML undergoing SCT in cCR. Patients with both cCR and MRD-WT1 negativity before SCT have a very good outcome with lower RR and improved OS. The pre allo-SCT MRD-WT1 stratification in AML is a valuable tool to identify patients at high risk of post-SCT relapse, and can influence conditioning regimen intensification and/or post-SCT preemptive strategies.

SDF1/CXCL12 is involved in recruitment of stem-like progenitor cells to orthotopic murine malignant mesothelioma spheroids.

BACKGROUND/AIM: Tumor progression is influenced by the microenvironment. We found stem cells are recruited to malignant mesothelioma spheroids. We aimed to determine if stem cell recruitment depends on the chemokine SDF1, and if inhibition of the cognate receptor CXCR4 affects tumor growth. MATERIALS AND METHODS: The kinetics of stem cell recruitment was determined using immunofluorescence staining, BrdU incorporation and eGFP transgenic mice. Chemokines were identified using PCR array. Inhibitors of CXCR4 were used to determine the effect on cell migration and tumor progression. RESULTS: The increasing number of stem cells found in tumor spheroids over time is attributed to cell recruitment. Stem cell migration in vitro was enhanced by exogenous SDF1 and abrogated by CXCR4 inhibition and. CXCR4 inhibition reduced tumor burden in vivo. CONCLUSION: SDF1 is a candidate chemokine for recruitment of stem cells to malignant peritoneal mesothelioma and a potential target for therapy.

Kinetics of host cell recruitment during dissemination of diffuse malignant peritoneal mesothelioma.

UNLABELLED: Diffuse malignant mesothelioma is an aggressive tumor which displays a median survival of 11.2 months and a 5-year survival of less than 5% emphasizing the need for more effective treatments. This study uses an orthotopic model of malignant mesothelioma established in syngeneic, immunocompetent C57Bl/6 mice which produce malignant ascites and solid tumors that accurately replicate the histopathology of the human disease. Host stromal and immune cell accumulation within malignant ascites and solid tumors was determined using immunofluorescent labeling with confocal microscopy and fluorescence-activated cell sorting. An expression profile of cytokines and chemokines was produced using quantitative real-time PCR arrays. Tumor spheroids and solid tumors show progressive growth and infiltration with host stromal and immune cells including macrophages, endothelial cells, CD4(+) and CD8(+) lymphocytes, and a novel cell type, myeloid derived suppressor cells (MDSCs). The kinetics of host cell accumulation and inflammatory mediator expression within the tumor ascites divides tumor progression into two distinct phases. The first phase is characterized by progressive macrophage and T lymphocyte recruitment, with a cytokine profile consistent with regulatory T lymphocytes differentiation and suppression of T cell function. The second phase is characterized by decreased expression of macrophage chemotactic and T-cell regulating factors, an increase in MDSCs, and increased expression of several cytokines which stimulate differentiation of MDSCs. This cellular and expression profile suggests a mechanism by which host immune cells promote diffuse malignant mesothelioma progression. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12307-010-0048-1) contains supplementary material, which is available to authorized users.

Short-term arginine deprivation results in large-scale modulation of hepatic gene expression in both normal and tumor cells: microarray bioinformatic analysis.

BACKGROUND: We have reported arginine-sensitive regulation of LAT1 amino acid transporter (SLC 7A5) in normal rodent hepatic cells with loss of arginine sensitivity and high level constitutive expression in tumor cells. We hypothesized that liver cell gene expression is highly sensitive to alterations in the amino acid microenvironment and that tumor cells may differ substantially in gene sets sensitive to amino acid availability. To assess the potential number and classes of hepatic genes sensitive to arginine availability at the RNA level and compare these between normal and tumor cells, we used an Affymetrix microarray approach, a paired in vitro model of normal rat hepatic cells and a tumorigenic derivative with triplicate independent replicates. Cells were exposed to arginine-deficient or control conditions for 18 hours in medium formulated to maintain differentiated function. RESULTS: Initial two-way analysis with a p-value of 0.05 identified 1419 genes in normal cells versus 2175 in tumor cells whose expression was altered in arginine-deficient conditions relative to controls, representing 9-14% of the rat genome. More stringent bioinformatic analysis with 9-way comparisons and a minimum of 2-fold variation narrowed this set to 56 arginine-responsive genes in normal liver cells and 162 in tumor cells. Approximately half the arginine-responsive genes in normal cells overlap with those in tumor cells. Of these, the majority was increased in expression and included multiple growth, survival, and stress-related genes. GADD45, TA1/LAT1, and caspases 11 and 12 were among this group. Previously known amino acid regulated genes were among the pool in both cell types. Available cDNA probes allowed independent validation of microarray data for multiple genes. Among genes downregulated under arginine-deficient conditions were multiple genes involved in cholesterol and fatty acid metabolism. Expression of low-density lipoprotein receptor was decreased in both normal and tumor cells. CONCLUSION: Arginine-sensitive regulation appears to be an important homeostatic mechanism to coordinate cell response and nutrient availability in hepatic cells. Genes predicted as arginine-responsive in stringent microarray data analysis were confirmed by Northern blot and RT-PCR. Although the profile of arginine-responsive genes is altered and increased, a considerable portion of the "arginome" is maintained upon neoplastic transformation.

Deoxycholic acid activates protein kinase C and phospholipase C via increased Ca2+ entry at plasma membrane.

BACKGROUND & AIMS: Secondary bile acids like deoxycholic acid (DCA) are well-established tumor promoters that may exert their pathologic actions by interfering with intracellular signaling cascades. METHODS: We evaluated the effects of DCA on Ca2+ signaling in BHK-21 fibroblasts using fura-2 and mag-fura-2 to measure cytoplasmic and intraluminal internal stores [Ca2+], respectively. Furthermore, green fluorescent protein (GFP)-based probes were used to monitor time courses of phospholipase C (PLC) activation (pleckstrin-homology [PH]-PLCdelta-GFP), and translocation of protein kinase C (PKC) and a major PKC substrate, myristolated alanine-rich C-kinase substrate (MARCKS). RESULTS: DCA (50-250 micromol/L) caused profound Ca2+ release from intracellular stores of intact or permeabilized cells. Correspondingly, DCA increased cytoplasmic Ca2+ to levels that were approximately 120% of those stimulated by Ca2+-mobilizing agonists in the presence of external Ca2+, and approximately 60% of control in Ca2+-free solutions. DCA also caused dramatic translocation of PH-PLCdelta-GFP, and conventional, Ca2+/diacylglycerol (DAG)-dependent isoforms of PKC (PKC-betaI and PKC-alpha), and MARCKS-GFP, but only in Ca2+-containing solutions. DCA had no effect on localization of a novel (PKCdelta) or an atypical (PKCzeta) PKC isoform. CONCLUSIONS: Data are consistent with a model in which DCA directly induces both Ca2+ release from internal stores and persistent Ca2+ entry at the plasma membrane. The resulting microdomains of high Ca2+ levels beneath the plasma membrane appear to directly activate PLC, resulting in modest InsP 3 and DAG production. Furthermore, the increased Ca2+ entry stimulates vigorous recruitment of conventional PKC isoforms to the plasma membrane.

Divalent cations regulate acidity within the lumen and tubulovesicle compartment of gastric parietal cells.

BACKGROUND & AIMS: Until recently, it has not been possible to evaluate factors that regulate the acidity of the microenvironment within the tubulovesicles and luminal (TV/L) spaces of the gastric gland. The goal of this study was to develop a method for monitoring the mechanisms that regulate acidity in the TV/L compartment. METHODS: Isolated rabbit gastric glands (intact or permeabilized with S. aureus alpha-toxin) were loaded with a recently characterized fluorescent dye, LysoSensor Yellow-Blue DND 160 (Molecular Probes, Eugene, OR), which localizes to highly acidic compartments and can be used to monitor acidity ratiometrically. RESULTS: In resting glands, the pH of the TV/L compartment was approximately 3.4. Moderate alkalizations ( approximately 0.5 to 1.0 pH unit alkalization) were observed during exposure to inhibitors of the apical H(+)/K(+) ATPase (omeprazole and SCH28080), thereby unmasking a stable, low-level leak of H(+) ions from the TV/L compartment. Similar changes were observed in alpha-toxin permeabilized glands following depletion of ATP in the cytoplasm. In intact and permeabilized glands, we used the cell-permeant, divalent cation chelator, tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) to probe the effects of lowering divalent cation content of the TV/L compartment. Exposure to relatively low concentrations (20 micromol/L, 50 micromol/L) of TPEN reversibly promoted H(+) leakage. At these concentrations, simultaneous inhibition using SCH28080 led to marked enhancement of the rate of alkalization. CONCLUSIONS: The effects of low-dose TPEN suggests that acidity within the TV/L compartment of the gastric gland may be regulated, at least in part, by its content of divalent cations such as Zn(2+), for which TPEN has high affinity.