Penetrance of biallelic SMARCAL1 mutations is associated with environmental and genetic disturbances of gene expression.

Biallelic mutations of the DNA annealing helicase SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1) cause Schimke immuno-osseous dysplasia (SIOD, MIM 242900), an incompletely penetrant autosomal recessive disorder. Using human, Drosophila and mouse models, we show that the proteins encoded by SMARCAL1 orthologs localize to transcriptionally active chromatin and modulate gene expression. We also show that, as found in SIOD patients, deficiency of the SMARCAL1 orthologs alone is insufficient to cause disease in fruit flies and mice, although such deficiency causes modest diffuse alterations in gene expression. Rather, disease manifests when SMARCAL1 deficiency interacts with genetic and environmental factors that further alter gene expression. We conclude that the SMARCAL1 annealing helicase buffers fluctuations in gene expression and that alterations in gene expression contribute to the penetrance of SIOD.

Comparison of the ABC and ACMG systems for variant classification.

The ABC and ACMG variant classification systems were compared by asking mainly European clinical laboratories to classify variants in 10 challenging cases using both systems, and to state if the variant in question would be reported as a relevant result or not as a measure of clinical utility. In contrast to the ABC system, the ACMG system was not made to guide variant reporting but to determine the likelihood of pathogenicity. Nevertheless, this comparison is justified since the ACMG class determines variant reporting in many laboratories. Forty-three laboratories participated in the survey. In seven cases, the classification system used did not influence the reporting likelihood when variants labeled as "maybe report" after ACMG-based classification were included. In three cases of population frequent but disease-associated variants, there was a difference in favor of reporting after ABC classification. A possible reason is that ABC step C (standard variant comments) allows a variant to be reported in one clinical setting but not another, e.g., based on Bayesian-based likelihood calculation of clinical relevance. Finally, the selection of ACMG criteria was compared between 36 laboratories. When excluding criteria used by less than four laboratories (<10%), the average concordance rate was 46%. Taken together, ABC-based classification is more clear-cut than ACMG-based classification since molecular and clinical information is handled separately, and variant reporting can be adapted to the clinical question and phenotype. Furthermore, variants do not get a clinically inappropriate label, like pathogenic when not pathogenic in a clinical context, or variant of unknown significance when the significance is known.

A correlation between cytotoxicity and reductase-mediated metabolism in cell lines treated with doxorubicin and daunorubicin.

The role of metabolism in daunorubicin (DAUN)- and doxorubicin (DOX)-associated toxicity in cancer patients is dependent on whether the parent drugs or major metabolites, doxorubicinol (DOXol) and daunorubicinol (DAUNol), are the more toxic species. Therefore, we examined whether an association exists between cytotoxicity and the metabolism of these drugs in cell lines from nine different tissues. Cytotoxicity studies using MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide] cell viability assays revealed that four cell lines [HepG2 (liver), HCT-15 (colon), NCI-H460 (lung), and A-498 (kidney)] were more tolerant to DAUN and DOX than the five remaining cell lines [H9c2 (heart), PC-3 (prostate), OVCAR-4 (ovary), PANC-1 (pancreas), and MCF-7 (breast)], based on significantly higher LC50 values at incubation times of 6, 24, and 48 hours. Each cell line was also assessed for its efficiency at metabolizing DAUN and DOX. The four drug-tolerant cell lines converted DAUN/DOX to DAUNol/DOXol more rapidly than the five drug-sensitive cell lines. We also determined whether exposure to DAUN or DOX induced an increase in metabolic activity among any of these nine different cell types. All nine cell types showed a significant increase in their ability to metabolize DAUN or DOX in response to pre-exposure to the drug. Western blot analyses demonstrated that the increased metabolic activity toward DAUN and DOX correlated with a greater abundance of eight aldo-keto and two carbonyl reductases following exposure to either drug. Overall, our findings indicate an inverse relationship between cytotoxicity and DAUN or DOX metabolism in these nine cell lines.

SMARCAL1 deficiency predisposes to non-Hodgkin lymphoma and hypersensitivity to genotoxic agents in vivo.

Schimke immuno-osseous dysplasia (SIOD) is a multisystemic disorder with prominent skeletal, renal, immunological, and ectodermal abnormalities. It is caused by mutations of SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1), which encodes a DNA stress response protein. To determine the relationship of this function to the SIOD phenotype, we profiled the cancer prevalence in SIOD and assessed if defects of nucleotide excision repair (NER) and nonhomologous end joining (NHEJ), respectively, explained the ectodermal and immunological features of SIOD. Finally, we determined if Smarcal1(del/del) mice had hypersensitivity to irinotecan (CPT-11), etoposide, and hydroxyurea (HU) and whether exposure to these agents induced features of SIOD. Among 71 SIOD patients, three had non-Hodgkin lymphoma (NHL) and one had osteosarcoma. We did not find evidence of defective NER or NHEJ; however, Smarcal1-deficient mice were hypersensitive to several genotoxic agents. Also, CPT-11, etoposide, and HU caused decreased growth and loss of growth plate chondrocytes. These data, which identify an increased prevalence of NHL in SIOD and confirm hypersensitivity to DNA damaging agents in vivo, provide guidance for the management of SIOD patients.

Broad genomic workup including optical genome mapping uncovers a DDX3X: MLLT10 gene fusion in acute myeloid leukemia.

In acute myeloid leukemia (AML), treatment decisions are currently made according to the risk classification of the European LeukemiaNet (ELN), which is based on genetic alterations. Recently, optical genome mapping (OGM) as a novel method proved to yield a genome-wide and detailed cytogenetic characterization at the time of diagnosis. A young female patient suffered from a rather unexpected aggressive disease course under FLT3 targeted therapy in combination with induction chemotherapy. By applying a "next-generation diagnostic workup" strategy with OGM and whole-exome sequencing (WES), a DDX3X: MLLT10 gene fusion could be detected, otherwise missed by routine diagnostics. Furthermore, several aspects of lineage ambiguity not shown by standard diagnostics were unraveled such as deletions of SUZ12 and ARPP21, as well as T-cell receptor recombination. In summary, the detection of this particular gene fusion DDX3X: MLLT10 in a female AML patient and the findings of lineage ambiguity are potential explanations for the aggressive course of disease. Our study demonstrates that OGM can yield novel clinically significant results, including additional information helpful in disease monitoring and disease biology.

Establishing analytical validity of BeadChip array genotype data by comparison to whole-genome sequence and standard benchmark datasets.

BACKGROUND: Clinical use of genotype data requires high positive predictive value (PPV) and thorough understanding of the genotyping platform characteristics. BeadChip arrays, such as the Global Screening Array (GSA), potentially offer a high-throughput, low-cost clinical screen for known variants. We hypothesize that quality assessment and comparison to whole-genome sequence and benchmark data establish the analytical validity of GSA genotyping. METHODS: To test this hypothesis, we selected 263 samples from Coriell, generated GSA genotypes in triplicate, generated whole genome sequence (rWGS) genotypes, assessed the quality of each set of genotypes, and compared each set of genotypes to each other and to the 1000 Genomes Phase 3 (1KG) genotypes, a performance benchmark. For 59 genes (MAP59), we also performed theoretical and empirical evaluation of variants deemed medically actionable predispositions. RESULTS: Quality analyses detected sample contamination and increased assay failure along the chip margins. Comparison to benchmark data demonstrated that > 82% of the GSA assays had a PPV of 1. GSA assays targeting transitions, genomic regions of high complexity, and common variants performed better than those targeting transversions, regions of low complexity, and rare variants. Comparison of GSA data to rWGS and 1KG data showed > 99% performance across all measured parameters. Consistent with predictions from prior studies, the GSA detection of variation within the MAP59 genes was 3/261. CONCLUSION: We establish the analytical validity of GSA assays using quality analytics and comparison to benchmark and rWGS data. GSA assays meet the standards of a clinical screen although assays interrogating rare variants, transversions, and variants within low-complexity regions require careful evaluation.

Naturally occurring variants of human CBR3 alter anthracycline in vitro metabolism.

Doxorubicin (DOX) and daunorubicin (DAUN) are anthracycline anticancer agents; however, considerable interpatient variability exists in their pharmacokinetics. This interpatient variability is attributed in part to altered metabolism by nonsynonymous single-nucleotide polymorphisms (ns-SNPs) in genes encoding the carbonyl reductases. This study examines the effect of seven naturally occurring ns-SNPs in the CBR3 gene on in vitro metabolism of anthracyclines to doxorubicinol and daunorubicinol. Kinetic assays measure metabolite levels by high-performance liquid chromatography separation with fluorescence detection by use of purified, histidine-tagged, human CBR3 wild type and variant enzymes. The V224M, C4Y, and V93I variants resulted in significantly reduced maximal reaction velocity (V(max)) for both anthracyclines compared with the wild-type enzyme, whereas the M235L variant had significantly reduced V(max) for DOX only. Significant increases in substrate affinity were found for the V244M variant with DAUN, as well as the C4Y and V93I variants with DOX. The catalytic efficiency values for the V244M, C4Y, and V93I variants were significantly lower than the wild type for DAUN and DOX. Furthermore, DOX was observed to be a better substrate than DAUN for the wild-type enzyme and its variants. HapMap analysis indicated that a haplotype carrying the C4Y and V244M mutations may occur in some individuals in the 11 ethnic populations studied in the HapMap project. Our preparation of the double mutant indicated a significant reduction in activity compared with the wild-type enzyme and single-mutant preparations. These findings suggest that commonly occurring ns-SNPs in human CBR3 significantly alter the in vitro metabolism of DOX and DAUN.

Histone deacetylase inhibitors induce growth arrest, apoptosis, and differentiation in clear cell sarcoma models.

Clear cell sarcoma is an aggressive malignancy occurring most commonly in the distal extremities of young adults, characterized by t(12;22)(q13;q12) creating the chimeric fusion oncoprotein EWS-ATF1. We assessed growth inhibition and differentiation effects of histone deacetylase inhibitors MS-275 and romidepsin (depsipeptide, FK228) on clear cell sarcoma cells and evaluated drug sensitivity among related translocation-associated sarcomas and other cell models. Three clear cell sarcoma cell lines, seven other sarcomas, six nonsarcoma malignant cell lines, and two nonneoplastic mesenchymal cell models were treated with MS-275 or romidepsin. Growth inhibition was assayed by monolayer 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Induction of cell cycle arrest and apoptosis were assessed by propidium iodide/Annexin V flow cytometry in monolayer and spheroid cultures and by immunoblotting analysis. Expression levels of key genes involved in mesenchymal differentiation and of EWS-ATF1 were measured by quantitative real-time PCR in clear cell sarcoma cells treated with histone deacetylase inhibitors. MS-275 and romidepsin inhibited growth in clear cell sarcoma cells by inducing cell cycle arrest and apoptosis in a time- and dose-dependent manner. Sarcomas showed greater sensitivity than other tumor types, with clear cell sarcomas most sensitive of all, whereas nonmalignant mesenchymal cells were highly resistant. MS-275 at 1 micromol/L and romidepsin at 1 nmol/L induced histone H3 acetylation, cell cycle arrest, apoptosis, and differentiation in clear cell sarcoma cells within 24 hours. Histone deacetylase inhibitors increased expression of SOX9, MYOD1, and PPARG and decreased EWS-ATF1 expression in clear cell sarcoma cells. Histone deacetylase inhibitors show promising preclinical activity in multiple clear cell sarcoma models.

S100A12 Serum Levels and PMN Counts Are Elevated in Childhood Systemic Vasculitides Especially Involving Proteinase 3 Specific Anti-neutrophil Cytoplasmic Antibodies.

Objectives: Chronic primary systemic vasculitidies (CPV) are a collection of rare diseases involving inflammation in blood vessels, often in multiple organs. CPV can affect adults and children and may be life- or organ-threatening. Treatments for adult CPV, although effective, have known severe potential toxicities; safety and efficacy of these drugs in pediatric patients is not fully understood. There is an unmet need for biologic measures to assess the level of disease activity and, in turn, inform treatment choices for stopping, starting, or modifying therapy. This observational study determines if S100 calcium-binding protein A12 (S100A12) and common inflammatory indicators are sensitive markers of disease activity in children and adolescents with CPV that could be used to inform a minimal effective dose of therapy. Methods: Clinical data and sera were collected from 56 participants with CPV at study visits from diagnosis to remission. Serum concentrations of S100A12, C-reactive protein (CRP) and hemoglobin (Hb) as well as whole blood cell counts and erythrocyte sedimentation rate (ESR) were measured. Disease activity was inferred by physician's global assessment (PGA) and the pediatric vasculitis activity score (PVAS). Results: Serum concentrations of standard markers of inflammation (ESR, CRP, Hb, absolute blood neutrophil count), and S100A12 track with clinically assessed disease activity. These measures-particularly neutrophil counts and sera concentrations of S100A12-had the most significant correlation with clinical scores of disease activity in those children with vasculitis that is associated with anti-neutrophil cytoplasmic antibodies (ANCA) against proteinase 3. Conclusions: S100A12 and neutrophil counts should be considered in the assessment of disease activity in children with CPV particularly the most common forms of the disease that involve proteinase 3 ANCA. Key messages: - In children with chronic primary systemic vasculitis (CPV), classical measures of inflammation are not formally considered in scoring of disease activity. - Inflammatory markers-specifically S100A12 and neutrophil count-track preferentially with the most common forms of childhood CPV which affect small to medium sized vessels and involve anti neutrophil cytoplasmic antibodies (ANCA) against proteinase-3.

Clinical practice variation and need for pediatric-specific treatment guidelines among rheumatologists caring for children with ANCA-associated vasculitis: an international clinician survey.

BACKGROUND: Because pediatric antineutrophil cytoplasmic antibody-associated vasculitis is rare, management generally relies on adult data. We assessed treatment practices, uptake of existing clinical assessment tools, and interest in pediatric treatment protocols among rheumatologists caring for children with granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). METHODS: A needs-assessment survey developed by an international working group of pediatric rheumatologists and two nephrologists was circulated internationally. Data were summarized with descriptive statistics. Pearson's chi-square tests were used in inferential univariate analyses. RESULTS: The 209 respondents from 36 countries had collectively seen ~1600 children with GPA/MPA; 144 had seen more than two in the preceding 5 years. Standardized and validated clinical assessment tools to score disease severity, activity, and damage were used by 59, 63, and 36%, respectively; barriers to use included lack of knowledge and limited perceived utility. Therapy varied significantly: use of rituximab rather than cyclophosphamide was more common among respondents from the USA (OR = 2.7 [1.3-5.5], p = 0.0190, n = 139), those with >5 years of independent practice experience (OR = 3.8 [1.3-12.5], p = 0.0279, n = 137), and those who had seen >10 children with GPA/MPA in their careers (OR = 4.39 [2.1-9.1], p = 0.0011, n = 133). Respondents who had treated >10 patients were also more likely to continue maintenance therapy for at least 24 months (OR = 3.0 [1.4-6.4], p = 0.0161, n = 127). Ninety six percent of respondents believed in a need for pediatric-specific treatment guidelines; 46% supported adaptation of adult guidelines while 69% favoured guidelines providing a limited range of treatment options to allow comparison of effectiveness through a registry. CONCLUSIONS: These data provide a rationale for developing pediatric-specific consensus treatment guidelines for GPA/MPA. While pediatric rheumatologist uptake of existing clinical tools has been limited, guideline uptake may be enhanced if outcomes of consensus-derived treatment options are evaluated within the framework of an international registry.

Early Outcomes in Children With Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

OBJECTIVE: To characterize the early disease course in childhood-onset antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and the 12-month outcomes in children with AAV. METHODS: Eligible subjects were children entered into the Pediatric Vasculitis Initiative study who were diagnosed before their eighteenth birthday as having granulomatosis with polyangiitis (Wegener's), microscopic polyangiitis, eosinophilic granulomatosis with polyangiitis (Churg-Strauss), or ANCA-positive pauci-immune glomerulonephritis. The primary outcome measure was achievement of disease remission (Pediatric Vasculitis Activity Score [PVAS] of 0) at 12 months with a corticosteroid dosage of <0.2 mg/kg/day. Secondary outcome measures included the rates of inactive disease (PVAS of 0, with any corticosteroid dosage) and rates of improvement at postinduction (4-6 months after diagnosis) and at 12 months, presence of damage at 12 months (measured by a modified Pediatric Vasculitis Damage Index [PVDI]; score 0 = no damage, score 1 = one damage item present), and relapse rates at 12 months. RESULTS: In total, 105 children with AAV were included in the study. The median age at diagnosis was 13.8 years (interquartile range 10.9-15.8 years). Among the study cohort, 42% of patients achieved remission at 12 months, 49% had inactive disease at postinduction (4-6 months), and 61% had inactive disease at 12 months. The majority of patients improved, even if they did not achieve inactive disease. An improvement in the PVAS score of at least 50% from time of diagnosis to postinduction was seen in 92% of patients. Minor relapses occurred in 12 (24%) of 51 patients after inactive disease had been achieved postinduction. The median PVDI damage score at 12 months was 1 (range 0-6), and 63% of patients had ≥1 PVDI damage item scored as present at 12 months. CONCLUSION: This is the largest study to date to assess disease outcomes in pediatric AAV. Although the study showed that a significant proportion of patients did not achieve remission, the majority of patients responded to treatment. Unfortunately, more than one-half of this patient cohort experienced damage to various organ systems early in their disease course.

Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin prevents synovial sarcoma proliferation via apoptosis in in vitro models.

Synovial sarcoma is a soft tissue malignancy with a poor prognosis; many patients will die from this disease within 10 years of diagnosis, despite treatment. Gene expression profiling and immunohistochemistry studies have identified oncogenes that are highly expressed in synovial sarcoma. Included in this group are receptor tyrosine kinases such as epidermal growth factor receptor, insulin-like growth factor receptor 1, fibroblast growth factor receptor 3, KIT, and HER2. Inhibitors of these growth-promoting receptors are likely to inhibit proliferation of synovial sarcoma; however, the effect of receptor tyrosine kinase inhibitors on synovial sarcoma is largely unknown. We assessed the ability of the following receptor tyrosine kinase inhibitors to halt proliferation and induce apoptosis in synovial sarcoma monolayer and three dimensional spheroid in vitro models: gefitinib (Iressa), NVP-AEW541, imatinib mesylate (Gleevec), SU5402, PRO-001, trastuzumab (Herceptin), and 17-allylamino-17-demethoxygeldanamycin (17-AAG). Gefitinib, NVP-AEW541, and imatinib inhibited proliferation only at relatively high concentrations, which are not clinically applicable. 17-AAG, which destabilizes multiple receptor tyrosine kinases and other oncoproteins through heat shock protein 90 inhibition, prevented proliferation and induced apoptosis in synovial sarcoma monolayer models at concentrations achievable in human serum. 17-AAG treatment was also associated with receptor tyrosine kinase degradation and induction of apoptosis in synovial sarcoma spheroid models. 17-AAG was more effective than doxorubicin, particularly in the spheroid models. Here we provide in vitro evidence that 17-AAG, a clinically applicable drug with known pharmacology and limited toxicity, inhibits synovial sarcoma proliferation by inducing apoptosis, and thus has potential as a systemic therapy for this disease.

Comparing Presenting Clinical Features in 48 Children With Microscopic Polyangiitis to 183 Children Who Have Granulomatosis With Polyangiitis (Wegener's): An ARChiVe Cohort Study.

OBJECTIVE: To uniquely classify children with microscopic polyangiitis (MPA), to describe their demographic characteristics, presenting clinical features, and initial treatments in comparison to patients with granulomatosis with polyangiitis (Wegener's) (GPA). METHODS: The European Medicines Agency (EMA) classification algorithm was applied by computation to categorical data from patients recruited to the ARChiVe (A Registry for Childhood Vasculitis: e-entry) cohort, with the data censored to November 2015. The EMA algorithm was used to uniquely distinguish children with MPA from children with GPA, whose diagnoses had been classified according to both adult- and pediatric-specific criteria. Descriptive statistics were used for comparisons. RESULTS: In total, 231 of 440 patients (64% female) fulfilled the classification criteria for either MPA (n = 48) or GPA (n = 183). The median time to diagnosis was 1.6 months in the MPA group and 2.1 months in the GPA group (ranging to 39 and 73 months, respectively). Patients with MPA were significantly younger than those with GPA (median age 11 years versus 14 years). Constitutional features were equally common between the groups. In patients with MPA compared to those with GPA, pulmonary manifestations were less frequent (44% versus 74%) and less severe (primarily, hemorrhage, requirement for supplemental oxygen, and pulmonary failure). Renal pathologic features were frequently found in both groups (75% of patients with MPA versus 83% of patients with GPA) but tended toward greater severity in those with MPA (primarily, nephrotic-range proteinuria, requirement for dialysis, and end-stage renal disease). Airway/eye involvement was absent among patients with MPA, because these GPA-defining features preclude a diagnosis of MPA within the EMA algorithm. Similar proportions of patients with MPA and those with GPA received combination therapy with corticosteroids plus cyclophosphamide (69% and 78%, respectively) or both drugs in combination with plasmapheresis (19% and 22%, respectively). Other treatments administered, ranging in decreasing frequency from 13% to 3%, were rituximab, methotrexate, azathioprine, and mycophenolate mofetil. CONCLUSION: Younger age at disease onset and, perhaps, both gastrointestinal manifestations and more severe kidney disease seem to characterize the clinical profile in children with MPA compared to those with GPA. Delay in diagnosis suggests that recognition of these systemic vasculitides is suboptimal. Compared with adults, initial treatment regimens in children were comparable, but the complete reversal of female-to-male disease prevalence ratios is a provocative finding.

Single-nucleotide polymorphisms in reductase genes are not associated with response to daunorubicin-based remission induction.

BACKGROUND: To improve the quality of care for patients with acute myeloid leukemia (AML), biomarkers predictive of response to the standard daunorubicin-based induction therapy are needed. Genetic variants affecting daunorubicin metabolism are attractive candidates for such biomarkers. METHODS: We have previously shown that 13 of the naturally occurring nonsynonymous single-nucleotide polymorphisms (SNP) in the reductase genes affect daunorubicin metabolism in vitro. Here, we test these SNPs individually and jointly for association with response to one cycle of daunorubicin-based chemotherapy in a sample of 189 patients with acute myelogenous leukemia. RESULTS: Of the 13 SNPs included in this study, only 5 passed quality control filters. No association was found between these 5 SNPs and response to one cycle of daunorubicin-based induction therapy in either individual or joint effect tests. CONCLUSIONS: Despite their showing in vitro effect on metabolic rate of daunorubicin, the nonsynonymous SNPs in the reductase genes on their own are not significant contributors to the observed variability in response to daunorubicin therapy and thus, as singularities, are not useful biomarkers of this outcome. IMPACT: The results of this investigation provide important information for studies on personalization of anthracycline-based therapies.

A discovery study of daunorubicin induced cardiotoxicity in a sample of acute myeloid leukemia patients prioritizes P450 oxidoreductase polymorphisms as a potential risk factor.

Anthracyclines are very effective chemotherapeutic agents; however, their use is hampered by the treatment-induced cardiotoxicity. Genetic variants that help define patient's sensitivity to anthracyclines will greatly improve the design of optimal chemotherapeutic regimens. However, identification of such variants is hampered by the lack of analytical approaches that address the complex, multi-genic character of anthracycline induced cardiotoxicity (AIC). Here, using a multi-SNP based approach, we examined 60 genes coding for proteins involved in drug metabolism and efflux and identified the P450 oxidoreductase (POR) gene to be most strongly associated with daunorubicin induced cardiotoxicity in a population of acute myeloid leukemia (AML) patients (FDR adjusted p-value of 0.15). In this sample of cancer patients, variation in the POR gene is estimated to account for some 11.6% of the variability in the drop of left ventricular ejection fraction (LVEF) after daunorubicin treatment, compared to the estimated 13.2% accounted for by the cumulative dose and ethnicity. In post-hoc analysis, this association was driven by 3 SNPs-the rs2868177, rs13240755, and rs4732513-through their linear interaction with cumulative daunorubicin dose. The unadjusted odds ratios (ORs) and confidence intervals (CIs) for rs2868177 and rs13240755 were estimated to be 1.89 (95% CI: 0.7435-4.819; p = 0.1756) and 3.18 (95% CI: 1.223-8.27; p = 0.01376), respectively. Although the contribution of POR variants is expected to be overestimated due to the multiple testing performed in this small pilot study, given that cumulative anthracycline dose is virtually the only factor used clinically to predict the risk of cardiotoxicity, the contribution that genetic analyses of POR can make to the assessment of this risk is worthy of follow up in future investigations.

Single-nucleotide polymorphisms in aldo-keto and carbonyl reductase genes are not associated with acute cardiotoxicity after daunorubicin chemotherapy.

BACKGROUND: Evidence suggests that interpatient variability in anthracycline metabolic rate may contribute to the cardiotoxicity associated with anthracycline-based chemotherapy. Therefore, polymorphisms in the anthracycline metabolizing enzymes have been proposed as potential biomarkers of anthracycline-induced cardiotoxicity (AIC). METHODS: We have previously shown that 13 of the naturally occurring nonsynonymous single-nucleotide polymorphisms (nsSNP) in the aldo-keto reductases (AKR) and carbonyl reductases (CBR) reduce anthracycline metabolic rate in vitro. Here, we test these SNPs individually and jointly for association with daunorubicin-induced cardiotoxicity in patients with acute myeloid leukemia (AML). RESULTS: Five of the 13 nsSNPs exhibiting an in vitro effect on anthracycline metabolism were detected among the 185 patients with AML. No association was found between the SNPs and daunorubicin-induced cardiotoxicity in either individual or joint effect analyses. CONCLUSIONS: Despite the shown in vitro effect of nsSNPs in reductase genes on anthracycline metabolic rate, on their own these SNPs do not explain enough variability in cardiotoxicity to be useful markers of this adverse event. IMPACT: The results of this study provide important information for biomarker studies on side effects of anthracycline chemotherapy.

Validation of a sequential extraction and liquid chromatography-tandem mass spectrometric method for determination of dihydrotestosterone, androstanediol and androstanediol-glucuronide in prostate tissues.

Androgens are key mediators of prostate development and function, a role that extends to the development of prostate diseases such as benign prostatic hyperplasia (BPH) and prostate cancer. In prostate, DHT is the major androgen and reduction and glucuronidation are the major metabolic pathways for DHT elimination. A streamlined method for quantitation of dihydrotestosterone (DHT), 5α-androstan-3α,17ß-diol (3α-diol), and 3α-diol glucuronide (diol-gluc) was established and validated for use with archived prostate tissue specimens to facilitate examination of the roles of the underlying metabolism. This involved a sequential 70/30 hexane/ethyl acetate (hex/EtOAc) extraction of steroids, followed by an ethyl acetate extraction for diol-gluc. Derivatization of the hex/EtOAc fraction with2-fluoro-1-methylpyridinium p-toluene-4-sulfonate (FMP) was used to enhance sensitivity for hydroxyl steroids and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was utilized for analysis of both fractions. The method was validated with calibration standards followed by recovery assessment from spiked samples of BPH and normal prostate. Lower limits of quantitation (LLOQ) were 50 pg/g, 20 pg/g and 100 pg/g for DHT, 3α-diol and diol-gluc, respectively for extracts from 50mg equivalents of tissue. Prepared samples were stable for up to three weeks at 4 °C and 37 °C. The method provides excellent sensitivity and selectivity for determination of tissue levels of DHT, 3α-diol, and diol-gluc. Furthermore, this protocol can easily be extended to other hydroxyl steroids, is relatively straightforward to perform and is an effective tool for assessing steroid levels in archived clinical prostate samples.

Biomarkers for severity of spinal cord injury in the cerebrospinal fluid of rats.

One of the major challenges in management of spinal cord injury (SCI) is that the assessment of injury severity is often imprecise. Identification of reliable, easily quantifiable biomarkers that delineate the severity of the initial injury and that have prognostic value for the degree of functional recovery would significantly aid the clinician in the choice of potential treatments. To find such biomarkers we performed quantitative liquid chromatography-mass spectrometry (LC-MS/MS) analyses of cerebrospinal fluid (CSF) collected from rats 24 h after either a moderate or severe SCI. We identified a panel of 42 putative biomarkers of SCI, 10 of which represent potential biomarkers of SCI severity. Three of the candidate biomarkers, Ywhaz, Itih4, and Gpx3 were also validated by Western blot in a biological replicate of the injury. The putative biomarkers identified in this study may potentially be a valuable tool in the assessment of the extent of spinal cord damage.

Histone deacetylase inhibitors reverse SS18-SSX-mediated polycomb silencing of the tumor suppressor early growth response 1 in synovial sarcoma.

Synovial sarcoma is a soft tissue malignancy characterized by the fusion of SS18 to either SSX1, SSX2, or SSX4 genes. SS18 and SSX are transcriptional cofactors involved in activation and repression of gene transcription, respectively. SS18 interacts with SWI/SNF, whereas SSX associates with the polycomb chromatin remodeling complex. Thus, fusion of these two proteins brings together two opposing effects on gene expression and chromatin structure. Recent studies have shown that a significant number of genes are down-regulated by the SS18-SSX fusion protein and that the clinically applicable histone deacetylase (HDAC) inhibitor romidepsin inhibits synovial sarcoma growth. Therefore, we set out to identify direct targets of SS18-SSX among genes down-regulated in synovial sarcoma and investigated if romidepsin can specifically counteract SS18-SSX-mediated transcriptional dysregulation. Here, we report that the tumor suppressor early growth response 1 (EGR1) is repressed by the SS18-SSX protein through a direct association with the EGR1 promoter. This SS18-SSX binding correlates with trimethylation of Lys(27) of histone H3 (H3K27-M3) and recruitment of polycomb group proteins to this promoter. In addition, we found that romidepsin treatment reverts these modifications and reactivates EGR1 expression in synovial sarcoma cell models. Our data implicate polycomb-mediated epigenetic gene repression as a mechanism of oncogenesis in synovial sarcoma. Furthermore, our work highlights a possible mechanism behind the efficacy of a clinically applicable HDAC inhibitor in synovial sarcoma treatment.

cDNA microarray-based translational research in soft tissue sarcoma.

The authors discuss application of cDNA microarray technology in translational research to identify diagnostic markers and therapeutic targets in adult soft tissue sarcoma. Recent results in synovial sarcoma are used to highlight the applicability of this technology for marker and target discovery, as well as the need for preclinical validation of putative therapeutic targets.