Urinary miRNAs as Biomarkers for Noninvasive Evaluation of Radiation-Induced Renal Tubular Injury.

Radiation nephropathy is one of the common late effects in cancer survivors who received radiotherapy as well as in victims of radiation accidents. The clinical manifestations of radiation nephropathy occur months after exposure. To date, there are no known early biomarkers to predict the future development of radiation nephropathy. This study focuses on the development of urinary biomarkers providing readout of acute responses in renal tubular epithelial cells. An amplification-free hybridization-based nCounter assay was used to detect changes in mouse urinary miRNAs after irradiation. After a single LD50 of total-body irradiation (TBI) or clinically relevant fractionated doses (2 Gy twice daily for 3 days), changes in urinary levels of microRNAs followed either an early pattern, peaking at 6-8 h postirradiation and gradually declining, or later pattern, peaking from 24 h to 7 days. Of 600 miRNAs compared, 12 urinary miRNAs showed the acute response and seven showed the late response, common to both irradiation protocols. miR-1224 and miR-21 were of particular interest, since they were the most robust acute and late responders, respectively. The early responding miR-1224 also exhibited good dose response after 2, 4, 6 and 8 Gy TBI, indicating its potential use as a biomarker for radiation exposure. In situ hybridization of irradiated mouse kidney sections and cultured mouse primary renal tubular cells confirmed the tubular origin of miR-1224. A significant upregulation in hsa-miR-1224-3p expression was also observed in human proximal renal tubular cells after irradiation. Consistent with mouse urine data, a similar expression pattern of hsa-miR-1224-3p and hsa-miR-21 were observed in urine samples collected from human leukemia patients preconditioned with TBI. This proof-of-concept study shows the potential translational utility of urinary miRNA biomarkers for radiation damage in renal tubules with possible prediction of late effects.

[DEFINITION OF INTERRELATION BETWEEN AMINOACID COMPOSITION OF URINE AND BONE TISSUE DENSITY AT CHILDREN WITH ACUTE LEUKAEMIA FOR ONCOHEMATOLOGICAL PATHOLOGY RISK GROUP FORMATION].

Age and sexual indexies of densitometry at patients with acute leukemia (AL) and healthy children are presented. 31% of children with AL during the initial period of disease had manifestations of the osteopenic syndrome. At patients with AL more often than at healthy children anomalies of development of front part of skull are defined. The partial contribution of free and peptides-connencted oxyproline in urine at AL patients differs in comparison with control group that is caused by modification or deficiency of the corresponding enzymes. 30% of patients with AL had raised concentration of free oxyproline in urine, and lowered glycine concentration that testifies to the increased disintegration of collagen and deficiency of tile plastic material necessary for collagene-forming processes. The obtained data should be considered for forming of risk group on oncohematological pathology at children.

Genomic applications in the clinic: use in treatment paradigm of acute myeloid leukemia.

In recent years, research in genomics has resulted in the rapid uncovering of the molecular pathogenesis of acute myeloid leukemia (AML). The identification of the genetic determinants of response to standard-but also to experimental-treatment is increasingly used for patient counseling, to guide clinical decision making, and for resource-efficient care provision at diagnosis, during consolidation treatment and follow-up, and after relapse. Gene mutations now allow us to explore the enormous diversity among cytogenetically defined subsets of AML, in particular the large subset of cytogenetically normal AML. Nonetheless, there are several challenges in evaluating the prognostic value of a specific mutation in the concert of the various concurrent mutations and determining the relative prognostic value of the genetic profile during the disease course. In particular, changes in the genetic profile in relapse compared with that at diagnosis will increasingly affect the treatment strategy at relapse, but also will give us the possibility of learning which treatment strategy during frontline therapy is best to prevent them.

Inhibition of OCTN2-mediated transport of carnitine by etoposide.

OCTN2 is a bifunctional transporter that reabsorbs filtered carnitine in a sodium-dependent manner and secretes organic cations into urine as a proton antiport mechanism. We hypothesized that inhibition of OCTN2 by anticancer drugs can influence carnitine resorption. OCTN2-mediated transport inhibition by anticancer drugs was assessed using cells transfected with human OCTN2 (hOCTN2) or mouse Octn2 (mOctn2). Excretion of carnitine and acetylcarnitine was measured in urine collected from mice and pediatric patients with cancer before and after administration of etoposide. Five of 27 tested drugs (50-100 µmol/L) inhibited hOCTN2-mediated carnitine uptake by 42% to 85% (P < 0.001). Of these inhibitors, etoposide was itself a transported substrate of hOCTN2 and mOctn2. Etoposide uptake by hOCTN2 was reversed in the presence of excess carnitine. This competitive inhibitory mechanism was confirmed in an in silico molecular docking analysis. In addition, etoposide inhibited the transcellular apical-to-basolateral flux of carnitine in kidney cells. Etoposide was also associated with a significant urinary loss of carnitine in mice (~1.5-fold) and in patients with cancer (~2.4-fold). Collectively, these findings indicate that etoposide can inhibit hOCTN2 function, potentially disturb carnitine homeostasis, and that this phenomenon can contribute to treatment-related toxicities.

Higher urinary excretion of inorganic phosphate during early induction chemotherapy predicts a good prognosis in childhood acute leukemia.

The rapidity of response to induction therapy is emerging as an important prognostic factor in children with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Urine inorganic phosphate (IP) and uric acid (UA) may increase in patients with acute leukemia who undergo their induction chemotherapy, owing to the breakdown of tumor cells. The crystallization of UA or calcium phosphate in renal tubules can result in acute tumor lysis syndrome (ATLS). Some reports indicate that patients who experience ATLS have a better prognosis than those who do not. We investigated the relationship between urinary IP and UA excretion and treatment outcome in children with acute leukemia. Participants included 93 patients with ALL and 31 patients with AML. Urine samples were collected and measured for the first 3 days of induction chemotherapy. Among patients with ALL, urinary IP excretion was significantly higher in patients without relapse than in those with relapse and correlated with long-term outcome. Among patients with AML, urinary IP excretion was significantly higher in patients without induction failure (IF) than those with IF. We propose that higher urinary IP excretion could be a useful prognostic marker for determining favorable outcomes in patients with acute leukemia.

High serum and urine lysozyme levels in a dog with acute myeloid leukemia.

A 2-year-old, female German Shepherd Dog with facial nerve paralysis was diagnosed with acute myelomonocytic leukemia based on clinical, cytologic, and immunologic findings. Proteinuria (urine proteincreatinine ratio = 1.5) occurred in the absence of renal failure. Qualitative assessment of proteinuria by sodium dodecyl sulfate-agarose gel electrophoresis revealed a broad band with a molecular weight of approximately 15 kDa that was compatible with lysozyme (LZM). A diagnosis of tubular proteinuria was made, and a chemical evaluation of LZM in serum and urine samples was performed using a turbidimetric assay. The LZM concentrations were 24.5 mg/l (reference interval: 2.5-8.0 mg/l) and 274.5 mg/l (reference interval: <2 mg/l) in serum and urine, respectively.

Determinants of urinary 8-hydroxy-2'-deoxyguanosine in Chinese children with acute leukemia.

The 8-hydroxy-2'-deoxyguanosine (8-OHdG), an oxidized nucleoside of DNA, not only is a widely used biomarker for the measurement of endogenous oxidative DNA damage, but might also be a risk factor for many diseases including cancer. Elevated level of urinary 8-OHdG has been detected in patients with various malignancies. In the present study, the level of urinary 8-OHdG was examined in 116 Chinese children with acute leukemia (94 acute lymphoid leukemia, ALL, 22 acute myeloid leukemia, AML), and its correlation with urinary metal elements was investigated. Our result showed that the level of urinary 8-OHdG in children with acute leukemia before treatment was significantly elevated compared with that in normal controls (11.92 +/- 15.42 vs. 4.03 +/- 4.70 ng/mg creatinine, P < 0.05). In particular, urinary 8-OHdG was higher in children with acute leukemia aged under 3 years (20.86 +/- 21.75 ng/mg creatinine) than in those aged 3-15 years (8.09 +/- 9.65 ng/mg creatinine), whereas no differences were shown in terms of gender, parental smoking and education, household income, place of residence, and use of paracetamol. In addition, urinary 8-OHdG levels were similar among different subtypes of acute lymphoid leukemia (ALL) patients. Furthermore, linear regression analysis revealed a significant correlation between urinary 8-OHdG and urinary Cr, but not Fe or As, in group aged <3 years compared with group aged 3-15 years (P = 0.041), indicating that the metal elements may be involved in increasing urinary 8-OHdG level in younger children with acute leukemia. Our results suggest that children with acute leukemia undergo an increased risk of oxidative DNA damage, which may be correlated with high level of Cr exposure in Chinese children with acute leukemia.

Urinary level of nickel and acute leukaemia in Chinese children.

The 8-hydroxy-2'-deoxyguanosine (8-OHdG), an oxidized nucleoside of DNA, not only is a widely used biomarker for the measurement of endogenous oxidative DNA damage but might also be a risk factor for many diseases including cancer. Metal exposure may play an important role in oxidative DNA damage among children. However, few studies on urinary 8-OHdG and metals have been conducted in children with acute leukemia. In the present study, urinary Ni and 8-OHdG were examined in 116 children with acute leukaemia (94 acute lymphoid leukaemia [ALL] and 22 acute myeloid leukaemia [AML]) and 51 healthy child controls. Our result showed that urinary Ni in acute leukaemia patients (ALL: 68.40 +/- 133.98, AML: 41.48 +/- 76.31 ng/mg creatinine) was significantly higher than that in controls (62.47 +/- 124.90 vs 17.63 +/- 46.17 ng/mg creatinine, P < 0.05). Similarly, the pretherapy level of urinary 8-OHdG in patients (ALL: 11.83 +/- 16.23, AML: 12.36 +/- 11.36 ng/mg creatinine) was significantly elevated compared with controls (11.92 +/- 15.42 vs 4.03 +/- 4.70 ng/mg creatinine, P < 0.05). Moreover, urinary 8-OHdG and urinary Ni showed a weak but significant association with increased risk of childhood leukaemia. The present study suggests that Ni may be an etiologic factor for childhood acute leukaemia by oxidative DNA damage.

Specific immunoassays for detection of intact and cleaved forms of the urokinase receptor.

BACKGROUND: The cell surface receptor (uPAR) for urokinase plasminogen activator (uPA) is a strong prognostic marker in several types of cancer. uPA cleaves the three-domain protein uPAR(I-III) into two fragments: uPAR(I), which contains domain I; and uPAR(II-III), which contains domains II and III. Established immunoassays measure a combination of uPAR forms. Our aim was to design immunoassays for specific quantification of the individual forms of uPAR. METHODS: Using appropriate combinations of epitope-mapped monoclonal antibodies (Mabs) for capture and europium-labeled detection Mabs, we designed two-site sandwich time-resolved fluorescence immunoassays (TR-FIAs): TR-FIA 1 to measure uPAR(I-III) alone; TR-FIA 2 to measure both uPAR(I-III) and uPAR(II-III); and TR-FIA 3 to measure uPAR(I). To avoid detection of uPAR(I-III) in TR-FIA 3, we used a combination of the peptide uPAR antagonist AE120 and a domain I antibody, R3. AE120 blocks the binding of R3 to uPAR(I-III). In contrast, AE120 does not interact with liberated domain I and therefore does not interfere with the binding of R3 to uPAR(I). RESULTS: The limits of quantification (CV <20%) determined by adding the proteins to uPAR-depleted plasma were <3 pmol/L in all three assays. The interassay CVs in plasma with added analytes were <11%, and recoveries were between 93% and 105%. Cross-reactivities of purified proteins in the three TR-FIAs were no more than 4%. Studies on chymotrypsin cleavage of uPAR and size-exclusion chromatography of plasma with and without added protein further supported the specificity of the assays. CONCLUSIONS: The three novel TR-FIAs accurately quantify uPAR(I-III) alone, uPAR(I-III) together with uPAR(II-III), and uPAR(I), respectively, in biological samples, including plasma, and thus are well suited for studies of the diagnostic and prognostic value of individual uPAR forms in cancer patients.

Increased excretion of urine coproporphyrins during daunorubicin administration in patients affected by acute myelogenous leukemia.

Increased erythrocyte porphyrin values and high urine porphyrin levels have been reported in leukemic patients, but it is not clear whether the alteration in porphyrin metabolism is due to the leukemia or its treatment. The aim of this study was to compare porphyrin levels in leukemic patients undergoing chemotherapy or not. We analysed porphyrin values in patients with acute emyelogenous leukemia, who had or had not received chemotherapy according to Gale. Erythrocyte and urine porphyrin levels were increased as a result of the leukemic process, but urine coproporphyrins were further increased by daunorubicin treatment. These higher urine coproporphyrin levels were attributed to the activity of daunorubicin, which is known to interfere with the coproporphyrinogen decarboxylation process leading to the accumulation and high excretion of coproporphyrins in urine.

Evidence of enhanced iron excretion during systemic phosphorothioate oligodeoxynucleotide treatment.

BACKGROUND: Phosphorothioate oligonucleotides, in general, possess properties that could be utilized in the development of therapeutic heavy metal chelators. METHODS: Iron excretion was measured in 16 patients participating in studies to test the safety of OL(1)p53, a 20-mer phosphorothioate oligonucleotide complementary to p53 mRNA. Patients were given OL(1)p53 at doses of 0.05 to 0.25 mg/kg/h for 10 days by continuous intravenous infusion. Urine was collected during the study and analyzed for iron, copper, cadmium, and zinc. RESULTS: We found that phosphorothioate oligonucleotides have a high affinity for iron as well as several other clinically relevant toxic metals. Analysis of patient urine following administration of OL(1)p53 reveals a 7.5-fold increase in iron excretion at low doses (0.05 mg/kg/h). CONCLUSIONS: Phosphorothioate oligonucleotides may have therapeutic potential as heavy metal chelators. Low doses of phosphorothioate oligonucleotide facilitated the excretion of iron. Renal clearance of iron-phosphorothioate oligonucleotide complexes most likely involves secretion into proximal tubules.

Urinary polyamine evaluation for effective diagnosis of various cancers.

With a newly modified analytical method, the concentrations of free and acetylated urinary polyamines were simultaneously determined in a control group (32 cases) and patients with various types of cancers (104 cases, 20 males and 84 females) by gas chromatography-nitrogen-phosphorus detection. Significant concentration differences between normal subjects and various cancer patients were found. The various types of cancers (advanced gastric carcinoma, ovarian cancer, acute myelocyte leukemia, non-Hodgkin's lymphoma) gave unique patterns of urinary polyamine profile as well as significant differences of concentration. To indirectly evaluate the possible involvement of enzymes, precursor-to-product concentration ratios were compared between controls and patients with various types of cancers.

Urinary excretion of proteolyzed alpha1-antitrypsin: specificity, quantitation, and relation to therapy response in patients with acute myeloid leukemia.

During remission induction chemotherapy, a 41-kDa cleavage product of alpha1-antitrypsin (alpha1-AT41) can be found in the urine of patients with acute myeloid leukemia. By using immunoblotting with antibodies against this protein, 27 patients with acute myeloid leukemia were screened for the excretion of this fragment and the amount of alpha1-AT41 compared with treatment response assessed by therapy-induced cytoreduction in the bone marrow and time to reach remission. Patients with acute lymphoblastic leukemia, malignant lymphomas, and solid tumors receiving chemotherapy, patients with nonmalignant diseases like sepsis and kidney dysfunction, and healthy subjects were probed to evaluate the specificity of this phenomenon. In 74% of the acute myeloid leukemia patients, the truncated inhibitor was detected. Mean concentration of peak excretion was found to be 6.7 microgram/mg creatinine (range, 1.1-41 microgram/mg). Among the patients treated with induction chemotherapy, those who responded completely (<5% residual marrow blast cells) exhibited significantly higher alpha1-AT41 concentrations than the nonresponders (P < 0.03). Patients who showed a partial response (6-25% residual blasts) excreted intermediate values of the protein. The probability of median time to reach remission was 40 days in patients excreting the truncated inhibitor in measurable amounts compared to 100 days in patients negative for alpha1-AT41 (P < 0.02). The 41-kDa fragment was also found in one of 10 patients with acute lymphoblastic leukemia and in 3 of 18 lymphoma patients but not in those with solid tumors, infections, or kidney disease or in healthy individuals.

Increased urinary excretion of human cytomegalovirus in children with malignancy: detection by polymerase chain reaction.

Human cytomegalovirus (HCMV) is one of the most important agents causing opportunistic infections in immunocompromised hosts. In this study, we examined the urinary excretion of HCMV in children with malignancy using polymerase chain reaction (PCR). Urine samples were collected from on-therapy, off-therapy patients with malignancy, and healthy controls. A simple DNA extraction method using glass powder was employed, and inhibitory effect of urine on PCR was prevented. For PCR, a pair of primers from the HCMV major immediate early gene sequence was used. Among patients who received intensive chemotherapy, 52.0% had urinary HCMV excretion after the chemotherapy course. In contrast, off-therapy patients and healthy controls showed a lower incidence of urinary HCMV excretion (20.4 and 8.7%, respectively). The incidence of HCMV urinary excretion in the on-therapy group was significantly higher than healthy controls (P < 0.05). In the on-therapy group, the total white blood cell count of the virus excreters was lower than that of non-excreters. The incidence of HCMV excretion was high in on-therapy patients. Most of the virus excreters were seropositive, so their viruria was thought to be caused by reactivation. Repeated monitoring of virus excretion by this rapid and simple method may be useful to detect HCMV infection early and to control it in such patients.

Elevated levels of oncopterin, N2-(3-aminopropyl)biopterin, a new pterin compound, in urine from patients with solid and blood cancers.

The concentrations of oncopterin, N2-(3-aminopropyl)biopterin, a new pterin compound, were determined in urine from various cancer patients by HPLC on a reverse-phase or ion-exchange column. The concentration of oncopterin increased after acid hydrolysis, indicating that it exists as an amide in urine. The oncopterin concentrations were very low in the urine of healthy controls. Among the urine samples examined, those from cases of solid cancers, e.g., hepatomas, prostatic cancer and bladder cancer, exhibited very high levels; and those from cases of blood cancers, e.g., myelomas, acute myelocytic leukemia, and lymphomas, showed moderate increases. Oncopterin may thus be a new biochemical marker of some types of cancer.

Proteolytic inactivation of alpha 1-proteinase inhibitor in vivo: detection, characterization and quantitation of the main fragment excreted in the urine of leukemia patients.

During the search for a therapy response parameter in patients with acute myeloid leukemia, we observed the appearance of a 41 kDa glycoprotein band in the urines of these patients under therapy. To investigate the nature of this molecule and to develop a specific detection system, the protein was isolated and antibodies were raised. Urines and sera of patients and healthy subjects were screened for crossreacting proteins by immunoblotting. Only the leukemia patients showed the urinary 41 kDa protein plus a 53 kDa band. In all sera, including those from healthy donors, a 53 kDa protein was intensely stained. Isolation of the plasma protein and sequence analysis of the urinary protein revealed that alpha 1-proteinase inhibitor is the crossreacting plasma protein and that the 41 kDa molecule is proteolytically modified alpha 1-PI, which has lost its antitryptic activity. Cleavage occurred in the N-terminal part as well as in the reactive site loop of the inhibitor. The 41 kDa truncated inhibitor was also found in the leukemic blast cells. A densitometric method is described for the quantitation of the molecule in the nanomolar range.

Clustering of adverse drug events: analysis of risk factors for cerebellar toxicity with high-dose cytarabine.

BACKGROUND: Cerebellar toxicity is a severe, therapy-limiting adverse reaction of cytarabine given in high doses. The Food and Drug Administration received a report of an increased frequency of cerebellar toxicity at the University of Wisconsin Hospital and Clinics after a switch from the product (Cytosar-U) manufactured by The Upjohn Co., Kalamazoo, Mich., to the generic form made by Quad Pharmaceuticals, Inc., Indianapolis, Ind. PURPOSE: To compare the incidence of cerebellar toxicity in Quad-treated patients with Upjohn-treated patients, a record-based cohort study was conducted at the University of Wisconsin Hospital and Clinics between January 1986 and August 1989. METHODS: The incidence of cerebellar toxicity was studied in 63 leukemia patients according to the manufacturer of the product received (34 Upjohn only, 25 Quad only, and four both manufacturers). The relative risk of cerebellar toxicity was adjusted for other known risk factors. RESULTS: Patients in the manufacturer-defined treatment groups did not differ significantly with respect to age, sex, type of leukemia, disease stage, calculated creatinine clearance, presence of abnormal liver function tests, or total dose received. The crude relative risk of cerebellar toxicity comparing the Quad product with the Upjohn product was 5.0 (95% confidence interval = 1.8-13.7). Adjustment for potential confounders did not alter the association. Other risk factors for cerebellar toxicity, independent of manufacturer, were age greater than 50 years, type of leukemia, disease stage, total dose greater than or equal to 20 g/m2, abnormal pretreatment liver function, and reduced creatinine clearance. CONCLUSION: This study found a significantly higher incidence of cerebellar toxicity with high-dose cytarabine manufactured by Quad Pharmaceuticals when compared with the incidence of cerebellar toxicity with the Upjohn product. Further research at independent institutions would be necessary to allow generalization of this finding. In addition, our findings suggest that a dose reduction in high-dose cytarabine therapy may be indicated for patients with reduced glomerular filtration rates.

Aggressive chemotherapy for acute leukaemia frequently causes intestinal protein leakage.

Cytostatic drugs are known to produce disturbances in intestinal absorption of carbohydrates. To further explore the gastrointestinal (GI) toxicity of cytostatic therapy, 37 patients with acute leukemia were investigated during and/or after remission induction courses by the use of the differential sugar absorption test (DSAT) and the intestinal clearance of alpha-1-antitrypsin (ClAAT). The ratio of the lactulose to the mannitol excretion in the urine was found abnormal in 44% of the tests. The ClAAT was increased in 74% of tests. The tests results differed considerably from patient to patient and depended on the chemotherapy course; correlation between the tests was low, probably indicating the unrelated pathophysiological processes were measured. After haematological regeneration, abnormal test results normalised. It is concluded that aggressive chemotherapy not only causes a reduction in the absorption of sugars, but commonly also protein leakage. These GI side-effects are reversible, and the application of both tests in combination provides a practical and reproducible method for investigation of GI toxicity in patients treated with cytostatic drugs.

Detection of urinary glycoprotein GP 41 in leukemic patients and in healthy donors.

Urinary glycoprotein patterns from leukemic patients and from healthy donors were found to be identical, when the detection was carried out with the same quantities of protein. Two dimensional polyacrylamide gel electrophoresis revealed seven independent glycoproteins in the molecular weight region of 41,000 dalton, regardless of the origin of the urine, indicating, that these glycoproteins do not reflect any specificity of cytoreduction in the course of chemotherapy.

Urinary 6-keto-PGF1 alpha level in patients with childhood leukemia/lymphoma; a possible indicator of vascular damage.

To determine the effect of anti-neoplastic chemotherapy on the vascular system(s) of children with leukemia/lymphoma, urinary excretion of 6-keto-PGF1 alpha was measured by radioimmunoassay (RIA). In 4 patients receiving therapy, 6-keto-PGF1 alpha increased to a mean of 148 (range; 126-170)% during therapy, then returned to pre-treatment level 3-5 days later. In 18 long-term survivors who had completed therapy, 6-keto-PGF1 alpha was determined to be a mean of 275 (range; 52-905) ng/g creatinine, and in the healthy control children the mean was 146 (range; 71-348) ng/g creatinine. These results were contrary to our hypothesis that chemotherapy might cause a decreased synthesis of PGI2, a precursor of 6-keto-PGF1 alpha, and suggest that increased urinary 6-keto-PGF1 alpha reflects a vascular response to acute exposure to chemotherapeutic drugs and possible vascular damage due to long-term intensive chemotherapy in pediatric patients with leukemia/lymphoma.