A Rapid High-Performance LC-MS/MS Method for Therapeutic Drug Monitoring of Voriconazole, Posaconazole, Fluconazole, and Itraconazole in Human Serum.

BACKGROUND: To achieve therapeutic efficacy and prevent toxicity simultaneously, therapeutic drug monitoring has been increasingly adopted for antifungal agents with narrow therapeutic indexes. We herein report the development and validation of an accurate, simple, fast, and cost-effective clinical test with high-performance LC-MS/MS to simultaneously quantify voriconazole, posaconazole, fluconazole, and itraconazole in human serum. METHODS: Mixed with extraction solution and internal standard, 100 µL serum samples were centrifuged for protein precipitation. Diluted supernatant was injected onto a Phenomenex® Luna C8 (2) 50 × 2 mm (3 µm) column and was analyzed with a Prominence Shimadzu high-performance liquid chromatography (HPLC) system coupled with a SCIEX QTRAP 4000 mass spectrometer in a positive ionization mode with multiple reaction monitoring. The total analytical run time was 3 min. RESULTS: The assay is linear for voricoanzole (0.01-10 µg/mL), posaconazole (0.02-40 µg/mL), fluconazole (0.2-200 µg/mL), and itraconazole (0.02-20 µg/mL). The intraday CVs ranged from 1.9% to 3.8% (n = 20); the interday CVs ranged from 2.7% to 5.4% (n = 20). Method comparison study (n = 39 or 40) demonstrated good correlation with reference laboratories (R >0.99) with average biases ranging from -7.2% to 17.5%. The recoveries for each analyte were above 90%, and matrix effects ranged from 95% to 112%. CONCLUSIONS: The method is acceptable for routine therapeutic drug monitoring of these antifungal agents in clinical laboratories for better therapeutic outcome.

Congenital B-lymphoblastic leukemia with a cryptic MLL rearrangement and post-treatment evolution to mixed phenotype acute leukemia.

Congenital leukemia is a rare event with a poor prognosis. We report a case of congenital leukemia with a cryptic rearrangement of MLL demonstrable only with RT-PCR. Interestingly, with treatment, the patient showed lineage plasticity of the leukemia with the development of monocytic lineage blasts after presenting with B-cell lineage blasts. This was heralded by the development of a new clonal cytogenetic abnormality. This case highlights the primitive nature of the leukemic cells in congenital leukemia, and emphasizes that RT-PCR for MLL rearrangements may identify a subset of cases which are otherwise negative by karyotyping, FISH, and chromosomal microarrays.

A sensitive LC-MS/MS method for the quantification of urinary 8-iso-prostaglandin F2α (8-iso-PGF2α) including pediatric reference interval.

BACKGROUND: Oxidative stress has been implicated in numerous diseases, including arthritis, atherosclerosis, Alzheimer's disease, cancer, diabetes, hypertension, and inflammation. 8-iso-prostaglandin F2α, a member of the F2 isoprostane family, has been well-accepted as a valuable biomarker for the assessment of oxidative stress. METHODS: We report the development and validation of an ultra-sensitive LC-MS/MS assay for urinary 8-iso-PGF2α measurements in pediatric population. RESULTS: The assay was linear from 0.024 to 20nmol/l (R(2)=0.99). Recoveries were above 85% and matrix effects were below 5%. The variability was determined at nmol/l concentration: the intra-day variability (%CV) ranged from 3.9% to 4.5% (n=20); and the inter-day variability ranged from 4.3% to 5.7% (n=20). The accuracy of our laboratory developed test was evaluated with a clinical reference laboratory (n=39), and a correlation coefficient of 0.9257 was observed. Reference interval were established to be <0.5ng/mg creatinine in a group of pediatric population (2months-18years, n=123). CONCLUSIONS: The precision of the assay will allow for accurate assessment of oxidative stress, and is acceptable for patient testing, particularly in pediatric population.

Quantification of glypican 3, ß-catenin and claudin-1 protein expression in hepatoblastoma and paediatric hepatocellular carcinoma by colour deconvolution.

AIMS: To identify an immunohistochemical panel for paediatric malignant epithelial liver tumours. METHODS AND RESULTS: Forty-five hepatoblastomas (HBs), 13 paediatric hepatocellular carcinomas (HCCs) and two hepatocellular malignant neoplasms not otherwise specified (NOS) were chosen for immunohistochemical staining of glypican 3 (GPC3), ß-catenin, claudin-1, delta-like protein (DLK), and forkhead box protein G1 (FOXG1). Immunostaining was quantitatively analysed with NIH imagej software coupled with colour deconvolution. Different subtypes of HB and HCC showed distinct staining patterns of GPC3, ß-catenin, and claudin-1. Moreover, GPC3, ß-catenin and claudin-1 all showed higher expression in classic HCC and embryonal HB than in fetal HB; GPC3 showed complete negativity in small-cell undifferentiated (SCU) HB and fibrolamellar HCC (FLC); ß-catenin showed the strongest expression in SCU HB but the weakest expression in FLC. A panel of these three immunomarkers was useful for the diagnosis of hepatocellular malignant neoplasms NOS. The expression of DLK and FOXG1 was inconstant among fetal and embryonal HB and classic HCC. CONCLUSIONS: A panel of GPC3, ß-catenin and claudin-1 is helpful for differentiating HB subtypes and distinguishing HB from HCC.

Simple, Fast, and Simultaneous Detection of Plasma Total Homocysteine, Methylmalonic Acid, Methionine, and 2-Methylcitric Acid Using Liquid Chromatography and Mass Spectrometry (LC/MS/MS).

Cobalamin (Vitamin B12) plays an essential role both in the conversion of methylmalonyl-CoA to succinyl-CoA and in the synthesis of methionine (Met) from homocysteine (Hcy). Elevations of total homocysteine (tHcy), Met, methylmalonic acid (MMA), and 2-methylcitric acid (2MCA) are indicative of disorders in these related pathways, and can clinically present as methylmalonic acidemia, cobalamin defects or deficiency, propionic acidemia, homocystinuria, and hypermethioninemia. We have developed a fast, sensitive, and simple method for the simultaneous detection of plasma tHcy, MMA, Met, and 2MCA using liquid chromatography mass spectrometry (LC/MS/MS). All analytes were directly determined without the need of derivatization. Both positive and negative modes were used to achieve the best sensitivity and specificity. The two stereo isomers of 2MCA (2S, 3S) and (2R, 3S) were successfully separated and were designated as 2MCA1 and 2MCA2. The assays were linear up to a concentration of 800 µMol/l for tHcy, 2,000 µMol/l for Met, 80 µMol/l for MMA, 40 µMol/l for 2MCA1, and 40 µMol/l for 2MCA2 (80 µMol/l for total 2MCA), respectively. The recovery was between 84.42 % and 120.05 %. The intra-assay coefficient of variations (CVs) ranged from 2.1 % to 6.9 % (n = 20), and the inter-assay CVs ranged from 2.7 % to 11.6 % (n = 20). Reference intervals were established and verified (n = 125). A total of 15 patients with variable disorders in related pathway were successfully confirmed. The assay can be performed either in diagnostic laboratories or as second-tier, follow-up test in newborn screening laboratories.A fast, sensitive, and simple LC/MS/MS method was developed successfully for the simultaneous detection of plasma total homocysteine, methylmalonic acid, methionine, and 2-methylcitric acid for diagnosis of disorders in related pathways.

Subcutaneous Panniculitis-like T-cell lymphoma in two pediatric patients: an HIV-positive adolescent and a 4-month-old infant.

Subcutaneous Panniculitis-like T-cell lymphoma (SPTCL) is a rare subtype of childhood non-Hodgkin lymphoma. Subcutaneous Panniculitis-like T-cell lymphoma has an aggressive variant associated with the hemophagocytic syndrome (HPS). Patients without HPS show resolution of the disease with prednisone or immunosuppressive therapy unlike other T-cell lymphomas. One HIV-positive adolescent and one infant with multiple subcutaneous masses are presented and the literature is reviewed. Lesional cells were consistent with SPTCL alpha-beta type. Our cases, without HPS, showed complete resolution of their lesions when treated with non-aggressive therapies. Patients with SPTCL alpha-beta should be treated conservatively.

Randomized comparison of low-dose involved-field radiotherapy and no radiotherapy for children with Hodgkin's disease who achieve a complete response to chemotherapy.

PURPOSE: Current standard therapy for children and adolescents with Hodgkin's disease includes combination chemotherapy and low-dose involved-field radiation (LD-IFRT). Because radiation may be associated with adverse late effects, the Children's Cancer Group (CCG) investigated whether radiation could be omitted in patients achieving a complete response to initial chemotherapy without jeopardizing the excellent outcome obtained with combined-modality therapy. PATIENTS AND METHODS: Between January 1995 and December 1998, 829 eligible patients were enrolled onto CCG 5942. A total of 501 patients who achieved an initial complete response after risk-adapted combination chemotherapy were randomized to receive LD-IFRT or no further treatment. Event-free survival (EFS) and overall survival were assessed from the date of study entry or the date of randomization, as appropriate. RESULTS: The projected 3-year EFS from study entry for the entire cohort was 87% +/- 1.2%. Among patients who achieved a complete response to initial chemotherapy, 92% +/- 1.9% of those randomized to receive LD-IFRT were alive and disease free 3 years after randomization, versus 87% +/- 2.2% for patients randomized to receive no further therapy (stratified log-rank test; P =.057). With an "as-treated" analysis, 3-year EFS after randomization for the radiation cohort was 93% +/- 1.7% versus 85% +/- 2.3% for patients receiving no further therapy (stratified log-rank test; P =.0024). Three-year survival estimates for patients treated with and without LD-IFRT were 98% +/- 1.1% for patients who received radiation and 99% +/- 0.5% for patients who did not receive radiation. CONCLUSION: LD-IFRT after an initial complete response to risk-adapted chemotherapy improved EFS. At this time, there is no survival advantage for LD-IFRT, but follow-up remains short.

BCR/ABL P190 transgenic mice develop leukemia in the absence of Crkl.

The Bcr/Abl fusion protein directly causes chronic myelogenous leukemia and Philadelphia-chromosome positive acute lymphoblastic leukemia. Multiple independent studies have implicated Crkl, a small adapter protein, in transduction of oncogenic signals of Bcr/Abl and Crkl tyrosine-phosphorylation is used as a diagnostic tool for Philadelphia-positive leukemia. To evaluate the contribution of Crkl to this type of leukemia, we generated mutant mice that lack Crkl expression. We found that the overall survival of P190 BCR/ABL crkl-/- mice was comparable to that of genetically matched P190 BCR/ABL crkl +/+ mice. Both genotypes developed lymphoid lineage leukemia/lymphoma. Western blot analysis of -/- and +/+ lymphomas showed that the related Crk protein was tyrosine phosphorylated and could be found complexed with Bcr-Abl P190. These data indicate that possible therapeutic approaches that target Crkl may be complicated by the presence of pathways that compensate for lack of Crkl function.

Bethesda proposals for classification of nonlymphoid hematopoietic neoplasms in mice.

The hematopathology subcommittee of the Mouse Models of Human Cancers Consortium recognized the need for a classification of murine hematopoietic neoplasms that would allow investigators to diagnose lesions as well-defined entities according to accepted criteria. Pathologists and investigators worked cooperatively to develop proposals for the classification of lymphoid and nonlymphoid hematopoietic neoplasms. It is proposed here that nonlymphoid hematopoietic neoplasms of mice be classified in 4 broad categories: nonlymphoid leukemias, nonlymphoid hematopoietic sarcomas, myeloid dysplasias, and myeloid proliferations (nonreactive). Criteria for diagnosis and subclassification of these lesions include peripheral blood findings, cytologic features of hematopoietic tissues, histopathology, immunophenotyping, genetic features, and clinical course. Differences between murine and human lesions are reflected in the terminology and methods used for classification. This classification will be of particular value to investigators seeking to develop, use, and communicate about mouse models of human hematopoietic neoplasms.

Bethesda proposals for classification of lymphoid neoplasms in mice.

A consensus system for classification of mouse lymphoid neoplasms according to their histopathologic and genetic features has been an elusive target for investigators involved in understanding the pathogenesis of spontaneous cancers or modeling human hematopoietic diseases in mice. An international panel of scientists with expertise in mouse and human hematopathology joined with the hematopathology subcommittee of the Mouse Models for Human Cancers Consortium to develop criteria for definition and classification of these diseases together with a standardized nomenclature. The fundamental elements contributing to the scheme are clinical features, morphology, immunophenotype, and genetic characteristics. The resulting classification has numerous parallels to the World Health Organization classification of human lymphoid tumors while recognizing differences that may be species specific. The classification should facilitate communications about mouse models of human lymphoid diseases.