Evaluation of Dose-Intense Ifosfamide, with and Without Edatrexate, in Adults with Sarcoma

Purpose. To define the maximally tolerated dose (MTD) of ifosfamide when given with G-CSF on an every other week schedule, and to define the MTD of edatrexate that can be given every two weeks with an intense schedule of ifosfamide

The turnover of folate coenzymes in murine lymphoma cells

To estimate the turnover of 5-CH3-H4-folate in murine lymphoma cells L1210, L1210R (a methotrexate-resistant subline), and L5178Y, suspensions of whole cells were allowed to concentrate 5-[14C]CH3-[9,3',5'-3H]H4-folate; analysis of cell extracts showed that, for each cell line, 81 to 85% of the total cell [14C]CH3 groups were transferred to nonfolate compounds within 5 min and 82 to 91% at time intervals up to 60 min. The initial transfer of 14C appeared to be into [14C]methionine, but insoluble cell materials were also progressively 14C labeled. Of the total cell 3H, more than 87% remained identified as 5-CH3-[3H3]H4-folate at 60 min, showing that within this period most of the [3H2]H4-folate derived from 5-CH3-[3H3]H4-folate returned to maintain the labeling of the pool of 5-CH3-[3H3]H4-folate. To estimate the flux of folates through the pathway of thymidylate biosynthesis, L1210 and L1210R cells were allowed to concentrate either 5-CH3-[9,3',5'-3H]H4-folate in the presence of methotrexate or 5-HCO-[6-3H]H4-folate. Of total 3H taken up as 5-HCO-[6-3H]H4-folate, 28% appeared to be transferred to thymidylate in 60 min by L1210 cells and 52% by L1210R cells. In methotrexate-treated L1210 cells, 23% of the total 3H taken up as 5-CH3-[3H3]H4-folate was accumulated in 60 min as [3H3]H2-folate, a product of thymidylate biosynthesis. However, in cells of the methotrexate-resistant L1210R line, no [3H3]H2-folate was accumulated by the use of 2 mM methotrexate despite the demonstrated high flux of folates through the pathway of thymidylate biosynthesis. These data show the significance, for methotrexate resistance, of the 11-fold increase of dihydrofolate reductase in L1210R cells

Conformational Polymorphism of cRNA of T-Cell-Receptor Genes as a Clone-Specific Molecular Marker for Cutaneous Lymphoma

A novel molecular assay for the detection and characterization of monoclonal lymphoid populations in clinical specimens was developed. The assay is based on the principle that upon non-denaturing polyacrylamide gel electrophoresis RNA molecules separate into several metastable conformational forms. These conformational polymorphisms strictly depend on the nucleotide sequence of the individual molecule. Using DNA from formalin-fixed, paraffin-embedded tissue of patients with mycosis fungoides, highly variable junctional sequences of rearranged T-cell receptor gamma genes were amplified by polymerase chain reaction. Subsequently, the polymerase chain reactions products were transcribed into complementary RNA and analyzed by non-denaturing polyacrylamide gel electrophoresis. In clinical specimens with a monoclonal lymphoid population, a clone-specific pattern of bands was identified representing conformational polymorphisms of cRNA molecules of rearranged T-cell receptor gamma genes of the predominant lymphoid clone. Three biopsies from one patient taken from different sites of the body over 3 years yielded an identical pattern of bands. This methodology provides a novel and rapid tool for the molecular identification and characterization of clonal lymphoid populations in clinical specimens. It is likely to be of special value for studies on the clonal evolution of lymphoid disorders of the skin

MiR-24 Tumor Suppressor Activity Is Regulated Independent of p53 and through a Target Site Polymorphism

MicroRNAs (miRNAs) are predicted to regulate approximately 30% of all human genes; however, only a few miRNAs have been assigned their targets and specific functions. Here we demonstrate that miR-24, a ubiquitously expressed miRNA, has an anti-proliferative effect independent of p53 function. Cell lines with differential p53 status were used as a model to study the effects of miR-24 on cell proliferation, cell cycle control, gene regulation and cellular transformation. Overexpression of miR-24 in six different cell lines, independent of p53 function, inhibited cell proliferation and resulted in G2/S cell cycle arrest. MiR-24 over expression in cells with wt-p53 upregulated TP53 and p21 protein; however, in p53-null cells miR-24 still induced cell cycle arrest without the involvement of p21. We show that miR-24 regulates p53-independent cellular proliferation by regulating an S-phase enzyme, dihydrofolate reductase (DHFR) a target of the chemotherapeutic drug methotrexate (MTX). Of interest, we found that a miR-24 target site polymorphism in DHFR 3′ UTR that results in loss of miR-24-function and high DHFR levels in the cell imparts a growth advantage to immortalized cells and induces neoplastic transformation. Of clinical significance, we found that miR-24 is deregulated in human colorectal cancer tumors and a subset of tumors has reduced levels of miR-24. A novel function for miR-24 as a p53-independent cell cycle inhibitory miRNA is proposed

Single Plasma Concentrations of 1′‐Hydroxymidazolam or the Ratio of 1′‐Hydroxymidazolam: Midazolam Do Not Predict Midazolam Clearance in Healthy Subjects

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/97152/1/009127002237986.pd

Clinical actionability of comprehensive genomic profiling for management of rare or refractory cancers

Background. The frequency with which targeted tumor sequencing results will lead to implemented change in care is unclear. Prospective assessment of the feasibility and limitations of using genomic sequencing is critically important. Methods. A prospective clinical study was conducted on 100 patients with diverse-histology, rare, or poor-prognosis cancers to evaluate the clinical actionability of a Clinical Laboratory Improvement Amendments (CLIA)-certified, comprehensive genomic profiling assay (FoundationOne), using formalin-fixed, paraffin-embedded tumors. The primary objectives were to assess utility, feasibility, and limitations of genomic sequencing for genomically guided therapy or other clinical purpose in the setting of a multidisciplinary molecular tumor board. Results. Of the tumors from the 92 patients with sufficient tissue, 88 (96%) had at least one genomic alteration (average 3.6, range 0–10). Commonly altered pathways included p53 (46%), RAS/RAF/MAPK (rat sarcoma; rapidly accelerated fibrosarcoma; mitogen-activated protein kinase) (45%), receptor tyrosine kinases/ligand (44%), PI3K/AKT/mTOR (phosphatidylinositol-4,5-bisphosphate 3-kinase; protein kinase B; mammalian target of rapamycin) (35%), transcription factors/regulators (31%), and cell cycle regulators (30%). Many low frequency but potentially actionable alterations were identified in diverse histologies. Use of comprehensive profiling led to implementable clinical action in 35% of tumors with genomic alterations, including genomically guided therapy, diagnostic modification, and trigger for germline genetic testing. Conclusion. Use of targeted next-generation sequencing in the setting of an institutional molecular tumor board led to implementable clinical action in more than one third of patients with rare and poor-prognosis cancers. Major barriers to implementation of genomically guided therapy were clinical status of the patient and drug access. Early and serial sequencing in the clinical course and expanded access to genomically guided early-phase clinical trials and targeted agents may increase actionability. Implications for Practice: Identification of key factors that facilitate use of genomic tumor testing results and implementation of genomically guided therapy may lead to enhanced benefit for patients with rare or difficult to treat cancers. Clinical use of a targeted next-generation sequencing assay in the setting of an institutional molecular tumor board led to implementable clinical action in over one third of patients with rare and poor prognosis cancers. The major barriers to implementation of genomically guided therapy were clinical status of the patient and drug access both on trial and off label. Approaches to increase actionability include early and serial sequencing in the clinical course and expanded access to genomically guided early phase clinical trials and targeted agents

Efficient Generation of Multipotent Mesenchymal Stem Cells from Umbilical Cord Blood in Stroma-Free Liquid Culture

BACKGROUND: Haematopoiesis is sustained by haematopoietic (HSC) and mesenchymal stem cells (MSC). HSC are the precursors for blood cells, whereas marrow, stroma, bone, cartilage, muscle and connective tissues derive from MSC. The generation of MSC from umbilical cord blood (UCB) is possible, but with low and unpredictable success. Here we describe a novel, robust stroma-free dual cell culture system for long-term expansion of primitive UCB-derived MSC. METHODS AND FINDINGS: UCB-derived mononuclear cells (MNC) or selected CD34(+) cells were grown in liquid culture in the presence of serum and cytokines. Out of 32 different culture conditions that have been tested for the efficient expansion of HSC, we identified one condition (DMEM, pooled human AB serum, Flt-3 ligand, SCF, MGDF and IL-6; further denoted as D7) which, besides supporting HSC expansion, successfully enabled long-term expansion of stromal/MSC from 8 out of 8 UCB units (5 MNC-derived and 3 CD34(+) selected cells). Expanded MSC displayed a fibroblast-like morphology, expressed several stromal/MSC-related antigens (CD105, CD73, CD29, CD44, CD133 and Nestin) but were negative for haematopoietic cell markers (CD45, CD34 and CD14). MSC stemness phenotype and their differentiation capacity in vitro before and after high dilution were preserved throughout long-term culture. Even at passage 24 cells remained Nestin(+), CD133(+) and >95% were positive for CD105, CD73, CD29 and CD44 with the capacity to differentiate into mesodermal lineages. Similarly we show that UCB derived MSC express pluripotency stem cell markers despite differences in cell confluency and culture passages. Further, we generated MSC from peripheral blood (PB) MNC of 8 healthy volunteers. In all cases, the resulting MSC expressed MSC-related antigens and showed the capacity to form CFU-F colonies. CONCLUSIONS: This novel stroma-free liquid culture overcomes the existing limitation in obtaining MSC from UCB and PB enabling so far unmet therapeutic applications, which might substantially affect clinical practice

An Outcome-based Approach for the Creation of Fetal Growth Standards: Do Singletons and Twins Need Separate Standards?

Contemporary fetal growth standards are created by using theoretical properties (percentiles) of birth weight (for gestational age) distributions. The authors used a clinically relevant, outcome-based methodology to determine if separate fetal growth standards are required for singletons and twins. All singleton and twin livebirths between 36 and 42 weeks’ gestation in the United States (1995–2002) were included, after exclusions for missing information and other factors (n = 17,811,922). A birth weight range was identified, at each gestational age, over which serious neonatal morbidity and neonatal mortality rates were lowest. Among singleton males at 40 weeks, serious neonatal morbidity/mortality rates were lowest between 3,012 g (95% confidence interval (CI): 3,008, 3,018) and 3,978 g (95% CI: 3,976, 3,980). The low end of this optimal birth weight range for females was 37 g (95% CI: 21, 53) less. The low optimal birth weight was 152 g (95% CI: 121, 183) less for twins compared with singletons. No differences were observed in low optimal birth weight by period (1999–2002 vs. 1995–1998), but small differences were observed for maternal education, race, parity, age, and smoking status. Patterns of birth weight-specific serious neonatal morbidity/neonatal mortality support the need for plurality-specific fetal growth standards