Hepatobiliary circulation and dominant urinary excretion of homogentisic acid in a mouse model of alkaptonuria.

Altered activity of specific enzymes in phenylalanine-tyrosine (phe-tyr) metabolism results in incomplete breakdown of various metabolite substrates in this pathway. Increased biofluid concentration and tissue accumulation of the phe-tyr pathway metabolite homogentisic acid (HGA) is central to pathophysiology in the inherited disorder alkaptonuria (AKU). Accumulation of metabolites upstream of HGA, including tyrosine, occurs in patients on nitisinone, a licenced drug for AKU and hereditary tyrosinaemia type 1, which inhibits the enzyme responsible for HGA production. The aim of this study was to investigate the phe-tyr metabolite content of key biofluids and tissues in AKU mice on and off nitisinone to gain new insights into the biodistribution of metabolites in these altered metabolic states. The data show for the first time that HGA is present in bile in AKU (mean [±SD] = 1003[±410] µmol/L; nitisinone-treated AKU mean [±SD] = 45[±23] µmol/L). Biliary tyrosine, 3(4-hydroxyphenyl)pyruvic acid (HPPA) and 3(4-hydroxyphenyl)lactic acid (HPLA) are also increased on nitisinone. Urine was confirmed as the dominant elimination route of HGA in untreated AKU, but with indication of biliary excretion. These data provide new insights into pathways of phe-tyr metabolite biodistribution and metabolism, showing for the first time that hepatobiliary excretion contributes to the total pool of metabolites in this pathway. Our data suggest that biliary elimination of organic acids and other metabolites may play an underappreciated role in disorders of metabolism. We propose that our finding of approximately 3.8 times greater urinary HGA excretion in AKU mice compared with patients is one reason for the lack of extensive tissue ochronosis in the AKU mouse model.

Investigating diagnostic sequencing techniques for CADASIL diagnosis.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a cerebral small vessel disease caused by mutations in the NOTCH3 gene. Our laboratory has been undertaking genetic diagnostic testing for CADASIL since 1997. Work originally utilised Sanger sequencing methods targeting specific NOTCH3 exons. More recently, next-generation sequencing (NGS)-based technologies such as a targeted gene panel and whole exome sequencing (WES) have been used for improved genetic diagnostic testing. In this study, data from 680 patient samples was analysed for 764 tests utilising 3 different sequencing technologies. Sanger sequencing was performed for 407 tests, a targeted NGS gene panel which includes NOTCH3 exonic regions accounted for 354 tests, and WES with targeted analysis was performed for 3 tests. In total, 14.7% of patient samples (n = 100/680) were determined to have a mutation. Testing efficacy varied by method, with 10.8% (n = 44/407) of tests using Sanger sequencing able to identify mutations, with 15.8% (n = 56/354) of tests performed using the NGS custom panel successfully identifying mutations and a likely non-NOTCH3 pathogenic variant (n = 1/3) identified through WES. Further analysis was then performed through stratification of the number of mutations detected at our facility based on the number of exons, level of pathogenicity and the classification of mutations as known or novel. A systematic review of NOTCH3 mutation testing data from 1997 to 2017 determined the diagnostic rate of pathogenic findings and found the NGS-customised panel increases our ability to identify disease-causing mutations in NOTCH3.

Genetic variants associated with exercise performance in both moderately trained and highly trained individuals.

Adaptation to exercise training is a complex trait that may be influenced by genetic variants. We identified 36 single nucleotide polymorphisms (SNPs) that had been previously associated with endurance or strength performance, exercise-related phenotypes or exercise intolerant disorders. A MassARRAY multiplex genotyping assay was designed to identify associations with these SNPs against collected endurance fitness phenotype parameters obtained from two exercise cohorts (Gene SMART study; n = 58 and Hawaiian Ironman Triathlon 2008; n = 115). These parameters included peak power output (PP), a time trial (TT), lactate threshold (LT), maximal oxygen uptake (VO2 max) in recreationally active individuals and a triathlon time-to-completion (Hawaiian Ironman Triathlon cohort only). A nominal significance threshold of α < 0.05 was used to identify 17 variants (11 in the Gene SMART population and six in the Hawaiian Ironman Triathlon cohort) which were significantly associated with performance gains in highly trained individuals. The variant rs1474347 located in Interleukin 6 (IL6) was the only variant with a false discovery rate < 0.05 and was found to be associated with gains in VO2 max (additional 4.016 mL/(kg min) for each G allele inherited) after training in the Gene SMART cohort. In summary, this study found further evidence to suggest that genetic variance can influence training response in a moderately trained cohort and provides an example of the potential application of genomic research in the assessment of exercise trait response.

Correction to: Assessing the effect of nitisinone induced hypertyrosinaemia on monoamine neurotransmitters in brain tissue from a murine model of alkaptonuria using mass spectrometry imaging.

The original publication of this article contained an incorrect version that did not include some final reviewers' suggestions, was inadvertently received for production and published. The original article has been corrected.

Assessing the effect of nitisinone induced hypertyrosinaemia on monoamine neurotransmitters in brain tissue from a murine model of alkaptonuria using mass spectrometry imaging.

OBJECTIVE: Nitisinone induced hypertyrosinaemia is a concern in patients with Alkaptonuria (AKU). It has been suggested that this may alter neurotransmitter metabolism, specifically dopamine and serotonin. Herein mass spectrometry imaging (MSI) is used for the direct measurement of 2,4-diphenyl-pyranylium tetrafluoroborate (DPP-TFB) derivatives of monoamine neurotransmitters in brain tissue from a murine model of AKU following treatment with nitisinone. METHODS: Metabolite changes were assessed using MSI on DPP-TFB derivatised fresh frozen tissue sections directing analysis towards primary amine neurotransmitters. Matched tail bleed plasma samples were analysed using LC-MS/MS. Eighteen BALB/c mice were included in this study: HGD-/- (n = 6, treated with nitisinone-4 mg/L, in drinking water); HGD-/- (n = 6, no treatment) and HGD+/- (n = 6, no treatment). RESULTS: Ion intensity and distribution of DPP-TFB derivatives in brain tissue for dopamine, 3-methoxytyramine, noradrenaline, tryptophan, serotonin, and glutamate were not significantly different following treatment with nitisinone in HGD -/- mice, and no significant differences were observed between HGD-/- and HGD+/- mice that received no treatment. Tyrosine (10-fold in both comparisons, p = 0.003; [BALB/c HGD-/- (n = 6) and BALB/c HGD+/- (n = 6) (no treatment) vs. BALB/c HGD-/- (n = 6, treated)] and tyramine (25-fold, p = 0.02; 32-fold, p = 0.02) increased significantly following treatment with nitisinone. Plasma tyrosine and homogentisic acid increased (ninefold, p = < 0.0001) and decreased (ninefold, p = 0.004), respectively in HGD-/- mice treated with nitisinone. CONCLUSIONS: Monoamine neurotransmitters in brain tissue from a murine model of AKU did not change following treatment with nitisinone. These findings have significant implications for patients with AKU as they suggest monoamine neurotransmitters are not altered following treatment with nitisinone.

Signal conditioning for compensating nonlinearity and nonrepeatability of an optical frequency scanning laser implemented in a C-OFDR system.

Reported systems using coherent optical frequency-domain reflectometry, C-OFDR, rely on the linearity of the scanning rate of the tunable laser and sometimes on its repeatability. In this work, we present the implementation of signal-conditioning algorithms for a fiber temperature sensor system based on coherent optical frequency-domain reflectometry. Postacquisition signal conditioning removes any nonlinearity and nonrepeatability effects and allows synchronization of the whole system. A low reflectivity, 0.1%, fiber Bragg grating, placed in a reference interferometer, is implemented for removing the nonrepeatability of the optical source. The spatial resolution achieved by the temperature fiber sensor is 0.36 mm, with repeatability of 0.032°C, while using telecommunication-grade components.

BDNF and TNF-α polymorphisms in memory.

Here, we investigate the genetic basis of human memory in healthy individuals and the potential role of two polymorphisms, previously implicated in memory function. We have explored aspects of retrospective and prospective memory including semantic, short term, working and long-term memory in conjunction with brain derived neurotrophic factor (BDNF) and tumor necrosis factor-alpha (TNF-α). The memory scores for healthy individuals in the population were obtained for each memory type and the population was genotyped via restriction fragment length polymorphism for the BDNF rs6265 (Val66Met) SNP and via pyrosequencing for the TNF-α rs113325588 SNP. Using univariate ANOVA, a significant association of the BDNF polymorphism with visual and spatial memory retention and a significant association of the TNF-α polymorphism was observed with spatial memory retention. In addition, a significant interactive effect between BDNF and TNF-α polymorphisms was observed in spatial memory retention. In practice visual memory involves spatial information and the two memory systems work together, however our data demonstrate that individuals with the Val/Val BDNF genotype have poorer visual memory but higher spatial memory retention, indicating a level of interaction between TNF-α and BDNF in spatial memory retention. This is the first study to use genetic analysis to determine the interaction between BDNF and TNF-α in relation to memory in normal adults and provides important information regarding the effect of genetic determinants and gene interactions on human memory.

Epigenetic inheritance at the agouti locus in the mouse.

Epigenetic modifications have effects on phenotype, but they are generally considered to be cleared on passage through the germ line in mammals, so that only genetic traits are inherited. Here we describe the inheritance of an epigenetic modification at the agouti locus in mice. In viable yellow ( A(vy)/a) mice, transcription originating in an intra-cisternal A particle (IAP) retrotransposon inserted upstream of the agouti gene (A) causes ectopic expression of agouti protein, resulting in yellow fur, obesity, diabetes and increased susceptibility to tumours. The pleiotropic effects of ectopic agouti expression are presumably due to effects of the paracrine signal on other tissues. Avy mice display variable expressivity because they are epigenetic mosaics for activity of the retrotransposon: isogenic Avy mice have coats that vary in a continuous spectrum from full yellow, through variegated yellow/agouti, to full agouti (pseudoagouti). The distribution of phenotypes among offspring is related to the phenotype of the dam; when an A(vy) dam has the agouti phenotype, her offspring are more likely to be agouti. We demonstrate here that this maternal epigenetic effect is not the result of a maternally contributed environment. Rather, our data show that it results from incomplete erasure of an epigenetic modification when a silenced Avy allele is passed through the female germ line, with consequent inheritance of the epigenetic modification. Because retrotransposons are abundant in mammalian genomes, this type of inheritance may be common.

Ochronosis in a murine model of alkaptonuria is synonymous to that in the human condition.

OBJECTIVE: Alkaptonuria (AKU) is a rare genetic disease which results in severe early onset osteoarthropathy. It has recently been shown that the subchondral interface is of key significance in disease pathogenesis. Human surgical tissues are often beyond this initial stage and there is no published murine model of pathogenesis, to study the natural history of the disease. The murine genotype exists but it has been reported not to demonstrate ochronotic osteoarthropathy consistent with the human disease. Recent anecdotal evidence of macroscopic renal ochronosis in a mouse model of tyrosinaemia led us to perform histological analysis of tissues of these mice that are known to be affected in human AKU. DESIGN: The homogentisate 1,2-dioxygenase Hgd(+/)(-)Fah(-)(/)(-) mouse can model either hereditary tyrosinaemia type I (HT1) or AKU depending on selection conditions. Mice having undergone Hgd reversion were sacrificed at various time points, and their tissues taken for histological analysis. Sections were stained with haematoxylin eosin (H&E) and Schmorl's reagent. RESULTS: Early time point observations at 8 months showed no sign of macroscopic ochronosis of tissues. Macroscopic examination at 13 months revealed ochronosis of the kidneys. Microscopic analysis of the kidneys revealed large pigmented nodules displaying distinct ochre colouration. Close microscopic examination of the distal femur and proximal fibula at the subchondral junctions revealed the presence of numerous pigmented chondrocytes. CONCLUSIONS: Here we present the first data showing ochronosis of tissues in a murine model of AKU. These preliminary histological observations provide a stimulus for further studies into the natural history of the disease to provide a greater understanding of this class of arthropathy.

Pedigree derived mutation rate across the entire mitochondrial genome of the Norfolk Island population.

Estimates of mutation rates for various regions of the human mitochondrial genome (mtGenome) vary widely, depending on whether they are inferred using a phylogenetic approach or obtained directly from pedigrees. Traditionally, only the control region, or small portions of the coding region have been targeted for analysis due to the cost and effort required to produce whole mtGenome Sanger profiles. Here, we report one of the first pedigree derived mutation rates for the entire human mtGenome. The entire mtGenome from 225 individuals originating from Norfolk Island was analysed to estimate the pedigree derived mutation rate and compared against published mutation rates. These individuals were from 45 maternal lineages spanning 345 generational events. Mutation rates for various portions of the mtGenome were calculated. Nine mutations (including two transitions and seven cases of heteroplasmy) were observed, resulting in a rate of 0.058 mutations/site/million years (95% CI 0.031-0.108). These mutation rates are approximately 16 times higher than estimates derived from phylogenetic analysis with heteroplasmy detected in 13 samples (n = 225, 5.8% individuals). Providing one of the first pedigree derived estimates for the entire mtGenome, this study provides a better understanding of human mtGenome evolution and has relevance to many research fields, including medicine, anthropology and forensics.

Investigation of Mitochondrial Related Variants in a Cerebral Small Vessel Disease Cohort.

Monogenic forms of cerebral small vessel disease (CSVD) can be caused by both variants in nuclear DNA and mitochondrial DNA (mtDNA). Mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is known to have a phenotype similar to Cerebral Autosomal Dominant Arteriopathy with Sub-cortical Infarcts and Leukoencephalopathy (CADASIL), and can be caused by variants in the mitochondrial genome and in several nuclear-encoded mitochondrial protein (NEMP) genes. The aim of this study was to screen for variants in the mitochondrial genome and NEMP genes in a NOTCH3-negative CADASIL cohort, to identify a potential link between mitochondrial dysfunction and CSVD pathology. Whole exome sequencing was performed for 50 patients with CADASIL-like symptomology on the Ion Torrent system. Mitochondrial sequencing was performed using an in-house designed protocol with sequencing run on the Ion GeneStudio S5 Plus (S5 +). NEMP genes and mitochondrial sequencing data were examined for rare (MAF < 0.001), non-synonymous variants that were predicted to have a deleterious effect on the protein. We identified 29 candidate NEMP variants that had links to either MELAS-, encephalopathy-, or Alzheimer's disease-related phenotypes. Based on these changes, variants affecting POLG, MTO1, LONP1, NDUFAF6, NDUFB3, and TCIRG1 were thought to play a potential role in CSVD pathology in this cohort. Overall, the exploration of the mitochondrial genome identified a potential role for mitochondrial related proteins and mtDNA variants contributing to CSVD pathologies.

Selective expression of CD45 isoforms on functional subpopulations of CD34+ hemopoietic cells from human bone marrow.

We have found that the small population of cells in human marrow that are characterized by their expression of CD34 can be readily subdivided into two apparently nonoverlapping subpopulations of approximate equal size, one expressing CD45RO and one CD45R. Functional studies of these subpopulations revealed that all of the primitive erythroid colony-forming cells (BFU-E) are CD34+ CD45RO+. Similarly, more primitive cells that give rise to both erythroid and granulopoietic colony-forming cells after being maintained for 5 wk on confluent irradiated long-term marrow culture feeder layers, also show this phenotype. In contrast, most granulopoietic colony-forming cells are CD34+ CD45RO- cells. The differential expression of CD45 isoforms on distinct functional subpopulations of hemopoietic cells is consistent with the concept that these molecules play an important role in the differentiation or activation of primitive, normally quiescent, hemopoietic cells. The presence of CD45RO and the lack of CD45R on human cells capable of initiating hemopoiesis in the long-term marrow culture system correspond to the reported lack of CD45R on transplantable hemopoietic stem cells in rodents and may be a useful addition to strategies for human stem cell purification, or for purging CD45R+ leukemic cells.

Glucocorticoid receptor haploinsufficiency causes hypertension and attenuates hypothalamic-pituitary-adrenal axis and blood pressure adaptions to high-fat diet.

Glucocorticoid hormones are critical to respond and adapt to stress. Genetic variations in the glucocorticoid receptor (GR) gene alter hypothalamic-pituitary-adrenal (HPA) axis activity and associate with hypertension and susceptibility to metabolic disease. Here we test the hypothesis that reduced GR density alters blood pressure and glucose and lipid homeostasis and limits adaption to obesogenic diet. Heterozygous GR(betageo/+) mice were generated from embryonic stem (ES) cells with a gene trap integration of a beta-galactosidase-neomycin phosphotransferase (betageo) cassette into the GR gene creating a transcriptionally inactive GR fusion protein. Although GR(betageo/+) mice have 50% less functional GR, they have normal lipid and glucose homeostasis due to compensatory HPA axis activation but are hypertensive due to activation of the renin-angiotensin-aldosterone system (RAAS). When challenged with a high-fat diet, weight gain, adiposity, and glucose intolerance were similarly increased in control and GR(betageo/+) mice, suggesting preserved control of intermediary metabolism and energy balance. However, whereas a high-fat diet caused HPA activation and increased blood pressure in control mice, these adaptions were attenuated or abolished in GR(betageo/+) mice. Thus, reduced GR density balanced by HPA activation leaves glucocorticoid functions unaffected but mineralocorticoid functions increased, causing hypertension. Importantly, reduced GR limits HPA and blood pressure adaptions to obesogenic diet.

Ten things your emergency department should consider to prepare for pandemic influenza.

Pandemic influenza remains a potential major threat to global public health. It is essential for emergency departments to be involved in planning for the management of such a major event. It is also important for emergency departments to be clear on their internal arrangements for staff and for patient care. This paper outlines 10 suggestions for UK emergency departments based on the recent experience of emergency departments in Hong Kong and elsewhere.

Ion torrent high throughput mitochondrial genome sequencing (HTMGS).

The implementation and popularity of next generation sequencing (NGS) has led to the development of various rapid whole mitochondrial genome sequencing techniques. We summarise an efficient and cost-effective NGS approach for mitochondrial genomic DNA in humans using the Ion Torrent platform, and further discuss our bioinformatics pipeline for streamlined variant calling. Ion 316 chips were utilised with the Ion Torrent semi-conductor platform Personal Genome Machine (PGM) to perform tandem sequencing of mitochondrial genomes from the core pedigree (n = 315) of the Norfolk Island Health Study. Key improvements from commercial methods focus on the initial PCR step, which currently requires extensive optimisation to ensure the accurate and reproducible elongation of each section of the complete mitochondrial genome. Dual-platform barcodes were incorporated into our protocol thereby extending its potential application onto Illumina-based systems. Our bioinformatics pipeline consists of a modified version of GATK best practices tailored for mitochondrial genomic data. When compared with current commercial methods, our method, termed high throughput mitochondrial genome sequencing (HTMGS), allows high multiplexing of samples and the use of alternate library preparation reagents at a lower cost per sample (~1.7 times) when compared to current commercial methodologies. Our HTMGS methodology also provides robust mitochondrial sequencing data (>450X average coverage) that can be applied and modified to suit various study designs. On average, we were able to identify ~30 variants per sample with 572 variants observed across 315 samples. We have developed a high throughput sequencing and analysis method targeting complete mitochondrial genomes; with the potential to be platform agnostic with analysis options that adhere to current best practices.

Retroviral gene transfer to primitive normal and leukemic hematopoietic cells using clinically applicable procedures.

Clinical uses of gene transfer to bone marrow transplants require the establishment of a reproducible method for infecting large numbers of very primitive hematopoietic cells at high efficiency using cell-free retrovirus-containing media. In this study we report the results of experiments with preparations of a high-titer (2-5 x 10(7)/ml) helper-free recombinant neo(r) retrovirus that indicate this goal can now be achieved based on measurements of gene transfer efficiencies to cells referred to as long-term culture initiating cells (LTC-IC) because they give rise to clonogenic cells after greater than or equal to 5 wk in long-term culture (LTC). Intermittent, repeated exposure of normal human marrow mononuclear cells to virus-containing supernatant over a 3-d period of cell maintenance on an IL-3/granulocyte colony-stimulating factor (G-CSF) producing stromal layer resulted in gene transfer efficiencies to LTC-IC of 41%; a level previously obtainable only using co-cultivation infection techniques. Marrow cells enriched greater than or equal to 500-fold for LTC-IC (1-2% pure) by flow cytometry showed gene transfer efficiencies of 27% when infected in a similar fashion over a shorter period (24 h), but in the presence of added soluble IL-3 and G-CSF without stromal feeders, and this increased to 61% when Steel factor was also present during the infection period. By using a less highly enriched population of LTC-IC obtained by a bulk immunoselection technique applicable to large-scale clinical marrow harvests, gene transfer efficiencies to LTC-IC of 40% were achieved and this was increased to 60% by short-term preselection in G418. Southern analysis of DNA from the nonadherent cells produced by these LTC over a 6-wk period provided evidence of clonal evolution of LTC-IC in vitro. Leukemic chronic myelogenous leukemia LTC-IC were also infected at high efficiency using the same supernatant infection strategy with growth factor supplementation. These data demonstrate the feasibility of using cell-free virus preparations for infecting clinical marrow samples suitable for transplantation, as well as for further analysis of human marrow stem cell dynamics in vitro.

A globin enhancer acts by increasing the proportion of erythrocytes expressing a linked transgene.

Enhancer elements have been shown to affect the probability of a gene establishing an active transcriptional state and suppress the silencing of reporter genes in cell lines, but their effect in transgenic mice has been obscured by the use of assays that do not assess expression on a cell-by-cell basis. We have examined the effect of a globin enhancer on the variegation of lacZ expression in erythrocytes of transgenic mice. Mice carrying lacZ driven by the alpha-globin promoter exhibit beta-galactosidase (beta-Gal) expression in only a very small proportion of embryonic erythrocytes. When the transgenic construct also contains the (alphaHS-40 enhancer, which controls expression of the alpha-globin gene, expression is seen in a high proportion of embryonic erythrocytes, although there are variations between transgenic lines which can be attributed to different sites of integration. Analysis of beta-Gal expression levels suggests that expressing cells in lines carrying only the alpha-globin promoter express as much beta-Gal as those in which the transgene also contains alphaHS-40. A marked decline in transgene expression occurs as mice age, which is mainly due to a decrease in the proportion of cells expressing the transgene. Thus, a globin enhancer can act to suppress variegation of a linked transgene; this result is consistent with a model in which enhancers act to establish and maintain an active domain without directly affecting the transcriptional rate.

Leukocyte count as a predictor of death during remission induction in acute myeloid leukemia.

Acute myeloid leukemia (AML) presenting with a high leukocyte count has been associated with an increase in induction mortality and poor results in a number of other survival measures. However, the level at which an elevated leukocyte count has prognostic significance in AML remains unclear. In this report on a series of 375 adult (non-M3) AML patients undergoing induction chemotherapy at a single institution, leukocyte count analyzed as a continuous variable is shown to be a better predictor of induction death (ID) and overall survival (OS) than a leukocyte count of > or = 100 x 10(9)/L, a value characteristically associated with "hyperleukocytosis" (HL). In this patient cohort, a presenting leukocyte count of > or = 30 x 10(9)/L had high sensitivity and specificity for predicting ID, and both performance status (PS) and leukocyte count more accurately predicted for ID than age. Considering these parameters in newly-diagnosed AML patients may facilitate the development of strategies for reducing induction mortality.

A quinazoline-based HDAC inhibitor affects gene expression pathways involved in cholesterol biosynthesis and mevalonate in prostate cancer cells.

Chronic inflammation can lead to the development of cancers and resolution of inflammation is an ongoing challenge. Inflammation can result from dysregulation of the epigenome and a number of compounds that modify the epigenome are in clinical use. In this study the anti-inflammatory and anti-cancer effects of a quinazoline epigenetic-modulator compound were determined in prostate cancer cell lines using a non-hypothesis driven transcriptomics strategy utilising the Affymetrix PrimeView® Human Gene Expression microarray. GATHER and IPA software were used to analyse the data and to provide information on significantly modified biological processes, pathways and networks. A number of genes were differentially expressed in both PC3 and DU145 prostate cancer cell lines. The top canonical pathways that frequently arose across both cell lines at a number of time points included cholesterol biosynthesis and metabolism, and the mevalonate pathway. Targeting of sterol and mevalonate pathways may be a powerful anticancer approach.

A randomized trial of two dose levels of cyclophosphamide, methotrexate, and fluorouracil chemotherapy for patients with metastatic breast cancer.

This study was designed to assess the role of dosage of chemotherapy for treatment of metastatic breast cancer. One hundred thirty-three patients without prior chemotherapy for metastatic disease were randomly allocated to receive two different dose levels of cyclophosphamide (C), methotrexate (M), and fluorouracil (F), administered intravenously (IV) every 3 weeks. Patients were stratified by sites of disease (visceral, bone, or soft-tissue dominant) and by interval from primary surgery to first recurrence. Doses on the higher-dose arm were 600 mg/m2 (C,F) and 40 mg/m2 (M) with escalation if possible; doses on the lower-dose arm were 300 mg/m2 (C,F) and 20 mg/m2 (M) without escalation. Patients who failed to respond to lower-dose CMF were crossed over to the higher-dose arm. Patients randomized to the higher-dose arm had longer survival measured from initiation of chemotherapy (median survival, 15.6 months v 12.8 months, P = .026 by log-rank test), but the effect of dose was of borderline significance (P approximately 0.12) when adjusted for a chance imbalance between the two arms in the time from first relapse to randomization, using the Cox proportional hazards model. Response rates (International Union Against Cancer [UICC] criteria) for patients with measurable disease were higher-dose arm: 16/53 (30%) and lower-dose arm: 6/53 (11%), (P = .03). Only one of 37 patients responded on crossover from the lower- to the higher-dose arm. Patients experienced more vomiting, myelosuppression, conjunctivitis, and alopecia when receiving higher doses of chemotherapy. A series of 34 linear analogue self-assessment scales were used to make detailed quality of life assessments on a subset of 49 patients. These scales confirmed greater toxicity in the immediate posttreatment period, but also a trend to improvement in general health and some disease-related indices, in patients receiving higher-dose chemotherapy. This trial suggests that better palliation is achieved by using full-dose chemotherapy.