Some problems related to the design and analysis of clinical trials.

The evaluation of new cancer treatments requires demonstration of their efficacy in clinical trials. Many nonrandomized clinical series have suggested benefit of a new treatment compared with historical controls or with controls from other institutions that received standard treatment. Often, however, the new treatment is not found to be beneficial when evaluated in a subsequent randomized trial. The interpretation of clinical trials has been confounded by many problems, several of which were described or summarized in the writings of Raymond S. Bush: These include (a) invalid comparison of nonrandomized controls because of patient selection or stage migration, (b) inappropriate endpoints, and in particular the failure to evaluate quality of life and complications of treatment, (c) inadequate power to detect clinically meaningful differences in randomized trials, and (d) multiple comparisons and retrospective analysis used to make inappropriate conclusions from randomized trials. The present paper reviews briefly some of these methodological issues that may lead to inappropriate interpretation of clinical trials, with particular reference to trials of combined modality therapy.     

Sequence variation in the 3'-untranslated region of the dopamine transporter gene and attention-deficit hyperactivity disorder (ADHD).

The dopamine transporter gene (DAT1) has been reported to be associated with attention-deficit hyperactivity disorder (ADHD) in a number of studies [Cook et al. (1995): Am J Human Genet 56(4):9993-998; Gill et al. (1997): Mol Psychiatry 2(4):311-313; Waldman et al. (1998): Am J Human Genet 63(6):1767-1776; Barr et al. (2001): Biol Psychiatry 49(4):333-339; Curran et al. (2001): Mol Psychiatry 6(4):425-428; Chen et al. (2003): Mol Psychiatry 8(4):393-396]. Specifically, the 10-repeat allele of the 40-bp variable number of tandem repeats (VNTR) polymorphism located in the 3' untranslated region (UTR) of the gene has been found to be associated with ADHD. There is evidence from in vitro studies indicating that variability in the repeat number, and sequence variation in the 3'-UTR of the DAT1 gene may influence the level of the dopamine transporter protein [Fuke et al. (2001): Pharmacogenomics J 1(2):152-156; Miller and Madras (2002): Mol Psychiatry 7(1):44-55]. In this study, we investigated whether DNA variation in the DAT1 3'UTR contributed to ADHD by genotyping DNA variants around the VNTR region in a sample of 178 ADHD families. These included a MspI polymorphism (rs27072), a DraI DNA change (T/C) reported to influence DAT1 expression levels, and a BstUI polymorphism (rs3863145) in addition to the VNTR. We also screened the VNTR region by direct resequencing to determine if there was sequence variation within the repeat units that could account for the association. Our results indicate that DAT1 is associated with ADHD in our sample but not with alleles of the VNTR polymorphism. We did not find any variation in the sequence for either the 10- or 9-repeat alleles in the probands screened nor did we observe the reported DraI (T/C) variation. Our results therefore refute the possibility of the reported DraI variation or alleles of the VNTR as the functional variants contributing to the disorder.     

Quantitation of Antibody to Non-Hemagglutinating Viruses by Single Radial Hemolysis: Serological Test for Human Coronaviruses

A single radial hemolysis test was developed for quantitation of specific antibody to non-hemagglutinating viruses. With the human coronaviruses as models, this test utilizes the binding properties of the chromic cation to attach viruses to glutaraldehyde-treated sheep erythrocytes. The most satisfactory system consisted of stabilizing washed sheep erythrocytes with 0.0073% glutaraldehyde for 15 min at 23 degrees C, binding a high concentration of virus to a 25% erythrocyte suspension with 0.0016% chromic chloride for 20 min at 23 degrees C, stopping the reaction with phosphate-saline, and finally mixing the treated, rewashed cells with complement and agarose at 45 degrees C to prepare a slide gel. The gel mix, which was dispensed in plastic plates (23 by 73 mm) in 3-ml volumes, consisted of 1% agarose, 0.1% sodium azide, 5% reconstituted complement, and 0.82% treated cells. Wells 2 mm in diameter were loaded with 5 mul of antiserum, incubated for 18 h at 4 degrees C for diffusion of antiserum and fixation of complement, and then incubated for 8 to 24 h at 37 degrees C for development of hemolysis zones. The diameter of a zone was linearly related to antibody concentration, as determined by conventional serological tests. This single radial hemolysis test was applicable to human and animal coronaviruses and to selected serotypes of the adenovirus, picornavirus, rhabdovirus, and rotavirus groups.     

The bowel microflora: an important source of urinary tract pathogens

The large bowel is home to a complex microbial community that is present throughout the life of the human host. Relatively few microbial species detected in faeces in relatively low numbers have been implicated as major aetiological agents of urinary tract infections. The impact of these few species on human health is considerable, especially when recurrent urinary tract infections are considered, and ways must be found to reduce their pathogenic activities. One approach may be to learn about the ecology of the bowel ecosystem and devise ways by which the numbers of enterobacteria, in particular, can be restricted. This, in turn, would decrease the dose of potential urinary tract pathogens present in the faeces.     

In vivo interaction of anti-cancer drugs with misonidazole or metronidazole: cyclophosphamide and BCNU.

The addition of misonidazole (MISO) or metronidazole (METRO) to treatment with cyclophosphamide (CY) increased delay to regrowth of 2 experimental tumours. The effect was observed for large an small tumours, was present for doses of MISO that are ineffective for killing hypoxic cells, and required that it be given with, or shortly before CY. Mice receiving combined treatment had more weight loss and myelosuppression than those receiving CY alone, and the Therapeutic Index was lower. MISO caused a marked increase in growth delay when combined with BCNU to treat the KHT sarcoma. This effect was observed for small and large tumours, required simultaneous administration of drugs, and also led to increased host toxicity. There was no therapeutic advantage from combined treatment. Survival of aerobic or anoxic Chinese hamster ovary (CHO) cells was assessed after exposure in vitro to serum from mice that had received CY or BCNU alone. MISO alone, or combined treatment. Results of these experiments suggest that (1) MISO delays the excretion or breakdown of active metabolites of CY, and (2) at a dose that does not kill hypoxic cells, it may selectively "sensitize" hypoxic cells (but not aerobic cells) to the action of BCNU. The presence of other undetermined interactions of BCNU and MISO is inferred from the increased toxicity to (aerobic) normal tissue. Misonidazole or metronidazole should be used with caution in patients who are receiving BCNU or cyclophosphamide.