Incorporating blood-based liquid biopsy information into cancer staging: time for a TNMB system?

Tissue biopsy is the standard diagnostic procedure for cancer. Biopsy may also provide material for genotyping, which can assist in the diagnosis and selection of targeted therapies but may fall short in cases of inadequate sampling, particularly from highly heterogeneous tumors. Traditional tissue biopsy suffers greater limitations in its prognostic capability over the course of disease, most obviously as an invasive procedure with potential complications, but also with respect to probable tumor clonal evolution and metastasis over time from initial biopsy evaluation. Recent work highlights circulating tumor DNA (ctDNA) present in the blood as a supplemental, or perhaps an alternative, source of DNA to identify the clinically relevant cancer mutational landscape. Indeed, this noninvasive approach may facilitate repeated monitoring of disease progression and treatment response, serving as a means to guide targeted therapies based on detected actionable mutations in patients with advanced or metastatic solid tumors. Notably, ctDNA is heralding a revolution in the range of genomic profiling and molecular mechanisms to be utilized in the battle against cancer. This review will discuss the biology of ctDNA, current methods of detection and potential applications of this information in tumor diagnosis, treatment, and disease prognosis. Conventional classification of tumors to describe cancer stage follow the TNM notation system, heavily weighting local tumor extent (T), lymph node invasion (N), and detectable metastasis (M). With recent advancements in genomics and bioinformatics, it is conceivable that routine analysis of ctDNA from liquid biopsy (B) may make cancer diagnosis, treatment, and prognosis more accurate for individual patients. We put forward the futuristic concept of TNMB tumor classification, opening a new horizon for precision medicine with the hope of creating better outcomes for cancer patients.

Cancer cell proliferation is inhibited by specific modulation frequencies.

BACKGROUND: There is clinical evidence that very low and safe levels of amplitude-modulated electromagnetic fields administered via an intrabuccal spoon-shaped probe may elicit therapeutic responses in patients with cancer. However, there is no known mechanism explaining the anti-proliferative effect of very low intensity electromagnetic fields. METHODS: To understand the mechanism of this novel approach, hepatocellular carcinoma (HCC) cells were exposed to 27.12 MHz radiofrequency electromagnetic fields using in vitro exposure systems designed to replicate in vivo conditions. Cancer cells were exposed to tumour-specific modulation frequencies, previously identified by biofeedback methods in patients with a diagnosis of cancer. Control modulation frequencies consisted of randomly chosen modulation frequencies within the same 100 Hz-21 kHz range as cancer-specific frequencies. RESULTS: The growth of HCC and breast cancer cells was significantly decreased by HCC-specific and breast cancer-specific modulation frequencies, respectively. However, the same frequencies did not affect proliferation of nonmalignant hepatocytes or breast epithelial cells. Inhibition of HCC cell proliferation was associated with downregulation of XCL2 and PLP2. Furthermore, HCC-specific modulation frequencies disrupted the mitotic spindle. CONCLUSION: These findings uncover a novel mechanism controlling the growth of cancer cells at specific modulation frequencies without affecting normal tissues, which may have broad implications in oncology.

Selecting patients for cytotoxic therapies in gastroenteropancreatic neuroendocrine tumours.

Gastroenteropancreatic neuroendocrine tumours (GEP-NET) have heterogenic clinical presentations. The majority of GEP-NET tumours have an indolent behaviour, but patients will eventually develop symptoms of tumour progression or hormone secretion that may require systemic medical interventions. Cytotoxic chemotherapy has been tested in GEP-NETs since the 80s, but treatment recommendations are controversial in many instances. Patient selection is mandatory for optimal use of chemotherapy. Important prognostic factors such as primary tumour site, tumour differentiation, tumour staging and proliferation index have been identified and validated in retrospective and prospective series. The combination of those factors and the natural history of GEP-NET provide valuable information with respect to treatment planning. In this report we provide treatment recommendations to improve systemic therapy in patients with advanced GEP-NETs based on a comprehensive review of the literature.

Treatment of advanced hepatocellular carcinoma with very low levels of amplitude-modulated electromagnetic fields.

BACKGROUND: Therapeutic options for patients with advanced hepatocellular carcinoma (HCC) are limited. There is emerging evidence that the growth of cancer cells may be altered by very low levels of electromagnetic fields modulated at specific frequencies. METHODS: A single-group, open-label, phase I/II study was performed to assess the safety and effectiveness of the intrabuccal administration of very low levels of electromagnetic fields amplitude modulated at HCC-specific frequencies in 41 patients with advanced HCC and limited therapeutic options. Three-daily 60-min outpatient treatments were administered until disease progression or death. Imaging studies were performed every 8 weeks. The primary efficacy end point was progression-free survival 6 months. Secondary efficacy end points were progression-free survival and overall survival. RESULTS: Treatment was well tolerated and there were no NCI grade 2, 3 or 4 toxicities. In all, 14 patients (34.1%) had stable disease for more than 6 months. Median progression-free survival was 4.4 months (95% CI 2.1-5.3) and median overall survival was 6.7 months (95% CI 3.0-10.2). There were three partial and one near complete responses. CONCLUSION: Treatment with intrabuccally administered amplitude-modulated electromagnetic fields is safe, well tolerated, and shows evidence of antitumour effects in patients with advanced HCC.

The German Mouse Clinic: a platform for systemic phenotype analysis of mouse models.

The German Mouse Clinic (GMC) is a large scale phenotyping center where mouse mutant lines are analyzed in a standardized and comprehensive way. The result is an almost complete picture of the phenotype of a mouse mutant line--a systemic view. At the GMC, expert scientists from various fields of mouse research work in close cooperation with clinicians side by side at one location. The phenotype screens comprise the following areas: allergy, behavior, clinical chemistry, cardiovascular analyses, dysmorphology, bone and cartilage, energy metabolism, eye and vision, host-pathogen interactions, immunology, lung function, molecular phenotyping, neurology, nociception, steroid metabolism, and pathology. The German Mouse Clinic is an open access platform that offers a collaboration-based phenotyping to the scientific community (www.mouseclinic.de). More than 80 mutant lines have been analyzed in a primary screen for 320 parameters, and for 95% of the mutant lines we have found new or additional phenotypes that were not associated with the mouse line before. Our data contributed to the association of mutant mouse lines to the corresponding human disease. In addition, the systemic phenotype analysis accounts for pleiotropic gene functions and refines previous phenotypic characterizations. This is an important basis for the analysis of underlying disease mechanisms. We are currently setting up a platform that will include environmental challenge tests to decipher genome-environmental interactions in the areas nutrition, exercise, air, stress and infection with different standardized experiments. This will help us to identify genetic predispositions as susceptibility factors for environmental influences.

Impairment of gastric acid secretion and increase of embryonic lethality in Foxq1-deficient mice.

The mouse Foxq1 gene, also known as Hfh1, encodes a winged helix/forkhead transcription factor. In adult mice, Foxq1 is highly expressed in kidney and stomach. Here, we report that Foxq1 is expressed during prenatal and postnatal stomach development and the transcripts are restricted to acid secreting parietal cells. Mice homozygous for a deletion of the Foxq1 locus on a 129/Sv x C57BL/6J hybrid genetic background display variable phenotypes consistent with requirement of the gene during embryogenesis. Approximately 50% of Foxq1-/- embryos die in utero. Surviving homozygous mutants are normal and fertile, and have a silky shiny coat. Although the parietal cell development is not affected in the absence of Foxq1, there is a lack of gastric acid secretion in response to various secretagogue stimuli. Ultrastructural analysis suggests that the gastric acid secretion defect in Foxq1-deficient mice might be due to impairment in the fusion of cytoplasmic tubulovesicles to the apical membrane of secretory canaliculi.

Age at first birth and the risk of breast cancer in BRCA1 and BRCA2 mutation carriers.

An early age at first full-term birth is associated with a reduction in the subsequent development of breast cancer among women in the general population. A similar effect has not yet been reported among women who carry an inherited BRCA1 or BRCA2 mutation. We conducted a matched case-control study on 1816 pairs of women with a BRCA1 (n = 1405) or BRCA2 (n = 411) mutation in an attempt to elucidate the relationship between age at first full-term pregnancy and the risk of developing breast cancer. Information about the age at first childbirth and other pregnancy-related variables was derived from a questionnaire administered to women during the course of genetic counselling. There was no difference in the mean age at first full-term birth in the cases and controls (24.9 years vs. 24.8 years; P = 0.81, respectively). Compared to women whose first child was born at or before 18 years of age, a later age at first full-term birth did not influence the risk of developing breast cancer (OR = 1.00 per year; 95% CI 0.98-1.03; P-trend = 0.67). Stratification by mutation status did not affect the results. These findings suggest that an early first full-term birth does not confer protection against breast cancer in BRCA mutation carriers. Nonetheless, BRCA mutation carriers opting for a prophylactic oophorectomy as a breast and/or ovarian cancer risk-reducing strategy should complete childbearing prior to age 40 when this prevention modality is most effective.

TGFBR1*6A is not associated with prostate cancer in men of European ancestry.

The TGFBR1*6A (*6A) variant in exon 1 of the TGFBR1 gene has been postulated as a putative tumor susceptibility allele in several studies. We have performed a case-control study in 537 men with histologically verified prostate cancer and in 488 unrelated controls to investigate the association of *6A with prostate cancer. Our results revealed that the frequency of the (*)6A allele does not differ in men with prostate cancer compared to healthy controls, even in a subset of age-matched cases and controls. There is no compelling evidence for an association of the *6A variant with prostate cancer.

Isolation and characterization of the human forkhead gene FOXQ1.

We have isolated a human genomic and cDNA clone that encodes a protein of 403 amino acids and belongs to the family of the FOX transcription factors (previously called HNF-3/forkhead transcription factors). The 2.7-kb transcript of the human FOXQ1 gene is expressed predominantly in the stomach, trachea, bladder and salivary gland. Additionally, overexpression of human FOXQ1 was shown in colorectal adenocarcinoma and lung carcinoma cell lines. The FOXQ1 gene is located on chromosome 6p23-25. Databank analysis shows 82% homology with the mouse Foxq1 gene (formerly Hfh-1L) and with a revised sequence of the rat FoxQ1 gene (formerly HFH-1). The DNA-binding motif, named HNF-3/forkhead domain, is well conserved, showing 100% identity in human, mouse, and rat. The human protein sequence contains two putative transcriptional activation domains, which share a high amino acid identity with the corresponding mouse and rat domains.

Role of transforming growth factor beta in cancer.

Transforming growth factor beta (TGF-beta) is an effective and ubiquitous mediator of cell growth. The significance of this cytokine in cancer susceptibility, cancer development and progression has become apparent over the past few years. TGF-beta plays various roles in the process of malignant progression. It is a potent inhibitor of normal stromal, hematopoietic, and epithelial cell growth. However, at some point during cancer development the majority of transformed cells become either partly or completely resistant to TGF-beta growth inhibition. There is growing evidence that in the later stages of cancer development TGF-beta is actively secreted by tumor cells and not merely acts as a bystander but rather contributes to cell growth, invasion, and metastasis and decreases host-tumor immune responses. Subtle alteration of TGF-beta signaling may also contribute to the development of cancer. These various effects are tissue and tumor dependent. Identifying and understanding TGF-beta signaling pathway abnormalities in various malignancies is a promising avenue of study that may yield new modalities to both prevent and treat cancer. The nature, prevalence, and significance of TGF-beta signaling pathway alterations in various forms of human cancer as well as potential preventive and therapeutic interventions are discussed in this review.

MAD2 haplo-insufficiency causes premature anaphase and chromosome instability in mammalian cells.

The mitotic checkpoint protein hsMad2 is required to arrest cells in mitosis when chromosomes are unattached to the mitotic spindle. The presence of a single, lagging chromosome is sufficient to activate the checkpoint, producing a delay at the metaphase-anaphase transition until the last spindle attachment is made. Complete loss of the mitotic checkpoint results in embryonic lethality owing to chromosome mis-segregation in various organisms. Whether partial loss of checkpoint control leads to more subtle rates of chromosome instability compatible with cell viability remains unknown. Here we report that deletion of one MAD2 allele results in a defective mitotic checkpoint in both human cancer cells and murine primary embryonic fibroblasts. Checkpoint-defective cells show premature sister-chromatid separation in the presence of spindle inhibitors and an elevated rate of chromosome mis-segregation events in the absence of these agents. Furthermore, Mad2+/- mice develop lung tumours at high rates after long latencies, implicating defects in the mitotic checkpoint in tumorigenesis.

TbetaR-I(6A) is a candidate tumor susceptibility allele.

We have previously described a type I transforming growth factor (TGF)-beta receptor (TbetaR-I) polymorphic allele, TbetaR-I(6A), that has a deletion of three alanines from a nine-alanine stretch. We observed a higher than expected number of TbetaR-I(6A) homozygotes among tumor and nontumor DNA from patients with a diagnosis of cancer. To test the hypothesis that TbetaR-I(6A) homozygosity is associated with cancer, we performed a case-control study in patients with a diagnosis of cancer and matched healthy individuals with no history of cancer and who were identical in their gender and their geographical and ethnic background to determine the relative germ-line frequencies of this allele. We found nine TbetaR-I(6A) homozygotes among 851 patients with cancer. In comparison, there were no TbetaR-I(6A) homozygotes among 735 healthy volunteers (P < 0.01). We also observed an excess of TbetaR-I(6A) heterozygotes in cancer cases compared to controls (14.6% versus 10.6%; P = 0.02, Fisher's exact test). A subset analysis revealed that 4 of 112 patients with colorectal cancer were TbetaR-I(6A) homozygotes (P < 0.01). Using mink lung epithelial cell lines devoid of TbetaR-I, we established stably transfected TbetaR-I and TbetaR-I(6A) cell lines. We found that, compared to TbetaR-I, TbetaR-I(6A) was impaired as a mediator of TGF-beta antiproliferative signals. We conclude that TbetaR-I(6A) acts as a tumor susceptibility allele that may contribute to the development of cancer, especially colon cancer, by means of reduced TGF-beta-mediated growth inhibition.

Molecular analysis of SALL1 mutations in Townes-Brocks syndrome.

Townes-Brocks syndrome (TBS) is an autosomal dominantly inherited malformation syndrome characterized by anal, renal, limb, and ear anomalies. Recently, we showed that mutations in the putative zinc finger transcription factor gene SALL1 cause TBS. To determine the spectrum of SALL1 mutations and to investigate the genotype-phenotype correlations in TBS, we examined 23 additional families with TBS or similar phenotypes for SALL1 mutations. In 9 of these families mutations were identified. None of the mutations has previously been described. Two of these mutations are nonsense mutations, one of which occurred in three unrelated families. Five of the mutations are short deletions. All of the mutations are located 5' of the first double zinc finger (DZF) encoding region and are therefore predicted to result in putative prematurely terminated proteins lacking all DZF domains. This suggests that only SALL1 mutations that remove the DZF domains result in TBS. We also present evidence that in rare cases SALL1 mutations can lead to phenotypes similar to Goldenhar syndrome. However, phenotypic differences in TBS do not seem to depend on the site of mutation.

Type I transforming growth factor beta receptor maps to 9q22 and exhibits a polymorphism and a rare variant within a polyalanine tract.

In a search for mutations of the type I transforming growth factor beta receptor (TbetaR-I), we mapped the gene to 9q22 and found a common polymorphism [TbetaR-I(6A)] and a rare variant [TbetaR-I(10A)] of TbetaR-I, causing an in-frame deletion of three alanines and an in-frame insertion of one alanine, respectively, in the receptor's extracellular domain. The biological relevance of the polymorphism TbetaR-I(6A) was investigated. When TbetaR-I(6A) was transiently transfected into TbetaR-I-deficient cells, the growth-inhibitory effects of transforming growth factor beta were restored. TbetaR-I(6A) and TbetaR-I(10A) frequency were assessed in 108 tumor samples and 80 nontumor samples from patients with a diagnosis of cancer, as well as in 118 normal blood donors of comparable ethnic composition. The frequency of TbetaR-I(6A) heterozygotes was fairly similar in normal blood donors (8%), in nontumor DNA of patients with a diagnosis of cancer (10%), and in tumor samples (14%). However, the frequency of TbetaR-I(6A) homozygotes among nontumor (4%) and tumor (8%) samples obtained from patients with a diagnosis of cancer was higher than that predicted by the Hardy-Weinberg law. The clinical and biological significance of TbetaR-I(6A) homozygosity needs to be further investigated.

Bright light and LEET effects on circadian rhythms, sleep and cognitive performance.

Shift work and jet lag can disrupt cicadian rhythms, with detrimetnal effects on alertness, performance and sleep. This study examined the effects of two interventions to adapt circadian rhythms, sleep and performance to a 10-h phase delay of the work-rest cycle. Bright light was administered from 2200 to 0200 each night to promote phase delay of circadian rhythms. Low energy emission therapy (LEET) was administered for 20 min prior to daytime sleep periods to promote sleep. Twelve subjects received bright light, 12 subjects received LEET, 11 received both interventions and 10 control subjects received only placebo treatments. Bright light accelerated phase delay of the circadian melatonin rhythms after the work-rest schedule shift. Further, subjects who received bright light had greater total sleep time (TST) and improved sleep continuity. LEET treatment produced a trend (p = 0.16) for increased TST, but LEET did not affect the melatonin circadian rhythm. After the schedule shift, cognitive performance measures showed few significant differences. Some minor improvements in cognitive performance were producced by light treatments but not by LEET.

Electroencephalographic changes following low energy emission therapy.

Low energy emission therapy (LEET) is a novel approach to delivering low levels of amplitude-modulated electromagnetic fields to the human brain. The sleep electroencephalogram (EEG) effects of a 15-min LEET treatment were investigated in a double-find cross-over study to assess sleep induction. Fifty-two healthy volunteers were exposed to both active and inactive LEET treatment sessions, with a minimum interval of 1 week between the two sessions. Baseline EEGs were obtained, and 15-min posttreatment EEGs were recorded and analyzed according to the Loomis classification. A significant increase in the duration of stage B1 sleep (0.58 +/- 2.42 min [mean +/- SD], p = 0.046), decreased latency to the first 10 sec epoch of sleep (-1.23 +/- 5.32 min, p = 0.051) and decreased latency to sleep stage B2 (-1.21 +/- 5.25 min, p = 0.052) were observed after active treatment. Additionally, establishment of slow waves with progression from stages B to C was significantly more pronounced after active LEET treatment (p = 0.040). A combined analysis of these results with those of an identical study performed in Denver showed that LEET had a significant effect on afternoon sleep induction and maintenance with shorter sleep latencies (decreased latency to the first 10 sec epoch of sleep; -1.00 +/- 5.51 min, p = 0.033; decreased latency to sleep stage B2; -1.49 +/- 5.40 min, p = 0.003), an increased duration of stage B2 (0.67 +/- 2.50 min, p = 0.003), an increase in the total duration of sleep (0.69 +/- 4.21 min, p = 0.049), and a more prominent establishment of slow waves with progression to a deeper sleep stage (p = 0.006). It is concluded that the intermittent 42.7 HZ amplitude modulation of 27.12-MHz electromagnetic fields results in EEG changes consistent with shorter sleep latencies, longer sleep duration, and deeper sleep in healthy subjects.

Effects of low energy emission therapy in chronic psychophysiological insomnia.

The treatment of chronic psychophysiological insomnia presents a challenge that has not been met using currently available pharmacotherapy. Low energy emission therapy (LEET) has been developed as a potential alternative therapy for this disorder. LEET consists of amplitude-modulated electromagnetic fields delivered intrabuccally by means of an electrically conducting mouthpiece in direct contact with the oral mucosa. The effect of LEET on chronic psychophysiological insomnia was assessed with polysomnography (PSG) and sleep rating forms on a total of 106 patients at two different centers. Active or inactive LEET was administered for 20 minutes in late afternoon three times a week for a total of 12 treatments. Primary efficacy endpoints evaluating the results were changes from baseline in PSG-assessed total sleep time (TST) and sleep latency (SL). Secondary endpoints were changes in sleep efficiency (SE), sleep stages, and reports by the subjects of SL and TST. There was a significant increase in TST as assessed by PSG between baseline and post-treatment values for the active treatment group (76.0 +/- 11.1 minutes, p = 0.0001). The increase for the inactive treatment group was not statistically significant. The TST improvement was significantly greater for the active group when compared to the inactive group (adjusted for baseline TST; p = 0.020. R1 = 0.20). There was a significant decrease in SL as assessed by PSG between baseline and post-treatment values for the active treatment group (-21.6 +/- 5.9 minutes, p = 0.0006), whereas the decrease noted for the inactive treatment group was not statistically significant. The difference in SL decrease between the two treatment groups was marginally significant (adjusted for baseline SL and center, p = 0.068, R2 = 0.60). The number of sleep cycles per night increased by 30% after active treatment (p = 0.0001) but was unchanged following inactive treatment. Subjects did not experience rebound insomnia, and there were no significant side effects. The data presented in this report indicate that LEET administered for 20 minutes three times a week increased TST and reduced SL in chronic psychophysiological insomnia. LEET is safe and well tolerated and it effectively improved the sleep of chronic insomniacs given 12 treatments over a 4-week period by increasing the number of sleep cycles without altering the percentage of the various sleep stages during the night. The therapeutic action of LEET differs from that of currently available drug therapies in that the sleep pattern noted in insomniacs following LEET treatment more closely resembles nocturnal physiological sleep. This novel treatment may offer an attractive alternative therapy for chronic insomnia.

Platelets and plasminogen activation.

Platelets serve as a site for assembly of the proteins of the plasminogen activator system. Once bound to the platelet surface, tissue-type plasminogen activator manifests enhanced catalytic activity. Plasmin, once formed, also binds to the platelets surface and, at low concentrations, renders the platelet dysfunctional by cleaving glycoprotein IIIa selectively in the presence of bound fibrinogen. At higher concentrations (approximately 1 caseinolytic unit/ml), plasmin activates the platelet directly. Activated platelets also bind plasminogen and tissue-type plasminogen activator, and manifest enhanced catalytic efficiency of plasminogen activation. These observations suggest that plasminogen activation by tissue-type plasminogen activator is an autocatalytic process on the platelet surface, and that unique reciprocating mechanisms govern the interaction between platelets and the components of the plasminogen activator system.

Structural changes in platelet glycoprotein IIb/IIIa by plasmin: determinants and functional consequences.

Plasmin exposure modulates platelet aggregation responses, but a direct effect of plasmin on the platelet fibrinogen receptor, glycoprotein IIb/IIIa (GPIIb/IIIa), has never been conclusively shown in a plasma milieu. To examine this issue, we incubated platelets in platelet-rich plasma with plasmin and measured the effect of this treatment on platelet aggregation, fibrinogen binding, and the structural integrity of GPIIb/IIIa. Plasmin treatment reduced maximal reversible fibrinogen binding in a dose-dependent fashion, and this reduction in binding was accompanied by a correlative reduction in the maximal rate of aggregation. Immunoblots performed with polyclonal antibodies against GPIIb/IIIa showed that GPIIIa had been cleaved by plasmin, but this cleavage was detected only after subsequent degradation of the solubilized GPIIb/IIIa with Staphylococcus aureus V8 (Glu-C) endoprotease. Peptide sequence analysis showed that cleavage occurred at the lys444-pro445 bond in the first cysteine-rich repeat domain of GPIIIa a unique proteolytic event observed only in the presence of plasma fibrinogen. These observations suggest that plasmin modifies GPIIIa by a unique proteolytic event in plasma that is dependent on fibrinogen binding and, consequently, is accompanied by significant reductions in fibrinogen binding and aggregation response.

Sleep inducing effect of low energy emission therapy.

The sleep inducing effect of a 15 min treatment with either an active or an inactive Low Energy Emission Therapy (LEET) device emitting amplitude-modulated electromagnetic (EM) fields was investigated in a double-blind cross-over study performed on 52 healthy subjects. All subjects were exposed to both active and inactive LEET treatment sessions, with an interval of at least 1 week between the two sessions. LEET consists of 27.12 MHz amplitude-modulated (sine wave) EM fields emitted intrabuccally by means of an electrically conducting mouthpiece in direct contact with the oral mucosa. The estimated local peak SAR is less than 10 W/kg in the oral mucosa and 0.1 to 100 mW/kg in brain tissue. No appreciable sensation is experienced during treatment, and subjects are therefore unable to tell whether they are receiving an active or an inactive treatment. In this study the active treatment consisted of EM fields intermittently amplitude-modulated (sine wave) at 42.7 Hz for 3 s followed by a pause of 1 s during which no EM fields were emitted. During the inactive treatment no EM fields were emitted. Baseline EEGs were obtained and 15 min post-treatment EEGs were recorded and analyzed according to the Loomis classification. A significant decrease (paired t test) in sleep latency to stage B2 (-1.78 +/- 5.57 min, P = 0.013), and an increase in the total duration of stage B2 (1.15 +/- 2.47 min, P = 0.0008) were observed on active treatment as compared with inactive treatment.(ABSTRACT TRUNCATED AT 250 WORDS)