Resting energy expenditure in adolescents with Down syndrome: a comparison of commonly used predictive equations.

BACKGROUND: Adolescents with Down syndrome (DS) are two to three times more likely to be obese than their typically developing peers. When preventing or treating obesity, it is useful for clinicians to understand an individual's energy intake needs. Predictive resting energy expenditure (REE) equations are often recommended for general use in energy intake recommendations; however, these predictive equations have not been validated in youth with DS. The aim of this study was to compare the accuracy of seven commonly used predictive equations for estimating REE in adolescents who are typically developing to REE measured by indirect calorimetry in adolescents with DS. METHODS: Adolescents with DS participated in a 90-min laboratory visit before 10:00 a.m. after a 12-h overnight fast and a 48-h abstention from aerobic exercise. REE was measured via indirect calorimetry, and estimated REE was derived using the Institute of Medicine, Molnar, Muller and World Health Organization equations. Mean differences between the measured and predicted REE for each equation were evaluated with equivalency testing, and P-values were adjusted for multiple comparisons using the Holm method. RESULTS: Forty-six adolescents with DS (age: 15.5 ± 1.7 years, 47.8% female, 73.9% non-Hispanic White) completed the REE assessment. Average measured REE was 1459.5 ± 267.8 kcal/day, and the Institute of Medicine equations provided the most accurate prediction of REE with a 1.7 ± 11.2% (13.9 ± 170.3 kcal/day) overestimation. This prediction was not statistically different from the measured REE [P-value = 0.582; 95% confidence interval (CI): -64.5, 36.7], and the difference between the measured and predicted REE was statistically equivalent to zero (P-value = 0.024; 90% CI: -56.1, 28.3). CONCLUSIONS: The results suggest that the Institute of Medicine equation may be useful in predicting REE in adolescents with DS. Future research should confirm these results in a larger sample and determine the utility of the Institute of Medicine equation for energy intake recommendations during a weight management intervention.

Weight status and associated comorbidities in children and adults with Down syndrome, autism spectrum disorder and intellectual and developmental disabilities.

BACKGROUND: Little is known about body weight status and the association between body weight and common comorbidities in children and adults with Down syndrome (DS), autism spectrum disorder (ASD) and other intellectual and developmental disabilities (IDDs). METHODS: Data were extracted from the University of Kansas Medical Center's Healthcare Enterprise Repository for Ontological Narration clinical integrated data repository. Measures included demographics (sex, age and race), disability diagnosis, comorbid health conditions, height, weight and body mass index percentiles (BMI%ile; <18 years of age) or BMI (≥18 years of age). RESULTS: Four hundred and sixty-eight individuals with DS (122 children and 346 adults), 1659 individuals with ASD (1073 children and 585 adults) and 604 individuals with other IDDs (152 children and 452 adults) were identified. A total of 47.0% (DS), 41.9% (ASD) and 33.5% (IDD) of children had overweight/obese (OW/OB), respectively. Children with DS were more likely to have OW/OB compared with children with IDD or ASD [odds ratio (OR) = 1.91, 95% confidence interval (CI): (1.49, 2.46); OR = 1.43, 95% CI: (1.19, 1.72)], respectively. A total of 81.1% (DS), 62.1% (ASD), and 62.4% (IDD) of adults were OW/OB, respectively. Adults with DS were more likely to have OW/OB compared with those with IDD [OR = 2.56, 95% CI: (2.16, 3.02)]. No significant differences were observed by race. In children with ASD, higher OW/OB was associated with significantly higher (compared with non-OW/OB) occurrence of sleep apnoea [OR = 2.94, 95% CI: (2.22, 3.89)], hypothyroidism [OR = 3.14, 95% CI: (2.17, 4.25)] and hypertension [OR = 4.11, 95% CI: (3.05, 5.54)]. In adults with DS, OW/OB was significantly associated with higher risk of sleep apnoea and type 2 diabetes [OR = 2.93, 95% CI: (2.10, 4.09); OR = 1.76, 95% CI: (1.11, 2.79) respectively]. Similarly, in adults with ASD and IDD, OW/OB was significantly associated with higher risk of sleep apnoea [OR = 3.39, 95% CI: (2.37, 4.85) and OR = 6.69, 95% CI: (4.43, 10.10)], type 2 diabetes [OR = 2.25, 95 % CI: (1.68, 3.01) and OR = 5.49, 95% CI: (3.96, 7.61)] and hypertension [OR = 3.55, 95% CI: (2.76, 4.57) and 3.97, 95% CI: (3.17, 4.97)]. CONCLUSION: Findings suggest higher rates of OW/OB in individuals with DS compared with ASD and IDD. Given the increased risk of comorbidities associated with the increased risk of OW/OB, identification of effective interventions for this special population of individuals is critical.

Induction of differentiation in HL60 cells by the reduction of extrachromosomally amplified c-myc.

Oncogene amplification in tumor cells results in the overexpression of proteins that confer a growth advantage in vitro and in vivo. Amplified oncogenes can reside intrachromosomally, within homogeneously staining regions (HSRs), or extrachromosomally, within double minute chromosomes (DMs). Since previous studies have shown that low concentrations of hydroxyurea (HU) can eliminate DMs, we studied the use of HU as a gene-targeting agent in tumor cells containing extrachromosomally amplified oncogenes. In a neuroendocrine cell line (COLO 320), we have shown that HU can eliminate amplified copies of c-myc located on DMs, leading to a reduction in tumorigenicity in vitro and in vivo. To determine whether the observed reduction in tumorigenicity was due to differentiation, we next investigated whether HU could induce differentiation in HL60 cells containing extrachromosomally amplified c-myc. We compared the effects of HU, as well as two other known differentiating agents (dimethyl sulfoxide and retinoic acid), on c-myc gene copy number, c-myc expression, and differentiation in HL60 cells containing amplified c-myc genes either on DMs or HSRs. We discovered that HU and dimethyl sulfoxide reduced both c-myc gene copy number and expression and induced differentiation in cells containing c-myc amplified on DMs. These agents failed to have similar effects on HL60 cells with amplified c-myc in HSRs. By contrast, retinoic acid induced differentiation independent of the localization of amplified c-myc. These data illustrate the utility of targeting extrachromosomal DNA to modulate tumor phenotype and reveal that both HU and dimethyl sulfoxide induce differentiation in HL60 cells through DM elimination.

Preparation of uniform, intact DNA samples from resected tumor tissues for pulsed-field gel electrophoretic analyses.

Preparation of high molecular weight DNA from resected tumor tissues suitable for pulsed-field gel electrophoresis (PFGE) can be complicated by the presence of nonviable cells and lymphocytes. We have developed a simple procedure to reduce the level of degraded DNA in PFGE DNA samples prepared from resected tumor tissues. The procedure employs a single, three component Percoll step gradient centrifugation and can be performed on several tumor samples simultaneously. Analyses of DNAs from 15 tumor specimens (7 solid tumors and 8 aspirated fluids) demonstrate that the technique enriches the integrity of PFGE DNA samples. Morphologic evaluation of 9 specimens suggested that both cellular debris and contaminating normal lymphocytes are removed from starting cell populations during the enrichment procedure. Fractionation of cells also reduced cell clumping, allowing for the formation of more uniform PFGE DNA samples.

Preclinical leads for innovative uses for etoposide.

Amplification of oncogenes in human tumors has been associated with a poor prognosis. Microscopically visible amplified oncogenes can be located either within chromosomes in homogeneously staining regions, or in an extrachromosomal compartment in double minutes (DMs). The DMs are composed of submicroscopic circular DNA (episomes), which have multimerized to form the microscopically visible DMs. When amplified oncogenes are located in an extrachromosomal location, they are vulnerable to loss from the cell. In this study we have found that the topoisomerase II inhibitor etoposide, in concentrations easily achievable clinically, causes a significant decrease in the number of DM-containing amplified oncogenes in three different human tumor cell lines. The elimination of amplified oncogenes from the cell could be accompanied by less aggressive tumor behavior.

Elimination of extrachromosomally amplified MYC genes from human tumor cells reduces their tumorigenicity.

Oncogene amplification has been observed in a broad spectrum of human tumors and has been associated with a poor prognosis for patients with several different types of malignancies. Importantly, at biopsy, the amplified genes localize to acentric extrachromosomal elements such as double-minute chromosomes (DMs) in the vast majority of cases. We show here that treatment of several human tumor cell lines with low concentrations of hydroxyurea accelerates the loss of their extrachromosomally amplified oncogenes. The decreases in MYC copy number in a human tumor cell line correlated with a dramatic reduction in cloning efficiency in soft agar and tumorigenicity in nude mice. No effect on gene copy number or tumorigenicity was observed for a closely related cell line containing the same number of chromosomally amplified MYC genes. One step involved in the accelerated loss of extrachromosomal elements is shown to involve their preferential entrapment of DMs within micronuclei. The data suggest that agents that accelerate the loss of extrachromosomally amplified genes could provide valuable tools for moderating the growth of a large number of human neoplasms.

Hydroxyurea accelerates loss of extrachromosomally amplified genes from tumor cells.

Gene amplification is one mechanism mediating the resistance of tumor cells to antineoplastic agents and the overexpression of a variety of oncogenes in diverse tumor types. There is mounting evidence that acentric extrachromosomal elements such as double minute chromosomes are common intermediates in the amplification process. These acentric structures partition unequally at mitosis and can be lost in the absence of selection. In the present study, we used human and hamster cell lines documented to contain extrachromosomally amplified drug resistance genes to investigate the feasibility of enhancing the loss rate of the extrachromosomally amplified genes to make the cells more sensitive to drug treatment. The results show that treatment of each of these lines with hydroxyurea accelerates the loss of the extrachromosomally amplified drug resistance genes. Loss of these extrachromosomal genes was associated with a corresponding increase in the drug sensitivity in the cases examined. The mechanism of accelerated loss does not appear to involve a differential effect on the replication of extrachromosomal DNA sequences. The results suggest that hydroxyurea treatment may provide a valuable tool for the general accelerated elimination of extrachromosomally amplified genes.

Double minutes arise from circular extrachromosomal DNA intermediates which integrate into chromosomal sites in human HL-60 leukemia cells.

Amplification of oncogenes has been found to be an important prognostic factor in behavior of patients' malignancies. In this study we have used new gel electrophoresis techniques to follow the location of amplified c-myc oncogene sequences in HL-60 promyelocytic leukemia cells. In passages 46-62 of the cells, the cells contain amplified c-myc sequences on submicroscopic circular extrachromosomal DNA (episomes). With increased passages in culture (passages 63-72) the cells lose the episome c-myc sequences with a shift of those sequences to double minutes. With additional passage in culture, the c-myc shifts from the double minutes to a chromosomal site der(5)t(5;17)(q11.2;q?11.2). Concomitant with the shift of the c-myc sequences into the chromosomal compartment is a phenotypic change of a shortened cell-doubling time. These studies provide the first molecular evidence of a progression from a submicroscopic location for amplified oncogene sequences to a chromosomal location for the amplified sequences. This molecularly documented model can now be used to test various strategies to prevent incorporation of extrachromosomally located oncogene sequences into chromosomal sites. Prevention of integration of the oncogene sequences into chromosomal sites could modulate progression of patients' tumors.

Selection of cancer chemotherapy for a patient by an in vitro assay versus a clinician.

One hundred thirty-three patients with advanced metastatic cancer were randomized to receive single-agent chemotherapy selected by either a medical oncologist or an in vitro capillary cloning system. Thirty-six of the 65 patients (55%) who were randomly assigned to selection of a drug by the clinician actually received a drug; these patients were able to be evaluated for clinical response. Of these 36 patients, one had a partial tumor response (3%). Only 19 of the 68 patients (28%) who were randomly assigned to selection of a drug by the capillary system actually received a drug; these patients were able to be evaluated for clinical response. Of these 19 patients, four (21%) had partial tumor responses. In the assessable patients (36 in the clinician's choice group, 19 in the capillary cloning group), the partial response rate was superior for drug selection by the capillary cloning system (P = .04). For all patients randomly assigned to a group (65 in the clinician's choice group, 68 in the capillary cloning group), the response rate was not significantly different (1.5% and 5.9%, respectively; P = .37). When overall survival rates for patients in the two groups were compared, there was no difference. We conclude that drug sensitivity testing in capillary tubes can improve the response rate for patients with advanced malignancies. This improved response rate, however, does not translate into improved survival times for these patients.

A radiometric method for evaluation of chemotherapy sensitivity: results of screening a panel of human breast cancer cell lines.

We have used a radiometric method to screen for chemotherapy sensitivity among a panel of human breast cancer cell lines. This method utilizes the inhibition of conversion of [14C]glucose to 14CO2 as an index of cytotoxicity. Nine different breast cancer cell lines were exposed for 1 h to 4 different concentrations of several antineoplastic agents with and without documented clinical activity against breast cancer. Cytotoxic effects were analyzed as a function of the ratio of the concentration required to inhibit cell growth to 50% of control to 1/10 of the known peak plasma concentration in humans for each particular drug. The drug-induced inhibition of 14C production by breast cancer tumor cells correlated strongly with the drug-induced antiproliferative effect (P less than 0.002) and with the inhibition of colony formation in a soft agar cloning assay (P less than 0.05). The HS578T cell line and one of the MCF7 cell lines exhibited a chemosensitivity pattern consistent with the clinical responsiveness of human breast cancer to the agents tested. Most of the other cell lines exhibited resistance to clinically active agents, especially the cell lines obtained from patients exposed to prior chemotherapy. These results suggest that this radiometric method measures a drug-induced metabolic effect that correlates with the antiproliferative activity of antineoplastic agents on breast cancer cells. The HS578T and the MCF7-KO cell lines, tested in this system, could be a useful screen for new anticancer compounds with activity against human breast cancer.

Alteration of EGF-receptor binding in human breast cancer cells by antineoplastic agents.

Polypeptide growth factors bind to membrane receptors on human breast cancer cells and stimulate cell proliferation, suggesting that they may be important in growth regulation. Inhibition of the stimulatory effects of these factors might result in antineoplastic activity. Since cytotoxic drugs have been shown to alter cell membrane characteristics, we have examined the effects of a variety of antitumor drugs on the binding of epidermal growth factor (EGF) to the membrane receptor of human breast cancer cells. Twenty-four standard or investigational cytotoxic drugs were screened at a concentration of one-tenth the achievable peak plasma level for their ability to inhibit binding of 125I-EGF to its receptor in MCF-7 human breast cancer cells. Although at this concentration statistically significant inhibition of binding was observed with 11 drugs, the maximum inhibition observed was only 27%. Five agents, representing classes of drugs with different modes of action, were then studied in more detail. Of these, preincubation with 5-fluorouracil, 4-hydroperoxy-cylophosphamide and doxorubicin inhibited MCF-7 colony formation in a dose-dependent manner, but these drugs had no effect on EGF-binding even at a concentration of 10 times the peak plasma level. Preincubation of cells with vinblastine and cisplatin, however, resulted in both reduced colony survival and a parallel reduction in EGF receptor binding. Membrane integrity, as measured by trypan blue exclusion, was not altered. Scatchard analysis of EGF binding demonstrated that the major effect of cisplatin was a reduction in binding affinity. We conclude that cisplatin and vinblastine at high concentrations can inhibit the binding of EGF to human breast cancer cells offering an additional possible mechanism for their antiproliferative activity.

Simultaneous in vitro drug sensitivity testing on tumors from different sites in the same patient.

A human tumor cloning assay was used to analyze drug sensitivity profiles for 99 pairs of tumor samples taken simultaneously from the same patient. These pairs included two areas of the same primary (12 pairs), primary tumor versus metastasis (29 pairs), and metastasis versus metastasis (45 pairs). In addition, to assess variability in the culture and sensitivity techniques, 13 specimens were divided equally and processed twice. One hundred fourteen evaluable drug sensitivity tests were performed on the specimen pairs. Drug sensitivity profiles for the same specimen processed twice demonstrated good agreement (P = 0.03). Sensitivity profiles from two different areas of the same primary were also quite similar (P = 0.002). However, when one sampled a primary tumor and its metastasis for drug sensitivity testing, the agreement (in terms of percent survival) was poor (P = 0.84). Agreement was also poor for drug sensitivity profiles of one metastasis versus another metastasis (P = 0.39). This heterogeneity in drug sensitivity profiles of primary versus metastasis and of metastasis versus metastasis could account for some of the false positives and false negatives noted in clinical correlation trials with the cloning assay. It may also have implications for adjuvant treatment programs where chemosensitivity of the primary tumor can be determined, but will probably not be predictive for chemosensitivity of the micrometastases.

Improved plating efficiencies for human tumors cloned in capillary tubes versus Petri dishes.

As now constituted, the human tumor cloning assay performed in Petri dishes has several limitations including: (a) not all patients' tumors form colonies in the assay; (b) the plating efficiencies (number of colonies formed/number of cells plated) are low; and (c) a large number of tumor cells are required to perform drug sensitivity testing. In this study the use of capillary tubes, as vessels in which to clone human tumors, is compared to the use of 35-mm Petri dishes. In 100-microliters capillary tubes the optimal plating efficiencies are found with 50,000 cells/vessel (500,000 cells/ml), while in 35-mm Petri dishes the optimal plating efficiencies are found with 500,000 cells/vessel (250,000 cells/ml). In head to head comparisons of plating efficiencies of 183 human tumors (18 different histological types), the median plating efficiency was 5-fold higher (range, 1.16-37.00) for the capillary tubes than for the Petri dishes. This improved plating efficiency was noted for nearly all of the histological tumor types examined. The improved plating efficiencies noted with the capillary system indicate that the Petri dish method may be too selective and not reflect the total number of clonogenic units in a human tumor. In addition, the higher plating efficiencies noted with the capillary system may be exploited to solve some of the problems noted with the conventional Petri dish method.

In vitro chemosensitivity testing of human neuroblastoma cell lines in soft agar: a tumor-specific drug screening model.

Tumor-specific screening of investigational anticancer drugs is a new approach to cytotoxic drug screening. A tumor-specific drug screening model has been designed using established human neuroblastoma cell lines. In vitro drug sensitivity was studied using a modification of the human tumor stem cell assay. Eleven cell lines were tested initially for colony formation in soft agar. Four cell lines formed only small cell clusters, while three cell lines formed colonies at a very low rate. The four remaining lines formed colonies with satisfactory efficiency. These cell lines were exposed to three concentrations each of cis-platinum, vincristine, doxorubicin, L-phenylalanine mustard, and ethidium chloride and suspended in soft agar. Colony formation was tabulated and sensitivities determined for each cell line. A heterogeneous pattern of in vitro sensitivity was observed. Limitations and pitfalls as well as potential advantages of using such a tumor-specific drug screening system are discussed.

Direct effects of the hypoxic cell sensitizer misonidazole on colony formation in a human tumor cloning assay.

The human tumor cloning assay as described by Hamburger and Salmon was utilized to study the direct antitumor effects of the hypoxic cell sensitizer misonidazole (MISO). Cells from 106 tumor specimens directly obtained from patients were exposed to MISO at clinically achievable drug concentrations (0.5 mM). Of 30 evaluable tumors, seven specimens (23%) showed a less than or equal to 50% decrease of TCFU's. In vitro sensitivity to MISO was noted in human breast cancer, renal cancer, non small-cell lung cancer, and adenocarcinoma of unknown primary site. A dose response relationship was demonstrated in a subset of experiments including 6 patient's tumors and one human breast cancer cell-line. An analysis relating MISO sensitivity or resistance to the results obtained with other, simultaneously tested standard anticancer drugs indicated that tumors exhibiting a less than or equal to 50% decrease of TCFU's in the presence of MISO were also likely to be sensitive to other cytotoxic drugs. In summary, our data suggest that the 'nitroimidazoles' may exert clinically significant direct antitumor effects in individual tumors. The human tumor cloning assay may have potential to evaluate these direct effects of MISO-analogues and other new radiosensitizers currently being tested in clinical trials.

Selection of chemotherapy for patient treatment utilizing a radiometric versus a cloning system.

From the 1950s to the 1970s, a number of in vitro systems that measured inhibition of glucose metabolism were used to predict the responsiveness of patients' tumors to chemotherapy. In vitro-in vivo correlations were excellent, with true positive predictions ranging from 68% to 96% and true negative predictions of 95% to 100%. The radiometric system is a new in vitro technique that measures the conversion of 14C-glucose to 14CO2. The system already has been utilized to screen prospective new antineoplastic agents for cytotoxicity. The present study was undertaken to determine if the radiometric system might be used to predict correctly the responsiveness of an individual patient's tumor to single-agent or combination-agent chemotherapy. Fifty-six tumor specimens were divided and tested for drug sensitivity in the radiometric system and a conventional human tumor clonning system. Overall, there was a significant correlation between in vitro and in vivo results for the conventional cloning system (P = 0.03). However, there was no significant relationship between in vitro and in vivo results for the radiometric system. The radiometric system consistently failed to predict the tumor's clinical sensitivity to single agents. A radiometric system is not useful in predicting the responsiveness of a patient's tumor to single agent chemotherapy and is not a replacement for the more biologically attractive human tumor cloning system.

Use of a radiometric system to screen for antineoplastic agents: correlation with a human tumor cloning system.

A rapid, semiautomated radiometric system is described for screening for new antineoplastic agents. This radiometric system utilizes inhibition of conversion of [14C]glucose to 14CO2 as an index of cytotoxicity. In this study the radiometric system (BACTEC 460) was first optimized using a variety of human and animal tumor cell lines. Overall, there was a clear linear relationship between the number of cells seeded and the production of 14CO2 from [14C]glucose. The BACTEC System easily detected antitumor activity of compounds from all four classes of antineoplastic agents (doxorubicin, vinblastine, cis-platinum, and methotrexate.) Human tumor cell lines were used to compare the antitumor activity of the same four agents measured by the BACTEC System versus the antitumor activity of the same agents measured by a conventional cloning system. For all cell lines tested there was good agreement in comparison of percentage of survival measured by the BACTEC System versus the standard cloning system. This agreement was better for a continuous exposure to drug in both systems (r = 87, P = less than 0.001) than for a 1-h exposure to the drug (r = 0.35, P = 0.036). In addition to determining the effect of drugs on tumor cells, the BACTEC System was successfully utilized to determine the cytotoxic effect on normal bone marrow buffy coat cells. By utilizing a comparison of suppression of 14CO2 production by normal versus tumor cells, a measurement of differential cytotoxicity could be made. Based on these findings, the radiometric BACTEC System represents a simple and rapid method to detect cytostatic or cytocidal activity of new compounds. It is ideal for use as a prescreen for testing a large number of new chemical entities against a large number of human tumor cell lines.

Direct cloning of human malignant melanoma.

An in vitro soft agar technique was used to culture human malignant melanoma cells from 61 solid tumors, 17 lymph nodes, 11 effusions, and four bone marrow specimens from 93 patients with malignant melanoma. Colonies grew in soft agar from 64 (69%) of the 93 specimens. Fifty-five percent of the specimens cultured formed greater than or equal to 30 colonies per 500,000 nucleated cells plated. Light microscopy, electron microscopy, tumor marker, and athymic nude mouse studies provided evidence the colonies were composed of malignant melanoma cells. Drug sensitivity studies utilizing the cloning technique showed similarities between in vitro results and the general clinical experience noted with the same drugs. The human tumor cloning system represents a new model for future basic biology and clinical studies of human malignant melanoma.

Activity of mitoxantrone in a human tumor cloning system.

We have utilized a recently developed human tumor cloning system to screen for antitumor effects in vitro of a new anthracenedione derivative, Mitoxantrone. The object was to determine if the system is useful for pinpointing the types of tumors in patients which should be studied in early Phase II clinical trials. Tumors from 267 patients were placed in culture (20 different histological tumor types). One hundred seventy tumors both grew and formed enough colonies for drug sensitivity assays. Excellent in vitro antitumor activity was noted for Mitoxantrone against human adenocarcinoma of the lung, small cell lung cancer, melanoma, and biliary tree cancer. Good antitumor activity was noted against breast cancer, ovarian cancer, non-Hodgkin's lymphoma, head and neck cancer, squamous cell lung cancer, soft tissue sarcoma, gastric cancer, and hepatomas. The drug showed no in vitro activity against colon cancer. These data indicate that Mitoxantrone has a wide spectrum of in vitro antitumor activity. A comparison of these in vitro results with the results of Phase II clinical trials with the drug should allow an evaluation of the utility of the human tumor cloning system for predicting clinical antitumor activity of a new compound.