Therapeutic synergism of hyperthermia-cis-platinum in a mouse tumor model.

A small-animal model was developed as a guide to whole-body hyperthermia in cancer patiens. Anesthetized DBA/2 mice were secured to a platform, and their hindlimbs were immersed in a 42.3 degrees C water bath for 30-60 minutes. Hindlimb hyperthermia reulted in steady-state rectal and femoral bone marrow and muscle temperatures of 42 degrees C and upper extremity muscle and esophagus temperatures of 41 degrees C. With this hyper thermia technique, the mouse spleen colony assay could be used to quantitate the lethality of hyperthermia and/or cis-dichloro-diammineplatinum(II) (cis-platinum) on clonogenic bone marrow and leukemia cells. Hyperthermia prior to cis-platinum administration increased cis-platinum inhibition of leukemia colony formation as much as 2 logs; however, antileukemia synergism ws greatest when cis-platinum administration immediately preceded hyperthermia and no evidence existd of synergism against normal bone marrow colonies. Correlative in vivo drug uptake studies showed a marked increase in leukemia cell uptake of 195mPt-cis-platinum at elevated temperatures, which suggested a potential mechanism for the apparent antileukemia synergism of cis-platinum and heat.

Enhancement of regression of cervical intraepithelial neoplasia II (moderate dysplasia) with topically applied all-trans-retinoic acid: a randomized trial.

BACKGROUND: Retinoids enhance differentiation of most epithelial tissues. Epidemiologic studies have shown an inverse relationship between dietary intake or serum levels of vitamin A and the development of cervical dysplasia and/or cervical cancer. Pilot and phase I investigations demonstrated the feasibility of the local delivery of all-trans-retinoic acid (RA) to the cervix using a collagen sponge insert and cervical cap. A phase II trial produced a clinical complete response rate of 50%. PURPOSE: This randomized phase III trial was designed to determine whether topically applied RA reversed moderate cervical intraepithelial neoplasia (CIN) II or severe CIN. METHODS: Analyses were based on 301 women with CIN (moderate dysplasia, 151 women; severe dysplasia, 150 women), evaluated by serial colposcopy, Papanicolaou cytology, and cervical biopsy. Cervical caps with sponges containing either 1.0 mL of 0.372% beta-trans-RA or a placebo were inserted daily for 4 days when women entered the trial, and for 2 days at months 3 and 6. Patients receiving treatment and those receiving placebo were similar with respect to age, ethnicity, birth-control methods, histologic features of the endocervical biopsy specimen and koilocytotic atypia, and percentage of involvement of the cervix at study. Treatment effects were compared using Fisher's exact test and logistic regression methods. Side effects were recorded, and differences were compared using Fisher's exact test. RESULTS: RA increased the complete histologic regression rate of CIN II from 27% in the placebo group to 43% in the retinoic acid treatment group (P = .041). No treatment difference between the two arms was evident in the severe dysplasia group. More vaginal and vulvar side effects were seen in the patients receiving RA, but these effects were mild and reversible. CONCLUSIONS: A short course of locally applied RA can reverse CIN II, but not more advanced dysplasia, with acceptable local side effects. IMPLICATIONS: A derivative of vitamin A can reverse or suppress an epithelial preneoplasia, lending further support to the notion that chemoprevention of human cancer is feasible.

Quantitation of the number of cells within tumor colonies in semisolid medium and their growth as oblate spheroids.

The number of cells within tumor colonies has not been determined accurately in prior reports because, in all but small clusters, cells grow too closely and stacked to allow direct counting of cells by inverted microscopy. Therefore, we stained colonies formed in agar from 38 tumor cell samples of diverse histological origin, removed them with a micropipet, and directly counted the number of cells. The number of cells within colonies increased geometrically with colony diameter and inversely with the size of the cells within the colonies. The relationship can be described using linear regression: [In(no. of cells/colony) = 0.87 - 2.80 In (colony cell diameter) + 2.38 In (colony diameter)] which gave an R2 of 0.92. Measurements of colonies in agar showed that they grew as oblate spheroids rather than spheroids. In an average sample, 60-micron-diameter colonies contained eight to 10 cells; the range for the 38 samples was 1.2 to 48.5. These results precisely define colonies in terms of cell number. They allow calculation of the total number of cells formed from clonogenic cells, a more complete estimate of proliferation than conventional cloning efficiencies which only measure initial proliferation. Furthermore, because of the dependence on the size of the colony cells, if colonies are defined only by a specific diameter then they do not contain similar numbers of cells. Calculations which assume spherical colonies, rather than the oblate spheroid shape we found, greatly overestimate the number of cells within colonies. Our data can increase the accuracy of quantitation in the clonogenic system and thus improve the interpretation of the proliferation of clonogenic tumor cells.

Similar self-renewal properties for different sizes of human primary melanoma colonies replated in agar.

Clonogenic assays currently define colonies as multicellular growth units above an arbitrarily designated cutoff size rather than by the biological function of different-sized growth units. To define the cutoff size between clusters and colonies in terms of the biological function of the cells within the growth units, we directly measured the self-renewal and proliferative capacity of cells from different-sized melanoma colonies. Primary colonies formed from cells of two patients were removed, pooled according to size, and replated, and the frequency and size distribution of the secondary colonies were analyzed. Cells from primary melanoma colonies that resulted from four to eight population doublings had similar extensive proliferative and self-renewal characteristics. The results demonstrated that self-renewal was not limited to cells in large colonies and suggested that the cutoff may be below 16 cells/growth unit. These data support the use of relatively small multicellular growth units to define colonies and measure highly proliferative human melanoma tumor cells. In addition, these methods may allow the determination of the cutoff size for other tumor types in terms of the biological function of cells rather than arbitrarily designating a cutoff size.

Correlation between amounts of cellular membrane components and sensitivity to hyperthermia in a variety of mammalian cell lines in culture.

The weight ratio of either cholesterol or phospholipid to protein contents in 7 different cell lines, growing exponentially at 37 degrees, correlates positively with increasing resistance of the cells to subsequent hyperthermic cell killing. The relative heat resistance of each cell line is derived from survival curves obtained when the different cell lines are exposed to 43 degrees. Cholesterol and phospholipid amounts in the particulate fraction correlate with survival sensitivity to 43 degrees when the values are expressed per mg protein but not when expressed per cell number. Also, cholesterol:phospholipid molar ratios and the amount of protein in the particulate fraction do not display linear correlations with sensitivity of the respective cell lines to 43 degrees-induced cell killing. The relative degree of fatty acid saturation at 37 degrees also is independent of whether cells show a higher degree of heat resistance. These data suggest that lipid (both cholesterol and phospholipid):protein weight ratios correlate with increasing resistance of cells to an elevation in temperature. The major implication of these data is that major membrane components can influence and perhaps predict cellular survival to hyperthermia.

Beyond tamoxifen: the retinoid X receptor-selective ligand LGD1069 (TARGRETIN) causes complete regression of mammary carcinoma.

Recently, we reported that LGD1069, a high-affinity ligand for the retinoid X receptors (RXRs), was shown to have an efficacy equivalent to that of tamoxifen (TAM) as a chemopreventive agent in the N-nitroso-N-methylurea-induced rat mammary carcinoma model. Furthermore, LGD1069 was very well tolerated during 13 weeks of chronic therapy with no classic signs of "retinoid-associated" toxicities. Due to the high efficacy and benign profile of this RXR agonist as a suppressor of carcinogenesis, we examined its role as a therapeutic agent on established mammary carcinomas. In the rat mammary carcinoma model, N-nitroso-N-methylurea was used to induce tumors, and the tumors were allowed to grow to an established size prior to initiation of treatment. LGD1069-treated animals showed complete regression in 72% of treated tumors and had a reduced tumor load compared to control. In addition, the combination of LGD1069 and TAM showed increased efficacy over either agent alone. Histopathological analysis showed a reduction of LGD1069-treated tumor malignancy, an increase in differentiation, and a sharp decrease in cellular proliferation compared to vehicle-treated control tumors. These data demonstrate that the RXR-selective ligand LGD1069 is a highly efficacious therapeutic agent for mammary carcinoma and enhances the activity of TAM.

In vitro chemosensitivities of human tumor stem cells to the Phase II drug 4'-(9-acridinylamino)methanesulfon-m-anisidide and prospective in vivo correlations.

A potential application of the human tumor stem cell colony assay is to guide Phase II clinical investigations by identifying classes of tumors (or individual patients) which are sensitive in vitro to a new antitumor compound. We have tested human tumor stem cells from 140 tumor biopsies representing 20 different tumor types for chemosensitivity to the Phase II drug 4'-(9-acridinylamino)methanesulfon-m-anisidide. In vitro sensitivity was defined as a reduction in the number of tumor colony-forming cells to 30% of the control or less after a 1-hr exposure to one-tenth of the pharmacologically achievable plasma concentration of 4'-(9-acridinylamino)methanesulfon-m-anisidide. In vitro sensitivity was found in 29 cases: non-Hodgkin's lymphoma (2 of 2); cervical carcinoma (1 of 1); sarcoma (3 of 6); neuroblastoma (1 of 2); acute myelogenous leukemia (6 of 16); chronic myelogenous leukemia (1 of 3); melanoma (8 of 34); uterine carcinoma (1 of 5); lung carcinoma (1 of 9); ovarian carcinoma (4 of 36); and breast carcinoma (1 of 11). Prospective in vitro-in vivo correlations in eight patients with various tumor types showed that three of three patients sensitive in vitro to 4'-(9-acridinylamino)methanesulfon-m-anisidide responded in vivo, while five of five patients resistant in vitro had no clinical response. The results provide support for further evaluation of the utility of the human tumor stem cell colony assay for targeting Phase II clinical trials.

Relation of in vitro colony survival to clinical response in a prospective trial of single-agent chemotherapy for metastatic melanoma.

We have used the effect of therapeutic agents on clonogenic growth in agar to discriminate between active and inactive agents for malignant melanoma. We report a prospective study of single-agent chemotherapy for metastatic melanoma. Forty-five separate in vitro/in vivo correlative trials were conducted in 34 patients. A number of agents were used in these evaluations, including actinomycin D, Amsacrine, bisantrene, mitoxantrone, BCNU, vinblastine, vindesine, 5-fluorouracil, MGBG, etoposide, interferon, tamoxifen, and 13-cis-retinoic acid. At the "cut-off" concentration, a colony survival less than 30% was designated as "sensitivity" and greater than 30% as "resistance." Clinical sensitivity was designated to include complete, partial, and mixed responses and was predicted in eight of 18 trials (44%). Clinical resistance (nonresponse) was predicted correctly in 24 of 27 cases (89%). Using Fisher's exact test the association of in vitro and in vivo results was significant (P = .05). These results offer further support for the concept that clonogenic assays may help select useful agents for clinical trials in metastatic melanoma.

Double-blind crossover study of the antiemetic efficacy of high-dose dexamethasone versus high-dose metoclopramide.

Nausea and vomiting remain common and debilitating side effects of therapy with many anticancer drugs. Recent reports have shown that both metoclopramide and dexamethasone are effective drugs for the treatment of severe nausea and vomiting caused by cis-platinum. A double-blind crossover study comparing the antiemetic properties of high-dose oral and intravenous regimens of metoclopramide and dexamethasone in outpatients was carried out. Standardized patient questionnaires and interviews were used to evaluate response. Dexamethasone and metoclopramide protected against more than five episodes of emesis in 48% and 40% of patients, respectively. Nausea persisted for less than six hours in 45% of patients on dexamethasone and in 37% on metoclopramide. The antiemetic efficacy of both regimens was retained through repeated courses of chemotherapy. Side effects were minimal with dexamethasone; however, 33% of patients experienced unacceptable extrapyramidal side effects to metoclopramide. Patient preference was significantly in favor of dexamethasone: 70% of patients chose to continue dexamethasone compared to 22% who preferred metoclopramide and 8% who chose other antiemetics. Dexamethasone was the preferred antiemetic in this patient population due to minimal side effects.

Prognostic factors for survival of patients treated systemically for disseminated melanoma.

PURPOSE: The current American Joint Commission on Cancer (AJCC) staging system distinguishes between soft tissue and visceral metastases in advanced (stage IV) melanoma. We sought to verify these staging criteria and to identify prognostic variables that could be used to evaluate the impact of systemic therapy on long-term survival during the prior decade. PATIENTS AND METHODS: We conducted a retrospective study of patients with advanced cutaneous melanoma enrolled in clinical trials between 1979 and 1989 at The University of Texas M.D. Anderson Cancer Center. Pretreatment age, sex, number of organs with metastases, serum levels of lactate dehydrogenase (LDH) and albumin, and period of enrollment were analyzed using a Cox proportional hazards model of survival. RESULTS: In univariate and multivariate analyses that involved 318 stage IV patients, normal serum levels of LDH and albumin, soft tissue and/or single visceral organ metastases (especially lung), female sex, and enrollment late in the decade were independent positive predictors for survival. In multivariate analyses, the current AJCC criteria did not significantly predict outcome. Systemic treatment response did not bias these results, and only 4% of patients had a complete response. Patients who lived more than 2 years (11%) had a mix of favorable prognostic characteristics and a high frequency of systemic or surgically induced complete response. CONCLUSION: This study supports the use of stratification parameters that reflect the favorable prognostic impact of soft tissue or single visceral organ metastases and normal serum levels of LDH and albumin at time of enrollment in advanced melanoma trials. Improved survival over the prior decade probably reflects advances in diagnostic and palliative interventions.

Factors predicting for response and survival in adults with advanced non-Hodgkin's lymphoma.

Knowledge of the prognostic factors that characterize a disease can assist in planning and analyzing clinical trials. The present study was conducted to determine the characteristics related to response and survival in patients with stage III and IV non-Hodgkin's lymphoma who were treated with combinations of cyclophosphamide, vincristine sulfate, and prednisone. Considering each characteristic individually and using stepwise regression analysis, tumor bulkiness, prior therapy, sex, and pretreatment lymphocyte count were selected as the four most important prognostic variables. Tumor architecture (diffuse or nodular pattern) and cell type, hemoglobin level, and symptoms although not important in predicting response were found to be important in predicting survival. The hemoglobin level had only marginal importance in predicting response. Factors found not be important were age, stage, symptoms, cell type, nodularity, marrow involvement, prior extensive radiotherapy, and bone involvement. A logistic regression equation has been derived that can be used to predict response rate.

Oral melphalan kinetics.

The systemic availability of melphalan after oral administration is not well known. Most patients are put on a fixed oral dosage regimen. We have studied the disposition of melphalan in 14 patients after single oral doses. Five were also studied after receiving the same dose intravenously. Oral melphalan had a mean plasma terminal phase half-life (t1/2) of 90 +/- 17 min. The mean area under the plasma concentration:time curve (CXT) was 53 +/- 33 micrograms . min/ml. Urinary excretion of oral melphalan averaged 10.9 +/- 4.9% during the first 24 hr. The CXT ratio (oral:intravenous) for the 5 patients studied after both oral and intravenous melphalal (0.6 mg/kg) ranged between 0.25 and 0.89 and averaged 0.56. After oral dosing in 14 fasting patients, the time at which melphalan first appeared in the plasma varied between 15 min and 6 hr. In a myeloma patient who took oral melphalan, no melphalan was found in plasma or urine up to 24 hr. Some instances of failure of tumor response to oral melphalan may be due to inadequate bioavailability rather than inherent tumor resistance.

Kinetics of intravenous melphalan.

We have studied the disposition and elimination of melphalan after intravenous administration in 9 patients with cancer. High-pressure liquid chromatography and 14C-melphalan were used to assay drug concentration in plasma and urine. Composite plasma t1/2alpha was 7.7 +/- 3.3 and t1/2beta was 108 +/- 20.8 min for 8 of the patients. The mean 24-hr urinary excretion of melphalan was 13.0 +/- 5.4% of the administered dose. In 2 patients, 80% to 100% of the measured 14C counts in plasma and urine samples at each study interval, up to 24 hr after drug administration, could be accounted for by the sum of parent compound, monohydroxy and dihydroxy products, and methanol nonextractable radioactivity (i.e., protein-bound activity). These data and evidence of rapid disappearance from plasma at 37 degrees in vitro suggest that spontaneous degradation, and not enzymatic metabolism, is the major determinant of the t1/2 of melphalan in vivo.

Effects of long-term intake of retinol on selected clinical and laboratory indexes.

BACKGROUND: Chemopreventive agents developed to be used in a moderate-risk but otherwise healthy population need to be both efficacious and to have minimal adverse effects. OBJECTIVE: The objective of this study was to evaluate the adverse effects of long-term retinol intake in a skin cancer chemoprevention trial in a large population at moderate risk for skin cancer. DESIGN: Participants (n = 2297) were randomly assigned to receive retinol [7576 retinol equivalents (RE), or 25000 IU] or a placebo daily. The adverse effects of retinol intake were studied by monitoring 14 clinical symptoms and laboratory indexes. The median follow-up time was 3.8 y. RESULTS: No adverse effects concerning the 14 symptoms were observed. Significant differences in alkaline phosphatase (P < 0.0001), triacylglycerol (P < 0.0001), cholesterol (P = 0.04), and HDL (P = 0.01) were observed over time between the 2 groups. After 49 mo of follow-up, alkaline phosphatase was 7% higher, triacylglycerol was 11% higher, cholesterol was 3% higher, and HDL was 1% lower in the retinol group than in the placebo group. CONCLUSIONS: Because a 1% increase in cholesterol concentrations has been reported to be associated with a 2% increase in coronary artery disease risk, long-term ingestion of 7576 RE vitamin A/d should be considered with caution. However, further studies are needed to confirm this finding.

Predictors of inactivation and reasons for participant inactivation during a skin cancer chemoprevention study.

Maintaining good compliance is a major challenge in long-term cancer chemoprevention trials. Minimizing the number of inactive participants during a trial is an important factor in maximizing compliance. Identifying reasons for and predictors of inactivation is the first step in being able to reduce participant inactivation. In this skin cancer chemoprevention trial, the 2,297 participants were randomized to receive 25,000 IU of retinol daily or a placebo. Median follow-up time was 3.8 years. The reason for inactivation was determined for each participant who stopped taking the study capsules. Six hundred and seventy-seven (29.7%) participants became inactive during the 5-year study. There was no significant difference between the number of participants inactivating by treatment group or sex. The most common reasons for inactivation were illness of subject, spouse, or a close relative (18.6%) and experience of a clinical symptom consistent with vitamin A ingestion (17.1%). Participants in the vitamin A group (10.1%) more frequently cited symptoms coded as "not consistent with vitamin A" as the reason for inactivation compared with those in the placebo group [5.4% (P < 0.05)]. The inactivation rate was highest in the first month of the trial and declined thereafter. A low education level (hazard ratio, 1.59) and unmarried status (hazard ratio, 1.29) were the only significant predictors of inactivation. These findings may be useful in developing targeted strategies to decrease inactivation and thereby increase compliance in future chemoprevention trials. However, these findings need to be confirmed because published research in this area is very limited.

Design and recruitment for retinoid skin cancer prevention (SKICAP) trials. The Southwest Skin Cancer Prevention Study Group.

The retinoid skin cancer prevention (SKICAP) trials are a set of double-blind, randomized, placebo-controlled clinical trials. The SKICAP-actinic keratoses (AK) trial tests the hypothesis that daily supplementation of retinol (25,000 IU) for 5 years reduces the incidence of skin cancers in high-risk individuals, those with a history of greater than ten clinically or pathologically diagnosed AK and, at most, one prior pathologically confirmed cutaneous squamous cell carcinoma (SCC) or basal cell carcinoma (BCC). The SKICAP-SCC/BCC (S/B) trial tests the hypothesis that daily supplementation of retinol (25,000 IU) or 13-cis-retinoic acid (5 or 10 mg) for 3 years reduces skin cancer incidence in very high-risk individuals, those with a history of at least four pathologically confirmed SCCs or BCCs. Between 1984 and 1988, 2800 participants were enrolled at two clinics on the SKICAP-AK trial; and between 1985 and 1990, a total of 719 participants were enrolled at four clinics on the SKICAP-S/B trial. The initial recruitment strategy was referral by dermatologists, but low accrual necessitated the use of other strategies to achieve enrollment goals, which included involving additional clinics and using paid trial-specific advertisements in print and electronic media. Thirteen % of the SKICAP-AK participants and 36% of the SKICAP-S/B participants were enrolled through dermatologist referral, whereas paid advertisements resulted in enrollment of 87% of SKICAP-AK and 43% of SKICAP-S/B participants. A population-based skin cancer registry was used to identify and enroll the remaining 21% of the SKICAP-S/B participants.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of retinol in preventing squamous cell skin cancer in moderate-risk subjects: a randomized, double-blind, controlled trial. Southwest Skin Cancer Prevention Study Group.

We conducted a randomized, double-blind, controlled trial to examine the efficacy of retinol supplementation on the incidence of first new nonmelanoma skin cancer in moderate-risk subjects. A total of 2297 free-living subjects were enrolled; subjects resided in Arizona (median age, 63 years) and had a history of more than 10 actinic keratoses and at most 2 squamous cell carcinoma (SCC) or basal cell carcinoma (BCC) skin cancers. Subjects were randomly assigned to receive oral retinol (25,000 IU) or placebo supplementation daily for up to 5 years. The primary end points for the trial were time to first new SCC or BCC. During a median follow-up time of 3.8 years, we found that 526 subjects had a first new skin cancer. Comparing retinol-supplemented subjects with placebo-supplemented subjects showed a hazard ratio for first new SCC of 0.74 (95% confidence interval, 0.56-0.99; P = 0.04). The hazard ratio of first new BCC for the retinol-supplemented subjects compared with those receiving placebo was 1.06 (95% confidence interval, 0.86-1.32; P = 0.36). Potentially adverse symptoms that were judged to be associated with retinol were rare (approximately 1% higher in the retinol group than in the control group). Therefore, we concluded that daily supplementation with 25,000 IU of retinol was effective in preventing SCC, although it did not prevent BCC.

Trial of retinol and isotretinoin in skin cancer prevention: a randomized, double-blind, controlled trial. Southwest Skin Cancer Prevention Study Group.

The objective of this study was to examine the effect of retinol and isotretinoin on the incidence of nonmelanoma skin cancer in high-risk subjects. A total of 525 participants with a history of at least four basal cell carcinomas (BCCs) and/or cutaneous squamous cell carcinomas (SCCs) were entered into a randomized, double-blind, placebo-controlled trial, performed in free-standing study clinics. Participants were randomly assigned to receive oral retinol (25,000 units), isotretinoin (5-10 mg), or placebo supplementation daily for 3 years. The time to first new occurrence of BCC or cutaneous SCC was used as the outcome measure. During the study period, 319 BCCs and 125 cutaneous SCCs were diagnosed clinically and pathologically. There were no differences between those who received retinol, isotretinoin, or the placebo, with regard to the time to first occurrence or to the total number of tumors noted. No beneficial effects were noted with regard to the prevention of nonmelanoma skin cancer with either retinol or isotretinoin.

Retinoids in prevention of skin cancer.

Two chemoprevention randomized clinical trials were begun in 1984 to evaluate retinoids in the prevention of skin cancers. Moderate risk subjects with a history of at least 10 actinic keratoses and at most two prior skin cancers were enrolled in the SKICAP-AK trial and randomized to 25,000 IU retinol or placebo daily for 5 years. High risk subjects with a history of at least four prior skin cancers were enrolled in the SKICAP-S/B trial and randomized to receive 25,000 IU retinol, 5-10 mg isotretinoin or placebo daily for 3 years. Data from the SKICAP-AK trial indicate that retinol reduces incidence of first new squamous cell skin cancers but had no effect on the incidence of first new basal cell skin cancer. The effect of retinoids had no significant benefit on squamous or basal cell skin cancers in the high risk subjects on the SKICAP-S/B trial, although intervention duration was less than planned. Daily retinol was effective in preventing squamous cell cancers in moderate risk subjects.

Comparison of two physical activity questionnaires, with a diary, for assessing physical activity in an elderly population.

Measurement of physical activity in epidemiological studies is usually achieved by means of a questionnaire. Little work has been done to determine which questionnaire format has greater validity in an elderly population. In this study of elderly subjects, physical activity as reported in two self-administered questionnaires (A and B), which differed in format and length, were compared to activity reported in a 4 day diary. As compared with the diary, moderate/heavy activity was more accurately reported in Questionnaire A (mean difference 5 min), the longer more detailed questionnaire, than B (mean difference 170 min). Light activity was under reported in Questionnaire A (mean difference 68 min) and over reported in B (mean difference 88 min) as compared with the diary. In contrast, time spent sitting was more accurately reported in Questionnaire B (mean difference 40 min) than in A (mean difference 230 min) as compared with the diary. The longer more detailed questionnaire was the more accurate instrument for assessing moderate/heavy activity in this elderly population. The shorter questionnaire was more accurate for assessing time spent sitting.