Development and Validation of a Risk-Adapted Scoring Model for Metachronous Upper Tract Urothelial Carcinoma following Radical Cystectomy.

PURPOSE: The incidence and risk factors for metachronous upper tract urothelial carcinoma (UTUC) following radical cystectomy (RC) remain incompletely defined, which has limited the ability to individualize postoperative surveillance. MATERIALS AND METHODS: A retrospective review of 2 institutional registries was performed to identify patients undergoing RC for urothelial carcinoma. Multivariable Cox proportional hazard models for metachronous post-RC UTUC were developed in one institutional data set and validated in the second institutional data set. A post-RC UTUC risk score was then developed from these models. RESULTS: A total of 3,170 RC patients were included from the training cohort and 959 RC patients from the validation cohort. At a median followup after RC of 4.6 years (IQR 2.1-8.7), 167 patients were diagnosed with UTUC. On multivariable analysis in the training cohort, risk factors for metachronous UTUC were the presence of positive urothelial margin (HR 2.60, p <0.01), history of bacillus Calmette-Guérin treatment prior to RC (HR 2.20, p <0.01), carcinoma in situ at RC (HR 2.01, p <0.01) and pre-RC hydronephrosis (HR 1.48, p=0.04). These factors had similar discriminative capacity in the training and validation cohorts (C-statistic 0.71 and 0.73, respectively). A UTUC risk score was developed with these variables which stratified patients into low (0 points), intermediate (1-3 points), and high risk (4+ points) for post-RC UTUC, with respective 5-year UTUC-free survivals of 99%, 96%, 89% in the training cohort and 98%, 96%, and 91% in the validation cohort. CONCLUSIONS: We developed and validated a risk score for post-RC UTUC that may optimize UTUC surveillance protocols after RC.

A comparative analysis of chromosomal aberrations in cultured human lymphocytes due to fluoroquinolone drugs at different expression periods.

Fluoroquinolones (FQ) are broad-spectrum antibacterial agents widely used for the treatment of infections with various types of gram negative and gram positive bacteria. Specifically, gatifloxacin (GFX) is under development as a component in a new antituberculosis fixed-dose drug combination. In the context of this project, GFX was also tested for genotoxic activity in human peripheral lymphocytes, and the induction of chromosomal aberrations by GFX in PHA-M stimulated cultured human lymphocytes, investigated under conditions of conventional and increased expression times, was further compared to the analogous effects induced by some other second- and third-generation FQ antibacterial agents, namely ofloxacin (OFX), ciprofloxacin (CFX) and sparfloxacin (SFX). OFX did not induce any significant chromosomal aberrations in human lymphocytes. CFX and SFX exhibited slight to moderate clastogenic potential at cytotoxic concentrations (150, 175, 200 and 225 microg/ml), and GFX, a third-generation FQ, induced a clear, concentration-dependent increase in the frequency of chromosomal aberrations at cytotoxic concentrations (150, 200 and 250 microg/ml). These effects were not apparent when metaphases were analysed at the conventionally used sampling time of 24 h, but only after prolongation of the expression time between treatment and harvesting to a sampling time of 36 h (4 h exposure and 32 h expression period). Also, an increased incidence of numerical aberrations (polyploidy and endoreduplication) was seen with GFX at non-cytotoxic concentrations (12.5, 25, 50 and 75 microg/ml). These effects can be attributed to the slight cross-reactivity of FQs between their inhibitory activity towards their intended targets, the prokaryotic type II topoisomerase enzymes DNA gyrase and topoisomerase IV, and the analogous mammalian enzyme topoisomerase II. We have also observed the formation of polycentrics, i.e., chromosomes with five to six centromeres, a rarely reported structural aberration, in GFX-treated cells. The significance of these observations with respect to the conventional conduct of such studies and to the interpretation of the effects is discussed.

Retarding fluoride accumulation in Amaranthes viridis through liming and implications of phosphorous treatment.

With an objective to retard fluoride being taken up by the plants from soil, a study was carried out on Amaranthes viridis. Four groups of treatment were carried out vis-à-vis fluoride alone, fluoride and calcium, fluoride and phosphorous and fluoride, calcium and phosphorous together at three different concentration levels vis-à-vis 1, 10 and 25 mg/kg soil of each. Sampling was carried out first on day 45 and at the end of reproductive phase on leaf and seed for accumulation of fluoride in the plants. It was observed that fluoride accumulation in plants could be averted through soil amendment by calcium treatment in the form of calcium carbonate thereby reducing the risk of human and livestock exposure to abnormal levels of fluoride through food chain other than protecting plants from getting affected. At the same time, fertilizing the soil contaminated with fluoride by superphosphate would aggravate fluoride accumulation and exacerbate fluorosis problem in human and livestock through food chain. Therefore it is recommended to use acid water-soluble orthophosphate or anhydrous dicalcium phosphate or soluble pyrophosphate fertilizers as an alternative.

Low levels of p53 mutations in Indian patients with osteosarcoma and the correlation with fluoride levels in bone.

The pathogenesis of osteogenic sarcoma is not known. Recently, chronic fluoride exposure has been incriminated as having a possible etiologic role by causing a nonspecific osteoblast proliferation. We were interested in exploring the possible relationship between fluoride bone content and p53 mutations. We analyzed p53 mutations in various exons in tissue of osteosarcoma, and correlated the findings with the bone fluoride levels in Indian patients. We analyzed tissue samples from 20 osteosarcoma patients for possible genetic alterations including mutations, and we assessed the extent of fluoride accumulation in bone. Fragments displaying an altered electrophoretic mobility were confirmed as having mutated sequences. Mutation was observed in samples of two cases (10% incidence). Eighteen samples showed bone fluoride levels between 1000 and 27,000 ppm, whereas the 2 mutated samples showed fluoride levels of 64,000 and 89,000 ppm, respectively. The high levels of bone fluoride levels and the similarity of the mechanisms of action between fluoride-induced DNA damage and chemically-induced p53 mutations lead us to propose that high fluoride bone content might have been one of the major factors causing osteosarcoma.

Cytogenetic and molecular genetic studies on Indian patients with chronic lymphocytic leukemia.

This is the first cytogenetic and molecular genetic study to find any specific genetic abnormalities in Indian patients with chronic lymphocytic leukemia (CLL). Cytogenetic studies on 18 patients indicated that their karyotypes were relatively simple and trisomy 12 was seen on karyotype evolution in one patient. Fluorescence in situ hybridization (FISH) revealed abnormal clones of trisomy 12 in nine cases and RB gene deletion in 14 of the 29 cases analyzed. Three patients had both clones. Immunoglobulin genes were rearranged in all the cases and TCR beta in none of the 18 cases Southern blotted. BCL-1 was rearranged in one case. No rearrangement of BCL-2 gene was seen in any case. Genetic changes in Indian CLL were more similar to Western CLL than to Japanese CLL, even though India is supposed to be a low incidence area. Therefore, factors (such as HLA and other genetic markers) other than these routine parameters must be studied to explain the low incidence of CLL in India.

Molecular abnormalities of bcr gene in some Indian patients with chronic myeloid leukaemia.

The DNAs from 7 unselected Philadelphia chromosome-positive (Ph') chronic myeloid leukaemia (CML) patients were analysed for the distribution of breakpoints in the breakpoint cluster region (bcr) gene by Southern blot. Breakpoints were detected between the regions of exons '2' and '4' of the bcr gene in 5 out of 7 patients. In two patients, breakpoints occurred around exon 4 of the bcr gene. This study presents some information on the nature of breakpoint distribution in the bcr gene of Indian CML patients.

Increased sister chromatid exchange (SCE) frequencies in lymphocytes from traffic policemen exposed to automobile exhaust pollution.

Sister chromatid exchange (SCE) frequencies were determined in peripheral lymphocytes from traffic policemen drawn from various busy traffic points of Madras metro in India. These policemen were under constant exposure to automobile exhaust pollution during their 8 h work schedule. Analysis of SCE frequencies revealed a significantly greater number of SCE/cell in these policemen as against the matched control population. Sixty percent of these subjects who remained in the service after 5 years showed insignificant but higher values of SCE than in the initial analysis.

Sister chromatid exchange frequency is elevated and cell proliferation is delayed in bone marrow cells of starved and marginally malnourished rats.

The frequency of sister chromatid exchanges (SCE) and the cell proliferation kinetic were analyzed in bone marrow (BM) cells of Wistar rats subjected to starvation and marginal malnutrition (MN) after 24h of 5-bromodeoxyuridine (BdUr) implantation. SCE were analyzed in a minimum of 18 consecutive second division metaphases and for cell proliferation, 100 consecutive metaphases were analyzed and classified into the first, second and third or subsequent replication cycles. Rats subjected to starvation and MN exhibited significantly higher mean SCE per lymphocyte in bone marrow than the well nourished rats. Further, they also showed a longer proliferation kinetic in BM cells. These observations indicate that starvation and MN per se resulted in greater SCE and prolongation of the cell cycle in experimental animals. On rehabilitation, the cells with high SCE frequency and prolonged cell cycle in both starved and MN rats were comparable to control groups.

Analysis of sister chromatid exchange, micronucleus and chromosomal aberration frequencies in rodents exposed to mosquito coil smoke by inhalation route.

Mosquito coil smoke emitting from a mosquito repellent, was tested for its mutagenic effect in bone marrow cells from mouse and rat after 4 h acute inhalation exposure. Coil smoke with suspended particulate concentrations of 99-129 mg/m3, significantly elevated the frequencies of sister chromatid exchanges in bone marrow cells and micronuclei in polychromatic erythrocytes. Analysis of chromosomal aberrations in metaphases also revealed a significantly higher incidence of chromosomal aberration frequency in exposed rats and mice.

Chromosomal abnormalities in starved and marginally malnourished rats and in utero upon rehabilitation.

The effects of starvation and marginal malnutrition (MN) on the lymphocytes of rats were evaluated by chromosomal analysis before and after rehabilitation. The effect of parental starvation or malnutrition on chromosomal aberrations in the foetus was also studied. Wistar rats, 30-35 days old, were starved for 5 days or fed a minimally restricted or a severely restricted diet for three weeks. At the end of the period of starvation or malnutrition, lymphocytes were isolated and chromosomal analysis was performed. Starved and severely restricted rats showed significantly higher mean chromosomal aberrations than the controls. These aberrations returned to a normal level when the experimental groups were rehabilitated for a month, indicating that the damage was transient. A chromosomal aberration study done on foetal cells from rehabilitated rats which had previously been starved or fed a severely restricted diet showed significantly increased values, indicating that some damage was permanent. A low number of implantations was also recorded in these experimental groups. These observations clearly indicate that young animals exposed to conditions like starvation or chronic malnutrition are prone to permanent damage of the genetic system.

Analysis of karyotype, SCE, and point mutation of RAS oncogene in Indian MDS patients.

Thirty Indian patients diagnosed as having primary myelodysplastic syndrome as per the French-American-British classification were investigated, on admission, for the frequencies of nonrandom karyotype abnormalities, sister chromatid exchange, and point mutations of the RAS oncogene. Successful karyotype analysis was possible in 24 patients, of whom 9 (37.5%) showed nonrandom karyotypic changes. Anomalies of chromosomes 5, 7, and 8 were detected in their bone marrow (BM). In addition, two new anomalies, del(8)(q22) and +19, were observed for the first time in our series. Six MDS patients were studied for SCE in either BM or peripheral blood. These data revealed a normal SCE incidence. Of the 10 MDS patients studied for point mutations of NRAS 12 and 61 and KRAS 12 and 61, one patient exhibited a base substitution at position 1 of the 12th codon of the KRAS gene. These data, gathered for the first time on the Indian patients, throw some light on the nature of genetic changes in MDS of our country.

Lack of genotoxicity of menthol in chromosome aberration and sister chromatid exchange assays using human lymphocytes in vitro.

The possible genotoxic effect of menthol was investigated by analysing the frequencies of sister chromatid exchange (SCE) and chromosomal aberrations in cultured human lymphocytes exposed to menthol. Phytohaemagglutinin-stimulated human lymphocyte cultures grown in the presence of menthol at final concentrations of 0.1, 1 or 10 mm, with or without S-9, had frequencies of polyploid cells and structural chromosomal aberrations similar to those seen in the solvent controls. Furthermore, menthol, either in the presence or absence of S-9, did not alter the SCE frequency in human chromosomes. These results suggest that menthol does not have a chromosomal-damaging effect in human lymphocytes.

Induction of sister chromatid exchanges in NIH 3T3 cells transformed by DNA from mice given cyclophosphamide.

The rationale behind this experiment was to investigate the role of sister chromatid exchange (SCE) in the transformation process in vitro. High molecular weight DNA was extracted from bone marrow cells of mice given 20 mg cyclophosphamide/kg body weight. 30 mug of this DNA was used to transfect NIH 3T3 in Eagle's medium seeded at 0.7 x 10(6) cells/plate. Morphologically visible foci were picked up after 16-21 days. The foci were trypsinized, washed and allowed to grow in the presence of bromodeoxyuridine (25 mum) for 72 hr in the dark. Analysis of SCE per chromosome indicated a 20-fold increase in the frequency of exchanges as compared with the induction seen in negative controls. These data suggest that the transformation process of NIH 3T3 cells by DNA from mice treated with cyclophosphamide is associated with an increased induction of SCEs.

Increased sister chromatid exchange frequency in bone marrow cells of myelodysplastic syndromes.

The frequency and distribution of sister chromatid exchange (SCE) was determined in bone marrow and peripheral lymphocytes of patients with preleukemic myelodysplastic syndromes. Patients with refractory anemia (RA), RA with excess of blasts (REAB), RA with ring sideroblasts, and RA in transformation presented a 2-3-fold increase in SCE frequency in the bone marrow cells. In contrast, lymphocytes from these patients showed only a marginal increase in SCE. Analysis of interchromosomal distribution of SCE indicated a preferential involvement of chromosomes in group C in patients with RA with excess of blasts. Furthermore, the SCE in patients was not found to be influenced by the karyotype status.

Elevated fetal chromosomal damage in malnourished pregnant rats.

Cytogenetic studies in fetal cells from pregnant rats given a protein-restricted diet revealed a significant induction of structural chromosomal aberrations and sister chromatid exchanges.

Defective ultraviolet-induced DNA repair in bone marrow cells and peripheral lymphocytes of patients with refractory anemia with excess of blasts.

The extent of ultraviolet (UV) irradiation-induced DNA repair was measured in bone marrow cells and peripheral lymphocytes of patients with refractory anemia with excess of blasts (RAEB). Bone marrow cells from RAEB, when exposed to a 2 J/m dose of UV, exhibited 50% lower incorporation of tritiated thymidine than those of control subjects. A similar finding was observed in the peripheral lymphocytes. These data suggest that bone marrow cells and peripheral lymphocytes from RAEB are deficient in repair of UV-induced lesions by DNA. Furthermore, this impaired DNA repair efficiency in RAEB was not related to the presence or absence of a karyotype abnormality.

Further studies on sister-chromatid exchange frequency in users of hormonal contraceptives.

Women using an estrogen-progestogen combination contraceptive exhibited an increased frequency of sister-chromatid exchange (SCE), supporting our previous observation. These elevated SCE frequencies tended to decline 3 months after the discontinuation of the pill. Women using a progestogen injectable contraceptive, on the other hand, had unaltered SCE rates. The analysis, in vitro; of mitomycin-C-induced SCE frequencies in women using either the combination or the progestogen injectable contraceptives showed significantly higher rates of induced SCE.

UV-induced chromosome aberrations, sister-chromatid exchanges and cell survival in cultured lymphocytes from malnourished children.

Cultured lymphocytes from children with kwashiorkor and from normal children were examined for their susceptibility to ultraviolet (UV)-induced chromosome aberrations, sister-chromatid exchanges and cell survival. Cells from kwashiorkor exhibited increased chromosome aberrations, but not sister-chromatid exchanges, when exposed to higher doses of UV. Furthermore, when cells from these patients were exposed to higher doses of UV, there was a significant reduction in viability. These results indicate that, as compared to normals, cells from kwashiorkor were more sensitive to the lethal effects of UV.