Lynne M. Ball

PCNA Ki-67 dissociation in childhood acute lymphoblastic leukaemia. An Immunofluorescent Laser Confocal Scanning Microscopical study.

Cell proliferation rates of diagnostic marrow aspirate cells of 21 children with Acute Lymphoblastic Leukaemia and 16 controls were compared using immunocytochemical labelling of PCNA and Ki‐67 antigen as assessed by Confocal Laser Scanning Microscopy.

Demonstration of Differences in Drug Resistance by Direct Testing of DNA Excision Repair Activity Following Standard and Liposomal Daunorubicin Exposure in Normal Paediatric Marrow Using High Resolution CLSM

BACKGROUND High resolution Confocal Laser Scanning Microscopy (CLSM) may be applied to testing of drug resistance in vitro in clinical setting. Rapid analysis of DNA damage by precise quantitation of excised DNA in bone marrow samples exposed to potential treatment moieties directly after isolation but the relative sensitivity of the integrated method is as yet untested. AIMS To test the clinical applicability of SCGE/high resolution CLSM for differences in drug resistance in marrow cells. METHODS Cells from normal bone marrow samples were exposed for identical periods and at 4 concentrations to either 1 hour of standard Daunorubicin (.5, 1, 1.5, 2 micrograms/ml) or 8 hours DaunoXome (courtesy of NeXstar Inc, USA) (.05, .1, .15, .2 microgram/ml). After 2 and 6 hours recovery, cells were harvested for SCGE, randomization, analysis of tail length, total excised DNA and fragment size distribution using high resolution CLSM. RESULTS Tail length and fragment size distribution was not, but total excised DNA was significantly increased after 0.1 microgram/ml Liposomal Daunorubicin (DaunoXome) compared to 1.0 microgram/ml Daunorubicin. CONCLUSION SCGE/high resolution CLSM effectively demonstrated differences in Daunorubicin resistance of human marrow cells to alternative formulations. The method has potential for use in clinical testing of neoplastic cell drug resistance.

Microsatellite Instability Assessment in Prediction of Drug Resistance in Childhood Burkitt’s and Large Cell Diffuse Malignant Non-Hodgkin Lymphoma (MNHL)

BACKGROUND Genomic instability may, especially with DNA directed treatment, be associated with increased therapeutic response; absence may be associated with drug resistance. In childhood MNHL, drug response is variable. At present the degree of presence of microsatellite variation, i.e., intrinsic DNA instability is not known. AIMS To determine presence and range of microsatellite variability in common childhood MNHL. METHODS 1.3.1. Study Populations. Consecutive, unselected (1976-96) cases of childhood Large Cell diffuse, N = 16; (9T,7B), age range 1y5m-16y8m; Burkitts Lymphoma, n = 13, age range 4y2m-14y. Non-malignant/pre-treatment tissue of 20 cases, 13 LC, 7 Burkitts MNHL. 1.3.2. Molecular Pathology. Routine DNA extraction, amplifications at loci D3S 1304 and D3S1537 (both closely distal to VHL, tumour suppressor gene); ELN gene D7S1870; IFNA D1S243 (1p36) which show microsatellite variation. Isotopic labelling in amplification, non-denaturing gel electrophoresis, autoradiography. RESULTS Microsatellite variability was found 3/16 LC and 2/13 Burkitts MNHL. LC MNHL, 4 abnormal areas: n = 1, 3 abnormal areas: n = 1, 2 abnormal areas n = 1; Burkitts MNHL, 3 abnormal areas: n = 1, 1 abnormal area n = 1. No variability was found in the normal (constitutional) DNA of any of the 20 patients studied. CONCLUSIONS Microsatellite variability occurred in 5/29 patients with common types of childhood MNHL, indicating a limited contribution to reduced drug resistance through this mechanism.

Differential kinetics of drug resistance in human leukaemic cells measured by SCGE/CLSM.

BACKGROUND New analogues of DNA directed chemotherapy moieties are available for comparative efficacy testing in human neoplastic disease. In addition to MTT testing direct assessment of DNA excision repair activity after direct exposure of marrow cells may provide information on relative DNA effects in vitro. AIMS To assess the ability of SCGE/high resolution CLSM to detect differences in drug resistance between human neoplastic cell lines in the DNA excision repair response to chemotherapy. METHODS Eight human leukaemia samples (4 childhood, 4 adult) were exposed to 1 hour of single concentrations of daunorubicin, DaunoXome (courtesy NeXstar Pharmaceuticals Inc, USA), cyclophosphamide and 4-hydroperoxycyclophosphamide (4-HC, courtesy Dr. M. Colvin, Duke University, USA), followed by SCGE/high resolution CLSM with quantitation of total excised DNA. Differences between cases/drug moieties/exposures were analysed. RESULTS Although generally equal effect dose levels for DaunoXome were lower than for standard daunorubicin, patients/individual neoplastic cells differed considerably in optimal dose levels. Conventional cyclophosphamide in comparison to 4-HC showed inconsistent results indicating considerable differences in the level of drug resistance to the conventional product. CONCLUSIONS Direct testing for drug resistance patterns in DNA directed drug moieties by SCGE/CLSM reveals individual variability of human malignant cell lines warranting comparison with results of MTT testing and in-vivo patient response.

HIGH RESOLUTION CONFOCAL LASER SCANNING MICROSCOPY ANALYSIS OF DNA EXCISION REPAIR CAPABILITY IN SMALL VOLUME MARROW SAMPLES EXPOSED TO DNA DIRECTED TREATMENT MOIETIES

BACKGROUND Assessment of resistance to drug moieties in tissue culture is complicated by limited sample, clonal selection and alteration of cycling fraction and cycle duration in clonally mixed lesions. DNA damage assessment by single cell gel electrophoresis (SCGE) of excised DNA is limited by non-linear analysis in fluorescent light microscopy. Confocal Laser Scanning Microscopy (CLSM) with high N.A. magnification allows for quantitation of total excised DNA fragment size distribution but is still limited by the large volume required for labour intensive SCGE, precluding multi-exposure clinical testing. AIMS To optimise sample requirement for SCGE and CLS. METHODS Standard slide mounted bed gels were punched with multiple coded 6 mm wells and filled with suspensions of cells subjected to drug/concentration variations. After SCGE, 30 microns frozen sections were prepared of each well and mounted in ethidium bromide solution on multi-well hydrophilic slides to allow for short working distance of high resolution CLSM in a Zeiss Axiovert L410 SM. Testing for feasibility, reproducibility and consistency used both cultured standard leukaemic cell lines, normal human control marrow and clinical samples. RESULTS AND CONCLUSION Multiple well SCGE followed by frozen section, high resolution CLSM allows for rapid analysis of high numbers of multiple drug exposure permutations clinically required.