Margaret Yhap

The impact of prophylactic fresh-frozen plasma and cryoprecipitate on the incidence of central nervous system thrombosis and hemorrhage in children with acute lymphoblastic leukemia receiving asparaginase

Asparaginase (ASP) therapy is associated with depletion of antithrombin (AT) and fibrinogen (FG). Potential toxicities include central nervous system thrombosis (CNST) and hemorrhage. Historical practice at the Izaak Walton Killam Health Centre (IWK) involves measuring AT and FG levels after ASP administration and transfusing fresh-frozen plasma (FFP) or cryoprecipitate (CRY) to prevent thrombotic and hemorrhagic complications. To determine whether this reduced these complications in children with acute lymphoblastic leukemia (ALL), incidence, outcome, and clinical characteristics of ASP-related CNST in ALL patients at IWK were compared with a similar cohort from BC Childrens Hospital (BCCH), where prophylaxis was not performed. Costs associated with preventative versus expectant management were estimated. From 1990 to 2005, 240 patients were treated at IWK and 479 at BCCH. Seven BCCH patients developed venous CNST (1.5%), compared with none at IWK. CNST occurred exclusively during induction. Six patients received anticoagulation and continued ASP. All 7 patients remain in remission. National Cancer Institute high-risk ALL predicted CNST risk (P = .02), whereas sex, age, race, and body mass index did not. Neither FFP nor CRY protected against CNST, suggesting prophylaxis is unwarranted for unselected ALL patients. However, prophylactic replacement for HR patients in induction may be cost-effective.

Osteoporosis at presentation of childhood ALL: management with pamidronate.

Vertebral fractures at diagnosis of childhood acute lymphoblastic leukemia (ALL) are an uncommon but recognized problem. Clinical issues associated with pathological fractures in these children include pain control and the potential for further treatment-associated fractures and long-term bony morbidity. The authors report the successful use of pamidronate in two children who presented with vertebral compression fractures at diagnosis of ALL. Both patients had pain and low bone mineral density at baseline. In addition to standard chemotherapy, pamidronate (1 mg/kg, IV) was given bimonthly. Initial rapid symptom relief and gradual improvement of bone mineral density was demonstrated in both patients.

Using MS-MLPA as an efficient screening tool for detecting 9p21 abnormalities in pediatric acute lymphoblastic leukemia.

Characterization of recurrent genetic lesions in childhood acute lymphoblastic leukemia (ALL) has enabled therapeutic stratification with improved outcomes. The tumor suppressor genes, CDKN2A and CDKN2B, encoding p16INK4a, p14ARF, and p15INK4b have been localized to 9p21. Abnormalities of 9p21 have been reported in 10–30% of childhood ALL using conventional cytogenetics and fluorescence in situ hybridization (FISH). The incidence of 9p21 using more sensitive techniques, such as methylation specific multiplex ligation‐dependent probe amplification (MS‐MLPA), remains uncertain, and thus also the prognostic significance.

Routine bone marrow examination in the initial evaluation of paediatric Hodgkin lymphoma: the Canadian perspective

Bone marrow examination (BME) in paediatric Hodgkin lymphoma (HL) was evaluated, as evidence from adult HL suggests it may be unnecessary. An internet‐based survey was used to examine the practice of Canadian paediatric oncologists regarding BME in children and the impact of routine BME was evaluated in patients with HL treated at our institution. Sixteen of 17 paediatric oncology centres were represented. Forty‐three percent of eligible doctors completed the survey. Routine BME for stages III and IV disease was consistent nationally. By contrast, 54% and 70% of respondents reported performing routine BME for stages I and II HL respectively. Respondents were more likely to report performing routine BME in low‐stage HL if trained outside Canada (P = 0·04, stage I; P = 0·07, stage II) or practicing at smaller centres (P = 0·05, stage I; P = 0·03, stage II). At our institution, 62 patients were eligible for analysis. Only four patients (6·5%) had a positive BME. Anaemia was the only significant risk factor (P = 0·006). No patient with otherwise low stage was found to have marrow involvement. Comparison of staging with and without BME demonstrated no significant difference to final risk classification. BME in paediatric patients with low‐stage HL has extremely low yield and may be unnecessary.

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.