Spontaneous lymphocyte activation after autologous BMT for neuroblastoma

Clinical Oncology (1993) 5:262-266 © 1993 The Royal College of Radiologists

Clinical Oncology

Abstracts Abstracts of Papers Presented at the United Kingdom Children's Cancer Study Group Scientific Meeting, 11 November 1992, Royal Victoria Infirmary, Newcastle upon Tyne, UK

Do Taped Parental Interviews Improve Communication? O. B. Eden and I. Black, Department of Paediatric Haematology, Edinburgh, UK Relating distressing news to the parents of a child with cancer is difficult to do successfully. From a previous study we found that 17% of parents denied having a formal interview and 60% remained confused by some details despite repeated information giving sessions. The closest correlation was between confusion and poor parental adjustment to the child's disease. We have attempted to overcome this problem by taping parental interviews, thus enabling the parents to listen repeatedly to information as they reach a greater degree of emotional equilibrium and are ready to 'hear' what has been said. In a pilot study involving 14 consecutively diagnosed patients, the use of taped interviews was followed by a home visit at which an independent interviewer using a structured questionnaire, gauged parental response to the information they had been given; 35 % expressed concern that they had been unable to take in some or all of the information at the time of taping (compared with 60% in an earlier study), and parents appeared to be at a higher stage of coping than in the first study. Replaying of tapes varied from 0 to 50 times. Reasons for not using the tapes included complete contentment and satisfaction with information and conversely, that it was too harrowing to replay. In all 7/14 families used the tape for a wider audience, to disseminate information. We routinely now use taped interviews but must remain alert to identify those parents for whom reinforcement and clarification of information is necessary.

ICRF187: Cardioprotection from High Dose Anthracycline Toxicity in Children with Malignant Disease F. A. Bu'Lock, H. M. Gabriel, A. Oakhill, R. P. Martin and M. G. Mott, Bristol Royal Hospital for Sick Children, Bristol, UK Anthracycline cardiotoxicity continues to be a major source of clinical concern to those involved in treating childhood malignancy. A new compound, ICRF187, has been shown to be cardioprotective in adults, but there are no data on its use in children. Clinical and echocardiographic data from six children with advanced malignancies, treated with ICRF187 on a compassionate-use patient basis (CPs) are compared with those from six similar children treated concurrently without ICRF187 (CTLs). Of the six CPs, aged 3.9 to 17.5 years (mean 11.6), five had relapsed solid tumours and one had multiply relapsed acute myeloid leukaemia (AML). Mean cumulative anthracyeline dose before receiving further anthracycline with ICRF187 (RXCP) was 440 mg/m 2 (range 400-455); at the end of treatment it was 912 mg/ m 2 (range 650-1650). The six contemporary CTLs were selected on the basis of closest matching dose schedule to the CPs. Aged 1.9 to 16.5 years (mean 8.5), five had relapsed solid tumours and one had relapsed AML. Mean dose before retreatment (RX) was 435 mg/m 2 (range 300-600), and at the end of RX was 806 mg/m 2 (range 600-1150). There was no significant difference in dosage between the two groups. ICRF187 was administered 30 minutes before anthracycline, in a dose ratio of 20 to 1. Three CTLs developed severe congestive cardiac failure (CCF), from which one died. The other two died from relapse, with ongoing CCF. One more child died from relapsed disease. One of

the two survivors has developed significant (asymptomatic) left ventricular (LV) dysfunction. No CP developed CCF or later LV dysfunction, although five have now died from relapse. LV function was assessed from the LV ejection fraction (EF) measured by M-mode echo. There was no significant difference in EF between the two groups before treatment. EF fell in all CTLs, from a mean of 73% (range 61-83) pre-RX to 58% (range 46-68) post-RX (P=0.03). EF fell in only three CPs, with no overall change in group values; the mean EF was 68% (range 60-74) pre-RXCP and 67% (range 61-74) post-RXCP (P=0.92). The difference in EF between groups after treatment was statistically significant (P=0.02). Fall in EF was highly correlated to increasing dose in CTLs, with Pearson Correlation Coefficient ( r = - 0 . 8 6 (P=0.03). This relationship was abolished by ICRF187, r = - 0 . 2 6 (P=0.3). ICRF187 appears to have provided highly effective cardioprotection to this small group of children with end-stage disease, in comparison with the severe cardiotoxicity seen in a similar group treated using equivalent anthracycline doses alone. Further studies on 'potential survivors' are clearly indicated.

Spontaneous Lymphocyte Activation After Autologous BMT for Neuroblastoma K. P. Windebank 1, M. A. Holscher 2, M. L. Epstein 1 and J. H. Robinson 2, Departments of 1Child Health and 2Immunology, University of Newcastle upon Tyne, UK Six children with disseminated neuroblastoma were followed after autologous bone marrow transplantation. The first three children did not receive pre-transplant 'priming' with eyclophosphamide, whereas the second three did. Serial studies were performed, quantifying peripheral blood lymphocyte surface marker expression and corresponding in vitro cytotoxicity. After preparation by a whole blood lysis technique, surface marker expression was analysed on a FACSCAN. Samples were tested for simultaneous two-colour flourescence using a panel of FITC and PE labelled monoclonals to CD3 CD4, CD14, CD16, CD19, CD30, CD45, CD56 and HLA-DR. In parallel, peripheral blood mononuelear cells were purified and assayed for spontaneous cytotoxicity against cell lines K562 (NK activity) and Raji (LAK activity). Standard chromium release cytoxicity assays were performed and inter-assay variation was monitored and corrected by using batched frozen lymphocytes as controls. The first three children developed increased NK and LAK activity between 20 and 40 days post-transplant. This was associated with a recovering white cell count and a preponderance of lymphocytes bearing a CD3-, CD16-, CD56+ phenotype. The second three 'primed' children experienced more profound and prolonged neutropenia, making analysis technically more difficult, owing to the low number of ceils available for assay. However, these children did not develop spontaneous LAK activity as their white counts recovered. The changes induced prior to harvest by priming were serially studied in one case. Whilst the total white count rose slightly, the percentages of CD3-, CD16-, CD56+, NK cells fell with a corresponding drop in cytoxicity. These findings suggest that, under some conditions, LAK activity may be induced spontaneously during bone marrow

I

Abstracts reconstitution. In the patients described this activity appeared to be present when C D 3 - , C D 1 6 - , CD56+ cells were abundant. This phenotype mirrors that found when NK cells are activated in vitro by IL-2. The biological significance of these findings is unclear.

MDR1 mRNA in Resistant Neuroblastoma Bone Marrow

M. B. Phillips and C. R. Pinkerton, Children's Department, Royal Marsden Hospital, Sutton, UK Poor prognosis in neuroblastoma may be related to the overexpression of the multiple drug resistance (MDR1) mRNA and its product P-glycoprotein. A quantitative mRNA polymerase chain reaction (PCR) technique has been established to identify varying levels of MDR1 mRNA expression. Radioactive PCR is performed on eDNA derived from total cellular RNA of marrow from neuroblastoma patients using MDR1 and B2M specific primers and the products separated by polyacrylamide gel electrophoresis (PAGE). Comparison of sample MDR1/B2M ratios obtained from autograph analysis of PCR products with a previously extrapolated curve provides a quantitative value for MDR1 copy number. Nine patients with primary refractory (five) or relapsed (four) neuroblastoma have been studied for level of expression of MDR1 mRNA compared with normal controls. At the time of diagnosis in eight patients MDR1 mRNA ranged from 1 to 10 times that of normal controls. During treatment patients had values ranging from 1 to 1000 times that of controls and, in all patients studied, values increased sequentially during treatment. Response to chemotherapy given with MDR antagonists has, however, not clearly reflected the degree of mRNA expression. The identification and quantification of MDR1 mRNA at diagnosis may provide important prognostic information and ultimately influence the individual treatment regimen.

Childhood Cancer and Ethnic Origin: Are Asian Children at Greater Risk of Developing Cancer?

J. E. Powell, S. E. Parkes, A. H. Cameron and J. R. Mann, West Midland Regional Children's Tumour Research Group, The Children's Hospital, Birmingham, UK Age standardized incidence rates (ASRs) were calculated for childhood cancer diagnosed between 1982 and 1991 in the West Midlands Health Authority Region (WMHAR). Estimated population figures for the ethnic minorities in the region were derived from the 1986 and 1988 OPCS Labour Force Survey. In the WMHAR, ethnic minorities comprise 8% of the total population and nearly 14% of children under 15 years. Of the minority childhood population, 69% are Asian (Indian, Pakistani or Bangladeshi), 17% are Afro-Caribbean and 14% are classed as 'other' (including mixed) ethnic origin. Between 1982 and 1991, 1247 cases of malignant disease in children aged up to 14 years were recorded by the West Midland Regional Children's Tumour Research Group. Of these, 1049 were in white Caucasians, 138 in Asians, 28 in Afro-Caribbeans and 32 in children of 'other' ethnic origin. The ASRs for all cancers in these four groups are, respectively, 128, 152, 124 and 163 per million per year. Reticuloendothelial tumours and non-sarcomatous solid tumours were significantly more common in Asian children than Caucasians. ASRs for the ethnic minorities were compared with the white Caucasian populations for different diagnostic categories. No significant differences were found in the rates for the AfroCaribbean and 'other' categories since the populations were too small. A significant (P<0.05) deficit of rhabdomyosarcoma and an excess of malignant germ cell turnouts and lymphomas were seen in Asian children. Retinoblastoma incidence was nearly double that in Caucasians, though the difference was not significant. These findings are in agreement with a previous study of the UKCCSG National Register which examined patterns of malignancy in the ethnic minorities, but could not assess incidence. To our knowledge, these are the first population based ASRs to be published for ethnic minorities in the UK.

263 Is Non-Compliance an Underestimated Problem in Childhood Leukaemia?

H. /~/])avies 1, L. Lennard 2 and J. S. Lilleyman 1, 1University Department of Paediatric Haematology, Sheffield Children's Hospital, Sheffield and aUniversity Department of Pharmacology and Therapeutics, Royal Hallamshire Hospital, Sheffield, UK Forty children with lyrnphoblastic leukaemia (ALL) taking daily oral 6-mercaptopurine (6-MP) as part of their remission maintenance chemotherapy had the intracellular 6-MP metabolites 6thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (methyl-MP) measured while supposedly on full protocol dose. Variable intracellular metabolism can lead to low concentrations of one or the other, but rarely both. The aim of the present study was to see how frequently low levels of both could be detected and whether the finding could be correlated with any clinical data indicative of non-compliance. As an index of cytotoxicity, for each child the time spent on attenuated doses (due to cytopenias) was calculated as a percentage of the total time on 6-MP, both overall and in the first and second halves of treatment. All parents were asked to write down how their children took the tablets and whether they ever failed to do so. Fifteen patients (37.5%) had erythrocyte concentrations of both 6-TGN and methyl-MP below the respective group median values (low metabolite (LM) group). Compared with the remainder (normal metabolite (NM) group) they spent significantly less time overall on attenuated doses (13% versus 18.5%; P<0.02), a difference that was more evident in the second half of their treatment (8% versus 20%; P<0.001). Direct questioning of the parents revealed significant problems with tablet taking in seven of the 15 LM group. No such problems were encountered in the NM group. This study suggests that an important number of children with ALL are not obtaining an adequate cytotoxic effect of 6-MP because, for at least some of the time and for a variety of reasons, they are not taking it. The exact proportion is unclear but it is more than 10% and may be as high as 30%. The problem may contribute to otherwise unexplained relapse in patients with good prognosis disease.

Imamnotoxin Studies in a Model of Human T-ALL Developed in SCID Mice

B. J. Morland 1, D. J. Flavell2 and J. A. Kohler z, iBirmingham Children's Hospital and 2Southampton General Hospital, UK We have developed an in vivo model of human T-acute lymphoblastic leukaemia (T-ALL) in a colony of SCID mice in order to study an immunotoxin targeting the plant toxin saporin to the T-cell marker CD7. Whereas most in vivo studies of leukaemia in animals rely on the measurement of sold tumour growths transplanted subcutaneously into nude mice, the SCID mouse xenograft model mimics more closely the pattern of leukaemia seen in humans. Intravenous injection of SCID mice with the human T-ALL cell line HSB2 was undertaken and pilot studies demonstrated disseminated disease occurring in animals starting with bone marrow infiltration and leading to multi-organ involvement, including meningeal disease. Histological confirmation of human T-lymphocyte disease in these animals was obtained using immunocytochemical methods; in addition, paraffin blocks from affected animals were used to obtain DNA which, when amplified using the polymerase chain reaction (PCR), demonstrated the presence of human specific T-cell receptor genes. By inoculating SCID mice with graded doses of HSB2 cells, survival curves were produced enabling an optimum experimental model to be established with survival times long enough to allow in vivo therapy studies to be designed. An anti-CD7/saporin immunotoxin was constructed with a hindered disulphide bond which was shown to have selective cytotoxicity against CD7 positive T-ALL cell lines in vitro. An in vivo therapeutic study was designed in which SCID mice were injected with 10 6 HSB2 ceils and 8 days later were given a single i.v. injection of immunotoxin equivalent to 20% of the LDs0 dose. Survival was prolonged in the treated animals (80% alive at day 100 compared with 40% survival in controls). We conclude that the SCID mouse may provide a useful tool for the study of new therapies directed against human leukaemia. The