Search

Patients post haematopoietic stem cell transplant or CAR-T cell therapy face significant risk of morbidity and mortality from SARS-CoV19, due to their immunosuppressed state. As case numbers in Australia and New Zealand continue to rise, guidance on management in this high-risk population is needed. Whilst we have learnt much from international colleagues who faced high infection rates early in the pandemic, guidance relevant to local health system structures, medication availability and emerging therapies is essential to equip physicians to optimally manage our patients. This article is protected by copyright. All rights reserved.

Background The Philadelphia chromosome t(9;22), a reciprocal translocation between chromosomes 9 and 22, results in the gene fusion BCR-ABL1, and occurs in 2-3% of childhood acute lymphoblastic leukemia (ALL). It is detected using cytogenetic and molecular techniques: karyotype, fluorescence in-situ hybridization (FISH) for t(9;22) and reverse transcription polymerase chain reaction (RT-PCR) for BCR-ABL1. Detection has implications for treatment, with the addition of tyrosine kinase inhibitors to chemotherapy regimens improving outcome. Low level BCR-ABL1 transcripts have been reported in blood of healthy individuals. We have observed this finding in bone marrow in newly diagnosed ALL in the absence of the t(9;22) by karyotype or FISH. The significance of low level positivity at diagnosis has not been determined in the setting of childhood Philadelphia chromosome negative (Ph-) ALL. Here we report, for the first time, the molecular evolutionary characteristics of children and adolescents with low level BCR-ABL1 positivity found at diagnosis to relapse. Methods We reviewed 327 patients aged 0-17 years diagnosed with ALL or Acute Leukemia of Ambiguous lineage (ALAL) at The Children's Hospital at Westmead, Sydney, Australia from 1 January 2010 to 30 June 2020. Those positive for the BCR-ABL1 gene fusion by RT-PCR, and negative for t(9;22) by karyotype or FISH were included. Demographics, cytogenetics at diagnosis and relapse, and outcome data were extracted from the medical record. Qualitative BCR-ABL1 analysis was performed using multiplex RT-PCR, followed by nested PCR, on RNA extracted from diagnostic bone marrow (sensitivity 5x10-6). If positive, quantitation was performed using real-time RT-PCR with results expressed as the ratio of BCR-ABL1 over ABL1 (sensitivity 1x10-5). Each PCR included positive and negative controls. Results Of 313 (96%) evaluable patients diagnosed with ALL or ALAL at our institution in the study period, 54 (17%) were positive by RT-PCR for BCR-ABL1 in diagnostic bone marrow. Seven patients were excluded as they had Ph+ ALL-specific treatment after the detection of t(9;22) by karyotype, FISH or other methods. Forty-seven (15%) children with Ph- ALL had low level BCR-ABL1 detected by qualitative PCR. Demographic and cytogenetic characteristics for these patients are summarized in Table 1. All were diagnosed with ALL, the majority (77%) of precursor B-cell lineage including 2 with infant ALL. The e1a2 transcript was identified in 43 (91%) patients, with other transcript types as follows: e4a2 in 1 (2%), e13a2 in 1 (2%), and splicing variants in 2 (4%). BCR-ABL1 quantitation was performed in 43 (91%) and was quantifiable only in 12 (28%) patients, with a median of 0.0008% (range 0.0003 - 0.095%). Forty-five (96%) patients were treated with Berlin-Frankfurt-Munster ALL chemotherapy regimens. The two infant ALL patients were treated on the Interfant06 trial. One received a bone marrow transplant (BMT) in first remission then died after relapse; the other relapsed and died before BMT. Seven (15%) of 47 relapsed, occurring at a median of 21 months (range 2 - 41 months) after diagnosis. Characteristics of these patients are presented in Table 2. Four patients were tested for BCR-ABL1 by RT-PCR in relapse marrow samples; all were negative. No patient with low level BCR-ABL1 positivity at initial diagnosis was diagnosed with Ph+ ALL at relapse. There was no difference in 5-year relapse-free (80% vs 83%, P = .451) or overall survival (86% vs 91%, P = .368) between children with low level BCR-ABL1 positivity (n=47) and those without (n=259). Conclusion BCR-ABL1 low level positivity detected by RT-PCR in the bone marrow of children with newly diagnosed ALL is a relatively common finding, and did not adversely affect outcome for patients treated for Ph- ALL using a contemporary risk-adapted approach. Importantly, this finding did not influence the molecular evolutionary characteristics at the time of relapse in our patient group. Disclosures No relevant conflicts of interest to declare.

Introduction Pulmonary graft versus host disease is a pathological process arising in peripheral airways causing significant morbidity and mortality post-Bone Marrow Transplantation (BMT). Current screening and diagnosis focuses on spirometry change from baseline to identify Bronchiolitis Obliterans Syndrome (BOS), and an at-risk pre-BOS stage BOS0p (≥10% fall FEV1). Sensitive peripheral airway function tools exist but remain under-utilized and utility is unclear. Aim To investigate longitudinal peripheral airway function change, detected by Multiple Breath Washout (MBW) and Forced Oscillation Technique (FOT), in pediatric BMT subjects with/without BOS and BOS0p. Methods Children aged ≥3 years were recruited. Testing occurred at baseline, monthly during 1st year and 6-monthly to 3yrs: MBW (LCI, Scond and Sacin, ExhalyserD®, Ecomedics), FOT (Rrs and Xrs at 5Hz, AX, Fres, tremoFlo®, Thorasys) and, if feasible, spirometry, plethysmography and DLCO. Groups (BOS, BOS0p, unaffected) compared at baseline, 12, 24 and 36 months. Results Of 24 recruited subjects (mean±SD (range) age 10.2±4.2 (3.3-18.1) years at BMT, 54% AML/ALL diagnosis, 71% male), 1 withdrew and 6 died (5 within 12 months). BOS and BOS0p alone were detected in 3 (12.5%) and 8 (33.3%), respectively. BOS was associated with pattern of marked abnormality, significant increases from baseline were observed in LCI, Scond, Sacin, Xrs, AX and Fres. One subject developed BOS3 at 105 days (1st feasible post-BMT test). MBW/FOT abnormality was detected 26 and 207 days prior to BOS≥1 in other cases. LCI trajectory increased with increasing BOS severity. At 3 years, statistically significant differences between BOS0p vs unaffected were observed for LCI (p=0.009), Sacin(p=0.03), Xrs (p=0.004) and approached significance for AX (p=0.09) and Fres (p=0.09). Conclusion BOS and its earlier at-risk stage BOS0p were associated with significant peripheral airway abnormality detectable on MBW and FOT. MBW and FOT abnormality predated BOS≥1 in 2/3 BOS cases.

Introduction: Peripheral airway abnormalities are common in children post Bone Marrow Transplantation (BMT). Lung Clearance Index (LCI) was abnormal in 50% of a paediatric post BMT cohort at our institution.(1) Little is known about the progression of peripheral airway function in children post BMT. Adult data has reported increasing LCI values over time post BMT.(2) Aim: To evaluate the change in lung function (including peripheral airway function) over time in our paediatric post BMT cohort. Methods: Follow up assessment of the previously studied cohort, performed 12-24 months after initial testing. Protocol included Multiple Breath Washout (SF 6 based), conventional lung function and Liverpool respiratory questionnaire (LRQ). Lung function data presented as median (range) for z scores or raw values. SF6 LCI ULN – 7.38. Maximum score for LRQ 128. Statistical analysis: Wilcoxon signed rank test. Results: 17 tested to date: 76% males; age 15.3(5.6-20.0) years, 6.3 (1.6- 12.8) years post BMT and 1.2(0.9-1.8) years since last assessment. Proportion with abnormal LCI was similar at baseline and on follow up testing (47%vs 35%, p=0.3) as were FEV1 z-scores (18% vs 18%). No significant change in LCI over time: 0.18 (-3.93 to 2.05). No significant change in spirometry z-score change over time; FEV1 0.16 (-0.77 to 0.71); FVC -0.08 (-0.80 to 0.81); FEV1/FVC 0.20 (-0.45 to 0.43); FEF25-75; -0.03 (-0.69 to 0.53). No significant change in LRQ score over time; 0(-28 to 30). Conclusion: Across the overall cohort respiratory symptoms, peripheral airway function and spirometry did not worsen. However within individual subjects considerable variation was observed.

Aim: Pulmonary complications are common following Bone Marrow Transplantation (BMT) but peripheral airway involvement may be underestimated by conventional lung function measures, such as spirometry. This study aimed to characterize peripheral airway function in children post BMT using Multiple Breath Washout (MBW) in comparison to standard lung function. Methods: Cross sectional study of children following BMT at a single tertiary centre. Inclusion criteria included age ≥3 years, allogenoeic BMT, similar conditioning regimens and stability at the time of testing. Assessment included MBW (performed using simultaneous inert gas washout with SF 6 and Helium) and conventional lung function. Non-parametric data displayed as median (range). Results: 24 subjects have been recruited to date: 16 (67%) male, age 14.3 (4.3-18.6) years, tested at 4.8 (0.7-12.1) years post BMT. Spirometry was feasible in 15/24 (63%) of the cohort, with abnormal values present in 5/24 (21%) for FEV 1 and 4/15 (17%) for FEF 25-75 . MBW was feasible in all subjects. Abnormal MBW parameters were observed in a greater proportion (18/24, 75%) vs. either FEV 1 or FEF 25-75 (p 6 MBW indices was greatest with S cond (0.034 (0.004-0.089), Upper limit of normal (ULN) 0.023, abnormal in 18/24, (75%) followed by LCI (7.52 (5.03-23.59), ULN 7.35, abnormal in 14/24, 58%) and S acin (0.105 (0.056-0.463), ULN 0.121, abnormal in 8/24, 33%). Conclusion: MBW offered improved feasibility, compared to spirometry, for lung function assessment. Peripheral airway involvement was present in the majority of the cohort, and was underappreciated by spirometry.

Introduction Telomeres are specialized DNA structures found at the end of linear chromosomes. In humans, they contain the repetitive sequence (TTAGGG)nalong with its complementary strand and associated proteins. With each cell division, the telomere shortens due to incomplete replication of the lagging strand, a phenomenon known as the end replication problem. Oxidative damage, as well as the action of nucleases also leads to telomere attrition. Once the telomere reaches a critically short length, cells enter senescence or apoptosis. In the general population, telomere length (TL) varies greatly, and is seen to decline with age. Patients with Dyskeratosis Congenita (DC) have inherently short telomeres, which leads to multiple organ dysfunction. These patients suffer increased organ toxicity when given standard myeloablative conditioning regimens during hematopoietic stem cell transplantation (HSCT). This is most likely due to the reduced ability of their cells to replicate and recover following cytotoxic treatment. It is not known whether patients without DC, who have telomere lengths at the lower end of the normal spectrum also suffer increased rates of toxicity. Thus we undertook a retrospective analysis to determine if there was an association between short TL and increased transplant-related mortality (TRM) following HSCT in children. Method Medical records of patients who underwent allogeneic HSCT at the Children's Hospital at Westmead (Sydney, Australia) between 2002 to 2013 were examined and demographic, prior treatment, transplant, toxicity and transplant information was extracted. The primary outcome measured was TRM, while overall survival, relapse and graft versus host disease (GVHD) being secondary outcomes of interest. Stored DNA, extracted from bone marrow, taken within 3 months prior to HSCT was used for telomere length testing, as a surrogate marker for TL in other tissues in the body. Quantitative PCR was carried out to determine relative telomere length, which was converted to an age adjusted TL (AATL) by subtracting the expected relative TL (i.e. 50th percentile for age of the patient) from the measured relative TL, so that patients of all ages could be analyzed together. Results Between 2002 and 2013, there were 121 patients who underwent allogeneic HSCT at our institution. Of these, 78 had DNA stored, with appropriate consent and ethics approval to be used in our study. Most children were undergoing HSCT for hematological malignancies (acute lymphoblastic leukemia 52%, acute myeloid leukemia 39%), with 55% having relapse or refractory disease. Matched sibling donors made up 46% of cases. At a median follow up of 3.2 years, 64% of patients remained alive. The single leading cause of death was relapse (48%) with 52% of deaths attributable to transplant-related causes. The median AATL for the entire cohort was -0.21 (range -0.61-0.28). Telomere length was not associated with relapse or refractory disease. There was no correlation between AATL and specific organ toxicity, relapse or overall survival. When the cohort was divided into 2 groups based on AATL, there was a trend (p=0.055) towards increased TRM in patients who had AATL below the median for the cohort (Figure 1). Multivariate analysis adjusting for age at transplant, CMV status, disease status and HLA matching showed a reduced risk (HR 0.33; p value 0.072) of TRM in patients with AATL above the median for the cohort. Conclusion AATL was shorter in patients pre-HSCT compared with age matched controls. There was a trend towards increased TRM in patients with shorter telomere length, however most likely due to small sample size, this did not achieve significance. If extended to a larger cohort of patients, TL may prove to be a significant marker for children at increased risk of TRM prior to HSCT. This could influence the choice to use less myeloablative conditioning in these patients. Support: NHMRC GNT1056667 Disclosures No relevant conflicts of interest to declare.

Introduction: Fludarabine phosphate is commonly used as part of conditioning regimens in children undergoing allogeneic haematopoietic stem cell transplantation (HSCT) for both malignant (n = 47) and non-malignant (n = 39) diseases, but its use has been associated with engraftment failure or mixed chimerism1. The aim of this study was to examine whether suboptimal engraftment is associated with fludarabine pharmacokinetics. Material (or patients) and methods: The clinical pharmacokinetics of fludarabine phosphate was evaluated by studying the free concentrations of the active metabolite, (9B-D-arabinosyl-2 fludarabine, F-Ara-A). Eighty-six HSCTs were studied in 84 children 0.1 to 19 years. Glomerular filtration rate (GFR) was determined by measuring the plasma clearance of diethylenetriaminepentacetic acid (DTPA). The children in this study were treated according to specific HSCT conditioning protocols which varied with respect to the total administered fludarabine dose and the number of days over which it was administered (range: 3 to 6). A weight-based dose of 1 to 2 mg/kg was used in 13 transplants where the patient weighed Results: Mixed chimerism or graft rejection occurred following 10 (of 86) transplants and, of these, 8 were being treated for malignant diseases. First dose free F-Ara-A AUC was significantly lower for these transplants compared with the remainder: median 3.0 (2.4 - 3.8) mg/L.h versus 4.1 (3.3 - 5.2) mg/L.h, p = 0.038, but the difference in clearance was not significant: 6.3 (5.2 - 11.0) L/h versus 4.6 (2.7 - 8.1) L/h, p = 0.06. The recovery of neutrophils also correlated significantly with first dose free AUC (r = -0.22, p= 0.044) but not with clearance (r=0.193, p = 0.075). Recovery of platelets was not significantly associated with any estimate of free F-Ara-A exposure. Within the fludarabine dose range of 25 to 40 mg/m2, there was a linear association with median free F-Ara-A AUC (r 2= 0.977), which was represented by the equation median F-Ara-A AUC (mg/l.h) = 0.1306 x fludarabine dose (mg/m2). There was a significant negative correlation between clearance and GFR (r = -0.31, p < 0.005) and GFR was significantly higher in patients with suboptimal engraftment: median 126 (116 - 152) versus and 111 (92 - 133) ml/min/1.73 m2 (p = 0.015). Suboptimal engraftment was significantly associated with poorer overall survival using Kaplan Meier survival analysis (p = 0.002 using log rank test). Conclusions: The data suggests that patients with good renal function are at greater risk of suboptimal engraftment since they have lower exposure to free F-Ara-A. These children may therefore require higher doses of fludarabine. We found that mg/m2 fludarabine dose was linear with free F-Ara-A AUC between the 25 and 40 mg/m2 dose range. 1.Lee JH, Joo YD, Kim H, Ryoo HM, Kim MK, Lee GW, et al. Randomized trial of myeloablative conditioning regimens: busulfan plus cyclophosphamide versus busulfan plus fludarabine. J Clin Oncol 2013 Feb 20; 31(6): 701-709. Disclosures No relevant conflicts of interest to declare.