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PCR of TCR/Ig gene rearrangements is considered the method of choice for minimal residual disease (MRD) quantification in BCP-ALL, but flow cytometry analysis of leukemia-associated immunophenotypes (FCM-MRD) is faster and biologically more informative. FCM-MRD performed in 18 laboratories across seven countries was used for risk stratification of 1487 patients with BCP-ALL enrolled in the NOPHO ALL2008 protocol. When no informative FCM-marker was available, risk stratification was based on real-time quantitative PCR. An informative FCM-marker was found in 96.2% and only two patients (0.14%) had non-informative FCM and non-informative PCR-markers. The overall 5-year event-free survival was 86.1% with a cumulative incidence of relapse (CIR5y) of 9.5%. FCM-MRD levels on days 15 (HzR 4.0, p< 0.0001), 29 (HzR 2.7, p< 0.0001), and 79 (HzR 3.5, p< 0.0001) associated with hazard of relapse adjusted for age, cytogenetics, and WBC. The early (day 15) response associated with CIR5yadjusted for day 29 FCM-MRD, with higher levels in adults (median 2.4 × 10−2versus 5.2 × 10−3, p< 0.0001). Undetectable FCM- and/or PCR-MRD on day 29 identified patients with a very good outcome (CIR5y= 3.2%). For patients who did not undergo transplantation, day 79 FCM-MRD > 10−4associated with a CIR5y= 22.1%. In conclusion, FCM-MRD performed in a multicenter setting is a clinically useful method for MRD-based treatment stratification in BCP-ALL.

The NOPHO ALL2008 is a population-based study using an unmodified pediatric protocol in patients 1–45 years of age with acute lymphoblastic leukemia. Patients with T-ALL were given a traditional pediatric scheme if fast responding (minimal residual disease (MRD) < 0.1% day 29), or intensive block-based chemotherapy if slow responding (MRD > 0.1% day 29). Both treatment arms included pediatric doses of high-dose methotrexate and asparaginase. If MRD ≥ 5% on day 29 or ≥0.1% after consolidation, patients were assigned to allogeneic hematopoietic stem cell transplantation. The 5-year overall survival of the 278 T-ALL patients was 0.75 (95% CI 0.69–0.81), being 0.82 (0.74–0.88) for patients 1.0–9.9 years, 0.76 (0.66–0.86) for those 10.0–17.9 years, and 0.65 (0.55–0.75) for the older patients. The risk of death in first remission was significantly higher in adults (12%) compared with the 1–9 years group (4%). The MRD responses in the three age groups were similar, and only a nonsignificant increase in relapse risk was found in adults. In conclusion, an unmodified pediatric protocol in patients 1–45 years is effective in all age groups. The traditional pediatric treatment schedule was safe for all patients, but the intensive block therapy led to a high toxic death rate in adults.

Minimal residual disease (MRD) measured by PCR of clonal IgH/TCR rearrangements predicts relapse in T-cell acute lymphoblastic leukemia (T-ALL) and serves as risk stratification tool. Since 10% of patients have no suitable PCR-marker, we evaluated flowcytometry (FCM)-based MRD for risk stratification. We included 274 T-ALL patients treated in the NOPHO-ALL2008 protocol. MRD was measured by six-color FCM and real-time quantitative PCR. Day 29 PCR-MRD (cut-off 10−3) was used for risk stratification. At diagnosis, 93% had an FCM-marker for MRD monitoring, 84% a PCR-marker, and 99.3% (272/274) had a marker when combining the two. Adjusted for age and WBC, the hazard ratio for relapse was 3.55 (95% CI 1.4–9.0, p= 0.008) for day 29 FCM-MRD ≥ 10−3and 5.6 (95% CI 2.0–16, p= 0.001) for PCR-MRD ≥ 10−3compared with MRD < 10−3. Patients stratified to intermediate-risk therapy on day 29 with MRD 10−4–<10−3had a 5-year event-free survival similar to intermediate-risk patients with MRD < 10−4or undetectable, regardless of method for monitoring. Patients with day 15 FCM-MRD < 10−4had a cumulative incidence of relapse of 2.3% (95% CI 0–6.8, n= 59). Thus, FCM-MRD allows early identification of patients eligible for reduced intensity therapy, but this needs further studies. In conclusion, FCM-MRD provides reliable risk prediction for T-ALL and can be used for stratification when no PCR-marker is available.

Adults with acute lymphoblastic leukemia (ALL) do worse than children. From 7/2008 to 12/2014, Nordic and Baltic centers treated 1509 consecutive patients aged 1–45 years with Philadelphia chromosome-negative ALL according to the NOPHO ALL2008 without cranial irradiation. Overall, 1022 patients were of age 1–9 years (A), 266 were 10–17 years (B) and 221 were 18–45 years (C). Sixteen patients (three adults) died during induction. All others achieved remission after induction or 1–3 intensive blocks. Subsequently, 45 patients (12 adults) died, 122 patients relapsed (32 adults) with a median time to relapse of 1.6 years and 13 (no adult) developed a second malignancy. Median follow-up time was 4.6 years. Among the three age groups, older patients more often had higher risk ALL due to T-ALL (32%/25%/9%, P<0.001), KMT2A rearrangements (6%/5%/3%, P<0.001) and higher day 29 residual leukemia for B-lineage (P<0.001), but not T-ALL (P=0.53). Event-free survival rates (pEFS5y) were 89±1% (A), 80±3% (B) and 74±4% (C) with significant differences only for non-high risk groups. Except for thrombosis, pancreatitis and osteonecrosis, the risk of 19 specified toxicities was not enhanced by age above 10 years. In conclusion, a pediatric-based protocol is tolerable and effective for young adults, despite their increased frequency of higher risk features.

Asparaginase (ASP)-associated pancreatitis (AAP) occurs during acute lymphoblastic leukemia treatment. Among 1285 children (1.0–17.9 years) diagnosed during July 2008–December 2014 and treated according to the Nordic/Baltic ALL2008 protocol, 86 (cumulative incidence=6.8%) developed AAP. Seventy-three cases were severe (diagnostic AAP criteria persisting >72 h) and 13 mild. Cases were older than controls (median: 6.5 vs 4.5 years; P=0.001). Pseudocysts developed in 28%. Of the 20 re-exposed to ASP, 9 (45%) developed a second AAP. After a median follow-up of 2.3 years, 8% needed permanent insulin therapy, and 7% had recurrent abdominal pain. Germline DNA on 62 cases and 638 controls was genotyped on Omni2.5exome-8-v1.2 BeadChip arrays. Overall, the ULK2variant rs281366 showed the strongest association with AAP (P=5.8 × 10−7; odds ratio (OR)=6.7). Cases with the rs281366 variant were younger (4.3 vs 8 years; P=0.015) and had lower risk of AAP-related complications (15% vs 43%; P=0.13) compared with cases without this variant. Among 45 cases and 517 controls <10 years, the strongest associations with AAP were found for RGS6variant rs17179470 (P=9.8 × 10−9; OR=7.3). Rs281366 is located in the ULK2gene involved in autophagy, and RGS6regulates G-protein signaling regulating cell dynamics. More than 50% of AAP cases <10 years carried one or both risk alleles.

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Intestinal dysfunction is frequent in cancer and during anti‐cancer treatment. Glucagon‐like peptide‐2 (GLP‐2) is secreted in a nutrition‐dependent manner from the intestinal enteroendocrine L‐cells. It accelerates crypt cell proliferation and nutrient absorption, inhibits enterocyte apoptosis and decreases mucosal permeability. Lack of GLP‐2 may increase the risk of malabsorption and intestinal bacterial translocation. The aim of this study is to evaluate meal stimulated secretion of GLP‐2 in children with cancer undergoing anti‐cancer treatment. Plasma‐GLP‐2 analysis after an overnight fast and 1 hour after intake of a mixed test meal. Data on gastrointestinal toxicity, blood neutrophile counts and food records were included in the analysis. Forty‐four meal stimulation tests were performed in 25 children (median age, 6.0 years; range, 2.5‐19) during anti‐cancer treatment. Median GI toxicity score was 5 (range, 0‐15), and mean energy intake was 62.4% of recommended values. P‐GLP‐2 values increased from mean (SD) 38 (18) to 63 (51) pmol/l (P< 0.0001). Twelve of the meal stimulation tests (28%) resulted in a p‐GLP‐2 increase >2 fold, which is assumed to be the lower limit of normal values. The increase was strongly dependent on the energy intake (r = 0.62, P< 0.0001), while toxicity score and neutrophile count had no significant influence (multiple regression). In children treated with anti‐cancer therapy, GLP‐2 secretion seems to be normal if the enteral energy intake is sufficient. Insufficient GLP‐2 secretion could influence the gastrointestinal problems seen in the children with a low enteral energy intake.

Intestinal dysfunction is frequent in cancer and during anti-cancer treatment. Glucagon-like peptide-2 (GLP-2) is secreted in a nutrition-dependent manner from the intestinal enteroendocrine L-cells. It accelerates crypt cell proliferation and nutrient absorption, inhibits enterocyte apoptosis and decreases mucosal permeability. Lack of GLP-2 may increase the risk of malabsorption and intestinal bacterial translocation. The aim of this study is to evaluate meal stimulated secretion of GLP-2 in children with cancer undergoing anti-cancer treatment.

Approximately 70% of children treated for cancer are cured, however the treatment often impairs reproductive function. The aim of the present study was to assess ovarian reserve in women with an apparently normal ovarian function, who were cured of cancer in childhood.

Children with brain tumors are at high risk of developing growth hormone deficiency (GHD) after cranial irradiation (CI) if the hypothalamus/pituitary (HP) axis falls within the fields of irradiation. The biological effective dose (BED) of irradiation to the HP region was determined, since BED gives a means of expressing the biological effect of various irradiation treatment schedules in a uniform way. Hypothalamic versus pituitary damage as cause of GHD was distinguished in 62 patients by comparing the growth hormone (GH) peak response to an insulin tolerance test (ITT)/arginine stimulation test and the GH response to a growth hormone-releasing hormone (GHRH) stimulation test. Peak GH response to a GHRH test was significantly higher (median 7.3 mU/l; range: 0.5–79.0 mU/l) than that of an ITT/arginine test (median 4.7 mU/l; range: 0.01–75.0 mU/l) (p = 0.017). Peak GH after a GHRH test was significantly inversely correlated to follow-up time (rs = –0.46, p < 0.0001) and to BED (Rs = –0.28, p = 0.03), and both were found to be of significance in a multivariante regression analysis. We speculate that a significant number of patients developed hypothalamic radiation-induced damage to the GHRH secreting neurons, and secondary to this the pituitary gland developed decreased responsiveness to GHRH following CI in childhood.

To assess the effect of maintenance chemotherapy (MT) on growth factors and growth in children with acute lymphoblastic leukemia (ALL).

In a population-based study of 115 children with non-B-cell acute lymphoblastic leukaemia, we analysed the relation of the degree of leukopenia and risk of relapse to the degree of hepatotoxicity (as measured by serum aminotransferase (AT] during oral methotrexate (MTX) and 6-mercaptopurine (6MP) maintenance chemotherapy (MT). Hepatotoxicity was calculated as a mean of all AT-measurements (mATMT). Lack of hepatotoxicity was defined as a mATMT less than or equal to 40 IUl-1. A highly significant correlation was demonstrated between the mean AT during the first, second, and third year of MT (r greater than 0.70, P less than 0.00001). mATMT was not related to the mean WBC during MT (r = -0.03, P = 0.36), but was related to the rise in WBC following cessation of therapy (r = 0.24, P = 0.06). Patients with recurrent disease had significantly lower mATMT than patients staying in remission (P = 0.03 for both over-all and haematological relapse risk). Patients with a mATMT greater than 40IUl-1 had a lower risk of relapse than patients with a mATMT less than or equal to 40IUl-1 (4.5 year CCR: 0.70 and 0.50, P = 0.06; and 4.5 year haematological remission: 0.83 and 0.63, P = 0.03). The favourable outcome for patients with hepatoxicity could be demonstrated for all risk groups.

A linkage has been detected between the locus for cystic fibrosis (CF) and the proα2(I) collagen gene (COL1A2) which is located in the region q21.3→q22.1 of chromosome 7. Based on the combined linkage data derived from 50 informative two-generation nuclear families collected in Canada and Denmark, the distance between COL1A2 and CF is estimated to be 19 centiMorgans. Close linkage has also been detected between COL1A2 and the DNA marker D7S15 (formerly DOCRI-917) and the serum enzyme activity marker paraoxonase (PON), both of which have previously been found linked to CF. The results of the two-point and three-point linkage analyses indicate that the most probable order of these four genetic loci is COL1A2 – D7S15 PON – CF.

Two children with acute lymphoblastic leukaemia (ALL) were found to be thiopurine methyltransferase (TPMT)‐deficient by both genotype and phenotype. They were monitored with haematological parameters and red blood cell concentrations of 6‐thioguanine nucleotides (E‐6TGN) and methotrexate (E‐MTX, including MTX polyglutamates), in relation to the doses of 6‐mercaptopurine (6MP) and methotrexate (MTX), during their maintenance chemotherapy. Both patients developed severe pancytopenia at the standard protocol dose of 6MP. Even at 25% and 5%, respectively, of the protocol dose of 6MP, they achieved E‐6TGN values several‐fold above the population median, but without unacceptable bone‐marrow toxicity. Their high E‐6TGN values had only a minor influence on their E‐MTX values and their tolerance to oral MTX, but severe pancytopenia followed high‐dose MTX infusions. Due to the risk of fatal myelo‐suppression we recommend up‐front determination of TPMT activity in patients treated with 6MP or azathioprine.

We present a rapid, sensitive and specific high-performance liquid chromatographic method for the analysis of 6-mercaptopurine (6MP), 6-thioguanine nucleotides (6TGN) and 6-thiouric acid (6TU), with excellent chromatographic separation of the thiopurines. Thiopurines in plasma and erythrocytes were extracted by mercuricellulose and re-eluted with β-mercaptoethanol. For quantitative detection a reverse phase column (Lichrosorb RP-18 4 mmx30 cm) and a UV detector were used. Detection limits were 20 nmol/1 for 6MP in plasma, 250 nmol/1 for 6TGN in erythrocytes, and 10 nmol/1 and 15 nmol/1, respectively, for 6MP and 6TU in urine diluted 1:100 with β-mercaptoethanol. Within-run and between-run variations were less than 10%. Recovery of 6MP added to plasma, and 6TGN monophosphate added to haemolysed erythrocytes were 91% and 81%, respectively.

BackgroundNausea is a common and distressing side effect for children in chemotherapy. Antiemetic recommendations are based on limited literature and prospective evaluation of antiemetic efficacy is required. Smartphone applications (apps) may collect patient-reported outcomes with precision and effectiveness1. We developed a smartphone app to track nausea in pediatric cancer patients during chemotherapy.MethodsMedical researchers, pediatric oncologists and software engineers worked synergistically in the development. We translated the validated Pediatric Nausea Assessment Tool to score nausea severity2. We conducted three rounds of patient-feedback and modification.ResultsThe app has a definition module where the child centers the attention to the concept of nausea. The child can then express nausea severity with four faces and the child’s own definition of nausea is incorporated in the question2. The app includes a notification system to ensure high response rates. All participants felt that the app was user-friendly, intuitive and that time spent was acceptable.ConclusionThe app is a user-friendly tool to assess nausea in pediatric cancer patients that can ease future pediatric antiemetic trials.ReferencesStone AA, Shiffman S, Schwartz JE, et al. Patient compliance with paper and electronic diaries. Control Clin Trials2003 Apr;24(2):182–99.Dupuis LL, Taddio A, Kerr EN, et al. Development and validation of the pediatric nausea assessment tool for use in children receiving antineoplastic agents. Pharmacotherapy2006;26:1221–31.Disclosure(s)Nothing to disclose

Purpose: Because more than 70 of children with cancer become long-term survivors, more emphasis is put on reducing late effects. Cryopreservation of semen and the intracytoplasmic sperm injection technique makes it possible to obtain pregnancy with very poor sperm quality. Two new semen retrieval methods are described that are applicable in pubertal boys with a fertility potential, although not psychologically ready to produce a semen sample, who are likely to become infertile because of anticancer therapy.

We investigated the association between indicators of fetal growth and neonatal stress and CNS tumours diagnosed in the Denmark, Sweden, Norway and Finland in 1985-2006, using data from national cancer registries and medical birth registries. We included 3,443 children with primary CNS tumours and 16169 individually matched controls drawn randomly from the national population registries.We observed a U-shaped relation between risk for CNS tumors and birthweight, at >4.5 kg (OR 1.27; 95% CI, 1.03-1.55) and < 2.0 kg (OR, 1.50; 95% CI, 1.13-1.99), the latter being attenuated after adjustment for gestational age. Moreover, small-forgestational age (OR, 1.28; 95% CI, 0.98-1.66) and large-forgestationalage (OR, 1.26; 95% CI, 1.02- 1.55) were both associated with CNS tumors. The OR for preterm births was increased per 1-week decrease in gestational age (OR, 1.58; 95% CI, 1.04-2.44). Increased Ors were also observed for head circumference >38 cm (1.80; 95% CI, 1.18- 2.74), 5-minute Apgar score < 7 (1.44;95% CI, 0.98-2.12), and breech presentation (1.33; 95% CI, 1.04-1.69). The observed associations varied little by histologic subgroup.This study provides evidence that several intrauterine and neonatal factors are associated with higher risk of CNS tumours, indicating an early onset or, alternatively, potentially harmful exposures in the neonatal period that might be preventable.

Background:In adults PET scanning of CNS tumors with the tracer FDG (18F-flourodeoxyglucose) can provide information about the degree of malignancy, tumor extent, and dissemination. FDG PET can also be able to assess tumor response to therapy and to differentiate recurrence from necrosis. Although CNS tumors are the most common solid tumor in childhood, so far only few PET-studies have been reported. Pre-operative assessment of malignancy would facilitate surgical planning and the use of pre-operative chemotherapy.