Helena Mörse

Identification of cryptic aberrations and characterization of translocation breakpoints using array CGH in high hyperdiploid childhood acute lymphoblastic leukemia.

High hyperdiploidy, characterized by non-random trisomies, is the largest cytogenetic subgroup in childhood acute lymphoblastic leukemia (ALL). It is not known whether the gained chromosomes are sufficient for leukemogenesis or if additional genetic aberrations are necessary. However, the suboptimal chromosome morphology of hyperdiploid ALLs makes detection of structural abnormalities difficult if using cytogenetic techniques; alternative methods are, therefore, needed. We performed array comparative genome hybridization (CGH) analyses, with a resolution of 100 kb, of eight cases of high hyperdiploid childhood ALL to characterize structural abnormalities found with G-banding/multicolor fluorescence in situ hybridization (FISH) and to detect novel changes. The non-centromeric breakpoints of four rearrangements, including three translocations and one 1q duplication, were narrowed down to <0.2 Mb. Furthermore, four submicroscopic imbalances involving 0.6–2.7 Mb were detected, comprising two segmental duplications involving 1q22 and 12q24.31 in one case and two hemizygous deletions in 12p13.2–31 – including ETV6 – and in 13q32.3–33.1 in another case. Notably, FISH analysis of the latter revealed an associated reciprocal t(3;13)(q?;32.2–33.1). In conclusion, the array CGH analyses revealed putative leukemia-associated submicroscopic imbalances and rearrangements in 2/8 (25%) hyperdiploid ALLs. The detection and characterization of these additional genetic aberrations will most likely increase our understanding of the pathogenesis of high hyperdiploid childhood ALL.

Acute onset of ovarian dysfunction in young females after start of cancer treatment

Female childhood cancer survivors are at risk of ovarian failure and premature ovarian insufficiency. We hereby present an interim analysis of a prospective observational study of ovarian function during cancer treatment of young females in relation to clinical factors.

MLL/GRAF Fusion in an Infant Acute Monocytic Leukemia (AML M5b) with a Cytogenetically Cryptic ins(5;11)(q31;q23q23)

More than 30 fusions involving the MLL gene at 11q23 have been reported in acute myeloid leukemia (AML). Some of these chimeras are rather common, such as MLL/MLLT3(AF9), but many are quite rare, with some, for example, MLL/GRAF, described only in a single case. The MLL/GRAF fusion, in which the reciprocal hybrid was not expressed, suggesting that the former transcript was the leukemogenic one, was detected in a juvenile myelomonocytic leukemia with a t(5;11)(q31;q23). Here, we report a second case—an infant acute monocytic leukemia (AML M5b)—with an MLL/GRAF fusion. By conventional G‐banding, the karyotype was normal. However, Southern blot and fluorescence in situ hybridization analyses revealed that MLL was rearranged and that the 5′ part of the MLL gene was inserted into 5q in the vicinity of 5q31, which harbors GRAF. Reverse‐transcriptase polymerase chain reaction (PCR) showed that exon 9 of MLL was fused in‐frame with exon 19 of GRAF. Extralong genomic PCR with subsequent sequence analysis demonstrated that the breakpoints occurred in intron 9 of MLL, nine base pairs (bp) downstream from exon 9, and in intron 18 of GRAF, 117 bp downstream from exon 18. A 6‐bp insertion (ACACTC) of unknown origin was present at the junction. The putative MLL/GRAF fusion protein would retain the AT‐hook DNA‐binding domain, the DNA methyl transferase motif, the transcription repression domain of MLL, and the SH3 domain of GRAF. As expected, the reciprocal GRAF/MLL was neither expressed nor generated at the genomic level as a consequence of the ins(5;11)(q31;q23q23). On the basis of the now‐reported two cases with MLL/GRAF, we conclude that this transcript—but not the reciprocal one—characterizes a rare genetic subgroup of infant AML.

Plasma neutrophil lipocalin, elastase-alpha1-antitrypsin complex and neutrophil protease 4 in preterm infants with respiratory distress syndrome.

Respiratory distress syndrome (RDS) and chronic lung disease of prematurity (CLD) are associated with inflammation of the airways and interstitial tissue of the lung. It is hypothesized that RDS severity and the risk of developing CLD may be correlated with neutrophil gelatinase‐associated lipocalin (NGAL), a marker of leucocyte activity, human elastase‐α1‐antitrypsin complex (HEAT) or free and complexed neutrophil protease 4 (NP4), markers of proteolytic enzyme secretion from granulocytes. Thirty‐three preterm infants with RDS were enrolled in the study and plasma sampled between 3 and 14 days of life. NGAL, HEAT and NP4 concentrations varied widely in infants with RDS. Significant correlations between subsequent development of CLD and plasma concentrations of HEAT and NP4, respectively, were found on days 3–4 of life, p=0.006 and p=0.02, respectively.

Tracheobronchial aspirate fluid neutrophil lipocalin, elastase- and neutrophil protease-4-alpha1-antitrypsin complexes, protease inhibitors and free proteolytic activity in respiratory distress syndrome.

UNLABELLED This study aimed to determine whether the protease/protease inhibitor balance and neutrophil activity is of pathophysiological importance in the severity and resolution of respiratory distress syndrome (RDS) and the eventual development of neonatal chronic lung disease (CLD). Ventilated preterm infants with RDS (n = 43) were studied during their first week of life. Tracheobronchial aspirate fluid (TAF) concentrations of neutrophil lipocalin, the elastase- and neutrophil protease-4 (NP4) complex concentrations, and alpha1-antitrypsin (alpha1AT), antichymotrypsin (ACT) and secretory leucocyte protease inhibitor (SLPI) levels were analysed. Free proteolytic and elastolytic activities were also determined. CLD correlated with low alpha1AT (p = 0.02) and ACT (p = 0.02) levels at 3-4 d of age and low SLPI (p = 0.03) at 7-8 d of age. No correlations were found between CLD or severity of RDS (as judged from radiological examination) and neutrophil lipocalin, elastase- and NP4-alpha1AT complexes during the first week of life, with one exception: RDS X-ray severity and the elastase-alpha1AT complex concentration were correlated at 3-4d of age (p = 0.02). Free proteolytic activity occurred in the TAF of 7/30 infants tested on day 3-4 and free elastolytic activity in 1 patient. During the rest of the first week of life no free elastolytic or proteolytic activities were observed. Caesarean section was correlated with low levels of SLPI on day 3-4 (p = 0.01), NP4 (p = 0.03) and ACT (p = 0.05) on day 5-6. Gestational age was positively correlated with protease inhibitors and their complexes at 3-4 d of age. CONCLUSION Free proteolytic or elastolytic activity in the TAF of RDS infants in the first week of life occurred by way of exception. Elastase-/NP4-alpha1AT complex or neutrophil lipocalin levels were not correlated with the development of CLD. The correlation between CLD and low alpha1AT or ACT at 3-4 d and SLPI at 7-8 d of age may be due to either immaturity or complex formation. The severity of RDS as judged from radiological examination was correlated with elastase-alpha1AT complex on day 3-4. The main hypothesis, that TAF protease/protease inhibitor levels or imbalance and leucocyte activity are important factors indicating a high risk of severe RDS and subsequent CLD development, was principally not confirmed.

Complications of video-assisted gastrostomy in children with malignancies or neurological diseases

Aim: To test the hypothesis whether the administration of cytostatic drugs close to surgery in children with malignancies influences the rate of postoperative complications. Method: Included in the study were 27 children with malignancies and a control group of 27 neurologically impaired children. All the children had nutritional problems and underwent a video‐assisted gastrostomy (VAG) operation during the period 1997–2002. The children were postoperatively followed up. All complications were documented according to a protocol by a specially trained nurse and correlated to the time elapsed from completion of the last preoperative or the first postoperative cytostatic drug treatment. The complications in the two groups were compared. Results: The children with malignant diseases did not have more postoperative complications of the VAG than those having neurological defects. There was no correlation to complications regarding timing of the operation and administration of cytostatic drugs.

Severe gonadotoxic insult manifests early in young girls treated for Ewing sarcoma

Abstract We prospectively investigated anti-Müllerian hormone (AMH) as a measure of ovarian insult in young females during and after treatment for Wilms tumor (WT), osteosarcoma (OS), and Ewing sarcoma (ES). Twenty-one female childhood cancer patients, with a mean age of 7.9 years (range 0.6–17), entered the study. Levels of AMH, follicle-stimulating hormone (FSH), and luteinizing hormone were monitored at diagnosis and every 3 to 4 months during, and regularly for a mean of 2.6 years after treatment. A profound decline in AMH was seen in the majority of the 21 study patients 3 to 4 months after the beginning of treatment, the exception being patients with WT, of whom 60% showed no such decline. During the remaining treatment, all patients except those with WT not treated with whole abdominal radiotherapy or stem cell transplantation (SCT) had AMH below detection limit. After completion of treatment, patients with OS and WT (without whole abdominal radiotherapy and SCT) recovered in AMH and had FSH in the normal range. In contrast, ES patients showed no AMH recovery and highly fluctuating FSH in the first years of follow-up, except for the 2 youngest patients, who had a late, slow AMH recovery. In conclusion, young female ES patients already showed signs of severe ovarian dysfunction during the first years after cancer treatment similar to patients treated with SCT and abdominal radiotherapy, in contrast to females with WT and OS. Fertility counseling and information concerning fertility preservation procedures should be considered before starting to treat young females with ES.

Reliability of AMH in Serum after Long-term Storage at -80°C and an Extended Thawing Episode

Background: Measurement of anti-Mullerian hormone (AMH) is a valuable clinical tool for evaluating ovarian function. The present study aims to evaluate the reliability of AMH measurements obtained from samples kept for long-term storage with or without intermittent thawing. Methods and findings: Serum samples from 35 young female cancer patients were prospectively collected and stored at -80°C from 2007 until 2010. In 2011, AMH was analyzed with the DSL assay. During storage, the samples were exposed to a freezer error in 2013 that resulted in their being thawed up to 11°C for a maximum of 21 days and then refrozen. In 2014, the same samples (new aliquots) were analyzed with the Ansh-AMH assay. To test the reliability of the results from 2014, we conducted a thawing experiment on serum samples from 10 randomly selected females and compared the Ansh-AMH results for samples stored in a freezer with aliquots from the same samples that were stored at 11°C for 1, 3, 7, 14, and 21 days, respectively. Average AMH levels were 1.6 times higher when assayed with the Ansh-AMH compared with the DSL-AMH, which is in line with reported agreement between these types of assays. The same difference between the assays was found in samples that differed two years in storage time. The Ansh-AMH levels from ten serum samples without long-term storage were not influenced by exposure to 11°C for up to 21 days. Conclusions: The results indicate that long-term storage at -80°C and episodes of thawing have little impact on AMH levels analyzed with current methods. These data are reassuring and enable longitudinal studies to be planned that will analyze all collected serum samples simultaneously.