Comparison of different methods for telomere length measurement in whole blood and blood cell subsets: Recommendations for telomere length measurement in hematological diseases.

Different methods of telomere length measurement are used to identify patients with telomeropathies. In our lab, we established four different methods for telomere length measurement, terminal restriction fragment (TRF) analysis by Southern blot analysis, quantitative PCR (qPCR), quantitative telomere/centromere fluorescence in situ hybridization (T/C-FISH) and fluorescence in situ hybridization combined with flow cytometry (FlowFISH). The methods each have distinct properties and apart from this-according to our experience and data-may have an impact on the individual result. In this study, we therefore compared and validated these methods measuring 154 healthy individuals of different age groups (newborn-81 years). A linear decline was found for every method (Southern blotting 64 bp per year; qPCR 31 bp per year; T/C-FISH 36 bp per year; FlowFISH 50 bp per year). With the equation of the regression line the values of each method (T/S ratio, T/C value, RTL value) can be expressed in absolute kb. All methods showed acceptable accuracy. The analysis indicated good agreement between all methods, with the best agreement between T/C-FISH and FlowFISH. Here, FlowFISH was the most precise, accurate, and reproducible method compared to the other methods. Based on our data, we emphasize the influence of expertise and experience that is required to produce robust and reliable telomere length analyses. Furthermore, we want to provide the scientific community working in diagnostics and research with data-funded advice on how to choose the appropriate method to safely discriminate between natural variability and pathological telomere shortening in individual cases.

Childhood cancer predisposition syndromes-A concise review and recommendations by the Cancer Predisposition Working Group of the Society for Pediatric Oncology and Hematology.

Heritable predisposition is an important cause of cancer in children and adolescents. Although a large number of cancer predisposition genes and their associated syndromes and malignancies have already been described, it appears likely that there are more pediatric cancer patients in whom heritable cancer predisposition syndromes have yet to be recognized. In a consensus meeting in the beginning of 2016, we convened experts in Human Genetics and Pediatric Hematology/Oncology to review the available data, to categorize the large amount of information, and to develop recommendations regarding when a cancer predisposition syndrome should be suspected in a young oncology patient. This review summarizes the current knowledge of cancer predisposition syndromes in pediatric oncology and provides essential information on clinical situations in which a childhood cancer predisposition syndrome should be suspected.

ELANE mutant-specific activation of different UPR pathways in congenital neutropenia.

A number of studies have demonstrated induction of the unfolded protein response (UPR) in patients with severe congenital neutropenia (CN) harbouring mutations of ELANE, encoding neutrophil elastase. Why UPR is not activated in patients with cyclic neutropenia (CyN) carrying the same ELANE mutations is unclear. We evaluated the effects of ELANE mutants on UPR induction in myeloid cells from CN and CyN patients, and analysed whether additional CN-specific defects contribute to the differences in UPR induction between CN and CyN patients harbouring identical ELANE mutations. We investigated CN-specific p.C71R and p.V174_C181del (NP_001963.1) and CN/CyN-shared p.S126L (NP_001963.1) ELANE mutants. We found that transduction of haematopoietic cells with p.C71R, but not with p.V174_C181del or p.S126L ELANE mutants induced expression of ATF6, and the ATF6 target genes PPP1R15A, DDIT3 and HSPA5. Recently, we found that levels of secretory leucocyte protease inhibitor (SLPI), a natural ELANE inhibitor, are diminished in myeloid cells from CN patients, but not CyN patients. Combined knockdown of SLPI by shRNA and transduction of ELANE p.S126L in myeloid cells led to elevated levels of ATF6, PPP1R15A and HSPA5 RNA, suggesting that normal levels of SLPI in CyN patients might protect them from the UPR induced by mutant ELANE. In summary, different ELANE mutants have different effects on UPR activation, and SLPI regulates the extent of ELANE-triggered UPR.

Pediatric Colorectal Carcinoma is Associated With Excellent Outcome in the Context of Cancer Predisposition Syndromes.

INTRODUCTION: Colorectal carcinoma (CRC) is the second most common adult cancer in Germany, however, it is extremely rare in children and adolescents. In these patients, previous literature describes aggressive behavior and diagnosis at advanced stage. METHOD: Thirty-one patients with CRC age ≤ 18 years and treated between 1990 and 2012 have been identified through the structures and registries of the German Society for Pediatric Oncology and Hematology. RESULTS: The age range was 9-18 years (median 13.5 years); the median follow-up time was 43.9 months (range 1-124 months). Twenty-six patients (84%) were tested for a genetic tumor syndrome (GTS); of these, 11 patients (35% of all patients) tested positive (eight cases of Lynch syndrome, one patient with familial adenomatous polyposis, two patients with constitutional mismatch repair deficiency). An unfavorable histology was reported in 55% of the records (n = 17), a poor differentiation (grade III) in 68% of carcinoma (n = 21). Overall survival (OS) and event-free survival at 5 years was 52.0% and 65.6%, respectively. Five-year survival according to stage was 100% in stage II (n = 2), 100% in stage III (n = 13), and 12.9% in stage IV (n = 15; P < 0.001). Five-year OS in patients with and without a defined GTS was 100% and 36.5% (P = 0.019), respectively. CONCLUSION: Children and adolescents with CRC are frequently diagnosed in advanced stages and have an unfavorable prognosis. In this study, a high percentage of pediatric CRC patients presented with a tumor predisposition syndrome and showed an especially favorable OS.

The relationship between the site of metastases and outcome in children with stage IV Wilms Tumor: data from 3 European Pediatric Cancer Institutions.

The aim of this study was to analyze in detail the site of metastasis of stage 4 Wilms tumor (WT) and its correlation with outcome. The databases from 3 major European pediatric cancer institutions were screened for children with WT between 1994 and 2011. Of 208 children identified, 31 (14.9%) had metastases at diagnosis. The lung was affected in 29 children (93.5%) and the liver in 6 children (19.4%). Twenty-seven children (87.1%) had metastases isolated to 1 organ, with the lung being the most common site (80.7%). Five-year overall survival was significantly better in those children with distant disease in either lung or liver (95.8%) compared with those affected in both lung and liver (57.1%, P=0.028). Further, prognostic markers were the response of metastases to preoperative chemotherapy (P=0.0138), high-risk histology (P=0.024), and local stage (P=0.026). Five-year overall survival was 82.1% and 5-year event-free survival was 67.9%. The overall follow-up time was 74.1 and 87.2 (2 to 151) months among survivors, and the treatment-related complication rate was 16.7%. In conclusion, in our series of stage 4 WT, prognosis was excellent if histology was favorable, metastatic disease was isolated to either lungs or liver, and if metastases responded to preoperative chemotherapy.

Inflammatory bowel disease and mutations affecting the interleukin-10 receptor.

BACKGROUND: The molecular cause of inflammatory bowel disease is largely unknown. METHODS: We performed genetic-linkage analysis and candidate-gene sequencing on samples from two unrelated consanguineous families with children who were affected by early-onset inflammatory bowel disease. We screened six additional patients with early-onset colitis for mutations in two candidate genes and carried out functional assays in patients' peripheral-blood mononuclear cells. We performed an allogeneic hematopoietic stem-cell transplantation in one patient. RESULTS: In four of nine patients with early-onset colitis, we identified three distinct homozygous mutations in genes IL10RA and IL10RB, encoding the IL10R1 and IL10R2 proteins, respectively, which form a heterotetramer to make up the interleukin-10 receptor. The mutations abrogate interleukin-10-induced signaling, as shown by deficient STAT3 (signal transducer and activator of transcription 3) phosphorylation on stimulation with interleukin-10. Consistent with this observation was the increased secretion of tumor necrosis factor alpha and other proinflammatory cytokines from peripheral-blood mononuclear cells from patients who were deficient in IL10R subunit proteins, suggesting that interleukin-10-dependent "negative feedback" regulation is disrupted in these cells. The allogeneic stem-cell transplantation performed in one patient was successful. CONCLUSIONS: Mutations in genes encoding the IL10R subunit proteins were found in patients with early-onset enterocolitis, involving hyperinflammatory immune responses in the intestine. Allogeneic stem-cell transplantation resulted in disease remission in one patient.

Ultra-Sensitive CSF3R Deep Sequencing in Patients With Severe Congenital Neutropenia.

High frequency of acquired CSF3R (colony stimulating factor 3 receptor, granulocyte) mutations has been described in patients with severe congenital neutropenia (CN) at pre-leukemia stage and overt acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Here, we report the establishment of an ultra-sensitive deep sequencing of a CSF3R segment encoding the intracellular "critical region" of the G-CSFR known to be mutated in CN-MDS/AML patients. Using this method, we achieved a mutant allele frequency (MAF) detection rate of 0.01%. We detected CSF3R mutations in CN patients with different genetic backgrounds, but not in patients with other types of bone marrow failure syndromes chronically treated with G-CSF (e.g., Shwachman-Diamond Syndrome). Comparison of CSF3R deep sequencing results of DNA and cDNA from the bone marrow and peripheral blood cells revealed the highest sensitivity of cDNA from the peripheral blood polymorphonuclear neutrophils. This approach enables the identification of low-frequency CSF3R mutant clones, increases sensitivity, and earlier detection of CSF3R mutations acquired during the course of leukemogenic evolution of pre-leukemia HSCs of CN patients. We suggest application of sequencing of the entire CSF3R gene at diagnosis to identify patients with inherited lost-of-function CSF3R mutations and annual ultra-deep sequencing of the critical region of CSF3R to monitor acquisition of CSF3R mutations.

A novel germline POLE mutation causes an early onset cancer prone syndrome mimicking constitutional mismatch repair deficiency.

In a 14-year-old boy with polyposis and rectosigmoid carcinoma, we identified a novel POLE germline mutation, p.(Val411Leu), previously found as recurrent somatic mutation in 'ultramutated' sporadic cancers. This is the youngest reported cancer patient with polymerase proofreading-associated polyposis indicating that POLE mutation p.(Val411Leu) may confer a more severe phenotype than previously reported POLE and POLD1 germline mutations. The patient had multiple café-au-lait macules and a pilomatricoma mimicking the clinical phenotype of constitutional mismatch repair deficiency. We hypothesize that these skin features may be common to different types of constitutional DNA repair defects associated with polyposis and early-onset cancer.

Thoracoscopic biopsy in children with diffuse parenchymal lung disease.

Lung biopsy is necessary for establishing the diagnosis in patients with otherwise unclassified diffuse or localized parenchymal lung disease. This study aimed to assess the safety and accuracy of video-assisted thoracoscopic (VATS) lung biopsy in children with diffuse parenchymal lung disease (DPLD). In addition we aimed to evaluate the value of this technique with respect to the spectrum of diseases encountered, correlating histological diagnosis with treatment decisions and subsequent clinical outcome. Data from all patients (n = 21) who underwent surgical lung biopsy for suspected DPLD between March 2001 and August 2006 were collected prospectively. Median age was 3 years, 8 months (range 11 days to 15 years, 2 months). All lung biopsies were performed by VATS under general anesthesia. Median operative time was 45 min (range 25-100 min). Conversion to minithoracotomy due to cardiorespiratory difficulties was necessary in two young infants. There were no further intraoperative complications. In 8/21 children, a chest tube was inserted postoperatively for a median of 2 days (range 1-5 days). In one patient, prolonged air-leakage was managed thoracoscopically on postoperative day 9. There were no other postoperative complications. The specimens were of adequate volume and quality and a histopathological diagnosis was obtained for all patients. There was a broad spectrum of different diagnoses which led to specific therapeutic decisions. Subsequent medical treatment was beneficial in the majority of the patients. In conclusion, VATS is a safe and effective procedure for diagnosis of children with suspected DPLD. Diagnostic accuracy is high, morbidity rates are low, and patients may benefit from avoiding thoracotomy.

LigaSure in laparoscopic transperitoneal heminephroureterectomy in children: a comparative study.

BACKGROUND: This study was conducted to evaluate the feasibility of using the LigaSure vessel sealing system (Valleylab, Boulder, CO) in laparoscopic transperitoneal vs. open retroperitoneal heminephroureterectomy in children. MATERIALS AND METHODS: Seven consecutive patients with impaired renal duplex systems underwent laparoscopic heminephroureterectomies using LigaSure between April 2003 and April 2005. The operative time, complications, and hospital stay were analyzed prospectively. The data of 7 consecutive patients who had undergone open retroperitoneal heminephroureterectomy from 2001 to 2003 were analyzed for comparison purposes. The mean ages, underlying disease, and location of the affected kidney pole were not significantly different between these groups. RESULTS: There were no intraoperative complications during laparoscopic heminephroureterectomy and all procedures were completed laparoscopically. The mean operative time of 144 minutes (range, 90-210 minutes) for laparoscopic heminephroureterectomy was somewhat longer than in open heminephroureterectomy-mean time 110 minutes (range, 60-165 minutes) (P = 0.5). Complications of open retroperitoneal heminephroureterectomy included bleeding of the surface of the remaining kidney pole in one patient, requiring extensive hemostatic suturing. Postoperative recovery was uneventful in all laparoscopic procedures, whereas intermittent retention of urine was noticed in one patient undergoing the open procedure. CONCLUSION: Laparoscopic heminephroureterectomy using LigaSure is feasible in children and has a similar operative time compared to conventional heminephroureterectomy.

Exposure to carbon dioxide and helium reduces in vitro proliferation of pediatric tumor cells.

BACKGROUND: Minimally invasive techniques are increasingly applied to children with malignant tumors. We showed previously that CO(2) used for pneumoperitoneum modulates the function of macrophages and polymorphonuclear cells via direct effects and via acidification. Numerous in vitro and small animal model studies also confirmed an alteration of the behavior of several types of adult tumor cells by CO(2). The impact of CO(2) and other gases used for pneumoperitoneum on the behavior of various pediatric tumors has not yet been determined. METHODS: Cell lines of neuroblastoma (IMR 32, SK-N-SH, Sy5y), lymphoma (Daudi), hepatoblastoma (Huh 6), hepatocellular carcinoma (Hep G2), and rhabdomyosarcoma (Te 671) were incubated for 2 h. Incubation was performed with 100% CO(2), 100% helium, and 5% CO(2) as control. Cell proliferation was determined by the MTT-assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] by actively growing cells to produce a blue formazan product. The MTT-assay was performed before, directly after incubation, and daily for 4 days. Vitality of the cells was determined by trypan blue. The extracellular pH during incubation was measured during gas exposition every 10 min using Bayer Rapid Lab 855. RESULTS: CO(2) for 2 h significantly decreased the proliferation of neuroblastoma, lymphoma, hepatoblastoma, and hepatocellular carcinoma cells. This decrease persisted over 4 days in neuroblastoma, lymphoma, and hepatocellular carcinoma cells. The CO(2) had no impact on hepatoblastoma and rhabdomyosarcoma cells. Helium had a similar effect on neuroblastoma cells. After 4 days, a significant decrease of cell activity was found in two neuroblastoma cell lines and in hepatoblastoma cells. Helium had no effect on lymphoma and hepatocellular carcinoma cells. The extracellular pH was 6.2 during incubation with CO(2), and 7.6 during incubation with helium. CONCLUSION: CO(2) and helium may affect the proliferation of some pediatric tumor cell lines in vitro. However, some of these effects and the impact on the extracellular pH are differential. The role of pH modulation, hypoxia and direct effects of gases remain to be investigated before a general recommendation on the use of minimally invasive techniques in pediatric oncology can be given.

Laparoscopic resection of congenital choledochal cyst, hepaticojejunostomy, and externally made Roux-en-Y anastomosis.

The authors present a 3-month-old patient with a congenital choledochal cyst, which was asymptomatic until treatment. On laparoscopy, a type I choledochal cyst was confirmed and excised laparoscopically. A Roux-en-Y anastomosis was constructed after exteriorization of the small bowel via the infraumbilical trocar incision. A laparoscopic end-to-side hepaticojejunostomy was carried out. The operation lasted 4(1/2) hours, without intraoperative problems. Oral food intake was started on day 2 and well tolerated with bile stained stools. Symptoms of bowel obstruction occurred on day 8. On minilaparotomy, the Roux-en-Y anastomosis was found to be adherent to the mesenterium of the colon, leading to obstruction. After mobilizing the loop, the postoperative course was uneventful. We conclude that laparoscopic resection of congenital choledochal cyst and choledochojejunostomy was feasible in the youngest patient operated on so far. However, adhesive small bowel obstruction can also occur, as after conventional operation, when the bowel is exteriorized for Roux-en-Y hepaticojejunostomy.

Thoracoscopic resection of intra- and extralobar pulmonary sequestration in the first 3 months of life.

BACKGROUND: Thoracoscopic techniques have gained increasing acceptance in pediatric surgery, but experience with newborns and small children is limited. To our knowledge, a series of minimally invasive resection of pulmonary sequestration in newborns has not yet been reported in the literature. We report on 5 patients with pulmonary sequestration thoracoscopically. METHODS: From November 2000 to November 2002, 5 patients underwent thoracoscopic resection of pulmonary sequestration. Ages ranged from 4 to 91 days. Two patients had postnatal pulmonary symptoms. Preoperative diagnosis was dubious in 4 children. There were 4 extralobar and 1 intralobar pulmonary sequestrations. RESULTS: Thoracoscopy was performed with 3-mm instruments and 3 to 5 ports. All procedures were completed successfully. The median duration of the operation was 95 minutes (range, 63-117 minutes), and visualization was excellent. Anomalous blood vessels were clipped and/or ligated. Four patients were extubated immediately after the operation, 1, the day after. The postoperative course was uneventful in all children. At follow-up after 14 months (mean; range, 10-19 months), all patients were free of symptoms and had normal chest x-rays. CONCLUSION: Thoracoscopy is feasible for resection of intra- and extralobar pulmonary sequestrations during the first 3 months of life.

Laparoscopically assisted gastric pull-up for long gap esophageal atresia.

BACKGROUND: Laparoscopically assisted gastric pull-up procedure has been performed in adults for various conditions. The authors report the first patient, who underwent laparoscopically assisted esophageal replacement for long gap esophageal atresia. METHODS: The patient had Down's syndrome and long gap esophageal atresia without fistula. A gastrostomy was performed right after birth, and a suction drain was positioned in the upper esophageal pouch. Esophageal replacement took place at the age of 3 months. The laparoscopic operation included complete mobilization of the stomach, resection of the lower esophageal stump (Endo-GIA), pyloroplasty, and transhiatal dissection. After a right cervical approach, the gastric pull-up was performed through the posterior mediastinum, and the upper anastomosis was completed. Finally, a laparoscopic jejunostomy was performed. RESULTS: The duration of the operation was 4.5 hours. The intra- and postoperative courses were uneventful. Feeding via the jejunostomy was started on day 1. Gastric emptying of contrast media was documented by x-ray examination. Oral feeding was started on day 8 and is now, 3 months postoperative, well tolerated. CONCLUSIONS: This is the first report on laparoscopically assisted gastric pull-up for long gap esophageal atresia. The technique represents an option for the treatment of long gap esophageal atresia.