Value of flow cytometry for MRD-based relapse prediction in B-cell precursor ALL in a multicenter setting.

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 CIR5y adjusted for day 29 FCM-MRD, with higher levels in adults (median 2.4 × 10-2 versus 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 > 10-4 associated 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.

Spermatogonial quantity in human prepubertal testicular tissue collected for fertility preservation prior to potentially sterilizing therapy.

STUDY QUESTION: Does chemotherapy exposure (with or without alkylating agents) or primary diagnosis affect spermatogonial quantity in human prepubertal testicular tissue? SUMMARY ANSWER: Spermatogonial quantity is significantly reduced in testes of prepubertal boys treated with alkylating agent therapies or with hydroxyurea for sickle cell disease. WHAT IS KNOWN ALREADY: Cryopreservation of spermatogonial stem cells, followed by transplantation into the testis after treatment, is a proposed clinical option for fertility restoration in children. The key clinical consideration behind this approach is a sufficient quantity of healthy cryopreserved spermatogonia. However, since most boys with malignancies start therapy with agents that are not potentially sterilizing, they will have already received some chemotherapy before testicular tissue cryopreservation is considered. STUDY DESIGN, SIZE, DURATION: We examined histological sections of prepubertal testicular tissue to elucidate whether chemotherapy exposure or primary diagnosis affects spermatogonial quantity. Quantity of spermatogonia per transverse tubular cross-section (S/T) was assessed in relation to treatment characteristics and normative reference values in histological sections of paraffin embedded testicular tissue samples collected from 32 consecutive boy patients (aged 6.3 ± 3.8 [mean ± SD] years) between 2014 and 2017, as part of the NORDFERTIL study, and in 14 control samples (from boys aged 5.6 ± 5.0 [mean ± SD] years) from an internal biobank. PARTICIPANTS/MATERIALS, SETTING, METHODS: Prepubertal boys in Sweden, Finland and Iceland who were facing treatments associated with a very high risk of infertility, were offered the experimental procedure of testicular cryopreservation. Exclusion criteria were testicular volumes >10 ml and high bleeding or infection risk. There were 18 patients with a diagnosis of malignancy and 14 patients a non-malignant diagnosis. While 20 patients had the testicular biopsy performed 1-45 days after chemotherapy, 12 patients had not received any chemotherapy. In addition, 14 testicular tissue samples of patients with no reported testicular pathology, obtained from the internal biobank of the Department of Pathology at Karolinska University Hospital, were included as control samples in addition to reference values obtained from a recently published meta-analysis. The quantity of spermatogonia was assessed by both morphological and immunohistochemical analysis. MAIN RESULTS AND THE ROLE OF CHANCE: The main finding was a significant reduction in spermatogonial cell counts in boys treated with alkylating agents or with hydroxyurea for sickle cell disease. The mean S/T values in boys exposed to alkylating agents (0.2 ± 0.3, n = 6) or in boys with sickle cell disease and exposed to hydroxyurea (0.3 ± 0.6, n = 6) were significantly lower (P = 0.003 and P = 0.008, respectively) than in a group exposed to non-alkylating agents or in biobank control samples (1.7 ± 1.0, n = 8 and 4.1 ± 4.6, n = 14, respectively). The mean S/T values of the testicular tissue samples included in the biobank control group and the patient group exposed to non-alkylating agents were within recently published normative reference values. LIMITATIONS, REASONS FOR CAUTION: Normal testicular tissue samples included in this study were obtained from the internal biobank of Karolinska University Hospital. Samples were considered normal and included in the study if no testicular pathology was reported in the analysed samples. However, detailed information regarding previous medical treatments and testicular volumes of patients included in this biobank were not available. WIDER IMPLICATIONS OF THE FINDINGS: This study summarizes, for the first time, spermatogonial quantity in a prepubertal patient cohort just before and after potentially sterilizing treatments. Boys facing cancer and cytotoxic therapies are regarded as the major group who will benefit from novel fertility preservation techniques. There are no previous reports correlating spermatogonial quantity to cumulative exposure to alkylating agents and anthracyclines (non-alkylating agents) and no information about the timing of cytotoxic exposures among this particular patient cohort. For prepubertal boys in whom fertility preservation is indicated, testicular tissue should be obtained before initiation of chemotherapy with alkylating agents, whilst for those with sickle cell disease and treated with hydroxyurea, this approach to fertility preservation may not be feasible. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from The Swedish Childhood Cancer Foundation (PR2016-0124; TJ2016-0093; PR2015-0073, TJ2015-0046) (J.-B.S. and K.J.), the Jane and Dan Olssons Foundation (2016-33) (J.-B.S.), the Finnish Cancer Society (K.J.), the Foundation for Paediatric Research (J.-B.S.), Kronprinsessan Lovisas Förening För Barnasjukvård/ Stiftelsen Axel Tielmans Minnesfond, Samariten Foundation (J.-B.S.), the Väre Foundation for Paediatric Cancer Research (K.J.) and the Swedish Research Council (2012-6352) (O.S.). R.T.M. was supported by a Wellcome Trust Fellowship (09822). J.P.A.-L. and M.K. were supported by the ITN Marie Curie program 'Growsperm' (EU-FP7-PEOPLE-2013-ITN 603568). The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Results of NOPHO ALL2008 treatment for patients aged 1-45 years with acute lymphoblastic leukemia.

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.

Bone marrow fibrosis in childhood acute lymphoblastic leukemia correlates to biological factors, treatment response and outcome.

We retrospectively evaluated reticulin fiber density (RFD) in 166 diagnostic bone marrow (BM) biopsies and 62 biopsies obtained at treatment day 29 from children with acute lymphoblastic leukemia (ALL). Patients with B-cell precursor (BCP)-ALL showed higher RFD as compared to patients with T-cell ALL (P<0.001). RFD correlated negatively with white blood cell count (P=0.008) in BCP-ALL patients. Patients with high-hyperdiploid ALL (51-61 chromosomes), no high-risk criteria and low RFD showed a favorable outcome when compared to similar patients with high RFD (P=0.002). In BCP-ALL patients, RFD at diagnosis correlated to the levels of minimal residual disease (MRD) analyzed by flow cytometry on treatment day 29 (P=0.001). Accordingly, patients with MRD > or = 10(-4) presented higher RFD at diagnosis compared to patients with MRD < 10(-4) (P=0.003). BCP-ALL patients with low RFD at diagnosis and a rapid reduction of RFD on day 29 had a favorable outcome compared to patients with the same baseline RFD level at diagnosis but a slow RFD reduction (P=0.041). To our knowledge, these findings are novel and may indicate BM fibrosis as a new valuable prognostic marker in childhood ALL. Expanded use of BM biopsy both at diagnosis and during follow-up is suggested.

Obesity and nocturnal gastro-oesophageal reflux are related to onset of asthma and respiratory symptoms.

Several studies have identified obesity as a risk factor for asthma in both children and adults. An increased prevalence of asthma in subjects with gastro-oesophageal reflux (GOR) and obstructive sleep apnoea syndrome has also been reported. The aim of this investigation was to study obesity, nocturnal GOR and snoring as independent risk factors for onset of asthma and respiratory symptoms in a Nordic population. In a 5-10 yr follow-up study of the European Community Respiratory Health Survey in Iceland, Norway, Denmark, Sweden and Estonia, a postal questionnaire was sent to previous respondents. A total of 16,191 participants responded to the questionnaire. Reported onset of asthma, wheeze and night-time symptoms as well as nocturnal GOR and habitual snoring increased in prevalence along with the increase in body mass index (BMI). After adjusting for nocturnal GOR, habitual snoring and other confounders, obesity (BMI >30) remained significantly related to the onset of asthma, wheeze and night-time symptoms. Nocturnal GOR was independently related to the onset of asthma and in addition, both nocturnal GOR and habitual snoring were independently related to onset of wheeze and night-time symptoms. This study adds evidence to an independent relationship between obesity, nocturnal gastro-oesophageal reflux and habitual snoring and the onset of asthma and respiratory symptoms in adults.

[Can asthma deaths in young people be predicted and prevented?]. / Kan astmadöd hos unga förutses och förebyggas?

UNLABELLED: In spite of increased asthma prevalence, the mortality in the disease has decreased during the last two decades in Sweden and in some other countries. However, in the age group 15-24 years an increased death rate was noted at the end of the 1980s. Therefore we started a prospective study from 1994 with the aim of trying to analyse the precipitating factors of all deaths in the ages 1-34 years in which the death certificate alleged asthma or anaphylaxis as the main or contributing cause of death. We conducted a confidential telephone enquiry of the next of kin of the deceased using a modified questionnaire developed by the British Thoracic Association. When available, patient records and post mortem protocols were obtained. RESULTS: The mortality in asthma in the ages 1-34 years has decreased from around 5 per million/year to 0.5/million/year over the period 1987-1998. The main preventable factors found in this analysis are undertreatment, non-compliance, psychosocial factors including alcohol/drug abuse, food allergy and inhalation allergy. Lacking awareness of the risks and underestimation of the severity of the asthma both by the physician and the patient seem to be dominating factors.

Continuous cervical epidural anesthesia in reconstructive hand surgery.

Continuous cervical epidural anesthesia was used for 17 operations in 16 patients undergoing immediate reconstructive surgery after upper-extremity injuries or tumor resection and was continued for postoperative pain management. Routine hemodynamics, arterial blood gases, plasma bupivacaine levels, and skin temperatures were recorded before and after the block. The surgery time ranged from 3 to 18 hours. Postoperative pain management was maintained for up to 6 days. The blocks were adequate for surgery and postoperative pain treatment in all cases. There were no signs or symptoms indicating local anesthetic toxicity. Circulatory and respiratory integrity was well maintained. The patients were all ambulatory the day after surgery and could start physiotherapy immediately. This regional anesthesia technique may have significant advantages over branchial plexus block or general anesthesia for lengthy surgical procedures of the upper extremity.

In situ detection of myocardial infarction in pig by measurements of aspartate aminotransferase (ASAT) activity in the interstitial fluid.

Microdialysis probes permeable to large molecules (m.w. cut-off > 200 kD) were introduced into the myocardium of anaesthetized pigs in order to evaluate their potential for early detection of myocardial ischaemia and enzyme markers for infarction. The left anterior descending coronary artery was occluded for 30 min and the myocardium was reperfused for 3 h. The concentrations of aspartate aminotransferase (ASAT), lactate, glucose and selected free amino acids were measured. The levels in the interstitium of ischaemic and non-ischaemic myocardium were compared with those in plasma from the coronary sinus as well as from a peripheral vein. Twelve probes were inserted in six pigs and withdrawn after 8-72 hours of sampling. No complications occurred. Simultaneous 100% increase of ASAT and lactate was found in myocardial dialysates after 30 min of ischaemia. ASAT activity remained at that level until the end of reperfusion. The plasma peak ASAT level was not attained until after 3 h. Glutamate was the only amino acid which increased significantly in the myocardial interstitium during ischaemia, peaking after 30 min of reperfusion. Dialysates from the unaffected myocardium showed no effects on lactate, ASAT or glutamate. The use of myocardial microdialysis for pre- and postoperative recordings in man is discussed.

Nitrogen saturation induced during winter by experimental NH4NO3 addition to a forested catchment.

To investigate the potential risk of 'nitrogen saturation' in Scandinavian boreal forests, the authors are experimentally adding 30-50 kg N ha(-1) year(-1) as NH4NO3 in precipitation to an entire 0.52-ha 80-year-old spruce forested catchment at Gårdsjön, near Gothenburg on the Swedish west coast. NO3 concentrations in runoff increased from 0 to about 7 microeq liter(-1) (maximum pulse of 43 microeq liter(-1)). The increase occurred in winter; during the April-October growing season, NO3 concentrations were very low. The speed of the response suggests that these forests are already close to saturation.

Oral sotalol reduces the incidence of atrial fibrillation after coronary artery bypass surgery.

Episodes of atrial fibrillation or flutter frequently complicate the postoperative course after coronary bypass surgery. A hundred and one patients undergoing coronary artery bypass surgery were randomized to oral pre- and postoperative treatment with sotalol, a non-selective beta-blocking agent with class-III antiarrhythmic properties (50 patients), or to half the preoperative beta-blocking dose according to the routine of the department (51 patients). Thus, there was no equipotency regarding beta blockade in the two groups. The incidence of atrial fibrillation was 10% in the sotalol group and 29% in the comparison group, p = 0.028. In 10% of the sotalol patients the dose had to be reduced or stopped compared to in none the group given routine treatment. The patients who developed atrial fibrillation were older, but otherwise there was no statistically significant difference between the two groups. Sotalol was effective in reducing the incidence of atrial fibrillation. However, careful titration of the optimal dose should be performed in order to avoid side effects of the beta blockade.

Identification of Casuarina-Frankia strains by use of polymerase chain reaction (PCR) with arbitrary primers.

Free-living N2-fixing Frankia strains isolated from Casuarina sp. were investigated for genomic polymorphism. We used six 10-mer oligonucleotides as single arbitrary primers (AP) for the polymerase chain reaction (PCR) in order to amplify random DNA fragments in the genome of free-living Frankia strains. Agarose-gels of the amplified genomic DNA revealed that two of the six arbitrary primers showed polymorphism in the eight different Frankia genomes. Analysis of the AP-PCR products showed 9 polymorphic bands ranging from 4.1-0.60 kb. We conclude that single arbitrary primers can be used to amplify genomic DNA, and that polymorphism can be detected between the amplification products of the different Frankia genomes.

Biological response in vitro to pulsed high dose rate electrons from a clinical accelerator.

Several studies of biological response to ionizing radiation of high absorbed dose rates have been performed, often with conflicting results. The aim of this study was to establish whether a difference between irradiation at high dose rates and at more conventional dose rates could be verified. Pulsed 50 MeV electrons from a clinical accelerator were used both for the high dose rate experiments (mean dose rate: 3.8 x 10(2) Gy/s) and the reference experiments (mean dose rate: 9.6 x 10(-2) Gy/s). In this study V-79 cells were irradiated in vitro. The experiments were carried out under both oxic and anoxic conditions. No significant difference in relative biological effectiveness (RBE) or oxygen enhancement ratio (OER) was observed at the different dose rates investigated.