Correction: Patient-derived oral mucosa organoids as an in vitro model for methotrexate induced toxicity in pediatric acute lymphoblastic leukemia.

[This corrects the article DOI: 10.1371/journal.pone.0231588.].

Patient-derived oral mucosa organoids as an in vitro model for methotrexate induced toxicity in pediatric acute lymphoblastic leukemia.

We have recently established a protocol to grow wildtype human oral mucosa organoids. These three-dimensional structures can be maintained in culture long-term, do not require immortalization, and recapitulate the multilayered composition of the epithelial lining of the oral mucosa. Here, we validate the use of this model to study the effect of Leucovorin (LV) on Methotrexate (MTX)-induced toxicity. MTX is a chemotherapeutic agent used in the treatment of pediatric acute lymphoblastic leukemia. Although effective, the use of MTX often results in severe side-effects, including oral mucositis, which is characterized by epithelial cell death. Here, we show that organoids are sensitive to MTX, and that the addition of LV reduces MTX toxicity, in both a concentration- and timing-dependent manner. Additionally, we show that a 24 hour 'pretreatment' with LV reduces MTX-induced cell death, suggesting that such a pretreatment could decrease mucositis in patients. Taken together, we provide the first in vitro model to study the effect of MTX on wildtype oral mucosa cells. Our findings underscore the relevance of the clinically applied LV regimen and highlight the potential of this model to further optimize modifications in dosing and timing of Leucovorin on oral mucosa cells.

C-Halide bond cleavage by a two-coordinate iron(i) complex.

Iron(i) species are debated as key intermediates during the C-halide activation step in Kumuda type C-C cross-coupling catalysis. However, there is only limited knowledge on the reactivity of isolable iron(i) complexes towards organohalides. Using the known two-coordinate iron(i) complex K{18c6}[FeI(N(SiMe3)2)2] we disclose its proficiency to rapidly cleave different types of carbon halide bonds including even C-F bonds. Mechanistic studies indicate stepwise one-electron processes with the involvement of organoradicals.

Role of proton-coupled folate transporter in pemetrexed resistance of mesothelioma: clinical evidence and new pharmacological tools.

BACKGROUND: Thymidylate synthase (TS) has a predictive role in pemetrexed treatment of mesothelioma; however, additional chemoresistance mechanisms are poorly understood. Here, we explored the role of the reduced-folate carrier (RFC/SLC19A1) and proton-coupled folate transporter (PCFT/SLC46A1) in antifolate resistance in mesothelioma. PATIENTS AND METHODS: PCFT, RFC and TS RNA and PCFT protein levels were determined by quantitative RT-PCR of frozen tissues and immunohistochemistry of tissue-microarrays, respectively, in two cohorts of pemetrexed-treated patients. Data were analyzed by t-test, Fisher's/log-rank test and Cox proportional models. The contribution of PCFT expression and PCFT-promoter methylation to pemetrexed activity were evaluated in mesothelioma cells and spheroids, through 5-aza-2'-deoxycytidine-mediated demethylation and siRNA-knockdown. RESULTS: Pemetrexed-treated patients with low PCFT had significantly lower rates of disease control, and shorter overall survival (OS), in both the test (N = 73, 11.3 versus 20.1 months, P = 0.01) and validation (N = 51, 12.6 versus 30.3 months, P = 0.02) cohorts. Multivariate analysis confirmed PCFT-independent prognostic role. Low-PCFT protein levels were also associated with shorter OS. Patients with both low-PCFT and high-TS levels had the worst prognosis (OS, 5.5 months), whereas associations were neither found for RFC nor in pemetrexed-untreated patients. PCFT silencing reduced pemetrexed sensitivity, whereas 5-aza-2'-deoxycytidine overcame resistance. CONCLUSIONS: These findings identify for the first time PCFT as a novel mesothelioma prognostic biomarker, prompting prospective trials for its validation. Moreover, preclinical data suggest that targeting PCFT-promoter methylation might eradicate pemetrexed-resistant cells characterized by low-PCFT expression.

Malnutrition in Healthy Individuals Results in Increased Mixed Cytokine Profiles, Altered Neutrophil Subsets and Function.

Malnutrition is commonly associated with increased infectious disease susceptibility and severity. Whereas malnutrition might enhance the incidence of disease as well as its severity, active infection can in turn exacerbate malnutrition. Therefore, in a malnourished individual suffering from a severe infection, it is not possible to determine the contribution of the pre-existing malnutrition and/or the infection itself to increased disease severity. In the current study we focussed on two groups of malnourished, but otherwise healthy individuals: moderately malnourished (BMI: 18.4-16.5) and severely malnourished (BMI <16.5) and compared several immune parameters with those of individuals with a normal BMI (≥18.5). Our results show a similar haematological profile in all three groups, as well as a similar ratio of CD4+ and CD8+ T cells. We found significant correlations between low BMI and increased levels of T helper (Th) 1 (Interferon (IFN)-γ, (interleukin (IL)-2, IL-12), Th2 (IL-4, IL-5, IL-13), as well as IL-10, IL-33 and tumor necrosis factor-α, but not IL-8 or C reactive protein. The activities of arginase, an enzyme associated with immunosuppression, were similar in plasma, peripheral blood mononuclear cells (PBMC) and neutrophils from all groups and no differences in the expression levels of CD3ζ, a marker of T cell activation, were observed in CD4+ and CD8+T cells. Furthermore, whereas the capacity of neutrophils from the malnourished groups to phagocytose particles was not impaired, their capacity to produce reactive oxygen species was impaired. Finally we evaluated the frequency of a subpopulation of low-density neutrophils and show that they are significantly increased in the malnourished individuals. These differences were more pronounced in the severely malnourished group. In summary, our results show that even in the absence of apparent infections, healthy malnourished individuals display dysfunctional immune responses that might contribute to increased susceptibility and severity to infectious diseases.

Efficacy and safety of zero-fluoroscopy ablation for supraventricular tachycardias. Use of optional contact force measurement for zero-fluoroscopy ablation in a clinical routine setting.

BACKGROUND: Conventional catheter ablation of cardiac arrhythmias is associated with radiation risks for patients and laboratory personnel. Widespread use of zero-fluoroscopic catheter ablation in clinical routine is limited by safety concerns. This study investigated the feasibility of zero-fluoroscopy catheter ablation using a three-dimensional mapping system and optional catheter contact force technology for an all-comers collective. PATIENTS AND METHODS: The study comprised 184 patients; 91 patients, including 29 pediatric patients, underwent a zero-fluoroscopic electrophysiology (EP) study using the EnSite NavX system with real-time visualization of all electrodes. These patients were matched to a control group, which was treated using fluoroscopy in the same period. Inclusion criteria were documented supraventricular tachycardia or a history of symptomatic paroxysmal supraventricular tachycardia. Transseptal access, if necessary, was achieved under transesophageal echocardiographic guidance for ablation of left-sided arrhythmias. Radiofrequency (using optional contact force measurement) or a cryotechnique was used for ablation. RESULTS: We observed no major acute complications. There were no significant differences between the two groups in the follow-up period. CONCLUSION: Zero-fluoroscopic catheter ablation is generally feasible in right-sided cardiac arrhythmias. Safety concerns regarding left atrial substrates or children can be overcome with optional real-time contact force measurement.

The sodium iodide symporter is unlikely to be a thyroid/breast shared antigen.

PURPOSE: Anti-thyroid peroxidase (TPO) autoantibodies (TPOAb) seem to be protective for patients with breast cancer (BC). Thyroid and breast tissues both express the sodium iodide symporter (NIS), similarly both have a peroxidase activity, TPO and lactoperoxidase (LPO) respectively. We hypothesize a common immune response to a thyroid/breast shared antigen suggesting three putative mechanisms: (1) TPOAb react to both TPO and LPO, (2) TPO could be expressed in BC and (3) patients with TPOAb could have autoantibodies to NIS (NISAb). Previous studies excluded NISAb that block NIS activity in sera of patients with thyroid autoimmunity (TA) and/or BC. This study investigates neutral NISAb (binding without affecting function). METHODS: Clones of CHO cells stably expressing human NIS (hNIS; CHO-NIS) were isolated following transfection of hNIS in pcDNA3 vector. Expression of hNIS mRNA and surface protein was confirmed by PCR and flow cytometry respectively using a hNIS-mouse-monoclonal-antibody. CHO-NIS and controls transfected with the empty pcDNA3 vector (CHO-Empty) were incubated with 42 heat-inactivated human sera followed by an anti-human-IgG-AlexaFluor488-conjugate: 12 with BC, 11 with TA, 10 with both BC and TA and 9 with non-autoimmune thyroid diseases. The Kolmogorov-Smirnov Test was used to compare the fluorescence intensity obtained with CHO-NIS and CHO-Empty, using sera from six young males as a negative control population. RESULTS: None of the 42 sera were positive for NISAb. CONCLUSIONS: NISAb are rare and NIS is unlikely to be a common thyroid/BC shared antigen. We have recently demonstrated TPO expression in BC tissue and are currently investigating TPOAb cross-reactivity with TPO/LPO.

Antifungal prophylaxis with posaconazole vs. fluconazole or itraconazole in pediatric patients with neutropenia.

Pediatric patients with hemato-oncological malignancies and neutropenia resulting from chemotherapy have a high risk of acquiring invasive fungal infections. Oral antifungal prophylaxis with azoles, such as fluconazole or itraconazole, is preferentially used in pediatric patients after chemotherapy. During this retrospective analysis, posaconazole was administered based on favorable results from studies in adult patients with neutropenia and after allogeneic hematopoietic stem cell transplantation. Retrospectively, safety, feasibility, and initial data on the efficacy of posaconazole were compared to fluconazole and itraconazole in pediatric and adolescent patients during neutropenia. Ninety-three pediatric patients with hemato-oncological malignancies with a median age of 12 years (range 9 months to 17.7 years) that had prolonged neutropenia (>5 days) after chemotherapy or due to their underlying disease, and who received fluconazole, itraconazole, or posaconazole as antifungal prophylaxis, were analyzed in this retrospective single-center survey. The incidence of invasive fungal infections in pediatric patients was low under each of the azoles. One case of proven aspergillosis occurred in each group. In addition, there were a few cases of possible invasive fungal infection under fluconazole (n = 1) and itraconazole (n = 2). However, no such cases were observed under posaconazole. The rates of potentially clinical drug-related adverse events were higher in the fluconazole (n = 4) and itraconazole (n = 5) groups compared to patients receiving posaconazole (n = 3). Posaconazole, fluconazole, and itraconazole are comparably effective in preventing invasive fungal infections in pediatric patients. Defining dose recommendations in these patients requires larger studies.

Systemic mesenchymal stem cell administration enhances bone formation in fracture repair but not load-induced bone formation.

Mesenchymal stem cells (MSC) were shown to support bone regeneration, when they were locally transplanted into poorly healing fractures. The benefit of systemic MSC transplantation is currently less evident. There is consensus that systemically applied MSC are recruited to the site of injury, but it is debated whether they actually support bone formation. Furthermore, the question arises as to whether circulating MSC are recruited only in case of injury or whether they also participate in mechanically induced bone formation. To answer these questions we injected green fluorescent protein (GFP)-labelled MSC into C57BL/6J mice, which were subjected either to a femur osteotomy or to non-invasive mechanical ulna loading to induce bone formation. We detected GFP-labelled MSC in the early (day 10) and late fracture callus (day 21) by immunohistochemistry. Stromal cell-derived factor 1 (SDF-1 or CXCL-12), a key chemokine for stem cell attraction, was strongly expressed by virtually all cells near the osteotomy--indicating that SDF-1 may mediate cell migration to the site of injury. We found no differences in SDF-1 expression between the groups. Micro-computed tomography (µCT) revealed significantly more bone in the callus of the MSC treated mice compared to untreated controls. The bending stiffness of callus was not significantly altered after MSC-application. In contrast, we failed to detect GFP-labelled MSC in the ulna after non-invasive mechanical loading. Histomorphometry and µCT revealed a significant load-induced increase in bone formation; however, no further increase was found after MSC administration. Concluding, our results suggest that systemically administered MSC are recruited and support bone formation only in case of injury but not in mechanically induced bone formation.

Comparison of itraconazole, voriconazole, and posaconazole as oral antifungal prophylaxis in pediatric patients following allogeneic hematopoietic stem cell transplantation.

Oral antifungal prophylaxis with extended-spectra azoles is widely used in pediatric patients after allogeneic hematopoietic stem cell transplantation (HSCT), while controlled studies for oral antifungal prophylaxis after bone marrow transplantation in children are not available. This survey analyzed patients who had received either itraconazole, voriconazole, or posaconazole. We focused on the safety, feasibility, and initial data of efficacy in a cohort of pediatric patients and adolescents after high-dose chemotherapy and HSCT. Fifty consecutive pediatric patients received itraconazole, 50 received voriconazole, and 50 pediatric patients received posaconazole after HSCT as oral antifungal prophylaxis. The observation period lasted from the start of oral prophylactic treatment with itraconazole, voriconazole, or posaconazole until two weeks after terminating the oral antifungal prophylaxis. No incidences of proven or probable invasive mycosis were observed during itraconazole, voriconazole, or posaconazole treatment. A total of five possible invasive fungal infections occurred, two in the itraconazole group (4%) and three in the voriconazole group (6%). The percentage of patients with adverse events potentially related to clinical drugs were 14% in the voriconazole group, 12% in the itraconazole group, and 8% in the posaconazole group. Itraconazole, voriconazole, and posaconazole showed comparable efficacy as antifungal prophylaxis in pediatric patients after allogeneic HSCT.

Does thyroid peroxidase provide an antigenic link between thyroid autoimmunity and breast cancer?

Women with breast cancer (BC) and antithyroid peroxidase (TPO) autoantibodies (TPOAb) have a better prognosis than women lacking TPOAb. Sera from women with TPOAb displayed immunoreactivity to BC tissue by immunofluorescence that was not apparent in women without TPOAb. We hypothesize a BC/thyroid shared antigen that provides a target for humoral or cell-mediated immune activity; candidates include the sodium/iodide symporter (expressed in thyroid and BC), cross-reacting epitopes in TPO and lactoperoxidase (LPO) or TPO itself. As the association is with TPOAb, we investigated TPO expression in BC, breast peritumoral tissue (PT), other tissues (tumoral and not) and thyroid as positive control. Transcripts for known and novel TPO isoforms were detected in BC (n = 8) and PT (n = 8) but at approximately 10(4) -fold lower than in thyroid while in non-BC tumors (n = 5) they were at the limit of detection. TPO was expressed also in adipose tissue (n = 17), 10(3) -fold lower than in thyroid. Full length TPO (Mr 105-110 kDa) was detected in Western blots in the majority of examined tissues; preabsorption of the TPO antibody with recombinant TPO (but not LPO) reduced the signal, indicating specificity. The same occurred with some lower molecular weight bands, which could correspond to smaller TPO transcript isoforms, present in all samples. In conclusion, TPO is weakly expressed in BC and other tissues; this could partly explain the high frequency and protective role of TPOAb in BC patients. Further studies will investigate tissue specificity, function and immunogenicity of the novel TPO variants (some BC-specific) identified.

Juvenile metachromatic leukodystrophy 10 years post transplant compared with a non-transplanted cohort.

Metachromatic leukodystrophy (MLD) is a rare inborn error of metabolism leading to severe neurological symptoms and early death. Hematopoietic SCT (HSCT) is considered a treatment option, but results are inconsistent and comparison with natural history is practically missing. We compare a girl with juvenile MLD 10 years after allogeneic HSCT not only with her untreated sister, but also with a large cohort of untreated patients. The girl received HSCT at the age of 5 years when first motor signs appeared. Over 10 years she was stable with respect to her clinical course and gained cognitive abilities. Magnetic resonance imaging (MRI) showed clear regression of white matter changes and magnetic resonance spectroscopy (MRS) demonstrated a reversal of the initial choline increase and N-acetyl-aspartate (NAA) decrease. Only axonal demyelinating neuropathy showed some progression. Her gross motor function and MRI-scores were clearly better compared with her sister and the cohort of untreated patients. Difference to her sister became apparent only 4 years after HSCT. We conclude that HSCT, early in the course of disease, can lead to stabilization of juvenile MLD with a course clearly different from the natural history. HSCT may prevent disease progression, if performed sufficient time before loss of walking, which typically initiates rapid deterioration.

Is the risk of primary hyperparathyroidism increased in patients with untreated breast cancer?

BACKGROUND: An increased frequency of primary hyperparathyroidism (PHP) has been reported in patients with treated breast cancer (BC). PHP has been found in about 7% of BC patients after surgery and radio-, chemio- or hormonal therapy. AIM: To evaluate the frequency of PHP in untreated BC patients. SUBJECTS AND METHODS: We evaluated 186 women with BC and 233 women with thyroid cancer (TC, no.=122) or benign thyroid diseases (BTD, no.=111). In all patients, serum calcium, albumin, PTH, and 25-hydroxyvitamin D (25-OH vitD) were measured before any treatment. RESULTS: Serum calcium concentrations were significantly higher in BC than in TC and BTD groups (median values 9.5 mg/dl, 9.3 mg/dl and 9.3 mg/dl, respectively) but, according to a logistic regression model, calcium was not significantly different between the 3 groups when age was taken into account. In all patients, serum calcium was in the normal range, indicating that no case of overt PHP was present. Five patients (1 in BC, 2 in TC, and 2 in BDT groups) had serum calcium close to the upper limit of normal range, high PTH and low 25-OH vitD, indicating a possible PHP with hypercalcemia masked by concomitant 25-OH vitD deficiency. CONCLUSIONS: In untreated BC group, no patient had overt PHP and 1/186 (0.5%) presented a possible PHP masked by 25-OH vitD deficiency, a PHP frequency much lower than that observed in treated BC patients. These data suggest that the treatments of BC may be responsible for the increased frequency of PHP reported in previous studies.

Allo-SCT using BU, CY and melphalan for children with AML in second CR.

Based on the results from the AML-BFM 98 trial, hematopoietic SCT (HSCT) is recommended for children with AML in second CR only. Here, we retrospectively analyze interphase data of children who underwent HSCT after myeloablative conditioning with BU, CY, and melphalan (BuCyMel) for AML in second remission (CR2) between 1998 and 2009. Out of 152 children, transplant data were available on 109 individuals. Sixty out of 109 children (55%) received BuCyMel. Median age at HSCT was 12.2 years (range 3.0; 18.3). GVHD prophylaxis mostly consisted of CsA and short term MTX with or without antithymocyte globulin. Matched-sibling donors were used for 6/60 analyzed recipients, the remainder either received grafts from matched unrelated (30/60) or mismatched donors. OS after 5 years was 62% (s.e. 6%), relapse incidence 35% (18/60 children) and treatment-related mortality accounted for 12% (7/60) of fatal events. In conclusion, even taking into account possible selection bias in this retrospective analysis, HSCT in CR2 using BuCyMel resulted in a respectable OS. Based on this data the prospective, controlled and centrally monitored AML SCT-BFM 2007 trial has started to recruit patients in January 2010 aiming to generate valid outcome data for further strategy decisions.

Long-term follow-up of children conditioned with Treosulfan: German and Austrian experience.

We report the long-term follow-up of children transplanted with Treosulfan (TREO)-based conditioning in Germany and Austria. Nine centres reported a total of 109 transplantations. Patients were stratified according to the paediatric TRM risk score derived from the paediatric BMT registry (PRST) and compared with the historical transplant population of this registry. Underlying diseases were malignancies, immunodeficiencies, and haematologic and metabolic disorders. TREO total dose ranged from 21-42 g/m(2). Additional conditioning drugs included fludarabine, thiotepa, melphalan, CY and/or TBI. EFS at 3 years for non-malignant and malignant diseases was 88% and 49%, respectively. Leukaemia patients in remission had a survival of 51% at 3 years; nonremission patients relapsed and died within 18 months. TRM and OS in the low-risk groups 0 and 1 were similar to PRST controls. TRM in the high-risk groups 2 and 3 was markedly lower (9% vs 28% and 13% vs 53%, respectively) than in the PRST group, but OS was similar. In conclusion, TREO-based conditioning regimens in children resulted in excellent engraftment and long-term survival in nonmalignant disease. In high-risk malignancy, low acute toxicity was followed by low TRM but it did not translate into increased survival.

High prevalence of breast cancer in patients with benign thyroid diseases.

BACKGROUND: In patients with breast cancer (BC) a high prevalence of benign thyroid diseases (BTD) has been described, Hashimoto's thyroiditis accounting to a large extent for this association. The aim of this study was to evaluate the prevalence of BC in a large group of patients with BTD. PATIENTS: Clinical records of 622 consecutive patients with BTD were examined. BC prevalence in BTD patients was compared with BC frequency in general population living in the same country. RESULTS: BC prevalence in patients with BTD (38/622; 6.11%) was significantly higher (p=0.0002) compared to BC frequency in general population (2.07%). When patients were divided according to the age of menopause, in females older than 49 yr BC frequency in BTD was significantly higher than in age-matched population (7.6 vs 3.3%; p=0.006), while in females aged 30-49 yr BC frequency in BTD was higher, but not statistically significantly, than in agematched population (3.7 vs 0.5%; p=0.06). No significant difference in BC prevalence was found when patients were grouped according to the diagnosis of thyroid disorders: Graves' disease, Hashimoto's thyroiditis, nodular goiter associated or not associated with serum thyroid autoantibodies (TAb). No significant difference in BC frequency was observed between TAb+ (26/377; 6.9%) and TAb- (12/245; 4.9%) patients. The distribution of known risk factors for breast malignancies was similar in patients with or without BC. CONCLUSION: In patients with BTD the prevalence of BC is significantly higher than the expected, showing the usefulness of screening for breast malignancy of patients with BTD.