INVASIVE FUNGAL INFECTIONS IN CHILDREN TREATED FOR HEMATOLOGIC MALIGNANCIES - A FIVE-YEAR SINGLE CENTER EXPERIENCE.

Invasive fungal infections (IFI) are life-threatening complications of intensive chemotherapy treatment, with the incidence in pediatric patients ranging from 2% to 21%. In this article, we describe our 5-year experience of IFI in pediatric oncology patients and its clinical manifestations with radiological findings, treatment and outcome. A retrospective and descriptive survey of IFI in children with hematologic neoplasms was conducted at the Department of Oncology and Hematology, Zagreb Children's Hospital. Medical charts of children 0-17 years of age, of both sexes, treated for leukemias and lymphomas from January 2016 to December 2020 were reviewed. In a 5-year period, 60 patients were treated for hematologic malignancy, acute lymphoblastic leukemia (ALL) being the most prevalent diagnosis. IFI was verified in 9 (15%) children, predominantly in patients with ALL (75%). The specific causative agent was detected in one child, whereas other infections were classified as probable pulmonary aspergillosis. All the patients received standard prophylaxis with fluconazole and treatment with liposomal amphotericin B and voriconazole. The majority of our patients achieved recovery. IFI prevention, diagnosis and treatment remain a challenge. Uniform prophylaxis and therapy protocols, as well as environmental control are of vital importance for the development of better strategies in the prevention, early detection and treatment of IFI in pediatric hematology patients.

Nutritional status as a predictor of adverse events and survival in pediatric autologous stem cell transplant.

Nutritional status is recognized as an independent and modifiable risk factor of outcome in stem cell transplant. Our research aim was to evaluate the impact of body mass index (BMI) and serum albumin on the prevalence of adverse events and survival in autologous transplant in children. A retrospective study was conducted of autologous transplants performed between 2006 and 2017 in the Children's Hospital Zagreb, Croatia. Nutritional status was assessed at the times of diagnosis, procedure, and discharge using BMI (underweight, normal, obese) and serum albumin (grades 1-4). Adverse events (fever, gastrointestinal toxicity, electrolyte disturbances, dysglycemia) and outcome (3-year, relapse, mortality) were documented. Seventy-seven children (54.5% males, mean age 7.9 years) underwent autologous transplant, mostly for neuroblastoma. In terms of BMI and albumin, which showed significant positive correlation at diagnosis (p = 0.026) and transplant (p = 0.016), most participants were well nourished. Average post-transplant weight loss was 4%. Major toxicities were severe mucositis (72.7%) and hypophosphatemia (31.2%). Relapse and mortality rates were 35.1% and 42.9%, respectively. Hypokalemia (p = 0.041) and hypomagnesemia (p = 0.044) were more prevalent in the underweight group, while obese children experienced significantly less severe mucositis (p = 0.016) and hypophosphatemia (p = 0.038). There was no significant difference regarding outcome among children of different nutritional status, although undernourished children tended to have lower relapse and mortality rates. In conclusion, underweight children are significantly more prone to severe electrolyte disorders and mucositis, and although statistical significance was not reached, are more likely to survive.

[RECOMMENDATIONS FOR PERIOPERATIVE FASTING IN CHILDREN].

Perioperative fasting is a standard procedure for the preparation of patients for surgery. The current guidelines for perioperative fasting in children recommend adherence to the instructions, "2-4-6" i.e. taking clear liquids up to 2 hours, breast milk up to 4 hours, and other non-human milk and solids up to 6 hours prior to surgery. Oral fluid intake is allowed within the first 3 postoperative hours in most pediatric patients. Too long perioperative fasting is not recommended, and may be harmful, both for healthy children so for a specific group of pediatric patients such as cancer patients. It is possible to avoid the adverse effects of prolonged perioperative fasting by appropriate planning of operating programs, good coordination of anesthetic and surgical team and compliance to the guidelines. Although recent studies suggest an advantage of more liberal perioperative approach in relation to the current guidelines in children, for now there is no enough evidence to change existing recommendations. However, according to ongoing studies it is possible that soon there will be evidence enough to support additional shortening of perioperative fasting time interval.

Determination of l-Asparaginase Activity and Its Therapeutic Monitoring in Children with Hematological Malignancies in a Single Croatian Center.

BACKGROUND: Among malignant diseases which develop during childhood, hematological cancers, such as leukemias and lymphomas, are the most common. Outcomes have greatly improved due to the refinement of multiagent chemotherapy regimens that include enhanced asparaginase therapy. In this study, we aimed to evaluate our experiences related to the analytical and clinical significance of determining l-Asparaginase activity. METHODS: Since 2016, the Laboratory of the Children's Hospital Zagreb has routinely measured l-Asparaginase activity and to date, has measured more than 280 examples of activity in a total of 57 children with hematological malignancy treated at the Pediatric Oncology Department of the Children's Hospital Zagreb. Three asparaginase products were available: native E. colil-Asparaginase; a pegylated form of this enzyme; and a native product from Erwinia chrysanthemi. A retrospective data analysis was performed. RESULTS: Out of the fifty-seven children, seven had an allergic reaction (12.3%), five (8.8%) had silent inactivation, and seven (12.3%) developed acute pancreatitis. Allergic reactions and silent inactivation were more common in children treated with native E. colil-Asparaginase, while pancreatitis was more common in children treated with the pegylated form. CONCLUSIONS: The monitoring of l-Asparaginase activity may help to optimize therapy by identifying patients with 'silent inactivation', and/or by dose correction when l-Asparaginase activity is too high (slow elimination).

Familial hemophagocytic lymphohistiocytosis type 3 (FHL3) caused by deep intronic mutation and inversion in UNC13D.

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive, often-fatal hyperinflammatory disorder. Mutations in PRF1, UNC13D, STX11, and STXBP2 are causative of FHL2, 3, 4, and 5, respectively. In a majority of suspected FHL patients from Northern Europe, sequencing of exons and splice sites of such genes required for lymphocyte cytotoxicity revealed no or only monoallelic UNC13D mutations. Here, in 21 patients, we describe 2 pathogenic, noncoding aberrations of UNC13D. The first is a point mutation localized in an evolutionarily conserved region of intron 1. This mutation selectively impairs UNC13D transcription in lymphocytes, abolishing Munc13-4 expression. The second is a 253-kb inversion straddling UNC13D, affecting the 3'-end of the transcript and likewise abolishing Munc13-4 expression. Carriership of the intron 1 mutation was found in patients across Europe, whereas carriership of the inversion was limited to Northern Europe. Notably, the latter aberration represents the first description of an autosomal recessive human disease caused by an inversion. These findings implicate an intronic sequence in cell-type specific expression of Munc13-4 and signify variations outside exons and splice sites as a common cause of FHL3. Based on these data, we propose a strategy for targeted sequencing of evolutionary conserved noncoding regions for the diagnosis of primary immunodeficiencies.

Relationship between tumor vascularity and vascular endothelial growth factor as prognostic factors for patients with neuroblastoma.

Although the role of angiogenesis in tumor progression and response to treatment is generally well-characterized, for neuroblastomas clinical data regarding the contribution of angiogenesis and its predictive capacity remain unclear. The aim of this study was to evaluate whether tumor vascularity in the combination with expression of vascular endothelial growth factor (VEGF) represent prognostic factors for patients with neuroblastoma. Immunohistochemistry using anti-CD34 and anti-VEGF antibodies was used to analyze paraffin-embedded primary tumor tissues from 56 patients diagnosed with neuroblastoma. Tumor vascularity was estimated by calculating the tumor vascular volume fraction (TVVF), and VEGF expression was determined using semi-quantitative scoring. Statistical analyses including multivariate analysis were performed and compared with these two factors. Tumor vascularity had impact on survival of high VEGF expression neuroblastoma patients. Combination of high VEGF expression and TVVF value < or = 5% was independent predictor of overall survival (p-value = 0.0041, odds ratio (OR) (95% CI) = 8.67 (1.99-37.69) by the Cox proportional hazards model). This study revealed for the first time a group of extremely high-risk neuroblastoma with both high VEGF expression and poor vascularity. For these patients reduced rates of survival were observed (37% vs. 92.5%) (p < 0.0001). These patients did not experience a significant improvement following hematopoietic stem cell transplantation, and could be candidates for receiving novel therapies. These results indicate the importance of the mutual relationship between tumor vascularity and VEGF, because it gives better insight into the prognosis of patients with neuroblastoma.

Contraception-related deep venous thrombosis and pulmonary embolism in a 17-Year-old girl heterozygous for factor V leiden, prothrombin G20210A mutation, MTHFR C677T and homozygous for PAI-1 mutation: report of a family with multiple genetic risk factors and review of the literature.

We present the case of a 17-year-old girl who suddenly woke up with localized pain in the left groin and the inability to twist her leg. After comprehensive physician and laboratory examinations, deep venous thrombosis with consequent pulmonary embolism was ascertained. She had not experienced any recent trauma, but she had started to take oral contraceptives 6 months prior to the onset of the symptoms. Her parents and sisters had been asymptomatic throughout their lives, but the family history revealed a few thromboembolic accidents. Using DNA analysis, heterozygosity for factor V Leiden, prothrombin gene mutation G20210A and methylenetetrahydrofolate reductase C677T, as well as the homozygous 4G/4G genotype in the plasminogen activator inhibitor 1 were identified in our patient. Subsequently, DNA analysis was performed in all living family members, and multiple factors associated with thrombophilia were discovered. Our case confirms the multifactorial cause of thromboembolic events and emphasizes the importance of oral contraceptive use in the onset of venous thrombosis, especially in teenage females. In addition, this case indicates that teenage females with a family history of thrombosis who are making choices about contraception could most likely benefit from advanced thrombophilia testing.

Pre-B-cell acute lymphoblastic leukemia with bulk extramedullary disease and chromosome 22 (EWSR1) rearrangement masquerading as Ewing sarcoma.

We report a 2-year-old female with a subcutaneous tumor who was initially misdiagnosed as suffering from Ewing sarcoma with a positive EWSR1 rearrangement and EWS/FLI1 transcript. After finding lymphoblasts in peripheral blood, the diagnosis of acute lymphoblastic leukemia was established. This necessitated further analysis of the subcutaneous tumor. The tissue was positive for immature B-cell markers and an immunoglobulin heavy chain gene rearrangement, which confirmed the final diagnosis of common type acute lymphoblastic leukemia with bulk extramedullary disease. The patient was treated with chemotherapy and was in remission 30 months after the diagnosis.

Expression and prognostic value of the Ki-67 in Wilms' tumor: experience with 48 cases.

PURPOSE: Ki-67, tumor proliferation marker, is an important prognostic factor in a variety of cancers. In the present study, we investigated the expression and the prognostic value of Ki-67 in nephroblastoma. METHODS: Ki-67 expressions were investigated by immunohistochemistry on paraffin-embedded material in 48 children operated on because of nephroblastoma. Patients were treated according to SIOP protocol. The mean follow-up period was 5.4 years. A proliferation index was obtained by immunohistochemistry using anti-Ki-67 anti-body. RESULTS: The mean Ki-67 proliferation index in the blastemal type was 12.3%, and in the epithelial type, 21.4%. In the anaplastic type, Ki-67 proliferation index was: in the blastemal component 20%, in the stromal 21%, and in the epithelial 31%. In the mixed tumor type, Ki-67 proliferation index was assessed as: in the blastemal component 10%, in the epithelial 33% and in the stromal 31.5%. Proliferation index for the epithelium was significantly higher than those found for the blastema (P = 0.001). A correlation between Ki-67 and tumor stage found proliferation index significantly higher in stages I and II (P = 0.002). CONCLUSION: The results support the conclusion that Ki-67 is a relevant marker for assessing the proliferative activity and tumor cell dynamics of nephroblastoma, but it may not be a good clinical prognostic marker.

Juvenile myelomonocytic leukemia with PTPN11 mutation in a 23-month-old girl.

Juvenile myelomonocytic leukemia (JMML) is a rare clonal myeloproliferative disorder affecting young children. The natural course of JMML is rapidly fatal with 80% of patients surviving less than three years. Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment of JMML. We report a case of a 23-month-old girl who presented with an upper respiratory tract infection, fever, rash, diarrhea, hepatosplenomegaly and abdominal distention. Severe elevation of white blood cell count with monocytosis and myeloid progenitors in the peripheral blood was also detected. Bone marrow smear showed morphology suggestive of JMML, an unspecific immune phenotype and a normal karyotype. DNA analysis revealed a mutation in the PTPN11 gene. Therefore, the final diagnosis of JMML with somatic PTPN11 mutation was established. Following three months of cytostatic therapy with 6-mercaptopurine and low doses of cytarabine partial remission was achieved and allogeneic HSCT was successfully performed. Six months after the diagnosis, the girl was in a good condition and in a complete remission of JMML. Early diagnosis and allogeneic HSCT were crucial for successful treatment outcome.

Familial hemophagocytic lymphohistiocytosis in a 6-week-old male infant.

Familial hemophagocytic lymphohistiocytosis (FLH) is an autosomal recessively inherited multisystem disease. This defect in cellular cytotoxicity is a life threatening condition characterized by fever, rash, splenomegaly, cytopenias and neurologic manifestations. PRF1, UNC13D and STX11 gene defects underlie in about 40-50% of primary cases. Chemoimmunotherapy followed by hematopoietic stem cell transplantation improved disease outcome. We report a case of a 6-week-old boy who presented with a fever, diffuse rash, disseminated intravascular coagulation, hypofibrinogenemia, hypertrigliceridemia, hepatosplenomegaly, leukocytosis with 90% of lymphocytes, granulocytopenia, anemia, trombocytopenia, hyperferritinemia and pathological findings in cerebrospinal fluid. The patient had decreased frequency of NK cells and low NK cell activity in peripheral blood. Bone marrow aspiration analysis showed degenerative changes of histocyte cells, with preserved cytophages (lymphophages and erythrophages) consistent with hematophagocytic syndrome. Given that the molecular diagnosis of the known mutations in genes PRF1 and UNC13D showed a mutation in UNC13D, the diagnosis of familial hemophagocytic lymphohistiocytosis subtype 3 was established. HLH-2004 chemotherapy protocol was performed and partial remission with residual central nervous system disease was achieved. Hematopoietic stem cell transplantation was successfully performed with an unrelated HLA-matched donor. Familiar HLH is generally a progressive and fatal disease. Early diagnosis with molecular genetic analysis and chemoimmunotherapy followed by hematopoietic stem-cell transplantation is the best approach.

Parental type of personality, negative affectivity and family stressful events in children with cancer.

OBJECTIVE: Psychological interactions between parents,children and social environment are very important for childhood health. The type of personality and stressful events are probably also cancer risk factors. We investigated personality types A/B and D (negative affectivity and social inhibition) in parents of children with cancer (PCC), as well as social environmental factors, and family / children's stressful events before the appearance of cancer. SUBJECTS AND METHODS: Bortner Type A Scale for evaluating parental type A/B personality, and 14 question personality test (DS14) for parental type D personality (negative affectivity and social inhibition score) were performed. Questionnaire eligible information about stressful events and social environmental factors in children with cancer (CC) were analyzed. RESULTS: Analyzing 127 PCC and 136 parents of healthy children (PHC) we found no significant differences in A/B type personality and social inhibition. There was significant difference in negative affectivity. PCC had more negative affectivity than PHC. We found more stressful events before cancer appearance in the families of children with cancer (FCC) than in healthy families (FHC), and more children's stressful events in CC then in healthy ones (HC). There were more quarrels in FCC, while CC were more "easy good-mannered children" than HC. CONCLUSIONS: Our results support the hypothesis that stress is a cancer risk factor and the idea that impaired parental functioning may be a mechanism linking family stress with the aetiology of cancer.

Spiritual well-being, intrinsic religiosity, and suicidal behavior in predominantly Catholic Croatian war veterans with chronic posttraumatic stress disorder: a case control study.

We investigated relationships between spiritual well-being (SWB), intrinsic religiosity (IR), and suicidal behavior in 45 Croatian war veterans with chronic posttraumatic stress disorder and 32 healthy volunteers. Compared with the volunteers, the veterans had significantly lower SWB scores (p = 0.000) and existential well-being (EWB) scores (p = 0.000). Scores on the religious well-being (RWB) subscale (p = 0.108) and the IR scale did not differ significantly between the groups (p = 0.803). Veterans' suicidality inversely correlated with SWB (p = 0.000), EWB (p = 0.000), RWB (p = 0.026), and IR (p = 0.041), with the association being stronger for the EWB subscale than for the RWB subscale. Veterans who had attempted suicide at least once in their lifetime had significantly higher Suicidal Assessment Scale scores and lower EWB scores than veterans who never attempted suicide. Low EWB scores may imply an increased risk of suicidality. Some religious activities were more frequent among the veterans than among the healthy volunteers, possibly reflecting the veterans' increased help-seeking behavior due to poor EWB.

Lemierre Syndrome in Adolescent with Active Ulcerative Colitis.

Inflammatory bowel disease (IBD) is a well-recognized risk factor for thrombotic events in adults but data on children are scarce. In the great majority of adult patients, thrombotic events are usually deep vein thrombosis and pulmonary embolism. Other sites such as jugular veins are extremely rare. We present a case of Lemierre syndrome in an adolescent girl with active ulcerative colitis and discuss possible risk factors. This is the first reported case of severe Lemierre syndrome with thrombus extension to cranial veins in a patient with ulcerative colitis. Early recognition of Lemierre syndrome in patients who present with rapidly worsening symptoms of neck pain, fever and signs of pharyngitis is imperative because it increases a chance of favorable prognosis. It is important for pediatricians treating IBD patients not to underestimate possible thrombotic events in children with IBD. Recognition of additional risk factors is crucial for prompt diagnosis and adequate treatment.

Mast-cell sarcoma of the tibia.

The mast-cell sarcoma of a bone is described here for the first time. The tumour presented in a 4-year-old boy, with pain, oedema and deformation of his right lower leg. Radiological findings revealed a destructive tumourous mass. Histopathological examination showed the tumour to be composed of large, atypical cells, with hyperchromatic oval and polygonal nuclei. The cytoplasm around them was eosinophilic with many basophilic and toluidine-blue-positive granules. These atypical mast cells were positive for chloroacetate esterase, c-kit, tryptase and negative for myeloperoxidase. The primary disease quickly progressed to mast-cell leukaemia, and despite intensive chemotherapy the patient died 18 months after first symptoms.

Large volume leukapheresis is efficient and safe even in small children up to 15 kg body weight.

BACKGROUND: The collection of peripheral blood stem cells, although now a routine procedure, is still a challenge in low body weight children because of specific technical and clinical issues. For paediatric patients it is crucial to obtain an adequate number of CD34+ cells with the minimum number of procedures: this can be done using large volume leukapheresis (LVL). MATERIALS AND METHODS: We analysed the efficacy and safety of 54 autologous LVL performed in 50 children (33 [66%] males and 17 [34%] females), median age 2 years (range, 1-5) and median body weight 12 kg (range, 6-15). The procedures were performed with a COBE Spectra previously primed with red blood cells; ACD-A solution and heparin were used as anticoagulants. RESULTS: The target CD34+ cell dose (≥5×10/kg body weight) were collected with one LVL in 46 (92%) patients, while four (8%) patients needed another procedure. All our LVL were well tolerated. Side effects were observed in five (9.2%) patients and one procedure had to be discontinued because of catheter-related haemorrhage. The platelet count decreased significantly (p<0.001) after each procedure but without bleeding or need for transfusion support. DISCUSSION: Our experience confirms that LVL is efficient and safe even in small children, if the procedure is adjusted considering the weight and age of child. The most important factors are good venous access, adequate preparation of the child's electrolyte status, and surroundings in which the small child as well as parents feel comfortable, and can tolerate the procedure better. Although a median platelet loss of 50% can be expected, LVL is safe and reduces the overall number of procedures required. It can be recommended for peripheral blood stem cell collection even in small body weight children with malignant diseases, particularly those who mobilise low numbers of CD34+ cells.

Neonatal hyperimmune T-cell reaction mimicking T-cell non-Hodgkin's lymphoma following BCG and hepatitis B co-vaccination.

We describe a case of a 2-week-old male infant who presented with a rapidly enlarging inguinal mass after having received both the bacille Calmette-Guérin (BCG) and hepatitis B vaccines at birth. The clinical picture raised suspicion of a neoplasm, and an excision biopsy was performed. It showed complete effacement of the lymph node architecture by a diffuse proliferation of monomorphic, mitotically active, and medium-sized T-cell blasts with strong expression of CD99. Coalescent necrotizing granulomas were also seen. The lymph node culture was negative for BCG. Upon expert review and additional molecular diagnostics, the initial pathological diagnosis of lymphoblastic T-cell lymphoma was changed to ectopic BCG lymphadenitis and hyperimmune post-vaccinal reaction. The atypical T-cell proliferation was most likely a result of the adjuvant effects of the co-administered vaccines. Post-vaccinal reactions usually involve the injection site or result in localized lymph node enlargements in the areas draining the inoculation site. This case highlights the importance of the clinical context for accurate interpretation of the pathological findings. In the setting of post-vaccinal lymphadenopathy, a biopsy is rarely needed but, when performed, should be interpreted with great caution.

Vascular endothelial growth factor in children with neuroblastoma: a retrospective analysis.

BACKGROUND: Despite aggressive therapy, advanced stage neuroblastoma patients have poor survival rates. Although angiogenesis correlates with advanced tumour stage and plays an important role in determining the tumour response to treatment in general, clinical data are still insufficient, and more clinical evaluations are needed to draw conclusions. The aim of this study was to evaluate vascular endothelial growth factor (VEGF) expression in patients with neuroblastoma, determine whether it correlates with other prognostic factors and/or therapeutic response, and to assess should VEGF be considered in a routine diagnostic workup. MATERIALS AND METHODS: VEGF expression was determined by immunohistochemistry using anti-VEGF antibody in paraffin embedded primary tumour tissue from 56 neuroblastoma patients. Semiquantitative expression of VEGF was estimated and compared with gender, age, histology, disease stage, therapy, and survival. Statistical analyses, including multivariate analysis, were performed. RESULTS: VEGF expression correlated with disease stage and survival in neuroblastoma patients. Combination of VEGF expression and disease stage as a single prognostic value for survival (P-value = 0.0034; odds ratio (OR) (95%CI) = 26.17 (2.97-230.27) exhibited greater correlation with survival than individually. Hematopoietic stem cell transplantation significantly improved survival of the advanced stage patients with high VEGF expression. CONCLUSION: VEGF expression should be considered in a routine diagnostic workup of children with neuroblastoma, especially in those more than 18 months old and with advanced disease stage. High VEGF expression at the time of disease diagnosis is a bad risk prognostic factor, and can be used to characterize subsets of patients with an unfavourable outcome.