Children with malignancies and septic shock - an attempt to understand the risk factors

Abstract Objectives: To explain the high mortality of septic shock in children with cancer. Methods: A retrospective cohort from 2016 to 2020, of children aged 0 to 18 years, and septic shock. Results: The authors included 139 patients. Acute lymphocytic leukemia was the most frequent diagnosis (16.5%), and Gram-negative bacteria were the most frequent blood culture isolates (22.3%). There were 57 deaths in ICU (41%), 10 in the first 24 hours of shock (early death). A LASSO model with variables: neutropenia (coefficient 0.215), respiratory (0.81), hematological (1.41), and neurological (0.72) dysfunctions, age (-0.002) and solid tumor recurrence (0.34) generated AUC = 0.79 for the early death outcome. Survivors had significant differences in the PRISM-IV score (mean ± SD 10.9 ± 6.2 in the survivors, 14.1 ± 6.5 in the deceased, p = 0.004), and in the mean number of organ dysfunctions (3.2 ± 1.1 in the survivors, 3.8 ± 6.5 in the deceased, p < 0.001). A positive fluid balance in the first 24 hours of sepsis between 2% and 6% of body weight showed a reduction effect on the probability of death in ICU (hazard ratio 0.47, 95% CI 0.24-0.92, p = 0.027). The recurrence of any cancer was a predictor of in-hospital death, regardless of severity. Conclusions: Recurrence of any cancer is an important risk of sepsis-related death. A positive fluid balance between 20 and 60 mL/kg or 2% and 6% of body weight in the first 24 hours after the onset of sepsis is related to lower mortality.

Children with malignancies and septic shock - an attempt to understand the risk factors.

OBJECTIVES: To explain the high mortality of septic shock in children with cancer. METHODS: A retrospective cohort from 2016 to 2020, of children aged 0 to 18 years, and septic shock. RESULTS: The authors included 139 patients. Acute lymphocytic leukemia was the most frequent diagnosis (16.5%), and Gram-negative bacteria were the most frequent blood culture isolates (22.3%). There were 57 deaths in ICU (41%), 10 in the first 24 hours of shock (early death). A LASSO model with variables: neutropenia (coefficient 0.215), respiratory (0.81), hematological (1.41), and neurological (0.72) dysfunctions, age (-0.002) and solid tumor recurrence (0.34) generated AUC = 0.79 for the early death outcome. Survivors had significant differences in the PRISM-IV score (mean ± SD 10.9 ± 6.2 in the survivors, 14.1 ± 6.5 in the deceased, p = 0.004), and in the mean number of organ dysfunctions (3.2 ± 1.1 in the survivors, 3.8 ± 6.5 in the deceased, p < 0.001). A positive fluid balance in the first 24 hours of sepsis between 2% and 6% of body weight showed a reduction effect on the probability of death in ICU (hazard ratio 0.47, 95% CI 0.24-0.92, p = 0.027). The recurrence of any cancer was a predictor of in-hospital death, regardless of severity. CONCLUSIONS: Recurrence of any cancer is an important risk of sepsis-related death. A positive fluid balance between 20 and 60 mL/kg or 2% and 6% of body weight in the first 24 hours after the onset of sepsis is related to lower mortality.

PRISM 4-C: An Adapted PRISM IV Algorithm for Children With Cancer.

We evaluated the performance of PRISM IV for pediatric cancer patients, and adapted and calibrated the algorithm to calculate mortality probabilities for these patients. An ambidirectional cohort was used, and data were collected from March 2017 prospectively to April 2019, and retrospectively to November 2014. The derivation set for model building contained 500 patients, and a validation set of 503 patients. Risk variables for hospital death were tested in logistic regression models encompassing PRISM IV components. There were 128 deaths (12.7%), being 65 deaths in the validation set. In the validation set, the PRISM IV algorithm had an area under the receiver operating characteristic curve of 0.89, with P=0.13 by Hosmer-Lemeshow test, and predicted 33 of the 65 deaths for a standardized mortality rate of 1.8 (95% confidence interval, 1.4-2.9; P<0.001 by Mid-P test). Our modified algorithm had an area under the receiver operating characteristic curve of 0.93, with P=0.3 by Hosmer-Lemeshow test and an standardized mortality rate of 1.02 (95% confidence interval, 0.79-1.19). The modified algorithm predicted 63.7 of 65 deaths. The PRISM IV algorithm was a poor predictor of mortality in children with cancer. The modified algorithm improved the predictive performance.

CYP Genotypes Are Associated with Toxicity and Survival in Osteosarcoma Patients.

Purpose: Osteosarcoma is the malignant bone tumor most common in children and adolescents. Many cytochrome P-450 (CYP) members detoxify anticancer drugs used in osteosarcoma treatment, and thus, the aim of the present study was to investigate CYP polymorphisms in osteosarcoma patients. Methods: The present study investigated DNA from peripheral blood from 70 osteosarcoma patients treated with high doses of cisplatin, doxorubicin, and methotrexate. CYP1A2*1F (163C>A; rs762551); CYP2C9*3 (1075A>C; rs1057910); and CYP3A5*3 (6986A>G; rs776746) polymorphisms were investigated through real-time PCR using TaqMan probes. Results: The CYP2C9*3 allele did not present any association with clinical events. The CYP1A2 CC/AC genotypes were associated with ototoxicity occurrence (p = 0.041, odds ratio [OR] = 8.4) and high grades of ototoxicity (p = 0.039, OR = 10.7), when compared with patients carrying the CYP1A2 AA genotype. The CYP1A2 CC genotype was associated with high grades of diarrhea (p = 0.043, OR = 4.6) and fever (p = 0.041, OR = 7.1) in comparison with the CYP1A2 AA/AC genotypes. The CYP3A5 CC genotype was associated with weight loss (p = 0.009, OR = 3.8) and high grades of hepatotoxicity (p = 0.010, OR = 4.3) when compared with the CYP3A5 TT/CT genotypes. The CYP3A5 CC/CT genotypes were associated with high grades of vomit (p = 0.013, OR = 10.8), pulmonary relapse absence (p = 0.029, OR = 9.5), and better overall and event-free survivals (p = 0.017, hazard ratio [HR] = 3.1; p = 0.044, HR = 2.5; respectively) when compared with the CYP3A5 AA genotype. Conclusion:CYP1A2*1A and CYP3A5*3 alleles were associated with toxicity events. CYP3A5*3 allele was associated with better survival. Thus, CYP genotypes might be promising markers to tailoring treatment in osteosarcoma patients.

Frequency of Pathogenic Germline Variants in Cancer-Susceptibility Genes in Patients With Osteosarcoma.

Importance: Osteosarcoma, the most common malignant bone tumor in children and adolescents, occurs in a high number of cancer predisposition syndromes that are defined by highly penetrant germline mutations. The germline genetic susceptibility to osteosarcoma outside of familial cancer syndromes remains unclear. Objective: To investigate the germline genetic architecture of 1244 patients with osteosarcoma. Design, Setting, and Participants: Whole-exome sequencing (n = 1104) or targeted sequencing (n = 140) of the DNA of 1244 patients with osteosarcoma from 10 participating international centers or studies was conducted from April 21, 2014, to September 1, 2017. The results were compared with the DNA of 1062 individuals without cancer assembled internally from 4 participating studies who underwent comparable whole-exome sequencing and 27 173 individuals of non-Finnish European ancestry who were identified through the Exome Aggregation Consortium (ExAC) database. In the analysis, 238 high-interest cancer-susceptibility genes were assessed followed by testing of the mutational burden across 736 additional candidate genes. Principal component analyses were used to identify 732 European patients with osteosarcoma and 994 European individuals without cancer, with outliers removed for patient-control group comparisons. Patients were subsequently compared with individuals in the ExAC group. All data were analyzed from June 1, 2017, to July 1, 2019. Main Outcomes and Measures: The frequency of rare pathogenic or likely pathogenic genetic variants. Results: Among 1244 patients with osteosarcoma (mean [SD] age at diagnosis, 16 [8.9] years [range, 2-80 years]; 684 patients [55.0%] were male), an analysis restricted to individuals with European ancestry indicated a significantly higher pathogenic or likely pathogenic variant burden in 238 high-interest cancer-susceptibility genes among patients with osteosarcoma compared with the control group (732 vs 994, respectively; P = 1.3 × 10-18). A pathogenic or likely pathogenic cancer-susceptibility gene variant was identified in 281 of 1004 patients with osteosarcoma (28.0%), of which nearly three-quarters had a variant that mapped to an autosomal-dominant gene or a known osteosarcoma-associated cancer predisposition syndrome gene. The frequency of a pathogenic or likely pathogenic cancer-susceptibility gene variant was 128 of 1062 individuals (12.1%) in the control group and 2527 of 27 173 individuals (9.3%) in the ExAC group. A higher than expected frequency of pathogenic or likely pathogenic variants was observed in genes not previously linked to osteosarcoma (eg, CDKN2A, MEN1, VHL, POT1, APC, MSH2, and ATRX) and in the Li-Fraumeni syndrome-associated gene, TP53. Conclusions and Relevance: In this study, approximately one-fourth of patients with osteosarcoma unselected for family history had a highly penetrant germline mutation requiring additional follow-up analysis and possible genetic counseling with cascade testing.

Pembrolizumab in paediatric patients with advanced melanoma or a PD-L1-positive, advanced, relapsed, or refractory solid tumour or lymphoma (KEYNOTE-051): interim analysis of an open-label, single-arm, phase 1-2 trial.

BACKGROUND: Pembrolizumab is approved for the treatment of advanced cancer in adults; however, no information is available on safety and efficacy in paediatric patients. We aimed to establish the recommended phase 2 dose of pembrolizumab and its safety and antitumour activity in advanced paediatric cancer. METHODS: KEYNOTE-051 is an ongoing phase 1-2 open-label trial. In this interim analysis, children aged 6 months to 17 years were recruited at 30 hospitals located in Australia, Brazil, Canada, France, Germany, Israel, Italy, South Korea, Sweden, the UK, and the USA. Patients with melanoma or a centrally confirmed, PD-L1-positive, relapsed or refractory solid tumour or lymphoma, and a Lansky Play/Karnofsky Performance status score of 50 or higher, received intravenous pembrolizumab at an initial dose of 2 mg/kg every 3 weeks. Pharmacokinetics and dose-limiting toxicities were used to establish the recommended phase 2 dose, and the safety and antitumour activity of this dose were assessed. Primary endpoints were determination of dose-limiting toxicities at the maximum administered dose, safety and tolerability, and the proportion of patients with objective response to pembrolizumab for each tumour type according to the Response Evaluation Criteria in Solid Tumours version 1.1 or the International Neuroblastoma Response Criteria. Safety and efficacy were assessed in all treated patients who received at least one dose of pembrolizumab. Separate reporting of the cohort of patients with relapsed or refractory classical Hodgkin lymphoma was a post-hoc decision. The data cutoff for this interim analysis was Sept 3, 2018. This trial is still enrolling patients and is registered with ClinicalTrials.gov, number NCT02332668. FINDINGS: Of 863 patients screened between March 23, 2015, and Sept 3, 2018, 796 had tumours that were evaluable for PD-L1 expression (278 [35%] were PD-L1-positive); 155 eligible patients were enrolled and 154 had at least one dose of pembrolizumab. The median age of the enrolled patients was 13 years (IQR 8-15). Median follow-up was 8·6 months (IQR 2·5-16·4). No dose-limiting toxicities were reported in phase 1, and pembrolizumab plasma concentrations were consistent with those previously reported in adults; the recommended phase 2 dose was therefore established as 2 mg/kg every 3 weeks. Of the 154 patients treated, 69 (45%) experienced grade 3-5 adverse events, most commonly anaemia in 14 (9%) patients and decreased lymphocyte count in nine (6%) patients. 13 (8%) of the 154 patients had grade 3-5 treatment-related adverse events, most commonly decreased lymphocyte count in three (2%) patients and anaemia in two (1%) patients. 14 (9%) patients had serious treatment-related adverse events, most commonly pyrexia (four [3%]), and hypertension and pleural effusion (two [1%] each). Four patients (3%) discontinued treatment because of treatment-related adverse events, and two (1%) died (one due to pulmonary oedema and one due to pleural effusion and pneumonitis). Of 15 patients with relapsed or refractory Hodgkin lymphoma, two had complete and seven had partial responses; thus, nine patients achieved an objective response (60·0%; 95% CI 32·3-83·7). Of 136 patients with solid tumours and other lymphomas, eight had partial responses (two patients each with adrenocortical carcinoma and mesothelioma, and one patient each with malignant ganglioglioma, epithelioid sarcoma, lymphoepithelial carcinoma, and malignant rhabdoid tumour); the proportion of patients with an objective response was 5·9% (95% CI 2·6-11·3). INTERPRETATION: Pembrolizumab was well tolerated and showed encouraging antitumour activity in paediatric patients with relapsed or refractory Hodgkin lymphoma, consistent with experience in adult patients. Pembrolizumab had low antitumour activity in the majority of paediatric tumour types, and responses were observed in only a few rare PD-L1-positive tumour types, suggesting that PD-L1 expression alone is not sufficient as a biomarker for the selection of paediatric patients who are likely to respond to PD-1 checkpoint inhibitors. Final results of KEYNOTE-051, expected by September, 2022, with the possibility for extension, will report further on the activity of pembrolizumab in Hodgkin lymphoma, microsatellite instability-high tumours, and melanoma. FUNDING: Merck Sharp & Dohme, a subsidiary of Merck & Co.

Adherence and immune response to revaccination following hematopoietic stem cell transplantation at a pediatric onco-hematology reference center.

BACKGROUND: Revaccination after hematopoietic stem cell transplantation (HSCT) is necessary to compensate for the loss of immunological memory. The aims of this study were to evaluate the adherence to revaccination schedule and the humoral immune response to different vaccine antigens in HSCT pediatric and young adult patients. METHODS: Patients submitted to HSCT for over 3 years were recruited. After written informed consent, a questionnaire was filled in, the vaccination card was analyzed, a blood sample was collected and tested by ELISA for diphtheria, Haemophilus influenzae type b (Hib), hepatitis A, hepatitis B, tetanus, measles, rubella, and varicella antibodies. RESULTS: Sixty-three patients (mean age at HSCT, 10.7 years) were evaluated. Forty-one (65%) were male; 34 (54%) had allogeneic and 29 (46%), autologous HSCT. Complete adherence to diphtheria revaccination was found in 79.4% patients and seropositivity was found in 92% of those who completed the revaccination schedule; for Hib, 68.3% adherence and 95.3% seropositivity were observed; for hepatitis A, 63.5% adherence and 92.5% seropositivity; for 3 doses of hepatitis B, 86.8% adherence and 79.2% seropositivity; for tetanus, 79.4% adherence and 100% seropositivity; for measles and rubella, 17.5% adherence and 100% seropositivity; for varicella, 7.9% adherence and 100% seropositivity. The existence of a Vaccination Center for Special Immunobiologicals in patients' municipality was positively associated with completed vaccine schedule; on the other hand, chronic GVHD was negatively associated with revaccination adherence. CONCLUSION: Hematopoietic stem cell transplantation patients showed good seropositivity rates after complete vaccination schedule. However, a low coverage rate was observed for live attenuated antigens.

Genome-wide association study identifies the GLDC/IL33 locus associated with survival of osteosarcoma patients.

Survival rates for osteosarcoma, the most common primary bone cancer, have changed little over the past three decades and are particularly low for patients with metastatic disease. We conducted a multi-institutional genome-wide association study (GWAS) to identify germline genetic variants associated with overall survival in 632 patients with osteosarcoma, including 523 patients of European ancestry and 109 from Brazil. We conducted a time-to-event analysis and estimated hazard ratios (HR) and 95% confidence intervals (CI) using Cox proportional hazards models, with and without adjustment for metastatic disease. The results were combined across the European and Brazilian case sets using a random-effects meta-analysis. The strongest association after meta-analysis was for rs3765555 at 9p24.1, which was inversely associated with overall survival (HR = 1.76; 95% CI 1.41-2.18, p = 4.84 × 10-7 ). After imputation across this region, the combined analysis identified two SNPs that reached genome-wide significance. The strongest single association was with rs55933544 (HR = 1.9; 95% CI 1.5-2.4; p = 1.3 × 10-8 ), which localizes to the GLDC gene, adjacent to the IL33 gene and was consistent across both the European and Brazilian case sets. Using publicly available data, the risk allele was associated with lower expression of IL33 and low expression of IL33 was associated with poor survival in an independent set of patients with osteosarcoma. In conclusion, we have identified the GLDC/IL33 locus on chromosome 9p24.1 as associated with overall survival in patients with osteosarcoma. Further studies are needed to confirm this association and shed light on the biological underpinnings of this susceptibility locus.

Outbreak of Fusarium oxysporum infections in children with cancer: an experience with 7 episodes of catheter-related fungemia.

BACKGROUND: Fusarium species are widely spread in nature as plant pathogens but are also able to cause opportunistic fungal infections in humans. We report a cluster of Fusarium oxysporum bloodstream infections in a single pediatric cancer center. METHODS: All clinical and epidemiological data related to an outbreak involving seven cases of fungemia by Fusarium oxysporum during October 2013 and February 2014 were analysed. All cultured isolates (n = 14) were identified to species level by sequencing of the TEF1 and RPB2 genes. Genotyping of the outbreak isolates was performed by amplified fragment length polymorphism fingerprinting. RESULTS: In a 5-month period 7 febrile pediatric cancer patients were diagnosed with catheter-related Fusarium oxysporum bloodstream infections. In a time span of 11 years, only 6 other infections due to Fusarium were documented and all were caused by a different species, Fusarium solani. None of the pediatric cancer patients had neutropenia at the time of diagnosis and all became febrile within two days after catheter manipulation in a specially designed room. Extensive environmental sampling in this room and the hospital did not gave a clue to the source. The outbreak was terminated after implementation of a multidisciplinary central line insertion care bundle. All Fusarium strains from blood and catheter tips were genetically related by amplified fragment length polymorphism fingerprinting. All patients survived the infection after prompt catheter removal and antifungal therapy. CONCLUSION: A cluster with, genotypical identical, Fusarium oxysporum strains infecting 7 children with cancer, was most probably catheter-related. The environmental source was not discovered but strict infection control measures and catheter care terminated the outbreak.

Vancomycin Therapeutic Targets and Nephrotoxicity in Critically Ill Children With Cancer.

To obtain pharmacokinetic and pharmacodynamic data for vancomycin in a cohort of critically ill pediatric oncology patients, we analyzed 256 measurements of vancomycin concentrations in 94 patients. Variables were tested as possible risk factors for vancomycin-related nephrotoxicity or death for 28 days. We found the following: mean vancomycin trough serum concentration, 15.6 ± 12.4 µg/mL; mean vancomycin clearance, 0.16 ± 0.098 L/h/kg; and mean vancomycin distribution volume, 1.04 ± 0.11 L/kg. Only 13.6% of serum trough level measurements were between 15 and 20 µg/mL. The trough levels showed a strong correlation with the AUC (area under the curve of serum concentrations vs. time over 24 h to the minimum inhibitory concentration ratio), with a 94% positive predictive value for AUC/MIC ≥ 400, but only for MIC=1. The doses that are currently used (60 mg/kg/d) attained the therapeutic target (AUC/MIC ≥ 400) in only 56% of measurements, considering MIC=1. A serum trough level of ≥ 20 µg/mL was an independent risk for nephrotoxicity (P = 0.0008; odds ratio = 17.83). Vancomycin-related nephrotoxicity was a predictor of death for up to 28 days (P = 0.003, odds ratio = 7.68). Currently administered doses of vancomycin do not reach the therapeutic target for critical cancer patients, particularly if staphylococci isolates have a MIC>1.

A Genome-Wide Scan Identifies Variants in NFIB Associated with Metastasis in Patients with Osteosarcoma.

UNLABELLED: Metastasis is the leading cause of death in patients with osteosarcoma, the most common pediatric bone malignancy. We conducted a multistage genome-wide association study of osteosarcoma metastasis at diagnosis in 935 osteosarcoma patients to determine whether germline genetic variation contributes to risk of metastasis. We identified an SNP, rs7034162, in NFIB significantly associated with metastasis in European osteosarcoma cases, as well as in cases of African and Brazilian ancestry (meta-analysis of all cases: P = 1.2 × 10(-9); OR, 2.43; 95% confidence interval, 1.83-3.24). The risk allele was significantly associated with lowered NFIB expression, which led to increased osteosarcoma cell migration, proliferation, and colony formation. In addition, a transposon screen in mice identified a significant proportion of osteosarcomas harboring inactivating insertions in Nfib and with lowered NFIB expression. These data suggest that germline genetic variation at rs7034162 is important in osteosarcoma metastasis and that NFIB is an osteosarcoma metastasis susceptibility gene. SIGNIFICANCE: Metastasis at diagnosis in osteosarcoma is the leading cause of death in these patients. Here we show data that are supportive for the NFIB locus as associated with metastatic potential in osteosarcoma.

Carboplatin in the treatment of Ewing sarcoma: Results of the first Brazilian collaborative study group for Ewing sarcoma family tumors-EWING1.

BACKGROUND: Large cooperative group studies have shown the efficacy of risk-adapted treatment for Ewing sarcoma. However, validation and local adaptation by National cooperative groups is needed. A multicenter protocol to determine the efficacy and safety of a risk-adapted intensive regimen was developed by the Brazilian cooperative group. PROCEDURE: Patients <30 years old with Ewing sarcoma were eligible. Induction chemotherapy consisted of two cycles of ICE (ifosfamide, carboplatin, and etoposide) followed by two cycles of VDC (vincristine, doxorubicin, and cyclophosphamide), followed by local control. Patients with low risk (LR) disease (localized resectable with normal LDH) received 10 additional alternating courses of IE with VDC. For patients with high-risk (HR) disease (unresectable, pelvic, metastatic, or high LDH), two additional cycles of ICE were given. RESULTS: One-hundred seventy five patients (39% metastatic) were enrolled. Fifty-two patients (29.7%) were LR and 123 (70.3%) were HR. Overall response rate at end of induction was 27.4%. Five-year event-free survival (EFS) and overall survival (OS) estimates were 51.4% and 54.4%, respectively. Patients with localized disease had better outcomes than patients with metastases (5-year EFS 67.9% vs. 25.5%, and 5-year OS 70.3% vs. 29.1%, respectively). On multivariate analysis, the presence of metastatic disease was the only prognostic factor (P < 0.01). CONCLUSION: The VDC/ICE protocol was feasible, and considering the high tumor burden in our population, resulted in comparable results to those reported by cooperative groups in high-income countries. Further adaptation to maximize efficacy and minimize toxicity will be required.

Aberrant DNA methylation of ESR1 and p14ARF genes could be useful as prognostic indicators in osteosarcoma.

Osteosarcoma (OS) is the eighth most common form of childhood and adolescence cancer. Approximately 10%-20% of patients present metastatic disease at diagnosis and the 5-year overall survival remains around 70% for nonmetastatic patients and around 30% for metastatic patients. Metastatic disease at diagnosis and the necrosis grade induced by preoperative treatment are the only well-established prognostic factors for osteosarcoma. The DNA aberrant methylation is a frequent epigenetic alteration in humans and has been described as a molecular marker in different tumor types. This study evaluated the DNA aberrant methylation status of 18 genes in 34 OS samples without previous chemotherapy treatment and in four normal bone specimens and compared the methylation profile with clinicopathological characteristics of the patients. We were able to define a three-gene panel (AIM1, p14ARF, and ESR1) in which methylation was correlated with OS cases. The hypermethylation of p14ARF showed a significant association with the absence of metastases at diagnoses, while ESR1 hypermethylation was marginally associated with worse overall survival. This study demonstrated that aberrant promoter methylation is a common event in OS and provides evidence that p14ARF and ESR1 hypermethylation could be useful as a prognostic indicator for this disease.

Genome-wide association study identifies two susceptibility loci for osteosarcoma.

Osteosarcoma is the most common primary bone malignancy of adolescents and young adults. To better understand the genetic etiology of osteosarcoma, we performed a multistage genome-wide association study consisting of 941 individuals with osteosarcoma (cases) and 3,291 cancer-free adult controls of European ancestry. Two loci achieved genome-wide significance: a locus in the GRM4 gene at 6p21.3 (encoding glutamate receptor metabotropic 4; rs1906953; P = 8.1 × 10⁻9) and a locus in the gene desert at 2p25.2 (rs7591996 and rs10208273; P = 1.0 × 10⁻8 and 2.9 × 10⁻7, respectively). These two loci warrant further exploration to uncover the biological mechanisms underlying susceptibility to osteosarcoma.

Gene expression pattern contributing to prognostic factors in childhood acute lymphoblastic leukemia.

The present study evaluated the expression profile of 19 genes previously reported in microarray studies and associated with resistance or sensitivity to vincristine (RPLP2, CD44, TCFL5, KCNN1, TRIM24), prednisolone (F8A, CDK2AP1, BLVRB, CD69), daunorubicin (MAP3K12, SHOC2, PCDH9, EGR1, KCNN4) and l-asparaginase (GPR56, MAN1A1, CLEC11A, IGFBP7, GATA3). We studied 140 bone marrow samples at diagnosis from children with acute lymphoblastic leukemia (ALL) treated according to the Brazilian Childhood Leukemia Treatment Group (GBTLI) ALL-99 protocol. The expression profiles of the genes listed above were analyzed by real-time quantitative polymerase chain reaction (PCR) and then related to the clinical and biological prognostic factors. The results showed significant associations (p ≤ 0.05) between the expression levels of genes GPR56, BLVRB, IGFBP7 and white blood cell (WBC) count at diagnosis; GATA3, MAN1A1, CD44, MAP3K12, CLEC11A, SHOC2 and CD10 B-lineage ALL; TCFL5 and bone marrow status at day 14; MAP3K12 and TRIM24 and bone marrow status at day 28; and CD69, TCFL5 and TRIM24 genes and ETV6/RUNX1 positive ALL. The up-regulation of SHOC2 was also associated with better 5-year event-free survival (EFS) in univariate and multivariate analysis (p = 0.02 and p = 0.03, respectively). These findings highlight genes that could be associated with clinical and biological prognostic factors in childhood ALL, suggesting that these genes may characterize and play a role in the treatment outcome of some ALL subsets.

C-kit expression in human osteosarcoma and in vitro assays.

Biologic agents targeting oncogenes have encourage researchs trying to correlate the role of tyrosine kinase in the pathogenesis of tumours. Osteosarcoma is a high grade aggressive neoplasm with poor survival. Our aim was to investigate c-kit immunoexpression, its prognostic relevance for patients with osteosarcoma, and the effect of imatinib mesylate (STI571) on proliferation and invasion of the human osteosarcoma cell line.A retrospective immunohistochemical study was performed on archival formalin-fixed paraffin-embedded specimens from 52 patients with high-grade primary osteosarcoma of extremities treated at the Pediatric Oncology Institute (IOP, GRAAC) and archived in the Department of Pathology, Federal University of São Paulo. Only pre-chemotherapy specimens were analyzed. Strongly stained cytoplasm and membrane cells were taken as positive. Human osteosarcoma cells from line MG-63 were incubated and the inhibitory effect of imatinib mesylate (STI571) on cell proliferation and invasion was studied. In 24 cases (46.15%), c-kit was expressed by the cells and c-kit-positive tumors exhibited lower necrosis post-chemotherapy. No correlation was found between c-kit expression and overall and disease-free survival. Imatinib mesylate decreased the rates of cell growth of osteosarcoma cells in low doses and invasion in high doses C-kit-positive tumors had worse response to chemotherapy and imatinib mesylate can play a role in blocking or decreasing the rate of growth of osteosarcoma cells, but not the invasive capacity of these neoplastic cells. These data suggested that imatinib mesylate could be a therapeutic target of strategies against osteosarcoma tumors. Further studies are necessary to confirm this indication.

Glutathione S-transferase polymorphisms in osteosarcoma patients.

BACKGROUND: Osteosarcoma is the most common malignant bone tumor in children and adolescents. Multidrug resistance and poor clinical outcome are the problems that still affect osteosarcoma patients. The glutathione S-transferase supergene family includes several genes that encode enzymes involved in the detoxification of many xenobiotic agents, including carcinogens and anticancer drugs. The polymorphisms in these genes have already been associated both with cancer susceptibility and anticancer drugs resistance. OBJECTIVES: This study aims to investigate the genotype frequencies of GSTM1, GSTT1 and GSTM3 genes in 80 osteosarcoma patients and 160 normal control participants, and also the influence of these polymorphisms in the clinical outcome of osteosarcoma patients. METHODS: GSTM1 and GSTT1 deletion polymorphisms were examined through a multiplex-PCR and the GSTM3 polymorphism of three base pair-deletion at intron 6 using PCR-restriction fragments length polymorphism method. RESULTS: We found that GSTM1 null genotype is correlated to poor clinical outcome characterized by the increased lung relapse occurrence [odds ratio (OR)=2.71, P=0.036], while the presence of at least one GSTM1 allele is associated with a good response to treatment and better survival (OR=4.28, P=0.020 and hazards ratio=4.09, P=0.0078, respectively). The GSTT1 null genotype was correlated with a better overall survival (hazards ratio=7.15, P=0.0247), whereas GSTM3*B allele was associated with metastasis at diagnosis (OR=2.83, P=0.028). CONCLUSION: The findings of this study suggest that GST polymorphisms may have a role in treatment response and osteosarcoma progression.

mRNA expression profile of multidrug resistance genes in childhood acute lymphoblastic leukemia. Low expression levels associated with a higher risk of toxic death.

BACKGROUND: Increased activity of multidrug resistance (MDR) genes has been associated with treatment failure in acute leukemias, although with controversial reports. The objective of the present study was to assess the expression profile of the genes related to MDR: ABCB1, ABCC1, ABCC3, ABCG2, and LRP/MVP in terms of the clinical and biological variable and the survival of children with acute lymphoblastic leukemia (ALL). PROCEDURE: The levels of mRNA expression of the drug resistance genes ABCB1, ABCC1, ABCC3, ABCG2, and LRP/MVP were analyzed by quantitative real-time PCR using the median values as cut-off points, in consecutive samples from 140 children with ALL at diagnosis. RESULTS: Expression levels of the ABCG2 gene in the patient group as a whole (P = 0.05) and of the ABCG2 and ABCC1 genes in patients classified as being at high risk were associated with higher rates of 5-year event-free survival (EFS) (P = 0.04 and P = 0.01). Expression levels of the ABCG2 gene below the median were associated with a greater chance of death related to treatment toxicity for the patient group as a whole (P = 0.009) and expression levels below the median of the ABCG2 and ABCC1 genes were associated with a greater chance of death due to treatment toxicity for the high-risk group (P = 0.02 and P = 0.03, respectively). CONCLUSION: The present data suggest a low participation of the drug efflux genes in treatment failure in patients with childhood ALL. However, the low expression of some of these genes may be associated with a higher death risk related to treatment toxicity.

A simplified minimal residual disease polymerase chain reaction method at early treatment points can stratify children with acute lymphoblastic leukemia into good and poor outcome groups.

BACKGROUND: Minimal residual disease is an important independent prognostic factor in childhood acute lymphoblastic leukemia. The classical detection methods such as multiparameter flow cytometry and real-time quantitative polymerase chain reaction analysis are expensive, time-consuming and complex, and require considerable technical expertise. DESIGN AND METHODS: We analyzed 229 consecutive children with acute lymphoblastic leukemia treated according to the GBTLI-99 protocol at three different Brazilian centers. Minimal residual disease was analyzed in bone marrow samples at diagnosis and on days 14 and 28 by conventional homo/heteroduplex polymerase chain reaction using a simplified approach with consensus primers for IG and TCR gene rearrangements. RESULTS: At least one marker was detected by polymerase chain reaction in 96.4% of the patients. By combining the minimal residual disease results obtained on days 14 and 28, three different prognostic groups were identified: minimal residual disease negative on days 14 and 28, positive on day 14/negative on day 28, and positive on both. Five-year event-free survival rates were 85%, 75.6%, and 27.8%, respectively (p<0.0001). The same pattern of stratification held true for the group of intensively treated children. When analyzed in other subgroups of patients such as those at standard and high risk at diagnosis, those with positive B-derived CD10, patients positive for the TEL/AML1 transcript, and patients in morphological remission on a day 28 marrow, the event-free survival rate was found to be significantly lower in patients with positive minimal residual disease on day 28. Multivariate analysis demonstrated that the detection of minimal residual disease on day 28 is the most significant prognostic factor. CONCLUSIONS: This simplified strategy for detection of minimal residual disease was feasible, reproducible, cheaper and simpler when compared with other methods, and allowed powerful discrimination between children with acute lymphoblastic leukemia with a good and poor outcome.

TNF-alpha, TNF-beta, IL-6, IL-10, PECAM-1 and the MPO inflammatory gene polymorphisms in osteosarcoma.

The inflammatory microenvironment of tumors is characterized by the presence of cytokines and growth factor's network both in the supporting stroma and in tumor areas. These molecules may contribute to tumoral growth and progression, facilitating metastatic process. Therefore, cancer susceptibility and severity may be associated with the functional polymorphisms of inflammatory genes. We hypothesized that inflammatory gene polymorphisms may have important role for osteosarcoma patients. We studied -308G>A TNF-alpha, +252A>G TNF-beta, -174G>C IL-6, -1082A>G IL-10, +125C>G PECAM-1, and the -463A>G MPO inflammatory gene polymorphisms in 80 osteosarcoma patients and 160 control individuals using polymerase chain reaction-restriction-fragment length polymorphism method. We found that the patients with variant genotype (GG) of the +252A>G TNF-beta gene showed an event-free survival rate of 20% at 100 months. We suggest that the presence of the variant genotype (GG) of the +252A>G TNF-beta polymorphism, which leads to higher level of cytokine production, could be a facilitator mechanism in tumor progression leading to a poor event-free survival.