Real-world performance of integrative clinical genomics in pediatric precision oncology.

Despite significant improvement in the survival of pediatric cancer patients, treatment outcomes for high-risk, relapsed, and refractory cancers remain unsatisfactory. Moreover, prolonged survival is frequently associated with long-term adverse effects due to intensive multimodal treatments. Accelerating the progress of pediatric oncology requires both therapeutic advances and strategies to mitigate the long-term cytotoxic side effects, potentially through targeting specific molecular drivers of pediatric malignancies. In this report, we present the results of integrative genomic and transcriptomic profiling of 230 patients with malignant solid tumors (the "primary cohort") and 18 patients with recurrent or otherwise difficult-to-treat nonmalignant conditions (the "secondary cohort"). The integrative workflow for the primary cohort enabled the identification of clinically significant single-nucleotide variants, small insertions/deletions, and fusion genes, which were found in 55% and 28% of patients, respectively. For 38% of patients, molecularly informed treatment recommendations were made. In the secondary cohort, known or potentially driving alteration was detected in 89% of cases, including a suspected novel causal gene for patients with inclusion body infantile digital fibromatosis. Furthermore, 47% of findings also brought therapeutic implications for subsequent management. Across both cohorts, changes or refinements to the original histopathological diagnoses were achieved in 4% of cases. Our study demonstrates the efficacy of integrating advanced genomic and transcriptomic analyses to identify therapeutic targets, refine diagnoses, and optimize treatment strategies for challenging pediatric and young adult malignancies and underscores the need for broad implementation of precision oncology in clinical settings.

Personalized dendritic cell vaccine in multimodal individualized combination therapy improves survival in high-risk pediatric cancer patients.

A lot of hope for high-risk cancers is being pinned on immunotherapy but the evidence in children is lacking due to the rarity and limited efficacy of single-agent approaches. Here, we aim to assess the effectiveness of multimodal therapy comprising a personalized dendritic cell (DC) vaccine in children with relapsed and/or high-risk solid tumors using the N-of-1 approach in real-world scenario. A total of 160 evaluable events occurred in 48 patients during the 4-year follow-up. Overall survival of the cohort was 7.03 years. Disease control after vaccination was achieved in 53.8% patients. Comparative survival analysis showed the beneficial effect of DC vaccine beyond 2 years from initial diagnosis (HR = 0.53, P = .048) or in patients with disease control (HR = 0.16, P = .00053). A trend for synergistic effect with metronomic cyclophosphamide and/or vinblastine was indicated (HR = 0.60 P = .225). A strong synergistic effect was found for immune check-point inhibitors (ICIs) after priming with the DC vaccine (HR = 0.40, P = .0047). In conclusion, the personalized DC vaccine was an effective component in the multimodal individualized treatment. Personalized DC vaccine was effective in less burdened or more indolent diseases with a favorable safety profile and synergized with metronomic and/or immunomodulating agents.

Precision immuno-oncology approach for four malignant tumors in siblings with constitutional mismatch repair deficiency syndrome.

Constitutional mismatch repair deficiency (CMMRD) is a rare syndrome characterized by an increased incidence of cancer. It is caused by biallelic germline mutations in one of the four mismatch repair genes (MMR) genes: MLH1, MSH2, MSH6, or PMS2. Accurate diagnosis accompanied by a proper molecular genetic examination plays a crucial role in cancer management and also has implications for other family members. In this report, we share the impact of the diagnosis and challenges during the clinical management of two brothers with CMMRD from a non-consanguineous family harbouring compound heterozygous variants in the PMS2 gene. Both brothers presented with different phenotypic manifestations and cancer spectrum. Treatment involving immune checkpoint inhibitors significantly contributed to prolonged survival in both patients affected by lethal gliomas. The uniform hypermutation also allowed immune-directed treatment using nivolumab for the B-cell lymphoma, thereby limiting the intensive chemotherapy exposure in this young patient who remains at risk for subsequent malignancies.

N-of-1 Trials in Pediatric Oncology: From a Population-Based Approach to Personalized Medicine-A Review.

Pediatric oncology is a critical area where the more efficient development of new treatments is urgently needed. The speed of approval of new drugs is still limited by regulatory requirements and a lack of innovative designs appropriate for trials in children. Childhood cancers meet the criteria of rare diseases. Personalized medicine brings it even closer to the horizon of individual cases. Thus, not all the traditional research tools, such as large-scale RCTs, are always suitable or even applicable, mainly due to limited sample sizes. Small samples and traditional versus subject-specific evidence are both distinctive issues in personalized pediatric oncology. Modern analytical approaches and adaptations of the paradigms of evidence are warranted. We have reviewed innovative trial designs and analytical methods developed for small populations, together with individualized approaches, given their applicability to pediatric oncology. We discuss traditional population-based and individualized perspectives of inferences and evidence, and explain the possibilities of using various methods in pediatric personalized oncology. We find that specific derivatives of the original N-of-1 trial design adapted for pediatric personalized oncology may represent an optimal analytical tool for this area of medicine. We conclude that no particular N-of-1 strategy can provide a solution. Rather, a whole range of approaches is needed to satisfy the new inferential and analytical paradigms of modern medicine. We reveal a new view of cancer as continuum model and discuss the "evidence puzzle".

Improved osteosarcoma survival with addition of mifamurtide to conventional chemotherapy - Observational prospective single institution analysis.

PURPOSE: Conventional osteosarcoma is an orphan disease. Current treatment approaches include combining a three drug chemotherapy schedule and surgery. The 3- and 5-year event-free survival (EFS) in localized disease is roughly 65 and 60%, respectively. The registration study of mifamurtide reported survival benefit, but some methodological controversies have been insufficient for FDA market authorization in contrast to EMA. METHODS: prospective single centre survival analysis of a mifamurtide addition to conventional therapy in 23 patients over a 5.5 year enrolment period is reported and compared to a historical control of 26 patient with localized disease. Bias arising from observational methodology was addressed using Landmark analysis and time-dependent Cox models. Blood count dynamics were analysed during the treatment. RESULTS: The adverse event profile was as expected with no dose limiting toxicities. There were no local relapses observed, one patient died in the first complete remission due to doxorubicin cardiotoxicity, one patient had pulmonary metastatic relapse. The observed 3- and 5-year EFS was 87.4% (CI 72.4-100%) and 87.4% (CI 72.4-100%), progression free survival (PFS) was 92.9% (CI 80.3-100%) and 92.9% (CI 80.3-100%), overall survival was 94.1% (CI 83.6-100) and 80.7% (CI 58.3-100), respectively. Comparison to the historical control showed statistically significant better PFS for mifamurtide patients (Landmark analysis; p = 0.044). Risk of progression was 5-times lower for the mifamurtide group (Cox model; HR 0.21, p = 0.136). Only subtle differences in lymphocyte counts were observed across treatment. CONCLUSION: the PFS benefit of mifamurtide is reported herein. The addition of mifamurtide could be considered as a best treatment option for localized osteosarcoma.

Granulation tissue enriched by aspirin and omega-3 fatty acids in healing experimental periodontal lesion.

AIMS: Granulation tissue (GT) and specialized pro­resolving mediators such as lipoxins and resolvins are key elements in the successful resolution of periodontitis. Aspirin­triggered lipoxins and resolvins are even more powerful than their natural analogues. Their biosynthesis can be accelerated by omega-3 fatty acids. The aim of this study was to evaluate the use of GT enriched by aspirin and omega-3 fatty acids during the surgical treatment of periodontitis in an experimental animal model (rabbit). METHODS: In each of 24 rabbits, two experimental periodontal defects were created. In total, 47 defects were treated with open-flap debridement and one of three procedures: (1) GT extracted and soaked with aspirin and omega-3 fatty acids (ASA+OMEGA3 group); (2) GT soaked with saline (PLACEBO group); or (3) GT left untreated (CONTROL group). Then, the GT was replaced in situ. Primary evaluated criteria were the probing pocket depth (PPD) and the clinical attachment level (CAL). Necropsies were harvested 2, 6, and 12 weeks after surgery. The samples were used for histological and molecular biological assessment. RESULTS: A trend of greater PPD and CAL in the ASA+OMEGA3 group was observed at 6 weeks. However, there was no significant difference between them. During the observation period, tissue levels of FGF-7, IL-1ß and TIMP-1 showed a statistically significant decrease (P<0.05). For the other variables, the ASA+OMEGA3 group was comparable with the PLACEBO and CONTROL groups. CONCLUSION: This experiment did not demonstrate the superiority of the proposed approach. However, the enriched granulation tissue did not impair healing outcomes.

Assessment of Tumor Mutational Burden in Pediatric Tumors by Real-Life Whole-Exome Sequencing and In Silico Simulation of Targeted Gene Panels: How the Choice of Method Could Affect the Clinical Decision?

BACKGROUND: Tumor mutational burden (TMB) is an emerging genomic biomarker in cancer that has been associated with improved response to immune checkpoint inhibitors (ICIs) in adult cancers. It was described that variability in TMB assessment is introduced by different laboratory techniques and various settings of bioinformatic pipelines. In pediatric oncology, no study has been published describing this variability so far. METHODS: In our study, we performed whole exome sequencing (WES, both germline and somatic) and calculated TMB in 106 patients with high-risk/recurrent pediatric solid tumors of 28 distinct cancer types. Subsequently, we used WES data for TMB calculation using an in silico approach simulating two The Food and Drug Administration (FDA)-approved/authorized comprehensive genomic panels for cancer. RESULTS: We describe a strong correlation between WES-based and panel-based TMBs; however, we show that this high correlation is significantly affected by inclusion of only a few hypermutated cases. In the series of nine cases, we determined TMB in two sequentially collected tumor tissue specimens and observed an increase in TMB along with tumor progression. Furthermore, we evaluated the extent to which potential ICI indication could be affected by variability in techniques and bioinformatic pipelines used for TMB assessment. We confirmed that this technological variability could significantly affect ICI indication in pediatric cancer patients; however, this significance decreases with the increasing cut-off values. CONCLUSIONS: For the first time in pediatric oncology, we assessed the reliability of TMB estimation across multiple pediatric cancer types using real-life WES and in silico analysis of two major targeted gene panels and confirmed a significant technological variability to be introduced by different laboratory techniques and various settings of bioinformatic pipelines.

Comparative Analysis of Putative Prognostic and Predictive Markers in Neuroblastomas: High Expression of PBX1 Is Associated With a Poor Response to Induction Therapy.

The survival rate for patients with high-risk neuroblastomas remains poor despite new improvements in available therapeutic modalities. A detailed understanding of the mechanisms underlying clinical responses to multimodal treatment is one of the important aspects that may provide precision in the prediction of a patient's clinical outcome. Our study was designed as a detailed comparative analysis of five selected proteins (DDX39A, HMGA1, HOXC9, NF1, and PBX1) in one cohort of patients using the same methodical approaches. These proteins were already reported separately as related to the resistance or sensitivity to retinoids and as useful prognostic markers of survival probability. In the cohort of 19 patients suffering from high-risk neuroblastomas, we analyzed initial immunohistochemistry samples obtained by diagnostic biopsy and post-induction samples taken after the end of induction therapy. The expression of DDX39A, HMGA1, HOXC9, and NF1 showed varied patterns with almost no differences between responders and non-responders. Nevertheless, we found very interesting results for PBX1: non-responders had significantly higher expression levels of this protein in the initial tumor samples when compared with responders; this expression pattern changed inversely in the post-induction samples, and this change was also statistically significant. Moreover, our results from survival analyses reveal the prognostic value of PBX1, NF1, and HOXC9 expression in neuroblastoma tissue. In addition to the prognostic importance of PBX1, NF1, and HOXC9 proteins, our results demonstrated that PBX1 could be used for the prediction of the clinical response to induction chemotherapy in patients suffering from high-risk neuroblastoma.

Phospho-Protein Arrays as Effective Tools for Screening Possible Targets for Kinase Inhibitors and Their Use in Precision Pediatric Oncology.

The specific targeting of signal transduction by low-molecular-weight inhibitors or monoclonal antibodies represents a very promising personalized treatment strategy in pediatric oncology. In this study, we present the successful and clinically relevant use of commercially available phospho-protein arrays for analyses of the phosphorylation profiles of a broad spectrum of receptor tyrosine kinases and their downstream signaling proteins in tumor tissue samples. Although these arrays were made for research purposes on human biological samples, they have already been used by several authors to profile various tumor types. Our study performed a systematic analysis of the advantages and pitfalls of the use of this method for personalized clinical medicine. In certain clinical cases and their series, we demonstrated the important aspects of data processing and evaluation, the use of phospho-protein arrays for single sample and serial sample analyses, and the validation of obtained results by immunohistochemistry, as well as the possibilities of this method for the hierarchical clustering of pediatric solid tumors. Our results clearly show that phospho-protein arrays are apparently useful for the clinical consideration of druggable molecular targets within a specific tumor. Thus, their potential validation for diagnostic purposes may substantially improve the personalized approach in the treatment of relapsed or refractory solid tumors.

Individualization of Treatment Improves the Survival of Children With High-Risk Solid Tumors: Comparative Patient Series Analysis in a Real-Life Scenario.

Introduction: The individualization of treatment is attractive, especially in children with high-risk cancer. In such a rare and very heterogeneous group of diseases, large population-based clinical randomized trials are not feasible without international collaboration. We therefore propose comparative patient series analysis in a real-life scenario. Methods: Open cohort observational study, comparative analysis. Seventy patients with high-risk solid tumors diagnosed between 2003 and 2015 and in whom the treatment was individualized either empirically or based on biomarkers were analyzed. The heterogeneity of the cohort and repeated measurements were advantageously utilized to increase effective sample size using appropriate statistical tools. Results: We demonstrated a beneficial effect of empirically given low-dose metronomic chemotherapy (HR 0.46 for relapses, p = 0.017) as well as various repurposed or targeted agents (HR 0.15 for deaths, p = 0.004) in a real-life scenario. However, targeted agents given on the basis of limited biological information were not beneficial. Conclusions: Comparative patient series analysis provides institutional-level evidence for treatment individualization in high-risk pediatric malignancies. Our findings emphasize the need for a comprehensive, multi omics assessment of the tumor and the host as well whenever molecularly driven targeted therapies are being considered. Low-dose metronomic chemotherapy or local control of the disease may be a more rational option in situations where targeted treatment cannot be justified by robust evidence and comprehensive biological information. "Targeted drugs" may be given empirically with a realistic benefit expectation when based on robust rationale.

Clinical and laboratory characteristics of enteroviral meningitis in children, including qRT-PCR and sequencing analysis.

AIMS: Enteroviruses (EVs) are the most common agents of aseptic meningitis. Some serotypes can cause serious neuroinfection leading to death. The aim of this study was to determine the representation of EVs in the etiology of aseptic meningitis in children and to analyze the demographic, clinical, laboratory, and epidemiological characteristics of patients with EV meningitis. PATIENTS AND METHODS: This was a prospective study including 147 patients in three groups: EV meningitis, tick-borne encephalitis, and aseptic meningitis with unidentified agent. RESULTS: Boys with EV meningitis predominated over girls. The average patient age was 11 years. Compared to the control group, these patients suffered more from stiff back (P=0.010), vomiting and nausea (P=0.009). They had shorter symptom duration (P<0.001), higher C-reactive protein in blood (P<0.001), higher predominance of polynuclears (P=0.026), and greater lactate (P=0.003) in cerebrospinal fluid (CSF). The serotype seen most frequently (68%) was ECHO virus (ECV) 30. CONCLUSIONS: Enteroviruses play the most important role in the differential diagnosis of aseptic meningitis. Short symptom duration, slightly higher inflammatory parameters in blood, predominance of polynuclears, and elevated CSF lactate have predictive value in diagnosing this disease. ECV 30 (frequently the agent of epidemics in the Czech Republic) was the aseptic meningitis agent most often seen.

Comprehensive Molecular Profiling for Relapsed/Refractory Pediatric Burkitt Lymphomas-Retrospective Analysis of Three Real-Life Clinical Cases-Addressing Issues on Randomization and Customization at the Bedside.

In order to identify reasons for treatment failures when using targeted therapies, we have analyzed the comprehensive molecular profiles of three relapsed, poor-prognosis Burkitt lymphoma cases. All three cases had resembling clinical presentation and histology and all three patients relapsed, but their outcomes differed significantly. The samples of their tumor tissue were analyzed using whole-exome sequencing, gene expression profiling, phosphoproteomic assays, and single-cell phosphoflow cytometry. These results explain different treatment responses of the three histologically identical but molecularly different tumors. Our findings support a personalized approach for patient with high risk, refractory, and rare diseases and may contribute to personalized and customized treatment efforts for patients with limited treatment options like relapsed/refractory Burkitt lymphoma. SUMMARY: The main aim of this study is to analyze three relapsed Burkitt lymphoma patients using a comprehensive molecular profiling, in order to explain their different outcomes and to propose a biomarker-based targeted treatment. In cases 1 and 3, the tumor tissue and the host were analyzed prospectively and appropriate target for the treatment was successfully implemented; however, in case 2, analyses become available only retrospectively and his empirically based rescue treatment did not hit the right target of his disease.

Personalized Treatment of H3K27M-Mutant Pediatric Diffuse Gliomas Provides Improved Therapeutic Opportunities.

Diffuse gliomas with K27M histone mutations (H3K27M glioma) are generally characterized by a fatal prognosis, particularly affecting the pediatric population. Based on the molecular heterogeneity observed in this tumor type, personalized treatment is considered to substantially improve therapeutic options. Therefore, clinical evidence for therapy, guided by comprehensive molecular profiling, is urgently required. In this study, we analyzed feasibility and clinical outcomes in a cohort of 12 H3K27M glioma cases treated at two centers. Patients were subjected to personalized treatment either at primary diagnosis or disease progression and received backbone therapy including focal irradiation. Molecular analyses included whole-exome sequencing of tumor and germline DNA, RNA-sequencing, and transcriptomic profiling. Patients were monitored with regular clinical as well as radiological follow-up. In one case, liquid biopsy of cerebrospinal fluid (CSF) was used. Analyses could be completed in 83% (10/12) and subsequent personalized treatment for one or more additional pharmacological therapies could be recommended in 90% (9/10). Personalized treatment included inhibition of the PI3K/AKT/mTOR pathway (3/9), MAPK signaling (2/9), immunotherapy (2/9), receptor tyrosine kinase inhibition (2/9), and retinoic receptor agonist (1/9). The overall response rate within the cohort was 78% (7/9) including one complete remission, three partial responses, and three stable diseases. Sustained responses lasting for 28 to 150 weeks were observed for cases with PIK3CA mutations treated with either miltefosine or everolimus and additional treatment with trametinib/dabrafenib in a case with BRAFV600E mutation. Immune checkpoint inhibitor treatment of a case with increased tumor mutational burden (TMB) resulted in complete remission lasting 40 weeks. Median time to progression was 29 weeks. Median overall survival (OS) in the personalized treatment cohort was 16.5 months. Last, we compared OS to a control cohort (n = 9) showing a median OS of 17.5 months. No significant difference between the cohorts could be detected, but long-term survivors (>2 years) were only present in the personalized treatment cohort. Taken together, we present the first evidence of clinical efficacy and an improved patient outcome through a personalized approach at least in selected cases of H3K27M glioma.

Early Dynamics of Interleukin-6 in Cerebrospinal Fluid after Aneurysmal Subarachnoid Hemorrhage.

BACKGROUND: Subarachnoid hemorrhage (SAH) is a severe condition associated with high mortality. Early brain injury (EBI) plays an important role in the pathophysiology of SAH, and inflammation is a major contributor to EBI. Inflammation is a widely studied topic in both experimental and clinical conditions; however, just a few clinical studies have focused primarily on the early inflammatory response after SAH, and detailed information about the association between the dynamics of early inflammatory response with main clinical characteristics is lacking. This study analyzes the early dynamics of inflammatory response after SAH and evaluates the possible associations between the markers of early inflammatory response and main clinical characteristics. PATIENTS AND METHODS: A total of 47 patients with a diagnosis of aneurysmal SAH within the last 24 hours were enrolled in the study. All treatments, including treatment of aneurysm (surgery/coiling) and implantation of a drainage system (external ventricular drainage/lumbar catheter), were conducted in the same way as in other patients with this diagnosis. Blood and cerebrospinal fluid (CSF) samples were collected three times a day for 4 days. The dynamics of proinflammatory cytokines were assessed, and associations between levels of the proinflammatory cytokines interleukin (IL)-6, IL-1ß, or tumor necrosis factor (TNF)α and main clinical characteristics were evaluated using linear mixed-effect models. RESULTS: The CSF levels of IL-6 were massively increased initially after SAH (up to 72 hours) with an additional increase in later phases (after 72 hours), but there was high variability in IL-6 levels. A significant association was noted between the Glasgow Outcome Scale score and both overall levels of IL-6 (p = 0.0095) and their dynamics (p = 0.0208); the effect of the Hunt and Hess scale was borderline (p = 0.0887). No association was found between IL-6 levels and Fisher grade, modality of treatment (surgery, coiling, no treatment), and later development of cerebral vasospasm. Plasmatic levels of IL-6 increased slightly, but no significant association was found. The levels of IL-1ß and TNFα were within the physiologic range in both CSF and plasma. CONCLUSIONS: Early dynamics of IL-6 in CSF are associated with a patient́s outcome. But it is difficult to use IL-6 alone for outcome prediction due to its high variability. The question is whether the dynamics of IL-6 could be used in combination with other early markers associated with brain injury. More detailed research is required to answer this question.

Expression of nestin, CD133 and ABCG2 in relation to the clinical outcome in pediatric sarcomas.

BACKGROUND: Nestin, CD133 and ABCG2 are recently discussed as putative markers, co-expression of which might determine a cancer stem cell (CSC) phenotype in sarcomas. OBJECTIVE: Our study is focused on immunohistochemical analysis of nestin, CD133 and ABCG2 expression in rhabdomyosarcoma, Ewing sarcoma and osteosarcoma. Furthermore, we also analyzed the possible correlation of nestin, CD133 and ABCG2 expression levels with the patient outcome to identify potential prognostic values of these three putative CSC markers in the same cohorts. METHODS: Using immunohistochemistry, expression of nestin, CD133 and ABCG2 was analyzed in 24 rhabdomyosarcoma, 22 Ewing sarcoma and 10 osteosarcoma tissue samples and expression levels of these markers were correlated with clinical outcome. RESULTS: High nestin levels indicate poor prognosis in patients with Ewing sarcoma (P = 0.001), and high CD133 expression is associated with shorter survival in rhabdomyosarcoma patients (P = 0.002). In contrast, no significant relationship was found between ABCG2 expression and the clinical outcome. CONCLUSIONS: Our analysis represents the first complex study of these three putative CSCs markers together in three different types of pediatric sarcomas and showed their possible prognostic values in these tumors.

Pre-treatment VD levels and VDR receptors as potential predictors of occurrence and overall survival in paediatric patients with solid tumours-a single institution pilot study.

Recently, vitamin D has been recognized as an important player in the immune system, and multiple studies suggested its involvement in cancer, too. The aims of this study were to investigate selected single nucleotide polymorphisms (SNPs) in the VDR gene, BsmI (rs1544410; A > G), FokI (rs 2228570; C > T), TaqI (rs731236; T > C), ApaI (rs 7975232; C > T) and Cdx-2 (rs11568820; A > G), and to evaluate their possible predictive role for outcomes in patients with paediatric solid tumours. A total of 111 children with paediatric solid tumours were enrolled at the Department of Paediatric Oncology, University Hospital Brno (Brno, Czech Republic) along with a control population of 787 adults; all study subjects were available for genotyping of selected SNPs, and the prediagnostic levels of 25-hydroxycholecalciferol (25(OH)D3) and 1,25-dihydroxycholecalciferol (1,25(OH)2D3) were measured in the cases, too. In FokI, the heterozygote CT genotype was weakly associated with a decreased risk of paediatric solid cancer occurrence 0.82 (0.53-1.28), while the CC genotype was associated with a decreased risk of 0.58 (0.30-1.09), p = 0.09. The 1,25(OH)2D3 prediagnostic levels were indicative of the overall survival in the cases (ß = -0.012, HR 0.988, 95 % CI (0.978-0.998), while higher prediagnostic levels of 1,25(OH)2D3 were associated with a statistically significant increase in overall mortality. We observed multiple effects of the alleles of the investigated polymorphisms and of 1,25(OH)2D3 on overall survival, regardless of the underlying disease.

The meaning of sampling density in multiple repeat prostate biopsies.

INTRODUCTION: Extended transrectal ultrasound-guided prostate biopsy is a state-of-the-art tool for prostate cancer detection. Nevertheless, approximately 1/3 of cancers are missed when using this method and repeat biopsy sessions are often required. The aim of this study was to investigate how sampling density (a compound variable reflecting the number of biopsy cores and prostate volume) impacts on detection rate in multiple repeat TRUS-biopsies. MATERIAL AND METHODS: A total of 1007 consecutive patients undergoing their 1st, 2nd, 3rd and any further repeat prostate biopsies were included. The relationship between sampling density and other clinical variables (age, prostate-specific antigen level, free/total PSA ratio, digital rectal examination, number of previous biopsies) and cancer detection rate were assessed by interaction analysis. RESULTS: There were 562 primary re-biopsies, 267 second re-biopsies and 178 third and further re-biopsies included in the study. Detection rate was 25.4%, 25.8% and 25.3%, respectively. Interaction of sampling density with age was demonstrated in patients undergoing their first repeat biopsy (but not further re-biopsies). No interaction was observed with other variables investigated. CONCLUSIONS: A more extensive prostate sampling leads to a higher cancer detection rate on repeat prostate biopsies, as shown previously. However, this effect seems to be particularly pronounced in men younger than 65 years undergoing their first repeat prostate biopsy.

DHFR-mediated effects of methotrexate in medulloblastoma and osteosarcoma cells: the same outcome of treatment with different doses in sensitive cell lines.

Although methotrexate (MTX) is the most well-known antifolate included in many standard therapeutic regimens, substantial toxicity limits its wider use, particularly in pediatric oncology. Our study focused on a detailed analysis of MTX effects in cell lines derived from two types of pediatric solid tumors: medulloblastoma and osteosarcoma. The main aim of this study was to analyze the effects of treatment with MTX at concentrations comparable to MTX plasma levels in patients treated with high-dose or low-dose MTX. The results showed that treatment with MTX significantly decreased proliferation activity, inhibited the cell cycle at S-phase and induced apoptosis in Daoy and Saos-2 reference cell lines, which were found to be MTX-sensitive. Furthermore, no difference in these effects was observed following treatment with various doses of MTX ranging from 1 to 40 µM. These findings suggest the possibility of achieving the same outcome with the application of low-dose MTX, an extremely important result, particularly for clinical practice. Another important aspect of treatment with high-dose MTX in clinical practice is the administration of leucovorin (LV) as an antidote to reduce MTX toxicity in normal cells. For this reason, the combined application of MTX and LV was also included in our experiments; however, this application of MTX together with LV did not elicit any detectable effect. The expression analysis of genes involved in the mechanisms of resistance to MTX was a final component of our study, and the results helped us to elucidate the mechanisms of the various responses to MTX among the cell lines included in our study.