Minimal residual disease monitoring in childhood B lymphoblastic leukemia with t(12;21)(p13;q22); ETV6-RUNX1: concordant results using quantitation of fusion transcript and flow cytometry.

INTRODUCTION: The translocation t(12;21)(p13;q22) resulting in the fusion gene ETV6-RUNX1, is the most frequent gene fusion in childhood B lymphoblastic leukemia. In the Nordic Society of Paediatric Haematology and Oncology ALL-2008 treatment protocol, treatment stratification in B-lineage ALL is based on results of minimal residual disease (MRD) analysis with fluorescence-activated cell sorting (FACS). In this study, we determined whether RT-qPCR of the ETV6-RUNX1 fusion transcript can be a reliable alternative for MRD analysis. METHODS: Seventy-eight bone marrow samples from 29 children at diagnosis and day 15, 29, and 78 during treatment were analyzed for MRD with FACS and with quantitative reverse transcription polymerase chain reaction (RT-qPCR). Fusion transcript MRD was defined as the ETV6-RUNX1/GUSB ratio at the follow-up time point (day 15/29/78) divided with the ETV6-RUNX1/GUSB ratio at diagnosis (%). RESULTS: MRD analysis with FACS and with RT-qPCR of ETV6-RUNX1 fusion transcript showed strong correlation. All cases showed concordant results at the treatment stratifying time points day 29 and day 78, when comparing the two methods with a cutoff set to 0.1%. CONCLUSION: RT-qPCR is a valuable addition and could also be an alternative to FACS in cases where FACS is not achievable for MRD analysis.

GEANT4 simulation of cyclotron radioisotope production in a solid target.

The use of radioisotopes in nuclear medicine is essential for diagnosing and treating cancer. The optimization of their production is a key factor in maximizing the production yield and minimizing the associated costs. An efficient approach to this problem is the use of Monte Carlo simulations prior to experimentation. By predicting isotopes yields, one can study the isotope of interest expected activity for different energy ranges. One can also study the target contamination with other radioisotopes, especially undesired radioisotopes of the wanted chemical element which are difficult to separate from the irradiated target and might result in increasing the dose when delivering the radiopharmaceutical product to the patient. The aim of this work is to build and validate a Monte Carlo simulation platform using the GEANT4 toolkit to model the solid target system of the South Australian Health and Medical Research Institute (SAHMRI) GE Healthcare PETtrace cyclotron. It includes a GEANT4 Graphical User Interface (GUI) where the user can modify simulation parameters such as the energy, shape and current of the proton beam, the target geometry and material, the foil geometry and material and the time of irradiation. The paper describes the simulation and presents a comparison of simulated and experimental/theoretical yields for various nuclear reactions on an enriched nickel 64 target using the GEANT4 physics model QGSP_BIC_AllHP, a model recently developed to evaluate with high precision the interaction of protons with energies below 200MeV available in Geant4 version 10.1. The simulation yield of the (64)Ni(p,n)(64)Cu reaction was found to be 7.67±0.074 mCi·µA(-1) for a target energy range of 9-12MeV. Szelecsenyi et al. (1993) gives a theoretical yield of 6.71mCi·µA(-1) and an experimental yield of 6.38mCi·µA(-1). The (64)Ni(p,n)(64)Cu cross section obtained with the simulation was also verified against the yield predicted from the nuclear database TENDL and compared to experimental yield obtained from literature.

Evaluation of physician eye lens doses during permanent seed implant brachytherapy for prostate cancer.

Treatment of low grade prostate cancer with permanent implant of radioactive seeds has become one of the most common brachytherapy procedures in use today. The implant procedure is usually performed with fluoroscopy image guidance to ensure that the seeds are deployed in the planned locations. In this situation the physician performing the transperineal implant is required to be close to the fluoroscopy unit and dose to the eye lens may be of concern. In 1991 the International Commission on Radiological Protection (ICRP) provided a recommended dose limit of 150 mSv yr(-1) for occupational exposures to the lens of the eye. With more long term follow-up data, this limit was revised in 2011 to 20 mSv yr(-1). With this revised limit in mind, we have investigated the dose to the lens of the eye received by physicians during prostate brachytherapy seed implantation. By making an approximation of annual workload, we have related the dose received to the annual background dose. Through clinical and phantom measurements with thermoluminescent dosimeters, it was found that the excess dose to the physician's eye lens received for a conservative estimate of annual workload was never greater than 100% of the annual background dose.

High-resolution array CGH analysis of salivary gland tumors reveals fusion and amplification of the FGFR1 and PLAG1 genes in ring chromosomes.

We have previously identified a subgroup of pleomorphic salivary gland adenomas with ring chromosomes of uncertain derivation. Here, we have used spectral karyotyping (SKY), fluorescence in situ hybridization (FISH) and high-resolution oligonucleotide array-CGH to determine the origin and content of these rings and to identify genes disrupted as a result of ring formation. Of 16 tumors with rings, 11 were derived from chromosome 8, 3 from chromosome 5 and 1 each from chromosomes 1, 6 and 9. Array-CGH revealed that 10/11 r(8) consisted of amplification of a 19 Mb pericentromeric segment with recurrent breakpoints in FGFR1 in 8p12 and in PLAG1 in 8q12.1. Molecular analyses revealed that ring formation consistently generated novel FGFR1-PLAG1 gene fusions in which the 5'-part of FGFR1 is linked to the coding sequence of PLAG1. An alternative mechanism of PLAG1 activation was found in tumors with copy number gain of an intact PLAG1 gene. Rings derived from chromosomes 1, 5, 6 or 9 did not result in gene fusions, but rather resulted in losses indicative of the involvement of putative tumor suppressor genes on 8p, 5p, 5q and/or 6q. Our findings also reveal a novel mechanism by which FGFR1 contributes to oncogenesis and further illustrate the versatility of the FGFR1 and PLAG1 genes in tumorigenesis.

Involvement of INK4A gene products in the pathogenesis and development of human osteosarcoma.

BACKGROUND: The INK4A tumor suppressor gene plays a crucial role in the regulation of the G1 cell cycle phase. It encodes two transcripts, p16 and p14 alternate reading frame (ARF), involved in retinoblastoma protein (pRb)- and p53- cell growth control pathways, respectively. METHODS: To define the role of gene status and molecule expression involved in the INK4A regulatory system, immunohistochemistry, immunoblotting, and polymerase chain reaction (PCR) analysis were performed on 35 primary high grade osteosarcomas (OS). RESULTS: Although p16 and p14ARF proteins were found negative or weakly detectable in 60% and 57% of the cases respectively, INK4A gene analysis of exons 1alpha, 1beta and 2 did not reveal any deletion or mutation. However, methylation status of the 5'CpG promoter region, assessed by methylation-specific PCR, was found in 12 out of 21 OSs with negative or weak p16 expression. A statistical analysis based on pRb/p16 and p53/p14ARF staining status showed that pRb and p16 co-expression was inversely correlated to tumor relapse and was a marker for a more favorable prognosis. A statistically significant inverse correlation was found between wt-p53 and p14ARF expression. In the group of wt-p53 tumors, the loss of p14ARF was associated with a decreased expression of p21 protein, suggesting a down-regulation of the transcriptional activity of p53. CONCLUSIONS: The current results suggest that, in OS, the altered expression of INK4A products plays a primary role in the deregulation of both pRb and p53 cell growth control pathways, contributing to tumor pathogenesis and development.

Changes in p14(ARF) do not play a primary role in human chondrosarcoma tissues.

The locus encoding the tumor suppressor p16 has been found to code for a second, different protein. This protein, p14(ARF), has been shown to protect p53 from degradation. Like p16, its gene is often altered in different cancers. In this study, the first unique exon, exon 1 beta, of p14(ARF), has been studied in 22 chondrosarcoma tissues using polymerase chain reaction, DNA sequencing and methylation-specific polymerase chain reaction. One chondrosarcoma was found to have exon 1 beta homozygously deleted, but neither mutations nor methylations were found in any of the chondrosarcomas. This indicates that genetic changes of p14(ARF) are a rare event in chondrosarcoma.

Alterations in the regulatory pathway involving p16, pRb and cdk4 in human chondrosarcoma.

The G1 regulatory pathway involving p16, pRb and cdk4 in the cell cycle has been investigated in human chondrosarcoma. The protein expression of p16, pRb and cdk4 was analyzed by Western blot in cultured cells from eight chondrosarcomas and in two chondrosarcoma cell lines. Both cell lines and one other sample were negative for p16. Moreover, one of the cell lines was pRb-negative and showed a high expression of cdk4 as well. In the other cell line and in three other samples pRb of expected size were detected in addition to a shorter form of the protein. To further investigate the reasons for down-regulation of the p16 protein, the p16-coding gene CDKN2 was analyzed by polymerase chain reaction (PCR), methyl-specific PCR (MSP) and sequencing in all tumor samples as well as in corresponding tumor tissues from three of the samples. The p16-negative samples were all found to have homozygous deletion of CDKN2. Another sample showed partial gene methylation and a heterozygous position in codon 148 was detected in one sample. The same base substitution was also found in two of the tissue samples. Finally, cytogenetic analysis of the samples with homozygously deleted CDKN2 revealed multiple structural abnormalities in all three cases. In conclusion, the p16/pRb/cdk4 pathway may play an important role in the pathogenesis of some chondrosarcomas.

Changes of the p16 gene but not the p53 gene in human chondrosarcoma tissues.

The role of two important tumour suppressor genes, p16 and p53, was evaluated in cartilaginous tumour tissues. Genomic DNA from 22 chondrosarcomas, 5 benign chondroid tumours, 1 sample of reactive proliferative cartilage and 2 samples of normal cartilage were analysed using polymerase chain reaction, single strand conformational polymorphism, DNA sequencing and methylation-specific polymerase chain reaction. The p16 gene was found to be partly methylated in 5 high-grade chondrosarcomas and homozygously deleted in 1 chondrosarcoma. Moreover, a polymorphism was detected in 3 malignant tumours, but not in benign tumours or normal cartilage. Analysis of the p53 gene revealed an unchanged structure in all samples. These findings show a role for p16, but not p53, in chondrosarcoma.

Alteration of pRb/p16/cdk4 regulation in human osteosarcoma.

Cell-cycle regulation depends on a fine balance between cyclin-cyclin-dependent kinase complexes and a family of kinase inhibitors that bind cyclin-cdk complexes and block their activity. To investigate the role of mechanisms regulating cell-cycle progression in human osteosarcomas (OS), pRb/p16/cdk4 expression was analyzed in 39 high-grade OS; 19 of these developed metastasis during follow-up. Positive reaction for functional pRB was shown by 18/39 (46%) OS, while 21/39 (54%) were negative. A higher probability of metastasis was seen in patients with negative pRb expression (p < 0.05). Furthermore, while functional pRb and D1 expression are inversely associated to metastasis occurrence, the presence of D1/cdk4 complex in our study was related to poor prognosis. We found that 10/18 pRb-positive and 14/21 pRb-negative tumors were p16-positive. No significant correlation was found between pRb and p16 expression. On the other hand, high cdk4 levels in p16-positive tumors as compared with p16-negative tumors resulted in a positive association between p16 and cdk4 expression (Chi squared = 5.98; p = 0.01). No extensive p16INK4A genomic alterations were found in tumors lacking p16-protein expression. To determine which mechanisms are involved in the down-regulation of p16 protein, the methylation status of the p16INK4 gene was evaluated on the 15 p16-negative tumors: 8 samples showed 5' CpG-island methylation; 4/8 had a complete methylation status, while in the remaining 4 the gene was only partially methylated. These data confirm the role of the pRb/p16/cdk4 pathway in OS development.

The helix-loop-helix transcription factors Id1 and Id3 have a functional role in control of cell division in human normal and neoplastic chondrocytes.

The expression and localization of the helix-loop-helix transcription factors Id1 and Id3, as well as localization of the E12-protein, were studied in cells isolated from human articular cartilage and chondrosarcoma. Serum withdrawal down-regulated Id1 and Id3 expression in chondrocytes but not the Id1 expression in chondrosarcoma cells. Antisense oligonucleotides directed against Id1 and Id3 decreased BrdU labeling in both cell types. E12 was localized to the nucleus in chondrocytes and non-confluent tumor cells and in confluent tumor cells, E12 had a cytoplasmic localization. This study suggests a functional role for Id1 and Id3 in the control of proliferation and differentiation of cartilage.

Peroneal nerve palsy after total knee arthroplasty.

The prognosis for peroneal palsy after total knee arthroplasty (TKA) is poorly defined. Twenty-six postoperative peroneal palsies occurred after 8998 TKAs performed between 1972 and 1985. Eighteen patients had complete and eight had incomplete peroneal palsies. Twenty-three had both motor and sensory deficits, and three had only motor deficits. At an average of 5.1 years (range, one to 11 years) after arthroplasty, recovery was complete for 13 palsies and partial for 12. Complete recovery was more likely in those palsies that were incomplete initially. Patients with palsies that were initially partial had significantly higher knee scores than those with complete palsies, and patients whose eventual recovery was complete had significantly higher knee scores than those with incomplete recovery. This new prognostic information should be useful for surgeons who encounter this unfortunate yet persistent complication of TKA.