J. W. G. Janssen

Rapid and reliable detection of N- ras mutations in acute lymphoblastic leukemia by melting curve analysis using LightCycler technology

We applied a new strategy for the detection of N-ras gene mutations based on LightCycler technology. We designed two sets of amplimers and internal hybridization probes representing N-ras codons 12/13 and codon 61, respectively. Genomic DNAs from 134 childhood acute lymphoblastic leukemia (ALL) patients (83 common ALL, nine pre-pre-B ALL, 19 pre-B ALL, 23 T-ALL) were amplified, followed by the analysis of the melting temperatures of the PCR products on the LightCycler. PCR products exhibiting an abnormal melting characteristic were directly sequenced. Sequence analyses unravelled nucleotide substitutions at codon 12 in 10 patients, at codon 13 in three, and at codon 61 in one case. The incidence of N-rasmutations (10%) is compatible with previous reports. The LightCycler technology facilitates the rapid analysis of other genes exhibiting hot spot mutations in human malignancies.

The fusion of TEL and ABL in human acute lymphoblastic leukaemia is a rare event

We have recently identified a common ALL patient which harboured a chromosomal fusion between the TEL gene on chromosome 12 and the ABL gene on chromosome 9. We designed an RT‐PCR assay to screen 186 adult ALL and 30 childhood ALL patients for this novel translocation. We were unable to identify any additional cases with a TEL/ABL fusion product.

High rate of chromosome abnormalities detected by fluorescence in situ hybridization using BCR and ABL probes in adult acute lymphoblastic leukemia

The value of dual-color fluorescence in situ hybridization (FISH) with BCR and ABL probes for the detection of the Philadelphia (Ph) translocation and of other alterations involving ABL and/or BCR was evaluated in adult acute lymphoblastic leukemia (ALL). One hundred and four patients were studied prospectively using interphase nuclei FISH, chromosome analysis (CA), and PCR assays for the chimeric BRC/ABL transcript. FISH detected a Ph translocation in 24 cases (23.1%), as was confirmed by CA and/or PCR. FISH revealed a false positive diagnosis of a Ph translocation in four cases (5% false positive rate). Among 54 cases with combined FISH, CA and PCR assays, FISH failed to establish a correct diagnosis in 3.7%, PCR in 5.6%, and CA in 7.4%. The combination of two screening methods led to discrepant results in 9.3% (FISH + PCR), 11.1% (FISH + CA), or 13% (CA + PCR) of the cases. In seven of 80 (8.8%) Ph-negative patients, gain of BCR and/or ABL was identified. Overall, FISH detected alterations of the BCR and/or ABL genes with an incidence of 29.8% of the current study. Due to the possibility of false positive diagnosis of a Ph translocation using dual-color FISH the combination with chromosome and/or RT-PCR analyses is recommended in adult ALL patients.

MYEOV : A candidate gene for DNA amplification events occurring centromeric to CCNDI in breast cancer

Rearrangements of chromosome 11q13 are frequently observed in human cancer. The 11q13 region harbors several chromosomal breakpoint clusters found in hematologic malignancies and exhibits frequent DNA amplification in carcinomas. DNA amplification patterns in breast tumors are consistent with the existence of at least 4 individual amplification units, suggesting the activation of more than 1 gene in this region. Two candidate oncogenes have been identified, CCND1 and EMS1/CORTACTIN, representing centrally localized amplification units. Genes involved in the proximal and distal amplicons remain to be identified. Recently we reported on a putative transforming gene, MYEOV, mapping 360 kb centromeric to CCND1. This gene was found to be rearranged and activated concomitantly with CCND1 in a subset of t(11;14)(q13;q32)‐positive multiple myeloma (MM) cell lines. To evaluate the role of the MYEOV gene in the proximal amplification core, we tested 946 breast tumors for copy number increase of MYEOV relative to neighboring genes or markers. RNA expression levels were studied in a subset of 72 tumors for which both RNA and DNA were available. Data presented here show that the MYEOV gene is amplified in 9.5% (90/946) and abnormally expressed in 16.6% (12/72) of breast tumors. Amplification patterns showed that MYEOV was most frequently coamplified with CCND1 (74/90), although independent amplification of MYEOV could also be detected (16/90). Abnormal expression levels correlated only partially with DNA amplification. MYEOV DNA amplification correlated with estrogen and progesterone receptor‐positive cancer, invasive lobular carcinoma type and axillary nodal involvement. In contrast to CCND1 amplification, no association with disease outcome could be found. Our data suggest that MYEOV is a candidate oncogene activated in the amplification core located proximal to CCND1.

Detection of chromosomal translocations in leukemia-lymphoma cells by polymerase chain reaction.

In recent years many chromosomal translocations involved in leukemia and lymphoma have been defined at the molecular level. In addition to advancing the understanding of pathological mechanisms underlying the transformation process, the cloning and sequencing of the genes altered by the translocations have provided new tools for diagnosis and monitoring of patients. In particular, the polymerase chain reaction (PCR) methodology yields rapid, sensitive and accurate diagnostic and prognostic information. As leukemias carrying certain translocations confer a higher risk of treatment failure, it is important to identify accurately all positive cases in order to give appropriate therapy. An important new initiative in the diagnostical setting and anti-leukemic therapy is the early detection of minimal residual disease (MRD). If MRD, implying an increased risk of relapse, is reliably detected during apparent clinical remission, alternative strategies could be applied early while the malignant cell burden is still minimal. The PCR assays are clearly more sensitive than other methods of MRD detection including morphology, immunophenotyping and cytogenetics; treatment failure is first detectable by PCR followed by cytogenetic relapse and finally clinical disease. PCR assays have been most often used in the MRD analysis of follicular lymphoma with t(14;18), chronic myeloid leukemia and acute lymphoblastic leukemia (ALL) with t(9;22), ALL with t(4;11), and acute myeloid leukemia (AML) with t(8;21) or t(15;17). PCR amplification is applicable to any other translocation provided the translocation is highly associated with the malignancy and the breakpoints are sufficiently clustered; a quickly increasing number of such specific molecular markers are now available for PCR assays. PCR still remains an experimental investigation for the detection of covert disease. However, the clinical relevance of MRD detection should be evaluated separately for each type of leukemia as significant prognostic differences between disease entities were found. This review describes the PCR assays available for the detection of leukemia cells with specific chromosomal translocations and summarizes the experience with the application of PCR techniques in monitoring patients during the course of the disease.

Prospective BCR-ABL analysis by polymerase chain reaction (RT-PCR) in adult acute B-lineage lymphoblastic leukemia: reliability of RT-nested-PCR and comparison to cytogenetic data.

The reliability of routine BCR-ABL RT-nested-PCR was evaluated in 1453 B-lineage ALL or hybrid leukemia at initial diagnosis by RT-nested-PCR. All BCR-ABL-positive (nu2009=u2009642) and 176 BCR-ABL-negative samples underwent a second RT-PCR. In 518 patients, karyotyping and/or FISH was compared to the BCR-ABL status. The second RT-PCR revealed in 155/642 initially positive samples a divergent result (153 BCR-ABL-negative, two other transcripts) that in most cases turned out to be caused by contaminations in the first RT-nested-PCR. Confirmatory RT-PCR detected 2/176 false negative first RT-nested-PCR results. Thirty-nine specimens remained ambiguous despite different RT-PCR approaches. As far as cytogenetic evaluation and FISH is available (nu2009=u200923), the majority but not all patients with an ambiguous RT-PCR result were Ph-negative (nu2009=u200918). RT-nested-PCR and cytogenetics yielded in 346 of 383 evaluable samples a concordant result. Differing results are given and account in part to the lower sensitivity of karyotyping. Taken together, confirmed RT-PCR detected BCR-ABL fusion transcripts consistently in 487 out of 1453 ALL samples (c-ALL: 43%, pre-B ALL: 34%, pro-B ALL: 5%, B-ALL: 0%, hybrid leukemia: 5/11). Since false positive initial RT-nested-PCR data were frequent, either confirmatory second RT-PCR or FISH analysis is warranted to guarantee sensitive and reliable results of utmost clinical relevance.

Novel interaction partners of the TPR/MET tyrosine kinase.

A large variety of biological processes is mediated by stimulation of the receptor tyrosine kinase MET. Screening a mouse embryo cDNA library, we were able to identify several novel, putative intracellular TPR/MET‐substrates: SNAPIN, DCOHM, VAV‐1, Sorting nexin 2, Death associated protein kinase 3, SMC‐1, Centromeric protein C, and hTID‐1. Interactions as identified by yeast two‐hybrid analysis were validated in vitro and in vivo by mammalian two‐hybrid studies, a far‐western assay and coimmunoprecipitation. Participation in apoptosis‐regulating mechanisms through interaction with DAPK‐3 and cell cycle control via binding to nuclear proteins such as CENPC and SMC‐1 are possible new aspects of intracellular MET signaling.

Multiparameter Approach in the Identification of Cross-Contaminated Leukemia Cell Lines

A common problem in cell culturing is cross-contamination with other cells or misidentification of cells. An effective cell culture quality and identity control is required in order to avoid inter- and intraspecies contamination of cell lines and their further propagation and dissemination. We present evidence that supposedly unrelated cell lines that we received from the original investigators are in fact related to the chronic myeloid leukemia cell line K-562. The sister cell lines SPI-801 and SPI-802 were originally established from a patient with T-cell acute lymphoblastic leukemia and displayed T-cell associated features. However, data from morphological evaluation, immunophenotyping, bcr-abl gene rearrangement analysis, DNA fingerprinting, Northern blot analysis of globin gene expression and esterase isoenzyme analysis clearly established that the three cell lines are related. Cytogenetic examination while not proving the common identity of the cells provided further evidence for the suspected common origin of all three cell lines. Chromosome banding, DNA fingerprinting and bcr-abl genotyping suggested further evolution of these clones during long-term cultivation. Quality and identity control is an essential feature of cell culture technique. Only regular monitoring for purity and integrity of cell lines will significantly reduce the incidence of cell line contamination and misidentification.

Control of MYEOV Protein Synthesis by Upstream Open Reading Frames

The myeov gene has been isolated by the tumorigenicity assay and is localized at chromosome 11q13, a frequent site for chromosomal rearrangements in various carcinomas and B-cell neoplasms. In addition, myeov is coamplified with cyclin D1 and overexpressed in carcinomas of various organs. The mechanisms of myeov regulation remain enigmatic. The 5′-untranslated region (5′-UTR) of the myeov gene is long, encompasses several upstream AUGs, and is predicted to fold in a strong secondary structure, suggesting that its translation might be regulated by an internal ribosomal entry site. Here we show that initial experiments using monocistronic and dicistronic reporter constructs supported this assumption. However, the application of in vitro transcription/translation assays, Northern blot analysis, and promoterless dicistronic constructs revealed promoter activity of the myeov 5′-UTR. DNA transfection of dicistronic DNA constructs, normal and mutated forms of myeov cDNA fragments cloned in a eukaryotic expression vector, and direct RNA transfection analysis revealed that upstream AUG triplets in the 5′-UTR of the myeov transcript abrogate translation. Alternative splicing mechanisms in specific cell types and/or developmental stage may evade this translation control. Control experiments suggest that the 5′-UTR from encephalomyocarditis virus, when inserted at the midpoint of a dicistronic vector, is also able to function as a cryptic promoter.

Duplex PCR facilitates the identification of immunoglobulin kappa (IGK) gene rearrangements in acute lymphoblastic leukemia

Duplex PCR facilitates the identification of immunoglobulin kappa (IGK) gene rearrangements in acute lymphoblastic leukemia