Allelic imbalance at the HER2/TOP2A locus in breast cancer.

BACKGROUND: Breast cancer is a heterogeneous disease with various histological features and molecular markers. These are utilized for the prediction of clinical outcome and therapeutic decision making. In addition to well established markers such as HER2 overexpression and estrogen and progesterone receptor (ER and PR) status, chromosomal instability is evolving as an important hallmark of cancers. The HER2/TOP2A locus is of great importance in breast cancer. The copy number variability at this locus has been proposed to be a marker for the degree of chromosomal instability. We therefore developed a Single Nucleotide Polymorphism (SNP) assay to evaluate allelic imbalance at the HER2/TOP2A locus in three different entities of primary breast tumors. METHODS: Eleven SNPs were carefully selected and detected by real time PCR using DNA extracted from paired (histologically normal and tumor) paraffin-embedded tissues. Primary breast tumors of 44 patients were included, 15 tumors with HER2 overexpression, 16 triple negative tumors, defined by the absence of HER2 overexpression and a negative ER and PR status and 13 ER and PR positive tumors without HER2 overexpression. As controls, histologically normal breast tissues from 10 patients with no breast tumor were included. RESULTS: Allelic imbalance was observed in 13/15 (87 %) HER2 positive tumors, the remaining 2 being inconclusive. Of the 16 triple negative tumors, 12 (75 %) displayed instability, 3 (19 %) displayed no instability, and 1 was inconclusive. Of the 13 hormone receptor positive tumors, 5 (38 %) displayed allelic imbalance, while 8 did not. CONCLUSIONS: We conclude that the SNP assay is suitable for rapid testing of allelic (im)balance at the HER2/TOP2A locus using paraffin-embedded tissues. Based on allelic imbalance at this locus, both triple negative and ER and PR positive breast tumors can be subcategorized. The clinical relevance of the allelic (im)balance status at the HER2/TOP2A locus in breast cancer is subject of future study. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2086062232155220.

Single nucleotide polymorphism (SNP)-based loss of heterozygosity (LOH) testing by real time PCR in patients suspect of myeloproliferative disease.

During tumor development, loss of heterozygosity (LOH) often occurs. When LOH is preceded by an oncogene activating mutation, the mutant allele may be further potentiated if the wild-type allele is lost or inactivated. In myeloproliferative neoplasms (MPN) somatic acquisition of JAK2V617F may be followed by LOH resulting in loss of the wild type allele. The occurrence of LOH in MPN and other proliferative diseases may lead to a further potentiating the mutant allele and thereby increasing morbidity. A real time PCR based SNP profiling assay was developed and validated for LOH detection of the JAK2 region (JAK2LOH). Blood of a cohort of 12 JAK2V617F-positive patients (n=6 25-50% and n=6>50% JAK2V617F) and a cohort of 81 patients suspected of MPN was stored with EDTA and subsequently used for validation. To generate germ-line profiles, non-neoplastic formalin-fixed paraffin-embedded tissue from each patient was analyzed. Results of the SNP assay were compared to those of an established Short Tandem Repeat (STR) assay. Both assays revealed JAK2LOH in 1/6 patients with 25-50% JAK2V617F. In patients with >50% JAK2V617F, JAK2LOH was detected in 6/6 by the SNP assay and 5/6 patients by the STR assay. Of the 81 patients suspected of MPN, 18 patients carried JAK2V617F. Both the SNP and STR assay demonstrated the occurrence of JAK2LOH in 5 of them. In the 63 JAK2V617F-negative patients, no JAK2LOH was observed by SNP and STR analyses. The presented SNP assay reliably detects JAK2LOH and is a fast and easy to perform alternative for STR analyses. We therefore anticipate the SNP approach as a proof of principle for the development of LOH SNP-assays for other clinically relevant LOH loci.

Sensitive detection and quantification of the JAK2V617F allele by real-time PCR blocking wild-type amplification by using a peptide nucleic acid oligonucleotide.

A single G-to-T missense mutation in the gene for the JAK2 tyrosine kinase, leading to a V617F amino acid substitution, is commonly found in several myeloproliferative neoplasms. Reliable quantification of this mutant allele is of increasing clinical and therapeutic interest in predicting and diagnosing this group of neoplasms. Because JAK2V617F is somatically acquired and may be followed by loss of heterozygosity, the percentage of mutant versus wild-type DNA in blood can vary between 0% and almost 100%. Therefore, we developed a real-time PCR assay for detection and quantification of the low-to-high range of the JAK2V617F allele burden. To allow the assay to meet these criteria, amplification of the wild-type JAK2 was blocked with a peptide nucleic acid oligonucleotide. JAK2V617F patient DNA diluted in JAK2 wild-type DNA could be amplified linearly from 0.05% to 100%, with acceptable reproducibility of quantification. The sensitivity of the assay was 0.05% (n = 3 of 3). In 9 of 100 healthy blood donors, a weak positive/background signal was observed in DNA isolated from blood, corresponding to approximately 0.01% JAK2V617F allele. In one healthy individual, we observed this signal in duplicate. The clinical relevance of this finding is not clear. By inhibiting amplification of the wild-type allele, we developed a sensitive and linear real-time PCR assay to detect and quantify JAK2V617F.

[Resistance to coumarin derivatives due to mutated vitamin-K enzyme]. / Genetische resistentie tegen cumarinederivaten. Gemuteerd vitamine K-epoxidereductase.

In 2006 a case report was published in this journal describing partial acenocoumarol- and phenprocoumon resistance in a 78-year-old man. A mutation in the VKORC1 gene was suggested to be the cause of the observed resistance. We examined the patient and found a new and hitherto unknown mutation in the VKORC1 gene which may well explain the observed resistance. The mutation concerns a polymorphism in exon 2 that predicts the substitution of tryptophan at position 59 of the VKOR protein by arginine, the p.Trp59Arg mutation. Meanwhile, we have detected the same p.Trp59Arg mutation in another patient with coumarin derivative resistance. The fact that this mutation did not occur in 100 individuals who responded well to coumarin derivatives, together with the knowledge that amino acid 59 is conserved among species, renders it likely that p.Trp59Arg was the cause of the partial acenocoumarol- and phenprocoumon resistance observed in these two patients.

A newly identified deletion of 970 bp at the alpha-globin locus that removes the promoter region of the alpha1 gene.

The most common causes of alpha-thalassemia (thal) are deletions that remove a part, or one or both of the functional alpha-globin genes. These deletions cause diminished expression of the alpha-globin protein, which may result in relatively low hemoglobin (Hb) and/or mean corpuscular volume (MCV) values. We here report the identification of a 970 bp deletion in the alpha1-globin gene that encompasses the entire promoter region of the alpha1-globin gene and 26 bp encoding the 5' end of the mRNA. Thus, the affected alpha1-globin gene is prone to be nonfunctional. We therefore nominated the newly identified deletion allele alpha-alphaDelta970. The MCV values of four related carriers of the alpha-alphaDelta970 allele were slightly lowered, consistent with the presence of three functional alpha-globin genes.