Simultaneous detection of TOP2A and HER2 gene amplification by multiplex ligation-dependent probe amplification in breast cancer.

HER-2/neu gene amplification, found in certain subtypes of (breast-) cancers, is an independent prognostic factor of poor outcome and determines eligibility for systemic treatment with trastuzumab. TopoII alpha (TOP2A) gene amplification seems to be predictive of response to a class of cytostatic agents called TopoII inhibitors, which include the anthracyclines. The observed increased efficacy of anthracyclines in HER2-positive tumors is thought to arise from the close proximity of both genes on chromosome 17, where the TopoII amplification status will determine the anthracycline sensitivity. This study aimed to validate a new polymerase chain reaction-based test, called multiplex ligation-dependent probe amplification (MLPA), as a simple and quick method to simultaneously assess HER-2/neu and TopoII alpha gene amplification status in paraffin-embedded breast cancer samples. To this end, MLPA results were compared with TopoII alpha, HER2 chromogenic in situ hybridization (CISH). We also assessed TopoII alpha protein expression by immunohistochemistry. Of 353 patients, 9% showed TopoII alpha amplification by MLPA and 13% of patients were HER2 amplified. TopoII alpha amplification was seen in 42% of HER2-amplified cases and showed no high level amplification without HER2 amplification. Eleven patients displayed TopoI alpha loss (3%). Concordance between MLPA and CISH was 91% for TopoII alpha and 96% for HER2. Correlation between amplification and overexpression of TopoII alpha was significant (P=0.035), but amplification did not always predict protein overexpression. Loss of the TopoII alpha gene was almost never associated with loss of its protein. In conclusion, MLPA is an easy and accurate method to simultaneously detect breast cancer HER-2/neu and TopoII alpha copy number status in paraffin-embedded tissue, and thus an attractive supplement or alternative to CISH.

HER-2/neu amplification testing in breast cancer by multiplex ligation-dependent probe amplification in comparison with immunohistochemistry and in situ hybridization.

BACKGROUND: Assessment of HER-2/neu status in invasive breast cancer is crucial to establish eligibility for trastuzumab and taxane based chemotherapy. Next to immunohistochemistry (IHC) to evaluate protein overexpression, a second line gene amplification test is required for cases with equivocal protein expression. This study aimed to validate a new PCR based test, called Multiplex Ligation-dependent Probe Amplification (MLPA), as a simple and quick method to assess HER-2/neu gene amplification status in invasive breast cancer. METHODS: MPLA results were compared with gene amplification status assessed by fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) as gold standard, and with protein overexpression by IHC in 518 breast carcinoma patients. RESULTS: About 10% of cases overexpressed HER-2/neu at the protein level (IHC), and 11% of cases showed gene-amplification by MLPA. A high concordance was found between FISH and CISH, MLPA and IHC, and MLPA and CISH. MLPA showed amplification in 7/36 (19%) of the equivocal IHC 2+ cases. However, of the IHC 0/1+ cases, 6/434 (1.4%) were also amplified by MLPA, and amplification was confirmed in all of these cases by FISH/CISH. On the other hand, one of the 48 (2%) IHC 3+ cases was normal by MLPA and lack of amplification was confirmed by FISH/CISH. CONCLUSION: MLPA is a fast, accurate and cheap method to detect breast cancer HER-2/neu amplification in small quantities of DNA extracted from paraffin blocks, and thereby a reliable alternative to FISH and CISH.

Highly diverse TCR delta chain repertoire in bovine tissues due to the use of up to four D segments per delta chain.

Tissue-specific distribution of gammadelta TCRs with limited TCR diversity is a common phenomenon in species with a low percentage of gammadelta T cells like humans and mice. We set out to investigate whether this is also the case in cattle (Bos taurus), a species with high percentages of gammadelta T cells. Using a method that was independent of variable (V) segment-specific primers, we generated 65 unique TCR delta chain sequences. We found no evidence for preferential use of certain Vdelta segments in lymph node, skin, spleen, small intestine, large intestine, and blood. The delta chain CDR3 length distribution was very wide in each tissue, which was confirmed by spectratyping. The highly variable CDR3 length was due to the use of up to four diversity (D) segments by one bovine delta chain. Human and murine delta chains contain only one or two D segments. The five functional Ddelta segments that we describe here were identified at cDNA and genomic level, and are the first ruminant D segments described. Fourteen TCR delta chain sequences used novel Vdelta1 segments, and one expressed a novel member of the Vdelta3 family. The number of known functional Vdelta segments in cattle including these new ones is 42 now, but the total number may be much higher. A high number of Vdelta segments in combination with the use of up to four out of five D segments, and the possibility of using non-template encoded (N) nucleotides on either side of these, makes the potential bovine delta chain repertoire much bigger than any known TCR chain. This situation is quite different from the situation in humans and mice, and suggests that the differences between gammadelta high and gammadelta low species in distribution, diversity, and function of gammadelta T cells may be substantial.