HER2 mRNA transcript quantitation in breast cancer

Purpose. The human epidermal growth factor receptor 2 (HER2) status in breast cancer is important for prognostic prediction and the determination of optimal treatment. Current methods rely on protein expression, as determined by immunohistochemistry (IHC), as well as gene amplification as determined by in situ hybridisation (ISH). We explored whether quantitative droplet digital PCR (ddPCR) can be used for the detection and absolute quantitation of HER2 mRNA. Methods. Digital droplet PCR (ddPCR) was performed for HER2 mRNA on 178 formalin-fixed paraffin-embedded (FFPE) breast cancer specimens. HER2 positive, equivocal and negative cases as defined by standard criteria were included and both core biopsies and tissue sections were assessed. Results. HER2 positive cases contained significantly higher levels of HER2 mRNA (169-1,000,000 copies/µl) by ddPCR compared with equivocal (112-139 copies/µl, p = 0.025) and negative cases (6.2-644 copies/µl. p < 0.001). A continuum of transcript quantity was observed but a cutoff of 490 copies/µl distinguished between HER2 positive and negative cases. Results were consistent between core biopsy and tissue sections. Conclusions. ddPCR can be used to quantify HER2 mRNA transcripts in FFPE breast cancer specimens. Our results highlight the potential of ddPCR on FFPE tissue to be used to accurately quantify HER2 transcripts. Validation in large cohorts will be required to determine a clinically applicable cutoff (AU)

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HER2 mRNA transcript quantitation in breast cancer.

PURPOSE: The human epidermal growth factor receptor 2 (HER2) status in breast cancer is important for prognostic prediction and the determination of optimal treatment. Current methods rely on protein expression, as determined by immunohistochemistry (IHC), as well as gene amplification as determined by in situ hybridisation (ISH). We explored whether quantitative droplet digital PCR (ddPCR) can be used for the detection and absolute quantitation of HER2 mRNA. METHODS: Digital droplet PCR (ddPCR) was performed for HER2 mRNA on 178 formalin-fixed paraffin-embedded (FFPE) breast cancer specimens. HER2 positive, equivocal and negative cases as defined by standard criteria were included and both core biopsies and tissue sections were assessed. RESULTS: HER2 positive cases contained significantly higher levels of HER2 mRNA (169-1,000,000 copies/µl) by ddPCR compared with equivocal (112-139 copies/µl, p = 0.025) and negative cases (6.2-644 copies/µl. p < 0.001). A continuum of transcript quantity was observed but a cutoff of 490 copies/µl distinguished between HER2 positive and negative cases. Results were consistent between core biopsy and tissue sections. CONCLUSIONS: ddPCR can be used to quantify HER2 mRNA transcripts in FFPE breast cancer specimens. Our results highlight the potential of ddPCR on FFPE tissue to be used to accurately quantify HER2 transcripts. Validation in large cohorts will be required to determine a clinically applicable cutoff.

Geometry optimization of molecular clusters and complexes using scaled internal coordinates.

Scaled internal coordinates are introduced for use in the geometry optimization of systems composed of multiple fragments, such as solvated molecules, clusters, and biomolecular complexes. The new coordinates are related to bond lengths, bond angles and torsion angles by geometry-dependent scaling factors. The scaling factors serve to expedite the optimization of complexes containing outlying fragments, without hindering the optimization of the intramolecular degrees of freedom. Trial calculations indicate that, at asymptotic separations, the scaling factors improve the rate of convergence by a factor of 4 to 5.

Technique for incorporating the density functional Hessian into the geometry optimization of biomolecules, solvated molecules, and large floppy molecules.

Traditional geometry optimization methods require the gradient of the potential surface, together with a Hessian which is often approximated. Approximation of the Hessian causes difficulties for large, floppy molecules, increasing the number of steps required to reach the minimum. In this article, the costly evaluation of the exact Hessian is avoided by expanding the density functional to second order in both the nuclear and electronic variables, and then searching for the minimum of the quadratic functional. The quadratic search involves the simultaneous determination of both the geometry step and the associated change in the electron density matrix. Trial calculations on Taxol indicate that the cost of the quadratic search is comparable to the cost of the density functional energy plus gradient. While this procedure circumvents the bottleneck coupled-perturbed step in the evaluation of the full Hessian, the second derivatives of the electron-repulsion integrals are still required for atomic-orbital-based calculations, and they are presently more expensive than the energy plus gradient. Hence, we anticipate that the quadratic optimizer will initially find application in fields in which existing optimizers breakdown or are inefficient, particularly biochemistry and solvation chemistry.