Impact of tissue sampling on accuracy of Ki67 immunohistochemistry evaluation in breast cancer

Background: Gene expression studies have identified molecular subtypes of breast cancer with implications to chemotherapy recommendations. For distinction of these types, a combination of immunohistochemistry (IHC) markers, including proliferative activity of tumor cells, estimated by Ki67 labeling index is used. Clinical studies are frequently based on IHC performed on tissue microarrays (TMA) with variable tissue sampling. This raises the need for evidence-based sampling criteria for individual IHC biomarker studies. We present a novel tissue sampling simulation model and demonstrate its application on Ki67 assessment in breast cancer tissue taking intratumoral heterogeneity into account.Methods: Whole slide images (WSI) of 297 breast cancer sections, immunohistochemically stained for Ki67, were subjected to digital image analysis (DIA). Percentage of tumor cells stained for Ki67 was computed for hexagonal tiles super-imposed on the WSI. From this, intratumoral Ki67 heterogeneity indicators (Haralick’s entropy values) were extracted and used to dichotomize the tumors into homogeneous and heterogeneous subsets. Simulations with random selection of hexagons, equivalent to 0.75 mm circular diameter TMA cores, were performed. The tissue sampling requirements were investigated in relation to tumor heterogeneity using linear regression and extended error analysis.Results: The sampling requirements were dependent on the heterogeneity of the biomarker expression. To achieve a coefficient error of 10 %, 5–6 cores were needed for homogeneous cases, 11–12 cores for heterogeneous cases; in mixed tumor population 8 TMA cores were required. Similarly, to achieve the same accuracy, approximately 4,000 nuclei must be counted when the intratumor heterogeneity is mixed/unknown. Tumors of low proliferative activity would require larger sampling (10–12 TMA cores, or 6,250 nuclei) to achieve the same error measurement results as for highly proliferative tumors.Conclusions: Our data show that optimal tissue sampling for IHC biomarker evaluation is dependent on the heterogeneity of the tissue under study and needs to be determined on a per use basis. We propose a method that can be applied to determine the sampling strategy for specific biomarkers, tissues and study targets. In addition, our findings highlight the benefit of high-capacity computer-based IHC measurement techniques to improve accuracy of the testing

Bimodality of intratumor Ki67 expression is an independent prognostic factor of overall survival in patients with invasive breast carcinoma

Proliferative activity, assessed by Ki67 immunohistochemistry (IHC), is an established prognostic and predictive biomarker of breast cancer (BC). However, it remains under-utilized due to lack of standardized robust measurement methodologies and significant intratumor heterogeneity of expression. A recently proposed methodology for IHC biomarker assessment in whole slide images (WSI), based on systematic subsampling of tissue information extracted by digital image analysis (DIA) into hexagonal tiling arrays, enables computation of a comprehensive set of Ki67 indicators, including intratumor variability. In this study, the tiling methodology was applied to assess Ki67 expression in WSI of 152 surgically removed Ki67-stained (on full-face sections) BC specimens and to test which, if any, Ki67 indicators can predict overall survival (OS). Visual Ki67 IHC estimates and conventional clinico-pathologic parameters were also included in the study. Analysis revealed linearly independent intrinsic factors of the Ki67 IHC variance: proliferation (level of expression), disordered texture (entropy), tumor size and Nottingham Prognostic Index, bimodality, and correlation. All visual and DIA-generated indicators of the level of Ki67 expression provided significant cutoff values as single predictors of OS. However, only bimodality indicators (Ashman’s D, in particular) were independent predictors of OS in the context of hormone receptor and HER2 status. From this, we conclude that spatial heterogeneity of proliferative tumor activity, measured by DIA of Ki67 IHC expression and analyzed by the hexagonal tiling approach, can serve as an independent prognostic indicator of OS in BC patients that outperforms the prognostic power of the level of proliferative activity

Genotyping a second growth coast redwood forest : a high throughput methodology

The idea that excitonic (electronic) coherences are of fundamental importance to natural photosynthesis gained popularity when slowly dephasing quantum beats (QBs) were observed in the two-dimensional electronic spectra of the Fenna–Matthews–Olson (FMO) complex at 77 K. These were assigned to superpositions of excitonic states, a controversial interpretation, as the strong chromophore–environment interactions in the complex suggest fast dephasing. Although it has been pointed out that vibrational motion produces similar spectral signatures, a concrete assignment of these oscillatory signals to distinct physical processes is still lacking. Here we revisit the coherence dynamics of the FMO complex using polarization-controlled two-dimensional electronic spectroscopy, supported by theoretical modelling. We show that the long-lived QBs are exclusively vibrational in origin, whereas the dephasing of the electronic coherences is completed within 240 fs even at 77 K. We further find that specific vibrational coherences are produced via vibronically coupled excited states. The presence of such states suggests that vibronic coupling is relevant for photosynthetic energy transfer

Exciton relaxation in nanotubular TPPS4 aggregates in water solution and in polymeric matrix

Exciton properties in meso-Tetra (4-sulphonatophenyl)porphin e (TPPS4) aggregates in water solution and in PVA matrix were investigated at different temperatures by means of transient absorption and fluorescence spectroscopy. Analysis of the steady state and transient absorption spectra shows that excitons delocalized over about 10 TPPS molecules are formed under the aggregate excitation to the higher energy absorption band, however they localize on one or two molecules after relaxation to the lowest energy excited state. The transient absorption and time resolved fluorescence show different relaxation kinetics, which is interpreted in terms of competition between the exciton relaxation to the nonradiative state and quenching by the quenching centers reached during thermally activated exciton diffusion. Exciton-exciton annihilation starts at high excitation intensity when more than I exciton per 20 molecules is created indicating that excitations visit about 20 molecules during their lifetime. The exciton relaxation becomes faster in optically annealed samples, when optically destroyed molecules create additional quenching centers. (c) 2006 Elsevier B.V. All rights reserved